(03406) Preparation and Primary Application of Monoclonal Antibodies against a Novel Ribosome-inactivating Protein Moschatin from Pumpkin Seeds

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, [email protected]