抗杀螟硫磷生物素化纳米抗体的制备及其在免疫检测方法中的应用

本研究制备了一种抗杀螟硫磷生物素化纳米抗体并将其应用于免疫分析方法检测食品中杀螟硫磷残留。将杀螟硫磷人工抗原免疫羊驼,四次免疫后从外周血单核细胞获取羊驼的重链抗体可变区(VHH)基因,构建了纳米抗体基因文库,库容量为10~7 cfu。通过噬菌体表面展示技术进行四轮淘筛,得到9株不同序列的纳米抗体,优选了灵敏度最高的Nbsm6克隆。将Nbsm6基因克隆到p INQ载体中进行定向生物素化并可溶性表达与纯化,用于构建间接竞争酶联免疫分析方法。在最优工作条件下,该方法检测杀螟硫磷半抑制浓度(IC_(50))为2.0 ng/m L,线性范围(IC_(20)～IC_(80))为0.6～6.9 ng/m L,检测限(IC_(10))为0.3 ng/m L。选择白菜、生菜及橘子为样品进行添加回收实验,样品经Qu ECh ERS净化后稀释20倍可消除基质效应,添加回收率在88.9%～117.4%之间,变异系数(CV)在6.2%～16.3%之间。该方法灵敏度高,样品前处理方便,可用于杀螟硫磷的快速筛查。

Production of Anti-fenitrothion Biotinylated Nanobody and the Application in the Development of Immunoassay

In this study, an enzyme-linked immunoassay (ELISA) based on oriented biotinylated nano-antibody was developed for the determination of fenitrothion residues in food. The nano-antibody genes was obtained from peripheral blood mononuclear cells of the alpaca after four times of immunizations based on the artificial antigens of fenitrothion. The nano-antibody gene library was constructed with the capacity of 107 cfu. Nine clones of nanobodies with different amino acid sequences were isolated from the library after four rounds of panning by phage display technology, of which Nbsm6 showed the highest sensitivity. The Nbsm6 gene was cloned into the pINQ vector for biotinylation expression to prepare biotinylated nano-antibody and used for the development of indirect competitive ELISA. Under the optimization condition, the proposed assay showed an IC50 of 2.0 ng/mL, a detection linear range (IC20~IC80) of 0.6-6.9 ng/mL and a limit of detection (IC10) of 0.3 ng/mL. The assay was applied to determine fenitrothion in Chinese cabbage, lettuce and orange samples. The sample was extracted and purified by QuEChERS and then diluted by 20 time to eliminate matric effects. The recovery rate of addition was between 88.9%~117.4%, and the coefficient of variation (CV) was between 6.2%~16.3%. The proposed assay has high sensitivity and is convenient for sample pretreatment, thus can be used for rapid screening of fenitrothion.