Microcystins (MCs) are a group of highly potent cyanotoxins that are becoming more widely distributed due to increased global temperatures and climate change. Microcystin-leucine-arginine (MC-LR) is the most potent and most common variant, with a guideline limit of 1 μg/l in drinking water. We previously developed a novel avian single-chain fragment variable (scFv), designated 2G1, for use in an optical-planar waveguide detection system for microcystin determination. This current work investigates interactions between 2G1 and MC-LR at the molecular level through modelling with an avian antibody template and molecular docking by AutoDock Vina to identify key amino acid (AA) residues involved. These potential AA interactions were investigated in vitro by targeted mutagenesis, specifically, by alanine scanning mutations. Glutamic acid (E) was found to play a critical role in the 2G1-MC-LR binding interaction, with the heavy chain glutamic acid (E) 102 (H-E102) forming direct bonds with the arginine (R) residue of MC-LR. In addition, alanine mutation of light chain residue aspartic acid 57 (L-D57) led to an improvement in antigen-binding observed using enzyme-linked immunosorbent assay (ELISA), and was confirmed by surface plasmon resonance (SPR). This work will contribute to improving the binding of recombinant anti-MC-LR to its antigen and aid in the development of a higher sensitivity harmful algal toxin diagnostic.

中文翻译：

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使用计算机对接和体外诱变了解微囊藻毒素-LR抗体的结合相互作用。

微囊藻毒素（MC）是一组高效的氰毒素，由于全球温度升高和气候变化，它们的分布越来越广泛。微囊藻氨酸-亮氨酸-精氨酸（MC-LR）是最有效，最常见的变体，饮用水中的指导限值为1μg/ l。我们先前开发了一种新颖的禽类单链片段变量（scFv），命名为2G1，用于光学平面波导检测系统中的微囊藻毒素测定。这项当前工作通过使用禽类抗体模板进行建模并通过AutoDock Vina分子对接来鉴定涉及的关键氨基酸（AA）残基，从而在分子水平上研究2G1与MC-LR之间的相互作用。这些潜在的AA相互作用进行了体外研究通过定向诱变，特别是通过丙氨酸扫描突变。发现谷氨酸（E）在2G1-MC-LR结合相互作用中起关键作用，重链谷氨酸（E）102（H-E102）与MC-的精氨酸（R）残基形成直接键LR。此外，轻链残基天冬氨酸57（L-D57）的丙氨酸突变导致酶联免疫吸附测定（ELISA）观察到的抗原结合改善，并通过表面等离振子共振（SPR）得以证实。这项工作将有助于改善重组抗MC-LR与其抗原的结合，并有助于开发更高灵敏度的有害藻毒素诊断试剂。