Quantifying C1q Levels in Biological Samples

C1q levels in sera, BAL fluid and culture supernatants were measured using an in-house developed ELISA, as described previously^{18 (link)}. In brief, 96-well Maxisorp plates (Nunc) were coated overnight with mouse anti-human C1q (Nephrology department, LUMC) in coating buffer (0.1 M Na₂CO₃, 0.1 M NaHCO₃, pH9.6). The next day, plates were washed with PBS/0.05% Tween and blocked with PBS/1%BSA. After subsequent washing, samples (serum diluted 1:4000, BAL fluid 1:1, culture supernatant 1:1) were added to the plates. A serially diluted pool of normal human serum (NHS) was taken along as a standard. Bound C1q was detected by incubation with rabbit anti-human C1q, followed by goat anti-rabbit HRP (both Dako) and ABTS substrate. C1q concentration is calculated in μg/mL from a human C1q standard.