Hemolytic Assay

Classical pathway: Complement mediated lysis of erythrocytes releases hemoglobin that can be measured by absorbance spectrophotometry at 415 nm. Antibody-sensitized sheep erythrocytes (EA; CompTech; B200) were rinsed and diluted in GVB⁺⁺ buffer (0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.15 mM CaCl₂ 0.5 mM MgCl₂. 0.025% NaN_3, pH 7.3). 5 µM of Sez6L2-MH or other purified proteins were pre-incubated in a v-bottom 96 well plate for 15 minutes on ice with 0.275% normal human serum (NHS, CompTech) in a final 90 µl buffer solution equivalent to 40% GVB⁺⁺ and 50% PBS with 0.15 mM CaCl₂ and 0.5 mM MgCl₂ (PBS⁺⁺). Next, 10 µl of EAs (4x10⁸ cells/mL in GVB⁺⁺) were added to each sample and the plate was incubated at 37⁰C for 30 minutes. During this incubation, cells were suspended with a multichannel pipet every 10 minutes. The reaction was stopped by adding 100 µl of cold GVBE buffer (0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.025% NaN₃, 10 mM EDTA, pH 7.3). Remaining erythrocytes were pelleted by centrifugation at 500xg for 3 minutes. 150 µl of the supernatant was transferred to a flat-bottom 96-well plate and measured for absorbance at 415 nm using a microtiter plate reader (SpectraMax M5). The absorbance obtained with just buffer, NHS, and EAs was set at 100% lysis and the absorbance obtained without serum added or when GVBE was used in place of GVB⁺⁺ to inhibit complement was set at 0% lysis. Absorbance from full lysis in each experiment was also determined by replacing PBS with H₂0 (at 50% final sample volume). Prior to testing the efficacy of complement inhibitors, the amount of each lot of normal human serum necessary to lyse EAs to 80-90% of the absorbance obtained by H₂0 samples was determined by testing a range of NHS concentrations from 0-2%. These titrations were done to ensure that inhibitors were not overwhelmed by saturating levels of complement activity. The titrations revealed that 80-90% lysis was usually obtained with 0.25-0.4% serum.

Alternative Pathway: The alternative pathway hemolytic assays were performed similar to the classical pathway assay except that plain rabbit erythrocytes (Er, CompTech) were rinsed and diluted in GVB° (0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.025% NaN₃, pH 7.3). 5 µM Sez6L2-MH or other purified proteins were pre-incubated for 15 minutes on ice with 6.5-7% normal human serum in a final 95 µl buffer solution containing 10 mM MgCl₂ and 10 mM EGTA and the equivalence of 45% GVB° and 50% PBS. Next, 5 µl of Ers (5x10⁸ cells/mL in GVB°) were added to each sample and the plate was incubated at 37°C for 30 minutes and processed similar to the classical hemolysis experiments. The 415 nm absorbance obtained with just buffer, NHS, and Ers was set at 100% lysis.