Investigation of osteoprotegerin interactions with ligands and antibodies using piezoelectric biosensors

Osteoprotegerin (OPG, osteoclastogenesis inhibitory factor) is a secretory glycoprotein involved as a soluble factor in the regulation of bone mass. OPG and its ligand (RANKL) levels in serum indicate the osteoclast formation activity. Alterations of the RANKL/OPG concentration ratio may be the cause of bone loss in many imbalances including osteoporosis, hypercalcaemia, metastatic osteolytic lesions and rheumatic bone degradation. The interactions of OPG with several antibodies were studied using the piezoelectric quartz crystal sensor. Monoclonal anti-OPG antibodies (5H3, 4E6H9 and OPG1.3) were immobilised on the sensing surface modified with covalently attached monolayer of protein A. Binding of both OPG standard and recombinant OPGFc chimeric protein was followed in real time. All antibodies were able to bind OPG and OPGFc, though in the case of MAb 4E6H9 the immunocomplexes dissociated quickly in the absence of OPG. Alternatively, biorecognition layers with RANKL were used. Two versions of the piezoelectric sensor for OPG were developed. The direct immunosensor was based on the antibody 5H3 and the affinity sensor employed the immobilised RANKL. The RANKL sensor exhibited poor reproducibility of results. For the immunosensor, the measuring range was 1.2-35 U/L of OPG. One analysis was completed within 15 min; the sensors were used repeatedly using regeneration with glycine buffer (pH 2.0). The developed immunosensor seems promising for rapid determination of osteoprotegerin in serum.