Breaking the Sensitivity-Convenience Trade-off: A Camelid HCAb-Based Immunoassay for Multi-Residue Detection of Antibacterial Synergists in Foods with Minimized Pretreatment

The instability of conventional antibodies (ConAbs) in food matrix or target extraction reagents often limits the convenience or sensitivity of immunoassays. In this study, alpacas were immunized with water-soluble adjuvants to induce heavy chain antibodies (HCAbs) against antibacterial synergists (ASGs). Compared to ConAb C₅, the HCAb H₅ exhibited superior stability (binding activity >80% and IC₅₀ change <2-fold) under harsh conditions (63 °C, pH 3.0-11.0, 6.0 mol L^-1 NaCl, 30% methanol, 30% acetonitrile). An indirect competitive ELISA (icELISA) based on H5 showed IC₅₀ values of 1.24, 1.44, 1.60, and 2.11 μg L^-1 for trimethoprim (TMP), dimethoprim (DVD), ormetoprim (OMP), and baquiloprim (BQP), with cross-reactivity rates of 100%, 86.3%, 77.7%, and 56%, respectively. Sample preparation was simplified to minimal dilution steps─chicken, egg, pork, and milk samples were diluted only 2-, 4-, 4-, and 8-fold, respectively─and the entire pretreatment could be completed within 1-8 min. The limits of detection (LODs) ranged from 0.14 to 1.36 μg L^-1/μg kg^-1, with recoveries of 76.4-107.3%, and coefficients of variation (CV) below 14.1%. This is the first HCAb-based icELISA for ASGs, offering high sensitivity, robustness, and minimized pretreatment for complex food matrices.