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Updated: Oct 21, 2025
Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope
Published on: March 24, 2017
Rabbit Antidiethoxyphosphotyrosine Antibody, Made by Single B Cell Cloning, Detects Chlorpyrifos Oxon-Modified
Seda Onder1, Marco van Grol2, Alex Fidder2
1Department of Biochemistry, School of Pharmacy, Hacettepe University, Ankara 06100, Turkey.
Abstract:
Chronic low-dose exposure to organophosphorus pesticides is associated with the risk of neurodegenerative disease. The mechanism of neurotoxicity is independent of acetylcholinesterase inhibition. Adducts on tyrosine, lysine, threonine, and serine can occur after exposure to organophosphorus pesticides, the most stable being adducts on tyrosine. Rabbit monoclonal 1C6 to diethoxyphosphate-modified tyrosine (depY) was created by single B cell cloning. The amino acid sequence and binding constant (Kd 3.2 × 10-8 M) were determined. Cultured human neuroblastoma SH-SY5Y and mouse neuroblastoma N2a cells incubated with a subcytotoxic dose of 10 μM chlorpyrifos oxon contained depY-modified proteins detected by monoclonal 1C6 on Western blots. depY-labeled peptides from tryptic digests of cell lysates were immunopurified by binding to immobilized 1C6. Peptides released with 50% acetonitrile and 1% formic acid were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Lumos mass spectrometer. Protein Prospector database searches identified 51 peptides modified on tyrosine by diethoxyphosphate in SH-SY5Y cell lysate and 73 diethoxyphosphate-modified peptides in N2a cell lysate. Adducts appeared most frequently on the cytoskeleton proteins tubulin, actin, and vimentin. It was concluded that rabbit monoclonal 1C6 can be useful for studies that aim to understand the mechanism of neurotoxicity resulting from low-dose exposure to organophosphorus pesticides.
Insights
Chronic low-dose organophosphorus pesticide exposure may increase neurodegenerative disease risk. A new antibody detects diethoxyphosphate-modified tyrosine (depY) adducts on proteins, aiding neurotoxicity mechanism studies.
Area of Science:
- Toxicology
- Neuroscience
- Biochemistry
Background:
- Chronic low-dose organophosphorus pesticide (OPP) exposure is linked to neurodegenerative diseases.
- The neurotoxic mechanism is not solely due to acetylcholinesterase inhibition.
- OPP exposure can form stable adducts on tyrosine residues.
Purpose of the Study:
- To develop a tool for detecting specific organophosphorus pesticide adducts.
- To investigate the mechanism of neurotoxicity independent of acetylcholinesterase inhibition.
- To identify proteins modified by diethoxyphosphate adducts in neuronal cells.
Main Methods:
- Development of a rabbit monoclonal antibody (1C6) targeting diethoxyphosphate-modified tyrosine (depY).
- Detection of depY-modified proteins in human (SH-SY5Y) and mouse (N2a) neuroblastoma cells exposed to chlorpyrifos oxon using Western blotting.
- Immunopurification of depY-labeled peptides followed by LC-MS/MS analysis for identification.
Main Results:
- Monoclonal antibody 1C6 successfully detected depY-modified proteins in neuronal cell lines.
- LC-MS/MS identified 51 depY-modified peptides in SH-SY5Y cells and 73 in N2a cells.
- Adducts were predominantly found on cytoskeleton proteins, including tubulin, actin, and vimentin.
Conclusions:
- Rabbit monoclonal antibody 1C6 is a valuable tool for studying OPP-induced neurotoxicity.
- The findings provide insights into the non-acetylcholinesterase inhibitory mechanisms of OPP neurotoxicity.
- Identification of modified cytoskeleton proteins suggests their involvement in OPP-related neurodegenerative processes.
