The unlabeled control proteins bound neither the recombinant nor the hybridoma depY. for OP-peptides and 1 10?12 M for OP-proteins. The limit of detection measured on Western blots hybridized with 0.14 g/mL depY was 0.025 g human albumin conjugated to YGGFL-OP. DepY was specific for diethoxyphospho-tyrosine (chlorpyrifos oxon adduct) as it failed to recognize diethoxyphospho-lysine, phosphoserine, phospho-tyrosine, phospho-threonine, dimethoxyphospho-tyrosine (dichlorvos adduct), dimethoxyphospho-serine, monomethoxyphospho-tyrosine (aged dichlorvos adduct), and cresylphospho-serine. In conclusion, a monoclonal antibody that specifically recognizes diethoxyphospho-tyrosine adducts has been developed. The depY monoclonal antibody could be useful for identifying fresh biomarkers of OP exposure. Keywords: Chlorpyrifos oxon, dichlorvos, tyrosine adduct, antibody, mass spectrometry, Biacore, OctetRED96 Graphical Abstract Intro Organophosphorus pesticides can have the same harmful effects as chemical warfare providers because they share the same mechanism of acute toxicity, namely irreversible inhibition of acetylcholinesterase (AChE).1 Furthermore, organophosphorus pesticides are readily available to individuals intent on harming others. Taliban insurgents have poisoned schoolgirls in Afghanistan with parathion in several attacks. Proof of exposure can be obtained from blood samples drawn more than a month after the event through mass spectrometry analysis of diethoxyphospho-tyrosine adducts on blood proteins. A study by vehicle der Schans found that the adduct on tyrosine 411 of human being albumin was detectable in blood drawn 49 days after poisoning by chlorpyrifos.2 (+)-α-Lipoic acid The same blood sample contained no detectable adducts on butyrylcholinesterase (BChE) because fresh BChE molecules had replaced the inhibited BChE. It is expected that a monoclonal antibody specific for diethoxyphospho-tyrosine could be used to detect exposure inside a device similar to the one developed by Quick Pathogen Screening, Inc.3 for nerve agent exposure. Such a device is simple to use and requires no expensive mass spectrometers. A second software of a monoclonal antibody to diethoxyphosphorylated (OP) tyrosine would be for understanding chronic neurotoxicity from organophosphorus pesticide exposure. To day the only proteins known to be revised by organophosphorus pesticides in vivo are acetylcholinesterase, butyrylcholinesterase, carboxylesterase, and albumin. Acyl peptide hydrolase in reddish blood cells is definitely a possible target, but evidence for reaction with organophosphorus pesticides in vivo has been demonstrated only in rats and only with dichlorvos.4 The proposed monoclonal antibody could be used to immunopurify diethoxyphospo-tyrosine-containing proteins in preparation for analysis by mass spectrometry. This would test the hypothesis that organophosphorus toxicants Rabbit polyclonal to AREB6 improve many proteins and that the consequence of changes is definitely disruption of signaling pathways.5 The strategy we used to make our monoclonal antibody to diethoxyphospho-tyrosine is based on the strategy that produced the monoclonal antibody to phospho-tyrosine. 6 We conjugated 4 carrier proteins to 13 different (+)-α-Lipoic acid OP-tyrosine peptides. Five conjugates were used as immunogen, a different 5 for boosting, and a different three for screening. Materials and Methods The following were from Sigma-Aldrich: human being albumin, (accession “type”:”entrez-protein”,”attrs”:”text”:”P02768″,”term_id”:”113576″,”term_text”:”P02768″P02768) Fluka 05418; bovine albumin (“type”:”entrez-protein”,”attrs”:”text”:”P02769″,”term_id”:”1351907″,”term_text”:”P02769″P02769) A-2153 and A-8022; ovalbumin (“type”:”entrez-protein”,”attrs”:”text”:”P01012″,”term_id”:”129293″,”term_text”:”P01012″P01012) A-5503; lysozyme (“type”:”entrez-protein”,”attrs”:”text”:”P00698″,”term_id”:”126608″,”term_text”:”P00698″P00698) L-6876; casein (mixture of isoforms “type”:”entrez-protein”,”attrs”:”text”:”P02662″,”term_id”:”115646″,”term_text”:”P02662″P02662, PO2663, “type”:”entrez-protein”,”attrs”:”text”:”P02666″,”term_id”:”115660″,”term_text”:”P02666″P02666, “type”:”entrez-protein”,”attrs”:”text”:”P02668″,”term_id”:”115667″,”term_text”:”P02668″P02668) C-5890; aprotinin bovine (“type”:”entrez-protein”,”attrs”:”text”:”P00974″,”term_id”:”115114″,”term_text”:”P00974″P00974) A-1153; Anti O-Phospho-tyrosine monoclonal antibody clone PY20 (Sigma P-4110); O-Phospho-L-tyrosine P-9405; 2-[Nmorpholino] ethanesulfonic acid M-8250; O-Phenylenediamine dihydrochloride P-8287; 1- ethyl-3-(3-dimethylaminopropyl) carbodiimide.HCl, Fluka 03450; Paraoxon-ethyl D-9286. The following were from Thermo Scientific: Sulfo-NHS, N-hydroxysulfosuccinimide 24510; 3,3,5,5-tetramethylbenzidine remedy N301; Nunc-Immuno MaxiSorp surface flat bottom 96 well polystyrene plate; Immulon 2HB Thermo 3455. (+)-α-Lipoic acid The following were from Chem Services Inc: Chlorpyrifos oxon MET-674B; Dichlorvos PS-89. Peptides were purchased from American Peptide Co., Sigma-Aldrich, and Genscript. Mouse albumin (“type”:”entrez-protein”,”attrs”:”text”:”P07724″,”term_id”:”5915682″,”term_text”:”P07724″P07724) was from Innovative Study Inc., Novi, MI. Porcine tubulin (mixture of alpha and beta “type”:”entrez-protein”,”attrs”:”text”:”P02550″,”term_id”:”135435″,”term_text”:”P02550″P02550, “type”:”entrez-protein”,”attrs”:”text”:”Q2XVP4″,”term_id”:”116256086″,”term_text”:”Q2XVP4″Q2XVP4, (+)-α-Lipoic acid “type”:”entrez-protein”,”attrs”:”text”:”Q767L7″,”term_id”:”75045190″,”term_text”:”Q767L7″Q767L7, “type”:”entrez-protein”,”attrs”:”text”:”P02554″,”term_id”:”135490″,”term_text”:”P02554″P02554) was from Cytoskeleton Inc T240. Protein G agarose was from Protein Mods LLC, Madison, WI. Horse anti-mouse IgG (weighty and light chains) conjugated to HRP was from Cell Signaling 7076. CNBr-activated Sepharose fast circulation was from Amersham Biosciences 17-0981-01. OP-Tyrosine peptides Diethoxyphospho-tyrosine adducts created spontaneously when peptides were incubated with an excess of chlorpyrifos oxon (CPO) or paraoxon at high pH. For example 70 mg of peptide YGGFL were incubated in 12 mL of 1 1 M Tris pH 10.8 with a 33 fold molar excess of paraoxon (0.1 mL of 4.16 M paraoxon) for 3 days at 37C. During this time the pH decreased to pH 9. Unreacted paraoxon was separated from p-nitrophenol and peptides by extraction with chloroform. The yellow p-nitrophenol was separated from peptides on a C18 Alltech 900 mg cartridge.
