Monoclonal Antibodies against Small Molecule Natural Products and Their Applications, Eastern Blotting and Knockout ExtractReport as inadecuate




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Faculty of Pharmaceutical Science, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan





Abstract To determine the hapten number in hapten-carrier protein conjugate matrix-assisted laser desorption-ionization MALDI tof mass spectrometry was applied. Highly specific anti-ginsenoside Rb1 and Rg1 monoclonal antibodies MAbs were prepared. Ginsenosides were developed on thin layer chromatography TLC plates which were covered by a polyvinylidene difluoride PVDF membrane resulting in blotting. The membrane was treated with NaIO4 solution to release the aldehyde group on the sugar moiety of the ginsenosides. By treatment of the membrane with a protein solution the ginsenoside-protein conjugation as a Schiff-base occurred, which can function to fix it to the PVDF membrane. A part of the ginsenoside aglycone was reacted with anti-ginsenoside Rb1 MAb, secondary MAb conjugated with enzyme and finally a substrate was added, resulting in a specific and highly sensitive staining that we named Eastern blotting. Furthermore, it makes one-step isolation of ginsenoside Rb1 possible using an immuno-affinity column conjugated with anti-ginsenoside Rb1 MAb. Furthermore, immunoaffinity concentration was carried out allowing high sensitivity analysis of lower concentrations of ginsenoside Rb1 so that several unknown bands could be structurally determined. View Full-Text

Keywords: monoclonal antibody; natural product; ginseng saponin-ginsenoside; Eastern blotting; knockout extract monoclonal antibody; natural product; ginseng saponin-ginsenoside; Eastern blotting; knockout extract





Author: Yukihiro Shoyama

Source: http://mdpi.com/



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