Identification of a Family of Insulin-Like Growth Factor II Secreted by Cultured Rat Epithelial-Like Cell Line 18,54-SF: Application of a Monoclonal Antibody

Abstract

Somatomedin/insulin-like growth factor (IGF) like polypeptides (designated SMP) were purified from the serum-free conditioned medium of cultured rat epithelial-like cells, 18,54-SF. A monoclonal antibody (MAb) was produced against partially purified SMP. The antibody was immunoglobulin G1 relatively specific for multiplication-stimulating activity III-2 (rat IGF-II), with a Kd value of 5.6 .times. 10-9 M. The antibody showed 100% cross-reactivity with human IGF-II and 10% cross-reactivity with human IGF-I, but did not cross-react with insulin. For purification of SMP, therefore, immunoaffinity chromatography on Sepharose coupled with the MAb was used besides a procedure including ion exchange chromatography, gel filtration, and reverse phase HPLC. The purified SMP (at least five polypeptides) each gave a single peak on reverse phase HPLC and appeared as a single band with an apparent mol wt of 5000-8000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major components of SMP (designated HP1-SMP and HP3-SMP), which were purified about 100-fold from conditioned medium, stimulated DNA synthesis in human fibroblasts in culture and sulfation in chick embryonic cartilage in culture. These polypeptides showed almost the same cross-reactivity as multiplication-stimulating activity III-2 on RIA with the MAb. The partial amino acid sequences of HP1- and HP3- SMP were determined, and these polypeptides were identified with rat IGF-II.