TY - JOUR
T1 - Immunopurification of Acetylcholinesterase from Red Blood Cells for Detection of Nerve Agent Exposure
AU - Dafferner, Alicia J.
AU - Schopfer, Lawrence M.
AU - Xiao, Gaoping
AU - Cashman, John R.
AU - Yerramalla, Udaya
AU - Johnson, Rudolph C.
AU - Blake, Thomas A.
AU - Lockridge, Oksana
N1 - Funding Information:
*Oksana Lockridge. Mailing address: University of Nebraska Medical Center, 600 Saddle Creek Rd., Omaha, NE 68198-5900. Phone: 402 559 6032. Fax: 402 559 4651. E-mail: olockrid@unmc.edu. ORCID Oksana Lockridge: 0000-0002-8345-3640 Funding Supported by DLS/NCEH/CDC Contract 200-2015-87939 (to O.L.), Fred & Pamela Buffett Cancer Center Support Grant P30CA036727, and Centers for Disease Control and Prevention, Office of Public Health Preparedness and Response, and Defense Threat Reduction Agency 11-005-12430 (to T.A.B. and R.C.J.). Notes The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, or the U.S. Department of Health and Human Services. The authors declare no competing financial interest.
Publisher Copyright:
© 2017 American Chemical Society.
PY - 2017/10/16
Y1 - 2017/10/16
N2 - Nerve agents and organophosphorus pesticides make a covalent bond with the active site serine of acetylcholinesterase (AChE), resulting in inhibition of AChE activity and toxic symptoms. AChE in red blood cells (RBCs) serves as a surrogate for AChE in the nervous system. Mass spectrometry analysis of adducts on RBC AChE could provide evidence of exposure. Our goal was to develop a method of immunopurifying human RBC AChE in quantities adequate for detecting exposure by mass spectrometry. For this purpose, we immobilized 3 commercially available anti-human acetylcholinesterase monoclonal antibodies (AE-1, AE-2, and HR2) plus 3 new monoclonal antibodies. The monoclonal antibodies were characterized for binding affinity, epitope mapping by pairing analysis, and nucleotide and amino acid sequences. AChE was solubilized from frozen RBCs with 1% (v/v) Triton X-100. A 16 mL sample containing 5.8 μg of RBC AChE was treated with a quantity of soman model compound that inhibited 50% of the AChE activity. Native and soman-inhibited RBC AChE samples were immunopurified on antibody-Sepharose beads. The immunopurified RBC AChE was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. The aged soman-modified PheGlyGluSerAlaGlyAlaAlaSer (FGESAGAAS) peptide was detected using a targeted analysis method. It was concluded that all 6 monoclonal antibodies could be used to immunopurify RBC AChE and that exposure to nerve agents could be detected as adducts on the active site serine of RBC AChE.
AB - Nerve agents and organophosphorus pesticides make a covalent bond with the active site serine of acetylcholinesterase (AChE), resulting in inhibition of AChE activity and toxic symptoms. AChE in red blood cells (RBCs) serves as a surrogate for AChE in the nervous system. Mass spectrometry analysis of adducts on RBC AChE could provide evidence of exposure. Our goal was to develop a method of immunopurifying human RBC AChE in quantities adequate for detecting exposure by mass spectrometry. For this purpose, we immobilized 3 commercially available anti-human acetylcholinesterase monoclonal antibodies (AE-1, AE-2, and HR2) plus 3 new monoclonal antibodies. The monoclonal antibodies were characterized for binding affinity, epitope mapping by pairing analysis, and nucleotide and amino acid sequences. AChE was solubilized from frozen RBCs with 1% (v/v) Triton X-100. A 16 mL sample containing 5.8 μg of RBC AChE was treated with a quantity of soman model compound that inhibited 50% of the AChE activity. Native and soman-inhibited RBC AChE samples were immunopurified on antibody-Sepharose beads. The immunopurified RBC AChE was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. The aged soman-modified PheGlyGluSerAlaGlyAlaAlaSer (FGESAGAAS) peptide was detected using a targeted analysis method. It was concluded that all 6 monoclonal antibodies could be used to immunopurify RBC AChE and that exposure to nerve agents could be detected as adducts on the active site serine of RBC AChE.
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U2 - 10.1021/acs.chemrestox.7b00209
DO - 10.1021/acs.chemrestox.7b00209
M3 - Article
C2 - 28892361
AN - SCOPUS:85031091171
SN - 0893-228X
VL - 30
SP - 1897
EP - 1910
JO - Chemical Research in Toxicology
JF - Chemical Research in Toxicology
IS - 10
ER -