Preparation and use of a monoclonal antibody against luffin b

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 x 10(9) mol(-1) . L and 7.5 x 10(8) 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.