AlphaLICA-based homogeneous immunoassay for highly sensitive detection of streptomycin in infant formula

Abstract

This study developed a novel streptomycin immunodetection method based on the Amplified Luminescence Proximity Homogeneous Assay (AlphaLICA), termed STR-AlphaLICA, for the highly sensitive and rapid detection of streptomycin residues in infant formula. The method was established by conjugating streptomycin–bovine serum albumin (STR–BSA) to receptor microspheres and goat anti-mouse IgG antibody to donor microspheres, leveraging the principle of competitive binding between free streptomycin and the immobilized STR–BSA for specific monoclonal antibodies. In the homogeneous system, fluorescence signals are generated through singlet oxygen energy transfer upon 680 nm laser excitation, allowing direct detection without washing steps. The established standard curve demonstrated excellent linearity (r = 0.99977) within the range of 0.1–312.5 ng mL⁻¹, with a detection limit (LOD) of 0.169 ng mL⁻¹ and a quantification limit (LOQ) of 0.776 ng mL⁻¹. Validation results showed excellent intra- and inter-batch precision (CV < 5.02%) and spiked recovery rates ranging from 91.21% to 106.64%. Cross-reactivity with the structural analog dihydrostreptomycin was 69.87%, while responses to other common antibiotics remained below 0.05%, indicating high specificity. Furthermore, validation results in five complex matrices—including honey and shrimp meat—further demonstrate the method's excellent universality and reliability. In actual sample testing, the results showed significant correlation (r² = 0.9414) when compared with commercial ELISA kits, confirming the method's suitability for high-throughput screening and precise quantification of streptomycin residues in complex matrices such as infant formula. This provides new technical support for food safety monitoring.