ELISA_DrRashmi_Patel_CIMS_V02.pdf

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
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
2
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.
3
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).
4
ELISA TEST-KITS & INSTRUMENTS
ELISA Micro-Titre Plate
ELISA Reader Spectrophotometer
5
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
6
1. DIRECT ELISA
 Antigen coated to a multi well plate detected by an antibody that has been
directly conjugated to an enzyme.
7
 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.
8
2. INDIRECT ELISA
9
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.
10
3. COMPETITIVE ELISA
11
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.
12
4. SANDWICH ELISA
13
5. CAPTURE ELISA
14
 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.
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.
15
16
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)
7. ELISpot
17
18
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
8. IgG Avidity ELISA
19
 In acute infection, the avidity of the antibody to bind is weaker
and so it dissociates from the antigen-antibody complex.
8. IgG Avidity ELISA
20
In chronic infections, the binding is stronger, hence there is no
dissociation from the antigen.
Use of ELISA
21
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
BIBLIOGRAPHY
22
 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.
23
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ELISA_DrRashmi_Patel_CIMS_V02.pdf

  • 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 2
  • 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. 3
  • 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). 4
  • 5. ELISA TEST-KITS & INSTRUMENTS ELISA Micro-Titre Plate ELISA Reader Spectrophotometer 5
  • 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 6
  • 7. 1. DIRECT ELISA  Antigen coated to a multi well plate detected by an antibody that has been directly conjugated to an enzyme. 7
  • 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. 8
  • 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. 10
  • 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. 12
  • 14. 5. CAPTURE ELISA 14  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. 15
  • 16. 16 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)
  • 18. 18 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 19  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 20 In chronic infections, the binding is stronger, hence there is no dissociation from the antigen.
  • 21. Use of ELISA 21 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 22  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.
  • 23. 23