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.

1. Made By :
RAJESH SINGH
M. Pharm 1ST Year
RKT

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

3. Introduction
ELISAstands for enzyme-linked immunosorbent assay.
It is a common laboratory technique which is used to measure the concentration
of an analyte (antigens) in solution.
WhereAg-Ab interaction is monitored by enzyme measurement.
It is similar in principle to Radio ImmunoAssay (RIA) but It depends on an
enzyme rather than a radioactive label.

4. 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 ELISAcan be used to detect either the presence of antigens or
antibodies in a sample depending how the test is designed.
Principle

5. Antigen + Antibody Ag –Ab
Ag –Ab + Substrate Ag –Ab
Enzyme
linked
E
E E + S = Colour Change

6.  Testing sample
 Antibody
 Polystyrene microtiter plate
 Blocking buffer
 Washing buffer
 Substrate
 Enzyme
Materials Needed

7. Anumber 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)

8. 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.

9. Types Of ELISA
1. Non-Competitive
a) Direct ELISA
b) Indirect ELISA
c) Sandwich ELISA
2. Competitive
Direct Elisa

11. 1) Microwell Plate:
Flat bottom
polystyrene
plate,
contains 8 x 12
wells holding
350 μL each.
Equipments Used

12. 2) Multipipette :
An 8-channel 100 μL
pipette is a good help
for even small-scale
work.

13. 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.

14. 4) Microplate washer:
• These are very efficient
with unusually low carry-
over contamination.

15. • 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)
Applications

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