The document provides an overview of enzyme-linked immunosorbent assays (ELISA), covering its introduction, basic principles, and various types such as indirect, sandwich, and competitive ELISA. It explains how ELISA is used to detect antigens or antibodies in biological samples, outlining the mechanisms and processes involved in each type. Additionally, it mentions advancements in ELISA techniques and their applications in fields like medicine and biotechnology.

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ELISA
Submitted to :- Dr. Rikeshwar P Dewangan
Submitted by :- Shameer
Course/branch :- Master of pharmacy / pharmaceutical Analysis
Year/semester :- (2020) 1/1

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Contents
 Introduction
 Basic principle
 Types of ELISA

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Introduction to Enzyme Immunoassays
The commonly used EIAs are enzyme linked immunosorbent assays (ELISAs)
ELISA is a commonly used analytical biochemistry assay, first conceptualize &
develop Peter Perlmann and Eva Engvall at Stockholm university, Sweden
EIAs can be used for detection of either antigen or antibodies in serum and other
body fluids of the patient

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In EIA techniques, antigen or antibody labeled with enzyme are used.
The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect
the presences of ligand (commonly a protein) in a liquid sample using
antibodies directed against the protein to be measured.
Elisa have got its application in the field of medicine as a diagnostic tool,
plant pathology, and biotechnology and do also found its role in QC check
among the various industries

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Basic principle of ELISA
ELISA is performed in polystyrene plate consisting of 96 wells or
384 wells
The reagents are immobilized in ELISA test
The assay has a monoclonal antibody coat on a microtiter plate.
When the blood sample is added, the specific
antibody (primary antibody) adheres to the protein of interest
(e.g.cytokine)

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A secondary antibody binds to a different epitope on a protein.
The assay Is labeled with biotin.
o-phenyl-diamine dihydrochloride for peroxidase, p-nitrophenyl
phosphate for alkaline phosphatase
The reaction is detected by reading the optical density. Usually, a
standard curve based on known concentrations of antigen or antibody
is prepared from which the unknown quantities are calculated.

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Types of ELISA
 Indirect ELISA
 Sandwich ELISA
 Competitive ELISA
 ELISPOT assay

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Indirect ELISA
 Quantitative estimation (antibodies in the serum and other body fluids)
 Specimens are added to microtiter plate wells coated with antigen to
which specific antibodies are to be detected
 After a period of incubation, the well are washed. If antibodies was
present in the sample , antigen-antibody complex would have been
formed and will not get washed away.
 On the other, if the specific antibody was not present in the specimen,
there would not be any complex formation.

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 Next, an anti-isotype antibody conjugated with an enzyme is added
and incubated.
 After another washing step, a substrate for the enzyme is added.
 If there was complex formation in the initial step, the second ary
anti –isotype antibody would have bound to the primary antibody,
and there would be a chromogenic reaction between the enzyme
and substrate.
 After a stop solution is added to arrest the chromogenic reaction,
one can determine the amount of antigen-antibody complex formed
in the first step (optical density values of the wells)

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Sandwich ELISA
 Used of antigens detection
 The known antibody is coated and immobilized onto the wells of
microtiter plates.
 The test sample containing the suspected antigen is added to the
wells and is allowed to react with the antibodies in the wells.
 After the step of washing the well, a second enzyme-conjugated
antibody specific for a different epitope of the antigen is added and
allowed to incubate.

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 After removing any free secondary antibody by rewashing, the specific
substrate is added, and the ensuing chromogenic reaction is
measured.
 The chromogenic reaction is then compared with a standard curve to
determine the exact amount of the antigen present in the test sample.
 After removing any free secondary antibody by rewashing, the specific
substrate is added, and the ensuing chromogenic reaction is measured.
 The chromogenic reaction is then compared with a standard curve to
determine the exact amount of the antigen present in the test sample.

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Competitive ELISA
 Competitive ELISA is another technique used for the estimation of
antibodies present in a specimen, such as serum.
 Principle of the test is that two specific antibodies, one conjugated with
enzyme and the other present in test serum (if serum is positive for
antibodies), are used.
 Competition occurs between the two antibodies for the same
antigen. Appearance of color indicates a negative test (absence of
antibodies), while the absence of color indicates a positive
test (presence of antibodies).

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 In this test, the microtiter wells are coated with HIV antigen. The sera to
be tested are added to these wells and incubated at 37°C and then
washed.
 If antibodies are present in the test serum, antigen–antibody reaction
occurs
 The antigen– antibody reaction is detected by adding enzyme-labeled-
specific HIV antibodies.
 In a positive test, no antigen is left for these antibodies to act. Hence,
the antibodies remain free and are washed away during the process of
washing.

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 When substrate is added, no enzyme is available to act on it.
Therefore, positive result indicates no color reaction.
 In a negative test, in which no antibodies are present in the serum,
antigen in the coated wells is available to combine with enzyme-
conjugated antibodies and the enzyme acts on the substrate to
produce color.

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ELISPOT Assay
 ELISPOT assay is a modification of ELISA.
 It allows the quantitative determination of number of cells in a
population that are producing antibodies specific for a given
antigen or an antigen for which one has a specific antibody.
 These tests have found application widely in the measurement
of cytokines.

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Recent advancement
 Development of new ELISA with high sensitivity and selectivity
for bio analysis of bevecizumab: A monoclonal antibody used for
cancer immunotherapy
 Application of nano ELISA in food analysis

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Reference
 www.biochain.com
 Wikipedia
 Microbiology and immunology textbook of 2nd edition (elsevier)
 www.ncbi.nlm.nih.gov
 www.thermofisher.com
 www.rockland.inc
 www.rndsystem.com
 www.sciencedirect.com
 www.biotek.com

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