The absorbance ACP-196 research buy at 450 nm was measured with an ELISA plate reader (Multiskan EX, Labsystems). The purity of the commercial fibronectin used in these assays was examined by SDS-PAGE. ELISA experiments with anti-fibrinogen antibodies revealed that the fibronectin was free of

fibrinogen contamination. ELISA assays Various concentrations of recombinant FnBPB A domain proteins in PBS were coated onto Nunc 96-well microtitre dishes for 18 h at 4°C. Wells were washed and blocked with BSA for 2 h as described above. Following three washes with PBST, 100 μl of anti-FnBPB A domain antibodies diluted in BSA-PBST (1.8 μg polyclonal IgG ml-1; 2.5 μg monoclonal IgG ml-1) were added to each well and incubated for 1 h at room temperature with shaking. Polyclonal antibody raised against the isotype I N23 domain of FnBPB was obtained by immunizing specific pathogen-free rabbits ABT-737 nmr with rFnBPB37-480 from S. aureus 8325-4. Monoclonal antibody 12E11 was generated by immunizing mice with recombinant isotype I FnBPB37-480. After 1 h incubation the wells were washed three times with PBST. Goat anti-rabbit IgG-HRP conjugated antibodies or goat anti-mouse IgG-HRP conjugated antibodies (Dako, Denmark), each diluted 1:2000 in BSA-PBST, were added to the wells and incubated for 1 h. After washing three times with PBST, bound HRP-conjugated antibodies were detected as described above. Analysis

of fibrinogen, elastin and fibronectin binding by surface plasmon resonance Surface plasmon resonance (SPR) was preformed using the BIAcore ×100 system (GE Healthcare). Human fibrinogen (4EGI-1 cell line Calbiochem), aortic elastin (Enzyme Research Laboratories) and fibronectin (Calbiochem) were covalently immobilized on CM5 sensor chips using amine coupling. This was performed using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), followed by N-hydroxysuccinimide (NHS) and ethanolamine hydrochloride, as described by the manufacturer. Fibrinogen (50 μg/ml), elastin (50 μg/ml) and fibronectin (50 μg/ml) were Glycogen branching enzyme dissolved in 10 mM sodium acetate at pH 4.5 and immobilized on separate

chips at a flow rate of 30 μl/min in PBS (Gibco). Each chip contained a second flow cell, which was uncoated to provide negative controls. All sensorgram data presented were subtracted from the corresponding data from the blank cell. The response generated from injection of buffer over the chip was also subtracted from all sensorgrams. Equilibrium dissociation constants (Kd) were calculated using the BIA ×100 evaluation software version 1.0. Acknowledgements We wish to acknowledge support from Trinity College Dublin for a postgraduate scholarship (for FMB). The work was supported by Grant 08/IN.1/B1845 from Science Foundation Ireland to TJF and Fondazione CARIPLO (Italy) and Fondo di Ateneo per la Ricerca (Pavia, Italy) to PS References 1. van Belkum A, Verkaik NJ, de Vogel CP, Boelens HA, Verveer J, Nouwen JL, Verbrugh HA, Wertheim HF: Reclassification of Staphylococcus aureus nasal carriage types.