木薯粉及制品中高灵敏度AFB1快速检测方法研究

High-sensitivity rapid detection method for aflatoxin B1 (AFB1) in cassava flour and based products

    摘要
  • 摘要: 【目的】基于酶联免疫吸附测定法（ELISA）检测原理，建立高灵敏度的直接竞争法检测木薯粉及其制品中的黄曲霉毒素B1（AFB1），为木薯食品安全提供技术支持。【方法】通过对AFB1抗原结构改造、免疫和细胞融合等过程获得抗原抗体，建立高灵敏度检测方法，并进行木薯粉及其制品样本的实例验证。【结果】制备的AFB1抗原和抗体，其抗体亚型均为IgG1，灵敏度最高的3F2细胞株产生的抗体对黄曲霉毒素G1、G2和B2交叉反应率分别为21.4%、1.9%和8.8%，且对其他毒素类无交叉反应，建立的ELISA线性区间为0.02~0.48 μg/kg，半数抑制浓度（IC50）为0.047 μg/kg，标准方程为y=-3.074x-4.3066（R2=0.9978）。以35%甲醇水溶液作为标准品稀释液，木薯食品原料用70%甲醇水溶液加0.2 g NaCl作为提取液，含油脂的木薯糕点用70%甲醇水溶液作为提取液，所有样本均为10倍稀释，检测限0.2~4.8 μg/kg，样品添加回收率81.5%~120.0%，变异系数均小于8.5%。应用验证试验表明，自建ELISA试剂盒对96份样本检出19份阳性样本，购买试剂盒检出7份阳性，大于2.0 μg/kg的样本检测结果完全符合，自建ELISA试剂盒方法灵敏度优于购买的ELISA试剂盒。【结论】本研究建立的直接竞争ELISA方法灵敏度高，操作简单，可广泛应用于木薯粉及其制品样本中AFB1的快速测定。

     

    Abstract: 【Objective】Based on the principle of enzyme-linked immunoassay(ELISA), to establish high-sensitive direct competitive ELISA for detecting aflatoxin B1(AFB1) in cassava flour and based products samples, provide technical support for cassava food processing and utilization.【Method】Antigen and antibody were obtained through structural modification of AFB1 antigen, immunization, cell fusion and others protocol. Then the highly sensitive rapid test was established, and it was carries on to test examples comparison with kits from market for verification for cassava flour and based products sample.【Result】The results showed that AFB1 antigen and antibody were successfully synthesized, whose antibody subtypes were all IgG1. One of the most sensitive 3F2 cell line had a cross-reactivity to aflatoxins G1, G2, and B2 for 21.4%, 1.9%, 8.8% respectively, and they had no cross-reactivity to other toxins. The linear range of ELISA was 0.02-0.48 μg/kg, half inhibitory concentration(IC50) was 0.047 μg/kg, the standard equation was y=- 3.074x-4.3066(R2=0.9978). With 35% methanol aqueous solution as standard diluent, cassava food raw materials were extracted with 70% methanol aqueous solution and 0.2 g NaCl, the oil-containing cassava pastries were using 70% methanol aqueous solution as the extract, all samples were 10 times diluted. It also found the detection limit was 0.2-4.8 μg/kg and, the recovery rate of cassava flour and based products samples addition was 81.5%-120.0%, whose coefficient of variation was less than 8.5%. While, the test results showed that, the selfbuilt ELISA kit detected 19 positive samples from 96 samples, and the purchased ELISA kit detected 7 positive samples. The detection results of samples more than 2.0 μg/kg were in complete agreement, self-developed ELISA kit was more sensitive than the ELISA kits from market.【Conclusion】The direct competitive ELISA method established in this study has high sensitivity, simple operation to be widely used for rapid determination for AFB1 in cassava flour and based products.

     

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