ELISA

ENZYME LINKED IMMUNO SORBENT ASSAY IS USED FOR DETECTION OF HIV,AND SOME ANTIGEN ,ANTIBODY INTRACTIONS

1. ELISA Enzyme Linked Immuno Sorbent Assay BY JAMMALA VAMSIKRISHNA ( M.sc. Microbiology)

2. Introduction to ELISA ELISA is a Immunoassay technique involving in the reactions with antigen and antibody ELISA test is mainly performed in micro well plates ELISA test was first screening test commonly employed for HIV

3. History of ELISA 1798 - First demonstration of vaccination smallpox vaccination (Edward Jenner) 1900 - Antibody formation theory (Paul Ehrlich) 1938 - Antigen-Antibody binding hypothesis (John Marrack) 1948 - antibody production in plasma B cells 1959-1962 - Discovery of antibody structure 1960 - Radioimmunoassay was first described in a scientific paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960 1971 - Peter Perlmann and Eva Engvall at Stockholm University invented ELISA

4. Components of ELISA Antigen : ELISA plate coated with A60 antigen and antibodies Primary Antibody : Human Serum IgG ,IgA,IgM Secondary Antibody : peroxide label anti human IgA,IgG Enzymes : Horse R addish Peroxide

5. Principles of ELISA Antibodies are immobilized on micro well plates The unbounded material is washed out Chromgenic substance are added to develop colour Resulting colour is determined in Spectrophotometer

6. Types of ELISA There are 3 types of ELISA Direct Elisa Indirect Elisa Competitive E lisa

7. Direct ELISA (Sand witch) : It uses the method of directly labeling the antibody itself. Micro well plates were counted with a sample containing the target antigen and binding of labelled antibody is quantited by calorymetry

8. Advantages : quick methodology since only one antibody is used Cross reactivity of secondary antibody s eliminated Disadvantages Labeling of every primary antibody is time consuming and expensive No flexibility in choice of primary antibody label from experiment to another

9. Indirect ELISA The Indirect ELISA utilizes an unlabeled primary antibody in conjunction with a label secondary antibody Since the label secondary antibody is directed against all antibodies of given spices

10. Advantages Wide Varity of label secondary antibody are available commercially Immuno reactivity of primary antibody is not affected by labeling Dis advantages Cross reactivity may occur with secondary antibody resulting in non specific signal An extra incubation step is required in the procedure

11. Sandwich model Plate is coated with capture antibody Sample is added and antigens is present bind to capture antibody Detecting antibody is added and bind to antigen Substrate is added and is counted by enzyme to detectable form

12. Competitive ELISA In this unlabeled antibody is incubated in the presence of its antigen This bound antigen or antibody complex are then added antigen coated well The plate is washed unbounded antibody is removed The secondary antibody specific to primary antibody is added A substrate is added and remaining enzyme is elect a chromomeric

13. Advantages The sample volume can be increase to improve the test sensitivity in clinical(saliva and urine),food(bulk milk)and water samples One give is left unsenstized to measure the non specific reaction of the sample

14. Applications Screening donated blood for evidence of viral contaminated by HIV1 and HIV2(presence of anti HIV antibody) Hepatitis C (presence of antibody) Hepatitis B (testing for birth antibody and viral antigen)

15. Measuring the hormonal level HCG (a test for pregnancy) LH(determine the time of ovulation) TSH,T3 and T4 (for thyroid function) Detecting infections Sexually transmitted agent like HIV,Syphills and Chlamydia Hepatitis B and c virus

16. Thank you