The Edinburgh (E) strain of
Trichuris muris was used in all experiments. Female mice (6-12 weeks old) were orally infected with 400 embryonated eggs. Mice were culled by cervical dislocation at day 32 pi, blood was collected by cardiac puncture for serum recovery, and caecum/proximal colon was dissected to inspect for worm presence by stereomicroscope. Levels of parasite-specific serum IgG1 and IgG2a Ab were by ELISA: briefly, ELISA plates were coated with
T. muris excretory/secretory (E/S) antigen at 5 μg/ml. Serum was diluted 1/40, and parasite-specific IgG1, IgG2a and IgE detected with
biotinylated anti-mouse IgG1 (Biorad),
biotinylated anti-mouse IgG2a (BD PharMingen), and
anti-mouse IgE (BioLegend), respectively. To generate
T. muris E/S antigen, live adult worms were incubated at 37°C for 24 hours in
RPMI-1640 medium (Gibco, UK) supplemented with 500 U/ml
penicillin, 500 μg/ml
streptomycin and 2 mM
L-glutamine (all Gibco, UK). Supernatants were removed, centrifuged at 2000 x g for 15 minutes, filtered through a
0.22 μm filter (Millipore, UK), concentrated using a 10 kD molecular weight cut off Centriprep concentrator (Amicon, UK) and dialysed against PBS over a 24-hour period. The supernatant was subsequently filtered again and protein concentration determined before use.
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