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 timeof-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-aminopterinthymidine-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 crossreactivities were observed with other related steroidal glycosides. The full range of the assay extends 26 ng/ml to 1.5 m mg/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.

Quality Control of Bupleurum Species by Newly Established Eastern Blotting

“…Immunizations, hybridizations and purifications of MAbs against SSa (1G6) and saikosaponins (3G10) and their developments of ELISA system for determination of SSa and total saikosaponins have been established (Zhu, 2004. Cross-reactivities are the most important factor in phytochemical investigations in which there are many structurally related compounds (see Table 1).…”

“…In addition, because Kampo medicines are manufactured as highly standardized ethical drugs in Japan, a method of comprehensive analysis of the compounds for scientific quality control is urgently needed. Recently we prepared a specific MAb against SSa (Zhu et al, 2004) and a MAb having wide cross-reactivity to saikosaponins , and developed an enzyme-linked immunosorbent assay (ELISA) system for quantitative measurement of SSa and/or total saikosaponins. However, it is difficult to gain the each content of SSa, SSb 2 , SSc and SSd by these competitive ELISA methods.…”

Pharmacologically Active Saikosaponin inBupleurum falcatumDetected by Competitive ELISA and Eastern Blotting Using Monoclonal Antibodies

“…We prepared anti-SSa MAb 1G6 having higher specificity against SSa, and established competitive ELISA for the determination of SSa concentration in Bupleuri radix and traditional Chinese medicines (Zhu et al, 2004). Fig.…”

“…Competitive ELISA using two kinds of monoclonal antibodies (MAbs), i.e. monospecifi c to saikosaponin a (SSa) and multispecifi c to saikosaponins, have also been developed and their reliability and accuracy evaluated for the quality control of Bupleuri radix (Zhu et al, 2004(Zhu et al, , 2006. Recently, competitive ELISA and eastern blotting using MAbs have been developed and used in breeding of Panax ginseng , Glycyrrhiza uralensis (Shan et al, 2001), Artemisia annua and Bacopa monnieri (Phrompittayarata et al, 2007) at a very young growth stage of these plants.…”

生薬学研究40年

“…薬用人参の ginsenoside Rb1, 27) Rg1, 28) Re， 29) 甘草の glycyrrhizin， 30) サフランの crocin， 31) 大 黄 の sennoside A ， 32) B ， 33) 柴 胡 の saikosaponin a， 34) berberine， 35) 芍薬の paeoni‰orin 36 IU/l と有意な低下が認められた（ p＜0.05） ． …”