To develop ELISA assays that can detect antibodies against HBoV species, we first sought to identify HBoV-positive andnegative sera samples using Western blot analysis. To obtain negative sera samples, we tested the reactivity of sera samples collected from infants aged 0-6 months who visited Beijing Children's Hospital for regular health check-ups against HBoV VLPs. Only the samples that were negative for HBoV1, 2, 3, and 4 VLPs simultaneously were selected as the negative samples. The negativity of those samples was confirmed using a competition ELISA (cELISA) assay, in which the absorbance value at 450 nm did not change significantly with or without competition assays ( Figure 3 ). Serum samples identified by cELISA as positive for individual HBoV species were obtained from 3 children and 4 adults and used as positive controls. However, we did not obtain a serum sample that was positive only for HBoV4 based on the assay; we used a sample from an adult that was IgG positive for HBoV1, HBoV2 and HBoV4. To determine the concentration of VLPs required for exhaustive antibody competition of HBoV1-4, the serum samples were monocompeted with concentrations of HBoV1, 2, 3, or 4 VLPs ranging from 0-32 mg/mL ( Figure 3 ). We found that HBoV VLPs concentration of 16 mg/mL was the effective concentration to perform the VLP-based competition assays.