ABAP: antibody-based assay for peptidylarginine deiminase activity.
Members of the family of peptidylarginine deiminases (PADs, EC 3.5.3.15) catalyze the posttranslational modification of peptidylarginine into peptidylcitrulline. Citrulline-containing epitopes have been shown to be major and specific targets of autoantibodies produced by rheumatoid arthritis patients. Recently, the citrullination of histone proteins by PAD enzyme was reported to influence gene expression levels. These findings greatly increase the interest in the PAD enzymes and their activities. A few procedures to monitor PAD activity in biological samples have been described previously. However, these assays either have low sensitivity or are rather laborious. Here we describe a reliable and reproducible method for the determination of PAD activity in both purified and crude samples. The method is based on the quantification of PAD-dependent citrullination of peptides, immobilized in microtiter plates, using antibodies that are exclusively reactive with the reaction product(s). Our results demonstrate that this antibody-based assay for PAD activity, called ABAP, is very sensitive and can be applied to monitor PAD activity in biological samples.
Zendman
Albert J W
AJ
Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen NL-6500 HB, The Netherlands.
Raijmakers
Reinout
R
Nijenhuis
Suzanne
S
Vossenaar
Erik R
ER
Tillaart
Marloes van den
Mv
Chirivi
Renato G S
RG
Raats
Jos M H
JM
van Venrooij
Walther J
WJ
Drijfhout
Jan W
JW
Pruijn
Ger J M
GJ
D000918:Antibody Specificity; D001172:Arthritis, Rheumatoid / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D001323:Autoantibodies / Q000378:metabolism*; D015153:Blotting, Western; D002384:Catalysis; D002956:Citrulline / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006657:Histones / Q000378:metabolism; D006801:Humans; D006867:Hydrolases / Q000032:analysis / Q000378:metabolism*; D007124:Immunoenzyme Techniques / Q000379:methods*; D011499:Protein Processing, Post-Translational; D000076342:Protein-Arginine Deiminases; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001323:Autoantibodies; D006657:Histones; D002956:Citrulline; D006867:Hydrolases; D000076342:Protein-Arginine Deiminases