Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are irreversibly inhibited by organophosphorus pesticides through formation of a covalent bond with the active site serine. Proteins that have no active site serine, for example albumin, are covalently modified on tyrosine and lysine. Chronic illness from pesticide exposure is not explained by inhibition of AChE and BChE. Our goal was to produce a monoclonal antibody that recognizes proteins diethoxyphosphorylated on tyrosine. Diethoxyphosphate-tyrosine adducts for 13 peptides were synthesized. The diethoxyphosphorylated (OP) peptides cross-linked to four different carrier proteins were used to immunize, boost, and screen mice. Monoclonal antibodies were produced with hybridoma technology. Monoclonal antibody depY was purified and characterized by ELISA, western blotting, Biacore, and Octet technology to determine binding affinity and binding specificity. DepY recognized diethoxyphosphotyrosine independent of the amino acid sequence around the modified tyrosine and independent of the identity of the carrier protein or peptide. It had an IC₅₀ of 3 × 10^–9 M in a competition assay with OP tubulin. K_d values measured by Biacore and OctetRED96 were 10^–8 M for OP-peptides and 1 × 10^–12 M for OP-proteins. The limit of detection measured on western blots hybridized with 0.14 μg/mL of depY was 0.025 μg of human albumin conjugated to YGGFL-OP. DepY was specific for diethoxyphosphotyrosine (chlorpyrifos oxon adduct) as it failed to recognize diethoxyphospholysine, phosphoserine, phosphotyrosine, phosphothreonine, dimethoxyphosphotyrosine (dichlorvos adduct), dimethoxyphosphoserine, monomethoxyphosphotyrosine (aged dichlorvos adduct), and cresylphosphoserine. In conclusion, a monoclonal antibody that specifically recognizes diethoxyphosphotyrosine adducts has been developed. The depY monoclonal antibody could be useful for identifying new biomarkers of OP exposure.

中文翻译：

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识别二乙氧基磷酸酪氨酸修饰的蛋白质和独立于周围氨基酸的肽的单克隆抗体

乙酰胆碱酯酶（AChE）和丁酰胆碱酯酶（BChE）通过与活性位点丝氨酸形成共价键而被有机磷农药不可逆地抑制。没有活性位点丝氨酸的蛋白质（例如白蛋白）在酪氨酸和赖氨酸上被共价修饰。不能通过抑制AChE和BChE来解释农药暴露引起的慢性疾病。我们的目标是生产一种单克隆抗体，该抗体可识别酪氨酸上二乙氧基磷酸化的蛋白质。合成了13种肽的二乙氧基磷酸-酪氨酸加合物。与四种不同的载体蛋白交联的二乙氧基磷酸化（OP）肽用于免疫，加强和筛选小鼠。单克隆抗体是用杂交瘤技术生产的。纯化单克隆抗体depY，并通过ELISA，蛋白质印迹，Biacore，和Octet技术来确定结合亲和力和结合特异性。DepY识别的二乙氧基磷酸酪氨酸不依赖于修饰酪氨酸周围的氨基酸序列，也不依赖于载体蛋白或肽的身份。它有一个ICOP微管蛋白竞争试验中的₅₀ ×3×10 ^–9M。Biacore和OctetRED96测得的OP肽的K _d值为10 ^–8 M，1×10 ^–12M为OP蛋白。在与0.14μg/ mL的depY杂交的Western印迹上测得的检测极限为与YGGFL-OP偶联的0.025μg人白蛋白。DepY对二乙氧基磷酸酪氨酸（毒死rif加成）特异，因为它无法识别二乙氧基磷酸赖氨酸，磷酸丝氨酸，磷酸酪氨酸，磷酸苏氨酸，二甲氧基磷酸酪氨酸（二氯代苯磺酸加合物），二甲氧基磷酸丝氨酸，单甲氧基磷酸酪氨酸（二氯代磷酸丙氨酸老化）和甲酚。总之，已经开发出一种特异性识别二乙氧基磷酸酪氨酸加合物的单克隆抗体。DepY单克隆抗体可用于鉴定OP暴露的新生物标记。