基于抗大豆苷单克隆抗体的Ic-ELISA方法的建立

目的: 建立大豆苷的快速免疫分析检测方法。方法: 该研究通过大豆苷单克隆抗体与葛根素、芦丁等共9种中药小分子的交叉反应证明该抗体具有特异性,并以制备出的大豆苷特异性单克隆抗体为基础,建立了大豆苷间接竞争酶联免疫分析方法(Ic-ELISA),并用此方法检测大豆中大豆苷的含量。结果: 抗体与葛根素的交叉反应率为115%,与芦丁、甘草酸、栀子苷等小分子无交叉反应。该方法线性范围为10~10 000 ng·mL^-1,孔内差和板间差均小于6.3%,平均回收率为105.4%%。采用该方法检测大豆中大豆苷的含量,所得结果与HPLC法的结果一致。结论: 该研究建立了大豆苷的Ic-ELISA方法,可为含大豆苷的中药及复方的质量控制分析提供更加快速灵敏的检测方法。

Objective: To establish a quick indirect competitive enzyme-linked immunosorbent assay (Ic-ELISA)method for the determination of daidzin. Methods: The study proved specificity of the monoclonal antibody against daidzin by the cross reaction against puerarin, rutin and so on,a total of 9 kinds of Chinese traditional medicine of small molecules.Ic-ELISA was developed by using monoclonal antibody against daidzin and then it was applied to daidzin measurement in the extracts of soybean. Results: The cross-reactivity of anti-daidzin monoclonal antibody against puerarin was 115%.There was no cross-reactivity with rutin,licorice acid,geniposide and so on.The standard curve of the established Ic-ELISA method was linear between 10-10 000 ng·mL^-1.The average recovery was 105.4% and the relative standard deviation (RSD)of measurements was <6.3% for the intra-well assay and the inter-plate assay.The analysis result of Ic-ELISA method was consistent with that of HPLC test in daidzin determination of soybean. Conclusion: Rapid Ic-ELISA method for the daidzin determination is well established and can be applied to the quality control of traditional Chinese herbal medicine/compound preparation which contains daidzin.