赭曲霉毒素A的高灵敏时间分辨荧光免疫分析

Ultrasensitive Time-resolved Fluoroimmunoassay of Ochratoxin A

采用时间分辨荧光免疫分析(TRFIA)技术建立快速的高灵敏度的赭曲霉毒素A(OTA)全自动检测方法.将OTA-BSA作为免疫分子免疫Balb/c小鼠,OTA-KLH为筛选用抗原包被板,用间接酶联免疫分析(ELISA)法筛选出专一针对OTA的高效价的阳性克隆3G9,用杂交瘤细胞制备抗OTA单克隆抗体.用OTA-BSA包被96孔板为固相抗原,与游离OTA共同竞争有限的抗OTA单克隆抗体,以稀土离子Eu3+标记的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OTA-TRFIA.该方法的灵敏度为0.03μg/L,测量范围为0.03￣1000μg/L,批内和批间变异分别为3.7%和5.3%,平均回收率为94.2%,与赭曲霉毒素B的平均交叉反应为3.7%,与黄曲霉毒素B1、牛血清白蛋白和苯基丙氨酸无交叉反应,说明抗体的特异性很好.8条不同时间进行的间接竞争OTA-TRFIA的效应点均值ED80、ED50、ED20分别为(0.33±0.02)μg/L、(1.44±0.08)μg/L和(5.22±0.12)μg/L,说明方法的稳定性好,样品经TRFIA和ELISA试剂盒同时检测OTA,两者的相关系数为0.925,结果相符.研究表明,OTA-TRFIA是目前报道的OTA检测中最灵敏的方法,该分析方法稳定性好,可测范围宽,具有很好的应用前景.

In order to provide a rapid, selectivity and very high sensitivity method for the determination of ochratoxin A (OTA), an indirect competitive time-resolved fluoroimmunoassay(TRFIA) was used. Tests were performed in a 96-well microplate using the self-produced toxin-specific monoclonal antibodies 3G9, obtained from mice immunized with ochratoxin A - bovine serum albumin (OTA-BSA). In indirect TRFIA format, OTA-BSA was coated onto the microtitre plate and incubated with standard toxin and anti-OTA antibody. A goat antimice IgG- Eu3+ conjugate was used to enable detection. The suitability of the assay for quantification of OTA was also studied. Results showed ascitic fluids could be used at a dilution exceeding 1:10 000 and the OTA detection limit to be 0.03 mu g /L for indirect competitive TRFIA formats. The 80%, 50% and 20% inhibition binding effective dose (ED80 ED50 ED20) of OTA were (0.33 +/- 0.02) mu g/L, (1.44 +/- 0.08) mu g/L and (5.22 +/- 0.12) mu g/L. The assay range was 0.03 - 1 000 mu g /L. The cross reactivity with ochratoxin B was 3.7% and the antibodies did not react with aflatoxin B1, phenylalanine and BSA. The within-run and between-run CVs of the OTA- TRFIA were 3.7% and 5.3% respectively. The mean recoveries from OTA-free cereals spiked with 1 200 mu g/kg OTA of cereals sample were 94.2%. Both OTA- TRFIA and OTA-ELISA test were applied for the quantitative measurement of OTA in the same cereals, and the coefficient of correlation was 0.925. It was shown that the newly developed TRFIA could be applied to detect the OTA contamination in cereals. The OTA-TRFIA provides very high sensitivity and optimal range, and it will be useful to screen OTA contamination easily, simply and economically when the number of samples is large.