Evaluating Complement Activation in Serum

Microtiter plates were coated with 5 µg/ml acBSA in PBS-buffer overnight at 4°C. Plates were blocked for 1 h with barbital-T and subsequently washed thrice in barbital-T. Serum diluted in barbital-T was added to the wells and incubated for 30 min at 37°C (45 min at 37°C for the detection of TCC). In order to evaluate whether the binding was dependent on calcium 10 mM EDTA or 10 mM EGTA with 5 mM Mg²⁺ was added to the serum. Plates were washed in barbital-T and subsequently incubated with anti-C4c (0.13 µg/ml), anti-C4c-biotin (2 µg/ml), anti-C3c (0.32 µg/ml) or anti-C5b-C9 (TCC) (1 µg/ml) for 2 h, shaking at room temperature. Plates were washed in barbital-T and incubated with HRP-conjugated streptavidin, anti-rabbit or anti-mouse IgG antibodies (all diluted 1:2000 in barbital-T) for 1 h at room temperature, shaking. Plates were washed and developed as described above.