The document provides an overview of the Enzyme-Linked Immuno-Sorbent Assay (ELISA) technique. It describes ELISA as an antigen-antibody reaction used to measure antibodies or antigens in blood. It then discusses the basic principles of ELISA, the different types of ELISA (direct, indirect, competitive, sandwich, etc.), how ELISA tests and instruments work, and applications of ELISA such as diagnosing infectious diseases.

1. Enzyme-Linked
Immuno-Sorbent Assay
(ELISA)
Presented By:
Dr. Rashmi Patel
1st Year PG Student
Dept. of Biochemistry
CIMS, Bilaspur
Moderator
Dr. Prashant Nigam
H.O.D. & Assoc. Professor
Dept. of Biochemistry
CIMS, Bilaspur

2. CONTENTS
Introduction
Basic Principles
ELISA Test Kits and Instruments
Types of ELISA
Direct, Indirect, Competitive, Sandwich,
Capture, Cylinder, ELISpot, IgG Avidity ELISA.
Use of ELISA
Bibliography
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3. INTRODUCTION
 ELISA is abbreviation for Enzyme-Linked Immuno-Sorbent Assay.
 ELISA is an antigen-antibody reaction.
 In 1971, ELISA was introduced by Peter Permann & Eva Engval at
Stockholm University in Sweden.
 It is a common Laboratory Technique which is used to measure the
concentration of antibodies or antigens in Blood.
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4. BASIC PRINCIPLE
 ELISA is plate based assay technique which is used for detecting and
quantifying substances such as Peptides, Proteins, Antibodies and
Hormones.
 An enzyme-conjugated with an antibody reacts with colourless substrate to
generate a coloured product.
 Such substrate is called chromogenic substrate. A number of enzymes have
been used for ELISA such as: Alkaline Phosphatase, Horse Radish
Peroxidase (HRP).
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5. ELISA TEST-KITS & INSTRUMENTS
ELISA Micro-Titre Plate
ELISA Reader Spectrophotometer
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6. TYPES OF ELISA
 ELISA is categorized into eight types based on binding structure between
the antigen and antibody.
1. Direct ELISA
2. Indirect ELISA
3. Competitive ELISA
4. Sandwich ELISA
5. Capture ELISA
6. Cylinder or Cassette ELISA
7. Enzyme-Linked ImmunoSpot Assay (ELISpot)
8. IgG Avidity ELISA
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7. 1. DIRECT ELISA
 Antigen coated to a multi well plate detected by an antibody that has been
directly conjugated to an enzyme.
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8.  Antibody can be detected and quantitatively determined by Indirect ELISA.
Antigen is coated
on the Micro-Titre
Well.
Add the
antibody
containing
sample to the
plate.
Incubate the
plate at 37o C
for 30 minutes
2. INDIRECT ELISA
Wash the plate
so that unbound
antibody is
removed.
Add secondary
antibody
conjugated to
an enzyme.
Incubate the
plate at 37o C
for 30 minutes
Wash the plate
so that unbound
enzyme linked
antibodies are
removed.
Specific substrate for the
enzyme is added. Enzyme
hydrolyses the substrate
to form coloured product.
The amount of coloured end-product is measured by Spectrophotometric plate reader.
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9. 2. INDIRECT ELISA
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10. 3. COMPETITIVE ELISA
 This test is used to measure the concentration of an antigen in a sample.
Wash the plate to
remove unbound
antibody.
Enzyme linked
secondary antibody,
specific to the primary
antibody is added.
Wash the plate so that
unbound enzyme
linked antibodies are
removed.
Add substrate which is then
converted by the enzyme
into the fluorescent signal.
Higher the concentration of antigen in the sample, lower will be the absorbance.
Antibody is 1st incubated
in the solution with
sample containing
antigen.
Antibody complex are then
added to the Micro-Titre well,
which are precoated with the
antigen.
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11. 3. COMPETITIVE ELISA
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12. 4. SANDWICH ELISA
 Antigen can be detected by Sandwich ELISA.
Antibody is
coated on the
Micro-Titre Well.
Add the antigen
containing
sample to the
plate.
Incubate the
plate at 37o C for
30 minutes
Wash the plate
so that unbound
antigen is
removed.
Add the enzyme
linked antibodies
which are also
specific to the
antigen.
Incubate the
plate at 37o C
for 30 minutes
Wash the plate
so that unbound
enzyme linked
antibodies are
removed.
Substrate is added to
the plate which is
hydrolysed by
enzyme to form the
coloured product.
The amount of coloured end-product is measured by Spectrophotometric plate reader.
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13. 4. SANDWICH ELISA
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14. 5. CAPTURE ELISA
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 This test is even more specific. Several variations of the ELISA technique
have been developed to provide simple diagnostic tests, including the card
and dipstick method suitable for clinical laboratory and bedside application.
 Example:
Then, the antigen
specific mouse
monoclonal
antibody, which is
conjugated with
HRP, is added.
The complex
binds to specific
antigens against
which the
antibody is being
detected.
Monoclonal
Antihuman IgM
antibodies are
coated on wells
which bind to IgM
from the patient’s
serum.
The complex is
detected based on
the enzyme-
substrate reaction
by a
spectrophotometer.

15. Test serum is added on the
nitrocellulose membrane and
allowed to filter into the absorbent
material placed below it in the
cassette base.
Antibody, which is
present in the
serum, will bind to
the appropriate
antigen.
 A simple modification of ELISA which has found wide application for testing
one or a few samples of sera at a time is the cylinder or cassette ELISA.
6. CYLINDER / CASSETTE ELISA
Washing to
remove the
unbound
antibody.
Then enzyme
labelled anti-
human
immunoglobulin
antibody is added.
Wash to remove
the unbound
conjugate.
Coloured spot developing at the
site of antigen against which the
antibody is present in the serum.
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This is a highly sensitive method to detect the secreted protein.
Example: Cytokines from cytokine-secreting cell,
IgG from immunoglobulin secreting cells
The secreted molecules are detected using a procedure similar to
ELISA.
7. Enzyme Linked ImmunoSpot Assay (ELISpot)

17. 7. ELISpot
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Avidity implies overall strength of binding between an antibody
and an antigen.
In this test an external destabilising agent such as urea is used to
dissociate binding between the specific IgG and the Antigen.
This differentiates acute infections from chronic infections, based
on principles of immuno-enzyme assay.
The test is used to detect infection of Rubella, Toxoplasma or
Cytomegalovirus in pregnant women.
8. IgG Avidity ELISA

19. 8. IgG Avidity ELISA
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 In acute infection, the avidity of the antibody to bind is weaker
and so it dissociates from the antigen-antibody complex.

20. 8. IgG Avidity ELISA
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In chronic infections, the binding is stronger, hence there is no
dissociation from the antigen.

21. Use of ELISA
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ELISA plays a major role in diagnosis of innumerable diseases; such as:
Infectious Diseases: Hepatitis, EBV, Cytomegalovirus IgM/IgG, Dengue
IgG, Influenza, TORCH panel, Hepatitis B, C etc.
Rotavirus detection in Fecal Specimen and enterotoxin of E.Coli in
Feces.
Syphilis IgG/IgM, H. Pylori IgG and Antigen Detection
Food Toxins : Campylobacter, Salmonella Antigen
Human Allergen: Specific IgG and IgA ELISA

22. BIBLIOGRAPHY
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 Harper’s Illustrated Biochemistry, 32nd Edition, By: Victor W. Rodwell, David A.
Bender, Kathleen M. Botham, Peter J. Kennelly, and P. Anthony Weil, McGraw Hill
Publication.
 Textbook of Biochemistry for Medical Students, 9th Edition, By: DM Vasudevan,
Sreekumari S., and Kannan Vaidyanathan, JAYPEE Publication.
 Textbook of Medical Biochemistry, 2nd Edition, By: Dr. S K Gupta, Avichal
Publication.
 Biochemistry, 5th Edition, By: U. Satyanarayana and U. Chakrapani, Elsevier
Publication.

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