title: Antibodies against C-reactive protein cross-react with 60-kilodalton heat shock proteins.
abstract: C-reactive protein (CRP) is an acute-phase reactant frequently used in histochemistry as a marker of ongoing inflammation. Furthermore, CRP is a powerful biomarker for the prediction of coronary artery disease risk. Heat-shock protein 60 (Hsp60) and CRP are complement-activating molecules, and the effect of their interactions on the regulation of complement activation was studied. However, during the first experiments, we learned that polyclonal anti-CRP antibodies cross-react with Hsp60. Therefore, the aim of our present study was to analyze the cross-reactivity of anti-CRP antibodies (Ab) with Hsp60 in solid-phase enzyme immune assays, in epitope studies using a series of overlapping synthetic peptides, and in Ouchterlony analyses. We found that three different commercial rabbit polyclonal antibodies and two monoclonal (9C9 and CRP-8) anti-CRP antibodies specifically recognize recombinant human Hsp60 and recombinant Mycobacterium tuberculosis Hsp65, respectively. Hsp60 was found to inhibit the binding of anti-CRP polyclonal Ab to Hsp60. Six epitope regions of Hsp60 were recognized by the anti-CRP antibodies, and one region (amino acids [AA] 218 to 232) was recognized by monoclonal antibodies CRP-8 and 9C9. This epitope region of Hsp60 displays 26.6% amino acid identity to CRP AA region 77 to 90. These data suggest that the B-cell epitopes shared between CRP and Hsp60 give rise to a true mimicry-based cross-reaction and the induction of cross-reactive antibodies. Our study underlines the importance of thorough study design and careful interpretation of results while using polyclonal anti-CRP antibodies for histochemistry, especially at low dilutions. Furthermore, analytical interference with Hsp60 in CRP assays should also be tested.
authors.affiliation: Third Department of Medicine, Semmelweis University, H-1125 Budapest, Kútvölgyi st. 4, Hungary.
authors.forename: Katalin
authors.initials: K
authors.lastname: Udvarnoki
authors.forename: László
authors.initials: L
authors.lastname: Cervenak
authors.forename: Katalin
authors.initials: K
authors.lastname: Uray
authors.forename: Ferenc
authors.initials: F
authors.lastname: Hudecz
authors.forename: Imre
authors.initials: I
authors.lastname: Kacskovics
authors.forename: Ralf
authors.initials: R
authors.lastname: Spallek
authors.forename: Mahavir
authors.initials: M
authors.lastname: Singh
authors.forename: George
authors.initials: G
authors.lastname: Füst
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Prohászka
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D001426:Bacterial Proteins / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D002097:C-Reactive Protein / Q000276:immunology* / Q000378:metabolism; D018834:Chaperonin 60 / Q000276:immunology* / Q000378:metabolism*; D018833:Chaperonins / Q000276:immunology / Q000378:metabolism; D003429:Cross Reactions; D006801:Humans; D051379:Mice; D009169:Mycobacterium tuberculosis / Q000276:immunology; D011817:Rabbits
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; D018834:Chaperonin 60; C074119:heat-shock protein 65, Mycobacterium; D002097:C-Reactive Protein; D018833:Chaperonins