College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China.
College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China.
J Immunol Methods. 2022 Jan;500:113184. doi: 10.1016/j.jim.2021.113184. Epub 2021 Nov 19.
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, 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 (IC), limit of detection (LOD) and linear range (IC-IC) of the ic-ELISA were 0.32 ng mL, 0.08 ng mL and 0.08-2.17 ng mL, 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.
免疫分析由于具有简单、高通量和低成本的优点，已被广泛用于检测小分子污染物。抗体是免疫分析的关键试剂，其质量直接决定了免疫分析的特点。本研究通过电融合制备了三唑磷的单克隆抗体(mAb)，并用于建立间接竞争酶联免疫吸附分析（ic-ELISA）。在最佳电融合条件下（细胞用蛋白酶处理，交变电场强度为 45 V cm，直流电压为 3 kV），融合效率为 1.104 ± 0.063‱，比化学融合法（0.255 ± 0.089‱）提高了 4 倍以上。获得了 3 株能稳定分泌抗三唑磷 mAb 的杂交瘤细胞系。细胞系 4G6F10 显示出最高的灵敏度，用于生成 mAb 并建立 ic-ELISA。经过优化，ic-ELISA 的 50%抑制浓度（IC）、检测限（LOD）和线性范围（IC-IC）分别为 0.32 ng mL、0.08 ng mL 和 0.08-2.17 ng mL。与三唑磷的类似物无明显交叉反应。在添加三唑磷的样品中，三唑磷的平均回收率为 77.5%-89.3%，相对标准偏差为 0.1%-9.2%。此外，ic-ELISA 对添加三唑磷的苹果样品的分析具有良好的重复性、重现性和准确性。
