Preparation and Use of a
Monoclonal Antibody against Luffin b
Yan Ming, Yang Xin-Xiu, Zhang Ru-Ping, Ping Bei-Fang, Li
Zhen, Zhang Zu-Chuan*
( Institute of Biochemistry and Cell biology, Shanghai Institutes for
Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031,
China )
Abstract Luffin b is
one of the most toxic single chain plant ribosome inactivating proteins. It has
been successfully used to prepare an immunotoxin against human melanoma cells.
Two strains of hybridomas (1E5 and 2E1) were screened out using cell fusion
technique which steadily secreted monoclonal antibodies against luffin b. These
antibodies specifically reacted with luffin b. The affinity constants of 1E5
and 2E1 monoclonal antibodies were determined to be 1.1×109 mol－1・L and 7.5×108 mol－1・L, by RIA,respectively. An
immunoaffinity gel composed with the 1E5 monoclonal antibody and Sepharose 4B
was prepared. The luffin b was successfully purified by one step immunoaffinity
chromatography from the crude extract of Luffa cylindrica seeds. An
immunoconjugate 1E5-HRP was also prepared and it was successfully used in
Western blotting for detection of recombinant luffin b from E.coli
total proteins.
Key words luffin b；monoclonal antibody；affinity chromatography；Western blot
*Corresponding author：Tel,86-21-64374430-281；Fax,86-21-64338357；e-mail,
[email protected]