4.6. AH-50 Assay

The AH-50 is an assay to test the functional capability of the alternative complement components to lyse rabbit red blood cells (RRBC). Rabbit erythrocytes (Complement Technology, Inc.) were used for this assay. The erythrocytes were washed by centrifuging at 1000× g for 5 min at 4 °C. The supernatant was discarded and the RRBC were diluted with GVB⁰ (without Ca²⁺ or Mg²⁺, pH 7.3). This basic buffer for complement assays contains 0.1% gelatin, 5 mM Veronal, 145 mM NaCl and 0.025% NaN₃. For the dilution series, GVB⁰ (47.5 mL) was mixed with 0.1M magnesium-ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (MgEGTA) (2.5 mL). As Mg²⁺ is required for the activation of the alternative complement pathway, addition of MgEGTA allowed this activation. First, the control tubes were prepared (double determination): 0% lysis with GVBE buffer (27.5 μL) and serum (10 μL) and 50% and 100% lysis with ddH₂O (43.5 μL and 37.5 μL, respectively), as well as a positive control with 27.5 μL GVB⁰+MgEGTA and 10 μL of any serum. Subsequently, serial dilutions of the test serum were made (1:2, 1:4, 1:8, 1:16, 1:32). Erythrocytes (12.5 μL) were added to each tube except for the 50% lysis control tube (6.25 μL). The tubes were incubated for 30 min at 37 °C. The hemolysis reaction was stopped by adding 200 µl ice-cold GVBE buffer (with EDTA, pH 7.3) (Complement Technology Inc.). GVBE buffer (again 0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.025% NaN₃, 10 mM EDTA) inhibited the complement activation and was subsequently centrifuged (1000× g, 3 min, 4 °C). The degree of hemolysis was quantified by measuring the absorbance of the hemoglobin released into the supernatant at 415 nm.