Monoclonal antibodies to the cell surface and a soluble form of the human nerve growth factor receptor

✍ Scribed by M. Clagett-Dame; C. Chung; M. V. Chao; P. S. DiStefano

• Publisher: John Wiley and Sons
• Year: 1990
• Tongue: English
• Weight: 921 KB
• Volume: 27
• Category: Article
• ISSN: 0360-4012
• DOI: 10.1002/jnr.490270425

✦ Synopsis

Abstract

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 (L__tk__^−^) 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 A A, 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 IgG~l, k~ subclass. All monoclonal antibodies bound to both NGF‐R and NGF‐Rt. Two monoclonal antibodies (VIIDl, 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.