Hupresin is a new affinity resin that binds butyrylcholinesterase (BChE) in human plasma and acetylcholinesterase (AChE) solubilized from red blood cells (RBC). Hupresin is available from the CHEMFORASE company. BChE in human plasma binds to Hupresin and is released with 0.1 M trimethylammonium bromide (TMA) with full activity and 10–15% purity. BChE immunopurified from plasma by binding to immobilized monoclonal beads has fewer contaminating proteins than the one-step Hupresin-purified BChE. However, when affinity chromatography on Hupresin follows ion exchange chromatography at pH 4.5, BChE is 99% pure. The membrane bound AChE, solubilized from human RBC with 0.6% Triton X-100, binds to Hupresin and remains bound during washing with sodium chloride. Human AChE is not released in significant quantities with non-denaturing solvents, but is recovered in 1% trifluoroacetic acid. The denatured, partially purified AChE is useful for detecting exposure to nerve agents by mass spectrometry. Our goal was to determine whether Hupresin retains binding capacity for BChE and AChE after Hupresin is washed with 0.1 M NaOH. A 2 mL column of Hupresin equilibrated in 20 mM TrisCl pH 7.5 was used in seven consecutive trials to measure binding and recovery of BChE from 100 mL human plasma. Between each trial the Hupresin was washed with 10 column volumes of 0.1 M sodium hydroxide. A similar trial was conducted with red blood cell AChE in 0.6% Triton X-100. It was found that the binding capacity for BChE and AChE was unaffected by washing Hupresin with 0.1 M sodium hydroxide. Hupresin could be washed with sodium hydroxide at least seven times without losing binding capacity.

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 IC of 3 × 10 M in a competition assay with OP tubulin. K values measured by Biacore and OctetRED96 were 10 M for OP-peptides and 1 × 10 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.

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Chronic illness from exposure to organophosphorus toxicants is hypothesized to involve modification of unknown proteins. Tyrosine in proteins that have no active site serine readily reacts with organophosphorus toxicants. We developed a monoclonal antibody, depY, that specifically recognizes diethoxyphospho-tyrosine in proteins and peptides, independent of the surrounding amino acid sequence. Our goal in the current study was to identify diethoxyphosphorylated proteins in human HEK293 cell lysate treated with chlorpyrifos oxon. Cell lysates treated with chlorpyrifos oxon were recognized by depY antibody in ELISA and capillary electrophoresis based Western blot. Tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry. Liquid chromatography tandem mass spectrometry identified 116 diethoxyphospho-tyrosine peptides from 73 proteins in immunopurified samples, but found only 15 diethoxyphospho-tyrosine peptides from 12 proteins when the same sample was not immunopurified on depY. The most abundant proteins in the cell lysate, histone H4, heat shock 70 kDa protein 1A/1B, heat shock protein HSP 90 β, and α-enolase, were represented by several diethoxyphospho-tyrosine peptides. It was concluded that use of immobilized depY improved the number of diethoxyphospho-tyrosine peptides identified in a complex mixture. The mass spectrometry results confirmed the specificity of depY for diethoxyphospho-tyrosine peptides independent of the context of the modified tyrosine, which means depY could be used to analyze modified proteins in any species. Use of the depY antibody could lead to an understanding of chronic illness from organophosphorus pesticide exposure.