Preparation and partial characterization of monoclonal antibodies specific for the nascent non-triple helical form of the type IV collagen alpha 1 chain

Makoto Morita; Hidemitsu Sugihara; Kazuhiro Tokunaka; Arihiro Tomura; Kan Saiga; Takamichi Sato; Yasutada Imamura; Toshihiko Hayashi

doi:10.1016/j.bbrep.2016.11.013

Preparation and partial characterization of monoclonal antibodies specific for the nascent non-triple helical form of the type IV collagen alpha 1 chain

Biochem Biophys Rep. 2016 Dec 8:9:128-132. doi: 10.1016/j.bbrep.2016.11.013. eCollection 2017 Mar.

Authors

Makoto Morita¹, Hidemitsu Sugihara¹, Kazuhiro Tokunaka¹, Arihiro Tomura¹, Kan Saiga¹, Takamichi Sato¹, Yasutada Imamura², Toshihiko Hayashi³

Affiliations

¹ Pharmaceutical Research Laboratories, Nippon Kayaku Co., Ltd., Tokyo 115-8588, Japan.
² Department of Chemistry and Life Science, School of Advanced Engineering, Kogakuin University, Hachioji, Tokyo 192-0015, Japan.
³ China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China.

Abstract

This report describes the preparation and partial characterization of monoclonal antibodies that are reactive specifically with the nascently produced non-triple helical form of the type IV collagen α1 chain, designated as NTH α1(IV). These antibodies were nonreactive with the α1 chain of the type IV collagen in the triple-helical conformation. Three antibodies, #141, #179 and #370, with different epitopes in NTH α1(IV) were found to be reactive with the nascent polypeptide secreted from human normal cells and a human carcinoma cell line. The antibodies with different epitopes may provide a key method for elucidating the physiological function and tissue distribution of NTH α1(IV), which is distinct from the chain derived from triple-helical type IV collagen.

Keywords: 2ME, 2-mercaptoethanol; Antibody; Basement membrane; CNT, control; FCS, Fetal Calf Serum; Hydroxyproline; IP, immunoprecipitation; NTH α1(IV), non-triple helical form of the type IV collagen α1 chain; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; Triple-helical conformation; Type IV collagen; WB, western blotting.