To minimize false positive results of the ELISA assay due to impurities in immunizing and coating antigens, the coating antigen and the protein used to prepare mouse antibodies were derived from a baculovirus expression system and a prokaryotic expression system. BALB/c mice were injected subcutaneously with the purified proteins obtained from E. coli. This study was carried out in accordance with the animal experiment regulations of the Chinese government. All animal experiments were performed in the facilities of the Institute of Laboratory Animal Sciences (ILAS), Chinese Academy of Medical Sciences (CAMS). All experimental procedures were approved (license number SCXKJ2009-0017) and supervised by the Animal Protection and Usage Committee of ILAS, CAMS. Sera collected from the treated mice were purified using protein-G sepharose columns (GE Healthcare, Waukesha, WI) and purified IgG antibody were quantified using Pierce BCA Protein Assay Kit (Thermo Scientific). The purified mouse IgG antibodies were serially diluted from 8 mg/mL to 0.008 mg/mL. Sera from pre-immune mice served as the negative control. Rabbit antiserum against human parvovirus B19 (a gift from Dr. Xia Xiao of National Institute for Viral Disease Control and Prevention, Chinese Center for Disease control and Prevention) and mice antiserum against influenza virus H5 hemaglutinin (HA) [39] were used as unrelated controls. The cross-reactivity between HBoV1, 2, 3, and 4 was tested using Western blot analysis and ELISA.