This document provides an overview of the ELISA (enzyme-linked immunosorbent assay) technique. It describes the basic principles of ELISA, including using an enzyme to detect antigen-antibody binding by converting a colorless substrate to a colored product. It outlines the different types of ELISA (qualitative, quantitative) and methods (direct, indirect, sandwich). It discusses materials needed, enzyme substrates used, the basic procedures for direct and indirect ELISA, applications of ELISA, advantages and disadvantages.

1. A
Presentation
on
ELISA
BABABHIMRAO AMBEDKAR UNIVERSITY,LUCKNOW
SUBMITTED BY, SUBMITTED TO,
AZAD SINGH Dr. SUDIPTA SAHA
M.PHARM(SEM.I) ASSIST.PROFESSOR

2. CONTENTS
 INTODUCTION
 PRINCIPLES
 TYPES
 APPLICATION
 Advantages
 Disadvantages
 REFRENCES

3. INTRODUCTION TO ELISA
 ELISA can be used to detect either presence of Antigens or
Antibodies.
 And also used for detecting quantifying substances such as peptides,
proteins, antibodies and hormones.
 Enzyme is used to detect the binding of Antibody – Antigen.
 Enzyme converts colorless substrate into colored product, indicating

4. PRINCIPLE
 Wet lab" analytic biochemistry assay, ELISA involves detection of an
"analyte" in a liquid sample by a method that continues to use liquid
reagents during the "analysis“.
 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.

5. TYPES OF ELISA
QUALITATIVE ELISA
Positive or Negative results
QUANTITATIVE ELISA
Optical density or fluorescent units of the sample
is interpolated into a standard curve, which is
typically a serial dilution of the target.

6. ELISA METHODS
INDIRECT ELISA
DIRECT ELISA
SANDWICH ELISA

7. MATERIALS NEEDED
Testing
Antibody/antigen
Polystyrene microtiter plate
Blocking buffer
Washing buffer
Substrate
Enzyme eg-Horseradish peroxidase , alkaline
phosphatase, lactoperoxidase, tetra methyl
benzidine

8. ENZYME SUBSTRATE
1)-Initially the substrate should be colorless
2)-After degradation by the enzyme it should be
strongly coloured.
ENZYME SUBSTRATE CHROMOGEN STOPPING
Alkaline Phosphates p-NPP p- NPP+di
ethandamine+
Mgcl2
1 M NaOH
Horse radish
Peroxidase
H2O2 Tetra
ethylbenzidine
+Phosphate-
Citrate buffer
H2SO4
Horse radish
Peroxidase
H2O2 O-
Phenylenediami
ne+ HCl
1 M HCl

9. DIRECT ELISA
well microtiter plate
being used for ELISA.

10. 1)-Antibody is adsorbed onto
microtiter plate.
2)-Add serum to test for
antigen
3)-Specific antigen binds if
contained

11. 1)-Remove test fluid and
unbound antigen
2)-Binding of antigen is
strong enough to withstand
rinsing

12. 1)-Remove test fluid
and unbound antigen
2)-Binding of antigen
is strong enough to
withstand rinsing

14. 1)-Add enzyme labeled
antibody
2)-Labeled antibody
binds to antigens
E
E
E
E
E
E
E
E

15. 1)-Wash sample to remove
unbound antibodies
EE E
E E
E
E
E

16. 1)-Wash sample to
remove unbound
antibodies
E
E
E
E
E
EEE

17. 1)-Wash sample to
remove unbound
antibodies
E E E

18. 1)-Add
substrate
2)-Measure
color changePositive Negative
E E E

19. INDIRECT ELISA

20. 1)-Antigen is
adsorbed onto
microtiter plate
2)-Add serum to test
for antibody

21. 1)-Antigen is adsorbed
onto
microtiter plat
2)-Add serum to test
for antibody

22. 1)-Antigen is adsorbed
onto microtiter plate
2)-Add serum to test for
antibody
3)-Wash sample

23. 1)-Antigen is adsorbed
onto microtiter plate
2)-Add serum to test
for antibody
3)-Wash sample

25. 1)-Add secondary
antibody
2)-Primary
antibody binds to
secondary
anitbody
E
E
E
E
E
E
E
E

26. EEE
E
E
E
E
E
1)-Add secondary
antibody
2)-Wash unbound
antibodies

27. 1)-Add
secondary
antibody
2)-Wash
unbound
antibodies
EEE
E
E
E
E
E

28. 1)-Add secondary
antibody
2)-Wash unbound
antibodies
EEE

29. 1)-Add
substrate
2)-Measure
color change
EEE
Positive Negative

30. Direct vs. Indirect ELISA
Advantages of Indirect ELISA:
1)-Immunoreactivity of primary antibody is not
affected by labeling.
2)-Wide variety of secondary antibodies available on
the market.
3)-Signal amplification (several epitopes).
Advantages of Direct ELISA:
1)-Quick methodology – only one AB.
2)-Cross-reactivity of second AB eliminated.
30

31. SANDWICH ELISA
The sandwich ELISA measure the amount of
antigen between two layers of antibodies (i.e.
capture and detection antibody).
The antigen to be measured must contain at least
two antigenic sites capable of binding to antibody,
since at least two antibodies act in the sandwich.

32. STEPS IN SANDWICH ELISA
1)-Prepare a surface to which a known quality of antibody is bound.
Apply the antigen-containing sample to the plate.
2)-Wash the plate, so that unbound antigen is removed.
3)-Apply the enzyme-linked antibodies which are also specific to the
antigen.
4)-Wash the plate , so that unbound enzyme-linked antibodies are
removed.
5)-Apply a chemical which is converted by the enzyme into a
fluorescent signal
6)-View the result : if it fluoresces, then the sample contained antigen.

34. APPLICATIONS
 Screening donated blood for evidence of viral contamination by-
 HIV-1 and HIV-2 (presence of anti-HIV antibodies)
 Hepatitis C (presence of antibodies)
 Hepatitis B (testing for both antibodies and a viral antigen)
 Measuring hormone levels
 HCG (as a test for pregnancy)
 LH (determining the time of ovulation)
 TSH, T3 and T4 (for thyroid function)
 Detecting infections
 Sexually-transmitted agents like HIV, syphilis and chlamydia
 Hepatitis B and C
 Detecting illicit drugs.
 Detecting allergens in food and house dust

35. ADVANTAGES OF ELISA
Reagent are relatively cheap and have a long shelf life
ELISA is highly specific and sensitive
No radiation hazards occur during labelling or disposal of
waste.
Easy to perform and quick procedures.
Equipment can be inexpensive and widely available.
Elisa can be used to a variety of infection.

36. DISADVANTAGES OF ELISA
Measurement of enzyme activity can be more
complex than measurement of activity of some
type of radioisotopes.
Enzyme activity may be affected by plasma
constituents.
Kits are commercially available but not cheap.

37. A NEW TECHNIQUE:-Reverse ELISA
A new techniques uses a solid phase made up of an
immunosorbent polystyrene rod with 4-12
protruding ogives . The entire device is immersed
in a test tube containing the collected sample and
the following steps (washing , incubation in
conjugate and incubation in chromogenous) are
carried out by dipping the ogives in microwells of
standard microplates pre-filled with reagents.

38. ADVANTEGE OF THIS TECHNIQUES
The ogives can each be sensitized to a different
regent , allowing the simultaneous detection of
different antibodies and different antigen for multi-
target assays.
The sample volume can be increased to improve the
the test sensitivity in clinical, food and
environmental samples.
One ogive is left unsensitized to measure the non –
specific reaction of the sample

39. REFERENCE
http://www.elisa-antibody.com/ELISA-
introduction/ELISA-Principle
http://www.sigmaaldrich.com/life-
science/cell-biology/Antibodies/Antibodies-
application/protocols/elisa.html