2. INTRODUCTION
Enzyme-linked immunosorbent assay, a rapid
immunochemical test that involves an enzyme used for
measuring a wide variety of tests of body fluids.
In 1971, Peter Perlmann and Eva Engvall at Stockholm
University in Sweden, and Anton Schuurs and Bauke van
Weemen in the Netherlands independently published
papers that synthesized this knowledge into methods to
perform ELISA.
The ELISA was the first screening test widely used for
HIV because of its high sensitivity.
3. BASIC PRINCIPLE OF
ELISA
ELISA involves detection of an analyte in a liquid
sample by a method that continues to use liquid
reagents during the analysis (i.e., controlled
sequence of biochemical reactions that will
generate a signal which can be easily quantified
and interpreted as a measure of the amount of
analyte in the sample) that stays liquid and
remains inside a reaction chamber or well needed
to keep the reactants contained.
4. CONT…
Use an enzyme to detect the
binding of antigen (Ag) antibody
(Ab).
The enzyme converts a colorless
substrate (chromogen) to a
colored product, indicating the
presence of Ag : Ab binding.
An ELISA can be used to detect
either the presence of antigens or
antibodies in a sample depending
how the test is designed.
ELISA was dveloped in 1971 and
became rapidly accepted.
8. TYPES OF ELISA
Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA
9. DIRECT ELISA
Antigen is coated onto the wells by passive adsorption and incubation.
Bovine serum albumin is used to block the other binding sites
The plates are washed with PBS-T three times to remove unbound
molecules.
Biotinylated Antibody (the enzyme conjugated antibody) IgG with
Horseradish peroxidase (HRP) is added and incubated.
The wells are washed again with PBS-T to remove unbound molecules.
Chromophore substrate (TMB) for enzyme is added which hydrolyses
to release a colour product.
The absorbance is measured by spectrophotometer or absorbance
microplate reader.
11. INDIRECT ELISA
Antigen is coated onto the wells by passive adsorption and incubation
The plate is washed with PBS to remove unbound antigens.
Bovine serum albumin is used to block the other protein binding sites
Primary sample antibody is added to the plate and incubated with the antigen.
The plates are washed with PBS to remove unbound molecules.
The secondary enzyme conjugated antibody is added and incubated with the
antigen.
Wells are washed to remove unbound molecules.
Chromophore substrate is added which detects the presence of the enzyme.
The absorbance is measured by spectrophotometer or absorbance microplate
reader.
13. SANDWICH ELISA
A "sandwich" ELISA is used to detect sample antigen.The steps are:
• A surface is prepared to which a known quantity of capture antibody is
bound.
• Any nonspecific binding sites on the surface are blocked.
• The antigen-containing sample is applied to the plate, and captured by
antibody.
• The plate is washed to remove unbound antigen.
• A specific antibody is added, and binds to antigen (hence the
'sandwich': the antigen is stuck between two antibodies). This primary
antibody could also be in the serum of a donor to be tested for
reactivity towards the antigen.
14. CONT…
• Enzyme-linked secondary antibodies are applied as detection
antibodies that also bind specifically to the antibody's Fc
region (nonspecific).
• The plate is washed to remove the unbound antibody-enzyme
conjugates.
• A chemical is added to be converted by the enzyme into a
color or fluorescent or electrochemical signal.
• The absorbance or fluorescence or electrochemical signal
(e.g., current) of the plate wells is measured to determine
the presence and quantity of antigen.
16. COMPETITIVE ELISA
This test is used to measure the concentration of an antigen in a sample.
• In this test, antibody is first incubated in solution with a sample containing
antigen.
• The antigen-antibody mixture is then added to the microtitre well which
is coated with antigen. The more the antigen present in the sample, the
less free antibody will be available to bind to the antigen-coated well.
• After the well is washed, enzyme conjugated secondary antibody specific
for isotype of the primary antibody is added to determine the amount of
primary antibody bound to the well.
• The higher the concentration of antigen in the sample, the lower the
absorbance.
18. dot-ELISA
The dot enzyme-linked immunosorbent assay (Dot-ELISA)
is a highly versatile solid-phase immunoassay for antibody
or antigen detection.
The assay uses minute amounts of reagent dotted onto
solid surfaces such as nitrocellulose and other paper
membranes which avidly bind proteins.
After incubation with antigen-specific antibody and
enzyme-conjugated anti-antibody, the addition of a
precipitable, chromogenic substrate causes the formation
of a colored dot on the solid phase which is visually read.
