DEVELOPMENT AND APPLICATION OF AN ENZYME-IMMUNOASSAY FOR KARASURIN-A,AN EFFECTIVE PROTEIN-COMPONENT OF TRICHOSANTHES-KIRILOWII MAX VAR JAPONICUM KITAM

A method was developed for specific estimation of the content of a non -enzymatic protein, karasurin A, in fractions taken during the extract ion and purification processes from a natural source. Anti-karokon ser um was elicited in rabbits immunized with fragments of karokon, a drie d root tuber of Trichosanthes kirilowii MAX. var. japonicum KITAM. Rab bit antibody specific for karasurin A was identified in anti-karokon s erum by the Western blotting method. After separation by SDS-PAGE, pro tein bands of purified karasurin A and extracted proteins from a medic inal herb which is a karasurin A source were reacted with anti-karokon serum followed by treatment with horseradish peroxidase (HRP)-labeled Fab' of goat anti-rabbit IgG, and then bound HRP-labeled second antib ody on protein bands was developed to brown by reaction with a substra te solution of the used enzyme. A novel selected antibody enzyme immun oassay (SAEIA) for karasurin A was developed using selective binding o f anti-karasurin A antibody in anti-karokon serum to solid phase karas urin A and HRP-labeled Fab' of the second antibody as the tracer. Spec ific estimation of the content of karasurin A in several fractions tak en during the isolation and purification processes of the protein were possible using the SAEIA method.