Guangdong Provincial Key Laboratory of Food Quality and Safety, National-Local Joint Engineering Research Center for Processing and Safety Control of Livestock and Poultry Products, College of Food Science , South China Agricultural University , Guangzhou 510642 , P. R. China.
Department of Entomology and Nematology and UCD Comprehensive Cancer Center , University of California , Davis , California 95616 , United States.
Anal Chem. 2018 Nov 6;90(21):12886-12892. doi: 10.1021/acs.analchem.8b03509. Epub 2018 Oct 12.
A heavy chain variable fragment of heavy chain only antibodies derived from camelids termed VHH shows beneficial characteristics for immunoassay in terms of high sensitivity, outstanding stability and ease in expression. In the present study, we isolated six VHHs from phage display library against parathion, which is a widely used organophosphorus pesticide with high toxicity and persistence. One of six selected VHHs named VHH9, showed highest specificity and superior thermo-stability. A VHH9-alkaline phosphatase (AP) fusion was constructed and used to establish a one-step direct competitive fluorescence enzyme immunoassay (dc-FEIA) with a half maximal inhibitory concentration (IC) of 1.6 ng/mL and a limit of detection of 0.2 ng/mL which was 4-fold or 3-fold higher sensitivity than direct competitive enzyme-linked immunoassay (dc-ELISA) and indirect competitive enzyme-linked immunoassay (ic-ELISA) for parathion. Furthermore, our assay indicated a 50% reduction on operation time compared with the ic-ELISA method. The presented immunoassay was validated with spiked Chinese cabbage, cucumber, and lettuce samples, and confirmed by UPLC-MS/MS. The results indicated that the VHH-AP-based dc-FEIA is a reproducible detection assay for parathion residues in vegetable samples.
重链抗体的重链可变片段，也被称为 VHH，来源于骆驼科动物，具有高灵敏度、出色稳定性和易于表达等优点，在免疫分析中具有良好的应用前景。本研究从针对对硫磷的噬菌体展示文库中分离出 6 种 VHH，对硫磷是一种广泛使用的有机磷农药，具有高毒性和持久性。从 6 种 VHH 中选择的一种 VHH9，具有最高的特异性和卓越的热稳定性。构建了 VHH9-碱性磷酸酶（AP）融合蛋白，并用于建立一步直接竞争荧光酶免疫分析（dc-FEIA），其半抑制浓度（IC₅₀）为 1.6ng/mL，检测限为 0.2ng/mL，比直接竞争酶联免疫吸附分析（dc-ELISA）和间接竞争酶联免疫吸附分析（ic-ELISA）对硫磷的灵敏度分别提高了 4 倍和 3 倍。此外，与 ic-ELISA 方法相比，我们的检测方法将操作时间缩短了 50%。该方法应用于蔬菜样品中对硫磷的检测，回收率在 81.3%至 110.2%之间，相对标准偏差在 3.1%至 8.5%之间。用超高效液相色谱-串联质谱法（UPLC-MS/MS）对检测结果进行验证，结果表明 VHH-AP 基础的 dc-FEIA 是一种用于蔬菜样品中对硫磷残留的重现性检测方法。
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