Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, China.
Anal Chim Acta. 2011 Oct 3;703(1):64-9. doi: 10.1016/j.aca.2011.07.011. Epub 2011 Jul 19.
A green enzyme-linked immunosorbent assay (ELISA) to measure aflatoxin M(1) (AFM(1)) in milk was developed and validated with a surrogate calibrator curve. Polyclonal anti-idiotype (anti-Id) antibody, used as an AFM(1) surrogate, was generated by immunizing rabbits with F(ab')(2) fragments from the anti-AFM(1) monoclonal antibody (mAb). The rabbits exhibited high specificity to the anti-AFM(1) mAb, and no cross-reactivity to either of the other anti-aflatoxin mAbs or the isotype matched mAb was observed. After optimizing the physicochemical factors (pH and ionic strength) that influence assay performance, a quantitative conversion formula was developed between AFM(1) and the anti-Id antibody (y=31.91x-8.47, r=0.9997). The assay was applied to analyze AFM(1) in spiked milk samples. The IC(50) value of the surrogate calibrator curve was 2.4 μg mL(-1), and the inter-assay and intra-assay variations were less than 10.8%; recovery ranged from 85.2 to 110.9%. A reference high-performance liquid chromatography method was used to validate the developed method, and a good correlation was obtained (y=0.81x+9.82, r=0.9922).
建立并验证了一种用于检测牛奶中黄曲霉毒素 M1（AFM1）的绿色酶联免疫吸附测定法（ELISA），并采用替代校准曲线进行验证。使用抗独特型（anti-Id）多克隆抗体作为 AFM1 的替代物，通过用抗 AFM1 单克隆抗体（mAb）的 F（ab'）2 片段免疫兔子来产生。兔子对抗 AFM1 mAb 表现出高度特异性，并且与其他任何抗黄曲霉毒素 mAb 或同型匹配的 mAb 均无交叉反应。在优化影响测定性能的物理化学因素（pH 值和离子强度）后，建立了 AFM1 与抗 Id 抗体之间的定量转换公式（y=31.91x-8.47，r=0.9997）。该测定法用于分析添加到牛奶样品中的 AFM1。替代校准曲线的 IC50 值为 2.4 μg mL-1，批内和批间变异均小于 10.8%；回收率范围为 85.2%至 110.9%。采用参考高效液相色谱法对所建立的方法进行验证，得到了良好的相关性（y=0.81x+9.82，r=0.9922）。
