Samples were collected from stage 46 metamorphs anaesthetised by bathing for 2 min in 0.22% MS-222 followed by decapitation. Samples were collected by aspiration and pelleting of RBCs prior to the collection of sera-like fluids. Adult blood samples were collected as per Zupanovic et al. [23] . Microtitre plates (Beckton-Dickonson) were coated with 50 mL/well of rAdglob (10 mg/mL) and positive control ovalbumin (4 mg/mL; Sigma) in Ringer's solution overnight at 4uC and washed (x3) in TBS with Tween-20 (0.05%; TBST). The plates were blocked for 2 h with 5% skim milk powder (Bio-Rad) in TBST at 37uC. Metamorph sera (treated and control) and adult toad aovalbumin control sera were serially diluted in 1% skim milk/TBS and applied to plates (50 mL/well). After incubation for 1 h at 37uC the plates were washed with TBST three times. Rabbit antisera against toad IgG (1:1000; Zupanovic et al. [23] ) was added in 1% skimmed milk/TBS, incubated for 1 h at 37uC and washed (x3) in TBST. Next, goat a-rabbit IgG (1:2000; KPL) was added to each well, incubated for 1 h at 37uC and again washed (x3) in TBST. HRP-conjugated streptavidin (50 mL; KPL) was incubated for 30 min at RT, washed three times and developed with peroxidase substrate solution (50 mL of TBM; KPL) and the absorbance measured (405 nm).