Synthesis Of Artificial Antigen Of Astragaloside Ⅳ And Preparation Of Monoclonal Antibody

Astragaloside is an important ingredient in Radix Astragali, whose commonly means of analysis is HPLC, while immunoassay method has advantage of simply pretreatment, highly sensitivity and specificity, which is expected to provide a new means of analysing astragaloside.Objective:1) To prepare the anti-astragaloside monoclonal antibodies, synthesize the artificial antigen of astragaloside and immunized Balb/C mice, then analyze the subtype and specificity of the antibodies.2) Prepare the immunoaffinity column, and establish the method of IAC-HPLC to identify monoclonal antibody.Method:1) Artificial antigen of astragaloside was synthed by means of sodium periodate oxidation, and identified by UV and TLC.2) Balb/C mice were immunized AST-BSA, after booster 3-4 times, titer and specificity of antiserum was tested by ELISA.3) PEG inducing the cell fusion, and screen the hybridoma cell lines that can stably secrete the antibodies, then prepare it in vivo.4) Identify the specificity of monoclonal antibodies by ic-ELISA, and appraise their subtypes.5) The antibodies are purified by octanoic acid, and coupled with agarose gel to prepare the immunoaffinity column (IAC). Identify the antibodies by detecting the diversification of knockout the component in the water extract of astragalus by HPLC, and establish the method of identification antiboties by IAC-HPLC.Results:1) UV spectroscopy and thin layer chromatography showed that astragaloside was successfully conjugated with BSA and OVA.2) After immuned AST-BSA, the mice can produce anti-astragaloside antibodies specifically, whose titer was up to 1:32 000, and cross-reactivity rate was<0.01% with the similarly structurally compounds. The results of ic-ELISA showed that the effect of detecting astragaloside was different in 0.01 M phosphate buffers (PBS, pH 7.4),0.05 M carbonate buffers (CBS, pH 9.6) and distilled water. In 0.05 M CBS, the anti-serum can detect the astragaloside specifically, Whlie in 0.01 M PBS, the detection was inhibited, and was greatly inhibited in distilled water.3) After monoclone three times, monoclonal cell lines can stably secrete specific antibodies, and were identified as IgG2a, with a κ light chain.4) The results of ic-ELISA showed the antibodies had a specificity with water extract of astragalus, and there were big effect on the sample matrices to detect astragaloside. This assay format was sensitive to varying pHs and ionic strengths. Results suggested that the rates of competitive inhibition are very low at N-butanol saturated water and methanol at 10%,13.42% and 6.07% respectively, while there was no significant competitive inhibition in total saponins of Astragalus, but not when used to increase the solubility of astragaloside, and the competitive inhibition curve of linear equations was y=-0.1161n(x)+1.5739.5) IAC-HPLC results showed that the antibodies obtained were anti-astragaloside monoclonal antibodies.Conclusion:1) With the advantage of simple and accurate, TLC is a quick and visual way of identification the synthesis of antigen.2) Matrix conditions have a greater impact on the specificity and sensitivity of monoclonal antibody.3) IAC-HPLC can be used to identify the antibodies, as well as studying the knockout of active substance and related pharmacology of TCM.