Staphylococcus aureus causes a variety of infections. Knowledge about the physiological role of most S. aureus antigens in colonization and infection is only limited. This can be studied by measuring antigen-specific antibody responses. In this study, we optimized the multiplex microsphere bead-based flow cytometry technique for mouse serum samples. We analysed immunoglobulin G (IgG) levels directed against 26 S. aureus proteins in a single small-volume mouse serum sample. We assessed possible cross reactivity. Furthermore, we analysed serum samples from mice with different types of S. aureus infections caused by different S. aureus strains. The results show that cross reactivity between proteins on microspheres and serum antibodies towards other proteins was limited. We found that lung-infected mice had a higher and broader IgG response than skin-infected mice. Clearly, the site of infection influences the IgG profile. Next, we compared sera from mice with intravenously-induced bacteraemia caused by different S. aureus strains. We showed different IgG responses depending on the causing S. aureus strain. It is concluded that the bead-based multiplex S. aureus antibody assay can be successfully applied to determine the immunogenicity of different S. aureus proteins in relation to the site of infection and the S. aureus strain causing the infection. (C) 2010 Elsevier B.V. All rights reserved.