| Rare ginsenosides are important active ingredients in ginsenosides, which mainly refers to ginsengoside Rg3(GsRg3), ginsengoside Rg2(GsRg2), ginsengoside Rh1(GsRh1), ginsengoside Rh2(GsRh2) and so on. They have low content or do not exist in the original medicine, they can be generated by processing or metabolic transformation, and have extensive pharmacological actions, of which the effect of antitumor has the most clinical value, the GsRh2and GsRg3etc. have been developed as anticancer drug. In addition, GsRg2increases cardiac function, improves blood rheology change, and protects the cardiovascular and cranial neuron. GsRhl has functions of immunoregulation, antitumor, improving memory and glucocorticoids resistance and so on.Their content in the original medicinal materials are so low that the separation and purification is complex and high cost. Currently, commonly used detection methods of rare ginsenosides include high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS). However, the requirement for tedious sample pretreatment, expensiveness of instrument, accompanying with high testing cost, these methods are far from satisfactory for analytical purposes in terms of sensitivity, simplicity, conveniency and rapidity, thus the further research of the rare ginsenosides is limited.Based on traditional Chinese medicine (TCM) small molecule monoclonal antibody (Mab), immunoassay (IA) is an emerging detection technology in recent years, it has the characteristics of high sensitivity, strong specificity, for the modernization of TCM research which include quality control and appraisal of Chinese herbs, separation and purification for effective component of Chinese herbs, metabolic process in the body and pharmacological efficacy research of Chinese herbal formulas, compared with the conventional physical and chemical analysis technology, the immunological analysis method has a unique advantage.In this study, the artificial antigens of GsRhl was synthesized, and then, anti-GsRh1monoclonal antibody (anti-GsRhl Mab) was got by means of the mice immune and cell fusion technology, On this basis, the GsRg2indirect competitive enzeyme-linked immunosorbent assay(ELISA) was established, and the method was applied to determine the content of GsRg2in Chinese herbal formulasand and human saliva. And try to study the establishment of immunoaffinity chromatography (IAC) column and its knockout based on the antibody. The main contents and results of this study:1. According to the molecular structure characteristics of GsRh1, bovine serum albumin (BSA) and ovalbumin (OVA) were chosen as carrier proteins, the immunizing antigen (GsRhl-BSA) and coating antigen (GsRhl-OVA) were synthesized by sodium periodate oxidation method. It was identified that the GsRhl successfully was conjugated with BSA and OVA via the detection of the UV spectrometry and TLC.2. Five BALB/c mice were immunized with the GsRh1-BSA, after the forth immunization, tail blood was acquired. The optimal blocking buffer and appropriate concentration of GsRh1-OVA were chosen to establish the best working conditions of the indirect ELISA method. GsRhl-OVA was used as coating antigen and the appropriate dilution ratio was1:4000which measured by the board method. The optimal blocking buffer was10mg/mL gelatin. The titer and specificity of the antibody in serum of immunized mice was detected respectively by indirect enzyme-linked immunosorbent assay (iELISA) and indirect competitive enzyme-linked immunosorbent assay (icELISA).3. Splenocytes of No.4mice whose titer and specificity are suitable were selected to fuse with SP2/0myeloma cells under the mediation of PEG. Limited dilution method was used to select hybridoma cell line which specially secreted anti-GsRhl Mab, the1G8C8cells whosecell culture had the higher titer and better specificity were adopt to expand by ascites preparation method, and a large number of Mabs were acquired.4. Ascites were purified by caprylic acid ammonium sulfate precipitation method. The characteristic of antibodies was identified by the indirect ELISA method and the indirect competitive ELISA method. The results showed:(1) the titer of ascites was up to1:2048000, the sensitivity of the Mabs against GsRhl were about125ng/mL, linear range26-512ng/mL. But the sensitivity of the Mabs against GsRg2was28ng/mL, which was higher than GsRh1, so, it is more appropriate that the Mabs was called against ginsenoside GsRg2Mab.(2) Analysis of cross-reactivity demonstrated that the antibody generated was specific for GsRhl (100%) with GsRg2(470.65%), GsRg3(13.88%), GsRh2(12.25%), GsRf (10.80%), with no reaction to R type hydroxyl ginsenosides. we accidently noticed that the five compounds has a highly relevant structure that they all have a (S)-type-hydroxyl group at C-20, and the position of antigenic determinant which the Mab (1G8C8) identified was indicated. 5. On the basis of acquired Mab, an optimum working concentration1:10000was chosen, GsRg2indirect competitive ELISA assay was established, the linear range was4-128ng/mL, the intra-assay and inter-assay were less than7.06%, the average recovery rate was101.65%, it was proved that the method was accurate, stable and reliable. The content of GsRg2in14kinds of commonly used formula containing ginseng were successfully measured by the method. Try to determine the content of GsRg2in human saliva, and analyze the metabolism of rare ginsenosides.6. With acquired Mab as a foundation, GsRg2immune affinity chromatography column and the knockout method of GsRg2was explored to establish.The experimental results showed that the anti-GsRg2MAb with high titer and high sensitivity was prepared successfully, and the GsRg2ELISA method was established. Because its unique advantages in the detection, such as the high sensitivity (up to the level of ng/mL), simple sample preparation (direct application after dilution) as well as low detecting amount (only50uL/hole), independent of complicated and expensive instruments and equipments etc., the method has a good application prospect on quality inspection of Chinese herbs and the study of Chinese herbs compound effective material base. |
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