Use of secondary antibodies for visualization of bound primary reagents in blotting procedures

Secondary antibodies are used for visualization of bound primary ligands in blotting procedures in several formats. The term “secondary antibody” usually denotes an anti‐immunoglobulin from a species different from the one producing the primary antibody. This secondary antibody is modified so as to produce a visible signal by conjugation with enzyme, fluorophore, radioisotope or suitable particulatematerial. The characteristics of these antibodies will influence both sensitivity and specificity of the assays in which they are used. Thorough knowledge of these characteristics is thus needed for critical evaluation of results. This paper discusses the production and reactivity of secondary antibodies, their immunochemistry and their possible side reactions. Alternative secondary reagents are also discussed with respect to specificity and sensitivity.