A Method for the Blocking of Endogenous Immunoglobulin on Frozen Tissue Sections in the Screening of Human Hybridoma Antibody in Culture Supernatants

Abstract

Endogenous immunoglobulin in tissue sections pose a problem in immuno-histochemical techniques employing homologous antibody as primary reagents and enzyme-labelled anti-immunoglobulin for the development. A method for the blocking of endogenous immunoglobulin in human tissue sections by incubation with monomeric pepsin fragments (Fab') of rabbit anti-human immunoglobulin before applying monoclonal antibody was evaluated for the screening of human monoclonal antibody. It was initially demonstrated that Fab' rabbit anti-human IgM and anti-IgG could block endogenous immunoglobulin in human IgM and IgG producing tumors thereby abolishing the binding of subsequently applied peroxidase-labelled anti-IgM or anti-IgG. Frozen sections of human colo-rectal adenocarcinomas show a variable background staining caused by the endogenous immunoglobulin. The background completely disappeared in the IgM system by preincubation with Fab' anti-IgM while the background was clearly reduced but not abolished in the IgG system. A human hybridoma supernatant containing IgM reactive with colo-rectal adenocarcinoma could easily be screened on frozen sections using this method. This approach should be generally useful for the screening of human antibody on human tissue sections.