Anti c3c antibody

The measurement of cofactor activity of cFH followed previous study.⁷ Briefly, the experiment was performed in fluid mixture 20 μL, which included cFH (0.5 μg, Merck, Kenilworth), factor I (50 ng, Merck), C3b (3 μg, Merck) and UMOD (8 μg). The mixture was incubated at 37℃ for 10, 20, 30 and 40 minutes, respectively. C3b and its cleavage products were detected by western blotting with a polyclonal anti‐C3c antibody (Dako Cytomation).

We tested whether uromodulin affected CFH–C3b binding. CFH was incubated with or without uromodulin in the buffer at 37°C for 1 h, and then the mixture was added to C3b‐coated wells. After 1 h of incubation at 37°C, bound CFH was detected with goat anti‐human CFH antibody, followed by appropriate secondary anti‐species AP‐conjugated Abs. After incubating the reaction for 1 h at 37°C, absorbance was measured at OD 405 nm.
The CFH‐mediated C3b inactivation assay, including CFH (1 μg), factor I (50 ng), C3b (3 μg) and uromodulin (0, 4, 8 and 16 μg), was conducted in a final volume of 20 μl and incubated at 37°C for 30 min. Experiments conducted in the absence of CFH were performed as negative controls. After incubation, the samples were heated for 5 min at 95°C in reducing buffer containing β‐mercaptoethanol and run on a 10% SDS‐PAGE gel. C3b and its cleavage products were detected by western blotting using a polyclonal anti‐C3c antibody (Dako Cytomation, Carpinteria, CA, USA).

The LP activity was measured in mouse plasma samples obtained at 4 days after TBI or sham injury. For ex vivo LP assessment, EDTA-plasma samples (2.5% final plasma concentration) were incubated on mannan-coated ELISA plates for 15 min to initiate activation followed by detection of C3c deposition as described elsewhere [22 (link)]. Briefly, EDTA-plasma samples were thawed on ice and suspended in barbital buffered saline (BBS; 4 mM barbital, 145 mMNaCl, 2 mM CaCl₂, 1mM MgCl₂, pH 7.4), to a final plasma concentration of 2.5%. Plasma solutions were incubated on the coated plate at 37 °C for 15 min. The plate was washed and incubated for 1 h 30 min at RT with a polyclonal anti-C3c antibody (Dako, A0062) diluted 1:5000 in washing buffer. After washing, the plate was incubated with an alkaline-phosphatase labelled goat anti-rabbit IgG antibody (Sigma A-3812) diluted 1:5000 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) and the absorption at 405 nm measured using the Infinite M200 spectrofluorimeter managed by Magellan software (Tecan, CH).