The use of alkaline-phosphatase-conjugated second antibody for the visualization of electrophoretically separated proteins recognized by monoclonal antibodies.

An immunoenzyme procedure is described for detection of proteins separated by SDS-polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred to nitrocellulose filters which were then sequentially incubated with mouse monoclonal antibody, alkaline-phosphatase-conjugated antibody to mouse immunoglobulins and a substrate mixture containing beta-naphthyl phosphate and Fast Blue B. The procedure has been successfully applied to several monoclonal antibodies of various heavy chain classes and can detect protein antigens at concentrations down to 0.5 ng/mm2.