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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (26): 144-150.

• 食品 营养 检测 安全 • 上一篇    下一篇

液相芯片竞争法检测黄曲霉毒素B1

宋慧君 刘淑艳 马惠蕊 蒋丹 曹远银   

  • 收稿日期:2011-04-12 修回日期:2011-05-17 出版日期:2011-10-15 发布日期:2011-10-15
  • 基金资助:

    中国质检总局资助项目

The Quantitative Analysis of AFB1 by Using the Microsphere Array Technology and Indirect Competition Theory

  • Received:2011-04-12 Revised:2011-05-17 Online:2011-10-15 Published:2011-10-15

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摘要：

采用间接竞争法原理和液相芯片技术平台，建立黄曲霉毒素B1（AFB1）液相芯片定量检测方法。选用AFB1单克隆抗体对AFB1定量检测方法进行探索，通过优化偶联抗原浓度，确定AFB1单抗临界饱和浓度和抗原抗体最佳孵育时间，建立AFB1液相芯片定量检测方法。本实验中，偶联微球得率介于70.08%~80.80%之间，最佳偶联抗原浓度为100 μg，同一用量偶联抗原和同一浓度单抗反应检测得到的荧光信号值随着孵育时间的增加呈现逐步升高并稳定的趋势，当孵育时间为24 h和48 h，单克隆抗浓度在2.0 ng/μL时检测得到的荧光信号值最大。本研究建立的标准曲线，以国际通用的IC50作为衡量标准，其灵敏度为2.33 ng/mL（0.1165 ng），线性方程为y=0.0006x+0.0014，抗体与AFB2、AFG1、ZEN的交叉反应率分别为9.34%、2.88%、<0.10%。本研究成功建立了AFB1液相芯片定量检测方法，检测限度符合国家对食品和饲料中的AFB1的限量标准，适用于大量样本的检测。

关键词: 北虫草新菌株, 北虫草新菌株, 培养基, 淫羊藿, 栽培技术

Abstract:

The method for quantitative analysis of AFB1 was established by using the microsphere array technology platform. Monoclonal anti-aflatoxin B1 was used in this study. Through optimizing the coupling antigen and determining critical saturated concentration of monoclonal anti-aflatoxin B1 as well as optimizing incubation time of antigen and antibody, the quantitative detection of AFB1 was established eventually. The results showed that, the yield of the coupled microballoon sphere was between 70.08%-80.80%, the best dosage of the coupling antigen was 100 μg. The median fluorescence intensity (MFI) detected by the same dosage of the coupling antigen and the identical concentration of the monoclonal anti-aflatoxin B1, increased along with the extending foster time and became stable finally. When the foster time was 24 h and 48 h, the MFI was the biggest with 2.0 ng/μL concentration of the monoclonal anti-aflatoxin B1. As referring to the international current standard of IC50, the standard curve showed that the detection sensitivity was 2.33 ng/mL (0.1165ng), the detection linear equation was y=0.0006x+0.0014, the cross reactivity rate of the monoclonal anti-aflatoxin B1 with AFB2, AFG1 and ZEN were 9.34%, 2.88% and<0.10%. The quantitative detection of AFB1 was established successfully in this study. The examination limitation of the standard curve was conformed to the China’s limitation standard of AFB1 in the food and feed. This method was suitable for the massive sample examination.