Development of monoclonal antibodies to parathyroid hormone-induced resorptive factors from osteoblast-like cells

Summary

Parathyroid hormone (PTH) induces osteoblastlike 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 dibutyral cAMP on resorption in organ culture. The effect of antibodies on bone resorption is not nonspecific, as hybridomas have no effect on increased ⁴⁵Ca release from organ culture induced by thyroxine. We conclude that we have isolated hybridomas that produce antibodies capable of binding to PTH-induced, osteoblast-produced factors. These antibodies are able to inhibit the increase in cellular bone resorption in organ cultures caused to either PTH or these factors.