Production of monoclonal and polyclonal antibodies against human osteocalcin sequences and development of a two-site ELISA for intact human osteocalcin

In this study we have raised polyclonal and monoclonal Antibodies against human osteocalcin sequences coupled to bovine serum albumin (BSA). The Antibodies were used for the development of a novel two-site ELISA on microtiter plates for intact human osteocalcin. The epitope, recognized by the monoclonal hybridoma product Os 31/2 that was used as the capture antibody, was located on the amino terminal sequence 1-29. The Antibodies used for detection recognized the sequence 1-49. This method failed to detect any tryptic Peptides derived from human osteocalcin sequence 1-49 nor did it detect peptide 1-29, thus demonstrating high specificity for the intact osteocalcin 1-49 only. This is a novel technique for the determination of intact osteocalcin. We have also validated the assay parameters for routine use. This direct and relatively simple procedure promises to be a powerful diagnostic and investigative tool.