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

Staphylococcus aureus (S. aureus) is globally recognized as a foodborne pathogen that causes food poisoning by secreting enterotoxins. In this study, a novel dual-recognition strategy for detecting S. aureus 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 S. aureus. However, porcine IgG can also bind to protein G, which poses the risk of generating false-positive signals from protein G-producing Streptococci. In contrast, canine IgG demonstrates a higher affinity for protein A than for protein G. When S. aureus 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 S. aureus 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 Streptococci, achieving specificity over non-target species. This approach highlights its potential for application in diverse sample matrices, including clinical and biological specimens.