ELISA for antigen- antibody test

1. Dr.APJ ABDUL KALAM TECHNICAL UNIVERSITY
(Formerly Uttar Pradesh Technical University ) LUCKNOW
SUBMITTED BY :-
AISHWARAY MUDGAL
M.PHARM Ist SEM
0211COL016
TOPIC :- ENZYME LINKED IMMUNOSORBENT ASSAY
Noida Institute of Engineering & Technology
( Pharmacy Institute )
SUBMITTED TO :-
Dr. Saumya Das
Associate Professor
Noida Institute of Engineering and
Technology (Pharmacy Institute),
Greater Noida.

2. ELISA INTRODUCTION :-
 It is an important immunological method for detecting and measuring antigens and
antibodies. It is a plate based assay technique designed for detecting and quantifying
substance such as peptides, proteins, antibodies and hormone.
 It is based on the same principle as that of radioimmunoassay.
 The main difference is that of enzyme immunoassay the antigen of antibody is conjugated
to an enzyme rather than a radioactive isotope.The enzyme is then detected by its ability
to convert a colourless substance into coloured one.
 Enzyme immunoassay have become very popular in view of their high sensitivity, safety,
economy and the simple instrument requirement.

3. SOME BASIC TERMS
 SOLID PHASE :- Usually microtiter plate well having 96 wells.
 ADSORPTION :- Process of having antigen or antibody ,diluted in buffer,so it attached to
solid phase on incubation.
 WASHING :- Simple flooding and emptying of wells with buffer solution to separate
bound reagent from unbounded reagent in ELISA.
 ANTIGEN :- Molecule entity responsible to produce antibody when introduced inside
body.
 ANTIBODY :- Protein produced in response to antigen.
 ENZYME CONJUGATE :- Enzyme irreversible attach to antibody (horse reddish peroxide
,alkaline phosphatase).
 CHROMOGEN :- Chemical alters colour as a result of enzyme interaction with substrate

4. PRINCIPLE OF ELISA
ELISA is based on following observations:
1. Antigens and antibodies can attach to polystyrene plastic plates or other
solid phase support and maintain immunological capabilities.
2. Antigens and antibodies can be bonded to exam and the resulting complexes
are fully functional both immunologically as well as enzymatically
3. Specity of antigen antibody reaction.

5. TYPES OF ELISA
1. Sandwich ELISA
2. Indirect ELISA
3. Direct ELISA
4. Competitive ELISA
Double-Antibody-Sandwich method
In this technique the wells and depression in a polystyrene plate are first coated with an antibody and the
sample containing antigen is then added and allow to react with the bound antibody. The wells is washed and
the second enzyme linked specific antibody is added and allowed to react. This result is an antibody(with
enzyme)-Antigen antibody sandwich. Finally the enzyme substrate is added for reaction with the enzyme. The
rate of enzyme action is directly proportional to the Amount of test antigen.

6. Enzyme activity may be followed by a colour change which can
which can be inspected visually or measured by colourimeter
this method has been used to assay of Hepatitis B-antigen.

7. Indirect Elisa Method
The principle of this test can be illustrated by outlining its application for detection of anti-HIV-1 and
anti-HIV-2 antibodies in the patient serum the wells of the polystyrene microtitre plate are coated
with antigen. Test anti serum is added and allow to incubate. If the antibodies in the antiserum are
homologous they will bind to immobilized antigen. The well is then washed and enzyme –labelled
antihuman antibodies are added to the system which link with the antibody –antigen complexes.
ELISA is a simple and versatile technique. ELISA kits are commercially available for the detection of
anti HIV, Hepatitis B surface antigen and rotavirus. ELISA kits have also been develop for detecting
Hepatitis-A-virus (from stool), Haemophilus influenza antigens (from spinal fluid), Toxoplasma
antigens(from serum),Entamoeba histolytica antigen(from faeces), Escherichia coli enterotoxin(from
stools)etc.

8. The enzyme substrate is added the rate of its degradation is
associated with colour change proportional to the
concentration of antibody present in the test sample the colour
change can be measured visually and by colourimeter.

9. DIRECT ELISA :-
• It uses a primary labeled anti-body that react directly with
antigen .
• It can be performed with the antigen that is directly
immobilized on assay plate.
• Not widely used but common for immuno-histochemical
Staining of cells & tissues.
COMPETITIVE :-
• Antibody coated microwell.
• Serum antigen & labeled antigen added together ….. Competition.
• Ab-Ag enzyme complex bound is inversely related to the concentration of antigen present in sample.
• Increased serum antigen results in reduced binding of Ag-enzyme conjugated with the antibody
producing less enzyme activity & (yellow) color formation.
• Used to determine small molecules like T3,T4 & Progesterone.

10. APPICATION OF ELISA:-
 Measuring hormone level in blood.
- HCG (pregnancy)
- LH (ovulation)
- TSH,T3,T4 (thyroid).
 Detection of infection.
- Sexually transmitted agents (HIV)
- Hepatitis B & C.
 Detection allergen in food.
 Used for rapid testing.
 Used in vaccine QC.
 For detecting genetically modified organism.

11. REFRENCES :-
o https://www.berthold.com/microplate/washers
o https://pubmed.ncbi.nlm.nih.gov
o https://www.slideshare.net/sabaahmed56/elisa-ppt-53678570
o https://medlineplus.gov/ency/article/003332.htm
o S.R. Sankar, Text book of pharmaceutical analysis, Third edition, Page
no. 28.1-28.8.

12. Thank you