直接竞争荧光免疫法测定中药材中的黄曲霉毒素B1

本文采用Mannich反应合成黄曲霉毒素B1(AFB1)人工抗原,免疫小鼠制备AFB1单克隆抗体。采用直接搅拌法将异硫氰酸荧光素(FITC)标记AFB1抗体,经Sephadex-50凝胶柱纯化,制得FITC-AFB1荧光标记抗体,分析其免疫学特性,从而建立了一种快速灵敏的直接竞争荧光免疫分析方法(FIA)以用于检测中药材中的AFB1含量。结果表明,AFB1-FITC标记抗体的结合比率为4.19。通过对检测体系多项影响因素的筛选优化,FIA检测方法的标准曲线方程为I=33.45 log C+25.55,R=0.9913,线性检测范围1~100 ng/m L,检测限0.69ng/m L,回收率90.4%~106.6%。该方法具有操作简单、快速灵敏、特异性高等特点,可用于中药材中AFB1的分析测定。

Determination of Aflatoxin B1 in Traditional Chinese Medicines by Direct Competitive Fluorescence Immunoassay

Objective: To develop a rapid and sensitive fluorescence immunoassay for the detection of AFB₁. Methods: Synthesis of AFB₁ artificial antigen by Mannich reaction, the monoclonal antibody with high affinity and specificity was obtained after immunization of Balb/c mice. The conjugates of AFB₁ antibody and FITC were prepared by direct label method. A direct competitive FIA method was employed. Results: The F/P ratio of labeling reaction was 4.19. After the optimal conditional experiment, the FIA standard curve was drawn up and the regression equation was obtained, I = 34.45 logC 25.55, correlation coefficient r=0.9913, and the range of linearity was from 1 to 100 ng.mL_-1. By FIA method, the recoveries of AFB₁ in traditional Chinese medicines samples were range from 90.4 to 106.6%. Conclusion: The FIA method has been successfully applied to the detection of AFB₁ in traditional Chinese medicines.