Question

What is the principle of indirect ELISA?

Answer 1

ELISA :

1. ELISA is a plate-based technique that is used for detecting and quantifying substances such as proteins, antibodies, and hormones.
2. An enzyme such as Alkaline phosphate conjugated with an antibody reacts with a colorless substrate to generate a signal in the form of a colored product such a substrate is termed chromogenic substrate.

Principle of ELISA:

1. The principle of ELISA is based on the formation of an antigen-antibody complex.
2. Antibodies in the sample are linked with an enzyme added to the well of the plate, by addition of a specific substrate to the well results in the formation of colored product.
3. In this technique, the sample is incubated in a well of the plate
4. Antibodies present in the sample is captured by a corresponding reaction between an antibody in the sample and coated antigen on the solid surface of the plate, after washing the unbound antibodies are removed with a washing buffer.
5. When a suitable substrate is added, the enzyme reacts to give color.
6. The color produced represents the quantity of antibodies or antigens present in the given sample.
7. The intensity of color produced (optical density) is measured at 450 nm on a spectrophotometer.

Indirect ELISA :

1. In this type of ELISA, an antigen is immobilized to the well of the plate, and the antibody specific to this antigen in the sample reacts with it to form an Ab-Ag complex (Antigen-Antibody complex).
2. The presence of primary antibody is detected by the addition of secondary antibody linked with enzyme, on the addition of substrate generates a signal in the form of the colored product.