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1. ELISA(Enzyme Linked Immunosorbent
Assay)
DR. MANOJ
VEDPATHAK

2. INTRODUCTION
 Type of heterogeneous EIA (Enzyme
Immunoassay)
 Based on the measurement of enzyme
labelled antigen, hapten or antibody.
 Use of an Immunosorbent, an absorbing
material specific for either antigen or
antibody.

3. INTRODUCTION CTD…
 96 well microtitre
plate suitable for
automation.
 Most widely used
in clinical
Absorbing material may be
particulate – cellulose/agarose or
solid phase – polystyrene, polyvinyl or
polycarbonate.

4. HISTORY
19
60
• RIA - Berson & Yalow
19
66
• Enzyme labelled conjugates
19
71
• Enzyme labelled Ag & Ab
19
77
• Nobel Prize to Yalow

5. HISTORY
 In 1971, Peter Perlmann and Eva
Engvall at Stockholm University in
Sweden, and Anton Schuurs and
Bauke van Weemen in the
Netherlands independently
published papers that synthesized
this knowledge into methods to
perform EIA/ELISA.

6. PRINCIPLE
 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.

7. Different antigen in sample
substrat
e
Colored
product
Primary antibody
Secondary antibody

8. COMPONENTS OF ELISA
 Antigen
 Antibody
 Enzyme
 Substrate-chromogen

9. Enzyme Substrate Chromoge
n
Horseradish
Peroxidase
Hydrogen
peroxide
Tetra methyle
benzidine
(TBM)
Urease Urea Bromocresol
β –
Galactosidase
O-Nitrophenyl-β-
D-
Galactopyranosi
de (ONPG)
O-Nitrophenyl-β-
D-
Galactopyranosi
de (ONPG)
Alkaline Para-Nitrophenyl Para-Nitrophenyl

10. TYPES OF ELISA
 Direct ELISA
 Indirect ELISA
 Sandwich ELISA
 Capture ELISA
 Competitive ELISA
 ELISPOT

11. ELISA Type Detection of Enzyme
labeled with
Direct ELISA Antigen Primary antibody
Indirect ELISA Antibody or
Antigen
Secondary
antibody
Sandwich ELISA Antigen Primary antibody -
Direct
Secondary
antibody - Indirect
Competitive ELISA Antigen or
Antibody
Secondary
antibody
ELISPOT Cells producing
antibody or
Primary antibody
Principles of different ELISA types

12. COMPARISON BETWEEN VARIOUS
TYPES OF ELISA
Direct
ELISA
Indirect
ELISA
Sandwich
ELISA
Competitive
ELISA

13. DIRECT ELISA
Well -----> Ag (test serum) -----> Primary Ab –
enzyme
-----> Substrate chromogen -----> color change

14. INDIRECT ELISA
Wells coated with Ag ----> Primary Ab (test serum) --
--> Secondary Ab - enzyme ----> Substrate-
chromogen ----> Color change

15. SANDWICH ELISA
Wells coated with capture Ab ----> Ag (test serum) --
--> Primary Ab - enzyme ----> Substrate-chromogen
----> Color change

16. COMPETITIVE ELISA
Ab-Ag (test serum) ----> Wells coated with Ag ---->
Secondary Ab - enzyme ----> Substrate-chromagen

17. ELISPOT
Wells coated with capture Ab ----> Cells (cytokines)
----> Anticytokine Ab - enzyme ----> Substrate
chrmogen ----> Color change
 Modification of
ELISA
 Allows
quantitative
detection of cells
producing
antibodies or
cytokines

18. POSITIVE COTROL NEGATIVE CONTROL
 Either endogenous
known protein or
peptide for the Ab using
in the test.
 Indicate procedure is
optimized and working.
 Verify – negative
results are valid.
 To check for non-
specific binding and
false positive results.
 To validate the results.

19. QUALITY COTROL
 Designed to detect, reduce and correct deficiencies
in order to improve the quality of the results.
 It is measure of precision or how well the
measurement system reproduces the same result
over time and under varying operating conditions.
 Quality control data can be analyzed by using
Levey – Jennings chart.

20. LEVEY – JENNINGS CHART
 Named after S. Levey and
E.R. Jennings
 It is graph that quality
control data is plotted on to
give visual indication whether
a laboratory test is working
well.
 The distance from mean is
measured in SD.
 On X-axis date and time or more usually the number of the
control run are plotted.
 A mark is made indicating how far away the actual result
from mean (which is the expected value for control).

21. FACTORS AFFECTS THE TEST RESULTS
 Pre-analytical :
Hemolysed sample
Grossly lipaemic sample
Repeated freezing and
thawing
Contaminated samples and
reagents
Improperly stored, expired
and deteriorated reagents

22. FACTORS AFFECTS THE TEST RESULTS
 Analytical :
Pipetting errors
Improper incubation time
Improper washing procedure
Equipment malfunction
Glove powder aerosol
Calculation error
 Post-analytical :
Transcription errors

23. FALSE POSITIVE RESULTS
 Autoimmune diseases
 Alcoholic hepatitis
 Primary biliary cirrhosis
 Leprosy
 Multiple pregnancies

24. FALSE NEGATIVE RESULTS
 Technical error
 During early stage of disease
 During end stage of disease

25. ADVANTAGES OF ELISA
 Economical
 Less time (2-3 hrs)
 Most sensitive immunoassay
 Specific

26. DISADVANTAGE OF ELISA
 Expensive equipments
 Takes more time compared to rapid
tests

27. APPLICATIONS OF ELISA
 Infectious diseases :
HIV detection, Hepatitis, EBV,
Dengue, CMV, Leptospira, Syphilis,
Chlamydia etc.
 Rotavirus detection in fecal
specimens
 Enterotoxin of E. coli in feces

28. APPLICATIONS OF ELISA
 Mycobacterial Antibody
 Human allergens – specific IgE &
IgA
 Food adulterant include E. coli,
Campylobacter and Salmonella
antigen
 Food toxins like penicillin,
aflatoxins, streptomycin etc.

29. APPLICATIONS OF ELISA
 Measuring hormone levels
HCG (as a test for pregnancy)
LH (determining the time of
ovulation)
TSH, T3 and T4 (for thyroid
function)

30. REFERENCES
 Ananthnarayan & Paniker’s Textbook of Microbiology 9th ed.(2013).
 Kuby; Immunology 7th ed.(2013).
 Mackie & McCartney; Practical Medical Microbiology 14th ed.(2015).
 Apurba Sastry ;Essentials of Medical Microbiology 1st ed.(2016).
 Koneman’s Color Atlas and Textbook of Diagnostic Microbiology 7th
ed.(2017).