Liposomes were prepared using dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), cholesterol and DNP‐cap‐PE, purchased from Avanti Polar Lipids (Alabaster, AL, USA). Lipid films were composed of DMPC–DMPG–cholesterol–DNP‐cap‐PE (45 : 5 : 49 : 1 mol%). Components were dissolved in chloroform–methanol (9 : 1 v/v) before drying under nitrogen gas and desiccation overnight. Films were rehydrated at 37°C for 30 min with a self‐quenching concentration of sulforhodamine B (20 mM; S1402 from Sigma Aldrich, St Louis, MO, USA) in PBS to a final lipid concentration of 0·8 mg/ml. The sulforhododamine B‐liposome mixture was sonicated for 5 min at 37°C in a water bath. Purification of liposomes was performed through size‐exclusion chromatography using a prepacked NAP‐25 column (17‐0852‐01; GE Healthcare, Little Chalfont, UK).
To analyze complement activity via membrane attack pore membrane attack complex (MAC)‐mediated dye leakage, purified liposomes were diluted ×10 in PBS and mixed with NHS (10% v/v final concentration) from Complement Technologies (Tyler, TX, USA). Sulforhodamine B fluorescence was measured with an excitation wavelength of 565 nm and emission wavelength of 585 nm using a CLARIOstar microplate reader (BMG Labtech, Offenburg, Germany). Fluorescence was measured at 21°C for 100 s before different antibodies (IgG1‐DNP and IgG1‐DNP‐RGY, both non‐modified and carbamylated) were added to final concentrations of 4·35 μg/ml/ml before assaying for a further 10 min. Total lysis was performed by adding 70% ethanol after assay. Experiments were performed in triplicate.
To analyze complement activity via membrane attack pore membrane attack complex (MAC)‐mediated dye leakage, purified liposomes were diluted ×10 in PBS and mixed with NHS (10% v/v final concentration) from Complement Technologies (Tyler, TX, USA). Sulforhodamine B fluorescence was measured with an excitation wavelength of 565 nm and emission wavelength of 585 nm using a CLARIOstar microplate reader (BMG Labtech, Offenburg, Germany). Fluorescence was measured at 21°C for 100 s before different antibodies (IgG1‐DNP and IgG1‐DNP‐RGY, both non‐modified and carbamylated) were added to final concentrations of 4·35 μg/ml/ml before assaying for a further 10 min. Total lysis was performed by adding 70% ethanol after assay. Experiments were performed in triplicate.