Electrofusion preparation of anti-triazophos monoclonal antibodies for development of an indirect competitive enzyme-linked immunosorbent assay.

Immunoassays have been widely used to detect small molecular contaminants due to the advantages of simplicity, high throughout and low-cost. Antibodies are essential reagents of immunoassays, their quality directly determines the characteristics of immunoassays. In this study, the monoclonal antibodies (mAbs) of triazophos were prepared by electrofusion, and used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Under the optimal electrofusion conditions (cells treatment with pronase, the alternating electric field strength of 45 V cm-1, the direct current voltage of 3 kV), the fusion efficiency was 1.104 ± 0.063‱, which was improved more than 4-fold compared with the chemical fusion method (0.255 ± 0.089‱). Three hybrid cell lines that can stably secrete the anti-triazophos mAbs were obtained. The cell line 4G6F10 showed the highest sensitivity, which was used to generate mAb and develop an ic-ELISA. After optimization, the 50% inhibition concentration (IC50), limit of detection (LOD) and linear range (IC10-IC90) of the ic-ELISA were 0.32 ng mL-1, 0.08 ng mL-1 and 0.08-2.17 ng mL-1, respectively. There was no significant cross-reactivity with the analogues of triazophos. The average recoveries of triazophos in spiked samples were 77.5%-89.3% with the relative standard deviations of 0.1%-9.2%. In addition, the ic-ELISA showed good repeatability, reproducibility and accuracy for the analysis of apple samples spiked with triazophos.

Development of immunoassays with high sensitivity for detecting imidacloprid in environment and agro-products using phage-borne peptides.

Imidacloprid is the most widely used neonicotinoid insecticide and has been reported to pose a threat to ecological security and human health. Therefore, simple-to-operate and highly sensitive methods for the detection of trace levels of imidacloprid are necessary. Here, we isolated two phage-borne peptides that compete with imidacloprid to bind the monoclonal antibody (mAb) 3D11 from phage display peptide libraries. A phage-enzyme-linked immunosorbent assay (P-ELISA) and two phage time-resolved fluoroimmunoassays (P-TRFIAs) for the detection of imidacloprid were developed using the phage-borne peptides as substitutes for chemically synthesized antigens. After systematic optimization, the half-maximum inhibition concentrations (IC50) of the P-ELISA, P-TRFIA-1, and P-TRFIA-2 were 0.067 ng mL-1, 0.085 ng mL-1, and 0.056 ng mL-1, respectively. Based on their IC50 values, the sensitivities of the P-ELISA and P-TRFIAs were more than four times greater than those of previous immunoassays. Additionally, the immunoassays showed satisfactory recovery in the detection of spiked samples and good correlation with high performance liquid chromatography (HPLC) for the detection of samples containing incurred residues.