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 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.
Kitagawa
T
T
Faculty of Pharmaceutical Sciences, Nagasaki University, Japan.
Yosida
M
M
Hu
J
J
Sakai
A
A
Bay
G
G
Fujiwara
K
K
Ogihara
Y
Y
Takeda
T
T
D000818:Animals; D004365:Drugs, Chinese Herbal / Q000032:analysis*; D007124:Immunoenzyme Techniques; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000032:analysis* / Q000276:immunology; D011817:Rabbits / Q000276:immunology
D004365:Drugs, Chinese Herbal; D010940:Plant Proteins; C069870:karasurin protein, Trichosanthes kirilowii; D009699:N-Glycosyl Hydrolases