Purified c3

Purified C3, C3b, C3a, iC3b, FH, and FI were obtained from Complement Technologies. Complement receptor 1 (CR1; CD35) was from Avant Immunotherapeutics. The mouse anti-C3b/iC3b [clone 3E7, (23 (link)) and specificity for iC3b confirmed in (24 (link))] was a generous gift from Dr. Ronald Taylor (University of Virginia Health Sciences Center) and was also purchased from MilliporeSigma. Rabbit anti-human C3a (A218) and goat anti-human C3 (A213) polyclonal antibodies (pAbs) were also purchased from Complement Technologies. Chicken anti-human C3 (GW20073F) pAb was obtained from MilliporeSigma.

To determine the C3 concentration after protein expression, an ELISA was performed. Two polyclonal antibodies were used, to eliminate the risk that a monoclonal antibody will recognize the mutants differently. Microtiter plates were coated with rabbit anti-human C3c antibody (Dako), diluted in 75 mM sodium carbonate, pH 9.6. The wells were washed with washing buffer (50 mM Tris–HCl, pH 8.0, 0.15 M NaCl, 0.1% Tween) and blocked with 3% fish gelatin (Norland Products) in washing buffer. The samples were diluted in blocking buffer, supplemented with 10 mM EDTA, and incubated on the plate for 1 h at 37°C. Purified C3 from Complement Technology was used as a standard. C3 was then detected using a goat anti-human C3 antibody (Quidel), followed by a HRP-conjugated rabbit anti-goat antibody (Dako). After 1 h incubation at RT for each of the antibodies, the plates were developed using OPD-tablets (Kem-En-Tec). The absorbance at 490 nm was measured spectrophotometrically.