Elisa (and its type)

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

z
Contents
 Introduction
 Basic principle
 Types of ELISA

z
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

z
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

z
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)

z
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.

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

z
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.

z
 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)

z
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.

z
 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.

z
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).

z
 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.

z
 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.

z
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.

z
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

z
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

Elisa (and its type)

In this document

More Related Content

What's hot

Similar to Elisa (and its type)

More from ShameerAbid

Recently uploaded

Elisa (and its type)