title: Novel dual-mode immunomagnetic method for studying reactivation of nerve agent-inhibited butyrylcholinesterase.
abstract: 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.
authors: 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.
authors: Carter W
authors: CW
authors: Abney
authors: Jennifer L S
authors: JL
authors: Knaack
authors: Ahmed A I
authors: AA
authors: Ali
authors: Rudolph C
authors: RC
authors: Johnson
mesh_terms: D002091:Butyrylcholinesterase / Q000097:blood / Q000378:metabolism*; D002800:Cholinesterase Inhibitors / Q000097:blood / Q000494:pharmacology*; D004789:Enzyme Activation; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D009946:Organothiophosphorus Compounds / Q000097:blood / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D002091:Butyrylcholinesterase; D002800:Cholinesterase Inhibitors; D009946:Organothiophosphorus Compounds; C009680:VX-agent