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 Raddish 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 Elisa

8. 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

9. • 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

10. 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

11. 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

12. • 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

13. 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

14. 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

15. 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)

16. 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

17. Thank you