Journal Article

Development and validation of novel biomarker assays for osteoarthritis

Kirwan, John, Blom, Ashley, Ourradi, Khadija, de Seny, Dominique, Sharif, Mohammed, Xu, Yunhe

2017

Overview

Osteoarthritis (OA) is the most common chronic joint disease usually diagnosed at relatively advanced stages when there is irreparable damage to the joint(s). Recently, we have identified two novel biomarkers C3f and V65 which appear to be OA-specific and therefore potential markers of early disease. We report the development of immunoassays for quantitative measure of these two novel biomarkers. Monoclonal and polyclonal antibodies were generated by immunising mouse and rabbits respectively with peptide-carrier conjugates of C3f and V65. Affinity purified antibodies were used for immunoassays development and assays validated using serum from OA patients and controls. The ELISAs developed showed spiked recovery of up to 96% for C3f and V65 peptides depending on serum dilutions with a coefficient of variation (CV) <10%. The intra- and inter-assay CVs for C3f and V65 were 1.3-10.8% and 4.2-10.3% respectively. Both assays were insensitive for measurements of the peptides in patients and the use of different signal amplification systems did not increase assay sensitivity. We have developed two immunoassays for measurements of C3f and V65 peptides biomarkers discovered by our earlier proteomic study. These assays could detect the endogenous peptides in serum samples from patients and controls but lacked sensitivity for accurate measurements of the peptides in patients. Our study highlights the difficulties and challenges of validating biomarker from proteomic studies and demonstrates how to overcome some of the technical challenges associated with developing immunoassays for small peptides.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Animals / Antibodies / Antibody Formation / Arthritis / Assaying / Biocompatibility / Biological markers / Biology and Life Sciences / Biomarkers / Biomarkers - blood / Blotting, Western / Coefficient of variation / Complement C3b - analysis / Damage detection / Diagnosis / Dilution / Disease / Enzyme-Linked Immunosorbent Assay - methods / Genetic aspects / Human health sciences / Humans / Immunoassay / Immunoassays / Mass spectrometry / Medicine and Health Sciences / Metabolism / Mice / Monoclonal antibodies / Osteoarthritis / Osteoarthritis - blood / Osteoarthritis - diagnosis / Osteoarthritis - immunology / Osteoarthritis/blood/diagnosis/immunology / Patients / Peptide Fragments - blood / Peptides / Physiological aspects / Polyclonal antibodies / Proteins / Rabbits / Research and Analysis Methods / Rheumatology / Rhumatologie / Sciences de la santé humaine / Scientific imaging / Sensitivity / Sensitivity and Specificity / Vitronectin - blood