Development of microfluidic-based assays to estimate the binding between osteocalcin (BGLAP) and fluorescent antibodies

Osteocalcin (bone gamma-carboxyglutamate protein; BGLAP) is a highly conserved molecule associated with mineralization of bone matrix. It regulates the dynamics of new bone formation and bone resorption. The synthesis of osteocalcin by osteoblasts is regulated by the active form of vitamin D. In this paper, we report the use of two fluorescent-based assays, one bead-based and a second involving an amino-silane surface pre-treatment, to obtain binding constants of 3.53×106 M-1 and 3.19×106 M-1, respectively, for the binding of BGLAP to fluorescently labeled monoclonal-anti BGLAP-clone 2D5. These simple microfluidic techniques demonstrate the feasibility of developing microsphere-based and surface electrostatically-attached binding assays to study peptide-antibody interactions. Both techniques utilize sub-microliter volumes of material an important consideration when studying bone turnover markers (BTMs) like BGLAP.