Complement Activation Binding Assay for HCV

In our standard binding assay, 3 mL of virus (1 to 3 × 10⁷ genomic copies for HCV genotype 1a), was mixed with 100 μL of serum (about 20-25 CH50 units) to initiate complement activation (22 (link)) and then incubated at 25°C for 30 minutes. Next, 2 mL of erythrocytes (5 × 10⁸ cells total) were added to the mixture and incubated for 15 minutes. The reaction was carried out in 15 mL sterile tubes with occasional mixing. After incubation, EDTA was added to a final concentration of 20 mM, and the reaction mixtures were immediately cooled down for 5 minutes in an ice bath. The cells were pelleted by centrifugation at 470 × g for 6 minutes at room temperature, and washed three times with 10 mL 1 × phosphate buffered saline (pH 7.4). The cell pellets were resuspended by pipetting, brought to a 200 μL volume with 1 × PBS, and then mixed with 600 μL of Blood RNA buffer (ZR Whole-Blood RNA MiniPrep kit, Zymo Research, Irvine, CA). Total RNA was isolated according to the manufacturer’s instructions, and each sample was eluted from the column with 50 μL of RNA diluent II, which contained nuclease-free water (Thermo Fisher Scientific, Waltham, MA) supplemented with 1.0 mM dithiothreitol and 200 units/mL RNasin^® (Promega, Madison, WI).