ELISA

1. KUVEMPU UNIVERSITY SEMINAR ON ELISA (Enzyme Linked ImmunoSorbent Assay) Presented by: Poornima R.

2. CONTENTS Introduction Principle Types Applications Advantages and disadvantages Summary Conclusion References

3. INTRODUCTION ELISA(Enzyme Linked ImmunoSorbent assay) is a widely used technique for detection of antigen (Ag) or antibody( Ab ). The technique was developed in 1971 by Peter Perlmann and Eva Engvall at Stockholm University, Sweden. A technique to prepare something like immunosorbent to fix antibody or antigen to the surface of a container was published by Wide and Jerker Porath in 1966 Eva Engvall Peter Perlmann

4. PRINCIPLE Principle is based on the formation of Ag- Ab complex , which is detected by chromogenic detection using enzyme conjugated secondary antibody. The conjugated enzyme acts on a specific substrate called chromogenic substrate, and generates a coloured reaction product. This product is qualitatively or quantitatively read using an ELISA plate reader. ELISA microplate reader

5. ELISA kits are commercially available, which can be conveniently used for laboratory purpose. Kit from REAGEN Kit from Forsight

6. TYPES OF ELISA Direct ELISA Indirect ELISA S andwich ELISA Competitive ELISA

7. 1.DIRECT ELISA It is used in the detection of antigen in the given biological sample. Microtiter wells are initially coated with antigen to be detected which is followed by an antibody linked to an enzyme conjugate. This follows the addition of substrate which produces colour detected using ELISA detector.

8. 2. INDIRECT ELISA It is used for detection of an antibody in the given sample. Microtiter wells are initially coated with antigen specific for antibody to be detected, followed by the addition of sample. Enzyme conjugated Secondary Antibody is added followed by the substrate which forms a coloured reaction product.

9. 3.SANDWICH ELISA It is used for detecting an antigen in the given sample. Microtiter wells are initially coated with monoclonal antibodies(called capture antibody) raised against antigen to be detected, followed by addition of sample. Any trace of antigen is detected by adding primary antibody (a MAb ),followed by enzyme conjugated secondary Ab and a chromogenic substrate; or by directly adding an enzyme conjugated primary Ab.

10. SANDWICH ELISA

11. 4.COMPETITIVE ELISA This variation of ELISA is used to quantitatively estimate the amount of antigen in the given sample. Ag and Ab are initially incubated so that they form Ag-Ab complex. This mixture is then added to microtiter wells coated with synthetic analogue of antigen to be detected, any free antibody binds to these antigens . This complex is estimated by enzyme conjugated secondary antibody by chromogenic detection .More the amount of antigen in the sample, lesser is the antibody available to bind to microtiter wells.

12. COMPETITIVE ELISA

13. APPLICATIONS Since ELISA can detect both antigen and antibody it is a useful tool for determining  serum antibody concentrations .  It has also found applications in the food industry in detecting potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs. The other uses of ELISA include: detection of  Mycobacterium  antibodies in tuberculosis detection of hepatitis B markers in serum detection of enterotoxin of  E. coli  in feces detection of HIV antibodies in blood samples

14. CONT...

15. CONT... ELISA in immunochromatography to detect HGC in urine sample:

16. ADVANTAGES Sensitive assay Equipments are widely available. No radiation hazards. Reagents are cheap with long shelf life. Qualitative and quantitative. ELISA can be used on most types of biological samples, such as plasma, serum, urine, and cell extracts

17. DISADVANTAGES Only monoclonal antibodies can be used as matched pairs Monoclonal antibodies can cost more than polyclonal antibodies Negative controls may indicate positive results if blocking solution is ineffective [secondary antibody or antigen (unknown sample) can bind to open sites in well] Enzyme/substrate reaction is short term hence color must be read as soon as possible.

18. SUMMARY Coat plate with Ag/Ab wash Add blocking buffer wash Add test samp le wash Add enzyme conjugated Antibody wash Add substrate Add stop solution Read absorbance at 450nm.

19. CONCLUSION Enzyme Linked ImmunoSorbent Assay (ELISA) is a novel technique useful in detecting (qualitatively and quantitatively) an antigen or antibody present in the given biological sample. Besides its disadvantages the technique is being widely used in diagnostics and drug screening. Chromogenic detection method used in ELISA is convenient and sensitive for any assay and is hazard free.

20. REFERENCES Kuby Immunology by Kindt, Goldsby and Osborne Medical Immunology by Gabriel Virella www.google.com www.wekipedia.org