title: A novel approach to enhance antibody sensitivity and specificity by peptide cross-linking.
abstract: Most current techniques employed to improve antigen-antibody signals in Western blotting and in immunohistochemistry rely on sample processing prior to staining (e.g., microwaving) or using a more robust reporter (e.g., a secondary antibody with biotin-streptavidin). We have developed and optimized a new approach intended to stabilize the complexes formed between antigens and their respective primary antibodies by cupric ions at high pH. This technique improves the affinity and lowers cross-reactivity with nonspecific bands of approximately 20% of antibodies tested (5/25). Here we report that this method can enhance antigen-antibody specificity and can improve the utility of some poorly reactive primary antibodies.
authors.affiliation: Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Building 37, Room 3132, Bethesda, MD 20814, USA.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Namiki
authors.forename: Julio C
authors.initials: JC
authors.lastname: Valencia
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Hall
authors.forename: Vincent J
authors.initials: VJ
authors.lastname: Hearing
mesh_terms: D000818:Animals; D000915:Antibody Affinity / Q000187:drug effects; D000918:Antibody Specificity / Q000187:drug effects; D000937:Antigen-Antibody Reactions / Q000187:drug effects; D001738:Biuret Reaction; D003300:Copper / Q000737:chemistry* / Q000494:pharmacology; D003429:Cross Reactions / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D051379:Mice; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D010455:Peptides; D003300:Copper
references.citation: Nat Genet. 2001 Aug;28(4):381-5
references.pmid: 11479596
references.citation: Pigment Cell Res. 2002 Jun;15(3):217-24
references.pmid: 12028586
references.citation: J Cell Sci. 2003 Aug 1;116(Pt 15):3203-12
references.pmid: 12829739
references.citation: Adv Protein Chem. 1967;22:257-424
references.pmid: 4882247
references.citation: Am J Pathol. 1986 May;123(2):195-203
references.pmid: 3518473
references.citation: Int J Cancer. 1987 Mar 15;39(3):414-8
references.pmid: 3102392
references.citation: J Biol Chem. 1991 Jan 15;266(2):1147-56
references.pmid: 1898730
references.citation: EMBO J. 1992 Feb;11(2):519-26
references.pmid: 1537333
references.citation: Biotechniques. 1992 Apr;12(4):496-9
references.pmid: 1503746
references.citation: Int J Pept Protein Res. 1996 Jul;48(1):48-55
references.pmid: 8844262
references.citation: J Am Soc Nephrol. 2005 Jun;16(6):1562-70
references.pmid: 15829703
references.citation: FEBS Lett. 2006 Mar 20;580(7):1865-71
references.pmid: 16516205
references.citation: J Biol Chem. 2006 Jul 28;281(30):21198-208
references.pmid: 16682408
references.citation: J Proteome Res. 2006 Nov;5(11):3135-44
references.pmid: 17081065
references.citation: Mol Pharmacol. 2007 Dec;72(6):1637-46
references.pmid: 17875604
references.citation: J Biol Chem. 2008 Feb 29;283(9):5486-95
references.pmid: 18166528