Acetylcholinesterase (AChE) monoclonal antibody generation and validation for use as a biomarker of glyphosate-based herbicide exposure in commercial freshwater fish

Monoclonal antibody specific to acetylcholinesterase (AChE) was extracted from the brain of hybrid catfish after exposure to glyphosate-based herbicide for 24 h. AChE was partially purified using hydroxyapatite and chromatography columns. The specific characteristics of AChE were studied by western blot using commercial polyclonal antibody (Rabbit anti-Fish AChE). It was found that the protein band had a molecular weight of 71 kDa. After mice were injected with AChE 4 times, the spleen showed a response to the induction. Polyclonal B cells from the mouse's spleen were taken and fused with myeloma cells to produce hybrid cells. After two fusions were performed, the clones specific to AChE were selected by dot blot, ELISA, immunohistochemistry and western blot techniques. Two clones, ACHE 33 and ACHE 99, which had the isotype of IgM were found. These two produced monoclonal antibodies specific to AChE in both denatured and native forms. The ACHE 33 monoclonal antibody clone from hybrid catfish could be cross-react with two commercial freshwater fishes, Nile tilapia and climbing perch, based on dot blot, immunohistochemistry, and western blot techniques. Moreover, AChE in Nile tilapia and climbing perch with glyphosate- based herbicide exposure gave a positive result with ACHE 33 as protein with molecular weight of 66 kDa. Based on our results, the produced monoclonal antibody showed specificity and could be applied to test AChE expression to assess glyphosate-based herbicide contamination in hybrid catfish, Nile tilapia and climbing perch. It could be also be a useful tool in indicating the quality of water resources.