Development of monoclonal antibodies to parathyroid hormone-induced resorptive factors from osteoblast-like cells.
Parathyroid hormone (PTH) induces osteoblast-like cells to secrete factors capable of increasing cellular bone resorption; these factors are extremely labile. We have partially isolated them from supernatants of PTH-stimulated ROS 17.2 cells using affinity chromatography. Products eluted from the matrix, carboxymethyl cellulose covalently linked to Cibacron Blue F3GA, (CM-AB) were used as immunogens in mice. Serum from these mice blocked the effect of PTH-stimulated supernatants in a bioassay for bone resorption (the bone rudiment system). Then, hybridomas were produced from spleen cells of these mice. Screening of these hybridoma cultures revealed a consistent blocking effect of supernatants at a dilution of 1:100 in the bioassay. At higher dilution (1:1000), however, fewer culture supernatants blocked this effect in the bioassay. Mixed hybridoma cultures have been cloned and subcloned. Evaluation of the resulting hybridoma supernatants revealed that supernatants from at least three clones were necessary to neutralize the effect of the PTH-induced, osteoblast-produced resorption factors in organ culture. Similarly, using sucrose density gradient analysis, it is necessary to use three antibodies to bind all the 35S-labeled protein from supernatants of PTH-stimulated ROS cultures (equivalent to the fraction used as immunogen in the mice). The effect of PTH in organ culture is blocked by this same group of hybridoma supernatants. The antibodies appear specific for osteoblast-like cell resorption factors, as they do not block the effect of PTH on cAMP accumulation in ROS cell cultures. On the other hand, they do block the effects of dibutyryl cAMP on resorption in organ culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Perry
H M
HM
Geriatric Research, Education, and Clinical Center, VA Medical Center, St. Louis, Missouri 63125.
Gurbani
S
S
D000818:Animals; D000911:Antibodies, Monoclonal; D001681:Biological Assay; D003470:Culture Media; D016207:Cytokines / Q000276:immunology* / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D051381:Rats; D019379:Teriparatide; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D003470:Culture Media; D016207:Cytokines; D010281:Parathyroid Hormone; D010446:Peptide Fragments; C025940:bone resorption factor; D019379:Teriparatide