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目的:合成中药活性成分芦丁的完全抗原并进行鉴定及免疫原性研究,同时建立快速检测芦丁的酶联免疫吸附(ELISA)分析方法。方法:采用高碘酸钠法制备芦丁完全抗原,用紫外扫描和SDS-PAGE的方法对其进行鉴定,以此抗原免疫健康雄性新西兰大白兔,制备抗血清。结果:紫外分析表明合成的完全抗原偶联比为13∶1,SDS-PAGE显示完全抗原与牛血清蛋白或卵清蛋白相比后移了。免疫新西兰大白兔得到特异针对芦丁的抗血清,芦丁抗体的效价为1∶4 000。经方法学考察,灵敏度IC50为5.37 mg.L-1,最低检测限为1 mg.L-1,平均添加回收率为102%,批内和批间RSD均小于10%,抗体与其他类似物的交叉反应率小于1%。结论:成功的合成了具有较好的免疫原性的芦丁完全抗原,并成功的建立了快速检测芦丁的酶联免疫吸附(ELISA)方法。
OBJECTIVE: To synthesize the complete antigen of rutin, the active ingredient of traditional Chinese medicine, to identify and study the immunogenicity of rutin. Meanwhile, a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the determination of rutin. Methods: Rutin antigen was prepared by sodium periodate method and identified by UV-scanning and SDS-PAGE. Anti-serum was used to immunize healthy male New Zealand white rabbits to prepare antisera. Results: UV analysis showed that the synthetic complete antigen to antigen ratio was 13: 1 and SDS-PAGE showed that the complete antigen was late shifted compared to bovine serum albumin or ovalbumin. New Zealand white rabbits immunized rutin antiserum specifically rutin antibody titers of 1: 4000. After the methodological study, the sensitivity IC50 was 5.37 mg.L-1, the lowest detection limit was 1 mg.L-1, the average added recovery was 102%, intra-assay and inter-assay RSD were less than 10% The cross-reaction rate was less than 1%. CONCLUSION: The rutin complete antigen with good immunogenicity has been successfully synthesized and the ELISA method for the rapid detection of rutin has been successfully established.