Contents lists available at ScienceDirect Clinica Chimica Acta journal homepage: www.elsevier.com/locate/cca Development of an in house ELISA for human intact osteocalcin and its utility in diagnosis and management of osteoporosis Hetal Bhadricha, M.Ikram Khatkhatay, Meena Desai ⁎ Department of Molecular Immunodiagnostics, ICMR-National Institute for Research In Reproductive Health, Jehangir Merwanji Street, Parel, Mumbai, India ARTICLE INFO Keywords: Osteocalcin Bone turnover marker Enzyme-linked immunosorbent assay (ELISA) Diagnosis Management Osteoporosis ABSTRACT Background: Serum Osteocalcin (OC) is a biomarker for evaluating bone turnover in humans. Commercial kits of OC in India are imported, hence the associated high cost prohibits their use in routine screening of osteoporosis. The present study describes the development, validation of human OC ELISA and establishes cut off values for its use in screening and management of women at risk for osteoporosis. Methods: A sandwich OC ELISA was developed using immuno-reagents prepared indigenously and validated for analytical sensitivity, specificity, accuracy and compared with commercial kit using Bland-Altman method. The utility of OC assay was evaluated by ROC analysis. Results: The new ELISA was sufficiently precise, accurate, matrix-free, sensitive and cost effective. The levels of OC were significantly different in women with osteopenia and osteoporosis (ANOVA, p < .0001) compared to women with normal BMD. ROC analysis demonstrated the cut off values of OC > 11.9 ng/mL for osteopenia and > 14.9 ng/mL for osteoporosis. The OC levels had maximum AUC of 0.831 in osteopenia and 0.932 in osteoporosis. Further, OC levels showed significant changes within 3 months in women monitored on therapy. Conclusion: The developed OC ELISA has great potential to be used as a biomarker for routine screening and management of osteoporosis in Indian women. 1. Introduction Osteoporosis is a common metabolic bone disorder characterized by a structural deterioration of bone tissue resulting in an increased risk of fragility fracture [1]. India is one of the leading countries affected by osteoporosis with estimates showing approximately 50 million people as osteoporotic or having low bone mass with one out of three women and one out of five men above the age of 50 years, at risk of osteo- porosis [2,3]. Moreover, it is more prevalent in postmenopausal women and being a silent disease, it is a major concern in India as the pre- valence rate is 35.7% in women over 50 years [4,5]. Currently, bone mineral density (BMD) measurement using dual- energy X-ray absorptiometry (DXA) scan is the gold standard for the diagnosis of osteoporosis [6–8]. However DXA scan has its own lim- itations being a static measure and expensive tool with limited avail- ability in the many parts of India. This necessitates the need for an alternative diagnostic method for screening women at risk with a marker for management and treatment for osteoporosis. In recent times, cellular bone matrix components have been identified and categorised as either bone formation or resorption markers [9,10]. These biomarkers provide the useful information on assessment of osteo- porosis at an earlier stage when the BMD measurement of DXA does not offer enough information to make the diagnosis [11]. Of late, these markers have been documented as tools in the clinical management of bone diseases. Therefore, a combination of BMD measurement by DXA and bone markers show the great potential to improve the early as- sessment of people with the high risk of osteoporosis. Osteocalcin (OC) also known as Bone Gla Protein is a non-col- lagenous, 49 amino acid long single chain protein (MW 5.8 kDa), con- taining three γ-carboxyglutamic acids. It is secreted solely by osteo- blasts and its biosynthesis is vitamin K dependent [12,13]. During bone formation, newly synthesized OC is incorporated into bone matrix, and a small fraction is secreted directly into the circulation, but its function remains elusive. Circulating OC concentrations have been broadly used for evaluating rate of bone turnover in metabolic bone diseases such as hyperparathyroidism, Paget's disease and renal osteodystrophy [14–17]. However its role in age related bone loss in women remains elusive. Several immunoassays for OC have been reported which include Immuno-radiometric assay (IRMA) and enzyme- immunoassays (EIA https://doi.org/10.1016/j.cca.2018.12.007 Received 3 April 2018; Received in revised form 15 October 2018; Accepted 7 December 2018 ⁎ Corresponding author at: Department of Molecular Immunodiagnostics, ICMR-National Institute for Research in Reproductive Health, Jehangir Merwanji Street, Parel, Mumbai 400012, India. E-mail address: mends153@gmail.com (M. Desai). Clinica Chimica Acta 489 (2019) 117–123 Available online 08 December 2018 0009-8981/ © 2018 Published by Elsevier B.V. T