His 6-tagged protein-binding assay (reactivity of HBsAg with anti-tag Ab [%])

The Ni-coated plate (HIS-Select High Capacitty (HC) Nickel coated plates; Sigma-Aldrich) was blocked with Super Block (Pierce), then dried and kept at 4°C until use. The PEG-precipitated culture supernatants or the cell lysates were loaded onto the 96-well Nickel-coated plate and incubated at 37°C for 1 h followed by washing three times with TBS-T (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween 20; Tris-buffered saline containing 0.1% Tween 20). The plate was then incubated for 30 min at 37°C with a mouse mAb (anti-Xpress^™ antibody; Invitrogen) and again washed three times with TBS-T. The plate was reacted with anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Dako) for 30 min at 37°C and washed with TBS-T three times. Finally, the color reaction was developed with a substrate solution (100 μl 0.0015% H₂O₂ plus 120 μg/ml TMB [3,3',5,5'-tetramethylbenzidine]). The optical density (OD) was measured at 450 nm with subtracted the reference wavelength (OD₆₃₀) by using a Spectra Max 190 microplate spectrophotometer (Molecular Devices) after stopping the reaction with 50 μl 3N H₂SO₄.