All analyses of variances employed the NCSS Quick Start 2001 software. Recombinant NcPDI was expressed in E. coli and purified by Ni2+-affinity chromatography, yielding a single protein band, migrating on an SDS–PAGE gel at approximately 55 kDa (Figure 1). Following loading of nanogels with recNcPDI, samples were subjected to ultracentrifugation, to determine how efficiently the recNcPDI antigen was associated with the nanogel particles. Silver stain and Western

blotting with a polyclonal rat anti-recNcPDI antiserum was used to identify recNcPDI antigen. The ultracentrifugation did not precipitate the nanogel-free protein (Figure 1a,b, lane 1 ‘supernatant’ compared with lane 2 ‘pellet’). In contrast,

when the recNcPDI-nanogel preparations were employed, all detectable recNcPDI was associated with the nanogels in the pellet (Figure 1a,b, lane 4 ‘pellet’ PD-0332991 supplier compared with lane 3 ‘supernatant’). Enzalutamide cost The recNcPDI antigen was also successfully incorporated into the chitosan/alginate-mannose nanogels (Figure 1a,b, lane 6 ‘pellet’ compared with lane 5 ‘supernatant’). It appeared that the majority of the recNcPDI detected when associated with the chitosan/alginate nanogels was reduced in size compared with the chitosan/alginate-mannose-associated material (Figure 1a,b, lane 4 compared with lane 6), but this may have been influenced by the recNcPDI association with the chitosan prior to the denaturation employed for the SDS–PAGE. Nevertheless, recNcPDI protein associated with the chitosan/alginate nanogels was still recognized by the anti-recNcPDI antiserum

(Figure 1b, lane 4). Following vaccination Phospholipase D1 with various formulations, either by i.p. or i.n. delivery, (see Table 1), mice were inspected daily for the presence of local reactions at the inoculation sites. No such reactions were found during the experiment (data not shown). The body weights of all mice were monitored at 3-day intervals, starting at the time of the first vaccination. They remained similar (at 22 ± 0·5 g), regardless of the vaccination procedure employed (data not shown). This indicated that vaccination had no adverse effects and suggested that all immunization procedures were safe and did not impose stressful conditions that would interfere with the general metabolic activity of the animals. Mice were then monitored in terms of the clinical signs (ruffled coat, hind limb paralysis, circular movements, apathy and inability to reach up for feeding). These were first detected in the saponin-treated control mice of group 1 (SAP) at day-9 PI (Table 2). Subsequently, all mice of groups 1 and 2 (SAP and 10PDI-SAP), which were vaccinated i.p., succumbed to infection prior to termination of the experiment. The last mouse to succumb was on day 32 PI.