A novel PAR2 chemiluminescence immunoassay as a biomarker for arthritic diseases

Purpose: Arthritis is a heterogeneous group of joint diseases, with the most common to be osteoarthritis (OA) and rheumatoid arthritis (RA). Increasing evidence has shown that protease-activated receptor 2 (PAR2) involved in many pathophysiological characteristics these like inflammatory responses, synovial hyperplasia, cartilage destruction, osteophyogenesis pain sensitization. PAR2 G-protein transmembrane coupled expressed different parts joint, lining, bone cartilage. It activated via cleavage its N-terminus by serine proteases (e.g., tryptase), unveiling an tethered ligand which binds leads activation. The aim this study was develop novel blood-based biomarker measuring activation fragment levels investigate association fragments disease. Methods: A monoclonal antibody against neo-epitope generated when cleaved then activated, produced. More specifically, enzyme-linked chemiluminescence immunoassay developed targeting C-terminal sequence released after N-terminal total protein. For optimal assay performance buffers, peptide concentrations, incubations times temperatures were tested. Briefly, protocol described as follows: coater added streptavidin pre-coated plates incubated at 20oC for 30min. Next, washing plates, standards, controls, samples HRP-labeled are 4oC overnight. Chemiluminescence substrate on signal detected reader 3-minute incubation substrate. Assay parameters including specificity, intra- inter-assay variation (CV%) linearity evaluated. selectivity towards epitope examined testing reactivity elongated truncated peptide. specificity investigated vitro human full-length recombinant protein matriptase performed. Biological validation serum 22 young healthy donors compared 23 OA patients 15 RA patients. Results: confirmed showing absence peptides (Figure 1). Inter-intra determined less than 15% (6.39% 5.82% accordingly) acceptable both plasma. Cleavage showed increased pro-fragment non-cleaved control. In serum, significantly higher but not OA, controls 2). Conclusions: high sensitivity good technical performance. donors, while only numeric non-statistical difference observed These data indicate may potential rheumatic diseases.View Large Image Figure ViewerDownload Hi-res image Download (PPT)