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