ELISA Assays for Complement Pathways

ELISAs were performed as described by Dekkers et al.⁷⁵ For the alternative pathway ELISA, Nunc Polysorp flat‐bottom plates (Thermo Scientific, Rockland, IL, USA) were coated overnight with 40 μg mL⁻¹ lipopolysaccharide from Salmonella typhosa (LPS, Sigma‐Aldrich, St. Louis, MI, USA) in phosphate‐buffered saline (PBS) at room temperature (RT). Plates were washed and incubated for 1 h at 37°C with 20% (v/v) NHS or FB‐or FD‐depleted serum, with or without purified FB (162.5 μg mL⁻¹) or FD (1.05 μg mL⁻¹) supplementation in Veronal Buffer (1.8 mm sodium barbital and 3.1 mm barbituric acid, pH 7.3–7.4; VB) with 0.1% (w/v) Tween‐20, 0.3% (w/v) BSA, 5 mm MgCl₂ and 10 mm EDTA. For the CP ELISA, Nunc Maxisorp flat‐bottom plates (ThermoFisher Scientific, Whaltam, MA, USA) were coated overnight with 5 or 0.5 μg mL⁻¹ agglutinated human IgG (AHG, Sanquin Reagents) in PBS at RT. Plates were washed and incubated for 1 h at RT with 25% (v/v) NHS with or without anti‐FB (202 μg mL⁻¹), anti‐FD (1.3 μg mL⁻¹) or anti‐C1q (31.75 μg mL⁻¹) in VB with 0.1% (w/v) Tween‐20, 0.3% (w/v) BSA, 1 mm CaCl₂ and 0.5 mm MgCl₂. ELISA was performed as described above. Biotinylated anti‐C3.19 was used as conjugate and ELISA was developed as described by Dekkers et al.,⁷⁵ with the exception of the usage of streptavidin‐HRP for the classical pathway ELISA.