Abstract
A method was developed for specific estimation of the content of a non-enzymatic protein, karasurin A, in fractions taken during the extraction and purification processes from a natural source. Anti-karokon serum was elicited in rabbits immunized with fragments of karokon, a dried root tuber of Trichosanthes kirilowii MAX. var. japonicum KITAM. Rabbit antibody specific for karasurin A was identified in anti-karokon serum by the Western blotting method. After separation by SDS-PAGE, protein bands of purified karasurin A and extracted proteins from a medicinal 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 antibody on protein bands was developed to brown by reaction with a substrate solution of the used enzyme. A novel selected antibody enzyme immunoassay (SAEIA) for karasurin A was developed using selective binding of anti-karasurin A antibody in anti-karokon serum to solid phase karasurin A and HRP-labeled Fab' of the second antibody as the tracer. Specific estimation of the content of karasurin A in several fractions taken during the isolation and purification processes of the protein were possible using the SAEIA method.
