Monoclonal antibodies that recognize antigens in human osteosarcoma cells and normal fetal osteoblasts

Four monoclonal antibodies (HOB-1-4) that react against human osteosarcoma cells (Saos-2) and human fetal osteoblasts in situ were developed by immunizing mice with Saos-2 cells. HOB-1 (IgG1, k) and HOB-2 (IgM, k) stain cytoplasmic antigens in Saos-2 cells, human authentic osteoblasts and occasional cells in liver, spleen and, in the instance of HOB-1, kidney and adrenal gland. On Western blotting of Saos-2 cell lysates, HOB-1 recognizes a single protein species of M(r) 59,000, while HOB-2 reacts with a different species of M(r) 57,000. HOB-3 (IgG1, k) stains a cell membrane associated antigen in Saos-2 cells and osteoblasts in situ. The reaction pattern of this antibody is virtually identical to that seen in alkaline phosphatase (ALP)-positive cells in all organs examined immunohistochemically, except for intestinal epithelium. Both immunoblotting and immunoprecipitation analyses confirm that the antigen detected by HOB-3 is ALP. HOB-4 (IgG1, k) reacts weakly against Saos-2 cells cultured under standard conditions, but strongly following the exposure of the cells to the secretory inhibitor monensin. On frozen section screening, this antibody reacts preferentially with the collagenous extracellular matrix not only of the periosteum but of other tissues and organs as well. The precise identity of the HOB-4 antigen remains to be established. We suggest that these four monoclonal reagents will be useful adjuncts in characterizing the osteoblastic phenotype.