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).
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