Detection of kanamycin residues in animal-derived foods using a homogeneous AlphaLICA immunoassay

Abstract

This study establishes a novel homogeneous immunoassay method based on the Amplified Luminescent Interfacial Homogeneous Detection (AlphaLICA) technology for detecting kanamycin residues in animal-derived foods (milk and honey), and systematically evaluates its analytical performance and application value. Using kanamycin-coupled bovine serum albumin (Kana-BSA) as the antigen and anti-kanamycin monoclonal antibody as the core recognition element, functionalized donor microspheres (conjugated with goat anti-mouse IgG) and receptor microspheres (conjugated with Kana-BSA) were prepared via the carbodiimide method. To construct a streamlined homogeneous competitive immunoassay workflow, a systematic optimization process was conducted, focusing on three pivotal parameters: the working concentration of microspheres, the concentration of the primary antibody, and the immunoreaction duration. The method's sensitivity, specificity, precision, accuracy, and linear range were rigorously validated. Practical performance was assessed through spiked sample detection and comparison with a commercial ELISA kit. This study successfully developed the Kana-AlphaLICA assay. Under optimized conditions, the linear range for kanamycin detection was 0.1–102.4 ng mL⁻¹, with limits of detection (LOD) and quantification (LOQ) at 0.023 ng mL⁻¹ and 0.083 ng mL⁻¹, respectively. The standard curve equation was logit(Y) = −2.20303X + 0.39361 (R² = 0.99818). The method demonstrated excellent specificity, with cross-reactivity rates below 0.04% against 12 structurally and functionally similar antibiotics. Spiked recovery rates in milk and honey matrices ranged from 93.31% to 108.88%, with intra- and inter-batch coefficients of variation (CV) below 6.20% and 7.86%, respectively. Testing of 18 spiked commercial samples demonstrated high correlation between this method and ELISA (R² = 0.9123), while exhibiting significant advantages in sensitivity (4.35-fold improvement) and linear range (12.79-fold expansion). The established Kana-AlphaLICA method offers high sensitivity, strong specificity, and simple, rapid operation, providing a reliable technical approach for high-throughput, highly sensitive screening of kanamycin residues in animal-derived foods.