Measurement of autoantibodies against osteoprotegerin in adult human serum: development of a novel ELISA assay

Piec, Isabelle ORCID:, Tang, Jonathan ORCID:, Washbourne, Christopher, Greeves, Julie, Jackson, Sarah, Ralston, Stuart, Riches, Philip and Fraser, William (2014) Measurement of autoantibodies against osteoprotegerin in adult human serum: development of a novel ELISA assay. In: American Society of Bone and Mineral Research, 2014-09-11 - 2014-09-15, George R Bush Convention Centre.

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Introduction: In 2009, neutralizing autoantibodies against OPG (α-OPGAb) blocking the inhibitory effect of OPG on RANK signaling pathway were identified in a man with celiac disease associated with severe osteoporosis. Although this finding was not reproduced in thirty patients presenting coeliac disease and low bone mineral density, Hauser et al (2013) recently detected the presence of α-OPGAb in patients presenting Rheumatoid Arthritis, Systemic Lupus Erythematosus, Spondyloarthritis and Osteoporosis. There is a growing focus on OPG autoantibodies as primary cause of high bone turnover in disorders with unknown etiology. Objective: To develop an enzyme linked immunosorbent assay (ELISA) for detection and quantification of α-OPGAb in patient serum samples. Method: A full-length human recombinant OPG is immobilized on a plate to allow capture of the antibodies from the sera. In a two-step reaction, the αOPGAb is detected using a biotinylated antibody and a horseradish peroxidase-labelled streptavidin. Substrate is incubated in a timed reaction and color development measured in a spectrophotometric microtiter plate reader. The concentration of human α-OPGAb in the samples is determined directly from a 4PL-fit standard curve. Results: Intra-assay imprecision was <5% at 274.4 ± 18.8 and 98.5 ± 2.9 ng/mL. Inter-assay imprecision was <20% at 324.2 ± 53.3 and 166.8 ± 30.6 ng/mL. Linear range was 0-500ng/mL. Lower and upper limit of quantification were 3.9 and 500 ng/mL. Cross reactivity was assessed against human sera containing raised thyroid antibody and RANKL to ensure assay specificity. Using the method presented, we established that the adult population would be considered positive with a titer above the cut-off limit (95%) of 68ng/mL. Our preliminary data suggested that 14% of our sample population (n=136) presented elevated α-OPGAb. Conclusion: We presented a novel ELISA assay for the detection and measurement of anti-OPG autoantibodies in human serum. The validated method showed excellent assay characteristics and is suitable for use in research and clinical hospital laboratories. In patients with severe form of osteoporosis, measurement of OPG autoantibodies could help clinicians identify appropriate treatment options for this particular subgroup of patients.

Item Type: Conference or Workshop Item (Poster)
Uncontrolled Keywords: sdg 3 - good health and well-being ,/dk/atira/pure/sustainabledevelopmentgoals/good_health_and_well_being
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
UEA Research Groups: Faculty of Medicine and Health Sciences > Research Groups > Musculoskeletal Medicine
Faculty of Medicine and Health Sciences > Research Centres > Metabolic Health
Depositing User: Pure Connector
Date Deposited: 21 Jan 2015 12:30
Last Modified: 19 Oct 2023 03:59


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