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.
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