Development of a luminex based competitive immunoassay for 2,4,6-trinitrotoluene (TNT)
Development of a luminex based competitive immunoassay for 2,4,6-trinitrotoluene (TNT)
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DOI：
10.1021/es062333n
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发表时间：
2007-04-15
影响因子：
11.4
通讯作者：
Charles, Paul T.
中科院分区：
文献类型：
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作者：
Anderson, George P.;Lamar, Jacqueline D.;Charles, Paul T.
此前，使用 Luminex 100 演示了 2,4,6-三硝基甲苯 (TNT) 的置换免疫测定。本次工作利用相同的专用流式细胞仪演示了 TNT 的高灵敏度和快速竞争性免疫测定。这需要将 TNT 类似物附着到微球表面。评估了各种接头；与各种较短的二胺接头相比，牛血清白蛋白提供的结合位点多出 3 倍以上。对于该测定，将 TNB 包被的微球添加到样品中；然后添加生物素化的抗 TNT 抗体和报告分子链霉亲和素-R-藻红蛋白。在没有TNT的情况下，微球表面形成高荧光复合物。 TNT 的存在导致剂量依赖性荧光减弱。评估了各种抗 TNT 抗体； Mab 30-1 给出了最强的响应，产生最低的检测限 (< 1.0 ng/mL) 和高达 1 μg/mL 的动态范围。其他因素，如反应时间、与其他硝基化合物的交叉反应性、TNT 污染土壤的丙酮提取物的评估、淡水和海水等环境基质的测试均已完成。最后，使用竞争形式成功地对 TNT 和三种蛋白质毒素进行了多重测定。
Previously, a displacement immunoassay for 2,4,6-trinitrotoluene (TNT) was demonstrated using the Luminex 100. The work presented utilized this same specialized flow cytometer to demonstrate a highly sensitive and rapid competitive immunoassay for TNT. This required a TNT analog to be attached to the microsphere surface. Various linkers were evaluated; bovine serum albumin provided over 3 times more binding sites in comparison to various shorter diamine linkers. For this assay TNB-coated microspheres were added to samples;then biotinylated anti-TNT antibody and the reporter molecule, Streptavidin-R-Phycoerythrin, were added. In the absence of TNT, a highly fluorescent complex was formed on the surface of the microsphere. The presence of TNT resulted in dose-dependent decreased fluorescence. Various anti-TNT antibodies were evaluated; Mab 30-1 gave the strongest response, yielding the lowest limit of detection (< 1.0 ng/mL) and a dynamic range up to 1 mu g/mL. Other factors such as reaction time, cross reactivity to other nitro-compounds, evaluation of acetone extracts of TNT contaminated soils, testing in environmental matrices such as fresh water and seawater were all completed. Finally, a multiplex assay for TNT and three protein toxins was successfully conducted using the competitive format.