1 M carbonate-bicarbonate selleckchem buffer, pH 9.6, coated onto a Nunc MaxiSorp® flat-bottom 96-well plate and incubated overnight at 4 °C. The plate
was washed with 0.05% PBS-Tween and blocked with 100 μL of 3% PBS-gelatin for 5 h at room temperature. Subsequently, 50 μL of twofold dilutions of the standard prepared in 1% PBS-gelatin, starting from a concentration of 32 ng mL−1, and 50 μL of the samples were added to the plate and incubated at 4 °C overnight. After washing, 50 μL of the secondary antibody diluted in 1% PBS-gelatin was added, and the plate was left at room temperature for 5 h, followed by the addition of 50 μL of 1:1000 streptavidin-peroxidase (KPL) prepared in 1% PBS-gelatin to each well and incubation at 37 °C for 30 min. The plates were developed with 100 μL well−1 of TMB (3,3′,5,5′-tetramethylbenzidine) substrate, the reaction was stopped by the addition of 100 μL well−1 of 0.2 M sulphuric acid, and A450 nm was measured. The ELISA for each cytokine was performed twice, and the samples and standards were tested in duplicates on each plate. Statistical analyses were performed using the graphpad Prism 4.0 software. The data generated from ELISAs were analysed by nonlinear regression, and interstrain comparison was performed by one-way anova. The role of surface-associated proteins and toxins of C. difficile in LY294002 datasheet infection and serum antibodies to them in determining the outcome of infection has been
clearly demonstrated (Pantosti et al., 1989; Mulligan et al., 1993; DNA ligase Péchiné et al., 2005a, b; Sánchez-Hurtado et al., 2008; Wright et al., 2008). Here, we demonstrate that toxins and surface-associated proteins from different C. difficile strains induce similar levels of production of pro-inflammatory cytokines by THP-1 macrophages. The SLPs, flagella and HSPs induced at 42 and 60 °C were extracted successfully from the five C. difficile strains, and the preparations were found to be free of endotoxin by the LAL assay. In the SLP extracts, two major bands were observed in preparations from all the five strains (Fig. 1a). As previously recorded, there was a wide variation in the molecular weights of the SLPs between
the different ribotypes (McCoubrey & Poxton, 2001; Spigaglia et al., 2011); strain 630, VPI 10463 and ribotypes 027, 001 and 106 were assigned S-layer types 5138, 5435, 5438, 5436 and 5037, respectively. In the flagella preparations, a prominent 39-kDa band (Delmée et al., 1990) was observed, which was the only band detected by Western blotting with rabbit antiserum prepared against whole UV-killed cells of C. difficile previously shown to react with flagella (McCoubrey & Poxton, 2001; Fig. 1b). A 58-kDa band was observed in HSP42 suggesting the presence of GroEL (Hennequin et al., 2001a; Fig. 1c), and three bands of approximately 66, 50 and 35 kDa were observed in HSP60 suggesting the presence of Cwp66 (Waligora et al., 2001; Fig. 1d).