Eastern Blotting and Immunoaffinity Concentration Using Monoclonal Antibody for Ginseng Saponins in the Field of Traditional Chinese Medicines
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- 作者：Hiroyuki Tanaka ; Noriko Fukuda ; Yukihiro Shoyama
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：2007
- 出版时间：May 16, 2007
- 年：2007
- 卷：55
- 期：10
- 页码：3783 - 3787
- 全文大小：270K
- 年卷期：v.55,no.10(May 16, 2007)
- ISSN：1520-5118
文摘
Ginsenosides separated by silica gel TLC blotted to a PVDF membrane that was treated with a NaIO4solution followed by bovine serum albumin (BSA) resulted in a ginsenoside-BSA conjugate on aPVDF membrane. The blotted spots were stained by anti-ginsenoside Rb1 (G-Rb1) and -Rg1 (G-Rg1) monoclonal antibodies (MAbs). The newly established immunostaining method, Eastern blotting,was applied for the determination of ginsenosides possessing protopanaxadiol and/or protopanaxatriolin the traditional Chinese medicine (TCM). This method developed a new way to separate theginsenoside molecule into two functional parts using a simple and well-known chemical reaction.The sugar parts were oxidized by NaIO4 to give dialdehydes, which reacted with amino groups of theprotein and covalently bound to the adsorbent PVDF membrane. The MAb bound to the aglyconpart of the ginsenoside molecule for immunostaining. Double staining of Eastern blotting forginsenosides using anti-G-Rb1 and -Rg1 MAbs promoted complete identification of ginsenosides inPanax species. The immunoaffinity concentration of G-Rb1 was deteremined by immunoaffinity columnconjugated with anti-G-Rb1 MAb leading to the knock-out extract, which will be useful for thepharmacological investigation. To concentrate and determine G-Rb1 in P. japonicus, the crude extractof P. japonicus was fractionated by immunoaffinity column conjugated with anti-G-Rb1 MAb. Twoginsenosides, chikusetsusaponins III and IV having protopanaxadiol as an aglycon, were identifiedby Eastern blotting, although it was expected that G-Rb1 might be a component of P. japonicus byenzyme-linked immunosorbent assay (ELISA) analysis.