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Immunopurification of Acetylcholinesterase from Red Blood Cells for Detection of Nerve Agent Exposure
Chemical Research in Toxicology ( IF 4.1 ) Pub Date : 2017-09-11 00:00:00 , DOI: 10.1021/acs.chemrestox.7b00209
Alicia J. Dafferner 1 , Lawrence M. Schopfer 1 , Gaoping Xiao 2 , John R. Cashman 3 , Udaya Yerramalla 4 , Rudolph C. Johnson 5 , Thomas A. Blake 5 , Oksana Lockridge 1
Affiliation  

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.

中文翻译:

从红细胞中免疫纯化乙酰胆碱酯酶以检测神经毒物暴露

神经药物和有机磷农药与乙酰胆碱酯酶(AChE)的活性位点丝氨酸形成共价键,从而抑制AChE活性和毒性症状。红细胞(RBC)中的AChE可以替代神经系统中的AChE。对RBC AChE上的加合物进行质谱分析可提供暴露的证据。我们的目标是开发一种免疫纯化人RBC AChE的方法,其数量足以通过质谱检测暴露。为此,我们固定了3种市售抗人乙酰胆碱酯酶单克隆抗体(AE-1,AE-2和HR2)以及3种新的单克隆抗体。表征单克隆抗体的结合亲和力,通过配对分析进行的表位作图以及核苷酸和氨基酸序列。用1%(v / v)Triton X-100从冷冻的RBC中溶解AChE。用抑制50%的AChE活性的梭曼模型化合物处理含有5.8μgRBC AChE的16 mL样品。天然和抑制人的RBC AChE样品在抗体-琼脂糖珠上免疫纯化。用胃蛋白酶消化经免疫纯化的RBC AChE,并在6600 Triple-TOF质谱仪上通过液相色谱串联质谱法进行分析。使用靶向分析方法检测老化的梭曼修饰的PheGlyGluSerAlaGlyAlaAlaSer(FGESAGAAS)肽。得出的结论是,所有6种单克隆抗体均可用于免疫纯化RBC AChE,并且在RBC AChE活性位点丝氨酸上的加合物可检测到神经毒剂的暴露。用一定量的抑制50%AChE活性的梭曼模型化合物处理8μgRBC AChE。天然和抑制人的RBC AChE样品在抗体-琼脂糖珠上免疫纯化。用胃蛋白酶消化经免疫纯化的RBC AChE,并在6600 Triple-TOF质谱仪上通过液相色谱串联质谱法进行分析。使用靶向分析方法检测老化的梭曼修饰的PheGlyGluSerAlaGlyAlaAlaSer(FGESAGAAS)肽。得出的结论是,所有6种单克隆抗体均可用于免疫纯化RBC AChE,并且在RBC AChE活性位点丝氨酸上的加合物可检测到神经毒剂的暴露。用一定量的抑制50%AChE活性的梭曼模型化合物处理8μgRBC AChE。天然和抑制人的RBC AChE样品在抗体-琼脂糖珠上免疫纯化。用胃蛋白酶消化经免疫纯化的RBC AChE,并在6600 Triple-TOF质谱仪上通过液相色谱串联质谱法进行分析。使用靶向分析方法检测老化的梭曼修饰的PheGlyGluSerAlaGlyAlaAlaSer(FGESAGAAS)肽。得出的结论是,所有6种单克隆抗体均可用于免疫纯化RBC AChE,并且在RBC AChE活性位点丝氨酸上的加合物可检测到神经毒剂的暴露。用胃蛋白酶消化经免疫纯化的RBC AChE,并在6600 Triple-TOF质谱仪上通过液相色谱串联质谱法进行分析。使用靶向分析方法检测老化的梭曼修饰的PheGlyGluSerAlaGlyAlaAlaSer(FGESAGAAS)肽。得出的结论是,所有6种单克隆抗体均可用于免疫纯化RBC AChE,并且在RBC AChE活性位点丝氨酸上的加合物可检测到神经毒剂的暴露。用胃蛋白酶消化经免疫纯化的RBC AChE,并在6600 Triple-TOF质谱仪上通过液相色谱串联质谱法进行分析。使用靶向分析方法检测老化的梭曼修饰的PheGlyGluSerAlaGlyAlaAlaSer(FGESAGAAS)肽。得出的结论是,所有6种单克隆抗体均可用于免疫纯化RBC AChE,并且在RBC AChE活性位点丝氨酸上的加合物可检测到神经毒剂的暴露。
更新日期:2017-09-11
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