Dr. Bhaskar GangulyM.V.Sc. Scholar,

Animal Biotechnology Center,CVASc, GBPUAT, Pantnagar – 263 145

ELISA{Enzyme Linked Immunosorbent Assay}

Direct ELISAEnzyme 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 Buffer1. 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 ELISAEnzyme-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 changeStop reaction with acid & Read plate.

Direct ELISAEnzyme-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 changeStop 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 bufferIncubate for 1 hr. at 37ºC

Antibody binds to Antigen No Antibody binds to Antigen

Wash to remove unbound Antibody

POSITIVE TEST SERUM NEGATIVE TEST SERUM

Indirect ELISA

Antibody detection...

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

diluted in blocking bufferIncubate for 1 hr. at 37ºC

Conjugate binds to Antibody No Antibody for Conjugate to bind

Wash to remove unbound Conjugate

POSITIVE TEST SERUM NEGATIVE TEST SERUM

Indirect ELISA

Antibody detection...

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

Allow color to develop for 10 minutes

Conjugate present

Enzyme present

Color

Stop reaction after 10 minutes by adding 1 M Sulphuric Acid

POSITIVE TEST SERUM NEGATIVE TEST SERUM

No Conjugate present

No Enzyme

No Color

Sandwich ELISA

Antigen detection

1. Trapping antibody (rabbit) added to plate in buffer

Incubate for 1 hr. at 37ºC or Overnight at 4ºCAntibody 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 4ºCAntibody traps (captures) AntigenWash 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 4ºCDetecting Antibody binds to AntigenWash 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 4ºCConjugate 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- ColorNegative 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