Binding of TNFs with OPG

Objective: Binding of Osteoprotegerin（OPG） to TNF-family ligands was studied. Methods: 1. Immunoprecipitaion with Western Blot, 2. ELISA, 3. Carbodiimide cross-linking, 4. Surface plasmon resonance. Results: This study showed the similar binding of OPG to TRAIL, TNFα, and RANKL. OPG binding to TNFα was confirmed by surface plasmon resonance. At pH 7.4 and 20℃, steady state binding had a Kd of 7.0 nmol/L, similar to OPG-TRAIL affinity. Binding of OPG to RANKL reached steady state slowly at 20℃, increased several folds with temperature from 4 to 40℃, and was sensitive to surfactants. Carbodiimide crosslinking produced OPG-TNFα complexes consistent with 1:1 OPG-TNFα binding. Conclusions: In human bone cells, multiple TNF-family proteins, including TNFα, are regulated by OPG, but physical conditions may affect functional binding.