Sensitized sheep erythrocytes

CP-specific inhibition of red blood cell lysis by Elp proteins was assessed by a hemolytic assay as described (28 ). In short, Elp truncations were added to sensitized sheep erythrocytes (Complement Technology) and 2% NHS (Innovative Research) in GHB⁺⁺ (10 mM Hepes [pH 7.3], 140 mM NaCl, 0.1% gelatin [w/v], 0.15 mM CaCl₂, and 0.5 mM MgCl₂). Assays were performed in at least triplicate and normalized to positive (100%, no inhibitor) and negative (0%, no serum) controls. Inhibitory curves were fitted using nonlinear regression to a normalized variable slope model in GraphPad, version 9 (Prism).

Sensitized sheep erythrocytes (Complement Technology Inc., Tyler, TX, USA) were washed once with tris buffered saline (TBS), centrifuged (3 min, 4°C, 500 g) and erythrocytes were re-suspended in GVB⁺⁺ buffer (with Ca²⁺ and Mg²⁺, pH 7.3) (Complement Technology Inc., Tyler, TX, USA). GVB⁺⁺ buffer contains 0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.025% NaN₃, 0.15 mM CaCl₂, and 0.5 mM MgCl₂, which allows the process of complement activation via the classical pathway. In order to determine 50 and 100% lysis, the erythrocytes were diluted with ddH₂O. The optical density was measured at 415 nm and OD values for 50 and 100% lysis were determined.
Serial dilution of porcine serum from 1:20 through 1:640 was prepared in GVB⁺⁺ buffer (with Ca²⁺ and Mg²⁺, pH 7.3). Then, Sensitized sheep erythrocytes were added to the diluted serum samples and incubated for 30 min at 37°C. Afterwards, ice cold GVBE buffer (with EDTA, pH 7.3) (Complement Technology Inc., Tyler, TX, USA) was added to the samples and samples were centrifuged (3 min, 4°C, 500 g). GVBE buffer contains 0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.025% NaN₃, and 10 mM EDTA, which inhibits the complement activation cascade. Afterwards, the supernatant was transferred and optical density from supernatant was measured at 415 nm.