Immunopurification of Acetylcholinesterase from Red Blood Cells for Detection of Nerve Agent Exposure

Alicia J. Dafferner, Lawrence M Schopfer, Gaoping Xiao, John R. Cashman, Udaya Yerramalla, Rudolph C. Johnson, Thomas A. Blake, Oksana Lockridge

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1897-1910
Number of pages14
JournalChemical Research in Toxicology
Volume30
Issue number10
DOIs
StatePublished - Oct 16 2017

Fingerprint

Acetylcholinesterase
Blood
Erythrocytes
Cells
Soman
Monoclonal Antibodies
Mass spectrometry
Nerve Agents
Serine
Mass Spectrometry
Catalytic Domain
Epitope Mapping
Covalent bonds
Poisons
Pepsin A
Liquid chromatography
Protein Sequence Analysis
Octoxynol
Neurology
Mass spectrometers

ASJC Scopus subject areas

  • Toxicology

Cite this

Immunopurification of Acetylcholinesterase from Red Blood Cells for Detection of Nerve Agent Exposure. / Dafferner, Alicia J.; Schopfer, Lawrence M; Xiao, Gaoping; Cashman, John R.; Yerramalla, Udaya; Johnson, Rudolph C.; Blake, Thomas A.; Lockridge, Oksana.

In: Chemical Research in Toxicology, Vol. 30, No. 10, 16.10.2017, p. 1897-1910.

Research output: Contribution to journalArticle

Dafferner, Alicia J. ; Schopfer, Lawrence M ; Xiao, Gaoping ; Cashman, John R. ; Yerramalla, Udaya ; Johnson, Rudolph C. ; Blake, Thomas A. ; Lockridge, Oksana. / Immunopurification of Acetylcholinesterase from Red Blood Cells for Detection of Nerve Agent Exposure. In: Chemical Research in Toxicology. 2017 ; Vol. 30, No. 10. pp. 1897-1910.
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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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