Ensaio de Imunossorventes Ligados A Enzimas (ELISA)

SPRINGER BRIEFS IN APPLIED SCIENCES AND
TECHNOLOGY  FORENSIC AND MEDICAL BIOINFORMATICS
Samira Hosseini
Patricia Vázquez-Villegas
Marco Rito-Palomares
Sergio O. Martinez-Chapa
Enzyme-linked
Immunosorbent
Assay (ELISA)
From A to Z
SpringerBriefs in Applied Sciences
and Technology
Forensic and Medical Bioinformatics
Series editors
Amit Kumar, Hyderabad, India
Allam Appa Rao, Hyderabad, India
More information about this series at http://www.springer.com/series/11910
Samira Hosseini Patricia Vázquez-Villegas
•
Marco Rito-Palomares Sergio O. Martinez-Chapa

•
Enzyme-linked
Immunosorbent Assay
(ELISA)
From A to Z
123
Samira Hosseini Marco Rito-Palomares
Escuela de Ingeniería y Ciencias Escuela de Medicina y Ciencias de Salud
Tecnologico de Monterrey Tecnologico de Monterrey
Monterrey, NL Monterrey, NL
Mexico Mexico
Patricia Vázquez-Villegas Sergio O. Martinez-Chapa

Escuela de Ingeniería y Ciencias Escuela de Ingeniería y Ciencias
Tecnologico de Monterrey Tecnologico de Monterrey
Monterrey, NL Monterrey, NL
Mexico Mexico
ISSN 2191-530X ISSN 2191-5318 (electronic)

SpringerBriefs in Applied Sciences and Technology
ISSN 2196-8845 ISSN 2196-8853 (electronic)
SpringerBriefs in Forensic and Medical Bioinformatics
ISBN 978-981-10-6765-5 ISBN 978-981-10-6766-2 (eBook)
https://doi.org/10.1007/978-981-10-6766-2
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© The Author(s) 2018

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Preface
ELISA: From A to Z was written aiming to provide Readers with a proper coverage
of all aspects of ELISA from fundamentals of immune system and the history of the
analytical assay prior to the invention of ELISA to the materials of choice for
fabrication of the platforms, possible biomolecular interactions, different protocols,
and evaluation parameters among the rest. This book guides Readers through dif-
ferent steps of the analytical assay while familiarizing them with the possible
sources of error in the assay. The book offers detailed insights on the immobi-
lization techniques used for protein attachment, different methods for evaluation
of the assay and calculation of the key important parameters in the analytical assay
such as sensitivity, specificity, accuracy, and limit of detection. Advantages and
shortages of the conventional ELISA as well as different attempts for improvement
of its performance are covered in this book. Merging and intergrading different
technologies with widely known ELISA have opened numerous windows of
opportunity to the advancement of this immunoassay. In that respect, the current
book provides the latest updates on integrated platforms such as ELISpot, plas-
monic ELISA, sphere-/bead-based ELISAs, paper-/fiber-based ELISAs as well as
ELISA in micro-devices.
Monterrey, Mexico Samira Hosseini

Patricia Vázquez-Villegas
Marco Rito-Palomares
Sergio O. Martinez-Chapa
v
Acknowledgements
Authors would like to acknowledge the financial support of Tecnológico de

Monterrey, Mexico, for the special grant (grant number: 002EICII01) awarded by
the Sensors and Devices Focus Group, School of Engineering and Sciences,
Tecnológico de Monterrey, Monterrey, Mexico.
vii
Contents
1 Fundamentals and History of ELISA: The Evolution of the

Immunoassays Until Invention of ELISA . . . . . . . . . . . . . . . . . . . . . 1
1.1 Evolution of the Immunoassays Until Invention of ELISA . . . . . . 1
1.1.1 Side Chain Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.2 Antigen-Antibody Binding Theory . . . . . . . . . . . . . . . . . . 2
1.1.3 Discovery of Antibody Structure . . . . . . . . . . . . . . . . . . . 3
1.1.4 Invention of Radioimmunoassay (RIA) . . . . . . . . . . . . . . . 3
1.1.5 Invention of Enzyme Linked Immunosorbent Assay
(ELISA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.2 Principles of the Immune System . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2.1 Antibody Production in Human Body . . . . . . . . . . . . . . . . 5
1.2.2 Different Types of Antibodies . . . . . . . . . . . . . . . . . . . . . 5
1.2.3 Antigen-Antibody Coupling . . . . . . . . . . . . . . . . . . . . . . . 10
1.3 Biomolecular Interactions Between Antibody and Antigen . . . . . . 11
1.3.1 Hydrogen Bonding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.3.2 Hydrophobic Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.3.3 Ionic Attraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.3.4 Van der Waals Forces . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2 General Overviews on Applications of ELISA . . . . . . . . . . . . . . . . . 19
2.1 Applications of ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.1.1 Food Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.1.2 Vaccine Development . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.1.3 Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.1.4 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.1.5 Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.1.6 Drug Monitoring and Pharmaceutical Industry . . . . . . . . . . 25
2.1.7 Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
ix
x Contents
3 Step by Step with ELISA: Mechanism of Operation, Crucial

Elements, Different Protocols, and Insights on Immobilization
and Detection of Various Biomolecular Entities . . . . . . . . . . . . . . . . 31
3.1 Mechanism of Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.2 Different Elements of the Assay . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.2.1 Solid Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.2.2 Adsorbents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.2.3 Washing Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.2.4 Blocking Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.2.5 Enzymes and Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.2.6 Stopping Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.2.7 Reading Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.2.8 Reading Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.2.9 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.3 Different Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.3.1 Direct ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.3.2 Indirect ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.3.3 Sandwich ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.3.4 Double Sandwich ELISA . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.3.5 Competitive ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.4 Initial Interaction of the Biomolecules with the Surface . . . . . . . . 47
3.5 Immobilization Techniques for Protein Attachment . . . . . . . . . . . . 49
3.5.1 Physical Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.5.2 Immobilization via Entrapment . . . . . . . . . . . . . . . . . . . . . 50
3.5.3 Covalent Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.5.4 Oriented Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . 52
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4 Evaluation of the Detection Results Obtained from ELISA . . . . . . . 57
4.1 Conducting a Reliable Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.1.1 Sources of Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.1.2 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4.2 Key Parameters in ELISA Evaluation . . . . . . . . . . . . . . . . . . . . . 62
4.2.1 Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
4.2.2 Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
4.2.3 Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
4.2.4 Limit of Detection (LOD) . . . . . . . . . . . . . . . . . . . . . . . . 64
4.3 Measurable Units in ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
5 Advantages, Disadvantages and Modifications of Conventional
ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
5.1 Significance of Conventional ELISA . . . . . . . . . . . . . . . . . . . . . . 68
5.2 Shortages of Conventional ELISA . . . . . . . . . . . . . . . . . . . . . . . . 68
Contents xi
5.3 Materials of Choice for Fabrication of ELISA Well Plates . . . . . . 69

5.4 Different Types of ELISA Well Plates . . . . . . . . . . . . . . . . . . . . . 70
5.5 Modified ELISA Platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
5.5.1 ELISA on Coated Platforms . . . . . . . . . . . . . . . . . . . . . . . 72
5.5.2 ELISpot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
5.5.3 Plasmonic ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
5.5.4 Sphere-/Bead-Based ELISA . . . . . . . . . . . . . . . . . . . . . . . 80
5.5.5 Paper-Based ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5.5.6 Fiber-Based ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
5.5.7 ELISA in Micro-Devices . . . . . . . . . . . . . . . . . . . . . . . . . 98
5.5.8 Other Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Chapter 1
Fundamentals and History of ELISA:
The Evolution of the Immunoassays Until
Invention of ELISA
Abstract Current chapter reviews background and history of the immunoassays

until invention of ELISA. In that perspective, important evolutions in the field such
as side-chain theory, antigen-antibody theory, discovery of the antibody structure,
invention of radioimmunoassay (RIA), and invention of enzyme linked
immunosorbent assay (ELISA) are reviewed. The chapter, also describes the
principles of the immune system such as antibody production in human body along
with different classes of antibodies, as well as antigen-antibody coupling and
specificity of such interaction. In that respect, the chapter also demonstrates sources
for biomolecular interaction between the biomolecules. Dominant forces that are
involved in physical interaction of the antigens and antibodies including hydrogen
bonding (H-bonding), hydrophobic interaction, ionic attraction, and Van der Waals
forces such as London dispersion force, dipole-dipole interaction, and ion-dipole
interaction are introduced in great details.
1.1 Evolution of the Immunoassays Until Invention

of ELISA
1.1.1 Side Chain Theory
In 1987 Paul Ehrlich, a German physician and scientist, published a communication

intended to explain how the cells interact with their surroundings, at the time where
little was known about the nature and constitution of living organisms [1]. This
publication was a concluding point to years of investigation that granted Ehrlich the
Nobel Prize in Physiology or Medicine in 1908 together with Élie Metchnikoff for
providing a theoretical basis for immunology. His theory hypothesized that cells
have “side chains” that bind to nutritious elements, which are necessary to keep
them alive. This theory was later known as “side chains theory”, which further
explained the specific interaction between antibodies and antigens in the blood [2,
3]. Ehrlich theorized that antibodies produced by white blood cells act as side
chains on the cell membrane. Side chains, he believed, can bind to the foreign
© The Author(s) 2018 1

S. Hosseini et al., Enzyme-linked Immunosorbent Assay (ELISA),
SpringerBriefs in Forensic and Medical Bioinformatics,
https://doi.org/10.1007/978-981-10-6766-2_1
2 1 Fundamentals and History of ELISA: The Evolution …
bodies via specific chemical structures that they possess on their binding sides. He
later named these chemical structures as “receptors”. Ehrlich proposed that binding
phenomenon between the receptor and an infectious agent was like the perfect fit
between a lock and a key [4]. Ehrlich also hypothesized that cells under the threat of
foreign micro-organisms grow extra side chains to capture the toxin elements.
These additional side chains that were designed to break off into the circulating
blood flow was identified as antibodies. Based on his theory, there exist many side
chains on the surface of white blood cells that can form chemical links with dif-
ferent antigens. For any certain antigen, there is at least one side chain with a
precise binding side that can stimulate the cell to produce and liberate more of the
same antibody type within the blood stream. It was these antibodies that Ehrlich
first defined as “magic bullets”; biomolecular entities that specifically target one
type of toxin or pathogen, no others, without harming the body [4, 5]. Known as
“the man with magic bullets”, Ehrlich described that the receptor’s specificity was
defined prior to its exposure to the antigen, thus it was the antigen that selected the
appropriate receptor [6]. Paul Ehrlich, regarded as the fathers of modern
immunology, was the first to suggest a model for an antibody molecule, a branched
structure consisted of multiple binding sites for capturing foreign agents (antigens)
[7]. This model matched with the ‘lock and key’ theory for enzymes, which was
originally proposed by Emil Fischer [6, 8].
1.1.2 Antigen-Antibody Binding Theory
Since advent of the side chain theory, opposing views by Paul Ehrlich and Jules
Bordet questioned the nature of this reaction. Ehrlich believed that the reaction was
purely chemical, while Bordet claimed that it was a physical adsorption occurring
on one component upon the other. Bordet’s theory suggested that the binding
reaction was of the colloid chemistry type, which relies on the surface character-
istics instead of the chemical nature of the reactants. Bordet, along with other
scientists including Svante Arrhenius and Thorvald Madsen, later described the
reaction as a reversible acid-base neutralization model. Karl Landsteiner, an
Austrian biologist, physician and immunologist, had opposing viewpoints than that
of Ehrlich’s. Landsteiner had later found evidences, which suggested that the
antigenic specificity highly depended on the antigen’s charge outlines, thus proving
that the reaction relied on both, physical surface characteristics as well as the
chemical nature of the antigen.
In 1934 John R. Marrack collected the existing knowledge of this area in his
renowned book “The Chemistry of Antigens and Antibodies” [9]. He also proposed
a new antigen-antibody reaction based upon a crystal lattice model. He suggested
that the relationship between an antigen and an antibody follows the correlation
between the molecules within a crystal network. The crystal molecules are linked
not via chemical valences, as Ehrlich’s proposed, but through the short-range forces
that surround a molecule. Such forces determine the specific selection of molecules
1.1 Evolution of the Immunoassays Until Invention of ELISA 3
in order to build a crystal matrix. Marrack also believed that antigen-antibody

reaction is less specific in comparison to the crystal formation, because the binding
site covers only a small segment of the whole molecule. Marrack suggested that
antibodies have more than one binding site, thus conjoined antigen-antibody would
form a lattice [9]. Marrack continued to work on the antigen-antibody reaction and
published a number of key important research articles, reviews, as well as the
second edition of his book, “The Chemistry of Antigens and Antibodies” [10–12].
Marrack’s ideas in regard to protein chemistry changed over time. Nonetheless, his
finding related to antibody-antigen reaction, which occurred at the right time in
the history of immunoassays, are still valid even eighty-three years after the pub-
lication of his book.
1.1.3 Discovery of Antibody Structure
In 1948, Swedish immunologist, Astrid Fagraeus discovered that plasma B cells are
directly involved in antibody generation [13]. Almost a decade later in 1957, an
Australian scientist, Frank Macfarlane Burnet expanded the ideas of David
Talmage, an American immunologist, and introduced the “clonal selection theory”
[14, 15]. This theory described that when an antigen enters the bloodstream or
tissue fluids it attaches to the surface of the lymphocytes with reactive sites cor-
responding to its antigenic determinants [14, 16]. Consequently, the cell is activated
thus undergo preferential proliferation to produce a variety of descendants. The
proliferation is only limited to the reactive clones that corresponded to the antigenic
determinants. Generated descendants result in liberation of soluble antibody in
bloodstream [14, 16].
The clonal selection theory laid the foundation for other scientists to advance this
field. In 1959, Gerald Edelman and Rodney Porter independently reported their
findings in regard to the molecular structure of the antibody [17, 18]. In 1972, the
Nobel Prize in Physiology or Medicine was jointly awarded to Edelman and Porter
“for their discoveries concerning the chemical structure of antibodies” [19]. The
first structure of an antibody fragment with atomic resolution was presented to the
scientific community in 1973 [20]. This finding was followed by another great leap,
when Georges Köhler and César Milstein in 1975 successfully generated mono-
clonal antibodies by continuous subculture of fused cells [21], which marked the
modern era of antibody research and discovery.
1.1.4 Invention of Radioimmunoassay (RIA)
Radioimmunoassay (RIA) was first introduced by American scientists, Solomon

Berson and Rosalyn Yalow, in 1960 for the measurement of endogenous plasma
[22]. The 1977 Nobel Prize in Physiology or Medicine was later awarded to Yalow
for “the development of the RIA for peptide hormones” [23]. Berson, however, had
not shared the award with Yalow due to his sudden death in 1972.
The radioactive labeled immunoassay techniques rapidly attracted the attention
of the researchers and clinicians. Various methods were subsequently developed
and ensuing decade RIAs for new analytes were reported. In 1968,
“immuno-radio-metric” was developed by Miles and Hales in which the antibodies
were labeled with radioactive agents instead of antigen for measuring insulin in
human plasma [24, 25].
In the initial stages of utilizing RIA as a widely applied immunoassay,
iodine-131 was used as label since there were no alternatives available at the time.
The possible health risks related to the application of radioactive materials were
somewhat reduced when iodine-125 (weak radiation) was introduced to the market.
Yet, health related issues for the laboratory personnel and radioactive wastes
remained as major concerns.
1.1.5 Invention of Enzyme Linked Immunosorbent Assay

(ELISA)
Throughout the evolving process of immunoassay, the idea of using enzyme labels
faced serious skepticism and incredulity. It was believed that enzymes are too large
for biomolecules to be used as labels and their presence would most likely cause
steric hindrance. Such concerned were addressed by careful planning and execution
of the experiments, which demonstrated the feasibility of enzymes as labels. The
success of the enzyme-linked immunoassays in the preliminary stages proved the
skeptics wrong and paved the path for further advancement of immunoassays.
Between 1966 and 1969, Avrameas et al., reported the successful
antigen-antibody coupling by using enzymes such as alkaline phosphatase, and
glucose oxidase among others [26, 27]. Avrameas and co-workers optimized the
labeling process and subsequent coupling via glutaraldehyde chemistry. The
enzyme-labeled biomolecules (antigen or antibody) were used to detect the com-
plimentary biomolecule through immunofluorescence [26, 27]. The Enzyme
immunoassay (EIA) was developed in Organon Research Laboratories in the
Netherlands by Anton Schuurs and Bauke van Weemen.
The enzyme linked immunosorbent assay (ELISA) was conceptualized at
Stockholm University, Sweden, by Peter Perlmann and Eva Engvall in 1971.
Perlmann and Engvall along with Schuurs and van Weemen received the German
scientific award of the “Biochemische Analytik” in 1976 for this invention [24]. The
first ELISA test had demonstrated quantitative measurement of rabbit antibody with
alkaline phosphatase as the reporter label [28]. ELISA has become more successful
than EIA in the commercialization aspect. Various types of solid-phase techniques
were applied for fabrication of microtiter plates [29, 30]. In developed microplates,
the antigen or antibody of interest would be non-covalently bound to the supporting
1.1 Evolution of the Immunoassays Until Invention of ELISA 5
material. The subsequent attachment of the complimentary biomolecules labeled with

the enzyme would lead to the generation of the detection signal [29, 30].
The impact of EIA and ELISA on diagnostic immunoassays and the healthcare
system is virtually unsurpassed. The number of analytical and clinical investiga-
tions performed worldwide based upon the knowledge of EIA and ELISA is
astronomical and the numbers of measurements using these immunoassays for
routine patient care are exceedingly large. There is almost no diagnostic laboratory
around the world, which has not encountered ELISA well plates [24].
1.2 Principles of the Immune System
1.2.1 Antibody Production in Human Body
Immunoglobulins (Igs), also known as antibodies, are manufactured proteins by the

immune system to help fighting against the foreign substances. By definition, any
foreign element that causes the immune system to respond to their presence by
producing antibodies can be considered as an antigen. Antigens are living organ-
isms from a wide variety of families including viruses, bacteria, fungi, chemicals,
pollen grains, or food allergens. Nonetheless, not all antigens are foreign bodies as
some may be produced within the body itself such as cancer cells. Highly antigenic
substances and certain chemicals such as the resin from the poison ivy plant, the
venoms from insect and reptile bites, solvents, formalin, and asbestos would most
likely trigger immune reaction in any type of body. Viral and bacterial infectious
agents also activate an immune response. Transplanted organs can sometimes be
rejected by the body following body’s attempt to differentiate the foreign element
that maintains within the organs or bloodstream. In this particular case, existing
proteins on the surface of the donated organ, can also act as antigens in the body of
the recipient.
1.2.2 Different Types of Antibodies
There exist five different antibody types with their specific configurations and
functions. These antibodies are IgG, IgA, IgM, IgD, and IgE. Capital letters in the
names of these antibodies refer to:
G: in the protein separation process, this antibody appears at the gamma band.
A: in the protein separation process, this antibody appears at beta and gamma
bands. It was initially named b2A and c1A, but later was renamed as alpha globulin.
M: this antibody is named macroglobulin due to its faster sedimentation process
than IgG.
D: in the protein separation process, this antibody appears after beta and gamma
bands. For that reason, its position is referred to as delta (d) band.
E: this antibody is produced only after exposure to certain allergic antigens that are
the cause of erythema disease (skin allergic reaction).
1.2.2.1 Immunoglobulin G (IgG)
Among different types of antibodies, IgG is considered to be the most common type
with approximately 75% of serum antibodies in humans [31]. They are produced
and released into the bloodstream by plasma B cells. IgG is the chief antibody
against microbes that act by covering them to accelerate their removal from the
immune system. Presence of IgG in body provides a long-lasting immunity against
the infectious agents. IgGs are relatively large molecules of tetrameric quaternary
structure (*150 kDa) composed of four peptide chains, two identical c heavy
chains and two identical light chains [32]. Each IgG has two binding sites for
coupling with the antigens (Fig. 1.1). They are mainly found in blood and extra-
cellular fluids.
IgGs can be very mobile. They have the ability to pass from the bloodstream or
between the cells into the organs or even to the skin where they neutralize the
Fig. 1.1 IgG structure [33]

1.2 Principles of the Immune System 7
invading microorganisms. This mobility in the nature of IgGs also allows them to
migrate through the mother’s placenta to her fetus, hence providing a temporary
defense in the body of the unborn child. Even after birth, IgGs, to a certain extent,
are transferred to the child’s body, through breastfeeding. Remaining of the
transferred IgGs via placental transmission will serve the child’s immune system
until the body starts producing antibodies.
1.2.2.2 Immunoglobulin A (IgA)
Immunoglobulin A (IgA) can be found in two isotypes, IgA-1 and IgA-2, which are
both glycosylated proteins [34]. Present in tears, saliva, mucus, and the secretions
of the respiratory, reproductive, digestive, and urinary tracts, IgAs play a vital role
in neutralizing bacteria and viruses and preventing them from entering the body or
accessing the internal organs.
IgAs can also be found in blood serum in very small concentrations. While
possessing some basic similarities, each IgA is specifically designed to defend the
body against a particular type of invader that might attack at different openings of
the body (Fig. 1.2).
Fig. 1.2 Model of human

IgA [35]
1.2.2.3 Immunoglobulin M (IgM)
Composed of five Y-shaped units, immunoglobulin M (IgM) is the largest antibody

among all, thus rather more effective against larger microorganisms (Fig. 1.3).
Present in the blood, IgM functions alike IgG in defending body against antigens.
However, due to its large size, it cannot cross the tissue membranes. IgMs are
generally responsible for an initial protection against invading microorganisms,
whereas the more effective protection will be offered by IgGs produced by the
plasma cells [36]. The ratio of IgM and IgG is in direct correlation with different
stages of the diseases. The number of IgMs at the early stages of the illness is
dominant. As the illness progresses, a greater number of IgGs would be present in
comparison to the IgMs.
Fig. 1.3 Structure of human IgM [37]

1.2.2.4 Immunoglobulin D (IgD)
Immunoglobulin D (IgD) is mostly present at the surface of the B cells as it assists

this class of cells to recognize specific types of antigens. For that reason, the
concentration of IgDs released in the blood serum is very small (0.25%). Figure 1.4
presents the structure of human myeloma IgD.
Normally, IgDs are co-expressed with IgMs on the cell surface. The approximate
molecular mass of an IgD is 185 kDa and it is active for 2.8 days [39]. IgDs exist in
various species from cartilaginous fish to human immunological [40]. IgD’s task in
B cells is signaling cells so they can be activated to take part in defense mechanism.
1.2.2.5 Immunoglobulin E (IgE)
Immunoglobulin E (IgE) antibodies are responsible for allergic reactions. They bind
to the surface of mast cells that often contain substances released during an allergic
reaction. IgEs are synthesized by plasma cells. IgEs consist of four peptide chains,
two heavy chains (e chain) and two light chains (Fig. 1.5) [41].
Their main function is defense against parasites such as Schistosoma mansoni,
trichinella spiralis, plasmodium falciparum, and fasciola hepatica [43–47]. IgEs are
Fig. 1.4 Structure of human

myeloma IgD [38]
Fig. 1.5 Structure of IgE

complexed with omalizumab
[42]
the main products of body in the case of type I hypersensitivity, which is expressed
in various allergic reactions, such as allergic asthma, most types of sinusitis, allergic
rhinitis, food allergies, specific types of chronic urticaria and atopic dermatitis [48].
While IgEs are considered to be the least abundant type (0.05% of antibodies) in
blood serum, they are capable of activating the most powerful inflammatory
responses in body [49].
1.2.3 Antigen-Antibody Coupling
Antibodies mainly possess Y shapes with different upper branches of the Y. These
differences are the structural variation on the amino acid structure at the binding
sites of the antibodies. Due to their specific amino acids, antibodies are able to
identify specific types of antigens from the binding sites that exist on the surface of
antigens. Through an immunological response to the presence of an antigen, the
antibody “wraps” its two “arms” around the antigen’s combining sites and captures
the foreign agent via the “lock” and “key” correlation to stop its progress.
1.2.3.1 Specificity of the Antigen-Antibody Coupling
In the structure of antibodies there are genes that direct the construction of specific
site for binding to the antigens. Such antigen-specific regions are located at the ends
of the Y-shaped arms of antibodies. The antibodies’ action against the antigens
varies with different types of antigens. With each Y-shaped arm, the antibody can
simultaneously attack two antigens. In the case of toxin antigens produced by
pathogenic bacteria the antibody-antigen coupling process occurs along with the
neutralization of the antigen’s toxin components. When attacking viruses (such as
influenza virus), antibodies prevent them from entering other cells. Another
defending mechanism is by calling forth other immune agents known as the plasma
complement system to assist antibodies in defeating the antigens. In the latest
strategy, the antibodies initially coat infectious agents and white blood cells sub-
sequently follow the action by overcoming the invaders.
1.3 Biomolecular Interactions Between Antibody

and Antigen
However, the mechanism of attack and defense might take place, it is important to
understand how the coupling at the actual binding sites occurs. What are the major
forces involved in attracting the antibody and the antigen towards each other? How
specific these interactions are?
The interaction between the antibody and the antigen happens via fundamental
forces between these biomolecular entities including hydrogen bonding
(H-bonding), ionic attraction (electrostatic interaction), hydrophobic interaction,
and Van der Waals forces. In this specific coupling, both sides (the antibody and the
antigen) actively play role. Below, major forces involved in antigen-antibody
interaction are briefly described.
1.3.1 Hydrogen Bonding
Hydrogen boding (H-bonding) is one of the most dominant forces that play a vital
role in physical attachment of different molecules. It occurs when the slight posi-
tively charged hydrogen atoms attract the negatively charged neighboring fluorine,
oxygen or nitrogen atoms (Fig. 1.6). H-bonds can form within each individual
molecule or between the hydrogen atoms of one molecule and electronegative
atoms of another molecule. H-bonding is known to be stronger than normal dipole
forces among the molecules but not nearly as strong as covalent bonds within a
molecule.
Fig. 1.6 Hydrogen bonding arises from the positive charge of the hydrogen atoms and the
negative charge of the neighboring fluorine, oxygen or nitrogen atoms
1.3.2 Hydrophobic Interaction
When nonpolar molecules are placed in the polar environment such as water they
interact with each other through a force that is known as hydrophobic interaction.
A single water molecule can create H-bonds with other water molecules (Fig. 1.7).
Such H-bonds would not form between the nonpolar and the water molecules.
Consequently, the orientation of the water molecules in close proximity of the
nonpolar molecules is quite ordered, which is not in favor of the entropy of the
system. Based on the second law of thermodynamics, the total entropy in an iso-
lated system increases over time. To increase the entropy, the nonpolar molecules
are released from the “cages” and form a nonpolar aggregate that also allows the
water molecules to liberate and possess less oriented position [50].
1.3.3 Ionic Attraction
In their amphoteric forms, biomolecules might react through ionic attraction or

electrostatic interaction. It is the primary interaction that occurs between the
oppositely charged ions, when one atom shares its valence electron(s) with another.
The atoms that gained the electron(s) are negatively charged (anions) and atoms that
lost electron(s) are positively charged (cations).
1.3 Biomolecular Interactions Between Antibody and Antigen 13
Fig. 1.7 The hydrophobic force brings together two non-polar agents
1.3.4 Van der Waals Forces
Van der Waals forces are series of interactions within the atoms and/or molecules.
These attractions and repulsions are the results of polarization fluctuation in the
nearby particles [51]. They are weaker than normal hydrogen, hydrophobic and
ionic bonds. Van der Waals attractions are short-range forces thus such bonds form
only between the closely located particles [51].
1.3.4.1 London Dispersion Force
Named after German-American physicist Fritz London, London dispersion forces are
generally formed between the molecules with instantaneous multipoles. Such mole-
cules do not normally possess the permanent multipole momentums rather offer
temporary dipoles due to the position of their adjacent atoms (Fig. 1.8). Although
weak in comparison to other forces such as H-bonding, hydrophobic interaction or
ionic attraction, London force is the strongest force under the category of Van der
Waals forces. Its strength, however, is proportional to the polarizability of the
molecule, which in turn, varies with the number of electrons and the space they
occupy. London dispersion force dominates the interaction of non-polar molecules.
1.3.4.2 Dipole-Dipole Interaction
Dipole-dipole interaction is the force between two atoms/molecules through their

electric or magnetic dipole momentums. When two dipolar molecules interact, the
negative side of the polar molecule interact with the positive side of the second
polar molecule (Fig. 1.9), thus dipole-dipole interaction occurs. This force is known
to be weaker than London dispersion force.
Fig. 1.8 London dispersion forces

1.3 Biomolecular Interactions Between Antibody and Antigen 15
Fig. 1.9 Dipole-dipole interaction
Fig. 1.10 Ion-dipole interaction
1.3.4.3 Ion-Dipole Interaction
As its name suggests, an ion-dipole interaction occurs when an ion and a neutral
molecule with temporary dipole attract each other (Fig. 1.10). This force particu-
larly forms in the solutions of ionic compounds within polar liquids. While known
as the weakest force among the family of Van der Waals forces, ion-dipole
attraction can become stronger if the charge on the ions increases, or if the mag-
nitude of the dipole in the polar molecule increases.
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Chapter 2
General Overviews on Applications
of ELISA
Abstract Current chapter reviews the applications of ELISA in various different

fields including food industry, vaccine development, immunology (autoimmunity
and humoral immunity), diagnosis (pregnancy, cancer and infectious diseases),
toxicology, drug monitoring, pharmaceutical industry, and transplantation.
Different examples related to each area are explained. ELISA was found to play
major roles in all the mentioned disciplines.
2.1 Applications of ELISA
Following the history of the immunoassays until the invention of ELISA, different
types of biomolecular entities involved in the assay procedure and the interaction
types between such molecules, which were described in previous chapter, the current
chapter provides a general overview on the broad spectrum of ELISA’s applications.
Several examples for applications of this widely applied technique in the areas of
food industry, vaccine development, immunology, diagnosis, toxicology, drug
monitoring, pharmaceutical industry, and transplantation are briefly reviewed.
2.1.1 Food Industry
ELISA plays a major role in food industry. It is the main platform for identifying
food allergens such as those present in milk, peanuts, walnuts, almonds, and eggs
[1]. Peng et al. developed a monoclonal antibody based sandwich ELISA for the
detection of ovalbumin in food, which is the most frequent cause of food allergy,
especially in children. ELISA can also be employed to corroborate the authenticity
of the food products [1]. This technique is of great help to avoid possible economic
losses caused by fraudulent substitution [2]. In the case of meat and meat-based
products, ELISA has proven to be a reliable technique that provides careful mon-
itoring of the product, especially when religious considerations in the choice of food

https://doi.org/10.1007/978-981-10-6766-2_2
20 2 General Overviews on Applications of ELISA
are concerned [2]. ELISA is also an essential technique for quality control of fish,
milk (as well as their sub products), genetically modified foods, irradiated foods, or
other harmful food components that can be transferred to human, such as bovine
spongiform encephalopathy [2]. Non-meat proteins such as soybean have valuable
nutritional properties. Nonetheless, due to the similarity to the mean product, they
are seldom added to the meat products undeclared. Careful monitoring of the
products with ELISA prevents such adulteration [2]. Unethical competitions for
higher economic gain often lead to the potential health hazard through the con-
sumed food and beverage.
Production of ELISA kits for food industry applications is challenging as a
selection of adequate control and standard samples is necessary to carefully cali-
brate the assay [3]. Additionally, ELISA can target different types of analytes in the
same food sample, thus the manufacturers should provide a complete set of kit
components for the potential target biomolecules [3].
2.1.2 Vaccine Development
ELISA serves as a great candidate for vaccine development. The sera sample from
immunized animal or human model can be tested to detect the presence of anti-
bodies against certain types of antigens, which were intentionally injected to the
host [4]. Normally different antigens are used to produce immune reactions in the
host, among which those that elect higher protection response with less adverse
effects can be selected [5].
The main challenge in application of ELISA in vaccine development is the
appropriate choice of positive and negative controls. In the experimental stage of
vaccine development and when dealing with unknown samples, it is particularly
difficult to achieve high analytical precision [5]. Nonetheless, ELISA technique has
proven to have a unique position in profiling of elicited immune responses, which
are widely performed for vaccine trials around the world [6, 7].
2.1.3 Immunology
The defender of the body, the immune system, can operate in cellular or humoral
(innate or adaptive) modes [8]. Measuring and monitoring the changes of the
immune response underlay the foundation for understanding immune disease.
Various studies have demonstrated ELISA as the gold standard method that is rapid
and cost-effective for such measurements and monitoring [9].
A great number of examples for ELISA applications in immunology are
reported, while some efforts were directed to optimize ELISA protocols further and
to validate/establish their accuracy, sensitivity and specificity to support the clinical
practice [10]. In this section, we describe some of these examples.
2.1 Applications of ELISA 21
2.1.3.1 Autoimmunity
Multiple infections, environmental factors, and mild immune system failings trigger
an autoimmune response through uncontrolled immune system activation [11]. The
body produces antibodies in response to different types of external pathogens.
These external pathogens can be particles or epitopes that penetrated the cells but
later have become part of the cells’ structure. In such situation, antibodies react
against the cells themselves thus resulting in an immunodeficiency-oriented
phenotype.
Pulmonary alveolar proteinosis (PAP) is an example of an autoimmune disease,
which is characterized by accumulation of surfactant in the alveolar system [10].
This disease has found to be associated with autoantibodies that are produced
against the granulocyte/macrophage-colony stimulating factor (GM-CSF). When a
pathogen enters the respiratory system, GM-SCF is needed in order to regulate the
infection [10]. To study PAP, radiology and cytology analyses can be of great
help. Additionally, ELISA can assist clinicians in identifying the thresholds asso-
ciated with the risk of PAP.
Bullous pemphigoid, an acute/chronic skin illness, is another example of
autoimmune disease known for its high mortality rate. Typically, it can be diag-
nosed through its clinical features and histopathological analysis. However, ELISA
has shown high sensitivity and specificity in detecting circulating autoantibodies
against the corresponding epitope to this illness [12]. Paper-based ELISA platforms
have demonstrated a rapid, cost-effective, and convenient diagnosis/monitoring
method of this disease [12].
The incidence of autoimmune diseases among individuals living with human
immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/
AIDS) have also been studied by variety of ELISA-based analytical platforms [13].
It has been found that infected patients with HIV had higher risk of developing
Sjögren syndrome, psoriasis, systemic lupus erythematosus, autoimmune haemo-
lytic anaemia and uveitis [14]. ELISA assay has revealed that low IgG antibody
responses to the pathogens may be the fundamental disorder in this kind of diseases
[15]. A number of ELISA based platforms for HIV detection were marketed and are
available for the end users in clinics and hospitals.
2.1.3.2 Humoral Immunity
ELISA has shown great potentials in studying the humoral response of the immune
system towards different classes of infections as well. Humoral immunity response
involves the substances (antibodies and other components) that exist in the body
fluids [16]. Monitoring and measurement of these components are of great
importance [16].
As an example, leprosy is a treatable infection that is considered to be a major
issue in developing countries [17]. ELISA has been widely applied to predict the
progression of this disease in infected individuals. In particular, paper-based ELISA
platforms and lateral flow test strips served the clinical practice for detection of this
disease in endemic areas with limited access to the centralized laboratories [17].
ELISA has also been employed for the detection of plasmatic antibodies against
antigenic peptides of human endogenous retrovirus (HERV), in line with the eti-
ology of multiple sclerosis [18]. The careful monitoring of the immuno biomarkers
associated with HERV is of great importance for analyzing the progression of the
illness, especially during the interferon beta (IFNb) therapy [18].
2.1.4 Diagnosis
In the area of diagnosis, ELISA has proven to be a capable platform applied

worldwide for detecting variety of disease types in human and animals. A number
of different commercial ELISA kits are available in the market for detection of HIV
[19], Influenza [20], Dengue fever [21–24], Ebola [25], Chagas disease [26],
Leishmaniasis [27], Lyme disease [28], West Nile virus [29], among others. Even in
plants pathology, ELISA technique is attracting increasing attention. ELISA has
successfully overcome the drawbacks of the previous serological analyses per-
formed in phyto-diagnosis [30].
In this section, a brief summary on the current diagnosis applications of ELISA
is provided.
2.1.4.1 Pregnancy Test
A number of different biomolecular entities including human chorionic gonado-

tropin (hCG), luteinizing hormone (LH), follicle stimulating hormone (FSH), estriol
(E3), and thyrotrophin-stimulating hormone (TSH) [31] can be expressed due to the
pregnancy. ELISA can detect some of these proteins from the maternal blood,
saliva, or urine at the early stages of the pregnancy [32]. HCG is one of the common
hormones that can be detected by ELISA during the first month after fertilization.
Another biomolecule associated with pregnancy is estriol (E3) that can be detected
with ELISA in the saliva at the 6th week of pregnancy.
Specific ELISA pregnancy tests were developed for animals as well [33]. ELISA
can also be used as a reliable method for measuring congenital infections such as
HIV or toxicoplasmosis during the pregnancy [34, 35].
To maximize detection sensitivity and accuracy for identifying pregnancy
complications in the early stages, marker panels were developed, which are capable
of monitoring/measuring multiple markers in the samples. The target biomolecules
are activin A, inhibin A, progesterone, A disintegrin and metalloprotease-12
(ADAM-12), pregnancy-associated plasma protein A (PAPP-A), pregnancy specific
B1-glycoprotein (SP1), placental-like growth factor (P-LGF), vascular endothelial
growth factor (VEGF), glycodelin (Glyc), and human corionic gonadotropin (hCG),
among others [36].
2.1.4.2 Cancer Detection
Highly sensitive detection of cancer provides with the early stage diagnostic, which
is crucial for patient survival. Cancer biomarkers, however, are some of the most
challenging biomolecular entities as target analytes. Advancements of ELISA
technique has promised its application in detection of cancer biomarkers.
Zhou et al. applied a gold nanoparticle layers (GNPL) in ELISA to amplify the
detection signal, which provided with a lower limit of detection (LOD). In this
technique, plasma spiked with carcinoembryonic antigen (CEA) were used as the
representative biomarker, proving that a straightforward and cost-effective
GNPL-based sandwich ELISA holds a clinical relevance.
Vazquez-Villegas et al. integrated chemically designed poly methacrylate
microspheres into the routine ELISA to detect microRNA-21 within this very
convectional platform that is typically incapable of microRNA recognition.
Presence of active functional groups on the surface of these spheres highly pro-
moted analyte-surface interaction via variety of physical forces, which has subse-
quently resulted in the detection of microRNA-21. This exogenous miRNAs in
blood serum were found to be inversely correlated to breast cancer incidence in
humans [37].
Sometimes the tested specimens are hard to be obtained. Therefore, even the
small sample volume is highly valuable. For instance, in the case of ovarian cancer,
the glycoprotein CA125 present in the serum is the appropriate choice of biomarker
for timely detection [38]. Scholler et al. developed a cost-effective ELISA-based
platform for CA125 detection that requires a few microliters of serum. This
microsphere integrated sandwich assay incorporates CA125 with other markers and
uses the immobilized antibodies on the surface of the spheres to capture the target
proteins. This platform has proven to be comparable to the commercially available
detection techniques, while requiring only 15 lL of the sample [38].
2.1.4.3 Detection of the Infectious Diseases
Even to date, ELISA-based infectious serology marks one of the most reliable
means for accurate diagnosis and prognosis. There is a broad range of developed
and marketed state-of-the-art assays for the detection of infectious agents. ELISA
has offered a high throughput detection in three classes of infectious diseases:
1. Sexually Transmitted Diseases (STDs) is a class of infectious diseases that has
targeted adults in developing countries. A number of different ELISA platforms
were designed and commercialized for sensitive and selective detection of STDs
including HIV, hepatitis, syphilis, chlamydia.
2. Regional or endemic diseases, often referred to as tropical diseases are wide
spread in tropical and subtropical regions. They might appear to be mild/
symptomless with serious and chronic consequences. Dengue, chagas,
borreliosis, and yellow fever are some of the examples of this class of fatal
diseases among others. While existing techniques lack timely detection of such
illnesses, advances in ELISA platforms have shown great promises in offering
early and effective diagnosis.
3. TORCH refers to Toxoplasma, “Other infections”, Rubella, Cytomegalovirus,
Herpes simplex, which is a group of viral pathogens that may result in prenatal
infections. This class of infectious diseases can be a potential threat to the
unborn children. Illnesses such as syphilis, hepatitis B, Epstein-Barr virus,
varicella-zoster virus, HIV fall under the category of “Other infections” that
might also result in serious consequences for the fetus. Commercialized ELISA
platforms successfully target these infectious agents in the current clinical
practice.
In Chap. 5 of this book, a thorough review on the latest advances of ELISA in
the area of diagnosis will be provided.
2.1.5 Toxicology
Toxicology involves studying the adverse effects of chemical compounds on the

living organisms. This area covers diagnosis and curing the effects of toxins
(antigenic agents from plant or animal origins) as well as toxicants (toxic substances
released into the environment). The correlation between the dosage of the toxic
materials and its effects on the exposed organism, routes of the exposure, origins of
the toxic substances and characteristics of the affected organs are the major con-
centrations in toxicology study. Few of the examples, among others, are mentioned
in this section as follows:
Competitive ELISA has a long history of being applied for detection of aflatoxin
B1, one of the known toxins from rice. Developed immunoassay for aflatoxin
monitoring is rapid, and straightforward, while offering desirable specificity and
sensitivity [39]. Competitive assay developed for this purpose was also reported to
have a considerably long shelf life (at least 12 months at room temperature) [39].
In another study, Bio-Quant direct ELISA was employed for regular screening of
drugs such as amphetamine and methamphetamine in biological fluids [40]. To
analyze cross-reactivity of the compounds, predetermined concentrations of com-
mon amphetamine-type substances, designer analogues, and putrefactive amines
were measured. Obtained data indicated that the Bio-Quant direct ELISA technique
was rapid and reliable for the presumptive screening of amphetamine and
methamphetamine in forensic samples [41].
2.1.6 Drug Monitoring and Pharmaceutical Industry
ELISA techniques have also found variety of applications in screening certain

classes of drugs in plasma. The conventional therapeutic drug monitoring
(TDM) strategies monitor drug levels in the plasma samples [42]. TDM also pro-
vides information regarding the treatment procedure allowing physicians to
examine if the medication is present in patient’s body [43]. However, the con-
ventional TDM technique is expensive and technically demanding.
As an alternative strategy, ELISA-based TDM has been introduced as a facile
and cost-effective method for measuring the concentrations of the drugs in plasma
samples. In particular, the aim of the study was to assess the plasma lopinavir
(LPV) levels of by TDM-ELISA in youths with perinatally acquired HIV [44].
In another study, an ELISA-based platform was employed for monitoring the
level of antidrug antibodies in patients receiving treatment for rheumatoid arthritis
and inflammatory bowel disease [45]. This strategy shows that ELISA incorporates
those features identified in the literature as important for the accurate analysis of
antidrug antibodies providing a relatively simple and low-cost assay for therapeutic
drug monitoring [45]. Offering a high specificity in immunoassays for therapeutic
proteins is an important consideration, when such assays are used to assess the
pharmacokinetics, bioequivalence and toxicokinetics studies [46, 47].
2.1.7 Transplantation
When transplantation is required, the pre-transplant cross-matching test represents

one of the most important steps for a successful relocation of the organ.
Complement-dependent cytotoxicity cross-match (CDC-CM) assay was developed
almost four decades ago to assess the compatibility of the given organ into the body
of the receptor. Selecting recipients without donor-specific antibodies is of crucial
importance to increase the survival rate in the patients who are subject to the
transplantation. In particular, CDC-CM plays a vital role for the recipients who
undergo treatments with special drugs/therapeutic antibodies or suffer from
autoimmune diseases.
CDC-CM test, however, requires lymphocytes isolation from the donors, which
typically has a limited availability [48]. ELISA-based cross-matching test has
demonstrated to be an adequate substitute procedure for such analysis. Schlaf et al.
reported an ELISA-based cross-matching approach for identifying donor-specific
anti-human leukocyte antibodies (HLA) by using deep-frozen blood or spleen
detergent lysate from a deceased donor [49]. This strategy permits the
cross-matching comparison to be frequently performed between the recipients’
anti-HLA antibody and the donors’ historically identified HLA types to monitor any
incompatibility between the examined samples [49].
ELISA-LATM (One Lambda Inc.) assay is another example of the ELISA-based

technology that has been tested on patients who underwent renal transplants. All
patients participated in this study had their pre-transplant sera analyzed by the
LATM assay prior to the actual transplantation. The clinical, biochemical, and
histopathological examinations were then performed to follow-up the progress of
the recipients [50]. From a total number of 164 studied patients, 149 received
organs from live donors and 15 from the deceased donors. In general, 36% of the
patients have experienced the organ rejection. This study shows that 100% of the
patients for whom ELISA-LATM test predicted the rejection chance, in fact
rejected the donor’s organs, while there were a number of patients for whom
ELISA-LATM fell short in predicating the chance of rejection. In an over view,
however, the technique proves promising, particularly in the case of transplantation
from the cadavers [50].
In the case of liver transplantation, the survival rate for ABO-incompatible
(ABO-I) recipients is relatively high. It is, therefore, of a great importance to
develop effective and rapid measurement of anti-A and anti-B antibodies in patients
prior to receiving the organs [51]. A novel class of ELISA has been developed to
monitor such antibodies in the recipients. Proposed ELISA method proved potent in
measuring anti-A and anti-B antibodies at the earlier stage than the previously
applied technique, agglutination. Therefore, this strategy is capable of contributing
to a timely treatment of humoral rejection due to ABO-I [51].
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Chapter 3
Step by Step with ELISA: Mechanism
of Operation, Crucial Elements, Different
Protocols, and Insights on Immobilization
and Detection of Various Biomolecular
Entities
Abstract Current chapter describes the essential components of ELISA including

the solid phase, the adsorbents (different types of target biomolecules),
and the washing and blocking agents used in assay procedure. The chapter
also reviews widely applied enzymes and substrates with their specific character-
istics. To complete the assay, the chapter offers information regarding the stopping
procedure and readout techniques such as colorimetric, fluorescence and lumines-
cence, along with their reading instruments. To secure a high specificity, the chapter
describes protocols for conducting different types of controls in the assay proce-
dure. These controls are namely: positive, endogenous, negative, standard, and
spike controls. The chapter subsequently describes available ELISA protocols
including direct, indirect, sandwich, double sandwich, and competitive assays.
Finally, this chapter is dedicated to reviewing immobilization techniques including
physical, covalent, oriented strategies as well as immobilization via entrapment. In
the case of covalent immobilization of the biomolecules, protein attachment via
zero-length cross linkers and spacers (linear or branched) are described.
3.1 Mechanism of Operation
Conducting an ELISA includes at least one antibody with specific immune response
against the antigen of interest. The bonding process of antigen to the solid substrate
can occur via different ways depending on the applied protocols. The detectable
agents (antigens) can be immobilized on a supporting substrate either through direct
non-specific adsorption or by specific capturing by another antibody. After
immobilization of antigen, the surface is normally coated with a blocking agent to
reduce the chance of non-specific bonding in the next steps of the assay [1].
Through a preferred protocol, the labeled antibody is added and subsequently

https://doi.org/10.1007/978-981-10-6766-2_3
32 3 Step by Step with ELISA: Mechanism of Operation, Crucial …
coupled with the antigen forming a complex that leads to the detection signal. The
labeled antibody can be enzyme-linked through a covalent method, or can be
further detected by a bio-conjugated secondary antibody. Between each step, the
plate is thoroughly washed with a washing buffer that is a mild detergent solution to
remove unbound proteins without leaving any negative effect on the bound proteins
[2]. The substrate is then added to the plate to develop the enzymatic reaction and to
produce the color from which the signal intensity can be measured. This signal (in
most cases) is a direct function of the antigens that are present on the surface [3–8].
Table 3.1 presents different concentrations (quantities) of the analytes to different
types of diseases. The Table also shows the applicable techniques for detection of
such diseases along with different means for signal readout. As can be seen, ELISA
remain as one of the most dominant techniques in bio-recognition of a wide range
of analytes.
Table 3.1 Analyte concentrations, the corresponding diseases and the readout techniques
Analyte Quantity Detection Read-out means Corresponding Ref.
concentration techniques diseases
Mili-molar 10−3 Paper strips Colorimetric/ Diabetes [9]
(quadrillion) enzymatic
chemistry
Micro-molar 10−6 ELISA Colorimetric/ Infectious [10]
(trillion) visual disease (HIV,
Nano-molar 10−9 Fluorescence dengue, [11]
(billion) chikungunya,
yellow fever,
Pico-molar 10−12 Luminescence [12]
etc.)
(million) Carbon Electrochemical [13]
nanotubes
Femto-molar 10−15 Nano-wires Electrical Alzheimer’s, [14]
(thousand) Bio-barcodes Scanometric/ prostate and [15]
surface plasmon breast cancers,
fluorescence cardiovascular
spectroscopy diseases
Quantum Fluorescence [16]
dots (micro/ resonance
nano energy transfer
particles) (FRET)
Atto-molar 10−18 Immuno-PCR Fluorescence [17]
(ten) Metal Scanometric or [18]
nanoparticles light scattering
Zepto-molar 10−21 Isothermal Fluorescence [19]
(<1) ramification
amplification
3.2 Different Elements of the Assay 33
3.2 Different Elements of the Assay
Even after four decades from its invention, ELISA continues to play a vital role in
clinical practice. This widely applied procedure has several different components.
In this section, main components of ELISA are described in a great detail.
3.2.1 Solid Phase
Different classes of ELISA carriers are commercially available in several formats

and supporting materials. The well-known platforms include microtiter well plates,
small balls and small tubes. Regularly, microplates are designed in the form of 96
well plates. These common well plates for detection purposes have 8 12 wells; a
characteristic that can be used for analysis of a large number of samples at the same
time. The 96-well plates are typically made from polystyrene (PS), which is a
cost-effective, highly transparent, and relatively hydrophilic material suitable for
protein adsorption. Alike other suitable candidate such as poly methyl methacrylate
(PMMA) and polycarbonate (PC), PS is a plastic material that can be used via
molding technique to fabricate varied shapes and designs of the detection platforms
including 96-well plates. PS, PMMA and PC are also great candidates for fabri-
cation of microfluidic platforms [20].
Solid support can also be available in the pre-coated form containing specific
types of antibodies or antigens present on the surface. Coated platforms should be
stored in low-temperature (2–8 °C) under dry condition up to six months.
3.2.2 Adsorbents
In the process of performing immunoassays, several different types of adsorbents

might be used depending on the ELISA protocols. Antibodies (produced in
response to antigenic stimuli) and antigens are the main adsorbents used in ELISA.
In most protocols, conjugated IgG, purified through affinity chromatography
techniques, are used. These antibodies are specific in immune-competence thus they
can involve in enzymatic reactions at low concentration. It is sometimes the case,
that an anti-species has to be use instead of normal antibodies. Anti-species are the
results of injected proteins into another species such as inserted guinea pig serum
into a rabbit’s body that causes the production of rabbit anti-guinea pig antibodies.
3.2.2.1 Target Biomolecules
A wide variety of biomolecular entities from different classes can be detected

through different ELISA protocols. Table 3.2 offers a general overview on the type
of the biomolecules detectable by ELISA including human antibodies, deoxyri-
bonucleic acids (DNAs), ribonucleic acid (RNA), hormones, tumor markers,
genetically modified organisms, infectious agents, as well as air and food allergens.
Table 3.2 also provides an example for each biomolecular family along with
specific characteristics of the biomolecule and its antigenic determinant.
3.2.3 Washing Agents
The washing process is one of the essential steps in every ELISA protocol. By
charging and emptying the wells with specific amount of washing buffer (typically
200 µl), unreacted reagents can be removed from the wells hence the accuracy of the
assay increases as only reacted analytes remain in the well plate (Fig. 3.1, step 2). It
is important to note that for the immunoassays only particular types of detergents
with carefully controlled concentrations can be used. The excess of applied detergents
dosage in preparation of the washing buffers can cause the biomolecules to denature
and lose their activities. Typical washing buffers consist of small concentration of
non-ionic detergents such as Twin-20 in phosphate buffered saline (PBS) solutions.
3.2.4 Blocking Agents
In order to achieve high selectivity and to avoid non-specific binding, blocking step
is required after immobilization of the first biomolecule on the well’s surface
(Fig. 3.1, step 3). This step is normally performed by using blocking buffer (200 µl)
for the period of 2 h. Common protein blockers include bovine serum albumin
(BSA), newborn calf serum (NBCS), casein, non-fat dry milk, normal whole serum,
and fish gelatin. Unlike washing agents, protein blockers attach to the surface
permanently.
3.2.5 Enzymes and Substrates
Enzymes are macro-biomolecular catalysts that can accelerate chemical reactions

with other types of molecules known as substrates. Upon reaction, the enzyme
chemically modifies the substrate into a measurable product thus signal readout is
possible. Applied enzymes in ELISA should meet basic requirements including
Table 3.2 Target biomolecules detectable in ELISA, biomolecular characteristics and antigenic determinant
Detectable Examples Characteristics Antigenic determinant Ref.
biomolecules
by ELISA
Human IgG Contains four polypeptide chains including two identical Expressed isotopic or allotopic determinant at different [21,
antibodies heavy (H) chains and two identical light (L) chains, linked regions of this IgG (i.e. hinge region, light chain or heavy 22]
together by inter-chain disulfide bonds chain)
DNA HPV-16 Palindromic DNA sequence correspond to the binding site The sequence of the A chain [23]
of the E2 transcriptional regulator of the human papilloma (GTAACCGAAATCGGTTGA) of the synthetic
virus, which can be used as antigen to produce a specific antibody corresponds to the binding site of HPV-16
anti-DNA response genome
RNA BC-200 BC-200 RNA (brain cytoplasmic 200 RNA) is a small The region of BC-200 RNA detected by the antibody is [24]
non-coding RNA expressed in cancer tumors such as localized among the residues of its nucleotides 60–110
breast, cervix, esophagus, lung, ovary, parotid, and tongue
carcinomas
Hormones HCG Human chorionic gonadotropin (hCG) is a placental Epitopes exist close to the receptor binding region [25,
hormone that stimulates secretion of the comprising the loop region ß Cys93-Cys100 and the a 26]
pregnancy-sustaining steroid progesterone. It is a member C-terminal peptide
of the family of glycoprotein hormones, which are
disulfide-rich heterodimers, with typical a-chains and
b-chains specific to their particular G-protein linked
receptors
Tumor VEGF Vascular endothelial growth factor (VEGF) is a The functional epitopes are: Met81, Gln89, Gly92, Arg82, [27,
markers multifunctional cytokine expressed at high levels by many Ile83, and Gly88. The most prominent structural feature at 28]
tumor cells in animals and humans the interface is the burial of Gly88 at the
antibody-combining site
Genetically EPSPS Modified protein from the CP4 strain of agrobacterium in The peptide sequences are identified as the B-cell epitopes [29]
modified soybeans, 5-enolpyruvylshikimate-3-phosphate synthase
organisms (EPSPS), is a genetically modified agent made at
Roundup Ready® soybeans
(continued)
35
Table 3.2 (continued)
36
Detectable Examples Characteristics Antigenic determinant Ref.

biomolecules
by ELISA
Infectious DENV An enveloped dengue virus (DENV) is from the Envelope dengue virus serotype 3 (EDENV3) is known to [30]
agents Flaviviridae family with the approximate size of 40– possess surface proteins with the single antigenic site that
60 nm, an isometric nucleocapsid of 25–30 nm and a includes residues such as K310, I312, P332, L389, and
*10.7 kb, linear, positive-sense RNA genome W391
Air allergens Dust mite Der p 2, with molecular weight of 25 kDa, is a cysteine Hydrophilic residues of the protein at positions 44–46 and [31]
protease from dust mite that is the responsible factor for 100 of the peptide sequence are the antigenic determinants
the antigenic response
Food Peanuts 614AA protein is a trimeric form heat stable allergic agent 23 different linear IgE-binding epitopes are located [32]
allergens (65 kDa) that comprises 12–16% of the total proteins in throughout the length of the protein
peanut extracts
3 Step by Step with ELISA: Mechanism of Operation, Crucial …
Fig. 3.1 Breakdowns of double sandwich assay, an overview on the performed steps in ELISA
high purity, high conversion rate, desirable specificity, high stability as well as
preserved activity and catalytic capacity after conjugation.
For every class of enzymes, a specific type of substrate is required. Substrates
have to offer ease of preparation and storability. Additionally, substrates should
react with relatively low concentrations of enzyme to promote the efficiency of the
assay.
3.2.5.1 Different Types of Enzyme
Among different enzymes, horseradish peroxidase (HRP) and alkaline phosphatase

(AP) are two of the most popular enzymes used for labeling the antibodies. These
enzymes are relatively inexpensive, and commercially available, while offering
high substrate turnover. The conjugation of the enzymes to the antibodies occurs
through the similar forces that play role in antibody-antigen interactions (described
in Chap. 1, Sect. 1.3). Table 3.2, summarizes detailed information about these
widely applied enzymes.
Horseradish peroxidase (HRP)
Horseradish peroxidase (MW * 44 kD) is one of the suitable candidates for con-
jugation to antibodies at the ratio of 4:1 (enzyme:antibody). HRP is a combination
Table 3.3 Detailed information about widely used enzymes

Enzyme Horseradish Alkaline phosphatase ß-D-galactosidase
peroxidase
Molecular weight 40,000 86,000 465,000 (4
(MW) subunits)
Enzyme to 4:1 2:1–3:1 1:1
antibody ratio
Expensiveness $ $$$ $$
Advantage – No steric hindrance – Signal enhancement – Enhanced
– Commercially – Increased sensitivity reaction rate in
available the presence of
alcohols
– Suitable for
assays in
hydrophobic
membrane
surfaces
Disadvantage – Incompatible with – Steric hindrance/high – Suffers from
many preservatives dose hook phenomenon antibody-induced
such as sodium – Inactivated by chelating inhibition
azide agents, acidic pH (<4.5)
and inorganic phosphates
Recommendation – Requires sterilized – Use of chelators and – Mostly used in
buffer acidic pH interferes the cell-related
reaction ELISA
procedures
of a porphyrin protein comprised of a colorless glycoprotein known as apoenzyme

(enzyme, with the maximum absorption peak at 275 nm) and a dark brown iron
porphyrin ring compound known as prosthetic group (heme, with the maximum
absorption peak at 403 nm). Due to the small size of this enzyme, it rarely causes
steric hindrance with neighboring proteins. It is inexpensive while varied types of
corresponding substrates, yielding soluble or insoluble reaction products, are
commercially available for this enzyme (Table 3.3).
The major drawback associated with HRP is its incompatibility with various
preservatives including sodium azide. Such preservatives are often used to reduce
the chance of microbial contamination in typical biological buffers. Even in low
concentrations, sodium azide can disrupt HRP’s activity. Other interfering
elements/compounds with HRP’s activity are metals found in water as well as the
endogenous peroxidases present in biological specimens. Using sterilized buffers
prepared with ultra-pure water and pretreating specimens that are suspected of
having high peroxidase levels can reduce such undesirable effects.
Alkaline phosphatase (AP)
Extracted from Escherichia coli, AP is approximately double in size
(MW * 80 kD), when compared to HRP. Due to its size, fewer number of enzymes
can be conjugated to the antibody of interest. This also means that the large AP
molecule can potentially cause steric hindrance in close proximity of the

antigen-antibody complexes. This phenomenon, in turn, reduces the efficiency of
the enzyme. Although more expensive, AP is reported to be more stable and
produces lower background signal in comparison to HRP. AP, however, can be
inactivated when exposed to chelating agents, inorganic phosphates, and/or acidic
environment (pH < 4.5). This enzyme can only be used in alkaline environment
thus any type of buffer with acidic nature must not come to contact with the enzyme
linked antibodies once they are immobilized inside the wells. For that reason,
chelators such as ethylenediaminetetraacetic acid (EDTA) with acidic nature can be
used as convenient and inexpensive stopping reagent for AP reactions.
ß-D-galactosidase
ß-D-galactosidase is a glycoside hydrolase enzyme with the approximate molecular
weight of 465 kDa that catalyzes through the cleavage of a glycosidic bond. Several
different substrates of b-galactosidases are known including ganglioside GM1, lac-
tosylceramides, lactose, and various glycoproteins. This enzyme has the major
application in the cell related ELISA protocols due to the lower endogenous enzyme
activity in varied cell types and low binding affinity between b-galactosidase and
antibodies conjugated to lymphoid and non-lymphoid cells [33, 34]. The cell-ELISA
protocols are versatile involving many cell types, including adherent and
non-adherent cells. b-D-galactosidase linked antibodies can be produced for such
assays by using the heterobifunctional crosslinking agents such as sulfo-SMCC
(sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate) [35].
3.2.5.2 Different Types of Substrate
As a result of a specific reaction with enzyme, substrate provides different means

for quantification of the analyte of interest in the assay. The substrate yields in a
colored, fluorescent, or luminescent measurable product that is proportional to the
amount of enzyme conjugated antibody. Such products can be soluble and/or
insoluble. Commonly used substrates that produce insoluble products are TMB
(3,3′,5,5′ tetramethylbenzidine), DAB (3,3′,4,4′ diaminobenzidine), and 4CN
(4-chloro-1-naphthol). The most common substrate that produces an insoluble
reaction product is BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/nitroblue
tetrazolium). It is recognized as an effective stable substrate with great fading
resistance. The most common substrates that produce soluble reaction products are
TMB (3,3′,5,5′ tetramethylbenzudine), ABTS (2,2′-azino-di [3-ethylbenzthiazoline]
sulfonate), and OPD (o-phenylenediamine).
In colorimetric measurement, the optical density value of the color produced in
the enzymatic reaction is recorded. In fluorescent detection, the reaction product is
excited by light of a particular wavelength thus the emitted light photons are
measured. Luminescence/chemiluminescent detection systems do not require a light
source for the excitement. The measurement is optically performed according to the
specific luminescence wavelength. Colorimetry and fluorescence strategies
typically offer a detection limit of 150 picoMolar (pM) for the analyte of interest,
while the chemiluminescent and luminescence approaches provide lower detection
limits (30–10 pM).
Table 3.4 provides detailed information regarding substrates used for different
enzymatic reactions. TMB is a dual function substrate that can produce both soluble
and insoluble reaction products. It is highly sensitive and ideal for on-line kinetic
analysis due to its rapid reaction rate. The blue color produced by TMB is mea-
surable at a wavelength of 650 nm. TMB also has applications in endpoint assays
by stopping the reaction with phosphoric acid. A yellow reaction product formed
upon acidification can be recorded at 450 nm.
ABTS is considered as a valuable substrate that serves multiple detection
strategies. Although less sensitive than TMB, it offers great adaptability for both
HRP and AP enzymes. Measurable at 405–410 nm, the reaction product of ABTS
is a blue-green compound. ABTS was found to be a suitable substrate for endpoint
assays as well. Its reaction can be stopped with SDS (sodium dodecyl sulfate),
without interfering the color or the absorbance range.
Slightly less sensitive than TMB and more sensitive than ABTS, the chro-
mogenic OPD is one of the most popular substrates for HRP. Its reaction product is
orange-brown in color and can be read spectrophotometrically at 450 nm.
Typically, 2,3-Diaminophenazine is the oxidation product of OPD catalyzed by
HRP.
Another substrate is p-NPP (p-nitrophenylphosphate) that produces a soluble
reaction product with intense yellow color measurable at 405–410 nm. The
advantageous feature about this substrate is that the color can be developed over an
extended period of time. It is partially due to the low reaction rate of p-NPP that it
may need 30–60 min to reach optimal color intensity.
Luminal (3-Aminophthalhydrazide), polyphenols/acridine esters, and polyphe-
nols are suitable substrates for chemiluminescent detection. Luminal or its
derivatives such as isoluminol (4-Aminophthalhydrazide) are perhaps the most
favorable substrates for luminescent detection strategies. Polyphenols are known for
their excellent signal to noise ratio but they tend to rapidly decay with light
exposure. Luminol is another commercially available substrate, which is normally
sold with enhancers such as phenols, naphthols, aromatic amines, or benzothia-
zoles. Such enhancers act as the enzyme protector, which subsequently allow the
reaction product to remain intact over a considerable time period after light
exposure. Normally, the light emission stabilizes within 2 min (measureable at
425 nm) and preserves the same quality of emission for approximately 20 min.
3.2.6 Stopping Process
The stopping process is performed by introducing compounds to the reaction that

prevent any further change in the developed colors as the results of the enzymatic
reaction. While application of stopping buffers should be according to the
Table 3.4 Detection strategies, related enzymes and substrates and their characteristics [36]
Enzyme Substrate Detection Characteristics
technique
AP p-NPP Colorimetric Yellow color measurable at 405–410 nm, increased sensitivity over an extended reaction time (30–
60 min), not recommended for kinetic analysis
4-MUP Excitation: 360 nm, emission: 440 nm, the pH has to be adjusted at *9.6, commercially prepared liquid
version produces higher background than freshly prepared solution
HRP TMB Rapid reaction rate, ideal for on-line kinetic analysis or endpoint analysis, blue color measurable at
650 nm and yellow color released upon acidification measurable at 450 nm
OPD Less sensitive than TMB, yellow color measurable at 450 nm
ABTS Low sensitivity, wide operating range, blue-green compound measurable ate 405–410 nm, suitable for
end point assays

HPA Fluorescence Excitation: 316 nm, emission: 414 nm, requires H2O2 to produce the fluorescent outcome
HPPA Excitation: 320 nm, emission: 404 nm, requires H2O2 to produce the fluorescent outcome
Luminal/luminol Luminescence Luminol oxidation reaction is typically carried out in an alkaline environment, light emission stabilizes
within 2 min, sustained emission lasts for approximately 20 min
Polyphenols and High signal to noise ratio, rapid light decay, only applicable in luminescent detectors capable of handling
acridine esters “flash” reactions
ß-D- 6-O-b-galactopyranosyl In this reaction, peroxidase replaces the in vivo enzyme, luciferase. The luminescence is stable, and there
galactosidase luciferin is virtually no loss in activity from 30 min to 4 h after adding the reagent
AMPPD These are alternative strategies in enzyme immunoassays instead of colorimetric and fluorescent
AMPGD detections, transformable to chemiluminescence by alkaline phosphatase and ß-D-galactosidase,
enhanced AMPPD luminescence and improved detection limit after addition of BSA [37], considerable
simplicity and high level of sensitivity allow development of rapid clinical assays
MUG The production of the fluorophore is monitored at emission/excitation wavelengths of 365/460 nm
TMB (3,3′,5,5′ tetramethylbenzudine); ABTS (2,2′-azino-di [3-ethyl-benzthiazoline] sulfonate); OPD (o-phenylenediamine); p-NPP (p-nitro-phenylphosphate); 4-MUP
(4-methylumbelliferyl phosphate); HPA (hydroxyphenil acetic acid); HPPA (3-p-hydroxyphenylpropionic acid); Luminal/luminol (3-Aminophthalhydrazide); AMPPD
(3-(2′-spiroadamantane)-4-methyl-4-(3′-phosphoryloxyphenyl-1,2-dioxetane, disodium salt)); AMPGD (3-(2′-spiroadamantane)-4-methoxy-4-(3′-ß-D-
galactopyranosyloxyphenyl-1,2-dioxetane)); MUG (4-methylumbelliferyl galactosidase)
41
recommended protocol for every kit, there are certain considerations, which are
general. The temperature for the preservation of the stopping solution is of great
importance. Stopping buffer has to meet the room temperature prior to the appli-
cation in the assays. Therefore, it is important to observe that the solution is
completely dissolved and has a clear appearance without signs of crystallinity
before it is used in the assay.
3.2.7 Reading Techniques
The signal measurement of the final product in ELISA can be done through several
different strategies including colorimetric, fluorescent, and/or luminescent readouts.
Current section describes each technique in a greater detail.
3.2.7.1 Colorimetric Assay
Colorimetric assays result in colored reaction products capable of absorbing light in

the visible range. The optical density measured for the reaction product is typically
proportional to the amount of analyte of interest. The specific wavelength is gen-
erally provided by the manufacturers of the products. The readout signals are
judged in contrast to the recorded background signals (signal-to-noise).
Colorimetric detection strategy took the conventional clinical practice to the next
level by making the visual judgment of the detection results possible. The presence
of target biomolecule can be confirmed by the naked eye, which laid the foundation
of paper-based bio-diagnostic devices including paper strips. A large number of
paper- and/or fiber-based bioanalytical platforms such as pregnancy test paper strips
are now available in the market that operate based on the principles of colorimetric
assay [38, 39].
Advancements in the area of hand-held bio-diagnostic would not be possible
without adventurous features of colorimetric assay. Particularly, it serves in remote/
rural areas and resource-constrained settings with limited access to centralized
laboratory facilities. Application of smart devices in recording the signal intensity
of the color and subsequent analysis of the data through designed applications or
mass transfer of the data to the centralized facilities have great promises to serves
millions of people. Latest strategies in mass transfer of the obtained data to the
clinical settings provide additional means for on-site bio-diagnosis with minimized
uncertainty of detection [38–40].
3.2.7.2 Fluorescent Assay
In fluorescent assays, the conjugate enzyme converts the substrate to the final
product called fluorophores (generally polyaromatic hydrocarbons or heterocycles).
Fluorophores become excited with light of a specific wavelength and emit

fluorescence when returning to the original energy state. Recorded fluorescence
units are typically proportional to the concentration of the analyte of interest. In
comparison to the colorimetric assay, a fluorescent assay is more sensitive.
However, it typically produces higher background signal due to the sensitive nature
of the fluorescent assay.
3.2.7.3 Luminescent Assay
In the case of luminescent immunoassays, the enzyme converts the substrate into a
reaction product that emits light photons (instead of developing a visible color or
emitting fluorescence) as it returns from an electronically excited state to the
original state.
Different types of luminescent assays including bioluminescence,
chemi-luminescence, photo-luminescence vary in the way the electron excitement
takes place. In bioluminescence, application of bioluminescent compounds such as
luciferin and firefly luciferase provides the emission. The light produced via a
chemical reaction (mainly excitation through oxidation and catalysis intermediates
formation) results in chemi-luminescence. While, photo-luminescence operates in
the same fashion as fluorescence strategy.
Both bioluminescence and chemi-luminescence are broadly used for various
types of immunoassays. Due to the highly sensitive nature of the luminescent
compounds, such assays result in greatly enhanced signals, while having a wide
dynamic range. Known as the most sensitive detection technique, luminescent
detection is benefited from intense and prolonged light emission, low background
signal, as well as signal multiplication and amplification. Luminescent is measured
in relative light units (RLU), typically proportionate to the concentration of the
analyte of interest.
3.2.8 Reading Apparatus
Microplate readers are the machineries that provide with the final detection out-
comes through recording the intensity/absorbance depending on the applied de-
tection method such as colorimetric, fluorescent, and/or luminescent techniques.
High throughput performance, compatibility, cost-effectiveness as well as
multi-mode operation are desirable criteria that readout instruments have to fulfill.
Some ELISA readers cover the entire ultraviolet-visible (UV-Vis) spectrum from
220 to 1000 nm. Obviously, equipment that offers additional features such as
fluorescence and luminescence coverage is more favorable. Incorporated software,
varied types of plate formats, plate shakers etc. could be additional assets to the
ELISA readout instruments.
3.2.9 Controls
In each conducted ELISA experiments, there are different types of controls that
serve specific purposes. Some of the main controls are as follows:
3.2.9.1 Positive Controls
Samples that certainly contain the analyte of interest are called positive controls.
They are made intentionally positive to control the performance of the assay.
3.2.9.2 Endogenous Positive Control
Endogenous positive replicates are normally used in the assay, when recombinant
proteins are part of the components of the ELISA. Recombinant proteins are known to
be challenging biomolecules as they may fold differently than those of endogenous
type thus prevent access to the binding sites. Application of endogenous positive
control is a way to validate if the recombinant proteins are functioning well in the assay.
3.2.9.3 Negative Controls
Negative replicates are samples that do not contain the analyte of interest. They
serve the purpose of checking the assay for its non-specific binding. In an ideal
case, a highly specific assay should produce no detection outcomes for negative
controls.
3.2.9.4 Standard Controls
Standard controls are positive replicates with predetermined analyte concentrations.

A careful design of standard samples results in calibration curve analysis, which can
subsequently offer detailed validation of the assay.
3.2.9.5 Spike Controls
Spike controls are standard controls that are prepared in serum instead of diluting
buffer. These samples are of particular importance, when the actual blood serum is
being tested. Together with normal standard samples, spike samples can evaluate
the performance of the assay.
3.3 Different Protocols 45
3.3 Different Protocols
Different ELISA protocols are typically used in the clinical practice depending on the
purpose of the assay, type of the analyzed samples, and the purity of the reagents. In
this section, main protocols for conducting ELISA including direct, indirect, sand-
wich, double sandwich, and competitive protocols are briefly described:
3.3.1 Direct ELISA
Direct ELISA is a two-step assay (without counting the washing and the blocking
steps). The first step involves attachment of the analyte of interest to the solid surface
of the well (Table 3.5). In the following step, an enzyme-labeled primary antibody is
introduced to the assay. Depending on the detection strategy, the substrate and
stopping agents are subsequently added to the well and the final readout signal can be
recorded (Table 3.5). This type of assay generally requires isolated and purified
samples (analytes) as other proteins in the sample might interact with the sold phase
thus introducing error in the assay. It should be noted that not all the primary
antibodies are possible to be enzyme labeled, thus this type of the assay is limited.
3.3.2 Indirect ELISA
Indirect ELISA follows the same steps as direct ELISA except for the labeled
antibody, which is a secondary antibody in indirect ELISA (Table 3.5). While this
assay offers better accessibility to a wide variety of secondary labeled antibodies, it
suffers from non-specificity of the assay as well.
3.3.3 Sandwich ELISA
Sandwich assay, as its name suggests, “wraps” the analyte of intrest in between the
primary and the secondary antibodies from both sides (Table 3.5). This strategy
offers a better control over specificity of the assay as the first immobilized bio-
molecule (primary antibody) can be highly purified. Nonetheless, if by any means,
the analyte of interest does not interact with the primary antibody, there would be a
chance for the secondary antibody to couple with the primary antibody and to
produce a false positive signal (Table 3.5).
Table 3.5 ELISA protocols, target biomolecules, number of steps in each assay (washing,
blocking, substrate addition and stopping steps are not calculated in the number of steps),
approximate duration of the assay as well as the advantages/disadvantages of each protocol
Protocol Target Steps Approx. Advantages Disadvantages
biomolecule length
Direct Ag 2 3 h and – Fast – Requires high Ag
10 min – Does not require the purity
secondary Ab – Conjugated primary
– Eliminates possible Ab is not widely
non-specific available
binding of
secondary antibody
Indirect Ab 3 4 h and – Availability of – An extra step is
10 min conjugated required in
secondary Ab for comparison to direct
this protocol protocol
– Immuno-reactivity – Chance of
of the primary Ab is non-specific
not compromised bindings are
– Multiple binding of relatively high
the secondary to
primary Ab offers
amplification of the
detection signal
Sandwich Ag 3 4 h and – Applicability in – An extra step is
10 min identification of a required in
wide variety of comparison to direct
target biomolecules protocol
– High specificity of – In the absence of Ag,
the protocol primary and
secondary Abs might
react and produce
false positive signal
Double sandwich Ag and Ab 4 5 h and – Applicability in –Lengthy and tedious
10 min identification of a procedure
wide variety of
target biomolecules
– High specificity of
the protocol
Competitive Ag Depends 4 h and – High specificity of – High reagents

on the 10 min (for the assay consumption
applied each – Lengthy and tedious
protocol experiment) procedure
3.3 Different Protocols 47
3.3.4 Double Sandwich ELISA
In this protocol, which is known as the most specific ELISA protocol, the analyte of
interest is sandwiched between two antibodies, which are produced in the bodies of
different hosts. Therefore, these antibodies are incapable of binding to each other
hence the chance of non-specific binding is minimized. As Table 3.5 illustrates, an
antigen is sandwiched between a capture antibody (the first biomolecule immobilized
in the well) and a primary antibody. The secondary antibody subsequently couples
with the primary antibody and the detection signal can be recorded. While suffering
from a lengthy and tedious procedure, this assay is the most reliable type of ELISA.
3.3.5 Competitive ELISA
Competitive ELISA offers slightly different strategy than the rest. In this protocol
two sets of experiments are performed in parallel. As Table 3.5 depicts, the first set
of the experiments follows the protocol of indirect ELISA, while the parallel
experiment introduces primary antibodies, which are already coupled with antigens
via prior incubation. Depending on the concentration of the antigen in the incu-
bation solution, some portions of the antibodies remain unbound. When these
complexes are added to the antigen-coated wells, there will be less chance for the
primary antibodies to react with antigens, as their binding sites are preoccupied.
This experiment in competition with the first set of experiments (where antibodies
are not pre-coupled with antigens) provides a comparative detection result. The
signal received from the pre-coupled antibodies is inversely correlated with the
presence of the analyte of interest. This assay is lengthy, tedious, and consumes
large samples volumes. Nonetheless, it provides a high degree of specificity.
3.4 Initial Interaction of the Biomolecules with the Surface
One of the significant challenges in manufacturing of ELISA platforms is the

suitable choice of solid material. The appropriate physic and chemistry of the solid
phase could encourage protein immobilization. Immobilized proteins on the surface
should preserve their integrity, native conformation, as well as their biological
function/activity [41]. Presence of active functional groups generated on the surface
of the supporting materials play a significant role in successful detection of the
analyte of interest [42]. PS, the main material of choice for fabrication of the
96-well plates, is known to be inert in its nature. Therefore, it does not possess
active functional groups that can involve biomolecules in substantial interaction
Fig. 3.2 Available active functionalities for biomolecular immobilization (a) insufficient con-
centration; (b) overly functionalized surface and; (c) optimal concentration [20]
with the surface. There are many factors that would impact protein activity in close
proximity with the surface [43]. A number of different treatment techniques have
been employed to modify the surface of the PS (or other similar fabrication
materials) and to generate desirable surface functionalities such as hydroxyl (−OH),
amine (−NH2), or carboxyl (−COOH) groups [43]. Additionally, the proper dis-
tribution of such functional groups is of great importance as well. Presence of such
functionalities can facilitate biomolecular immobilization via physical or chemical
means. The current section reviews some of these methods.
Figure 3.2 illustrates different concentrations of the surface functionalities and
the effectiveness of the protein immobilization in each case. In the first depiction,
insufficient number of functionalities is present on the surface (Fig. 3.2a). If surface
treatment method does not generate enough reactive functional groups, the proteins
can lose their activities in close proximity of the solid surface. In contrast, overly
functionalized surfaces can cause steric repulsion due to the protein deactivation
(Fig. 3.2b). The proper distribution of the surface functionalities, however, results
in an efficient and successful protein immobilization as Fig. 3.2c suggests.
Therefore, a close control over manufacturing of the ELISA well plates and
favorable surface properties of such platforms can play vital roles in potential
chemical and physical immobilization of the proteins on the surface [43].
3.5 Immobilization Techniques for Protein Attachment 49
3.5 Immobilization Techniques for Protein Attachment
Variety of techniques has been reported for immobilization of enzymes, antibodies,

antigens, and different types of proteins on the supporting surfaces [42–44].
Different immobilization techniques have been commonly applied in food industry,
organic compound removal from wastewater, in situ measurements of environ-
mental pollutants, metabolite control and biosensor applications [45]. Table 3.6
highlights the examples of well-known immobilization techniques and applications
of such techniques for biomolecular immobilization.
3.5.1 Physical Immobilization
Perhaps the most straightforward method for immobilization of the biomolecules

for subsequent bio-recognition is physical immobilization. This method relies on
the physical attachment of the protein to the surface, which is commonly used in
clinical practices such as ELISA. In this technique, immobilization mainly occurs
through different interactions between targeted biomolecules and solid substrates
[46, 47]. There are some key forces that can greatly influence the efficiency of the
immobilization among which three important forces play the vital roles in suc-
cessful immobilization. Namely ionic interaction (electrostatic interaction),
hydrophobic interaction and hydrogen bonding have great impact on the immobi-
lization efficiency [48]. Surface functional groups of the supporting substrates (such
Table 3.6 Biomolecular surface immobilization techniques and the biosensor applications
Immobilization techniques Examples of application
Physical attachment Detection of DENV by using ELISA assay [49], and early
detection of DENV using ELISA analytical kit [53]
Immobilization by entrapment Immobilization of glucose oxidase on polypyrrole for its
application in amperometric glucose sensors [54], and
sol-gel-entrapped glucose-oxidase immobilization for
sensing application [55]
Covalent immobilization via Modification of PS particles with amine substitute HSV
carbodiimide chemistry reporter probes via carbodiimide chemistry for ultimate
DNA detection [56]
Covalent immobilization via Attachment of trypsin by using glutaraldehyde as cross
zero-length cross-linker linker on ammonia plasma treated polyethylene films [57]
Covalent immobilization via Using amine spacers of different sizes such as PEI, HMD,
spacers PAH and DAP for enhancing antibody binding to polymer
based microfluidic devices [58]
Oriented immobilization CNBr activation of Sepharose (Seph) coupling to the
protein via the d-amino group of lysine [59]
as −COOH) can interact with functional groups of the proteins (such as −NH2) and
result in protein attachment through ionic attraction [49, 50].
According to the previous findings, the effect of ionic interaction on protein
immobilization in comparison to other two forces can be considered insignificant
[48]. Hydrophobic nature of the supporting substrate can offer protein immobi-
lization via hydrophobic interaction, while presence of surface −COOH groups (as
an example of the active functional groups) can result in hydrogen bonding with the
primary amines of the proteins [48]. Between these two major forces, hydrogen
bonding has proven to influence protein immobilization in a stronger manner than
hydrophobic interaction. Despite development of more complex immobilization
techniques, clinical practice still relies on the direct attachment of the targeted
biomolecules to the surface. Relative hydrophobicity of the bio-receptor surfaces
hand in hand with the existence of desirable surface functional groups could make
protein immobilization more effective.
3.5.2 Immobilization via Entrapment
The entrapment of the antibody/antigen inside the membrane or gel material is another
relatively simple method for immobilization. In this method, the permeability of the
material has drawn a great deal of importance as it can directly affect the sensitivity and
background noise of the resulting detection signal. The major drawback of such
techniques is that the unreacted entrapped primary antibody might not be elimi-
nated from the matrix of the material during the washing step due to the structure of the
supporting material, hence causing false signal as a consequence of coupling with
secondary antibody [51, 52]. Therefore, this method might result in an unacceptable
level of background signal originated from the substrate, in particular when more
complex protocols such as sandwich/double sandwich ELISAs are aimed.
3.5.3 Covalent Immobilization
Covalent immobilization of antibody/antigen is one of the most commonly applied

methods even in the routine laboratory procedures. In this method, the existing
stable functionalities are generally transformed to the semi-stable highly reactive
functional groups that can covalently bind to the proteins. Covalent immobilization
can be performed via cross-linkers of different categories.
3.5.3.1 Immobilization via Zero-Length Cross Linker
A great number of research works have reported application of zero-length cross

linkers such as glutaraldehyde (GA), 1-ethyl-3-(3-dimethylaminopropyl) carbodi-
imide (EDC), and N-hydroxysuccinimide (NHS) in covalent immobilization of the
proteins to the substrate [60, 61]. Functional conversion occurs quite differently
from one cross-linking agent to another, thus making reproducibility difficult. For
example, one of the most commonly used carbodiimide agents is the water-soluble
EDC for aqueous crosslinking. Carbodiimides such as EDC, can activate −COOH
functional groups for direct conjugation to primary amines of the biomolecule. The
available −COOH surface groups can be converted to unstable reactive
O-acylisourea ester groups (Fig. 3.3). This intermediate compounds, by association
of NHS can be converted to semi-stable NHS-ester groups (Fig. 3.3), which are
highly reactive toward −NH2 groups of the peptide sequences (Lys) in proteins,
thus resulting in covalent immobilization of the protein on the surface.
However, the versatility of the generated functional groups in EDC/NHS
chemistry makes reproducibility of this technique difficult. Previous reports indicate
the precautions and possible drawbacks of such approach as in some cases EDC/
NHS reaction results in formation of the anhydride functional groups (instead of
Fig. 3.3 Carbodiimide reaction and activation of the −COOH functional groups
NHS-ester groups), which are unreactive towards proteins [61]. Early cross-linking
inside the individual protein molecules might also happen when EDC/NHS treat-
ment is aimed for activation of the surface. Such undesirable effects can cause the
loss of protein activity that, in turn, results in poor detection signal and significant
loss of sensitivity [62].
3.5.3.2 Immobilization via Spacers
Larger cross-linkers are called “spacers molecules”. Spacers are molecules with
available functional groups for coupling with proteins. In particular, they offer
distance between proteins and the solid phase of the substrate, resulting in higher
spatial freedom for binding of proteins to the surface. Among all kinds of spacers,
amine bearing molecules such as PEI (polyethylenimine), HMDA (hexam-
ethylenediamine) and DAP (1,2-diaminopropane) are the most commonly used
intermediate compounds [58]. Linear amine spacers such as HMDA are smaller in
size therefore they offer less active functional groups in comparison to branched
spacers such as PEI. For that reason, larger spacers normally result in higher
immobilization rate due to the number of available active functionalities, which are
offered for protein attachment. As the first step, carbodiimide chemistry provides
the reactive intermediate functionalities suitable for covalent attachment of the NHS
ester groups to terminal −NH2 functionalities of the spacers. In a further step, the
aminated surface reacts with glutaraldehyde, yielding aldehyde groups that could
form imine linkages with primary amine groups of the proteins [62, 63]. GA is a
well-known amine-reactive homobifunctional cross-linker, frequently used in bio-
chemistry applications. Such experimental strategy results in generation of reactive
functional groups for more robust protein binding with high reproducibility
[58, 63].
3.5.4 Oriented Immobilization
All of the mentioned immobilization strategies, to this point, can be classified in the
category of random immobilization techniques, as there is no control over the
direction of the immobilized protein on the substrate. The lack of direct control in
random immobilization might result in loss of biological activity of the antibody/
antigen upon immobilization. Mainly, such loss can be attributed to the random
placement of the symmetrical macromolecules on the substrate [64]. Oriented
attachment of the proteins to the supporting surface and subsequent increase in
binding affinity can be achieved by chemical alteration of the surface functionalities
[64]. In this technique, proteins can be attached to the surface in a very orderly
manner due to this well-defined immobilization approach. This ordered way of
immobilization offers applications in many fields such as bioreactors, biosensors
and bioelectronics. The major advantage of this method over random methods is
a high reproducibility of the immobilization as biomolecules are immobilized from

their specific sites.
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Chapter 4
Evaluation of the Detection Results
Obtained from ELISA
Abstract This chapter presents the most commons errors that typically occur when
performing ELISA. Following every error, the chapter points out the possible
reasons behind such errors and possible methods to overcome the problems. The
chapter also describes key important parameters in assay evaluation including
sensitivity, specificity, accuracy and limit of detection (LOD). Offering methods for
calculation of the evaluation parameters, the chapter also provides insights and
considerations for assessing the reliability of the assay. Such analysis is essential to
upgrade a newly developed assay from the analytical relevance to the clinical
application. Finally, the chapter provides information in regard to the measurable
units in ELISA assay.
4.1 Conducting a Reliable Assay
Nowadays, many companies offer a wide range of ready-to-use ELISA kits that
include antibodies, blocking buffer, substrate solutions and other necessary com-
ponents. Such packages simplify the procedure and ensure a reliable assay just by
closely following the instructions. Reagent management is an important aspect of
conducting the assay. Any type of solution should be prepared only in the volume
necessary for carrying out the intended protocol. If there is an excess of reagent
solutions, they should be stored adequately.
Nonetheless, errors are inevitable even in the well-established commercially
available assays. In particular, when the protocol should be designed and conducted
from scratch for the economic reasons, or when a new protocol is being developed
based on adaptation from the previous protocols, there is a higher chance of error.
There exist common errors in conducting ELISA that can be avoided by having a
clear knowledge of the sources of such errors. The suppliers and distributors often
provide the users with the necessary guidelines and troubleshooting strategies to
overcome the errors and to carry out successful assays. In this chapter, we describe
some of these common errors that might occur, when performing ELISA and
corresponding solutions to minimize and/or correct such mistakes.

https://doi.org/10.1007/978-981-10-6766-2_4
58 4 Evaluation of the Detection Results Obtained from ELISA
4.1.1 Sources of Errors
When performing ELISA, one of the common sources of errors can be inconsis-
tency in the applied materials such as the type and the brand of the well plates,
pipettes, tips for pipettes, as well as chemicals and reagents used for buffer
preparation. Especially, when the results of each test were to be compared with the
previous or the next replicates, it is of great importance to follow the exact same
protocol and to use the same tools for conducing the assay. Another source of error
is in an exceeding amount of analyte in the sample, for which serial dilutions might
lead to inaccurate analyte concentration and misleading results [1]. There are other
considerations for conducting a successful assay as well.
The main problems are encountered when actual serum samples with multiple
proteins are tested. Even when the coupling is specific, contaminants or other
biomolecules similar to the target analyte could also compete and bind to each
counterpart or even to the solid surface by the forces described in Chap. 3, giving
raise to the false negative or the false positive signals. Such false signals, known as
the background noise can even exceed the actual detection signal especially
when highly diluted samples are used [1]. Appearance of such background signals
obviously causes failure in the assay. This problem can be solved by employing
more specific protocols such as competitive, double sandwich, or fluid phase
ELISA [2–4]. These protocols, however, are complex, time consuming and tedious.
Determining the background optical density (OD) in the absence of the analyte of
interest is therefore one of the mandatory controls in every assay [1].
False positive background signal in ELISA typically happens when proteins
non-specifically bind to the solid phase or other proteins. False positive signals
could also occur due to the direct or competitive inhibition of the target analyte by
other antibodies or components present in blocking or diluting buffers, or by
denaturation of its epitopes by ionic detergents [1]. Subtracting the background
values from the OD values is a routine step in ELISA data interpretation.
4.1.2 Troubleshooting
Apart from non-specific binding and background noise, there are several possible
errors that might be faced, while performing ELISA. High or low signal readouts,
incomplete color development, poor reproducibility, inconsistency in the control
readouts, and non-linear calibration curves are some examples. It is helpful then to:
• Write down the entire personalized protocol and deviations prior to conducting
the assay.
• Label and arrange the samples, the buffers, the well plates etc., and have a clear
mapping of the experiment in advance.
• Develop pipetting skills and a good concentration on the assay.
4.1 Conducting a Reliable Assay 59
• Do a careful checking of the equipment such as incubators, fridges, and readout

machines before planning an assay. Pipettes need to be calibrated on a regular
basis as well.
Table 4.1 reviews a detailed list of assay errors along with the possible reasons
behind these problems and the solutions to overcome the errors.
Table 4.1 Common errors in ELISA along with their possible reasons and solutions
Possible reason Possible solutions
High background signal
Concentration of secondary antibody Dilute the secondary antibody 10-fold or more
was very high
Well plate was not clean Ensure that the well plates are new and clean
Inappropriate blocking reagent was Follow the protocol and use the freshly prepared
used known blocking reagents for buffer preparation
Add the blocking protein to the washing solutions
There was no absorption of antigen Perform an overnight adsorption at 4 °C
Inconsistency in the blank readouts
Incubation was done with Make sure the coating buffer has the right components
inappropriate buffer
Well plate dried due to the lack of Control the solution level inside the wells during
aqueous media analyte binding step
Failure in the assay
Buffers were not thawed properly Make sure buffers are at room temperature before
starting the assay
Antigens did not bind to the wells Change the binding conditions according to
manufacturer’s instructions
Change the diluting/coating buffer based on the
protein type and the well plate
Before the actual assay, run a control protein assays
(such as Bradford, and bicinchoninic acid) to ensure
the coating
Some steps of the assay were Follow the protocol carefully
omitted
Plate reader was set at an incorrect Check the filter settings of the readout instrument
wavelength
Inconsistency in the readout outcomes
Samples were prepared with Use the buffers provided by manufacturer or refer to
different buffers the provided methodology
Buffers evaporated prior to readout Cover the plate with an adhesive plastic layer or the lid
when incubating
Biomolecules were denatured Use PBS buffer with pH * 7 as washing buffer to
avoid denaturation. Avoid the use of sodium azide in
buffer preparation
Samples were not homogeneous Mix the samples thoroughly
(continued)

Samples were used after multiple Prepare several aliquots
freeze-thaw cycles
Interfering elements were present in Ensure the use of washing buffer with 0.01–0.05% of
the assay detergent included and perform a second washing step
with the PBS buffer without detergents
Old or inappropriately stored Use fresh samples and store them correctly
samples were used
The washing step was incomplete Allow incubation of 5–10 min with washing buffer at
each washing step. Use shakers at the low speeds to
facilitate better washing
Components were improperly Thaw all the components completely and mix them
thawed thoroughly before use
Expired or improperly stored Check the expiration date and store the components
reagents were used appropriately
If buffers are more than 1 month old, filtrate them
before use
Samples were stored for an extended Preferably, prepare fresh reagents before each assay
period of time
Incorrect incubation times or Refer to the manufacturer’s guideline and verify the
temperatures were chosen correct incubation times and temperatures
Incorrect volumes were used Use calibrated pipettes and standard tips
Non-linear calibration curve
Partially thawed components were Thaw and re-suspend all components before preparing
used in the assay the reaction mixture
Cross contamination occurred Always change the tips from one set of sample/
concentration to another
Pipetting errors occurred in Avoid pipetting very small volumes
preparation of the standard solutions
Pipetting errors occurred in Prepare a stock reaction mixture
preparation of the reaction mixtures
Air bubbles were formed in the wells Pipette gently against the walls of the wells and/or
tubes
Conduct washing and blocking incubation overnight
Standard stock solutions had Refer to the manufacturer’s instruction for the
incorrect concentrations standard dilution procedure
Calculation were done mistakenly Recheck calculations after referring to manufacturer’s
instructions
Substitute reagents from older Use fresh components
preparations were applied
Unanticipated results
Samples were measured at incorrect Check the readout equipment and the filter settings
wavelengths
(continued)
4.1 Conducting a Reliable Assay 61

Empty plates produced high Perform a sensitivity test (read an empty well) before
background signal running the experiments. If the signal is recorded as
high, change the filter settings
Strips were chosen from different Always employ the strips from the same batch
batches
User forgot the exact location of the Always label the well plates carefully and clearly
samples in the well plate
Incubation time after TMB addition Avoid inconsistency in the incubation times. Using
was inconsistent multichannel pipette can be a great help to save time
and charge the wells with higher accuracy
Add stopping buffer (according to the manufacturer’s
guideline) once the incubation time is over
Samples contained interfering Repeat the assay with predetermined analyte
substances concentrations
Sample readouts were recorded Concentrate or dilute the samples to bring the readout
above or below the linear range within the linear range
Calculate the maximum number of the biomolecules
that are expected to bind to the wells and keep the
concentration within that range
The consistency of the assay is important to assure its reproducibility and

accuracy. All the aspect of an assay including preparation of the dilutions and
different analyte concentrations (standard samples for calibration curves), the
temperature, the pH of the buffers as well as the buffer compositions should strictly
follow the standard procedure.
Readout technique, in turn, plays a vital role. The routine method is absorbance
measurement from top of the well in normal ELISA well plates. Microplate readers
are equipped with the corresponding light filters for different wavelength mea-
surement. Spectrofluorometers can increase the sensitivity one order of magnitude
in comparison to the colorimetric measurement. However, there is a chance for
bleaching effect to occur in the fluorescence measurement. Luminometers or
multi-label readers can measure with even further sensitivity than both colorimetry
and fluorescence techniques. Depending on the employed readout technique, the
well plate should be chosen. For example, white and black plates can be used for
top absorbance readout and fluorescence measurements, respectively. Transparent
plastic, glass, quartz or other clear polymer-based well plates are suitable for
reading of absorption or fluorescence/luminescence emission from the bottom of the
wells. In Chap. 5 a more detailed description of well plate material and different
readout techniques are provided. Table 4.2, summarizes the recommendations on
the selection of ELISA well plates and signal measurement strategies for con-
ducting a reliable and accurate assay.
Table 4.2 Appropriate choices of well plates for effective measurement

Reading Absorbance Fluorescence Bio-/
chemi-luminescence
Top Transparent well plate Black well plate to avoid White well plate to
if the absorbance is auto fluorescence maximize the output
high (nonspecific fluorescence signal via light
White well plate to emission from undesirable reflection
maximize the sources)
absorbance signal via
light reflection
Bottom White well plate with Black well plate with
transparent bottom transparent bottom
Glassy bottomed well plate if confocal microscopy is the
means for analysis
Zero-crosstalk well plate for ultra-sensitive assays
4.2 Key Parameters in ELISA Evaluation
Every newly developed assay, or clinical laboratory test has to be carefully assessed
for its analytical performance and its capability for the intended application [5]. It is
vital to investigate the limitations of the novel assay strategies and to ensure their
suitability for bio-diagnosis. An ELISA protocol of any category results in a final
readout outcome that is considered as the raw data and needs further processing as
the raw data can be debated for its accuracy. While the readout outcome might
show a better performance of platform “A” over platform “B”, the results of the
controls may draw an unlike conclusion. Therefore, it is of great importance to
conduct the assay along with different controls for the careful evaluation of the
assay. Negative controls are particularly vital. They are the results of the assay
conducted in the absence of the targeted analyte. Cut-off values are calculated from
the recorded readout outcomes for negative controls by using the equation below
[6]:
Cut off value ¼ 2 averageðnegative controlsÞ
In an ideal case, the results of the negative controls are expected to be the
negative detection signal as the target analyte is absent in the assay. In reality,
however, it is rarely the case thus the chance for slight error has to be considered as
an inevitable part of the assay procedure. Precise measurement and subtraction of
the negative outcomes from the original data is a way to ensure the detection result
is reliable. Detection signals, on the other hand, can be considered as positive
detection results if only they are higher than twice of the average value of the
negative control readouts or what is known as “cut-off value” [7]. Negative repli-
cates are not the only control samples in the assay. Sometimes the assay results in
negative readout, while the target analyte existed in the samples. Therefore,
obtained results can be classified into categories as follows:
4.2 Key Parameters in ELISA Evaluation 63
True positive (TP); was detected as positive and contains the target analyte
True negative (TN); was detected as negative and does not contain the target
analyte
False positive (FP); was detected as positive but does not contain the target analyte
False negative (FN); was detected as negative but contains the target analyte.
4.2.1 Sensitivity
True/false positive and negative replicates can be used to calculate key parameters
including sensitivity and specificity. These evaluation parameters are the main
elements in evaluation of the assays [8–10]. Sensitivity of the assay is typically
calculated via equation below:
TP
Sensitivity ¼
TP þ FN
4.2.2 Specificity
Specificity of an analytical assay provides an understanding of how likely is the

possibility of non-specific binding in the conducted assay. It also shows how
effective the blocking procedure has been in the applied protocol. Specificity of the
assay can be calculated by using equation below:
TN
Specificity ¼
TN þ FP
There are two types of sensitivity and specificity: analytical as well as clinical
sensitivity and specificity. The analytical evaluation is mainly applicable when a
new assay or protocol is being developed. It proves how likely it is for a novel
strategy to open its way to the hospitals and clinics and to be applied as a reliable
technique. Diagnostic/clinical evaluation, on the other hand, is applied in routine
clinical practice, when dealing with actual samples (e.g. human blood serum) [11].
4.2.3 Accuracy
Another key important parameter in assay evaluation is accuracy. Accuracy is

typically calculated by associating the negative and positive outcomes (true and
false) in comparison to the total number of conducted replicates following equation

below:
TP þ TN
Accuracy ¼
Total number of conducted replicates
Additionally, conducted assays should be calibrated by running the assay with

predetermined concentrations of the targeted analyte. This leads to the calibration
curves. The linearity of the plotted calibrations is a measure for the precision of the
assay. The squared correlation coefficient (R2) of these plots provides with an
statistical model for studying the precision of the assay [12]. R2 ranges from 0 to 1.
When R2 has the closest value to 1, the plot is at its highest linearity thus the assay
is at its best precision level. Calibration analysis is one of the essential steps in assay
evaluation as it reveals the reliability of the assay.
4.2.4 Limit of Detection (LOD)
Limit of detection (LOD) is the lowermost quantity of a targeted analyte that the
assay can possibly detect with assured confidence. Limit of blank (LOB) is the
lowest recorded detection signal for the blank controls. It is expected that LOD
occupies greater values that LOB [5]. This specific parameter (LOD) is of great
importance especially when the early detection of any type of disease is concerned.
There are different ways of calculating LOD values from which following equation
is a widely used method [13]:
3 Standard deviation
LOD ¼
Slope of the calibration curve
It should be noted that LOD calculation through the introduced equation is valid
if only the number of positive replicates is higher than the number of negatives
replicates in the assay.
4.3 Measurable Units in ELISA
ELISA can be a qualitative or a quantitative method for detection. In the case of

quantitative measurements, the measurable units depend on target analyte.
Comercialized kits may provide the measurements based on the United State Food
and Drug Administration (FDA), the United States Pharmacopoeia, the World
Health Organization (WHO), or the European Pharmacopoeia. Those are adopted
common standard measuring systems, which are normally presented as units/ml
(sometimes referred to as International Units (IU/ml) or ELISA Units (EU/ml)).
4.3 Measurable Units in ELISA 65
The ELISA kits are also calibrated to meet the requirements of internationally
certified laboratories such as Center for Biologics Evaluation and Research (CBER)
associated with FDA [14]. In most cases, the presence and specific concentration of
an antigen is the target for the measurements. Such measurements take place
through the absorbance measurement. According to the Beer-Lambert law, con-
centration of a compound in a solution is proportional to the quantity of light
absorbed at its specific wavelength and at a constant optical path-length. Following
absorbance measurement, a simple calculation leads to the concentration of the
analyte via equation below:
A ¼ elc
where A is absorbance of the solution at the particular wavelength, e is the molar

absorptivity, l is the path length or deepness of the solution (which for common
spectrophotometers is 1 cm or less if a well plate reader is employed), and c is the
concentration of the substrate in the reaction solution (M or mol/dm3). Majority of
ELISA microplate readers correspond to this method for the calculation of the
analyte concentration. Introducing standard solutions with known analyte concen-
trations can be the basis of such calculations for the instrument. Measurement unit
are typically expressed in gram per liter (g/l), mol per liter (mol/l) and copies per
milliliter (copies/ml) for nucleotides, plaque forming units per milliliter (pfu/ml) for
infectious viruses, colony forming units per milliliter (cfu/ml) for cells and phages,
as well as unit per milliliter (u/ml) for specific enzymes. These measurement units
can be also reported over time (i.e. ng/ml/h). Relative units (RU/ml) or equivalent
units (Eq/ml) are normally used if no international standard references exist for the
conversion.
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4. Yunginger JW, Ahlstedt S, Eggleston PA, Homburger HA, Nelson HS, Ownby DR et al
(2000) Quantitative IgE antibody assays in allergic diseases. J Allergy Clin Immunol
105:1077–1084
5. Armbruster DA, Pry T (2008) Limit of blank, limit of detection and limit of quantitation. Clin
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immunosorbent assay specific to dengue virus type 1 nonstructural protein NS1 reveals
circulation of the antigen in the blood during the acute phase of disease in patients
experiencing primary or secondary infections. J Clin Microbiol 40:376–381
7. Linares EM, Pannuti CS, Kubota LT, Thalhammer S (2013) Immunospot assay based on
fluorescent nanoparticles for Dengue fever detection. Biosens Bioelectron 41:180–185
Synthesis and processing of ELISA polymer substitute: the influence of surface chemistry and
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effect and antibody immobilization on −COOH exposed surfaces designed for dengue virus
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13. Shrivastava A, Gupta V (2011) Methods for the determination of limit of detection and limit
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J Clin Microbiol 48:4459–4463
Chapter 5
Advantages, Disadvantages
and Modifications of Conventional ELISA
Abstract Nowadays ELISA is considered to be the troy horse for the routine
clinical practice. This widely applied technique offers specific detection of a wide
variety of target analytes in different kinds of samples. Since the invention of the
technique four decades ago, ELISA has rapidly found various applications in food
quality, environmental, biotechnological, and chemical disciplines among others. In
spite of its many advantages, ELISA has certain limitations such as tedious/
laborious assay procedure, and insufficient level of sensitivity in bio-recognition of
challenging biomolecular entities such as microRNAs. A great number of research
works has shown valuable attempts in addressing such shortages of ELISA through
modification strategies. This chapter is dedicated to reviewing some of the main
promising alternatives to the traditional ELISA. Paper- and fiber-based ELISAs,
have shown great potentials for point-of-care (POC) applications due to their
cost-effectiveness. Miniaturization of ELISA within micro-devices has increased
the number and type of samples that can be analyzed, while much lower sample
volume is required. Multiplexing was obtained as a result of micro and nano
fabrication strategies and the integration of the assay within lab-on-chip (LOC) and
lab-on-compact-disk (LOCD) devices. Taking advantage from a significantly vast
surface area of the spheres, ultra-sensitive diagnosis was achieved by using micro-/
nano-particles with different optical proprieties, sizes, synthetic variables and
compositions. ELISA on the spot made possible to measure the biomolecules
in vitro. Plasmonic ELISA offered detection strategies even with the aim of the
naked eyes. Finally, the digital era has opened new windows of opportunity for
ELISA, as the results of immunoassays can be recorded in remote/rural areas and
subsequently analyzed by digital technologies or in centralized laboratories via
mass data transfer.

https://doi.org/10.1007/978-981-10-6766-2_5
68 5 Advantages, Disadvantages and Modifications …
5.1 Significance of Conventional ELISA
ELISA is known as the most commonly applied method in the clinics and hospitals
all across the world. It has also been widely used for accurate detection and
quantification of biological agents (mainly proteins and polypeptides) in the
biotechnology industry and is becoming increasingly important in clinical, food
safety, and environmental applications [1]. It is believed that there is no laboratory
that has not encountered ELISA in one form or another. ELISA provides highly
reproducible, quantitative data that makes it an advantageous biotechnological tool
in scientific research and clinical diagnosis [2].
ELISA is reliable, sensitive and specific, compared to other immunoassays. The
sensitivity of this technique is due to the enzyme amplification strategies. This
technique can provide with a chromogenic signal even inside the cells. The
specificity is benefited from the antibody-antigen coupling approach, which occurs
through a very specific manner as the paratope of the antibody at the binding site
couples with the complementary epitope of the antigen (the lock and the key
approach). When ELISA is compared with other immunoassays for detection and
quantification of antibodies in blood serum and plasma samples, it was found to be
favorable due to the availability of the reagents, robust performance and great
accessibility in many laboratories [3].
Nonetheless, ELISA suffers from certain drawbacks. Different attempts were
made for development of the modified platforms to improve this technique and
overcome its shortages.
5.2 Shortages of Conventional ELISA
Some of the general shortages of the conventional ELISA are as follows:

• Tedious/laborious procedure
• Limited multiplexing options
• Necessity for centralized laboratory equipment
• Relatively high sample volume is required.
While ELISA is highly reproducible, such shortages might give rise to the
typical errors in the assay. Variation in optical density readout can be addressed by
right choice of controls in the assay including blank, zero concentration,
non-specific binding and maximum binding control replicates. Consistency in
handling ELISA comes with the practice. Typically, laboratory technicians develop
the necessary skills for conducting a highly reliable and reproducible assay. Chapter
4 of this book is dedicated to the sources of error in ELISA and possible solutions to
address the existing errors.
5.2 Shortages of Conventional ELISA 69
Moreover, the detection limit of conventional ELISA is barely less than

nanomolar concentration level, which is inadequate to reach the clinical threshold
of many protein biomarkers, especially in the early stage of diseases [4].
5.3 Materials of Choice for Fabrication of ELISA Well

Plates
In microplate production, the selection of well plate material is of great importance

since physical and chemical properties as well as optical characteristics of the end
product depend on the careful choice of the production material. Cost-effectiveness,
ease of fabrication, transparency (in the case of well plates used for optical
detection) and high stability of the materials also play vital roles in well plate
production.
Via any ELISA protocol, there is always an initial biomolecule that binds to the
surface either passively (via physical attachment) or immobilized through modifi-
cations of the surface (covalent attachment). The quality of the subsequent protein
coupling/binding closely relates to the successful immobilization of the initial
protein (whether it is an antibody or an antigen). It is this binding and immobi-
lization of different biomolecular entities that makes ELISA a reliable platform.
Successful attachment of this first biomolecule also permits elimination of the
unbound proteins from bound ones through washing procedure. The ability to
eliminate nonspecifically bound analytes makes ELISA a powerful tool for specific
measurement of the targets. Thus, the characteristics of the solid phase in ELISA
play the most significant role.
Polystyrene (PS) is a relatively hydrophobic material in its nature. Therefore, PS
potentially binds to a wide variety of proteins via physical attraction of the bio-
molecules to the surface. Carbohydrates and heavily glycosylated proteins do not
involve in hydrophobic interaction hence they have less tendency to attach to the PS
surface. In such cases, immobilization via covalent means is recommended. PS is a
very cost-effective material for mass fabrication of the well plates. Uncolored PS is
highly transparent and possesses favorable optical characteristics, especially suited
for colorimetric assays as well as other optical measurements. Visible wavelength
range (900–335 nm) measurements require clear plastic materials for which PS is a
highly favorable material.
More demanding applications such as assay development and high throughput
screening require use of ultra-pure PS with additional optical brighteners such as
titanium dioxide. This approach is normally used for white well plates to increase
the reflectivity of the surface. Furthermore, specific steel tools for molding such
plates are carefully polished to provide a smooth surface inside the wells. Hence it
is ensured that the emitted light within the wells reflects straight upwards, where the
sensitive optical detector is positioned during the intensity measurement.
For the fluorescence readout, carbon black is typically added to the PS substrate,
which results in effective platforms for fluorescence assays. In this case, the internal
surfaces of the well plates are also polished to insure maximum reflectivity. But the
main challenges arise when handling luciferase assays in which a high dynamic
range is normally observed across the plate. In such cases, a certain amount of
visible light might “leak” through the white plastic walls of the wells, which, in
turn, is a source of error in the assay. To overcome this problem “black and white”
PS well plates were developed in which black PS matrix has integrated individual
white cells fabricated via molding system. This design would prevent the crosstalk
of the wavelengths and the “leakage” phenomenon.
Different modification strategies have been used for covalent linking of bio-
molecules to the plastic surface of PS. For instance, a maleimide group can react
with a sulfhydryl group forming a covalent link between the plastic surface and a
protein/peptide. A hydrazine can react with aldehydes formed through periodate
oxidation of carbohydrates. N-Hydroxysuccinimide (NHS) from the family of
carbodiimides can also react with the amine groups on peptides or proteins. In
general, peptides can be immobilized on the PS surface either through the
zero-length cross-linkers such as carbodiimide agents or through the amine bearing
spacers by cross-linker such as disuccinimidyl suberate (DSS). Detailed strategies
for physical or covalent immobilization of the biomolecules to the surface were
thoroughly described in Chap. 3 (Sect. 3.5) of this book.
Readings in the range of 220–335 nm will require a UV-transparent material
such as quartz sheet or alternative polymers including cyclo-olefin co-polymer
(COP/COC). Polypropylene (PP) can be another alternative material for mass
fabrication of ELISA well plates. It is an amorphous, rigid, glassy polymer with
high optical clarity and low biomolecular binding ability. It has a high degree of
tolerance to the temperature change and is resistance to many of the laboratory
chemicals. ELISA well plates made from PP are mainly used for cell-related assays
via colorimetric, fluorescent and luminescent detection methods. Polyvinyl (PV) is
also a material of choice for micro plate fabrication. It has low binding capacity and
is mainly used for hydrophobic interactions. Majority of the well plates available in
the market are made from either PS or derivatives of PS.
5.4 Different Types of ELISA Well Plates
The well plate can also offer surfaces variants with different binding capacity for
reacting with hydrophilic and/or hydrophobic biomolecules or with the combination
of both. The color, the number of wells and the shape can also vary. Well plates of
6-, 12-, 24-, 48-, and 96-well formats are commercially available. The well plates
can be white, black, or transparent with or without transparent bottoms. Microplates
can be different in their bottom shape offering U, V, F, and C shapes in 96-well
format. The inner surface of the wells can be modified via chemical or plasma
treatments for better shelf life and storability of the platforms. Other types of
5.4 Different Types of ELISA Well Plates 71
treatments might be applied for pre-coating of the well plates or enhancing the
binding stability of the surface with the biomolecules.
The most common configuration is 96-well plate that has 8 rows and 12 col-
umns. Each well has an internal area of 2.5 cm2, which can be charged with the
volume of approximately 350 ll. Recently, micro plates with 384 and 1536 wells
were also developed with the overall dimensions same as the conventional 96-well
plates. Such plates have their main applications in high throughput screening.
Moreover, 96-well plates with half the volume of the conventional platforms were
also introduced to the market. Assay procedures in the half volume well plates are
identical to that of traditional plates, while requiring twice less sample volumes
thus savings the reagents considerably.
Another recent innovation has added fins to the inside of the wells in order to
increase the surface area for effective binding with proteins and higher adsorption.
Reports have shown a 10–20% increase in protein adsorption when using “Star
Wells” instead of conventional platforms. Another alternative strategy designed the
bottom of the wells in the rounded shapes instead of the flat bottom that is located in
90° angle with the walls. This approach was reported to help thorough washing of
the wells.
5.5 Modified ELISA Platforms
In bio-sensing domain, there is an increasing request for cost-effective, reliable,

portable, rapid, and high throughput detection platforms. The World Health
Organization (WHO) outlined seven key parameters for the development of bio-
analytical platforms: “(i) Affordability, (ii) Sensitivity, (iii) Specificity,
(iv) User-friendliness, (v) Rapid and robust, (vi) Equipment-free, and
(vii) Deliverable to those in need for such technologies” [5], referring to the
acronym ‘‘ASSURED’’. While some of these parameters promise the application of
such devices in resource-limited areas in major needs for extreme point of care
(EPOC), these seven requirements defined by WHO are of crucial importance for
development of any class of bioanalytical platforms [5].
For nearly half a decade, ELISA had a leading role in a revolution that resulted
in standardization, automation and multiplexing of robust immunoassays for
heterogeneous samples. Thousands of ELISA replicates are performed in clinic and
laboratories every day to detect a wide variety of biomolecular entities. It is a
reliable technique with acceptable sensitivity, specificity and accuracy. However,
when the early detection is concerned, ELISA lacks providing timely information
for early diagnostic of curtain types of diseases [6].
Numerous strategies were introduced to overcome the current problems of
ELISA and to enhance its performance. These strategies involved modification of
the existing microplates to increase the efficiency of the solid phase or introduction
of novel approaches based upon the concept of ELISA. For instance, microspot
technologies offered simple, sensitive and rapid diagnosis, which were based on the
principles of ELISA. Miniaturization technologies, in turn, presented a high degree

of sensitivity within the platforms that require considerably less sample volume for
the assay that can be performed in a much shorter time as well [6, 7]. The major
emphasis of this chapter is on covering some of the modifications of ELISA that
promise improvements in any aspect of this well-known assay.
5.5.1 ELISA on Coated Platforms
Apart from pre-coating of ELISA well plates either with capture antibodies or other
types of reagents, the inner walls of well plates can also be treated through diverse
modification strategies. Different means such as plasma treatment, UV treatment or
acid treatment were applied to generate desirable functional groups that can pro-
mote protein binding in a stronger manner. Such treatment approaches, however,
have proven to cause limited shelf life for the functionalized platforms. Aside from
the hazardous waste material that is produced via chemical treatment, there is a lack
of control over generation of functional groups in respect to the type and distri-
bution of the functionalities on the surface [8]. Furthermore, generated functional
groups (especially via plasma treatment) tend to lose their activity over time due to
the aging effect or reorientation phenomenon [8]. It is, consequently, of great
importance to develop modification strategies that offer specific types of func-
tionality with the desirable concentrations that can last long and do not lose activity
towards the proteins [8].
In a novel attempt, a co-polymer composition was developed by using two
monomers namely: methyl methacrylate (MMA) and methacrylic acid (MAA) [9].
This copolymer, poly(MMA-co-MAA), contained carboxyl (–COOH) groups due
to the presence of MAA in the structure. By the aim of spin coating technique, a
fine micro layer of this co-polymer was deposited on the surface and the coated
chips were integrated into the untreated conventional 96-well plate (Fig. 5.1). The
assay was performed via physical (direct attachment) and covalent (via carbodi-
imide chemistry) immobilizations of the proteins to the available –COOH groups of
the surface. This study has shown improved detection signal (*2-fold) in com-
parison to the conventional assay due to the presence of the functional groups that
have involved biomolecules in analyte-surface interaction [10, 11].
In a further attempt, surface functional groups were used for antibody immo-
bilization and subsequent detection of infectious agents via amine bearing spacers
(linear and branched spacers) [10, 11]. This technique has shown a considerable
improvement in the detection outcomes. The authors have also demonstrated that
proposed coated platforms and their surface functionalities are active even after
6 months from the date of sample preparation (Fig. 5.2) [10]. Therefore, this
strategy presents a solution to avoid aging effect or reorientation phenomenon. In
this method, created functionalities are part of the chemical history of the com-
pound thus they would not be affected by aging phenomenon [10].
5.5 Modified ELISA Platforms 73
Fig. 5.1 Summary of the experimental procedure: a poly(MMA-co-MAA) chemical structure

prepared via free-radical polymerization reaction; b spin-coating procedure by using poly
(MMA-co-MAA) solutions of different compositions deposited on silicon wafers; c SEM
cross-section image of the coated silicon wafer; d developed samples were cut into the chips with
dimensions of 4 mm 4 mm to fit in 96 well-plate; e physical attachment of dengue Ab to the
coated surface; f covalent immobilization of dengue Ab by means of carbodiimide chemistry [10]
Fig. 5.2 Detection range analysis performed on aged polymer coated surface via physical
immobilization and conventional ELISA (polystyrene) in a broad range of DV concentrations [10]
In this perspective, the question might be raised in regard to the direct coating of
the ELISA well plates with such co-polymer compositions. It is important to note
that plastic surface of the well plate can be damaged by the solvents applied for
such coating procedures and may cause irregularities in the inner walls of the well
plate. This effect changes the specific surface area from one well to another, which
results in the inconsistency of the outcomes. Moreover, solvents might impact the
transparency of the platforms and create chance of error in the readout especially if
the bottom parts of the wells lose clarity and transparency. Finally, such modifi-
cation might leave traces of the toxic chemicals inside the wells that can affect/
deactivate the proteins and cause failure in the assay. For the mentioned reasons, a
direct application of such co-polymer, as a coated layer, within ELISA well plate
seemed to be impractical.
As an alternative strategy, a new formulation for a co-polymer composition that
offers inherent functionalities can become the next material of choice for mass
fabrication of the well plates. While this new material could potentially possess
desirable characteristics of the current materials such as cost-effectiveness, trans-
parency, ease of fabrication etc., a slight alteration in its chemical structure could
result in significant enhancement of the well plate in biorecognition [11].
In the case of cell-based assays, different types of surface treatments/coatings
(known as tissue culture treatments) are normally performed to increase the adhesion
of cells to the solid phases. Some methods for performing these treatments include the
use of a high-voltage arc discharge into the wells or exposure to the plasma radiation.
Varying the source of plasma radiation or a combination of sources can lead to the
optimized surfaces modified for different cells, proteins or for other biomolecules to
adhere to the surface. Coating the inner walls of the well plates is another popular
strategy in cell-based ELISA protocols. Such coatings include streptavadin (for
binding of biotinylated compounds), collagen and poly-d-lysine. Well plates coated
with biomolecules normally have a restricted shelf life.
5.5.2 ELISpot
In 1983 Cecil Czerkinsky’s group in Gothenburg, Sweden described a method to enu-

merate the frequency of B hybridoma cells secreting an antigen specific immunoglobulin,
known as ELISpot [12]. Five years later, computer imaging was coupled with ELISpot
for analyzing and counting the spots. Later, membrane bottomed plates were developed
to perform ELISpot, and several studies validated the performance of the assay.
Comparison between ELISpot and other related immune cell assays granted ELISpot an
especial position as the basis for vaccination studies declared by WHO [13, 14].
Nowadays, ELISpot is an integral step for optimization of cell-based
antigen-specific anti-tumor therapy. Table 5.1 provides a list of commercially
Table 5.1 Different types of ELISpot along with their counting methods, study models, and other
specifics
Brand Spot counting Study model/ Targeted Specifics Ref.
method number of analyte
cells in the
plates
U-cytech Kodak 1D/software Mice spleens IFN-c and IL-4 The vaccine [109]
(Utrecht, package 3105 cells in response to a activated a high
Netherlands) per well liposome number of spots
encapsulated in the ELISpot
AE36 vaccine plate (16.67-fold
versus buffer
group)
Mabtech Via Peripheral IFN-c- (or IL-5- The sensitivity of [110]
(Stockholm, streptavidin-alkaline blood or IL-10) in ELISpot was
Sweden) phosphatase mononuclear response to found to be
substrate cells/ cephalosporin higher than
2.5 105 in conventional test
100 µl (40 vs. 8%,
p = 0.008)
NA Peripheral IFN-c– ELISpot [111]
blood releasing cells confirmed the
in response to diagnosis of
100 lg/mL DIHS in patients
sulfasalazine for whom the
diagnosis was
previously
doubtful
Dakewe Immunospot reader Splenocytes IFN-c—in Interferon [112]
(Shenzhen, and image analyzer response to expression level
China) DNA vaccine was increased
significantly in
the cells of DNA
vaccinated mice
ELISpot reader Splenocytes/ Humoral and IFN-c release was [113]
4 105 cells cellular immune significantly
per well responses via enhanced by
immunization adjuvant.
with The DNA vaccine
recombinant could induce both
DNA vaccine the humoral and
cellular immune
responses in mice
Oxford ELISpot reader BAL and Diagnosis of The predictive [114]
Immunotec pleural fluid/ TB in real-life value for TB in
(Abingdon, 2.5 105 clinical practice patients with
UK) fresh cells positive ELISpot
was 82.6% in
BAL and 92.3%
in pleural fluid
IFN-c Interferon gamma; TB tuberculosis; BAL Bronchoalveolar lavage; IL Isoleucine; AE36 A short
sequence of HER2 protein overexpressed in metastatic cancer; DISH Drug-Induced Hypersensitivity
Syndrome; DNA Deoxyribonucleic acid
available ELISpot platforms, which were presented in the recent years. ELISpot has
been widely used to assess the cytokine release of various types of cells with or
without stimuli. One of the main applications of ELISpot is to evaluate the immune
responses induced by the vaccines. For example, ELISpot technique can be applied in
the diagnosis of drug allergy in patients. This technique can determine whether the
problem is at the starting point or a severe case of a drug-related allergic reaction.
IFN-c-based assay is the most popular form of the ELISpot technique. IFN-c is a
T cell specific cytokine that is dramatically secreted by activated T cells. The IFN-c
ELISpot assay has been extensively used for the quantification of the immune
responses in vaccine discovery as well as for the prevention/treatment of various
types of diseases including acquired immune deficiency syndrome (AIDS) and
tuberculosis (TB). Owing to its advantageous features such as high specificity,
sensitivity and a wide detection range, ELISpot is a favorable, pivotal tool for
clinical trials-development, drug discovery, immunity monitoring, and cell-type
differentiation, among others. In general, ELISpot is a rapid, convenient, and
cost-effective assay that can be implemented in daily clinical routines. High sen-
sitivity, wide range of target analytes (e.g., cytokines and granzyme B), quantitative
measurement, high throughput, high content analysis, low limit of detection,
applicability to frozen/thawed biological samples and affordable price are the main
advantages of this technique [15]. It is believed that ELISpot is 100–200-fold more
sensitive than conventional ELISA in the detection of the secreted cytokines. It has
been successfully employed for detection of prostate cancer [16–18], breast cancer
metastasis [19], and T-cell-based cancer immunotherapy [20]. ELISpot could also
be upgraded to multi-color FluoroSpot [21] (Fig. 5.3).
5.5.3 Plasmonic ELISA
Metallic nanoparticles (e.g. gold and silver) exhibit optical absorption and scat-
tering properties in the visible-near-infrared (Vis-NIR) range, due to the
light-induced collective oscillation of surface electrons. This photon-driven
coherent oscillation of localized plasmon is known as localized surface plasmon
resonance (LSPR). The LSPR spectral position is in correlation with the size, shape,
composition, and inter-particle spacing of the nanoparticles. This spectral position
is also strongly dependent on the refractive index of the dielectric medium in the
surroundings of the metallic nanoparticles. For that reason, a slight change in the
morphology, assembly or local environment of such nanoparticles can result in
variation of LSPR peak, which is quantitatively controlled. These mechanisms
generate a shift in LSPR wavelength hence broadening the absorption spectra that,
in turn, displays a substantial color change [4]. This class of immunoassays is also
Fig. 5.3 Comparison of detection of Borrelia-specific T cells in peripheral blood by the iSpot
Lyme assay and conventional ELISPOT assay. a The frequency of rBorrelia antigen-induced
IFN-c spot was established under both conditions in peripheral blood mononuclear cells (PBMC)
of Borrelia positive patients. Data points obtained from the same donor with the iSpot Lyme assay
and conventional ELISPOT assay are connected by a line. Each data point represents the mean
spot forming unit (SFU) of triplicate antigen-stimulated wells minus the mean SFU of the
corresponding medium control wells. A non-parametric Mann-Whitney U test was used to
compare the matched results with a p-value of <0.05 considered statistically significant.
b Representative well images for test results obtained from one healthy control run in triplicate and
c from a Borrelia positive patient run in triplicate. d Size distribution of IFN-c ELISPOTs obtained
from the iSpot Lyme assay versus the conventional ELISPOT assay, as specified by the closed and
open circles, respectively [108]
known as “nano-ELISA”, which promises an ultra-sensitive and efficient detection

strategy. This special class of nano-ELISA, also referred to as plasmonic ELISA
(pELISA), is gaining an increasing popularity.
Compared to the conventional ELISA, in which the color is developed by
enzymatic catalysis of organic dye/chromogenic nature, in pELISA changes in the
color tonality occur through an enzyme-assisted interaction in nanoparticle solution
as a function of analyte concentration. Normal ELISA requires laboratory readout
machinery. In contrast, pELISA detection is based on the visual judgment of the
results [4]. Table 5.2 provides several examples of plasmonic ELISA along with
their mechanism, target analyte, detection techniques and other properties.
Table 5.2 Different types of plasmonic ELISAs along with their mechanism of operation, target analytes, detection techniques, and other key important assay
78
evaluation parameters
Type of Plasmonic mechanism Targeted analyte Detection Sensitivity/specificity/ Properties Ref.
plasmonic technique limit of detection (LoD)
ELISA
Triangular Glucose oxidation induces the Prostate-specific SPR/micro plate Linear response from Ultrasensitive, cost-effective, easy [24]
silver etching of AgNPRs into smaller antigen (PSA) reader 10 fg/mL to 100 pg/mL to operate, lower LOD by more
nano-prism nanoparticles, resulting in color with the LOD of than 5 orders of magnitude in
(AgNPR) shift 4.1 fg/mL comparison to conventional assay
Hydrogen peroxide is used to etch Cr (III) in aqueous SPR spectrum LOD of 3.13 ng/mL Equipment free detection via [115]
AgNPRs resulting in a color media collected by and sensitivity of visual judgment of the samples
change from blue to mauve micro-plate 6.25 ng/mL for visual
reader detection
Gold Hydrolysis of acetylthiocholine Treponema The absorption Linear response from Compatible with conventional [116]
nanoparticles alters the surface charge pallidum antibodies spectra (400– 1 pg/mL to 10 ng/mL ELISA, cost-effective, easy to
(AuNPs) distribution of the AuNPs and 800 nm) with the LOD of automate
leads to the color and absorption measured by 0.98 pg/mL
spectral response changes UV–Vis
Catalase (Cat) consumes H2O2 Methyltestosterone The absorbance LOD of 1.0 ng/mL with Equipment free detection via [117]
thus AuCl4− ions are generated in in spiked beef intensities of naked eye, LOD of visual judgment of the samples,
the reaction mixture, which sample reaction 0.01 ng/mL and linear higher sensitivity (50-fold) than
subsequently produce Au NPs. solutions were response from 0.03 to conventional ELISA, longer
The application of Au seeds recorded at 1.0 ng/mL with the assay procedure
generates a clearer color change 450 nm reader
Silica nanoparticles containing Ochratoxin A The absorption LOD of 1 ag/mL with Considerably low limit of [118]
poly acrylic acid brushes with (OTA), in samples spectra naked eye and 0.05 ag/ detection, reliable and robust
Cat-labelled OTA compete for of corn, wheat, rice, measured at mL by microplate detection platform, high clinical
antibodies. At low and high OTA coffee as well as in 562 nm. reader and agricultural relevance
concentrations, AuNPs make a blood serum Photographs of
blue or red solution, respectively the solutions
were taken by a
digital camera
(continued)
5 Advantages, Disadvantages and Modifications …
Type of Plasmonic mechanism Targeted analyte Detection Sensitivity/specificity/ Properties Ref.
plasmonic technique limit of detection (LoD)
ELISA
Gold ELISA controls the growth of HIV-1 capsid The absorbance LOD of 1 ag/mL with Low concentrations detection, [119]
(III) chloride gold nanoparticles and generates antigen p24 in at 550 nm was the naked eye naked eyed recognition
tri-hydrate solutions with distinct colors in whole serum recorded with
the presence of analyte plate reader
Nanoparticles growth was HIV-1 protein The absorbance LOD of 0.08 aM The tonality of the resulted [120]
dependent on the small changes in gp120 was measured at solution is the function of
the concentration of H2O2 550 nm by UV– controlled changes in H2O2

controlled by Cat. The growth Vis concentration
procedure stopped after adding
glutathione
Silver Silver nanocrystals are produced Low-abundance The absorbance LOD of 0.23 ng/mL Higher sensitivity, LOD 10-fold [121]
nanoparticles from alkaline phosphatase alpha fetal protein at 400 nm was lower than conventional ELISA
(AgNPs) mediated ascorbic acid reacting (AFP) in clinical measured by a
silver ions, which turns the sera micro-plate
colorless solution to yellow reader
Gold Iodine mediated etching of Human IgG in The extinction LOD of 100 pg/mL, Considerably low limit of [122]
nanorods AuNRs from rod to sphere shape, spiked bovine spectra were visual detection limit detection, shorter assay procedure
(AuNRs) leads to a change of the solution’s serum measured on a was found to be 3 ng/ due to avoiding additional
color from blue to red NanoDrop mL modification of antibodies,
(linear response auto-aggregation, and labeling of
at 670 nm) nanoparticle
79
Further advances in enhancing accuracy of pELISA were dedicated to the

quality of the naked-eye inspection. Guo et al., proposed a dual-color response that
improved the sensitivity and accuracy of the visual judgment of the detection
outcomes [22]. In their strategy, two distinct colors were observed in response to
different concentrations of analytes. The reaction solution gradually changed from
wine red to colorless, and subsequently to yellow with the concentration of the
analyte increases.
Through the enzyme-mediated growth of silver and gold nanoparticles, ELISA
leads to a rapid color emergence of the colloidal solutions with various degrees of
yellow, blue and red (Table 5.2) [23]. This strategy permits growing of nanopar-
ticles with a desired state of aggregation during a signal-generation step. In this
reaction, the concentration of hydrogen peroxide is the key factor to control the
state of nanoparticles aggregation [23].
By taking advantage from the unique optical properties of triangular silver
nanoparticles AgNPRs and the etching effect of hydrogen peroxide generated from
the enzymatic oxidation of glucose, Liang et al., successfully developed a simple
sensor for detecting cancer biomarkers in the clinical samples (Table 5.2) [24]. This
assay is based on an etching process in which the alteration in the shape and the size
of the nano-prisms can be detected via an SPR shift. The results indicated that this
plasmonic biosensor can detect biomarkers at a concentration as low as 4.1 fg/mL
without needing sophisticated experimental procedure or equipment [24].
Dou et al. (1997) were the first to adapt an ELISA-type assay by replacing the
chromogenic readout with surface enhanced Raman scattering (SERS) detection
[25]. Cao et al. (2002) prepared SERS-active nano-structures by seeded growth of
silver on gold and demonstrated that these structures are useful for multiplexed
nucleic acid detection [26, 27]. Several approaches were reported to indirectly
detect the presence of a complex and/or weakly scattering molecule by labeling it
with a small molecule with high intensity and characteristic spectral features [28,
29]. Figure 5.4 represents the extrinsic labeling process for ELISA-based detection
of different analytes.
5.5.4 Sphere-/Bead-Based ELISA
Application of micro- and nanos-spheres in immunoassays is universal as such

spherical platforms overcome some of the major shortages of the current
bio-receptive platforms [7, 30]. Some of the main advantages of the micro/nano
spheres are as follows: (i) amenability to screening and multiplexing; (ii) 3D
configuration that offers larger surface areas in comparison to 2D structures; and
(iii) high spatial freedom for analyte-surface interaction. Particularly, micro- and
nano-spheres have attracted considerable attention as the enhancers of
bio-recognition. Different studies have reported applications of micro-/nano-spheres
for detection of several categories of targeted biomolecules [7, 30]. Metallic par-
ticles such as silver nanoparticles (AgNPs), gold nanoparticles (AuNPs),
Fig. 5.4 Schematic of extrinsic SERS labeling methods: a SERS used to detect enzyme reaction
product, b Detection of a labeled DNA after hybridization to capture ssDNA bound to a silver
substrate, c Extrinsic Raman Labels (ERLs) formed by the co-addition of a Raman reporter and
protein to gold colloid, d Composite nanospheres with encapsulated Raman labels and protective
silica shell, e Silver-enhanced SERS detection with protein-modified gold probes acting as
nucleation sites for electroless silver deposition [27]
paramagnetic microspheres, as well as polymeric microspheres have rapidly found

important positions in bio-analytical applications [31–33].
The bead-based ELISA typically represents the sandwich assays and substitutes
the flat plastic surfaces. Due to the mobility of the particles, bead-based ELISA can
be applied in different classes of detection systems [34]. For instance, Qu et al.
(2014), employed spherical polyacrylic acid brushes as scaffold for HRP loading via
covalent immobilization (Fig. 5.5). By using EDC/NHS chemistry, a layer of capture
antibody was immobilized on the HRP loaded brushes. Via a sandwich assay, human
chorionic gonadotrophin (hCG) was detected, which is a biomarker for early preg-
nancy and trophoblastic tumor. This strategy significantly improved the detection
limit up to 267-fold owing to the 3D architecture of the nano-spheres [35].
It is believed that the bead-based immunoassays are capable of producing
enhanced detection signals when comparing to individual assay replicates. With
hundreds or thousands of beads reporting the capturing of the target analyte, bead-/
sphere-based assays are promising platforms for high throughput detection [6].
Hosseini et al., developed PMMA-based microspheres in different size ranges
through suspension polymerization reaction (Fig. 5.6). In a thorough study, authors
have charged wells of the conventional ELISA well plates with different dosages of
the developed microspheres and conducted the normal assay procedure as per-
formed in the clinical practices (Fig. 5.7) [6].
The polymeric microspheres were designed to not only offer a large surface area
but also to possess specific chemistry as shown in Fig. 5.8. Due to the presence of
MAA monomers in reaction mixture (2% and 4%), the surface of the microspheres
offered –COOH groups, known as one of the desirable active functional groups.
Spheres were further employed for immobilization of anti-dengue antibodies by
which enveloped dengue virus (DENV) was detected. Authors report both physical
and covalent immobilization of the biomolecules on the surface of the spheres by
Fig. 5.5 Ultra-sensitive sandwich ELISA using enzyme-loaded nano-spherical brushes as labels
[35]
Fig. 5.6 FESEM images of the methacrylic microspheres [30]
using EDC/NHS treatment followed by glutaraldehyde (GA) chemistry (Fig. 5.8).

This strategy produced 25-fold greater fluorescence signal for detection of DENV
in comparison to the conventional assay (Fig. 5.9). It is particularly important as
infectious diseases such as dengue fever are fatal illnesses, which have relatively
short period (7–14 days) that can result in the death of the patients if they enter to
the acute phases. Current clinical assays are capable of detecting dengue only from
the third day from the onset of the fever when body produces enough number IgGs
against the virus. Early detection of DENV is only possible by focusing on
bio-recognition of biomarkers such as NS1, which are available in the body since
the onset of the fever. Sphere-/bead-based strategies promise such advancements for
ultra-sensitive detections. Table 5.3 provides some of the examples of the sphere-/
bead-based detection platforms, which are commercially available.
Quantum dots (QDs) offer another interesting strategy in which the wavelength
and the intensity of the emitted light is defined by the chemical composition, and
shape of the QDs as well as their environment. The utilization of the QDs’ optical
properties is typically the key factor for designing such ultra-sensitive assays.
Enzymatic formation of QDs enables various physical methods such as fluores-
cence spectroscopy and electrochemistry to be employed for the signal readout thus
significantly improves the detection limit of the immunoassays [34]. However, the
Fig. 5.7 Integrated microspheres into the 96-well plate [7]
exploitation of the electrochemical characteristics of the QDs that can result in

quantification of enzymatically generated signal without any light source is yet to
be fully explored [36, 37]. In recent years, several voltammetry systems were used
for electrochemical characterization and quantification of QDs [38, 39]. Taking
advantage from the electrochemical properties of QDs would lead to the develop-
ment of the universal cost-effective platforms, which could be applicable to a wide
range of detection targets by using the enzymatic growth of QDs [34].
5.5.5 Paper-Based ELISA
Paper materials have proven to have various applications in areas such as tissue
engineering, controlled drug release, wound healing dressings, molecular separa-
tion, preservation of bioactive compounds, environmental analysis, and food/
beverage quality control [40–42]. By addressing some of the WHO’s requirements,
paper-based bio-sensing devices have also demonstrated to be capable and reliable
platforms for detecting various types of target analytes. Paper-based biosensors
have attracted substantial attention for their cost-effectiveness, large available
Fig. 5.8 Dengue antibody binding on microspheres with surface –COOH groups and detection of
DENV through sandwich ELISA method: (top) physical adsorption of antibodies on the surface of
the spheres and subsequent coupling of antigen; (middle) proposed chemical structure after
free-radical polymerization between MMA, MAA and cross-linker TEGDMA; (bottom) covalent
immobilization of antibody molecules through carbodiimide cross-linking for further interaction
with antigen [30]
surface area and highly porous structures [43]. Especially, power-free fluid trans-
portation through the capillary action is one of the most interesting features of the
paper materials that make them suitable candidates for hand held detection devices
[40].
Analytical use of paper materials dates back to early 17th century when cellulose
papers were used for chromatographic purposes, [44] and pH sensing [45, 46]. The
first paper-based dipstick for glucose measurement in urine samples was presented
in the 1950s. This platform was commercialized one decade later for the diabetes
tests [47]. Although known for their use in filtration purposes, nitrocellulose
membranes have also established their applications in molecular recognition in the
1970s [48]. The subsequent decades marked a substantial growth of paper
Fig. 5.9 Fluorescence microscopy images of the microspheres after detection of the Dengue
virus: a methacrylic microsphere (MMS) 2% and b MMS 4%; c recorded fluorescence detection
signal [30]
applications in serological lateral flow tests, especially for pregnancy tests [49].
Recent expansions in application of papers in equipment-free bio-analytical devices
that rely on visual judgment have introduced a new exhilarating chapter in the
design and fabrication of paper embedded platforms [50].
In the recent decades, various type of paper-based bio-analytical platforms were
developed, including dot-immunobinding assays (DIAs), microfluidic paper-based
analytical devices (lPADs), lateral flow immunoassay (LFIA), laminated
paper-based analytical devices (LPADs), immunospot, nitrocellulose pads
(NC-PADs) and paper-based ELISA (P-ELISA) well plates [40–42]. Fabrication of
such devices includes different techniques, such as plotting [51, 52], wax-printing
[53–57], inkjet-printing [58, 59], flexographic printing [60], computer-controlled
knife cutting [61], laser cutting [62, 63], vapor-phase polymer deposition [64–66],
photolithography [67–72], spraying [73], electrospinning [43, 44, 74], coating [75]
as well as other techniques [40, 59]. Papers have found increasing popularity and
their fabrication strategies, applications in bio-recognition, the storability and the
marketability have been the focus of various studies [40, 41, 59, 76–79].
Table 5.3 Different types of sphere-/bead-based detection platforms along with their target analytes, principles of operation and outcomes
Brand Target analyte Principle Outcomes Ref.
Polysciences, Human isoleucine (IL-10) Antibody coated polystyrene beads LOD of 12.5 ng/mL [123]
Inc., were pneumatically loaded into a The system performed all the
(Warrington, column and ELISA was performed by necessary ELISA steps in a quarter of
PA, USA) sequential addition of reagents the time required for corresponding
plate-based protocol
Invitrogen Total aflatoxins Antibodies were conjugated to LOD of 0.21 ng/g and a detection [124]
(Carlsbad, (AFB1 + AFB2 + AFG1 + AFG2) in carboxylic acid activated magnetic range of 0.22–19.8 ng/g
CA, USA) spiked and contaminated maize beads via carbodiimide chemistry Recoveries from 74.5–96.5% in spiked
samples The HRP labelled aflatoxins compete samples with coefficients of variation
for coupling with antibodies (CV) under 12.1%
Apolipoprotein A1, a biomarker highly Magnetic beads functionalized with LOD of 9.16 ng/mL and the detection [125]
correlated with bladder cancer epoxy groups were coated with range of 0–9000 ng/mL
antibodies and integrated within a No sample dilution is required
microfluidic platform equipped with a Relatively short assay procedure (30–
bubble removing mixer 40 min)
Readout signal is obtained by
fluoroscopy
Anti-mycobacterial IgG in plasma ELISA was performed by simultaneous Sensitivity of 80%, and specificity of [126]
actuation of antigen-coated 75%
tosyl-activated magnetic microbeads Low cost ($10 USD)
moved by the aim of six magnets below Low sample volume required
the platform and the sequential Quick and easy assay procedure
insertion of reagents (15 min)
(continued)
87
88
Brand Target analyte Principle Outcomes Ref.

R&D systems C-reactive protein (CRP) in human A low cost thin-film transistor LOD of 0.2 ng/mL [127]
(Austin, serum (TFT) nanoribbon sensor was used for Detaching the immobilized proteins
Texas, USA) detection of the inflammatory from the surface enhanced the
biomarker CRP via a miniaturized chip, detection signal
and magnetic bead-based ELISA Compared to classically fabricated
nanowires, the TFT nanoribbon
sensors are simple, easy to fabricate,
and cost-effective
Matrix metalloproteinases (MMP) and Analyte-specific antibodies are This assay was performed to [128]
myeloperoxidases (MPO) in tears of pre-coated on the surface of demonstrate the number of neutrophils
patients with ocular graft versus host color-coded micro-particles. Captured released enzymes (MMP and MPO)
disease (oGVHD) analytes are subsequently detected by positively correlated to oGVHD
using a cocktail of biotinylated (P < 0.001)
detection antibodies and a
streptavidin-phycoerythrin conjugate
BioRad Phosphorylated kinases level after Kinase specific microspheres were This assay demonstrated that kinase [125]
(Hercules, treatment with inhibitor drugs (this placed inside a conventional solid activity is down regulated
CA, USA) treatment has shown to induce phase ELISA microtiter plate (phosphorylated) 1- to 4-fold when the
apoptosis and necrosis in various types inhibitor is added during a 4 h
of tumors cells) treatment
Perhaps, one of the more acknowledged paper-based ELISA platforms is

Western blot. Its main application is to identify and separate proteins. In Western
blot, a combination of proteins is separated according to the molecular weights, and
type of the biomolecules by the aim of gel electrophoresis. A membrane that can
produce a band corresponding to each protein displays the results. This membrane
is loaded with labeled antibodies specific to the protein of interest. As the mem-
brane film is being developed, the labeled antibodies that bound to the protein of
interest produce the visible band. The thickness of the band in this assay is in direct
correlation with the concentration of the protein [80].
Wang et al. (2012) presented a paper-based chemiluminescent ELISA
(CL-ELISA), a versatile, ultrasensitive platform with promising applications.
Authors report sandwich CL-ELISA on the chitosan modified paper pads, intro-
ducing a new strategy to overcome the instability of the pure cellulose papers.
Typical cellulose papers lose the strength when saturated with aqueous media. In
particular, when the means for immobilization is through covalent attachment of the
biomolecules to the surface, there are additional steps of treatment and incubations
such as EDC/NHS and/or GA treatments involved. Such lengthy steps and incu-
bation in strong chemical and reagents typically compromise the nature of the
paper. Authors described that a higher binding-stability and a better wet-strength in
sandwich CL-ELISA were achieved when the papers were chitosan modified [81].
Based upon the principles of ELISA, a wide range of microfluidic paper-based
analytical device (lPAD) were designed and fabricated that offered highly
sensitive/selective detection options for complex biological samples [82].
Commercially available papers known as Whatman # 1 were used for the detection
of rabbit IgG antibody via indirect ELISA [83]. The lPAD in this study was
fabricated in the shape of paper mats with a set of electrodes on each side.
Micro-zones in this paper device were imaged by a scanner for the visual judgment
and the readout outcomes were also measured by cyclic voltammetry through
electrochemical detection.
Whatmann CHR # 1 papers were aluminum foil-baked for design and fabrication
of a lPAD with very small features and narrow barriers [84]. By the aim of wax
printing, nitrocellulose paper (NC) were applied for fabrication of a lPAD with
printed channels that function as timing valves. This platform was used for
detection of imidacloprid (small molecule pesticide) via competitive ELISA
method. In this technique, the color intensity was recorded by a smartphone and
images were processed by Image J software [85]. Via inkjet printing, NC paper was
also used for fabrication of a lPAD that successfully detected the hCG via a
sandwich assay (Fig. 5.10). In this strategy, a digital camera imaged the color
changes. The mean color intensity of the image at the selected area was quantified
by using the histogram function with the RGB channel in Adobe Photoshop [86].
Nylon membrane was also used for development of the bio-receptive platforms.
Farahmand et al., coated commercially available nylon membranes with poly
(MMA-co-MAA) synthesized via free-radical polymerization reaction [75]. This
technique not only has given more stability to the paper material to bear the time
consuming tedious sandwich ELISA, but also provided with surface –COOH
Fig. 5.10 Schematic illustration of the automated paper-based device with 2 different designs for
the sandwich ELISA. The prepared substances were included on the devices in (a) the control
zone, which contained the immobilized Ab that picks up free (Ag unbound) enzyme-linked
detection Ab to confirm that the test has operated correctly; (b) the test zone, which contained the
immobilized Abs specific to the target Ag (forming a sandwich ELISA) and showed a colored
band for positive test samples; (c) enzyme-linked detection antibody (the second antibody) that
was allowed to bind to the antigen; and (d) substrate mixture that reacted with the enzyme label to
generate the insoluble colored product. The outer shape of the device was cut from an NC
membrane [86]
groups that can highly promote analyte-surface interaction. Figure 5.11 shows the
frontal view and the cross-section images of the uncoated and coated nylon
membranes. Coated paper was cut into the circle shapes to fit at the bottom of the
96-well plate and ELISA was performed through physical as well as covalent
means to assess the performance of the assay in detection of DENV (Fig. 5.12)
[75]. Chemically designed coated nylon membranes have shown improvement
(4-folds) of the detection outcomes in comparison to the uncoated nylon or con-
ventional ELISA. Moreover, this strategy resulted in significant enhancement in
sensitivity, specificity, accuracy and LOD when compared to that of conventional
assay [75].
5.5.6 Fiber-Based ELISA
Significant potentials of fiber-based platforms promise a vibrant area of research to

the scientific society. Application of fibers in bio-analytical fields initiated by the
use of textile materials for chromatographic purposes [87]. Additionally,
Fig. 5.11 SEM images of polymer-coated membranes (a and b); cross-sections of the uncoated
nylon membrane control (c); and representative poly(MMA-co-MAA) coated membrane (d) [75]
Fig. 5.12 Polymer-coated nylon membranes for the dengue virus detection: a free-radical
synthesis of poly(MMA-co-MAA) with AIBN initiator; b dip-coating of nylon membranes in the
polymer solution; and c dengue antibody attachment to polymer-coated membrane and sandwich
colorimetric ELISA experiment with (left) EDC/NHS cross-linking, and (right) physical
adsorption of dengue Ab molecules [75]
high-performance liquid chromatography (HPLC) technique has introduced poly-

mer fibers as suitable candidates for protein separation due to their high capacity/
mass transfer rates, tunable chemistry, and non-denaturing and re-generable sur-
faces [88]. In medical diagnostic, carbon-based bio-receptors such as carbon micro/
nano fibers and composite carbon fibers provided substantial advancement in
bio-recognition of bio-entities via electrochemical detection [89–91]. Various
carbon-based biosensors were reported to enhance detection strategies conven-
tionally used [91–94]. Polymer fibers, on the other hand, have opened new win-
dows of opportunity to the new classes of fiber-based medical diagnostics
platforms. Table 5.4 presents some of the state-of-art developments of paper/fiber
platforms, in specific for their application in detection of antibodies, antigens,
whole viruses, bacteria, and different classes of proteins via ELISA.
Nano-coated nylon fibers were reported by Reukov et al., to possess positively
charged characteristics after surface modification. This class of fiber-based materials
were used for bacterial vaginosis detection and pregnancy diseases monitoring [74].
Triple-blend electrospun fiber mat were applied for the bio-recognition of colorectal
cancer [95]. Wu et al., presented electrospun poly(e-caprolactone) (PCL) fibers for
fluorescent detection of antibodies against human serum albumin (anti-HSA) [96].
Polyvinylidene fluoride (PVDF) nano-fiber membrane was reported for its appli-
cation in Western blot [97].
Electrospinning was also used for fabrication of polyhydroxybutyrate
(PHB) fibers. This material offered a flexible platform with high strength for tedious
lengthy assays. PHB fibers were dip-coated with poly(MMA-co-MAA) to introduce
–COOH reactive functional groups to the surface [43]. Figure 5.13 shows the
frontal and the cross-section views of the uncoated and coated electrospun PHB
fibers. Clear signs of interstitial coverage can be observed from the morphology of
the coated fibers. These fibers were used in sandwich ELISA for the highly sen-
sitive and selective detection of DENV [43]. Further thermal gravimetric analysis
(TGA) of the fibers after protein immobilization has shown a shoulder in TGA
spectrum that corresponds to the presence of capture antibodies on the surface of
the fibers (Fig. 5.14) [98].
In another study, the same strategy (electrospinning and dip-coating) was used
for fabrication of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) fibers
coated with poly(MMA-co-MAA) (Fig. 5.15) [44]. PHVB possess favorable
characteristics for the intended application in sandwich ELISA as it was found to be
a stronger material than PHB for such laborious procedure. Furthermore, PHBV is
generally more electronegative than PHB due to the presence of oxygen atoms in its
chemical structure. This fact to a certain extent can promote biomolecular attraction
via electrostatic interaction and hydrogen bonding. Therefore, the overall perfor-
mance of PHBV in detection was found to be better than PHB with 6 times greater
detection signal resulted from the same conducted assay [43, 44].
Figure 5.16 provides all the possible means through which PHBV fibers can
facilitate biomolecular interaction with capture antibodies against DENV. In gen-
eral, a suitable bio-receptor surface has to be benefited both from the physical
Table 5.4 Paper- and fiber-based bio-analytical platforms aimed for ELISA assays along with the type of target biomolecules, applied detection techniques,
and evaluation parameters of the method. [129]
Type of Target analyte Detection technique Sensitivity/specificity/limit of detection Ref.
paper/fiber (LoD)
Whatman # Anti-dengue IgM Fluorescence Specificity = 99% [130]
903 antibody The fluorescence spectra were measured in a spectrofluorometer with the excitation at LOD = 40 ng/mL in serum and 0.8 ng
360 nm and emission at 450 nm in serum dilution 1:10000 as positive
control
Whatmann Fetoprotein (AFP) Chemiluminiscence Detection range of 0.1–35 ng/mL for [131]
CHR # 1 Cancer antigen 125 Readout was performed by ultraweak luminescence analyzer AFP, 0.5–80 U/mL for CA125, and
(CA125) 0.1–7 ng/mL for CEA
Carcinoembryonic
antigen (CEA)
Haptoglobin in bovine The test zone was scanned using a flatbed desktop scanner and images were transformed into Sensitivity was reported to be low; [132]
serum 8-bit gray-scale LOD = 0.73 lg/mL
Rabbit IgG antibody Electrochemical (cyclic voltammetry) LOD 3.9 fM [133]
Micro-zones in the paper device were imaged by GE Typhoon Trio Scanner. The excitation
and emission wavelengths were 532 nm and 580 nm, respectively.
Whatman # 1 Ubiquitin and Colorimetric Changes on the paper disk were recorded with a mobile phone camera and Not specified [83]
enhanced green quantified by Adobe Photoshop CS2 software in grey mode to obtain the
fluorescent protein average intensity using a fixed quadrant
(eGFP)
Cardiac marker The color changes in the platform were scanned using a desktop scanner and Detection concentration = 50 ng/mL [134]
protein, myoglobin analyzed by Image J
Extracellular vesicles The test zone was scanned using a desktop scanner and the data were saved Not specified [135]
in 8-bit format. The intensity of the color was quantified using Image J
Auto-antibodies The color changes were recorded by a commercial desktop scanner and Sensitivity in serum = 81.8% [136]
analyzed by Adobe Photoshop software Sensitivity in blister fluids = 83.3%
Specificity in serum = 75%
Specificity blister fluids = 85.7%
(continued)
93
94
Type of Target analyte Detection technique Sensitivity/specificity/limit of detection Ref.

paper/fiber (LoD)
Dengue virus antigens The colorimetric results were recorded using a commercial Apple, iPhone 4S LOD 100 pg/mL [137]
and enveloped dengue Sensitivity >40 times than
virus conventional readout
Neuropeptide Y For the purpose of comparison, images were captured using a Canon EPS/ Pico to nanomolar range [138]
Rebel T3i/EOS 600D camera, HTC Droid Eris smartphone as well as HP
Color 4540 scanner/printer
Toxoplasma gondii The test zones were analyzed by a commercial desktop scanner and Adobe Sensitivity = 87%; Specificity = 96% [139]
antibody in serum Photoshop software. Digitalized images were converted to the CMYK color
mode, and the mean pixel intensity was determined using the histogram tool
Vascular endothelial The color changes were recorded by a desktop scanner and smart phone and Detection range = 0.01–100,000 pg/ [140]
growth factor (VEGF) analyzed by Adobe Photoshop software mL; LOD = 0.03 pg
PHB fibers Dengue enveloped Conventional readout was performed by ELISA reader Sensitivity = 100%; Specificity = 80% [43]
PHBV fibers virus Sensitivity = 97.49%; [44]
Specificity = 90.83%
Nylon Sensitivity = 100%; [75]
Specificity = 93.75%
Cellulose Antibody against Using a desktop scanner LOD = 54 fmol/zone [82]
HIV-1 envelope
antigen (gp41)
Whatman #42 T7 bacteriophage Color changes were recorded using a desktop scanner. The samples were Detection range = 100–109 pfu/mL [141]
then analyzed using standard image processing software
Whatman E. coli bacterial Color intensities were recorded by portable scanners or smartphones Detection concentration = 105 cells/mL [142,
Fusion 5TM 143]
Nitrocellulose Imidacloprid (small Color intensities were recorded by a smartphone and the images processed by LOD = 0.01 ppm [85]
molecule pesticide) Image J
Human chorionic A digital camera imaged color changes. The mean color intensity of the Detection concentration in [86]
gonadotropin (hCG) image at the selected area was quantified using the histogram function with urine = 4 ng/mL
the RGB channel in Adobe Photoshop CS3
Fig. 5.13 Representative morphology analysis of PHB fibers by FESEM: a PHB electrospun
fibers; b and c copolymer coated PHB fiber; d cross-section image of the copolymer coated PHB
fibers [43]
perspective (desirable hydrophobicity, porosity, and surface area) as well as the

chemistry of the surface (surface functionalities).
Whitesides’ research group was the first to develop a paper-based ELISA
96-well plate. The paper-based indirect ELISA developed in this study has shown
successful detection of rabbit IgG and HIV-1 envelope antigen gp41. This strategy
combined the sensitivity and selectivity of ELISA with the cost-effectiveness and
ease-of-use qualities of the paper material [82]. Several other paper-/fiber-based
microplates were developed as the continuation of this approach. While some of
these platforms were commercialized, majority of these paper-based well plates are
complex in their fabrication, assembling and application. Figure 5.17a represents an
example of a commercially available paper-based well plate. This multilayered
platform is hard to be assembled, while in attaching some layers of this well plate
double sided adhesive layer was used.
Application of the adhesive materials in the assay that deals with biomolecules is
known to affect these entities or may cause their total denaturation as glue is known
to be toxic to the biomolecular entities. Furthermore, the paper parts in the majority
of these platforms are located at the bottom of the well plate. This characteristic, in
turn, limits readout options as the light cannot pass through the well and absorbance
Fig. 5.14 Thermal analyses of the coated PHB fibers before and after protein immobilization,
along with a schematic representation of the sandwich ELISA that takes place on the surface of the
coated fibers and leads to the final detection signal; inset is the zoomed in region between 0 and
300 °C showing the onset of degradation for proteins, as well as PHB fibers [98]
measurement is impossible in the bottom-readout strategies. In a novel attempt,

Hosseini et al., introduced a fiber-based well plate that involves the fiber segments
from the top into the wells (Fig. 5.18). This platform that is known as intrant
ELISA (i-ELISA) modifies only the lid, while the well plate itself remains intact
[44]. By adding pillar segments to the lid of the well plate, fiber segments are
introduced to the platform as Fig. 5.18 suggests.
This strategy allows the chemically designed fiber surfaces to enter to the
reaction solution, play their roles, and exist in the last step of the assay making the
regular readouts possible along with the visual judgment of the detection results.
Figure 5.17 schematically represent the differences between i-ELISA and a repre-
sentative of the currently available paper-based ELISA well plates. I-ELISA offers a
2-pieces platform that can follow the standard protocol applied in the clinical
practices. It is especially easy to operate without needing any assembling process in
advance. The mass fabrication of i-ELISA can be done through molding technique
promising a relatively cost-effective bio-analytical microplate benefited from the
presence of fiber surfaces.
Fig. 5.15 Schematic presentation of the procedure for fabrication of the polymer‐coated PHBV
(poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate)) fibers: a SEM (scanning electron microscopy)
image of pristine electrospun PHBV fiber; b FFES (far-field electrospinning) technique for
fabrication of the fibers; c dip-coating of PHBV fibers in poly(MMA-co-MAA) (poly methyl
methacrylate-co-methacrylic acid) solutions of different co-polymer compositions; d SEM image
of representative polymer-coated PHBV fiber sample; e, f cross-section SEM images of the
uncoated and coated PHBV fibers [44]
Fig. 5.16 Major forces that contribute to the biomolecular interaction via surface functional
groups [44]
5.5.7 ELISA in Micro-Devices
Miniaturization of ELISA in microfluidic platforms offers various advantages

including: (i) shorter duration of the assay; (ii) use of smaller sample volumes; (iii)
enhanced sensitivity and selectivity; (iv) multifunctionality of the platforms;
(v) easy operation for onsite health check; (vi) portability and application in remote/
rural areas; and (vii) minimized human contact with dangerous species.
Conventional ELISA is a series of mixing, washing, incubating, and aspirating
steps that might result in inconsistencies [1]. Due to the automation option offered
by microfluidic devices, the chance for error can be curtailed to a considerable
extent. Table 5.5 provides a number of different examples for ELISA in micro-
fluidic devices.
The immobilization of the biomolecules can take place inside the channel, the
chamber or on the surface of the integrated platforms such as coated chips, papers,
or beads. Micro-devices are benefited from the miniaturized size and its subsequent
large surface-to-volume ratio [1]. Depending on the design, some examples of
Fig. 5.17 General comparison between a multilayer analytical 96-well plate as the representative
of the complex paper-assisted platforms and i-ELISA [44]
Fig. 5.18 Fabrication of

i-ELISA (intrant
enzyme-linked
immunosorbent assay)
platforms: a modification of
the lid of a 96-well plate; and
b fixation of PHBV
electrospun fiber mat and
intrusion into ELISA liquid
mixture [44]
micro-devices involve electric or magnetic forces to conduct the assay [99].

Different designs of the microfluidic platforms introduce passive or active valves to
guide the fluids to the channels or through a series of chambers. Al-Faqheri et al.,
developed a microfluidic compact disk (CD) platform that incorporated passive
check valves for liquid swapping. This platform operates based on the centrifugal
force thus the liquid transfer occurs, while the CD is rotating with a certain speed
[100].
Figure 5.19 presents the CD microfluidic, reported in this article, which was
used for detection of dengue antigen via ELISA [100].
Efficient analyte-surface interaction is a function of various different factors
including specific surface area, functionality of the bio-receptive surfaces and
accessibility of the surface to the entire sample volume. In particular, in the case of
micro-devices, the proper mixing of the reaction components plays a major role due
to the small size of the platform. Mixing strategies in micro-devices address this
issue through variety of different designs and fabrication strategies. Aeinehvand
et al., introduced a micro-balloon mixing system within a CD microfluidic platform
that allowed an effective mixing process [101]. Figure 5.20 shows the mixing
platform, which was evaluated by using an intensity histogram. This micromixer
operated based on the expansion and contraction of a micro-balloon that provided a
continuous reciprocation of the flow. Authors reported a sensitivity enhancement in
detection of infectious agents by one order of magnitude when using this
Table 5.5 A summary on the performed ELISA protocols inside the microfluidic devices
Target analyte Automation Disposability ELISA Equipment Detection technique LOD Time Features Ref.
type of the
assay
Staphylococcal Yes No Sandwich Computer, digital Chemiluminescence 0.1 ng/mL <2 h – Low cost [144]
enterotoxin B CCD camera – Multi-target
– High sensitivity
due to the
application of
carbon nanotubes
(CNTs)
IFN-c Yes Yes Sandwich Potentiostat, Amperometry 126.75 pg/ <5 h – Possible assay [145]
laptop mL parallelization
– Accurate reagent
fluidic control
Human No Yes Sandwich Fully integrated Visual colorimetric 0.01 ug/ 2h – Equipment-free [146]
C-reactive system mL – Visual analysis
protein – Ease of operation
– High sensitivity
– Low cost
Ochratoxine A Automated fluid No Indirect/ Peristaltic pump, Chemiluminescent 0.5 ng/mL 3 h, – Automated [147]
in wine and delivery competitive fluid dispenser, 46 min – USB
bear transimpedance communication
amplifier
(continued)
101
102
Target analyte Automation Disposability ELISA Equipment Detection technique LOD Time Features Ref.
type of the
assay
IgG antibodies Semi-automated No Direct Laser source Fluorescence, 0.182 pM/ 30 min – Multiple detection [148]
Pumps Colorimetric, cm2 – Potential
Computer Chemiluminescence disposability
Dengue Yes Yes Sandwich Electronic Absorbance NA 2 h, – Clinical relevance [149]
antibody IgG circuitries and 10 min – High accuracy and
platform for sensitivity
sensors and – Data analysis by
motors smartphone
Interferon-g No Yes Sandwich Microscope Fluorescence 20 pg/mL 4 h, – Oxygen plasma [150]
(IFN-g) 30 min treated
micro-channels
– Broad detection
range
– Shorter detection
time
Fig. 5.19 Microfluidic CD design to perform ELISA [100]
micro-device equipped with mixing system in comparison to the conventional

ELISA [101].
ELISA was fully automated on a PS-bead-based CD microfluidic for detection
of hepatitis B virus (HBV) in whole blood. The centrifugal force was used for
mixing PS beads in the mixing chamber to maximize the analyte-surface interac-
tion. The whole blood sample was added to the CD platform and the plasma
separation, incubation with antigen-antibody conjugated microbeads, washing
procedures and substrate addition/enzymatic reaction were all performed inside the
CD platform within 30 min (Fig. 5.21) [102]. In the final step, the CD device was
placed inside a blood analyzer equipped with an absorbance detector and the color
intensity was recorded (Fig. 5.22).
Custom-made microspheres with designed chemistry of the surface were inte-
grated into a CD microfluidic equipped with micro-mixing system [7]. Spheres of
different size categories were loaded into the mixing chamber (Fig. 5.23), where
mixing happened through the reciprocation of the flow by the aim of centrifugal
force and the design of a micro-balloon (Fig. 5.24). In particular, the mixing
mechanism was based on the change in the rotation direction of the CD that has led
the micro-balloon to pump the solution up and down in the chamber hence facil-
itating proper mixing. Taking advantage from the micro-balloon micro-mixing
strategy, Hosseini et al., reported considerable improvement in the sensitivity of the
assay (15-fold higher detection signal than conventional assay), which was per-
formed in a significantly shorter time (5 min incubation duration). This specific
design of the CD microfluidic closely mimicked the actual 96-well plate thus by
keeping the sample volume constant the platform proved to be capable of
ultra-sensitive detection of DENV with the LOD of 1.9 p.f.u/mL [7].
The chemistry of the synthesized microspheres, on the other hand, played a
dominant role in detection enhancement. As Fig. 5.25 suggests, available surface
Fig. 5.20 Evaluating the

mixing efficiency using
intensity histogram. Gray
scale images and 2D color
intensity histogram obtained
after second, eighth and
fourteenth mixing cycles
[101]
Fig. 5.21 a Disc design showing the detailed microfluidic layout and functions. b–g Schematic
diagram of the reaction principle of the ELISA on a disc [102]
functionalities of the surface promoted majority of the physical forces in

biomolecular interaction. It is expected for higher number of the proteins to connect
to the surface of the spheres as the means for protein immobilization is not only
limited to the available surface area but also specific chemistry of the surface.
5.5.8 Other Strategies
5.5.8.1 Smart Devices in ELISA
Rapidly growing application of smart devices introduced a new era in design and
fabrication of bio-analytical devices. Use of smartphones, desktop scanners, tablets
and cameras, nowadays, seem to be an inevitable part of modern clinical strategies.
Such technologies, particularly serve in the remote and resource-limited areas that
typically lack the centralized laboratory facilities. Ukita et al., manufactured a
microfluidic analytical system for colorimetric ELISA by using 3D printing tech-
nique. In this approach, a mini-centrifuge can be used for onsite analysis, while the
results were analyzed by the aim of a smartphone [103]. This inexpensive platform
Fig. 5.22 a A photo image of the blood analyzer. b Schematic diagram showing the inside of the
blood analyzer. A detector module is installed for the absorbance detection. c Top and bottom plate
of the disc before the bonding. d An example of UV bonding image. The black area is where the
UV adhesive is applied. e A photo image of the bonded disc after the valve formation. f A
schematic diagram showing the side view of the mixing chamber (noted as red dotted square in
part E). The PS beads are confined in the mixing chamber by the weir structure in the outlet of the
mixing chamber [102]
Fig. 5.23 Integration of the optimized microspheres into the microfluidic disk equipped with
micro-mixing system [7]
promises onsite bio-recognition of different diseases as well as environmental safety

monitoring as the platform can to be easily deployed and readily used by a wide
range of users [104].
Fig. 5.24 Liquid reciprocations inside a mixing chamber [7]
Fig. 5.25 Different immobilizing interactions between microspheres and antibodies at the
interface [7]
5.5.8.2 Digital ELISA
Digital ELISA is a technique for capturing proteins immobilized on the microscopic

beads placed on the arrays of 50 FL ELISA well plates. The proteins are labeled
with enzymes, therefore, bead associated enzymatic activity can be detected via
fluorescence imaging. This strategy can facilitate multiplexing by using the sub-
populations of the beads with their fluorescent signatures on the same array within a
microfluidic device, in a simultaneous manner. This strategy allows biomolecular
recognition to sub-femto-molar level. Combined with other appropriate technolo-
gies, the LOD can reach to the single molecule level [105].
5.5.8.3 Aptamers as Antibody Substitutes in ELISA
Aptamers are single stranded DNAs or RNAs that bind to a wide variety of bio-
molecules. Aptamers have shown great potentials for replacing antibodies in the
ELISA procedure. These molecules are easy to be chemically synthesis, which
reduces the time and the cost of antibody production. Aptamers are smaller than
antibodies and less immunogenic in their nature. They typically do not lose their
inherent configuration and would denature only with a drastic temperature rise
[106]. Therefore, aptamers are interesting candidates for application in
biorecognition.
5.5.8.4 Multiple and Portable ELISA
Multiple and portable ELISA is a novel technique that incorporates a multi-catcher

device with 8–12 immunosorbents protruding pins onto a central stick that can be
immersed into the sample. The washing steps and incubations with
enzyme-conjugated proteins are simply performed by dipping the pins in the
charged micro-wells with related reagents. The main benefit of these ready-to-use
lab kits is that they are cost-effective, allow screening for a large number of sam-
ples, and do not require trained technicians or sophisticated laboratory equipment.
Mentioned qualities make the multiple and portable ELISA a great candidate for
providing healthcare in resource-limited settings [107]. Clinical applications of this
platform include the detection of infectious diseases, bacterial toxins, oncologic
markers, as well as drug screening.
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Ensaio de Imunossorventes Ligados A Enzimas (ELISA)

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SPRINGER BRIEFS IN APPLIED SCIENCES AND

TECHNOLOGY  FORENSIC AND MEDICAL BIOINFORMATICS

Forensic and Medical Bioinformatics

Marco Rito-Palomares Sergio O. Martinez-Chapa

Patricia Vázquez-Villegas Sergio O. Martinez-Chapa

ISSN 2191-530X ISSN 2191-5318 (electronic)

© The Author(s) 2018

Printed on acid-free paper

This Springer imprint is published by Springer Nature

Monterrey, Mexico Samira Hosseini

Authors would like to acknowledge the ﬁnancial support of Tecnológico de

1 Fundamentals and History of ELISA: The Evolution of the

3 Step by Step with ELISA: Mechanism of Operation, Crucial

5.3 Materials of Choice for Fabrication of ELISA Well Plates . . . . . . 69

Abstract Current chapter reviews background and history of the immunoassays

1.1 Evolution of the Immunoassays Until Invention

1.1.1 Side Chain Theory

In 1987 Paul Ehrlich, a German physician and scientist, published a communication

© The Author(s) 2018 1

1.1.2 Antigen-Antibody Binding Theory

in order to build a crystal matrix. Marrack also believed that antigen-antibody

1.1.3 Discovery of Antibody Structure

1.1.4 Invention of Radioimmunoassay (RIA)

Radioimmunoassay (RIA) was ﬁrst introduced by American scientists, Solomon

1.1.5 Invention of Enzyme Linked Immunosorbent Assay

material. The subsequent attachment of the complimentary biomolecules labeled with

1.2 Principles of the Immune System

1.2.1 Antibody Production in Human Body

Immunoglobulins (Igs), also known as antibodies, are manufactured proteins by the

1.2.2 Different Types of Antibodies

1.2.2.1 Immunoglobulin G (IgG)

Fig. 1.1 IgG structure [33]

1.2.2.2 Immunoglobulin A (IgA)

Fig. 1.2 Model of human

1.2.2.3 Immunoglobulin M (IgM)

Composed of ﬁve Y-shaped units, immunoglobulin M (IgM) is the largest antibody

Fig. 1.3 Structure of human IgM [37]

1.2.2.4 Immunoglobulin D (IgD)

Immunoglobulin D (IgD) is mostly present at the surface of the B cells as it assists

1.2.2.5 Immunoglobulin E (IgE)

Fig. 1.4 Structure of human

Fig. 1.5 Structure of IgE

1.2.3 Antigen-Antibody Coupling

1.2.3.1 Speciﬁcity of the Antigen-Antibody Coupling

1.3 Biomolecular Interactions Between Antibody

1.3.1 Hydrogen Bonding

1.3.2 Hydrophobic Interaction

1.3.3 Ionic Attraction

In their amphoteric forms, biomolecules might react through ionic attraction or

1.3.4 Van der Waals Forces

1.3.4.1 London Dispersion Force

1.3.4.2 Dipole-Dipole Interaction

Dipole-dipole interaction is the force between two atoms/molecules through their

Fig. 1.8 London dispersion forces

Fig. 1.9 Dipole-dipole interaction

Fig. 1.10 Ion-dipole interaction

1.3.4.3 Ion-Dipole Interaction

1. Ehrlish P (1987) Klin Jahr:299

Abstract Current chapter reviews the applications of ELISA in various different

2.1 Applications of ELISA

2.1.1 Food Industry

© The Author(s) 2018 19

2.1.2 Vaccine Development