ELISA, principle and method by kk sahu

KAUSHAL SAHU
KAUSHAL SAHUKAUSHAL SAHU
Method of ELISA
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )
Synopsis
 What is ELISA.
 Principle.
 History.
 Types of ELISA method.
 1.Direct ELISA.
 2.Indirect ELISA.
 3.Sandwhich ELISA.
 Conclusion.
 References.
Definitions
 Antibodies (also known as immunoglobulins abbreviated
Ig) are gamma globulin proteins that are found in blood
and are used by the immune system to identify and
neutralize foreign objects, such as bacteria and viruses.
 Antigens
A substance that when introduced into the body
stimulates the production of an antibody
 Immunoassay
A laboratory technique that makes use of the binding
between an antigen and its homologous antibody in
order to identify and quantify the specific antigen or
antibody in a sample
INTRODUCTION
 ELISA, or enzyme-linked immunosorbent assay, is an
immunoassay technique involving the reaction of
antigen and antibody in vitro.
 ELISA is a sensitive and specific assay for the detection
and quantification of antigens or antibodies.
 ELISA tests are usually performed in microwell
plates.
 The ELISA test, or the enzyme immunoassay (EIA),
was the first screening test commonly employed for
HIV. It has a high sensitivity.
A 96-WELL MICROTITER PLATE USED FOR
ELISA
What is ELISA
 It is enzyme- linked immunosorbent assay.
 It is also called enzyme immunoassay.
 Is a biochemical technique used mainly in immunology
to detect the presence of an antibody or an antigen in a
sample.
 The technique is divided into 3 types.
 1-Direct ELISA.
 2- Sandwich ELISA.
 3- Indirect ELISA .
Principle of ELISA
 ELISA is a sensitive method for quantification of
protein, which is suitable for various application. The
basic principle of an ELISA is to use an enzyme to
detect the Ag-Ab binding (antigen- antibody binding).
The enzyme converts a colorless substrate (chromogen)
to a colored product, indicating the presence of Ag:Ab
binding.
History
 Antigen- antibody binding hypothesis was given by John
marrack 1938.
 A technique to prepare immunosorbent to fix ag or ab to
surface of a container was published by Wide & Porath in
1966.
 In 1971 Perlmann & engvall invented ELISA.
 In 1975 George kohler & Milstein invented 1st monoclonal
antibody.
Different antigen in sample
substrate
Colored
product
Primary antibody
Secondary antibody
HOW DOES ELISA WORK ?
 There are variations of the ELISA test but the most basic type
consist of an antibody attached to the solid surface. This antibody
has affinity for (will latch onto) the substance of interest.
 For example… Human Chorionic Gonadotropin (HCG), the
commonly measured protein which indicates pregnancy.
 A mixture of purified HCG linked to an enzyme and sample
(blood, urine, etc.) under test are added to the test system. If no
HCG is present in the test sample the only HCG with linked
enzyme will bind. The more HCG which is present in the test
sample the less enzyme linked HCG will bind.
Reagents
 Substrate: TMB (3,3',5,5', tetramethyl benzidine) The
enzyme acts as a catalyst to oxidize substrate in the
presence of Hydrogen peroxide to produce a blue color.
Reaction stopped with dilute acid to cause complex to
turn yellow.
 Enzyme: Horse Radish Peroxidase (HRPO)
 Antibody: IgG fraction of serum purified by afinity
chrometography.
 Solid phase: poly vinyl chloride.
 Dilute buffer: phosphate buffer, neutral ph.
Types of ELISA
 1.Direct ELISA.
 2. Indirect ELISA.
 3. Sandwhich ELISA.
DIRECT ELISA
The direct ELISA uses the method of directly labeling the
antibody itself.
 Micro well plates are coated with a sample containing the
target antigen,
 and the binding of labeled antibody is quantitated by a
colorimetric, chemiluminescent, or fluorescent end-point.
 DIRECT ELISA
1. Apply a sample of known
antigen to a surface.
2. Enzyme linked primary
antibody is applied to the plate.
3. Washed, After this wash, only
the antibody-antigen
complexes remain attached.
4. Apply a substrate which is
converted by the enzyme to
elicit a chromogenic signal.
Advantages of Direct Detection
 Quick methodology since only one antibody is used.
 Cross-reactivity of secondary antibody is eliminated.
Disadvantages of Direct Detection
 Immunoreactivity of the primary antibody may be reduced
as a result of labeling.
 Labeling of every primary antibody is time-consuming
and expensive.
INDIRECT ELISA
 The indirect ELISA utilizes an unlabeled primary
antibody in conjunction with a labeled secondary
antibody. Since the labeled secondary antibody is
directed against all antibodies of a given species (e.g.
anti-mouse), it can be used with a wide variety of
primary antibodies (e.g. all mouse monoclonal
antibodies).
 INDIRECT ELISA
 Antigen is added to plate.
 Added Blocking buffer.
 Suitable primary antibody is added.
 Secondary antibody- HRPO is then
added which recognizes and binds to
primary antibody.
 TMB substrate is added, is converted
to detectable form.
Advantages of indirect detection
 Wide variety of labeled secondary antibodies are available
commercially.
 Versatile, since many primary antibodies can be made in
one species and the same labeled secondary antibody can
be used for detection.
 Immuno reactivity of the primary antibody is not affected
by labeling.
 Sensitivity is increased because each primary antibody
contains several epitopes that can be bound by the labeled
secondary antibody, allowing for signal amplification.
Disadvantages of indirect detection
 Cross-reactivity may occur with the secondary antibody,
resulting in nonspecific signal.
 An extra incubation step is required in the procedure.
 SANDWICH ELISA
1. a. Plate is coated with suitable antibody.
b. Blocking buffer is added.
2. Sample is added to plate so antigen is bounded by capture antibody.
3. A suitable biotin labeled detection antibody is added to plate.
4. Enzyme HRPO is added and binds the biotin labeled detection antibody.
5. TMB substrate is added and converted by HRPO to colored product.
 MATERIALS NEEDED FOR ELISA KIT
 ELISA Plate.
 Positive control.
 Negative control.
 Dilution Buffer.
 Conjugate.
 TMB Substrate.
 Stop Solution.
 ELISA data is typically graphed with Optical density Vs Log concentration to produce a
sigmoidal curve.
 Known concentrations of antigen are used to produce a standard curve and then this
data is used to measure the concentration of unknown samples by comparison to the
linear portion of the standard curve.
 This can be done directly on the graph or with curve fitting software which is typically
found on ELISA plate readers. Which measure the colour density .
 APPLICATIONS
 Screening donated blood for evidence of viral contamination by
 HIV-1 and HIV-2 (presence of anti-HIV antibodies)
 Hepatitis C (presence of antibodies)
 Hepatitis B (testing for both antibodies and a viral antigen)
 Measuring hormone levels
 HCG (as a test for pregnancy)
 LH (determining the time of ovulation)
 TSH, T3 and T4 (for thyroid function)
 Detecting infections
 Sexually-transmitted agents like HIV, syphilis and chlamydia
 Hepatitis B and C
 Toxoplasma gondii
 Detecting allergens in food and house dust
CONCLUSION
 ELISA is a sensitive and specific assay for the
detection and quantification of antigens or
antibodies.
 ELISA tests are usually performed in
microwell plates.
REFERENCES
 Biophysical Chemistry – Principle and
technique- Upadhyay and Nath.
 www.wikipidia.com.
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ELISA, principle and method by kk sahu

  • 1. Method of ELISA By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )
  • 2. Synopsis  What is ELISA.  Principle.  History.  Types of ELISA method.  1.Direct ELISA.  2.Indirect ELISA.  3.Sandwhich ELISA.  Conclusion.  References.
  • 3. Definitions  Antibodies (also known as immunoglobulins abbreviated Ig) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.
  • 4.  Antigens A substance that when introduced into the body stimulates the production of an antibody  Immunoassay A laboratory technique that makes use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample
  • 5. INTRODUCTION  ELISA, or enzyme-linked immunosorbent assay, is an immunoassay technique involving the reaction of antigen and antibody in vitro.  ELISA is a sensitive and specific assay for the detection and quantification of antigens or antibodies.  ELISA tests are usually performed in microwell plates.  The ELISA test, or the enzyme immunoassay (EIA), was the first screening test commonly employed for HIV. It has a high sensitivity.
  • 6. A 96-WELL MICROTITER PLATE USED FOR ELISA
  • 7. What is ELISA  It is enzyme- linked immunosorbent assay.  It is also called enzyme immunoassay.  Is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample.  The technique is divided into 3 types.  1-Direct ELISA.  2- Sandwich ELISA.  3- Indirect ELISA .
  • 8. Principle of ELISA  ELISA is a sensitive method for quantification of protein, which is suitable for various application. The basic principle of an ELISA is to use an enzyme to detect the Ag-Ab binding (antigen- antibody binding). The enzyme converts a colorless substrate (chromogen) to a colored product, indicating the presence of Ag:Ab binding.
  • 9. History  Antigen- antibody binding hypothesis was given by John marrack 1938.  A technique to prepare immunosorbent to fix ag or ab to surface of a container was published by Wide & Porath in 1966.  In 1971 Perlmann & engvall invented ELISA.  In 1975 George kohler & Milstein invented 1st monoclonal antibody.
  • 10. Different antigen in sample substrate Colored product Primary antibody Secondary antibody
  • 11. HOW DOES ELISA WORK ?  There are variations of the ELISA test but the most basic type consist of an antibody attached to the solid surface. This antibody has affinity for (will latch onto) the substance of interest.  For example… Human Chorionic Gonadotropin (HCG), the commonly measured protein which indicates pregnancy.  A mixture of purified HCG linked to an enzyme and sample (blood, urine, etc.) under test are added to the test system. If no HCG is present in the test sample the only HCG with linked enzyme will bind. The more HCG which is present in the test sample the less enzyme linked HCG will bind.
  • 12. Reagents  Substrate: TMB (3,3',5,5', tetramethyl benzidine) The enzyme acts as a catalyst to oxidize substrate in the presence of Hydrogen peroxide to produce a blue color. Reaction stopped with dilute acid to cause complex to turn yellow.  Enzyme: Horse Radish Peroxidase (HRPO)  Antibody: IgG fraction of serum purified by afinity chrometography.  Solid phase: poly vinyl chloride.  Dilute buffer: phosphate buffer, neutral ph.
  • 13. Types of ELISA  1.Direct ELISA.  2. Indirect ELISA.  3. Sandwhich ELISA.
  • 14. DIRECT ELISA The direct ELISA uses the method of directly labeling the antibody itself.  Micro well plates are coated with a sample containing the target antigen,  and the binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end-point.
  • 15.  DIRECT ELISA 1. Apply a sample of known antigen to a surface. 2. Enzyme linked primary antibody is applied to the plate. 3. Washed, After this wash, only the antibody-antigen complexes remain attached. 4. Apply a substrate which is converted by the enzyme to elicit a chromogenic signal.
  • 16. Advantages of Direct Detection  Quick methodology since only one antibody is used.  Cross-reactivity of secondary antibody is eliminated. Disadvantages of Direct Detection  Immunoreactivity of the primary antibody may be reduced as a result of labeling.  Labeling of every primary antibody is time-consuming and expensive.
  • 17. INDIRECT ELISA  The indirect ELISA utilizes an unlabeled primary antibody in conjunction with a labeled secondary antibody. Since the labeled secondary antibody is directed against all antibodies of a given species (e.g. anti-mouse), it can be used with a wide variety of primary antibodies (e.g. all mouse monoclonal antibodies).
  • 18.  INDIRECT ELISA  Antigen is added to plate.  Added Blocking buffer.  Suitable primary antibody is added.  Secondary antibody- HRPO is then added which recognizes and binds to primary antibody.  TMB substrate is added, is converted to detectable form.
  • 19. Advantages of indirect detection  Wide variety of labeled secondary antibodies are available commercially.  Versatile, since many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection.  Immuno reactivity of the primary antibody is not affected by labeling.  Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification.
  • 20. Disadvantages of indirect detection  Cross-reactivity may occur with the secondary antibody, resulting in nonspecific signal.  An extra incubation step is required in the procedure.
  • 21.  SANDWICH ELISA 1. a. Plate is coated with suitable antibody. b. Blocking buffer is added. 2. Sample is added to plate so antigen is bounded by capture antibody. 3. A suitable biotin labeled detection antibody is added to plate. 4. Enzyme HRPO is added and binds the biotin labeled detection antibody. 5. TMB substrate is added and converted by HRPO to colored product.
  • 22.  MATERIALS NEEDED FOR ELISA KIT  ELISA Plate.  Positive control.  Negative control.  Dilution Buffer.  Conjugate.  TMB Substrate.  Stop Solution.
  • 23.  ELISA data is typically graphed with Optical density Vs Log concentration to produce a sigmoidal curve.  Known concentrations of antigen are used to produce a standard curve and then this data is used to measure the concentration of unknown samples by comparison to the linear portion of the standard curve.  This can be done directly on the graph or with curve fitting software which is typically found on ELISA plate readers. Which measure the colour density .
  • 24.  APPLICATIONS  Screening donated blood for evidence of viral contamination by  HIV-1 and HIV-2 (presence of anti-HIV antibodies)  Hepatitis C (presence of antibodies)  Hepatitis B (testing for both antibodies and a viral antigen)  Measuring hormone levels  HCG (as a test for pregnancy)  LH (determining the time of ovulation)  TSH, T3 and T4 (for thyroid function)  Detecting infections  Sexually-transmitted agents like HIV, syphilis and chlamydia  Hepatitis B and C  Toxoplasma gondii  Detecting allergens in food and house dust
  • 25. CONCLUSION  ELISA is a sensitive and specific assay for the detection and quantification of antigens or antibodies.  ELISA tests are usually performed in microwell plates.
  • 26. REFERENCES  Biophysical Chemistry – Principle and technique- Upadhyay and Nath.  www.wikipidia.com.