A method for the blocking of endogenous immunoglobulin on frozen tissue sections in the screening of human hybridoma antibody in culture supernatants.
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.
Nielsen
B
B
Institute of Pathology, University of Odense, Denmark.
Borup-Christensen
P
P
Erb
K
K
Jensenius
J C
JC
Husby
S
S
D000911:Antibodies, Monoclonal; D003110:Colonic Neoplasms / Q000276:immunology / Q000473:pathology; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007074:Immunoglobulin G / Q000032:analysis; D007075:Immunoglobulin M / Q000032:analysis; D007136:Immunoglobulins / Q000032:analysis*; D008223:Lymphoma / Q000276:immunology* / Q000473:pathology; D010954:Plasmacytoma / Q000276:immunology* / Q000473:pathology; D012004:Rectal Neoplasms / Q000276:immunology / Q000473:pathology
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007136:Immunoglobulins