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.
Highlights
In the recent rapid development of the molecular biosciences and their biotechnological applications, and owing to their specific affinity, immunoassay systems using monoclonal antibodies (MAbs) against drugs and small molecular weight bioactive compounds have become an important tool for studies on receptor binding analysis, enzyme assay, and quantitative and/or qualitative analytical techniques in animals or plants
For production of MAb, synthesis of hapten which is derived from immune antigen and linker bridge, and carrier protein conjugates is necessary
Since the enzyme-linked immunosorbent assays (ELISAs) for ginsenoside Rb1 was established for phytochemical investigations involving crude plant extracts, the assay specificity was checked by determining the cross-reactivity of the MAb with various related compounds
Summary
In the recent rapid development of the molecular biosciences and their biotechnological applications, and owing to their specific affinity, immunoassay systems using MAb against drugs and small molecular weight bioactive compounds have become an important tool for studies on receptor binding analysis, enzyme assay, and quantitative and/or qualitative analytical techniques in animals or plants. The immunoblotting method is based on the Western blotting technique that utilizes the antigen-antibody binding properties and provides a specific and sensitive detection of higher molecule analytes like peptides and proteins. As an application of MAb the MAb against ginsenosides have been prepared resulting in the development of a new technique that we have named the Eastern blotting method and the knockout extract preparation. They will be introduced in this paper
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