Enzyme-linked immunosorbent assay for detection of organophosphorylated butyrylcholinesterase: a biomarker of exposure to organophosphate agents.
Enzyme-linked immunosorbent assay for detection of organophosphorylated butyrylcholinesterase: a biomarker of exposure to organophosphate agents.
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DOI：
10.1016/j.aca.2011.03.013
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发表时间：
2011-05
影响因子：
6.2
通讯作者：
Limin Wang;Dan Du;Donglai Lu;Chiann-Tso Lin;J. Smith;C. Timchalk;Fengquan Liu;Jun Wang;Yuehe Lin
中科院分区：
文献类型：
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作者：
Limin Wang;Dan Du;Donglai Lu;Chiann-Tso Lin;J. Smith;C. Timchalk;Fengquan Liu;Jun Wang;Yuehe Lin
建立了一种检测有机磷酸化丁酰胆碱酯酶（OP-BChE）的夹心酶联免疫吸附试验（sELISA），该酶是人体暴露于有机磷杀虫剂和神经毒剂的潜在生物标志物。通过系统筛选不同来源的抗BChE抗体（anti-BChE）和抗磷酸丝氨酸抗体（anti-Pser），鉴定出一对OP-BChE加合物特异性抗体。选择的抗BChE（设置为捕获抗体）抗体识别磷酸化和非磷酸化BChE。因此，这些抗体可用于从样品基质中捕获BChE和OP-BChE。抗Pser抗体（作为检测抗体）用于识别OP-BChE加合物的OP部分。结合所选抗体对，优化了几个关键参数（如抗BChE和抗Pser的浓度以及封闭剂），以提高sELISA的灵敏度和选择性。在最佳条件下，sELISA的线性范围为0.03 nM ~ 30 nM，检测限为0.03 nM。此外，sELISA成功应用于体外生物样品（例如加标OP-BChE的大鼠血浆）中检测OP-BChE，加合物回收率良好（z> 99%）。这些结果表明，这种新的方法有很大的希望开发ELISA试剂盒，并提供了一个简单的和具有成本效益的工具，筛选/评估暴露于有机磷杀虫剂和神经毒剂。
A sandwich enzyme-linked immunosorbent assay (sELISA) has been developed for detection of organophosphorylated butyrylcholinesterase (OP-BChE), a potential biomarker for human exposure to organophosphate insecticides and nerve agents. A pair of antibodies specific to OP-BChE adduct were identified through systematic screening of several anti BChE antibodies (anti-BChE) and anti-phosphoserine antibodies (anti-Pser) from different sources. The selected anti-BChE (set as capture antibody) antibodies recognize both phosphorylated and nonphosphorylated BChE. These antibodies can therefore be used to capture both BChE and OP-BChE from the sample matrices. The anti-Pser(set as detecting antibody) was used to recognize the OP moiety of OP-BChE adducts. With the combination of the selected antibody pair, several key parameters (such as the concentration of anti-BChE and anti-Pser, and the blocking agent) were optimized to enhance the sensitivity and selectivity of the sELISA. Under the optimal conditions, the sELISA has shown a wide linear range from 0.03 nM to 30 nM, with a detection limit of 0.03 nM. Furthermore, the sELISA was successfully applied to detect OP-BChE usingin vitrobiological samples such as rat plasma spiked with OP-BChE with excellent adduct recovery (z> 99%). These results demonstrate that this novel approach holds great promise to develop an ELISA kit and offers a simple and cost-effective tool for screening/evaluating exposure to organophosphate insecticides and nerve agents.