Enhanced Specificity in Dual-Recognition Colorimetric Determination of <i>Staphylococcus aureus</i> by Incorporating Canine Immunoglobulin G into Vancomycin Functionalized Magnetic Beads

<i>Staphylococcus aureus</i> (<i>S. aureus</i>) is globally recognized as a foodborne pathogen that causes food poisoning by secreting enterotoxins. In this study, a novel dual-recognition strategy for detecting <i>S. aureus</i> was developed, employing vancomycin-functionalized magnetic beads (Van-MBs) for target capture and canine immunoglobulin G (IgG) to selectively and sensitively bind to staphylococcal protein A (SPA) on the bacterial surface. Dual-recognition strategies based on vancomycin and porcine IgG have previously been developed for the detection of <i>S. aureus</i>. However, porcine IgG can also bind to protein G, which poses the risk of generating false-positive signals from protein G-producing <i>Streptococci</i>. In contrast, canine IgG demonstrates a higher affinity for protein A than for protein G. When <i>S. aureus</i> is present in the sample matrix, the Van-MBs and biotin-labeled canine IgG successfully form a sandwich complex. Then, streptavidin-conjugated horseradish peroxidase (HRP) is added, which binds to the sandwich structure. HRP then catalyzes the oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB), resulting in the formation of a colorimetric signal that was proportional to the concentration of <i>S. aureus</i> within the concentration range of 6.3 × 10³–4.0 × 10⁵ CFU/mL with a detection limit of 1.9 × 10³ CFU/mL. By substituting porcine IgG with canine IgG, this method eliminates cross-reactivity with protein G-expressing <i>Streptococci</i>, achieving specificity over non-target species. This approach highlights its potential for application in diverse sample matrices, including clinical and biological specimens.