title: A new monoclonal antibody (Cox mAB 31A2) detects VP1 protein of coxsackievirus B3 with high sensitivity and specificity.
abstract: Human enteroviruses, e.g. coxsackieviruses, induce a variety of severe acute and chronic forms of disease, including myocarditis, meningitis and diabetes mellitus type 1. To visualize enterovirus infection with a diagnostic intent, many studies have applied a commercially available antibody (anti-CVB5 VP1, clone 5-D8/1, Dako, Hamburg, Germany) that identifies VP1 of different enteroviral serotypes. Many antibodies, however, have been found to bind non-specifically to proteins of cardiomyocytes and in the interstitial space, resulting in non-specific staining in immunohistochemistry. In this paper we show that the anti-CVB5 VP1 antibody, recognizing VP1 of coxsackieviruses and widely used in diagnostics and research, shows strong cross-reactivity with cellular proteins in the heart (and pancreas) of humans and mice, which calls for a more specific antibody to be used for diagnostic purposes. We observed by Western blot analyses of lysates from human heart tissue samples and HeLa cells two cross-reactive bands when using clone 5-D8/1. Peptide mass fingerprinting (MALDI-TOF) identified these proteins as creatine kinase (B-type) and tubulin, confirming that this mAb detects cellular proteins in addition to viral VP1. In order to overcome the problems of false positive VP1 staining we generated a new highly specific and sensitive monoclonal antibody (Cox mAB 31A2) that recognizes VP1 from CVB3. The new antibody was characterized and was found to function well in immunohistochemistry, immunofluorescence staining, Western blotting, ELISA and FACS analyses.
authors: Institute for Pathology, Department of Molecular Pathology, University Hospital of Tuebingen, Liebermeisterstrasse 8, D-72076, Tuebingen, Germany.
authors: Nicole
authors: N
authors: Ettischer-Schmid
authors: Mediagnost GmbH, D-72770, Reutlingen, Germany.
authors: Andrea
authors: A
authors: Normann
authors: Institute for Pathology, Department of Molecular Pathology, University Hospital of Tuebingen, Liebermeisterstrasse 8, D-72076, Tuebingen, Germany.
authors: Martina
authors: M
authors: Sauter
authors: Institute for Pathology, Department of Molecular Pathology, University Hospital of Tuebingen, Liebermeisterstrasse 8, D-72076, Tuebingen, Germany.|Interfaculty Institute of Biochemistry, University of Tuebingen, D-72076, Tuebingen, Germany.
authors: Lisa
authors: L
authors: Kraft
authors: Interfaculty Institute of Biochemistry, University of Tuebingen, D-72076, Tuebingen, Germany.
authors: Hubert
authors: H
authors: Kalbacher
authors: Institute for Pathology, Department of Molecular Pathology, University Hospital of Tuebingen, Liebermeisterstrasse 8, D-72076, Tuebingen, Germany.
authors: Reinhard
authors: R
authors: Kandolf
authors: Mediagnost GmbH, D-72770, Reutlingen, Germany.
authors: Bertram
authors: B
authors: Flehmig
authors: Institute for Pathology, Department of Molecular Pathology, University Hospital of Tuebingen, Liebermeisterstrasse 8, D-72076, Tuebingen, Germany. karin.klingel@med.uni-tuebingen.de.
authors: Karin
authors: K
authors: Klingel
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D003429:Cross Reactions; D004770:Enterovirus / Q000378:metabolism*; D004769:Enterovirus Infections / Q000821:virology*; D004797:Enzyme-Linked Immunosorbent Assay; D006321:Heart / Q000821:virology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D009205:Myocarditis / Q000821:virology*
chemical_list: D000911:Antibodies, Monoclonal
keywords: Coxsackievirus B3; Diagnostics; Monoclonal antibody; Myocarditis; VP1