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.
Mikrochim Acta. 2023 Dec 30;191(1):68. doi: 10.1007/s00604-023-06151-w.
Peptidomimetic and anti-immunocomplex peptides can be easily isolated from phage display libraries, and can be used as alternatives to chemical competing haptens to improve the sensitivity of small molecule immunoassay. In this work, 16 peptidomimetics and 7 anti-immunocomplex peptides of pendimethalin (PND) were obtained from cyclic 7-, 8-, 9-, and 10-residue peptide phage libraries. Peptidomimetic EJ-2 (CMFTGTDFPC) with the highest sensitivity in competitive phage enzyme-linked immunosorbent assay (ELISA) and immunocomplex peptide EF-30 (CNPGWPPIPC) with the highest sensitivity in noncompetitive phage ELISA were selected to prepare phage-free peptides with GGGSSK-biotin at the C-terminus. Competitive and noncompetitive lateral flow immunoassays (CLFIA and NLFIA) were developed by using the phage-free peptides. After optimization, the CLFIA and NLFIA showed visual limit of detections (vLODs) of 5 ng/mL and 2.5 ng/mL, respectively, which were improved two- and fourfold compared with a LFIA based on chemical hapten. The NLFIA showed better sensitivity than CLFIA in the detection of spiked samples, and can meet the detection requirements for agro-products regulated by EU and China. The detection results of CLFIA and NLFIA for blind samples were consistent with that of ultra performance liquid chromatography/tandem mass spectrometry.
从噬菌体展示文库中可以轻松分离出肽模拟物和抗免疫复合物肽,并可以替代化学竞争半抗原,从而提高小分子免疫分析的灵敏度。在这项工作中,从环 7、8、9 和 10 残基肽噬菌体文库中获得了 16 个苯达松(PND)肽模拟物和 7 个抗免疫复合物肽。在竞争噬菌体酶联免疫吸附测定(ELISA)中具有最高灵敏度的肽模拟物 EJ-2(CMFTGTDFPC)和在非竞争噬菌体 ELISA 中具有最高灵敏度的免疫复合物肽 EF-30(CNPGWPPIPC)被选择用于制备末端带有 GGGSSK-生物素的无噬菌体肽。通过使用无噬菌体肽开发了竞争和非竞争侧向流动免疫分析(CLFIA 和 NLFIA)。经过优化,CLFIA 和 NLFIA 的可见检测限(vLOD)分别为 5ng/mL 和 2.5ng/mL,与基于化学半抗原的 LFIA 相比,灵敏度提高了两倍和四倍。在检测加标样品时,NLFIA 的灵敏度优于 CLFIA,可满足欧盟和中国规定的农产品检测要求。CLFIA 和 NLFIA 对盲样的检测结果与超高效液相色谱/串联质谱法一致。