Enzyme linked immuno sorbant assay and their types

2. What is ELISA
History
Components Of the ELIA test
Applications
Mechanism and Reagents
Types of ELISA
Methods

3.  Also known as enzyme immuno asay(EIA)
 Enzyme-Linked immunosorbent assay or
ELISA is a biochemical technique which is
used to detect and quantify the antigen or
antibody in the sample.
 Diagnostic tool in medicine and plant
pathology
 Quality control check in Industries

4. The amount of compound corresponds to the
substrate converted to the colored product by
the enzyme linked antibody
Quick and sensitive method of quantifying
large number of samples
The ELISA test was the first screening test
commonly employed for HIV. It has high
sensitivity
Replaced radio immuno assay(RIA)

5.  HIV, which causes AIDS
 Lyme disease
 pernicious anemia
 Rocky Mountain spotted fever
 rotavirus
 Syphilis
 syphilis
 toxoplasmosis
 varicella-zoster virus, which causes chickenpox and shingles
 Zika virus

6. Measure antibody levels(Vaccine)
Detect viruses(Hepatitis,HIV
Detect Hormonal changes(Pregnancy)
Detect circulatory inflammatory
markers(cytokines

7. • Before the ELISA test, the only option was
radio immuno assay(RIA)
• Radio immuno assay was first described by
Rosalyn Sussman Yalow and Solomon Berson

8. • Finally in 1971, two Swedish scientists, Eva
Engvall and Peter Perlman invented the test
which is Called the ELISA test

9. Advantages of ELISA Over RIA
• Reproducible
• Minimal Reagents
• Qualitative and Quantitative
• Qualitative Eg. HIV testing
• Quantitative Eg.Drug monitoring
• Wells can be coated with Antigens or
Antibodies
• No radiation hazards

10. Components of ELISA
Testing Sample
Polystyrene microtitre plate
Antibody
Enzymetic Applications
Antigen
Blocking buffers and wash buffers
Detection System for ELISA
Substrate

11. Polystyrene microtitre plate
• Multiple samples can be conductd
• Prepared with reagents to optimize the
protein binding capacity
8 well
strips
10-well
plates
96-well
plates

12. Polystyrene microtitre plate

13. Antibody
 proteins that body produces in response to harmful
substances called antigens
Polyclonal
• identifying different epitopes on a given antigen
• antibodies that are secreted by different B cell
lineages within the body
Monoclonal
• specificity toward a single epitope that allows
fine detection and quantitation of small
differences in antigen.

14. Types of Antibody in ELISA

15. Antigen
1. Foreign substance which induces the
production of antibodies in the body.
2. the antigen (target macromolecule) is
immobilized on a solid surface (micro plate)
and then mixed with an antibody that is
linked to a reporter enzyme.

16. Relation of Antigen, Antibody, Enzyme
and Substrate

17. Blocking buffers and wash buffers
Blocking
Buffer
• That adsorbs to all remaining binding
surfaces of the plate.
Wash
buffers
• Washing steps are necessary to remove non-
bound reagents and decrease background,
thereby increasing the signal to noise rati

18. Detection System for ELISA
 Involves the introduction of an enzyme
substrate
Enzyme converts the substrate to a detectable
product
There are following detection strategies In ELISA
test:
Chromogenic
Assay
Fluorescent Assay Chemiluminescent
Assay

19. Mechanism
• Absorption of antigen to a solid surface which
is placed in contact with a dilution of serum
• Enzyme linked antibody detects antigen using
colorless substrate

20. Types of ELISA test
Direct ELISA Indirect ELISA
Sandwich ELISA
Competitive
ELISA
ELISA

21. Direct ELISA
• It is simple and quick Test
• Only an enzyme-labeled primary antibody is
used
• There are no secondary antibodies in Direct
ELISA test.

22. Steps of Direct ELISA test
Antigen is immobilized to the plate Plastic
plate(polystyrene).
Enzyme-labeled primary antibody
"directly" binds to the Antigen
Enzyme linked to the primary antibody
reacts with the colorless substrate and
then the substrate gives the visible signal

23. Advantages of Direct ELISA
Simple and
Quick
No cross-
reactivity
Reagents
saving test

24. Disadvantages of Direct ELISA
There is no signal amplification so
it reduces the sensitivity
higher background noise than
indirect ELISA
Immunoreactivity of the primary
antibody is affected by labeling of
the enzyme

25. Indirect ELISA
• Primary antibody and secondary antibody is
used.Primary antibody is not labeled with an
enzyme. Instead, the secondary antibody is
labeled with an enzyme.

26. Steps for Indirect ELISA
Primary antibody binds to the antigen
immobilized to the plate
Enzyme-labeled secondary antibody binds
to the primary antibody
Enzyme linked to the secondary antibody
reacts with its substrate to produce a
visible signal that can be measured

27. Advantages of Indirect ELISA
High signal
amplification
High
Sensitivity
High
flexibility

28. Disadvantages of Indirect ELISA
An extra incubation step is
required in the procedure
Complex protocol
Cross-reactivity from
secondary antibody

29. Sandwich ELISA
• Capture antibody is immobilized to the plate.
Detection antibodies include the unlabeled
primary detection antibody and the enzyme-
labeled secondary detection antibody

30. Steps For Sandwich ELISA

31. Advantages of Sandwich ELISA
for complex
and impure
samples
High
Flexibility
High
specificity

32. Disadvantages of Sandwich ELISA
The antigen of interest must
be large enough so that two
different antibodies can bind
to it at different epitopes
It's sometimes difficult to find two
different antibodies that recognize
different epitopes on the antigen
of interest and cooperate well in a
sandwich format.

33. Competitive ELISA
• It is a technique used for the estimation of
antibodies present in a specimen, such as
serum

34. Advantages of Competitive ELISA
High
specificity,
since two
antibodies
Suitable for
complex
samples,
Flexibility
and
sensitivity
than other
ELISA
methods

35. References
• 1. John R. Crowther, Methods in Molecular Biology,
The ELISA Guidebook. Second Edition. Humana Press, a
part of Springer Science + Business Media, LLC 2009.
• Engvall, Eva, and Peter Perlmann. Enzyme-linked
immunosorbent assay (ELISA) quantitative assay of
immunoglobulin G. Immunochemistry 8.9 (1971): 871-
874.
• 3. Lequin, Rudolf M. Enzyme immunoassay
(EIA)/enzyme-linked immunosorbent assay (ELISA).
Clinical chemistry 51.12 (2005): 2415-2418.

36. THANKU