Quantifying Residual Lectin Pathway Activity

A LP specific C3 deposition ELISA was performed to measure residual LP functional activity in patient plasma (38 (link)). A Maxisorp ELISA plate (NUNC™) was coated with 10 μg/mL mannan to test LP activation by MBL (38 (link), 39 (link)), or 25 μg/mL acetylated bovine serum albumin (acBSA) to test LP activation by ficolins (21 (link), 40 (link)) diluted in coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) and incubated overnight at 4°C. Residual protein binding sites were saturated by incubating the plate with 1% BSA-TBS blocking buffer (0.1% (w/v) BSA in 10 mM Tris–CL, 140 mM NaCl, 1.5 mM NaN₃, pH 7.4) overnight at 4°C. The plate was then washed with washing buffer (TBS with 0.05% Tween 20 and 5 mM CaCl₂). EDTA-plasma samples were thawed on ice and suspended in barbital buffered saline (BBS; 4 mM barbital, 145 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, pH 7.4), to a final plasma concentration of 6%. Wells receiving only BBS buffer were used as negative controls. Plasma solutions were incubated on the coated plate at 37°C for 1 h 30 min (40 μL/well). The plate was washed and incubated for 1 h 30 min at RT with a polyclonal anti-human C3c antibody (Dako, A0062) diluted 1:5,000 in washing buffer. After washing, the plate was incubated with an alkaline-phosphatase labeled goat anti-rabbit IgG antibody (Sigma A-3812) diluted 1:5,000 in washing buffer for 1 h 30 min at RT. Following washing, the assay was developed by adding 100 μL substrate solution (Sigma Fast p-Nitrophenyl Phosphate tablets, Sigma). The absorption at OD405 nm was then measured using the Infinite M200 spectrofluorimeter managed by Magellan software (Tecan, CH).