Immuno-PCR Assay for Homodimeric Osteoprotegerin

Abstract

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor is a recently cloned cytokine that specifically inhibits differentiation and maturation of osteoclasts (1)(2)(3)(4). OPG is present as a monomer (60 kDa) and a homodimer (120 kDa) in conditioned media from human fibroblasts and from CHO cells producing human OPG (3)(5). Notably, the homodimeric form of recombinant OPG exerted more potent biologic activity than the monomeric form in vivo (5)(6)(7). Which form of OPG predominates in human serum has not been definitively determined. Determining the amount of each form of OPG in patient serum may be useful in the diagnosis and treatment of metabolic bone diseases associated with abnormal osteoclast recruitment and function, such as osteopetrosis, osteoporosis, metastatic bone disease, Paget disease, rheumatoid arthritis, and periodontal disease. Yano et al. (8) have described ELISAs with tetramethylbenzidine for total OPG and the homodimeric form of OPG (detection range for both ELISAs, 65–500 ng/L). The homodimeric form of OPG, however, cannot be detected in human serum by conventional ELISAs because the concentrations are below the detection limit. Additionally, the monomeric form of OPG cannot be measured directly at the present time because no specific antibody for the monomeric form is available. We therefore devised a highly sensitive immuno-PCR assay to measure the homodimeric form of OPG in sera from healthy individuals and patients with osteoporosis. Blood samples were obtained from 11 healthy volunteers and 22 patients with osteoporosis. The immuno-PCR assay consisted of the following steps: an ELISA for antigen detection, followed by PCR for signal amplification. The ELISA for human OPG (hOPG) was performed according to a method described previously (8). Monomeric …