Question

why, in an immunostaining procedure for a specific antigen, do you normally use two antibodies sequentially? Support your answers with citations from the scientific literature

Answer 1

Immunostaining procedure is an antibody based method used to detect a specific protein or target protein in a sample, as antibodies are highly specific, antibody binds to a particular protein in the tissue section, antigen-antibody interaction is seen by using chromogenic detection, in it enzyme conjugated and antibody cleaves a substrate to produce a colored precipitate at the location of the protein, this can also be seen by fluorescent detection in it specific fluorescent dye or flurophore is conjugated to antibody and is observed under fluorescence microscopy, Immunostaining is used in assessment and progress of cancer.

The immunohistochemistry is done by two processes i) Direct method ii) Indirect method.

Direct Method:- Direct method saves time, it uses a detective marker (like HRP, or fluorescence) to label the first antibody; but it is not an effective method like indirect method as in this method the signal is not amplified.

Indirect Method:-

In indirect method an unlabeled primary antibody is used (first antibody) against a target antigen in tissue and a labeled secondary antibody is used . The second antibody reacts with the primary antibody (The secondary antibody is usually developed in an animal by inducing the animal with IgG from the host of primary antibody). Indirect method is more sensitive than direct method because here the detecting signal is amplified by binding more than one molecules of the second antibody to the first antibody molecule. The second antibody can be labeled by fluorescent dyes like FITC, PE and Cy5 . The secondary antibody can be also labeled by an enzyme like peroxidase, alkaline phosphatase or glucose oxidase.This method is more sensitive due to signal amplification by binding of several secondary antibodies to different antigenic sites on primary antibody on both F_c and F_ab fragments.

Due to high sensitivity indirect method is used more commonly used than the direct method.