Evaluating Complement Activation in Serum
Corresponding Organization : Rigshospitalet
Protocol cited in 6 other protocols
Variable analysis
- Addition of 10 mM EDTA or 10 mM EGTA with 5 mM Mg2+ to the serum
- Binding of serum to acBSA-coated plates
- Detection of C4c, C4c-biotin, C3c, and C5b-C9 (TCC) in the bound serum
- Coating of plates with 5 µg/ml acBSA in PBS-buffer overnight at 4°C
- Blocking of plates for 1 h with barbital-T
- Washing of plates thrice in barbital-T
- Incubation of serum diluted in barbital-T for 30 min at 37°C (45 min at 37°C for the detection of TCC)
- Incubation 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
- Incubation 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
- Not explicitly mentioned
- Not explicitly mentioned
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