Rapid detection and semi-quantification of IgG-accessible Staphylococcus aureus surface-associated antigens using a multiplex competitive Luminex assay

The surface characterization of Staphylococcus aureus is currently labor intensive and time consuming. Therefore, we developed a novel method for the rapid yet comprehensive characterization of S. aureus cell-surface-associated proteins and carbohydrates, based on a competitive Luminex assay. In this assay, various S. aureus bacteria or culture supernatant dilutions are exposed to human pooled serum. S. aureus specific IgGs from the serum are captured to the IgG-accessible staphylococcal antigens present on the bacterial surface, or in supernatant, in a dose dependent manner. The remaining non-captured IgGs are then quantified by incubation with Luminex beads to which the individual recombinant S. aureus surface proteins and carbohydrates are covalently linked. The signals obtained are inversely proportional to the antigen concentration measured, and binding patterns were dependent on the particular S. aureus isolate used, growth conditions and growth phase. For example, the detection of cell wall teichoic acid (WTA) and peptidoglycan (PG) carbohydrate structures increased significantly when bacteria entered the stationary growth phase, whereas the amounts of clumping factors (ClfA and ClfB) decreased significantly. Using this technique, the appearance of several S. aureus gene knockout strains were studied. We demonstrated that in an S. aureus srtA gene knockout strain, srtA-dependent cell-surface-associated proteins were detectable yet not covalently linked to the PG layer. In conclusion, we show that this multiplex competitive Luminex assay is a suitable method for the rapid and simultaneous determination of S. aureus IgG-accessible cell-surface-associated proteins and carbohydrates in various growth phases and growth conditions in different S. aureus strains.

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