For the detection of specific antibody secreting cells by ELISPOT, PVDF membrane-lined 96-well plates (Mabtech, Nacka Strand, Sweden) were coated with 100 ml of PBS containing VLPs (10 mg/ml), recombinant E2 (10 mg/ml) or anti IgG antibody (10 mg/ml) overnight at 4uC. Plates were washed 5 times with PBS and blocked for 2 hours using RPMI 1640 medium (Gibco, USA) supplemented with 20% BSA (Sigma, Australia). Wells were emptied before 5610 5 splenocytes in 200 ml of RF-10 medium was added and incubated for 36 hours at 37uC in an atmosphere of 5% CO 2 . Spot forming units representative of specific antibody-producing cells were developed as previously described for the detection of IFN-c-secreting cells except that biotinylated anti-IgG antibody and streptavidin-conjugated horseradish peroxidase (both from Mabtech, Nacka Strand, Sweden) were used as detecting reagents.