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A number of osteocalcin antisera recognize epitopes on proteins other than osteocalcin in cultured skin fibroblasts: Implications for the identification of cells of the osteoblastic lineage in vitro
Author(s) -
Bradbeer J.N.,
Virdi A.S.,
Serre C.M.,
Beresford J.N.,
Delmas P.D.,
Reeve J.,
Triffitt J.T.
Publication year - 1994
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1002/jbmr.5650090812
Subject(s) - osteocalcin , antiserum , polyclonal antibodies , staining , biology , osteoblast , microbiology and biotechnology , in vitro , western blot , antibody , endocrinology , medicine , chemistry , immunology , alkaline phosphatase , biochemistry , genetics , gene , enzyme
Rabbit antisera to bovine osteocalcin were produced independently in two laboratories and their specificities established by western blot analysis. By immunohistochemistry each of the five polyclonal antisera produced an intense cytoplasmic staining in human bone‐derived cells. Staining intensity was strongly attenuated by preabsorption of the antisera with osteocalcin. No staining was observed using nonimmune rabbit serum. However, the choice of skin cells as negative controls for osteocalcin synthesis yielded an unexpected positive staining pattern similar to that seen with the bone‐derived cells over a range of antiserum dilutions. This was not caused by the uptake of exogenous osteocalcin from the culture medium because a similar pattern of staining was observed when medium was supplemented with osteocalcin‐depleted fetal calf serum. Treatment with 1,25‐dihydroxyvitamin D 3 induced osteocalcin mRNA expression and osteocalcin secretion in cultures of bone‐derived cells but not in skin fibroblasts. The results demonstrate that these polyclonal antisera also recognize epitopes shared with other proteins synthesized in culture by skin fibroblasts. Furthermore, three mouse monoclonal antibodies to distinct regions of the osteocalcin molecule show differential staining of human bone‐derived cells, skin cells, and osteosarcoma cells (MG63). These observations indicate that the shared epitope resides in the central region of osteocalcin and are consistent with the specific synthesis of osteocalcin by bone cells alone. The observed nonspecificity of many osteocalcin antisera may compromise immunocytochemical studies of the osteoblast phenotype in studies in vitro when based solely on reactivity with inadequately characterized osteocalcin antisera.