Experimental animal models of different S. aureus infections have been developed, and mice are frequently used as models. For quantification of circulating antibody levels, conventional immunological techniques such as the Enzyme-Linked ImmunoSorbent Assay (ELISA) can be applied. This technique is time- and serum-consuming, and antibodies against Depsipeptide in vitro only one antigen can be measured in one separate ELISA. To assess levels of antibodies directed against a broad range of antigens, multiple mice need to be

bled to yield enough serum and this may confound observations due to inter-experiment variations. The microsphere bead-based flow cytometry technique (xMap, Luminex Corporation, Austin, TX, USA) permits the simultaneous analysis of antibodies for up to 100 different antigens from a single, small-volume

serum sample (Fulton et al., 1997). To our knowledge, this technique has as yet only been used for measuring antibodies against S. aureus proteins in human serum samples ( Martins et al., 2006, Verkaik et al., 2009a and Verkaik et al., 2010b). In the present study, we optimized the Luminex technology to quantify immunoglobulin G (IgG) antibodies directed against a broad panel of S. aureus proteins in mouse serum, and we assessed cross reactivity. In addition, this technique was applied to analyse serum samples from mice with different types of S. aureus infections PS-341 manufacturer caused by different S. aureus strains. Female BALB/cOlaHsd mice (6–8 weeks old, specified pathogen free) were immunized intranasally (5 mice per group) with monovalent Gram-positive Enhancer Matrix (GEM)-based vaccines containing clumping factor A (ClfA), extracellular fibrinogen-binding protein (Efb), or toxic shock syndrome toxin 1 (TSST-1). One dose of vaccine

consisted of 2.5 × 109 GEM-particles containing 8.0, 2.0, or 2.1 μg ClfA, Efb, or TSST-1, respectively, Etofibrate in a volume of 10 μL. Another group of mice was immunized subcutaneously (4 mice per group) with monovalent GEM-based vaccines containing endonuclease (Nuc), peptidoglycan hydrolase (LytM), or immunodominant staphylococcal antigen A (IsaA). One dose of vaccine consisted of 2.5 × 109 GEM-particles containing 25.0, 10.0, or 17.5 μg Nuc, LytM, or IsaA, respectively, in a volume of 100 μL. The immunization schedule consisted of three doses given at 10-day intervals. Animal experiments were performed with approval of the Animal Experimentation Committee of the University of Groningen, The Netherlands. Sera were collected before immunization and 2 weeks after the last immunization. Sera from mice with lung infection or skin infection caused by S. aureus strain LAC (USA300) were obtained from Dr. M.G. Bowden and prepared as described ( Brown et al., 2009b). In short, female BALB/c mice (6 weeks old, specified pathogen free) were inoculated intranasally (5 × 107 CFU in 20 μL, 9 mice) for lung infection or intradermally (1 × 107 CFU in 50 μL, 10 mice) for skin infection with S. aureus strain LAC.