Monoclonal antibodies to the cell surface and a soluble form of the human nerve growth factor receptor.
Monoclonal antibodies (designated IIIG5, VIID1, VIIIC8, and XIF1) have been produced that bind to the human nerve growth factor receptor (NGF-R) as well as to a soluble, truncated form of the receptor (NGF-Rt). The antibodies were generated against partially purified NGF-Rt from the conditioned medium of E9b cells, a transfected mouse fibroblast cell line (Ltk-) that expresses large numbers of the low affinity form of the human NGF-R on its cell surface (Chao MV, Bothwell MA, Ross AH, Koprowski H, Lanahan AA, Buck CR, Sehgal A [1986]: Science 232:518-521). Hybridomas were screened by radiometric immunosorbent assay (RISA) and by immunoprecipitation of solubilized cell surface receptor covalently cross-linked to [125-I]-NGF. Four positive lines were cloned by limiting dilution and were found to secrete monoclonal antibodies of the IgGl,k subclass. All monoclonal antibodies bound to both NGF-R and NGF-Rt. Two monoclonal antibodies (VIID1, XIF1) immunoblotted the NGF-R from E9b cell preparations resolved on non-reducing sodium dodecyl sulfate (SDS)-polyacrylamide gels. The antibodies immunoprecipitated NGF-R from both E9b cells and from SH-SY5Y human neuroblastoma cells. The monoclonal antibodies bound to monkey (rhesis and cynomolgus) NGF-Rt, but did not cross-react with NGF-R from chick or rat. Results of antibody competition studies demonstrated that three antibodies bound to a similar or overlapping epitope on the NGF-Rt and one monoclonal antibody (IIIG5) recognized a distinct receptor epitope. Antibodies that bound to different sites on the receptor were used to develop a sensitive 2-site RISA. The 2-site RISA can be used to rapidly quantitate NGF-R and NGF-Rt in large numbers of biological samples in the absence of added [125-I]-labeled NGF.
Clagett-Dame
M
M
School of Pharmacy, University of Wisconsin-Madison.
Chung
C
C
Chao
M V
MV
DiStefano
P S
PS
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001667:Binding, Competitive / Q000276:immunology; D002459:Cell Fusion; D003429:Cross Reactions; D000939:Epitopes; D006801:Humans; D015151:Immunoblotting; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface / Q000276:immunology*; D017475:Receptors, Nerve Growth Factor; D012711:Serum Albumin, Radio-Iodinated
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor; D012711:Serum Albumin, Radio-Iodinated