A novel immunoperoxidase procedure of using human monoclonal antibodies for the detection of cellular antigens in tissue sections

Abstract

A sensitive immunoperoxidase method was utilized to detect binding of human monoclonal antibodies to cellular antigens in formalin-fixed and paraffin-embedded tissue sections. The method was a 2-layer system utilizing biotin-labeled human monoclonal antibody and an avidin-biotin-horseradish peroxidase complex (ABC). A comparative study of this and a 4-layer peroxidase-antiperoxidase (PAP) staining method was made. In the PAP system, the monoclonal was followed by rabbit anti-human IgG, swine anti-rabbit IgG as the bridge-antibody and rabbit PAP complex. In both cases, the reaction was visualized by the use of aminoethylcarbazole (AEC) as chromogens. The former procedure was almost as sensitive and, most importantly eliminated an inherent problem associated with the latter namely the difficulty in distinguishing binding of the primary human monoclonal antibody from endogenous human Igs in tissue section. In addition, the 2-stage ABC procedure by comparison requires almost half the time.