Enzyme linked immunosorbent assay

The enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used to

measure antibodies, antigens, proteins and glycoproteins in biological samples.

Types

1. Direct ELISA 2. Indirect ELISA 3. Sandwich ELISA 4. Competitive ELISA

Direct ELISA

Antigen is bound to the surface of the slide

The test sample is poured on the substrate, if it contains the complimentary antibody then it
binds with the antibody

Following this rinsing is performed to remove unbound antibody

Next, the mixture containing a secondary antibody with bound flourphore (Enzyme) is added,
this antibody binds to the antibody antigen complex.

Following this rinsing is performed to remove any unbound antibody.

Finally substrate of the enzyme is added which changes color on reaction with enzyme thereby
indicating a positive result.

Indirect ELISA

1. Antigen is bound on the polystyrene surface

2. Sera samples are added to the wells

3. Negative and positive controls are important parts of a test

4. The wells are covered to allow antigen antibody in the incubation step.

5. Wash buffer is added to remove any unbound antibodies.

6. Now conjugated antibody (horse radish peroxide) is added which helps to detect and quantify
the primary antibodies

7. Rinsing is carried out using wash buffer

8. Substrate solution (TMB) is added to each well and incubated

Sandwich ELISA

A sandwich ELISA measures antigen between two layers of antibodies (capture and detection
antibody). The target antigen must contain at least two antigenic sites capable of binding to
antibodies.

Western Blot
Widely used technique for detection and analysis of proteins.

Blot is a membrane on which nucleic acids and proteins are immobilized.

Process of transferring macromolecules from gel to membrane followed by its detection of

these on the membrane is known as blotting.

5 main steps

Protein gel electrophoresis (Separation and characterization of proteins)

Protein transfer

Blocking

Antibody probing

Detection