- ELISA (Enzyme-Linked Immunosorbent Assay) is a plate-based assay technique used to detect substances like proteins, peptides, antibodies, and hormones. There are four main types of ELISA: direct, indirect, sandwich, and competitive. - Direct ELISA involves coating an antigen onto a plate and adding an enzyme-labeled primary antibody that binds directly to the antigen. Indirect ELISA uses an unlabeled primary antibody and enzyme-labeled secondary antibody. Sandwich ELISA uses two antibodies that bind to different sites on an antigen. Competitive ELISA measures antigen concentration based on competition between antigen in a sample and antigen coated on a plate for binding to an antibody. - ELISA has various applications like screening donated blood,

1. Enzyme Linked
Immunosorbant Assay
ELISA
-Kavya Rojesh (19HGMA13)
I M.Sc Human Genetics And Molecular Biology

2. History and Development of ELISA
• Before the development of the ELISA, the only option for
conducting an immunoassay was radioimmunoassay.
• As radioactivity poses a potential health threat, a safer
alternative was sought.
• 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
EIA/ELISA

3. • ELISA is a plate-based assay technique designed for detecting
and quantifying substances such as peptides, proteins,
antibodies and hormones.
ELISA
Competitive
Non-
Competitive
Direct
Indirect
Sandwich

4. Commonly used enzymatic markers
• Alkaline Phosphatase
– PNPP (p-Nitrophenyl Phosphate, Disodium Salt) yellow
• HRP (Horseradish Peroxidase)
– OPD (o-phenylenediamine dihydrochloride) Amber
– ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic
acid]-diammonium salt) Green
– TMB (3,3',5,5'-tetramethylbenzidine) Blue
• turns yellow after the addition of sulfuric or phosphoric acid.

5. Direct ELISA
• A direct ELISA is intended for the detection and quantification
of a specific analyte (e.g. antigens, antibodies, proteins,
hormones, peptides, etc.) from within a complex biological
sample.
• Of the four different ELISA formats, direct ELISA is the simplest
and quickest to perform

6. Procedure
• Antigen is immobilized onto the wells of a 96-well
polystyrene plate via passive adsorption.
• An enzyme-labeled primary antibody specific for the
target antigen is added to the wells and directly binds to
the antigen.
• A respective enzyme substrate a suitable substrate is
added, which upon reaction with the enzyme, produces a
visible colorimetric output that can be measured by a
spectrophotometer or absorbance microplate reader.

7. Advantages
• Less reagents and fewer steps are
required making this ELISA format
simple and quick while
minimizing potential user error
• Cross-reactivity of secondary
antibody is eliminated
Disadvantages
• Antigen immobilization is not
specific resulting in potentially
high background interference
• Primary antibody must be labeled
individually, which is time-
consuming and expensive
• No signal amplification
• Low flexibility – a specific
conjugated primary antibody is
needed for each target protein
• Immunoreactivity of the primary
antibody may be adversely
affected by labeling with enzymes

8. Indirect ELISA
• Antibody can be detected or quantitatively determined by indirect ELISA.
• In this technique, Antigen is coated on the microtiter well.
• Serum or some other sample containing primary antibody is allowed to react with
the coated antigen. (Any free primary antibody is washed away)
• Ag-1’Ab Complex is detected by adding an enzyme conjugated secondary antibody
that binds to the primary antibody. (Unbound secondary antibody is then washed away )
• A specific substrate for the enzyme is added to form colored products.
• The amount of colored end product is measured by absorbance of all the wells of
96-well plate.

9. Procedure of Indirect ELISA
• Coat the micro titer plate wells with antigen.
• Block all unbound sites to prevent false positive results.
• Add sample containing antibody (e.g. rabbit monoclonal
antibody) to the wells and incubate the plate at 37°c.
• Wash the plate, so that unbound antibody is removed.
• Add secondary antibody conjugated to an enzyme (e.g.
anti- mouse IgG).
• Wash the plate, so that unbound enzyme-linked
antibodies are removed.
• Add substrate which is converted by the enzyme to
produce a colored product.
• Reaction of a substrate with the enzyme to produce a
colored product.

10. Advantages:
• Increased sensitivity
• A wide variety of commercially
available secondary antibodies.
• Maximum immunoreactivity of the
primary antibody is retained because
it is not labeled.
• Versatile because many primary
antibodies can be made in one
species and the same labeled
secondary antibody can be used for
detection.
• Flexibility, Different primary
detection antibodies can be used
with a single labeled secondary
antibody.
• Cost savings, since fewer labeled
antibodies are required.
• Different visualization markers can be
used with the same primary
antibody.
Disadvantages:
• Cross-reactivity might occur with the
secondary antibody, resulting in
nonspecific signal.
• An extra incubation step is required
in the procedure.

11. Sandwich ELISA
• Antigen can be detected by sandwich ELISA.
• In this technique, antibody is coated on the microtiter well.
• A sample containing antigen is added to the well with the antibody
attached to the well, forming antigen-antibody complex. (the well is
washed)
• A 2’Ab specific for a different epitope on the antigen is added and allowed
to react with the bound antigen. (unbound secondary antibody is
removed)
• Finally substrate is added to the plate which is hydrolyzed by enzyme to
form colored products.

12. Procedure of sandwich ELISA
• Prepare a surface to which a known quantity of antibody is
bound.
• Add the antigen-containing sample to the plate and
incubate the plate at 37°c.
• Wash the plate, so that unbound antigen is removed.
• Add the enzyme-linked antibodies which are also specific to
the antigen and then incubate at 37°c.
• Wash the plate, so that unbound enzyme-linked antibodies
are removed.
• Add substrate which is converted by the enzyme to
produce a colored product.
• Reaction of a substrate with the enzyme to produce a
colored product.

13. Advantages
• High specificity, since two
antibodies are used the
antigen is specifically captured
and detected.
• Suitable for complex samples,
since the antigen does not
require purification prior to
measurement.
• Flexibility and sensitivity, since
both direct and indirect
detection methods can be
used.
Disadvantages
• Optimization in terms of
antibody becomes problematic
due to cross-reactivity issues.
• For recognition of a specific
epitope, only monoclonal
antibodies can be applied as
matched pairs.
• To procure monoclonal
antibodies it is a tedious
process in case of matched
pairs and are more expensive
than polyclonal antibodies.

14. 3. Competitive ELISA
• This test is used to measure the concentration of an antigen in a sample.
• Antibody is first incubated in solution with a sample containing antigen.
• The ag-ab 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.

15. Procedure
• Antibody is incubated with sample containing antigen.
• Antigen-antibody complex are added to the microtitre well
which are pre-coated with the antigen.
• Wash the plate to remove unbound antibody.
• Enzyme linked secondary antibody which is specific to the
primary antibody is added.
• Wash the plate, so that unbound enzyme-linked antibodies
are removed.
• Add substrate which is converted by the enzyme into a
fluorescent signal.

16. Advantages
• High specificity, since two antibodies are used.
• High sensitivity, since both direct and indirect
detection methods can be used.
• Suitable for complex samples, since the
antigen does not require purification prior to
measurement.

17. Applications Of ELISA
• Screening of Donated Blood for
Viral contaminations
– HIV-1 & HIV-2 (Ab)
– Hepatitis –C (Ab)
– Hepatitis-B (Both Ag & Ab)
• Measuring hormone Levels
– hCG (Pregnancy Test)
– LH (Ovulation)
– TSH, T3 & T4 (Thyroid Function)
Other Disease Diagnosis
• HIV, which causes AIDS
• Lyme disease
• pernicious anemia
• Rocky Mountain spotted fever
• rotavirus
• squamous cell carcinoma
• syphilis
• toxoplasmosis
• varicella-zoster virus, which
causes chickenpox and shingles
• Zika virus

18. Take Home Message

19. Thank You