Novel Dual-Mode Immunomagnetic Method for Studying Reactivation of Nerve Agent-Inhibited Butyrylcholinesterase

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• 作者：Carter W. Abney ; Jennifer L. S. Knaack ; Ahmed A. I. Ali ; Rudolph C. Johnson
• 刊名：Chemical Research in Toxicology
• 出版年：2013
• 出版时间：May 20, 2013
• 年：2013
• 卷：26
• 期：5
• 页码：775-782
• 全文大小：314K
• 年卷期：v.26,no.5(May 20, 2013)
• ISSN：1520-5010

文摘

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 卤 0.7 ng/mL decreased over 1 h to 10.6 卤 0.6 ng/mL when reactivated with 2-PAM and 14.4 卤 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.