Thiol-reactive compounds prevent nonspecific antibody binding in immunohistochemistry.
Nonspecific antibody binding is the primary source of confounding background in immunohistochemistry (IHC). Based on observed patterns of background staining, and the known spontaneous reduction of immunoglobulin disulfide bonds in vivo and in vitro, we tested the hypothesis that nonspecific antibody binding in IHC is mediated by sulfhydryl interactions. Coincubation of primary antibodies with reduced glutathione (GSH), L-cysteine, iodoacetic acid, Ellman's reagent and other thiophilic reagents in pH 8 tris-EDTA (TE) buffer inhibited background staining. In contrast, oxidized glutathione (GSSG) exerted no effect. When empirically optimized, coincubation of GSH with primary antibodies significantly improved IHC signal:noise ratio. Tissue preincubation with mercaptans, soft and borderline metals, and other sulfhydryl-reactive reagents also inhibited background staining, but at the expense of target sensitivity. ELISA results confirmed direct binding between murine serum antibodies and GSH in a nonantigen-dependent manner. In summary, thiol-reactive compounds prevent nonspecific antibody binding in IHC. We propose a mechanism whereby nonspecific background resulting from formation of disulfide bridges and other sulfhydryl bonds between primary antibodies and tissue side groups is interrupted by prior exposure to thiol-reactive reagents such as GSH. These findings provide a molecular basis to improve the specificity of IHC and other immunoassays, and hold implications for antibody-based clinical diagnostics and therapeutics.
Rogers
Arlin B
AB
Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. abr@mit.edu
Cormier
Kathleen S
KS
Fox
James G
JG
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000378:metabolism; D000918:Antibody Specificity; D003545:Cysteine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D005978:Glutathione / Q000737:chemistry; D007150:Immunohistochemistry / Q000379:methods*; D007202:Indicators and Reagents; D019807:Iodoacetic Acid / Q000737:chemistry; D008024:Ligands; D051379:Mice; D010084:Oxidation-Reduction; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D016707:Tissue Fixation
D000906:Antibodies; D007202:Indicators and Reagents; D008024:Ligands; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D005978:Glutathione; D003545:Cysteine; D019807:Iodoacetic Acid