全自动免疫磁珠纯化法与免疫亲和柱法对食品中黄曲霉毒素B1检测结果的影响比对

Effection and comparison of detection results of aflatoxin B1 in foods by fully automated immunomagnetic bead purification method and immunoaffinity column method

中文关键词:  黄曲霉毒素B1  免疫磁珠纯化法  免疫亲和柱法  高效液相色谱法-柱后衍生法

英文关键词:aflatoxin B1  immunomagnetic bead purification method  immunoaffinity column method  high performance liquid chromatography-post-column derivatization method

中文摘要:

      目的 采用全自动免疫磁珠纯化法与免疫亲和柱法对大米、小麦制品、花生制品等多种基质进行前处理, 检测其黄曲霉毒素B1 (aflatoxin, AFB1)含量, 并比较两种前处理方法的检测结果。方法 样品经甲醇:水(70:30, V:V)提取后, 一部分提取液采用免疫亲和柱法经AFB1专用免疫亲和柱净化, 另取一部分提取液采用搭配免疫磁珠试剂盒的全自动免疫磁珠纯化仪进行前处理, 最后都采用高效液相色谱法-柱后衍生法进行检测。结果 免疫磁珠纯化试剂盒效能和免疫亲和柱柱效均大于95%, 皆能满足实验要求。在质量浓度为1.00~50.00 ng/mL的线性范围内, 线性相关系数(r)为0.99993, 线性关系良好。在大米基质中进行3水平加标回收率实验(n=6), 两种前处理方法回收率范围为98.2%~106.0%, 相对标准偏差为1.0%~5.0%; 在小麦制品中进行单水平加标回收率实验(n=6), 回收率超过80%, 相对标准偏差为3.7%~7.2%。AFB1阳性花生制品、玉米粉基质质控样品采用两种前处理方法进行检测, 并对结果进行t检验, 显示所得结果无显著性差异; 对玉米粉基质质控样品采用两种前处理方法进行检测后所得结果进行Bland-Altman统计分析, 结果表明在检测该基质AFB1时两种方法可以相互替代。免疫亲和柱法净化步骤耗时长, 20批样品耗时约3~4 h; 只能人工操作, 产生大约1000 mL废液; 而免疫磁珠法采用全自动磁珠纯化仪, 净化步骤一次40 min, 可自动完成20批样品, 几乎无废液产生。结论 全自动免疫磁珠纯化法与免疫亲和柱法检测多种基质中的AFB1均能取得良好效果, 且免疫磁珠纯化法效率高、处理简便、产生废液少, 成本可降低大约50%。

英文摘要:

      Objective To compare the detection results of aflatoxin B1 (AFB1) content in various matrices such as rice, wheat products, and peanut products using 2 kinds of different pretreatment methods: Fully automated immunomagentic bead purification and immunoaffinity column pretreatment method. Methods After extraction with methanol:water (70:30, V:V), a portion of the extract was purified using the immunoaffinity column method with an AFB1-specific immunoaffinity column, while another portion of the extract was processed by a fully automated immunomagnetic bead purification system paired with an immunomagnetic bead kit. Finally, both purified extracts were analyzed using high performance liquid chromatography coupled with post-column derivatization for detection. Results The efficacy of the immunomagetic bead purification kit and the column efficiency of the immunoaffinity column were both greater than 95%, which could meet the experimental requirements. Within mass concentration range of 1.00–50.00 ng/mL, the linear correlation coefficient (r) was 0.99993, indicating a good linear relationship. The 3 level spiked recovery experiments were conducted in rice matrix (n=6), and the recovery rates of the 2 kinds of pretreatment methods ranged from 98.2%–106.0%, with a relative standard deviation range of 1.0%–5.0%; single level spiked recovery experiments were conducted in wheat products (n=6), with a recovery rate exceeding 80% and a relative standard deviation of 3.7%–7.2%. Meanwhile, the 2 kinds of methods were used to detected AFB1 positive peanut products and the quality control sample of corn flour matrix, their results were subjected to t-test, which showed no significant difference. Further, Bland-Altman statistical analysis was carried out on the results obtained from the quality control samples of corn flour matrix after 2 kinds of pretreatment methods were used. The results showed that the 2 kinds of methods could replace each other in the detection of AFB1 in this matrix. The purification step of the immunoaffinity column method was time-consuming, 20 batches of samples take about 3–4 hours. And could only be manually operated, which generated about 1000 mL waste liquid; while the immunomagetic bead method used a fully automatic magnetic bead purifier, which automatically completed 20 batches of samples in 40 minutes at one time, with almost no waste liquid generated. Conclusion The fully automated immunomagnetic bead purification method and the immunoaffinity column method both can achieve good results in detecting AFB1 in various matrices. Also, the immunomagnetic bead purification method is more efficient, easy to handle, generates less waste liquid, and the cost can be reduced by about 50%.