title: An immunohistochemical assay on human tissue using a human primary antibody.
abstract: Non human antibodies administered to human patients often generate anti-antibody responses, leading in extreme cases to anaphylactic shock. Completely human antibodies are therefore favored over their murine, chimeric and humanized counterparts. However, the accurate evaluation of human antibodies on human tissue samples cannot be achieved using indirect immunohistochemical methods because of endogenous immunoglobulins that are co-detected by the secondary antibodies. Direct detection is often used instead, but this lacks the signal amplification conferred by the secondary antibody and is therefore less sensitive. We developed a simple fluorescence-based indirect immunohistochemical method that allows human primary antibodies bound specifically to their target antigens in human tissue samples to be detected clearly and without interfering background staining. This approach involves a biotinylated human primary antibody (H10(Biotin)) and Cy3-conjugated streptavidin (Strep(Cy3)). We tested the protocol using a human carcinoembryonic antigen (CEA) specific IgG1 (H10). We identified an exposure time threshold that allowed the elimination of low Strep(Cy3) background staining, yet achieved sufficient signal amplification to make our approach four times more sensitive than comparable direct immunohistochemical procedures. The principle of this indirect immunohistochemical assay should be transferable to other species allowing the specific and sensitive detection of any primary antibody on homologous tissues.
authors.affiliation: a Fraunhofer Institute for Molecular Biology and Applied Ecology IME , Aachen , Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Peuscher
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Gassler
authors.forename: Ursula
authors.initials: U
authors.lastname: Schneider
authors.forename: Pascal
authors.initials: P
authors.lastname: Thom
authors.forename: Stefan
authors.initials: S
authors.lastname: Rasche
authors.forename: Holger
authors.initials: H
authors.lastname: Spiegel
authors.forename: Stefan
authors.initials: S
authors.lastname: Schillberg
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D016466:CHO Cells; D002232:Carbocyanines; D002272:Carcinoembryonic Antigen / Q000032:analysis*; D002277:Carcinoma / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology; D003110:Colonic Neoplasms / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology; D003412:Cricetulus; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry; D051379:Mice; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D019809:Streptavidin / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D002232:Carbocyanines; D002272:Carcinoembryonic Antigen; D007074:Immunoglobulin G; C089187:cyanine dye 3; D001710:Biotin; D019809:Streptavidin