Question

Why in an immunostaining procedure for a specific antigen do you normally use two antibodies sequentially?

Answer 1

The primary antibody is used to detect the proteins or biomarkers or antigens that is to be detected in the immunostaining procedure. The primary antibody is directly bound to the target antigen at the Fab domain. The Fab domain of the secondary antibody binds to the Fc domain of the primary antibody. Since the Fc domain is constant, only one type of secondary antibody is required to bind to many types of primary antibodies. This reduces the cost by labeling only one type of secondary antibody that catalyzes the detection reaction, rather than labeling various types of primary antibodies. Secondary antibodies also help increase sensitivity and signal amplification due to multiple secondary antibodies binding to a primary antibody.