Question

assume antigen is present, describe how the two antibodies and HRP are work to produce a color change in this immunoassay?

Answer 1

If the sample contains desired antigen, the primary antibody binds to them. The primary antibody is specific to the antigen present in the sample.

The secondary antibody is raised against the primary antibody. It binds to Fc or Fab regions of the primary antibody. The secondary antibody is conjugated to an enzyme such as HRP or AP.

So, when the secondary antibody binds to the primary antibody, the enzyme conjugated to the secondary antibody is available to perform its enzymatic reaction in the presence of a chromogenic substance.

The color is produced only when both bindings i.e primary antibody binding to the antigen and the secondary antibody binding to the primary antibody.

The amount of primary antibody bound to the antigen is directly proportional to the amount of antigen present in the sample. This indirectly dictates the amount of enzyme-conjugated secondary antibody.

So, the color produced by the action of HRP/AP on a chromogenic substance is directly proportional to the concentration of the antigen present in the sample.

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