Abney Carter W, Knaack Jennifer L S, Ali Ahmed A I, Johnson Rudolph C
Oak Ridge Institute for Science and Education Fellow at the Centers for Disease Control and Prevention , 4770 Buford Highway, MS F44, Chamblee, Georgia 30341, United States.
Chem Res Toxicol. 2013 May 20;26(5):775-82. doi: 10.1021/tx4000717. Epub 2013 May 8.
A novel immunomagnetic method has been developed for the simultaneous measurement of organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BuChE) and free OPNAs in serum. This new approach, deemed dual-mode immunomagnetic analysis (Dual-Mode IMA), combines immunomagnetic separation (IMS) and immunomagnetic scavenging (IMSc) and has been used to measure the effectiveness of cholinesterase reactivators on OPNA-inhibited BuChE in serum. BuChE inhibited by the nerve agent VX, uninhibited BuChE, and unbound VX were measured up to 1 h after the addition of oxime reactivators pralidoxime (2-PAM) and obidoxime. IMS experiments consisted of extracting BuChE and VX-BuChE serum adducts using antibutyrylcholinesterase monoclonal antibodies conjugated to protein-G ferromagnetic particles. In a parallel set of experiments using IMSc, BuChE-coated magnetic beads were used to extract free VX from protein-depleted serum. Adducts from both IMS and IMSc were analyzed using a published IMS liquid chromatography tandem mass spectrometry (IMS-LC-MS/MS) protocol, which has also been demonstrated with other OPNAs. By applying this Dual-Mode IMA approach, 2-PAM was observed to be more potent than obidoxime in reactivating VX-adducted BuChE. VX-BuChE peptide concentrations initially measured at 19.7 ± 0.7 ng/mL decreased over 1 h to 10.6 ± 0.6 ng/mL when reactivated with 2-PAM and 14.4 ± 1.2 ng/mL when reactivated with obidoxime. These experiments also show that previously published IMS-LC-MS/MS analyses are compatible with serum treated with oximes. Dual-Mode IMA is the first immunoaffinity method developed for the simultaneous measurement of OPNA adducted BuChE, unadducted BuChE, and free nerve agent in serum and is a promising new tool for studying reactivator effectiveness on cholinesterases inhibited by nerve agents.
已开发出一种新型免疫磁方法，用于同时测量血清中有机磷神经毒剂（OPNA）与丁酰胆碱酯酶（BuChE）的加合物以及游离OPNA。这种新方法被称为双模式免疫磁分析（Dual-Mode IMA），它结合了免疫磁分离（IMS）和免疫磁清除（IMSc），并已用于测量胆碱酯酶复活剂对血清中OPNA抑制的BuChE的作用效果。在添加肟类复活剂氯解磷定（2-PAM）和双复磷后，对被神经毒剂VX抑制的BuChE、未受抑制的BuChE以及未结合的VX进行了长达1小时的测量。IMS实验包括使用与蛋白G铁磁颗粒偶联的抗丁酰胆碱酯酶单克隆抗体提取BuChE和VX-BuChE血清加合物。在使用IMSc的一组平行实验中，用包被有BuChE的磁珠从去除蛋白质的血清中提取游离VX。使用已发表的IMS液相色谱串联质谱（IMS-LC-MS/MS）方案对IMS和IMSc得到的加合物进行分析，该方案也已在其他OPNA上得到验证。通过应用这种双模式IMA方法，观察到2-PAM在复活被VX加合的BuChE方面比双复磷更有效。最初测得的VX-BuChE肽浓度为19.7±0.7 ng/mL，用2-PAM复活时在1小时内降至10.6±0.6 ng/mL，用双复磷复活时降至14.4±1.2 ng/mL。这些实验还表明，先前发表的IMS-LC-MS/MS分析与用肟类处理的血清兼容。双模式IMA是第一种开发用于同时测量血清中OPNA加合的BuChE、未加合的BuChE和游离神经毒剂的免疫亲和方法，是研究复活剂对被神经毒剂抑制的胆碱酯酶作用效果的一种有前景的新工具。
