Rabbit anti human c1q

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.

To measure the concentration of C1q in serum samples, an in-house ELISA was performed as described before (25 (link)). Nunc MaxiSorp ELISA plates (ThermoScientific) were coated with 50 µl 2.5 µg/ml mouse anti-human C1q monoclonal antibody (mAb) 2204 (kind gift Prof. C. van Kooten, Dept Nephrology, LUMC) in 0.1M bicarbonate coating buffer (pH 9.6) and incubated overnight at room temperature. Wells were washed 3 times with PBS/0.05% Tween and blocked with 100 µl PBS/1% BSA and incubated for 1 hour at 37°C. After washing, 50 µl of serially diluted serum samples in PTB, as well as a standard curve from a pool of normal human serum were added to the wells, followed by incubation for 1 hour at 37°C and washing 3 times. Wells were then incubated with 50 µl 1:2000 diluted rabbit anti-human C1q (DAKO) for 1 hour at 37°C. After washing, 50 µl 1:2000 goat anti-rabbit-HRP (DAKO) was added for detection and wells were incubated for 1 hour at 37°C. Following the final washing sequence, wells were incubated with 50 µl ABTS/0.015% H₂O₂ and absorbance was read at 415 nm. C1q concentrations were calculated based on the standard curve of a reference serum.