title: Generation of monoclonal antibodies against peptidylarginine deiminase 2 (PAD2) and development of a PAD2-specific enzyme-linked immunosorbent assay.
abstract: The enzyme peptidylarginine deiminase 2 (PAD2) has been associated with inflammatory diseases, such as rheumatoid arthritis and neurodegenerative diseases including multiple sclerosis. To investigate the association of various diseases with extracellular PAD2, we raised monoclonal antibodies (mAbs) against rabbit PAD2 and evaluated their cross-reactivity with human PAD2 by indirect enzyme-linked immunosorbent assay (ELISA), western blotting and immunohistological staining of inflamed synovial tissue. Moreover, we established a sandwich ELISA detecting human PAD2, based on two different monoclonal antibodies, mAbs DN2 and DN6. The assay had a lower detection limit of 200pg/mL in serum and plasma samples, and showed dilution linearity and recovery ranging from 95 to 106%. The mAbs and the ELISA showed isotype specificity for PAD2. Circulating PAD2 was found in 8/28 (29%) serum samples from healthy donors. In conclusion, several of our mAbs proved useful in western blotting and immunohistochemistry, and the ELISA described here reliably measures PAD2 levels in blood. This allows investigation of PAD2 as a possible biomarker and further investigation of PAD2's involvement in various inflammatory diseases.
authors.affiliation: Institute for Inflammation Research, Department of Infectious Diseases and Rheumatology, Copenhagen University Hospital, Rigshospitalet, Denmark.
authors.forename: Dres
authors.initials: D
authors.lastname: Damgaard
authors.affiliation: Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, Denmark.
authors.forename: Yaseelan
authors.initials: Y
authors.lastname: Palarasah
authors.affiliation: Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, Denmark.
authors.forename: Karsten
authors.initials: K
authors.lastname: Skjødt
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska University Hospital and Karolinska Institutet, Sweden.
authors.forename: Anca I
authors.initials: AI
authors.lastname: Catrina
authors.affiliation: Department of Biomolecular Chemistry, Institute for Molecules and Materials, Nijmegen Center for Molecular Life Sciences and Netherlands Proteomics Centre, Radboud University Nijmegen, Nijmegen, The Netherlands.
authors.forename: Sanne M M
authors.initials: SM
authors.lastname: Hensen
authors.affiliation: Department of Biomolecular Chemistry, Institute for Molecules and Materials, Nijmegen Center for Molecular Life Sciences and Netherlands Proteomics Centre, Radboud University Nijmegen, Nijmegen, The Netherlands.
authors.forename: Ger J M
authors.initials: GJ
authors.lastname: Pruijn
authors.affiliation: Institute for Inflammation Research, Department of Infectious Diseases and Rheumatology, Copenhagen University Hospital, Rigshospitalet, Denmark. Electronic address: claus.henrik.nielsen@rh.regionh.dk.
authors.forename: Claus H
authors.initials: CH
authors.lastname: Nielsen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D001172:Arthritis, Rheumatoid / Q000097:blood / Q000175:diagnosis / Q000276:immunology; D015415:Biomarkers / Q000097:blood; D015153:Blotting, Western / Q000379:methods; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D006867:Hydrolases / Q000097:blood / Q000276:immunology*; D007150:Immunohistochemistry / Q000379:methods; D019636:Neurodegenerative Diseases / Q000097:blood / Q000175:diagnosis / Q000276:immunology; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; D006867:Hydrolases; C000620352:PADI2 protein, human; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases
keywords: Citrullination; ELISA; Monoclonal antibody; PAD2; Peptidylarginine deiminase