For IgM EIA, a µ-capture format was used (19) . Serum samples diluted 1:200 in PBS and 0.05% Tween (PBST) were applied in duplicate into wells of plates coated with goat anti-human IgM (Cappel/ICN Biomedicals, Costa Mesa, CA, USA) for 60 min at room temperature. After being rinsed 5 times with PBST, biotinylated HBoV VLPs were applied at a concentration of 25 ng/well and incubated for 45 min at 37°C. Bound antigen was visualized by using horseradish peroxidase-conjugated streptavidin (Dako, Glostrup, Denmark) at 1:12,000 in PBST plus 0.5% bovine serum albumin for 45 min at 37°C, followed by ophenylenediamine dihydrochloride (Dako) and H 2 O 2 for 15 min at 37°C. Cutoff values were calculated from IgM EIA absorbances of 5-year follow-up samples of 61 children who were IgG positive 5 years earlier. Cutoff absorbances for negative and positive IgM EIA results were 0.136 (mean + 3 SD) and 0.167 (mean + 4 SD), respectively. Parvovirus B19 serologic analysis was conducted by using commercial (Biotrin, Dublin, Ireland) and in-house EIAs (18, 20) .