Quantification of Serum C1q Levels by ELISA

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.