Fitc conjugated rabbit anti human c1q antibody

C1q binding was assessed using a flow cytometry-based assay modified from Pawluczkowycz et al. (38 (link)). CHO-K1 cells stably expressing either the SARS-CoV-1 or SARS-CoV-2 spike protein were used in the experiment. Cells were washed with PBS and stained with fixable viability dye (eBioScience Cat. 65-0866-014) on ice in the dark for 15 mins. Following wash with PBS, 300,000 cells were stained with appropriate antibodies after serial dilution. Purified human C1q (Complement Technology Cat. A099) at 3.5 mg/mL or 35 mg/mL was added to the mixture and incubated at 37°C for 1 h. Cells were washed twice with buffer (BD Cat. 554657) and resuspended in 10% heat-inactivated rabbit serum (Gibco Cat. 16120-099) and incubated on ice for 15 minutes to block non-specific binding. Cells were then incubated with FITC-conjugated rabbit anti-human C1q antibody (Dako, Cat. F0254, 1:100 dilution) at RT for 25 minutes. Cells were washed twice with buffer (BD Cat. 554657) and 10,000 events were detected using Cytoflex flow cytometer (Beckman). GraphPad Prism software was used to plot four-parameter dose-response curves and obtain EC50 values.

Wien-133 cells were opsonized with antibody serial dilutions for 15 min at 37 °C. Subsequently, cells and antibodies were put on ice, purified human complement component C1q (2.5 µg ml⁻¹) was added and incubated for 45 min. After washing, C1q binding was detected using a fluorescein isothiocyanate (FITC) conjugated rabbit antihuman C1q antibody (Dako) and quantified as the FITC geometric mean fluorescent intensity (gMFI) determined using an iQue Screener flow cytometer (Sartorius).