Made By :

RAJESH SINGH
M. Pharm 1ST Year

RKT
 Contents
 Introduction
 Principle
 Materials Needed
 Methodology
 Types Of ELISA
 Equipments Used In ELISA
 Applications
 References
 Introduction

ELISA stands for enzyme-linked immunosorbent assay.

It is a common laboratory technique which is used to measure the concentration

of an analyte (antigens) in solution.

Where Ag-Ab interaction is monitored by enzyme measurement.

It is similar in principle to Radio Immuno Assay (RIA) but It depends on an

enzyme rather than a radioactive label.
 Principle

ELISA use an enzyme to detect the bindingof antigen (Ag) antibody

(Ab).

The enzyme converts a colorless substrate (chromogen) to a colored

product, indicating the presence of Ag:Ab binding.

An ELISA can be used to detect either the presence of antigens or

antibodies in a sample depending how the test is designed.
Antigen + Antibody Ag – Ab
Enzyme E
linked

Ag – Ab + Substrate Ag – Ab

E E + S = Colour Change
 Materials Needed
 Testing sample
 Antibody
 Polystyrene microtiter plate
 Blocking buffer
 Washing buffer
 Substrate
 Enzyme
A number of enzymes have been employedfor ELISA, including
1. Alkaline phosphatase
2. Horseradish peroxidase

Substrates Used
1. OPD (O-phenylene diamine, Dihydrochloride)
2. PNPP (P- Nitrophenylene phosphase, Disodium salt)
 Methodology
 Take a polystyrene microtitre plate (96 wells) and add antigen containing sample.

 Antigen will be attached to the surface.

 Wash the microtitre plate by using Tris-phosphate buffer and detergent (tween
20).

 Add enzyme linked antibody (specified) to the microtitre plate.

 Washing of the plate by using buffer solution.

 Add the specified substrate.

 Then it will react with enzyme linked antibody and form a product which is blue
colour (Generally).

 Then H2SO4 will be added and colour will be changed to yellow.

 Then it will observe in the colorimetry.
 Types Of ELISA
1. Non-Competitive
a) Direct ELISA
b) Indirect ELISA
c) Sandwich ELISA
2. Competitive

Direct Elisa
 Equipments Used

1) Microwell Plate:
Flat bottom
polystyrene
plate,
contains 8 x 12
wells holding
350 μL each.
2) Multipipette :
An 8-channel 100 μL
pipette is a good help
for even small-scale
work.
3) Washing Device:
manually operated washing devices.
may be of use particularly when there is a risk that the
samples tested in ELISA contain infectious material, so must
be collected for subsequent disinfection.
4) Microplate washer:
• These are very efficient
with unusually low carry-
over contamination.
 Applications
• Serum antibody concentration.
• Detecting potential food allergens. (milk, peanut, walnut, almonds and
eggs)
• Tracking the spread of disease in disease outbreaks. (e.g. HIV, bird
flu, common COLD, STDs.)
• Detection of antigen. (e.g. pregnancy hormones, drug allergens,
GMO, mad cow disease)
• Detection of antibodies to past exposure to disease. (e.g. Lyme
disease, trichinosis, HIV, bird flu)
 References

• www.hhmi.org/biointeractive/immunology-virtual-lab.
• www.ncbi.nlm.nih.gov/pubmed/25926946.
• https://www.sinobiological.com/category/elisa-Enzyme-linked
• ELISA -A to Z .....from introduction to practice by Katsumi
WAKABAYASHI, Ph.D , Prof. Emer. Gunma University.