Emergency Response and Air Toxicants Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F44, Chamblee, Georgia 30341, United States.
Anal Chem. 2012 Nov 6;84(21):9470-7. doi: 10.1021/ac302301w. Epub 2012 Oct 19.
An automated high-throughput immunomagnetic separation (IMS) method for diagnosing exposure to the organophosphorus nerve agents (OPNAs) sarin (GB), cyclohexylsarin (GF), VX, and Russian VX (RVX) was developed to increase sample processing capacity for emergency response applications. Diagnosis of exposure to OPNAs was based on the formation of OPNA adducts to butyrylcholinesterase (BuChE). Data reported with this method represent a ratio of the agent-specific BuChE adduct concentration, relative to the total BuChE peptide concentration that provides a nonactivity measurement expressed as percent adducted. All magnetic bead transfer steps and washes were performed using instrumentation in a 96-well format allowing for simultaneous extraction of 86 clinical samples plus reference materials. Automating extractions increased sample throughput 50-fold, as compared to a previously reported manual method. The limits of detection, determined using synthetic peptides, were 1 ng/mL for unadducted BuChE and GB-, GF-, VX-, and RVX-adducted BuChE. The automated method was characterized using unexposed serum and serum pools exposed to GB, GF, VX, or RVX. Variation for the measurement of percent adducted was <12% for all characterized quality control serum pools. Twenty-six (26) serum samples from individuals asymptomatic for cholinesterase inhibitor exposure were analyzed using this method, and no background levels of OPNA exposure were observed. Unexposed BuChE serum concentrations measured using this method ranged from 2.8 μg/mL to 10.6 μg/mL, with an average concentration of 6.4 μg/mL.
为了提高应急响应应用中的样品处理能力,开发了一种自动化高通量免疫磁分离(IMS)方法,用于诊断接触有机磷神经毒剂(OPNAs)沙林(GB)、环已沙林(GF)、VX 和俄罗斯 VX(RVX)。OPNA 暴露的诊断基于 OPNA 加合物与丁酰胆碱酯酶(BuChE)的形成。该方法报告的数据代表特定于试剂的 BuChE 加合物浓度与提供非活性测量的总 BuChE 肽浓度的比值,以加合物百分比表示。所有磁珠转移步骤和洗涤均使用 96 孔格式的仪器进行,允许同时提取 86 个临床样本和参考材料。与之前报道的手动方法相比,自动化提取将样品通量提高了 50 倍。使用合成肽确定的检测限分别为未加合 BuChE 和 GB-、GF-、VX-和 RVX-加合 BuChE 的 1ng/mL。使用未暴露的血清和暴露于 GB、GF、VX 或 RVX 的血清池对自动化方法进行了表征。所有表征的质量控制血清池的加合物百分比测量的变异均<12%。使用该方法分析了 26 份来自未出现胆碱酯酶抑制剂暴露症状的个体的血清样本,未观察到 OPNA 暴露的背景水平。使用该方法测量的未暴露 BuChE 血清浓度范围为 2.8μg/mL 至 10.6μg/mL,平均浓度为 6.4μg/mL。