Abstract：
Organophosphorus nerve agents (OPNAs) covalently bind to tyrosine 411 of human serum albumin (HSA) and the formed adducts are stable biomarkers of OPNA exposure. The detection of these adducts has been limited to mass spectrometry techniques combined with protein digestion. Here, we developed indirect competitive ELISA (icELISA) methods to verify OPNA exposure by the detection of OPNA-phosphonylated adducts at tyrosine 411 residue (OPNA-HSA adducts), in which monoclonal antibodies (mAbs) against phosphonylation sites at tyrosine 411 were introduced. The two mAbs were prepared by the fourth generation of rabbit mAb technology using the phosphonylated peptides of LVRY(GD or VX)TKKVPQC as the haptens. These mAbs were screened using our developed competitive ELISA method and then selected based on their individual affinity and selectivity. As a result, we obtained two mAbs that recognized the HSA Tyr 411 adduct of GD (mAb-5G2) or VX (mAb-12B9), respectively. They shared the highest affinity exhibiting a Kd value of about 10-6 mol/L of the OPNA exposure concentration. They also had remarkable selectivity, which could especially recognize their individual OPNA-HSA adducts in a native state but did not recognize other OPNA-HSAs and unadducted HSAs. Especially for mAb-12B9, it could clearly distinguish VX-HSA and GB-HSA between which there was only one alkyl difference in their phosphonyl portion of the adducted sites. The two mAbs were then used to build the icELISA method for analysis of the serum samples exposed to OPNA. It was found that the detectable lowest GD- and VX-exposed concentrations in serum samples were respectively 1.0 × 10-6 mol/L and 10.0 × 10-6 mol/L. This study provides one novel approach and strategy for the retrospective detection of OPNA exposure, and the two mAbs have great potential to be extended for point-of-care testing of OPNA intoxication.
摘要：
有机磷神经毒剂（OPNA）与人血清白蛋白（HSA）的酪氨酸411共价结合，形成的加合物是OPNA暴露的稳定生物标志物。这些加合物的检测仅限于与蛋白质消化结合的质谱技术。这里,我们开发了间接竞争ELISA（icELISA）方法，通过检测酪氨酸411残基处的OPNA-磷酸化加合物（OPNA-HSA加合物）来验证OPNA暴露，其中引入了针对酪氨酸411处的磷酸化位点的单克隆抗体（mAb）。使用LVRY(GD或VX)TKKVPQC的磷酸化肽作为半抗原,通过第四代兔mAb技术制备两种mAb。使用我们开发的竞争性ELISA方法筛选这些mAb,然后基于它们的个体亲和力和选择性进行选择。因此,我们获得了两种单克隆抗体，它们识别GD（mAb-5G2）或VX（mAb-12B9）的HSATyr411加合物，分别。它们具有最高的亲和力，其Kd值为约10-6mol/L的OPNA暴露浓度。它们还具有显著的选择性，它可以特别识别其处于天然状态的单个OPNA-HSA加合物，但不识别其他OPNA-HSA和未加合的HSA。特别是对于mAb-12B9,它可以清楚地区分VX-HSA和GB-HSA,它们之间在它们的加合位点的膦酰基部分中只有一个烷基差异。然后将两种mAb用于构建icELISA方法,用于分析暴露于OPNA的血清样品。发现血清样品中可检测的最低GD-和VX-暴露浓度分别为1.0×10-6mol/L和10.0×10-6mol/L。本研究为OPNA暴露的回顾性检测提供了一种新的方法和策略，这两种单克隆抗体在OPNA中毒的即时检测中有很大的潜力。
