用双标记时间分辨荧光免疫法同时检测黄曲霉毒素B_1和赭曲霉毒素A

Simultaneous detection of aflatoxin B_1 and ochratoxin A by the dual-label time-resolved fluoroimmunoassay

目的采用双标记时间分辨荧光免疫分析(TRFIA)技术建立同时检测黄曲霉毒素B1(AFB1)和赭曲霉毒素A(OTA)的高灵敏分析方法。方法将AFB1-HRP、OTA-BSA包被96孔板为固相抗原,与游离AFB1、OTA共同竞争有限的抗AFB1多克隆抗体或抗OTA单克隆抗体,以稀土离子Eu3+标记的羊抗兔抗体、Sm3+标记羊抗鼠抗体进行示踪,采用间接竞争免疫分析方法在解离增强荧光免疫分析体系中建立AFB1/OTA-TRFIA。结果该方法检测AFB1的灵敏度为0.02μg/L,测量范围为0.02～100μg/L,批内和批间变异系数(CV)分别为3.2%和7.3%,平均回收率为88.1%;检测OTA的灵敏度为0.05μg/L,测量范围为0.05～50μg/L,批内和批间CV分别为2.9%和7.9%,平均回收率为89.9%,AFB1/OTA-TRFIA检测时,AFB1与OTA不相互干扰。样品经双标记AFB1/OTA-TRFIA与单标记AFB1-TRFIA、OTA-TRFIA同时检测,与AFB1-TRFIA两者检测AFB1结果的相关系数为0.972,与OTA-TRFIA两者检测OTA结果的相关系数为0.981,说明结果相符。结论AFB1/OTA-TRFIA灵敏度高,可测范围宽,稳定性好,一次操作可以同时得到AFB1、OTA的两个结果,是一种简便、快速、经济的可进行大批量样品筛查的方法。

Objective Using dual-label time-resolved fluoroimmunoassay （TRFIA） technology to establish a high sensitive method for simultaneously detect aflatoxin B1 （AFB1） and Ochratoxin A （OTA）. Methods AFB1-HRP and OTA-BSA coated 96-well plates were taken as the solid phase antigen to compete with free AFB1 and OTA for limited anti- AFB1 polyclonal antibody or anti-OTA monoclonal antibody. Then rare earth ion Eu^3＋ labeled goat anti-rabbit antibody and Sm^3＋ labeled goat anti-mouse antibody were applied as tracers to establish AFB1/OTA-TRFIA in dissociation enhanced fluorescence immunoassay system by using indirect competitive immunoassay. Results For this method, the sensitivity of AFB1 was 0.02μg/L with a measurement range of 0.02 - 100μg/L, intra-and inter-assay CV of 3.2% and 7.3% , and an average recovery rates of 88.1% . While the sensitivity of OTA was 0.05μg/L with a measurement range of 0.05 - 50μg/L, intra-and inter-assay CV of 2.9% and 7.9 %, and an average recovery rates of 89.9 %. In AFB1/OTA-TRFIA detection, the AFB1 and OTA did not interfere with each other. When samples were detected by dual-label AFB1/OTA-TRFIA, single label AFB1-TRFIA and OTA-TRFIA at the same time, the AFB1/OTA-TRFIA detection and AFB1-TRFIA detection for AFB1 were correlated by 0. 972, while the correlation coefficient of AFB1/OTA-TRFIA detection and OTA-TRFIA detection for OTA was 0.981, indicating that the results were consistent. Conclusion The study showed that AFBl/OTA-TRFIA, with high sensitivity, wide measuring range, good stability, and simultaneous detection of AFB1 and OTA, could be a simple, fast and economic method.