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目的为建立龙胆苦苷免疫检测方法,需合成龙胆苦苷(GTP)的免疫抗原和包被抗原。方法将琥珀酸酐与龙胆苦苷中的羟基进行反应,再利用混合酸酐法将龙胆苦苷半抗原与载体蛋白(BSA和OVA)偶联形成龙胆苦苷-载体蛋白人工抗原,进而利用紫外分光光度计法(UV)、红外光谱法(IR)、聚丙烯酰胺凝胶电泳法(SDS-PAGE)鉴定人工抗原,结合三硝基苯磺酸法(TNBS)测定人工抗原的偶联比率,并免疫小鼠,最后用间接ELISA方法检测免疫小鼠血清中抗体效价。结果 IR、UV和SDS-PAGE法检测结果均表明龙胆苦苷与载体蛋白偶联成功,TNBS结果表明GTP-BSA(免疫抗原)和GTP-OVA(包被抗原)偶联比分别为9∶1和6∶1,间接ELISA结果表明免疫小鼠血清中抗龙胆苦苷抗体效价均达到1∶12800以上。结论本实验成功合成并鉴定了龙胆苦苷人工抗原,为龙胆苦苷免疫分析提供了实践基础。
Objective To establish a method for the immunoassay of gentiopicroside, it is necessary to synthesize gentiopicroside (GTP) immunogen and coat antigen. Methods succinic anhydride and gentiopicroside hydroxyl reaction, and then mixed anhydride method gentiopicroside haptens and carrier protein (BSA and OVA) coupled to form gentiopicroside - carrier protein artificial antigen, and then use Artificial antigens were identified by ultraviolet spectrophotometry (UV), infrared spectroscopy (IR) and polyacrylamide gel electrophoresis (SDS-PAGE), and the coupling ratio of artificial antigens was measured by trinitrobenzene sulfonic acid method (TNBS) , And the mice were immunized. Finally, the antibody titers in serum of immunized mice were detected by indirect ELISA. Results The results of IR, UV and SDS-PAGE showed that gentiopicroside was successfully coupled with the carrier protein. The results of TNBS showed that the coupling ratios of GTP-BSA (immunogen) and GTP-OVA (coating antigen) 1 and 6:1, respectively. The results of indirect ELISA showed that the anti-gentiopicroside antibody titer reached above 1:12800 in the serum of immunized mice. Conclusion The experiment successfully synthesized and identified gentiopicroside artificial antigen, which provided the practical basis for gentiopicroside immunoassay.