Dr.

Bhaskar Ganguly
M.V.Sc. Scholar,
Animal Biotechnology Center,
CVASc, GBPUAT, Pantnagar – 263 145
 ELISA
{Enzyme Linked Immunosorbent Assay}

Direct ELISA
Enzyme labeled Antigen
Enzyme labeled Antibody
Indirect ELISA
Competitive ELISA
Sandwich ELISA
 ELISA Principles

96 well ELISA Plate:

Polystyrene
Polyvinyl
Protein binds passively in appropriate buffer {e.g. PBS}.
Plates are not re-usable.
Binding of protein occurs by ADSORPTION.
ELISA Principles...

After addition of reagents and incubation, the plates are washed in buffer to remove
unbound reagent because:
We want SOLID-PHASE Reaction, not Liquid-Phase Reaction!

After primary adsorption to the solid-phase other reagents are added in blocking buffer to
prevent non-specific binding to the plate.
E.g. BSA, Casein, Gelatin, Tween 20, Milk powder
Antigen or antibody can be covalently linked or labeled with enzyme.
E.g. Alkaline phosphatase, Horseradish peroxidase (HRPO)
The substrate for HRPO, Hydrogen peroxide is reduced to water and oxygen by the action of
the enzyme.

Produces color change due to substrate-enzyme interaction.

E.g. Orthophenylene diamine
Stops enzyme reaction after suitable color development
E.g. 1M Sulphuric acid
 Blocking Buffer
1. Antigen (or Ab) is adsorbed to the plate in adsorption/binding buffer.
2. When Antibody is added it binds to Antigen (specific) & to spaces on
the plate (non-specific).

If Antibody is added in blocking buffer it binds to antigen (specific) only

as blocking buffer blocks Non-specific binding to the plate.
 Direct ELISA
Enzyme-labeled Antigen
Ab adsorbed to plate in buffer
{2 hrs. at 37ºC or Overnight at 4ºC}
Wash to remove unbound Ab

Add Enzyme-labeled Ag (Conjugate) in

blocking buffer
{Incubate for 2 hrs. at 37ºC}
ANTIGEN binds to ANTIBODY.
Wash to remove unbound Ag

Add substrate/chromogen: color change

Stop reaction with acid & Read plate.
 Direct ELISA
Enzyme-labeled Antibody
Ag adsorbed to plate in buffer
{1 hr. at 37ºC or Overnight at 4ºC}
Wash to remove unbound Ag

Add Enzyme-labeled Ab (Conjugate) in

blocking buffer
{Incubate for 1 hr. at 37ºC}
ANTIBODY binds to ANTIGEN.
Wash to remove unbound Ab

Add substrate/chromogen: color change

Stop reaction with acid & Read plate.
 Indirect ELISA
Antibody detection
Used for the detection of antibodies.
Either by serum titration or spot test.

1. Antigen added to plate in buffer

{PBS or Carb/Bicarb}
Incubate for 1 hr. at 37ºC or Overnight at
4ºC

Antigen adsorbs to the plate

Wash to remove unbound antigen

Indirect ELISA
Antibody detection...

2. Add test serum (Bovine) diluted in blocking buffer

Incubate for 1 hr. at 37ºC

POSITIVE TEST SERUM NEGATIVE TEST SERUM

Antibody binds to Antigen No Antibody binds to Antigen

Wash to remove unbound Antibody

Indirect ELISA
Antibody detection...

3. Add Anti-species (Anti-Bovine) HRPO conjugate

diluted in blocking buffer
Incubate for 1 hr. at 37ºC
POSITIVE TEST SERUM NEGATIVE TEST SERUM

Conjugate binds to Antibody No Antibody for Conjugate to bind

Wash to remove unbound Conjugate

Indirect ELISA
Antibody detection...

4. Add Substrate (Hydrogen Peroxide) & Chromogen (OPD)

Allow color to develop for 10 minutes
POSITIVE TEST SERUM NEGATIVE TEST SERUM

Conjugate present No Conjugate present

Enzyme present No Enzyme
Color No Color
Stop reaction after 10 minutes by adding 1 M Sulphuric Acid
Sandwich ELISA
Antigen detection
1. Trapping antibody (rabbit) added to plate in
buffer
Incubate for 1 hr. at 37ºC or Overnight at 4ºC
Antibody adsorbs to plate
Wash to remove unbound antibody

2. Test sample added to plate in blocking

buffer
Incubate for 1 hr. at 37ºC or Overnight at 4ºC
Antibody traps (captures) Antigen
Wash to remove unbound Antigen
Sandwich ELISA
Antigen detection...
3. Detecting antibody (guinea pig) added to
plate in blocking buffer
Incubate for 1 hr. at 37ºC or Overnight at 4ºC
Detecting Antibody binds to Antigen
Wash to remove unbound antibody

4. Anti-Species (Anti-Guinea pig) Conjugate

added to plate in blocking buffer
Incubate for 1 hr. at 37ºC or Overnight at 4ºC
Conjugate binds to Guinea-pig antibody
Wash to remove unbound Antibody
Sandwich ELISA
Antigen detection...
5. Add Substrate (Hydrogen Peroxide) & Chromogen (OPD)
Allow color to develop for 10 minutes
-Conjugate present
-Enzyme present

Positive samples- Color

Negative samples- No color
Ideal for testing crude samples viz. feces and epithelium
 Competitive ELISA
IF WE ADD:
•Constant Anti-Species (Anti-Mouse) Conjugate
•Constant detecting Antibody (Mouse)
•Constant Antigen
Competitive ELISA...
POSITIVE TEST SERUM
Test Antibody (Bovine) binds to Antigen
Detecting Antibody (Mouse) cannot bind
Conjugate (Anti-Mouse) cannot bind
No enzyme present

NEGATIVE TEST SERUM

Test Antibody (Bovine) does not bind
Detecting Antibody (Mouse) binds to Antigen
Conjugate (Anti-Mouse) binds
Enzyme present