Abney_2013_Chem.Res.Toxicol_26_775

A novel immunomagnetic method has been developed for the simultaneous measurement of organophosphorus nerve agent (OPNA) adducts to butyrylcholinesterase (BCHE) 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 BCHE in serum. BCHE inhibited by the nerve agent VX, uninhibited BCHE, 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 BCHE and VX-BCHE serum adducts using antibutyrylcholinesterase monoclonal antibodies conjugated to protein-G ferromagnetic particles. In a parallel set of experiments using IMSc, BCHE-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 BCHE. VX-BCHE 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 BCHE, unadducted BCHE, and free nerve agent in serum and is a promising new tool for studying reactivator effectiveness on cholinesterases inhibited by nerve agents.