After overnight incubation, cells were washed gently with 200 μl of Dulbecco’s PBS (Sigma) and fixed with 70 μl ice-cold ethanol for 2 min. The ethanol was then removed and 70 μl crystal violet (1%, w/v, in ethanol; Pro-Lab) added to the fixed cells for 30 min at 22 °C. Plates were washed carefully in water to remove excess dye, dried at 37 °C and then 200 μl of 50% (v/v) ethanol added. Plates were incubated in a shaker incubator (37 °C; 300 rpm) for 2 h then read at 492 nm. ED50 Alectinib datasheet values were derived from the resulting toxin neutralisation

curves using 4 or 5-pl nonlinear regression models (SigmaPlot 12.0). The Syrian hamster model was performed as described previously using groups of 10 animals [30]. All hamsters were weighed and administered clindamycin (2 mg in 0.2 ml sterile H2O) by the orogastric route on Day 0. On Day 2, test animals were challenged (orogastrically) with between 102 and 103 colony forming units of selleck kinase inhibitor C. difficile spores in 0.2 ml DMEM. Animals were weighed daily and monitored 6 times/day for 15 days for disease symptoms (diarrhoea, weight loss, lethargy and tender abdomen) [19] and [32]. Survival curves were analysed by log rank tests (non-parametric distribution analysis, right censoring). For passive immunisation, ovine IgG was purified from antisera generated using TxA4 and TxB4 fragments. Doses (0.5–2 ml) were administered at

various times by the intraperitoneal route (see Fig. 4). The panel of TcdB-derived fragments is summarised in Fig. 1. Construct TxB5 contained the mutation Cys700 → Ser to reduce substantially the activity of the cysteine protease (CP) domain [33]. With the exception of antigen TxB2, levels of total protein expression

and levels of soluble expression were low without the addition of an N-terminal fusion protein. Several fusion protein candidates were screened and thioredoxin and NusA were found to promote the highest levels of soluble expression. Details of the design of antigen constructs are provided as supplemental data (Fig. S1). Purified fragments were analysed by SDS-PAGE (Fig. 2) and immunoblotting. For each construct, the principal protein band reacted strongly with antibodies raised to TcdB ALOX15 [18] (data not shown). Proteomic analysis of TxB4 by GeLC–MS/MS using in-gel tryptic digestion confirmed its identity and presence of >98% of the predicted construct sequence. End points in Vero-cell assays used to assess the levels of toxin-neutralising antibodies to fragments were determined by both microscopy (complete cell protection as the endpoint) and as an ED50 in which cell integrity was assessed using crystal violet staining (Fig. 3 and Table 1). While there was a generally good correlation between the two methods used to determine toxin-neutralising titres in the cell assay, there was little correlation between these and titres obtained by ELISA.