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

Hector Carmona; Hector Valadez; Yeoheung Yun; Jagannathan Sankar; Lissette Estala; Frank A Gomez

doi:10.1016/j.talanta.2014.10.025

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

Talanta. 2015 Jan:132:676-9. doi: 10.1016/j.talanta.2014.10.025. Epub 2014 Oct 22.

Authors

Hector Carmona¹, Hector Valadez¹, Yeoheung Yun², Jagannathan Sankar², Lissette Estala¹, Frank A Gomez³

Affiliations

¹ Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032-8202, USA.
² Department of Bioengineering, North Carolina Agricultural & Technical State University, 1601 E. Market Street, Greensboro, NC 27411, United States.
³ 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.

PMID: 25476363
DOI: 10.1016/j.talanta.2014.10.025

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.

Keywords: Binding assay; Fluorescent antibodies; Osteocalcin (BGLAP).

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Antibodies, Monoclonal / chemistry*
Fluoresceins
Fluorescent Dyes
Humans
Kinetics
Microfluidic Analytical Techniques*
Osteocalcin / chemistry*
Peptides / chemistry*
Protein Binding
Recombinant Proteins / chemistry
Silanes

Substances

Antibodies, Monoclonal
Fluoresceins
Fluorescent Dyes
Peptides
Recombinant Proteins
Silanes
Osteocalcin
4-carboxyfluorescein