Heng-Chuan XIA, Wei-Guo HU, Xin-Xiu YANG, Feng LI, and Zu-Chuan ZHANG*
( Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
the Chinese Academy of Sciences, Shanghai 200031, China )

Abstract Plant ribosome-inactivating proteins (RIPs) have multiple biological functions, and have been widely used in the studies on biomedical and agronomic applications. Moschatin is a novel single-chain RIP recently purified from pumpkin seeds, and it has been successfully applied to construct the immunotoxin that can selectively kill the cultured human melanoma cells. Six stable strains of hybridomas (2H8, 4A8, 5B6, 6F8, 4H10 and 6C2) that can secrete high specific monoclonal antibodies against Moschatin have been successfully prepared using hybridoma technique. The isotypes of these monoclonal antibodies are IgG₁, IgG₁, IgG₁, IgG₁, IgG_2a and IgG_M. Their affinity constants were determined to be 1.42×10⁸, 2.71×10⁸, 8.72×10⁷, 2.06×10⁸, 1.36×10⁸ and 1.51×10⁸ M^-1 in a sequent order, measured by non-competitive ELISA. The monoclonal antibody 4A8 has been used to detect Moschatin in Western blot. An immunoaffinity gel, which consisted of a monoclonal antibody 4H10 and Sepharose 4B, was prepared and used to purify Moschatin from pumpkin seeds crude extract.

Key words Moschatin; monoclonal antibody; affinity chromatography; Western blot

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Received: October 15, 2003 Accepted: November 18, 2003
This work was supported by a grant from Knowledge Innovation Program of the Chinese Academy of Sciences
*Corresponding author: Tel, 86-21-54921281; Fax, 86-21-54921011; E-mail, zhangzc@sunm.shcnc.ac.cn