Question

What are 2 completely different ways to perform an indirect ELISA?

What 2 different types of information do you get from these methods?

Answer 1

Indirect ELISA is a biochemical estimation assay which gives quantitative estimation for the presence of analyte in the sample. The ELISA plate is pre-coated with a primary antibody and the sample containing the antigen is added to it. The primary antibody and the secondary antibody are allowed to bind and react.

After a few minutes, another secondary antibody is added to this system. Now this secondary antibody can be attached to two different types of reporter molecules and the nature of these reporter molecules will determine the method of ELISA.

Method 1: The reporter can be an enzyme which causes bio-conversion of a substrate to give rise to a colorimetric product.

Method 2: The reporter can be a fluorescent probe which can directly emmit fluorescence and the same can be detected.

These two different methods give absorbance and fluorescence intensity as a result of binding. Both of these parameters are directly proportional to the amount of analyte present in the sample and thus the concentration of analyte can be calculated using a standard graph.