Department of Fisheries, Faculty of Agriculture and Technology, Rajamangala University of Technology Isan, Surin Campus, Surin 32000, Thailand.
Department of Science and Mathematics, Faculty of Agriculture and Technology, Rajamangala University of Technology Isan, Surin Campus, Surin 32000, Thailand.
Comp Biochem Physiol C Toxicol Pharmacol. 2021 Mar;241:108956. doi: 10.1016/j.cbpc.2020.108956. Epub 2020 Dec 17.
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.
从暴露于草甘膦除草剂 24 小时后的杂交鲶鱼脑中提取了一种针对乙酰胆碱酯酶 (AChE) 的单克隆抗体。使用羟磷灰石和色谱柱对 AChE 进行了部分纯化。使用商业多克隆抗体（兔抗鱼 AChE）通过 Western blot 研究了 AChE 的特异性特征。发现蛋白质条带的分子量为 71 kDa。在给小鼠注射 4 次 AChE 后，脾脏对诱导产生了反应。从小鼠脾脏中提取多克隆 B 细胞并与骨髓瘤细胞融合，产生杂交细胞。进行两次融合后，通过斑点印迹、ELISA、免疫组织化学和 Western blot 技术选择了针对 AChE 的克隆。发现了两个克隆，ACHE 33 和 ACHE 99，它们的同种型为 IgM。这两种抗体都可以特异性识别变性和天然形式的 AChE。来自杂交鲶鱼的 ACHE 33 单克隆抗体克隆可以与两种商业淡水鱼尼罗河罗非鱼和攀鲈通过斑点印迹、免疫组织化学和 Western blot 技术发生交叉反应。此外，暴露于草甘膦除草剂的尼罗河罗非鱼和攀鲈中的 AChE 与 ACHE 33 反应，产生分子量为 66 kDa 的蛋白质。根据我们的结果，所产生的单克隆抗体表现出特异性，可用于检测 AChE 表达，以评估杂交鲶鱼、尼罗河罗非鱼和攀鲈中的草甘膦除草剂污染。它也可以作为指示水资源质量的有用工具。
