The putative major virus capsid protein VP2 gene (nt 3443-5071) of the HBoV St 2 isolate (GenBank accession no. DQ000496) was cloned into a baculovirus vector pAcSG2 (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA) by standard procedures and confirmed by sequencing. The VP2-containing vector was transfected into Sf9 insect cells by using FuGENE 6 Transfection reagent (Roche, Basel, Switzerland). Two million adherent cells in T25 bottles were transfected in 1 mL of Insect Express media (Lonza, Basel, Switzerland) with a mixture of 2 µg plasmid, 250 ng linearized baculoGold DNA (Becton Dickinson Biosciences), and 15 µL FuGENE reagent. Fresh cells were infected 3 times every third day by using virus medium collected from the previous infection. VP2containing Sf9 cells were harvested on day 3, and cell pellets were resuspended in phosphate-buffered saline (PBS), pH 7.5, at a concentration of 2.1 × 10 7 cells/mL. Protease inhibitor (complete EDTA-free; Roche) was added (≈75 µL/mL), and cells were lysed by sonication (4 × 20 s). After subsequent centrifugation at 13,200 rpm for 3 min, VLPs were purified by 48-h CsCl gradient ultracentrifugation at 24,200 rpm (L-70 Ultracentrifuge; Beckman, Fullerton, CA, USA) at 4°C after fraction collection and dialysis against PBS. The product was concentrated in columns (Amicon Ultra-15 50,000 MWCO; Millipore, Billerica, MA, USA). Expressed HBoV VP2 was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular mass of ≈60 kDa and by electron microscopy to be spherical symmetric parvovirus-like particles ≈20 nm in diameter ( Figure 1 ). Before use as antigen, VLPs were biotinylated as described (17) .