SpyTagged Mimotope Peptide Mediated Competitive Antigen-Based Rapid Quantitative Immunoassays for Uniconazole Residue

Mimotope-based immunoassays offer an eco-friendly alternative to chemically synthesized antigens for the quantitative analysis of small molecules, but their use for practical on-site and high-throughput residue monitoring remains limited. Herein, we report the selection, production, and application of a phage display-derived mimotope targeting an anti-uniconazole monoclonal antibody (UCZ-mAb), with the aim of developing two complementary immunoassays that enable sensitive, eco-friendly detection of UCZ residues in agricultural samples. A 12-mer phage-displayed peptide library was screened to identify UCZ-specific mimotopes, and a selected sequence was genetically fused to SpyTag and expressed in <i>Escherichia coli</i> to generate a SpyTagged mimotope. Leveraging the SpyCatcher/SpyTag self-assembly system, the SpyTagged mimotope was directionally conjugated onto SpyCatcher-functionalized time-resolved fluorescence beads (TRFBs) and subsequently used as a signal-labeled competitive antigen in a lateral flow immunoassay (LFIA) designed for rapid on-site screening. In parallel, a wash-free magnetic separation immunoassay (MSIA) suitable for green, high-throughput screening in routine laboratories was established using self-assembled mimotope-TRFB probes. The LFIA and MSIA exhibited half-maximal inhibitory concentrations (IC₅₀) of 3.70-6.72 μg/kg and 16.4-18.3 μg/kg, respectively, in real samples. Spiked-sample recoveries ranged from 91.1 to 107.8% for LFIA and 92.6-115.7% for MSIA, demonstrating acceptable accuracy and precision. These results indicate that the SpyTagged mimotope-based LFIA and MSIA provide complementary, reliable, and sensitive platforms for on-site screening and high-throughput monitoring of UCZ residues in agricultural samples, while avoiding the drawbacks associated with traditional chemical antigen synthesis.