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
7.
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