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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.

Development of an Assay System for Saikosaponin a Using Anti-saikosaponin a Monoclonal Antibodies