title: 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.
authors.affiliation: Emergency Response and Air Toxicants Branch, Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, 4770 Buford Highway, MS F44, Chamblee, Georgia 30341, USA.
authors.forename: Jennifer L S
authors.initials: JL
authors.lastname: Sporty
authors.forename: Sharon W
authors.initials: SW
authors.lastname: Lemire
authors.forename: Edward M
authors.initials: EM
authors.lastname: Jakubowski
authors.forename: Julie A
authors.initials: JA
authors.lastname: Renner
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Evans
authors.forename: Robert F
authors.initials: RF
authors.lastname: Williams
authors.forename: Jurgen G
authors.initials: JG
authors.lastname: Schmidt
authors.forename: Marcel J
authors.initials: MJ
authors.lastname: van der Schans
authors.forename: Daan
authors.initials: D
authors.lastname: Noort
authors.forename: Rudolph C
authors.initials: RC
authors.lastname: Johnson
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D002091:Butyrylcholinesterase / Q000097:blood* / Q000302:isolation & purification / Q000378:metabolism; D002619:Chemical Warfare Agents / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D009946:Organothiophosphorus Compounds / Q000737:chemistry; D010434:Pepsin A / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D012524:Sarin / Q000737:chemistry; D053719:Tandem Mass Spectrometry
chemical_list: D000911:Antibodies, Monoclonal; D002091:Butyrylcholinesterase; D002619:Chemical Warfare Agents; D009946:Organothiophosphorus Compounds; D010434:Pepsin A; D010455:Peptides; D012524:Sarin; C009680:VX-agent