Linked Life Data

16534499

Source:http://linkedlifedata.com/resource/pubmed/id/16534499

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
5
pubmed:dateCreated
2006-4-20
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0023-6837
pubmed:author
pubmed:issnType
Print
pubmed:volume
86
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
526-33
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2006
pubmed:articleTitle
Thiol-reactive compounds prevent nonspecific antibody binding in immunohistochemistry.
pubmed:affiliation
Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. abr@mit.edu
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural