OUTLINE:

Immunoassay
Principle of immunoassay
History of immunoassay
Component of immunoassay
Classification of immunoassay
Types of immunoassay
Clinical application of immunoassay
Advantages of immune assay
Disadvantages of immunoassay
Advances in immunoassay
conclusion
IMMUNOASSAY
An immunoassay is a type of laboratory test that uses the principles of
immunology to detect the presence or concentration of a specific substance in a
sample. This type of assay typically involves the use of antibodies, which are
proteins produced by the immune system in response to the presence of a specific
substance (known as an antigen). The antibodies are highly specific, meaning that
they will only react with the specific antigen they were produced against.

(Marnet PG et al.,1994)
 PRINCIPLE
The principle behind the Immunoassay test is the use of an antibody that
will specifically bind to the antigen of interest.
 The antibodies used in the Immunoassay must have a high affinity for the
antigen. The antibodies used in the Immunoassay can either be monoclonal
or polyclonal antibodies.

(Marnet PG et al.,1994)
 HISTORY
The first immunoassay was described by Berson and Yalow in 1959. Their
work resulted in their receipt of the Nobel Prize in Medicine in 1977. Since
this introduction, immunoassays have evolved considerably. There have
been several milestones that have led to the proliferation of modern
immunoassays.
COMPONENT OF IMMUNOASSAY

Antibody

Antigen (Analyte)
Detectable Label
ANTIBODY
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped
protein used by the immune system to identify and neutralize foreign objects such as
pathogenic bacteria and viruses . The antibody recognizes a unique molecule of the
pathogen, called an antigen.

(Chen, K., and Cerutti, A. 2011)

schematic diagram of immunoglobulin
ANTIGEN
 substance that is capable of stimulating an immune response, specifically
activating lymphocytes, which are the body’s infection-fighting white blood
cell . In general, two main divisions of antigens are recognized: foreign antigens
(or heteroantigens) and autoantigens (or self-antigens.). Foreign antigens
originate from outside the body.

(Abbas AK et al., 2018).

 DETECTABLE LABEL
 Immunoassays employ a variety of different labels to allow for detection of antibodies
and antigens present.
 Labels are typically chemically linked or conjugated to the desired antibody or antigen.
 A label is a molecule that react as part of the assay, so a change in signal can be
measured in the blood-reagent solution
 Example of a label are; radioactive compound, an enzyme that causes change in color
of a solution, DNA Reporters, Fluorogenic Reporters, Electro chemiluminescent Tags
 A label can be applied during the manufacture of the reagent to either the antibody or
antigen
CLASSIFICATION OF IMMUNOASSAY
Competitive immunoassay: is divided into;
- competitive homogenous immunoassay
- competitive heterogenous immunoassay

Non competitive immunoassay

Competitive homogenous immunoassay:
In a competitive, homogeneous immunoassay, unlabeled analyte in a sample

competes with labeled analyte to bind an antibody. The amount of labelled, unbound
analyte is then measured. In theory, the more analyte in the sample, the more labelled
analyte gets displaced and then measured; hence, the amount of labelled, unbound
analyte is proportional to the amount of analyte in the sample.

(Carlson, Bruce 2014).

Competitive heterogenous immunoassay:
heterogeneous assays involve washing steps, allowing the separation of the bound

form from the free-labelled antibody. In this case, the presence of solid support is
important to perform automatic processes. This feature, together with the higher
versatility, higher specificity and higher sensitivity allow the heterogeneous
immunoassays to turn out as the most popular assay format.

(Carlson, Bruce 2014).

Noncompetitive immunoassay:

Noncompetitive immunoassay is divided into one-site noncompetitive immunoassay and two-site

noncompetitive immunoassay;

a. One-site noncompetitive immunoassay:

The unknown analyte in the sample binds with labelled antibodies. The unbound, labelled antibodies are

washed away, and the bound, labelled antibodies are measured. The intensity of the signal is directly
proportional to the amount of unknown analyte.

a. Two-site noncompetitive immunoassay:

 The analyte in the unknown sample is bound to the antibody site, then the labelled antibody is bound to
the analyte. The amount of labelled antibody on the site is then measured. It will be directly proportional
to the concentration of the analyte because the labelled antibody will not bind if the analyte is not present
in the unknown sample. (Carlson, Bruce 2014).
 Fig2: demonstration of competitive and noncompetitive immunoassay
 Source: springerlink
TYPES OF IMMUNOASSAY

Radioimmunoassay (RIA)
Enzyme-linked Immunosorbent Assay (ELISA)
Fluoroimmunoassay (FIA)
Surface-enhanced Raman Scattering (SERS)
Surface Plasmon Resonance (SPR)
Chemiluminescence Immunoassay (CLIA)
Electrochemiluminescence Immunoassay (ECLIA):
Radioimmunoassay (RIA):
 Radioimmunoassay (RIA) is one of the first methods employed for the detection
of biological targets. Yalow and Berson introduced the use of radio-label for the
recognition of insulin, thus developing RIA as analytical technique.

Fig 3: radioimmunoassay machine

Source: medical expo
 Enzyme-linked Immunosorbent Assay (ELISA)
The replacement of radiolabels with enzymes made immunoassay much simpler and more
popular, and today the enzyme-linked immunosorbent assay (ELISA) is the most widely used
immunoassay method. This immunoassay method is based on an antibody- or antigen-coated
solid surface with an enzyme involved in the signal generation process. (Engvall, E. 1972)

Fig 4: an ELISA machine

Source: medical expo
Fluoroimmunoassay (FIA)
Conn et al. developed fluorescently labeled immunoassay (FIA) in the 1940s. The
antigen or antibody is labeled with a fluorescent material that binds to the
appropriate antigen or antibody, and the fluorescence intensity is measured using a
fluorescent microscope or UV irradiation.

Fig 5: fluoroimmunoassay machine

Source: yusen med
Surface-Enhanced Raman Scattering (SERS):
 Another kind of optical immunoassays is based on the detection of the surface-enhanced Raman
scattering (SERS) as a signal readout. Raman scattering is the inelastic scattering of a photon after
its interaction with the matter, a phenomenon discovered by Raman in 1928 that allows for the
gaining of structural and quantitative information on molecules and materials.

Fig 6: surface-enhanced Raman scattering machine

Source: medical expo
Chemiluminescence Immunoassay (CLIA):

Another strategy to generate an optical readout alternative to fluorescence is the

chemiluminescence (CL), which arises from chemical reactions producing an
optical signal. By using labels showing chemiluminescence, it is possible to apply
this phenomenon to immunoassays. (professor Anthony Campbell 1983).

Fig 7: chemiluminescence immunoassay machine

Source: bio base
•Electrochemiluminescence Immunoassay (ECLIA):
 Electrochemiluminescence (ECL), or electrogenerated chemiluminescence, was first investigated by
Hercules, Visco and Bard in the 1960s by studying aromatic compounds. in 1972 opened new possibilities for
immunoassays, owing to the high electrochemical stability, the easy detection of the ECL emission and the
possibility to operate at a low potential offered by this metal complex.

Fig 8: Electrochemiluminescence immunoassay machine

Source: medical expo
 CLINICAL APPLICATION OF
IMMUNOASSAY
 Diagnosing and monitoring infectious diseases such as HIV, hepatitis, and tuberculosis.

 Detecting and monitoring autoimmune disorder, such as lupus and rheumatoid arthritis’

 Screening and monitoring for drug abuse, such as alcohol, marijuana, and opoids.

 Detecting and monitoring pregnancy and fertility hormone levels.

 Monitoring levels of therapeutic drugs, such as insulin and blood thinners

 Assessing Immune system function and response to vaccines

 Detecting and measuring hormones and other biomarkers for research and clinical

studies.
 Providing rapid and accurate diagnostic results for point-of-care testing in hospitals,

clinics, and other healthcare settings. (Twyman, SA. 2001)

ADVANTAGES OF IMMUNOASSAY
High sensitivity-low detection limit

High specificity-detect specific compound

Safe and simple

Fast test (between 5 minutes and 1 hour)

Cost effective
Test can yield quantitative or qualitative data
DISADVANTAGES OF IMMUNOASSAY
Negative result doesn’t always rule out the presence of a drug

May not be sensitive to be certain compounds

Some chemists are reluctant to use immunoassay due to its biological

basis and their unfamiliarity with it.

 CLINICAL ADVANCES IN IMMUNOASSAY
FLOW-INJECTION IMMUNOASSAY

Flow-injection immunoassay (FIIA) methods were recently introduced to enhance

the efficiency of immunochemical reaction, as well as to increase the performance
of the analysis. (De la Rica, et al., 2012).

CAPILLARY ELECTROPHORESIS IMMUNOASSAY

Capillary electrophoresis immunoassay (CEIA) has been recently introduced as a

sensitive analytical technique, particularly when combined with a sensitive
detection method. This technique is a combined use of the principles of the capillary
electrophoresis separation and the heterogeneous immunoassay.
(Findlay JW, et al. 2000)
CLONED ENZYME DONOR IMMUNOASSAY

Cloned enzyme donor immunoassay (CEDIA) methodology is a novel approach

which uses the DNA technology to produce homogenous enzyme immunoassays
for drugs. (Signo P, et al., 2005)

IMMUNOSENSORS

Immunosensors represent the most technological progress in the field of

immunoassay development. These sensors are analytical devices composed of an
immunochemical recognition element directly interfaced to a signal transducer,
which together relate the concentration of an analyte to a measurable response.
(Micheli L, et al. 2002)
CONCLUSION
 Immunoassay methods are bioanalytical methods in which quantitation of
analyte depends on its reaction with specific antibody. The response signal is
generated from a label attached to either the analyte or antibody. The property of
highly specific recognition of analytes by antibodies leads to the high selectivity
of these assays. The extreme affinity of analyte-antibody interaction results in
greater sensitivity of immunoassay methods. The instrumentation technology
leads to automation of immunoassays and consequently increasing their
throughput. Immunoassay methods are capable of quantifying wide variety of
compounds such as low molecular weight drugs, macromolecular biomolecules,
metabolites, and/or biomarkers which indicate disease diagnosis or prognosis.
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