A rapid aflatoxin B1 ELISA:: Development and validation with reduced matrix effects for peanuts, corn, pistachio, and soybeans
A rapid aflatoxin B1 ELISA:: Development and validation with reduced matrix effects for peanuts, corn, pistachio, and soybeans
复制标题
DOI：
10.1021/jf0354038
复制
发表时间：
2004-05-19
影响因子：
6.1
通讯作者：
Kennedy, IR
中科院分区：
文献类型：
--
作者：
Lee, NA;Wang, S;Kennedy, IR
在已经描述的黄曲霉毒素的竞争性elisa中，很少有充分验证降低基质效应的。利用黄曲霉毒素B-1 （AFB(1)）特异性多克隆抗体(由AFB（1)-肟偶联牛血清白蛋白（BSA）产生）和AFB(1)-和AFB(2)-酶偶联物，基于96微孔板（两个标准分析和两个快速分析）开发了四种直接竞争elisa，特别注意为一系列农产品提供相对无干扰的可靠检测。该抗体对AFB(1)具有特异性，在四种黄曲霉毒素(AFB(1)、AFB(2)、AFG（1)和AFG(2)）的混合物中仅检测AFB(1)，但当检测单个化合物时，与AFG(1)发生显著交叉反应（57-61%）。对于AFB(1)和AFB(2)-酶偶联物，标准测定法（长测定法）比快速测定法（短测定法）显示出更高的灵敏度，样品中IC50值分别为12 +/- 1.5和9 +/- 1.5杯/千克（样品提取物稀释1 / 5）。这些检测方法的检测范围较窄（样品中7.1 -55.5马克/千克），并且需要稀释样品提取物以克服溶剂和基质的干扰，这使得这些检测方法作为分析方法不太理想。快速分析显示，AFB(1)-和AFB(2)-酶偶联物的IC50值分别为21.6 +/- 2.7和12 mug/kg。这些检测具有理想的更广泛的检测范围（样品中4.2-99.9马克杯/千克），并且由于孵育时间较短和酶偶联物用量增加，没有高达80%的甲醇效应，基质干扰显著减少。因此，快速测定被格式化为无需提取液稀释即可执行。快速检测可在15分钟内完成，可能适用于快速决策分离低污染和高污染至关重要的接收区。利用AFB(1)-酶偶联物的快速检测进一步验证表明，在玉米、花生、开心果和大豆中添加AFB1的回收率相对较高，没有明显的基质效应。结果表明，该快速检测方法适用于玉米、花生、开心果、大豆等食品中黄曲霉毒素AFB(1)在现行法定最大残留限量为10杯/千克的情况下的检测。
Among the competitive ELISAs for aflatoxins that have been described, few have been adequately validated for reduced matrix effects. Using an aflatoxin B-1 (AFB(1))-specific polyclonal antibody (produced from AFB(1)-oxime conjugated to bovine serum albumin (BSA)) and AFB(1)- and AFB(2)-enzyme conjugates, four direct competitive ELISAs based on 96-microwell plates (two standard assays and two rapid assays) were developed, paying special attention to producing a robust assay relatively free of interferences for a range of agricultural products. The antibody was AFB(1)-specific, detecting only AFB(1) in a mixture of four aflatoxins (AFB(1), AFB(2), AFG(1), and AFG(2)), but showed significant cross-reaction with AFG(1) (57-61%) when an individual compound was tested. Standard assays (long assays) exhibited higher sensitivities than rapid assays (short assays) with IC50 values of 12 +/- 1.5 and 9 +/- 1.5 mug/kg in sample (with 1 in 5 dilution of sample extract) for AFB(1) and AFB(2)-enzyme conjugates, respectively. These assays have narrower detection ranges (7.1 -55.5 mug/kg in sample) and required dilution of sample extracts to overcome solvent and matrix interferences, making these assays less ideal as analytical methods. Rapid assays exhibited IC50 values of 21.6 +/- 2.7 and 12 mug/kg in sample for AFB(1)- and AFB(2)-enzyme conjugates, respectively. These assays have ideally broader detection ranges (4.2-99.9 mug/kg in sample) and showed no methanol effects up to 80% with significantly reduced matrix interferences as a result of the shorter incubation times and increasing the amounts of enzyme conjugate used. Therefore, the rapid assays were formatted to perform without a need for extract dilution. The rapid assays can be completed within 15 min, potentially suitable for receival bays where quick decision-making to segregate low and high contamination is critical. Further validation using the rapid assay with AFB(1)-enzyme conjugate indicated relatively good recoveries of AFB1 spiked in corn, peanuts, pistachio, and soybeans, which were free from significant matrix effects. It can be concluded that this rapid assay would be suitable for monitoring aflatoxin AFB(1) at current legal maximum residue limits of 10 mug/kg in food such as corn, peanuts, pistachio, and soybeans.