Serum samples were taken at 0, 4, 12, and 24 hours after each infusion. Serum HBV DNA levels were quantitated Opaganib by Amplicor HBV Monitor assay with a limit of detection 200 copies/mL (Roche Diagnostics, Branchburg, NJ). Serum HBsAg levels were determined by an automated immunoassay (IMX system; Abbott GmbH Diagnostika, Wiesbaden-Delkenhaim, Germany),

using a purified HBsAg preparation as standard. The limit of detection of this assay is 0.125 ng/mL. The PLC/PRF/5 cell line was established from hepatocellular carcinoma.14 These cells contain integrated HBV DNA fragments and produce 22-nm noninfectious HBsAg particles.15-17 The HBsAg production was shown to be constant on a per-cell basis during culture.18, 19 In the present study, PCL/PRF/5 cells were cultured in Dulbecco’s modified Eagle medium selleck inhibitor (DMEM; Invitrogen, Paisley, UK), supplemented with 10% fetal bovine serum (Invitrogen), 500 U/mL penicillin, 500 μg/mL streptomycin, and 2 mM L-glutamine. The cells were seeded in 24-well plates at 50,000 per well. After 48 hours, the cells were confluent, which was the starting time point (T0) of the experimental conditions outlined below. At T0, the supernatants were removed and replaced with medium only (DMEM/5% fetal bovine serum, control); medium plus HBV-Ab17 at two concentrations (0.2 and 0.5 mg/mL); medium plus HBV-Ab19 (0.2 and 0.5

mg/mL); HepeX-B (0.5 mg/mL); or medium plus isotype IgG (0.2 and 0.5 mg/mL) as a further control. After 48 hours (T48), the supernatants and the cells were collected separately. The cell lysates were tested for cellular

IgG and HBsAg by western blot. The HBsAg secreted in the supernatants was quantitated by enzyme-linked immunosorbent assay (ELISA). In this set of experiments, at T0 the supernatants medroxyprogesterone of PLC/PRF/5 cells were replaced with: (1) medium only; (2) isotype IgG control (0.5 mg/mL); (3) human AB serum, as another source of nonimmune IgG (0.5 mg/mL); (4) HBV-Ab17 (0.5 mg/mL); (5) HBV-Ab19 (0.5 mg/mL); or (6) HepeX-B (0.5 mg/mL). The cells were cultured continuously for 48 hours, during which period an aliquot of the supernatants was taken (without adding new medium) at 3, 6, 12, 24, 36, and 48 hours, and the HBsAg levels were determined by ELISA. The supernatants at T0 were replaced with the same controls or antibodies, as outlined above. During the first 24 hours, an aliquot of the supernatants was collected at 3, 6, 12, and 24 hours. After 24 hours (T24), the supernatants in all wells were replaced with fresh medium, without nonimmune IgG or anti-HBs, and the cells were kept in culture for a further 24 hours. During the second 24-hour period, aliquots were collected at the same time points as during the first period, i.e., at 27, 30, 36, and 48 hours, and the HBsAg levels were quantitated by ELISA. After 48 hours, the cells were trypsinized and washed two times in phosphate-buffered saline and resuspended in lysis buffer.