title: Development of an assay system for saikosaponin a using anti-saikosaponin a monoclonal antibodies.
abstract: For immunization, saikosaponin a (SSa) was conjugated with bovine serum albumin (BSA). The hapten number in an antigen conjugate was determined to be eleven by matrix-assisted laser adsorption/ionization time-of-flight mass spectrometry (MALDI-TOF Mass). Hybridomas secreting monoclonal antibodies (MAb) against SSa were produced by fusing splenocytes immunized with SSa-BSA conjugate and a hypoxanthine-aminopterin-thymidine-sensitive (HAT) mouse myeloma cell line, P3-X63-Ag8-653. A high specific MAb against SSa was selected from hybridomas using enzyme-linked immunosorbent assay (ELISA) analysis. Weak cross-reactivities occurred with saikosaponin c, b(2) and d, which are stereochemical and/or functional isomers of SSa, but no cross-reactivities were observed with other related steroidal glycosides. The full range of the assay extends 26 ng/ml to 1.5 microg/ml of SSa. Good correlation of SSa concentrations in a crude extract of Bupleuri radix between ELISA and HPLC methods was obtained after hydrolysis of acyl saikosaponins by treatment with a mild alkaline solution. The newly established ELISA has been applied for the quantitative assay of SSa in the Bupleuri radix and the Kampo medicines (TCM) prescribed with Bupleuri radix.
authors.affiliation: Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Shu-Hang
authors.initials: SH
authors.lastname: Zhu
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Shimokawa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Shoyama
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D029972:Bupleurum / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006241:Haptens / Q000737:chemistry; D017403:In Situ Hybridization; D020835:Medicine, Kampo; D051379:Mice; D008807:Mice, Inbred BALB C; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000032:analysis* / Q000276:immunology; D018517:Plant Roots / Q000737:chemistry; D012503:Saponins / Q000032:analysis* / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D006241:Haptens; D012503:Saponins; D009828:Oleanolic Acid; C025759:saikosaponin D