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Clinical and Vaccine Immunology, April 2007, p. 335-341, Vol. 14, No. 4
1071-412X/07/$08.00+0     doi:10.1128/CVI.00155-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Antibodies against C-Reactive Protein Cross-React with 60-Kilodalton Heat Shock Proteins

Katalin Udvarnoki,¹ László Cervenak,² Katalin Uray,³ Ferenc Hudecz,^3,4 Imre Kacskovics,^5, Ralf Spallek,^6,7 Mahavir Singh,^6,7 George Füst,^1,2 and Zoltán Prohászka^1,2*

Third Department of Medicine, Faculty of Medicine, Semmelweis University, Budapest, Hungary,¹ Research Group of Inflammation Biology and Immunogenomics, Hungarian Academy of Sciences, Budapest, Hungary,² Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Budapest, Hungary,³ Department of Organic Chemistry, L. Eötvös University, Budapest, Hungary,⁴ Department of Physiology and Biochemistry, Faculty of Veterinary Science, Szent István University, Budapest, Hungary,⁵ Department of Genome Analysis, Helmholtz Center for Infection Research,⁶ Lionex, Ltd., Braunschweig, Germany⁷

Received 25 April 2006/ Returned for modification 25 July 2006/ Accepted 31 January 2007

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.

Published ahead of print on 14 February 2007.

Present address: Department of Immunology, Institute of Biology, L. Eötvos University, Budapest, Hungary.