College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China; State & Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing 210095, China.
College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China; State & Local Joint Engineering Research Center of Green Pesticide Invention and Application, Nanjing 210095, China.
J Hazard Mater. 2022 Mar 5;425:128011. doi: 10.1016/j.jhazmat.2021.128011. Epub 2021 Dec 7.
Clothianidin is a widely used pesticide that has been banned from outdoor use by the European Union due to its toxicity. To improve the sensitivity and specificity of existing clothianidin immunoassays, we developed competitive and noncompetitive immunoassays for clothianidin based on phage-displayed peptides. Cyclic 8-, 9-, and 10-residue peptide libraries were constructed using an optimized phagemid pComb-pVIII to prevent the loss of theoretical library diversity. Twenty-eight peptidomimetics and two anti-immunocomplex peptides were isolated through a blended panning process and used to develop competitive and noncompetitive phage enzyme-linked immunosorbent assays (P-ELISAs), respectively. After optimization, the half inhibition concentration (IC) and half saturation concentration (SC) of competitive and noncompetitive P-ELISAs were 3.83 ± 0.23 and 0.45 ± 0.02 ng/mL, respectively. Competitive P-ELISA showed 2.6-18.2% cross-reactivity with imidaclothiz, nitenpyram and imidacloprid. Importantly, noncompetitive P-ELISA, which has the best specificity and great sensitivity for clothianidin, showed no cross-reactivity with the analogs. The average recoveries of competitive and noncompetitive P-ELISAs were 73.8-104.1% and 76.6-102.2%, respectively, while the relative standard deviations were ≤ 11.0%. In addition, the results of P-ELISAs in the analysis of blind samples were consistent with those of high-performance liquid chromatography.
噻虫啉是一种广泛使用的农药，由于其毒性已被欧盟禁止在户外使用。为了提高现有噻虫啉免疫分析的灵敏度和特异性，我们基于噬菌体展示肽开发了噻虫啉的竞争和非竞争免疫分析。使用优化的噬菌粒 pComb-pVIII 构建了 8、9 和 10 残基环肽文库，以防止理论文库多样性的损失。通过混合淘选过程分离了 28 个肽模拟物和 2 个抗免疫复合物肽，并分别用于开发竞争和非竞争噬菌体酶联免疫吸附测定（P-ELISA）。经过优化，竞争和非竞争 P-ELISA 的半抑制浓度（IC）和半饱和浓度（SC）分别为 3.83±0.23 和 0.45±0.02 ng/mL。竞争 P-ELISA 对噻虫脒、吡虫啉和噻虫嗪的交叉反应率为 2.6-18.2%。重要的是，具有最佳特异性和对噻虫啉高灵敏度的非竞争 P-ELISA 与类似物无交叉反应。竞争和非竞争 P-ELISA 的平均回收率分别为 73.8-104.1%和 76.6-102.2%，相对标准偏差均≤11.0%。此外，盲样分析中 P-ELISA 的结果与高效液相色谱法的结果一致。
