title: Development of microfluidic-based assays to estimate the binding between osteocalcin (BGLAP) and fluorescent antibodies.
abstract: 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 3.53×10(6) M(-1) and 3.19×10(6) 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.
authors.affiliation: Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032-8202, USA.
authors.forename: Hector
authors.initials: H
authors.lastname: Carmona
authors.affiliation: Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032-8202, USA.
authors.forename: Hector
authors.initials: H
authors.lastname: Valadez
authors.affiliation: Department of Bioengineering, North Carolina Agricultural & Technical State University, 1601 E. Market Street, Greensboro, NC 27411, United States.
authors.forename: Yeoheung
authors.initials: Y
authors.lastname: Yun
authors.affiliation: Department of Bioengineering, North Carolina Agricultural & Technical State University, 1601 E. Market Street, Greensboro, NC 27411, United States.
authors.forename: Jagannathan
authors.initials: J
authors.lastname: Sankar
authors.affiliation: Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032-8202, USA.
authors.forename: Lissette
authors.initials: L
authors.lastname: Estala
authors.affiliation: Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032-8202, USA. Electronic address: fgomez2@calstatela.edu.
authors.forename: Frank A
authors.initials: FA
authors.lastname: Gomez
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D046210:Microfluidic Analytical Techniques; D015675:Osteocalcin / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D012821:Silanes
chemical_list: D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010455:Peptides; D011994:Recombinant Proteins; D012821:Silanes; D015675:Osteocalcin; C045132:4-carboxyfluorescein
keywords: Binding assay; Fluorescent antibodies; Osteocalcin (BGLAP)