CH50 hemolysis assay

The CH50 hemolysis assay measures the complement activity of the antibody-dependent classical complement pathway by measuring the capability of the serum complement to lyse antibody-sensitized sheep red blood cells (RBC) [34]. We used a previously described established assay for CH50 determination [35–37]. In short, human serum is titrated to obtain the fraction of serum which causes lysis of 50% of the RBCs as measured by the hemoglobin released into the supernatant. Antibody-sensitized sheep erythrocytes (Complement Tech, Tyler TX) were centrifuged for 3 min at 1000 g. The supernatant was removed and replaced with an equal volume of fresh gelatin veronal buffered saline containing 0.15 mM calcium chloride and 0.5 mM magnesium chloride (GVB++; Complement Tech, Tyler Tx). The sheep erythrocytes were at a concentration of 5 x 10⁸ cells per ml. Human sera samples were diluted in 10 serial dilutions (2-fold each) in GVB++. Sheep erythrocytes (100 μl), 50 μl of GVB++ and 50 μl of the serum sample were added into 96-well tissue culture treated plates (USA Scientific) and mixed by pipetting up and down three times. The plate was incubated at 37°C for 1 h. After incubation the plate was centrifuged at 1000 g for 3 min and 100 μl of the supernatant was transferred to a new plate without disturbing the pellet. The absorbance of the hemoglobin released into the supernatant was read at 412 nm with the Infinite® 200 PRO (Tecan Life Sciences, Switzerland). Assay controls consisted of GVB++ buffer only as the background of the plate, sheep erythrocytes with GVB++ as the background of lysis, and sheep erythrocytes lysed with Triton X100 for total lysis. Additionally, we included the same sample (standard) for inter-assay standardization. Only assay results with a range of values +/- 20% within the standard sample were included in the final analysis.