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
4 1 Fundamentals and History of ELISA: The Evolution …

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.
6 1 Fundamentals and History of ELISA: The Evolution …

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]
8 1 Fundamentals and History of ELISA: The Evolution …

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 Principles of the Immune System 9

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]
10 1 Fundamentals and History of ELISA: The Evolution …

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 Principles of the Immune System 11

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.
12 1 Fundamentals and History of ELISA: The Evolution …

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
14 1 Fundamentals and History of ELISA: The Evolution …

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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