PRODUCTION AND CHARACTERIZATION OF AN ANTIBODY AGAINST THE HUMAN BONEGLA PROTEIN (BGP OSTEOCALCIN) PROPEPTIDE AND ITS USE IN IMMUNOCYTOCHEMISTRY OF BONE-CELLS/

We have generated and characterized an antibody that recognizes the C- terminal sequence of the propeptide of human bone GLA protein (BGP/ost eocalcin) (amino acid -26 to -1, with +1 being the amino terminus of t he mature protein). The range of sensitivity of the antibody, as deter mined by enzyme-linked immunosorbent assay (ELISA), was 0.5-250 ng/ml. The antibody effectively recognized pro-BGP in cell layer extracts of transformed cells (KT-005), but did not recognize mature, propetide-l ess BGP in the medium from the same cultures. Strong labelling was obt ained using this antibody in immunoperoxidase staining or immunofluore scence of both transformed and normal human bone cells in vitro. Monen sin significantly altered the intracellular pattern of labelling in im munofluorescence studies, indicating that the recognized antigen was a ssociated with the cellular secretory pathway. We also obtained a spec ific and strong staining of cells in tissue sections of human fetal bo ne. Antibodies against the mature protein strongly stained the mineral ization front, but did not stain cells to any appreciable level. Newly embedded osteocytes were the predominant cell type stained in such ma terial, suggesting that they may represent the major sourer of BGP in the intact tissue. These observations indicate that BGP synthesis is a late event in osteoblastic development and that antibodies generated against the propeptide sequence are a potentially powerful tool in the analysis of bone tumors and evaluation of osteoblastic differentiatio n.