We have attempted to develop monoclonal antibodies against the rat type II (insulin-like growth factor) IGF receptor. Assays that were devised to measure blocking and immunoprecipitating antibodies have been utilized to screen mouse sera and hybridoma supernatants. Mice immunized with cells or membranes containing the rat type II receptor have developed positive titers in their sera. However, when splen cells from these mice have been fused with plasmacytoma cells and the resulting hybridomas screened for the production of receptor antibodies, the incidence of positive responses has been low and the few positive hybridoma supernatants have been weak, either of low titer or low affinity. We are currently using in vitro immunization techniques with the rat type II receptor and switching from rat to human type II receptor as a source of antigen for conventional hybridoma technology. While we previously were unable to demonstrate IGF-dependent phosphorylation of the type II receptor in whole cell labeling experiments with rat embryo fibroblasts, we have been able to demonstrate IGF-dependent phosphorylation in a rat liver cell line (BRL-3A2). One difference between the rat embryo fibroblasts and the rat liver cell line is that rat embryo fibroblasts produce IGF-II, raising the possibility that endogenously produced IGF-II causes phosphorylation of the type II receptor. Earlier we had reported that fetal rat fibroblasts produced IGF-II, but not IGF-I, whereas postnatal fibroblasts produced predominantly IGF-I. We have examined IGF production by human fetal and postnatal fibroblasts. In contrast to rat fibroblasts, the levels of IGF are much lower and there are not dramatic differences between the relative amounts of IGF-I and IGF-II in fetal versus postnatal fibroblasts. Chemical crosslinking experiments demonstrate that the human fibroblasts produce binding proteins analogous to subunits of both the small and large IGF binding proteins in human serum.
