Abstract
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 be...Continue Reading
References
Feb 1, 1975·The Journal of Experimental Medicine·C B Laurell, E Thulin
May 31, 1977·Biochemistry·E J VictoriaS P Masouredis
Aug 1, 1990·European Journal of Immunology·H MartinM Loos
May 1, 1989·Journal of Clinical Microbiology·D B BudzkoD Armstrong
Jan 1, 1986·International Archives of Allergy and Applied Immunology·E SchauensteinW List
Feb 1, 1974·The Biochemical Journal·B M Buchwald, G E Connell
Jan 1, 1982·Journal of Immunological Methods·E SchauensteinF Dachs
Oct 1, 1994·Hearing Research·Y Sato, J Santos-Sacchi
Oct 1, 1996·Biochemistry and Molecular Biology International·E SchauensteinP Felsner
Jun 9, 1997·Journal of Biochemical and Biophysical Methods·R HorejsiW Steinschifter
Jan 27, 1999·Biochimica Et Biophysica Acta·R M JonesG J Hughes
Feb 4, 1999·American Journal of Clinical Pathology·J C IezzoniM H Stoler
Jun 8, 2002·Biotechnology Progress·Wei Zhang, Marta J Czupryn
Jun 19, 2004·Toxicologic Pathology·Jerrold M Ward
Jul 12, 2005·Veterinary Pathology·J A Ramos-Vara
Citations
Jan 24, 2009·Proceedings of the National Academy of Sciences of the United States of America·S E ErdmanJ G Fox
Mar 25, 2009·Infection and Immunity·Z ShenJ G Fox
Aug 20, 2008·Infection and Immunity·Megan E McBeeDavid B Schauer
Feb 19, 2009·Infection and Immunity·Laura B LemkeJames G Fox
Jun 18, 2011·Cancer Prevention Research·Alexander ShehJames G Fox
Dec 24, 2009·Toxicologic Pathology·J T PainterR A Herbert
May 25, 2010·BMC Neuroscience·Thomas G MastDebra A Fadool
Oct 27, 2010·PloS One·Megan E McBeeDavid B Schauer
Mar 18, 2011·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Allan WangJoanna L Jankowsky
Nov 6, 2007·The American Journal of Pathology·James G FoxTimothy C Wang
Oct 24, 2009·Gut·J G FoxA B Rogers
Feb 23, 2012·Scientific Reports·Igor BuchwalowMarkus Tiemann
Jan 25, 2014·Veterinary Pathology·J PangJ G Fox
Apr 9, 2017·Redox Biology·Zhen ZhangJian Yao
Jun 13, 2019·Mikrochimica Acta·Eva ValenciaRosario Pereiro
Nov 11, 2014·Helicobacter·Zeli ShenJames G Fox
Mar 24, 2011·Immunology·Mark T WharyJames G Fox
Aug 1, 2013·Science and Technology of Advanced Materials·Shao-Hsuan ChuagChien-Fu Chen
Oct 5, 2011·The American Journal of Pathology·Ian N HinesArlin B Rogers