Organosfatlara Maruziyet Sonrasında Oluşan Protein Eklenti Ürünlerini Tanıyan Monoklonal Antikorun Karakterizasyonu

Abstract

Organophosphorus (OP) pesticides are irreversible inhibitors of cholinesterases [acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)]. The OPs cause the acute toxic effect by covalent binding to a serine amino acid in the active site of AChE. Proteins that have no active site serine, such as albumin, are covalently modified by OPs on tyrosine and lysine. The covalent bond of OP compounds with tyrosine is more stable than with serine. Chronic illness from OP pesticides exposure is not explained by inhibition of AChE and BChE. New biomarkers are needed to elucidate the cause of chronic toxicity. The goal of this study was to design and characterize monoclonal antibody that recognizes protein adducts on tyrosine that would be useful in the identification of new biomarkers. Various diethoxyphosphate-tyrosine peptides were synthesized and cross-linked to 4 different carrier proteins. Monoclonal antibody called as depY were produced with hybridoma technology using the conjugated proteins. DepY antibody was purified and characterized by ELISA, Western Blot, Biocore, Octet technology to determine binding affinity and binding specificity. The results showed that depY recognizes diethoxyphospho-tyrosine modified proteins and peptides independent of the surrounding amino acid sequence. Furthermore, HEK293 cell lysates were treated with chlorpyrifos oxon. When tryptic peptides immunopurified with depY-Sepharose were analyzed by mass spectrometry, 116 diethoxyphosphate-tyrosine modified peptides from 73 proteins were identified. Consequently, the use of depY monoclonal antibody could be useful for identifying novel biomarkers of OP exposure and for analyzing modified proteins in any species. Keywords: Organophosphate, chlorpyrifos oxon, depY monoclonal antibody, diethyloxyphosphate-tyrosine, mass spectrometry. This thesis