Immunomagnetic Separation and Quantification of Butyrylcholinesterase Nerve Agent Adducts in Human Serum

Abstract

A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 microL serum samples. The particle-antibody-BuChE product was rinsed and directly digested with pepsin. Native and isotopically enriched nonapeptides corresponding to the pepsin digest products for uninhibited BuChE, and sarin, cyclohexylsarin, VX, and Russian VX nerve agent-inhibited BuChE were synthesized for use as calibrators and internal standards, respectively. Internal standards were added to the filtered digest sample, and the samples were quantified via high performance liquid chromatography-isotope dilution-tandem mass spectrometry. The ratio of adducted to total BuChE nonapeptides was calculated for each nerve agent-exposed serum sample using data collected in a single chromatogram. Nerve agent-inhibited quality control serum pools were characterized as part of method validation; the method was observed to have extremely low background noise. The measurement of both uninhibited and inhibited BuChE peptides compensated for any variations in the pepsin digestion before the internal standard peptide was added to the sample and may prove useful in individualizing patient results following a nerve agent exposure.

New insights on molecular interactions of organophosphorus pesticides with esterases

“…Both LC and MALDI-TOF MS have been used in the identification of OP exposures from either insecticides or nerve agents (Sun and Lynn, 2007;Thompson et al, 2010;Noort and Fidder, 2006;Li et al, 2008;Sun and Lynn., 2009;Li et al, 2010;Schopfer et al, 2010;Liyasova et al, 2011). In order to remove the limitations of affinity chromatography for biomarker purification, immunomagnetic beads coupled to a specific antiBChE antibody have been used in other studies that use LC-MS/MS to monitor OP exposures (Kim et al, 2010;Sporty et al, 2010;Marsillach et al, 2011;Aryal et al, 2012). The LC-MS/MS analysis of OP exposures has shown to have several advantages over the currently used colorimetric enzyme activity assays: 1) the analysis does not require a separate blood draw for the determination of baseline activity levels; 2) it provides a more accurate measurement of the percentage modification of the active-site serine, and; 3) it also provides some information about the OP that adducted the active-site serine (e.g., a methyl-or ethyl based insecticide).…”

Fe3O4 Magnetic Nanoparticle Peroxidase Mimetic-Based Colorimetric Assay for the Rapid Detection of Organophosphorus Pesticide and Nerve Agent

“…9,10 Laboratory-based GC/LC/MS analytical techniques are commonly used and show detection limits in the nanomolar concentration range. However, these techniques are time-consuming, expensive, have to be performed by a highly trained technician, and not suitable for rapid analyses under field conditions, especially in emergency cases.…”

A high‐throughput UHPLC–MS/MS method for the quantification of five aged butyrylcholinesterase biomarkers from human exposure to organophosphorus nerve agents

“…(Carter et al, 2013, Fidder et al, 2002, Knaack et al, 2012, Sporty et al, 2010) When adducted BChE peptides (FGESAGAAS) are fragmented using collision-induced dissociation, β-elimination of the OPNA adduct is observed, converting the adducted serine into a dehydroalanine ( m/z 778.3) (Figure 4). For the MeP-BChE, EtP-BChE, PrP-BChE, and P-BChE peptides, quantitation was based on the transition of each respective [M+H] + precursor ion to the product ion m/z 778.3.…”

“…OPNA-BChE adducts have relatively long half-lives (8–12 days), are abundant in serum and plasma, and can easily be extracted from blood since BChE is not membrane-bound. (Knaack et al, 2012, Pantazides et al, 2014, Sporty et al, 2010)…”