Construction and characterization of a class-specific single-chain variable fragment against pyrethroid metabolites
Construction and characterization of a class-specific single-chain variable fragment against pyrethroid metabolites
复制标题
针对拟除虫菊酯代谢物的类特异性单链可变片段的构建和表征
DOI：
10.1007/s00253-020-10728-3
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发表时间：
2020-07-15
影响因子：
5
通讯作者：
Liu, Xianjin
中科院分区：
文献类型：
--
作者：
Liu, Yuan;Liu, Dan;Liu, Xianjin
摘要拟除虫菊酯是一种广泛应用于城乡的杀虫剂。进入环境后，拟除虫菊酯通过各种生物或非生物方法迅速代谢或降解。本研究以3-苯氧基苯甲酸（3-PBA）杂交瘤为基础，构建了能同时检测3种拟除虫菊酯代谢产物的单链抗体（scFv）。通过分子对接，发现3-PBA与单链抗体之间存在氢键、疏水相互作用、CH-π相互作用和阳离子-π相互作用。所有参与形成氢键的接触残基都位于VH-CDR 2或其邻近区域，其中两个是最近种系序列的突变体。基于竞争性ELISA，计算出scFv对3-PBA、3-苯氧基苯甲醛（PBAld）和3-苯氧基苄醇（PBAlc）的半数最大抑制浓度（IC 50）分别为0.55、0.59和0.63 μgmL-1。与苯菊酯、溴氰菊酯、氰戊菊酯、高效氯氰菊酯和甲氰菊酯的交叉反应率分别为23.91%、13.41%、1.15%、1.00%和0.56%。scFv的广泛特异性可能是由于其半抗原设计。该单链抗体可用于含苯氧基苄基（PB）基团的拟除虫菊酯代谢物的类特异性免疫分析。此外，该单链抗体还可用于拟除虫菊酯的降解表征或PBAlc（PBAld）的单独检测，检测结果还需通过其他选择性方法确认。关键点·构建了可同时检测3-PBA、PBAlc和PBAld的单链抗体。·获得了抗体信息学和单链抗体的结合模式。讨论了其广泛特异性的原因。·与PB基团联用，可用于监测单一或多种拟除虫菊酯代谢产物。
AbstractPyrethroids are insecticides that are widely used in rural and urban areas worldwide. After entering the environment, pyrethroids are rapidly metabolized or degraded by various biological or abiotic methods. In this study, a single-chain variable fragment (scFv) which could simultaneously detect three pyrethroid metabolites was constructed based on a hybridoma raised against 3-phenoxybenzoic acid (3-PBA). By molecular docking, it showed that there were hydrogen bonds, hydrophobic interactions, CH-π interaction, and cation-π interaction between 3-PBA and its scFv. All the contact residues contributing to hydrogen bonds are located in VH-CDR2 or its neighboring region, and two of them were mutants of the closest germline sequence. Based on competitive ELISA, the half maximal inhibitory concentration (IC50) of the scFv for 3-PBA, 3-phenoxybenzaldehyde (PBAld), and 3-phenoxybenzyl alcohol (PBAlc) were calculated to be 0.55, 0.59, and 0.63 μgmL−1, respectively. The scFv also showed 23.91%, 13.41%, 1.15%, 1.00%, and 0.56% cross-reactivity with phenothrin, deltamethrin, fenvalerate, beta-cypermethrin, and fenpropathrin. The broad specificity of the scFv may be due to its hapten design. The scFv could be employed in class-specific immunoassays for pyrethroid metabolites with phenoxybenzyl (PB) group. It is also potentially used for characterizing degradation of pyrethroids or detecting PBAlc (PBAld) alone, and the detection results should be confirmed by other selective methods.Key points•A scFv which can simultaneously detect 3-PBA, PBAlc, and PBAld was constructed.•Antibody informatics and binding mode of the scFv were obtained.•The reason for its broad specificity was discussed.•It could be used to monitor single or multi-pyrethroid metabolites with PB group.