To measure antibodies specific to VP2 antigen of an individual HBoV species (HBoV1, 2, 3, or 4), antibodies in serum samples were absorbed with heterologous VLPs of the other three HBoVs, as previously described [22] . Briefly, HBoV VLPs were serially diluted from 32 mg/mL to 0.5 mg/mL to determine the concentration needed for effective competition between cross-reactive antibodies. For detection of specific HBoV antibodies, three heterologous HBoV VLPs of 16 mg/mL were added to a 1:200 dilution of plasma [17] and incubated for 2 hr at 4uC prior to performing the ELISA assay. In parallel with the heterologous competition, human sera samples were used to compete with VLP that was homologous to the immobilized antigen. The absorbance at 450 nm was residual absorbance value. Net absorbance values were calculated by subtracting the residual absorbance value from the raw absorbance value read at 450 nm [22] .