Generation of monoclonal antibodies against peptidylarginine deiminase 2 (PAD2) and development of a PAD2-specific enzyme-linked immunosorbent assay

TY - JOUR

T1 - Generation of monoclonal antibodies against peptidylarginine deiminase 2 (PAD2) and development of a PAD2-specific enzyme-linked immunosorbent assay

AU - Damgaard, Dres

AU - Palarasah, Yaseelan

AU - Skjødt, Karsten

AU - Catrina, Anca I

AU - Hensen, Sanne M M

AU - Pruijn, Ger J M

AU - Nielsen, Claus H

N1 - Copyright © 2013 Elsevier B.V. All rights reserved.

PY - 2014/3

Y1 - 2014/3

N2 - 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.

AB - 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.

KW - Animals

KW - Antibodies, Monoclonal

KW - Antibody Specificity

KW - Arthritis, Rheumatoid

KW - Biological Markers

KW - Blotting, Western

KW - Cross Reactions

KW - Enzyme-Linked Immunosorbent Assay

KW - Humans

KW - Hydrolases

KW - Immunohistochemistry

KW - Neurodegenerative Diseases

KW - Rabbits

KW - Reproducibility of Results

KW - Sensitivity and Specificity

KW - Species Specificity

U2 - 10.1016/j.jim.2013.12.008

DO - 10.1016/j.jim.2013.12.008

M3 - Journal article

C2 - 24384061

SN - 0022-1759

VL - 405

SP - 15

EP - 22

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

ER -