Rabbit anti human uromodulin polyclonal antibody

The commercial CFH, factor I, and C3b were purchased from Merck (Kenilworth, NJ, USA). Factor H short consensus repeat (SCR) units (SCR1–4, SCR7, SCR11‐14, and SCR19–20) were produced by Gene‐Script Corporation (Piscataway, Nanjing, China). Antibodies used in this study included a goat anti‐human CFH polyclonal antibody (Merck), a mouse anti‐human CFH monoclonal antibody (US Biological, Salem, MA, USA), a rabbit anti‐human uromodulin polyclonal antibody (Biomedical Technologies Inc., Stoughton, MA, USA), a mouse anti‐human uromodulin monoclonal antibody (Cedarlane, Burlington, NC, USA), and species appropriate secondary antibodies (anti‐mouse IgG‐alkaline phosphatase conjugated antibodies and anti‐rabbit IgG‐alkaline phosphatase conjugated antibodies) from Sigma‐Aldrich (St. Louis, MO, USA). TRITC‐conjugated rabbit anti‐goat IgG (Zhongshan Biotech, Guangdong, China) and FITC‐conjugated rabbit anti‐mouse IgG (Abcam, Cambridge, UK) were also used when necessary.

Binding of THP with CL‐11, MBL, and ficolins was detected by ELISA. The microplates were first coated with 4μg/ml recombinant CL‐11, MBL or ficolin1, 2, −3 (R&D) in 0.1 mol/L carbonate buffer (pH 9.6) at 4°C for overnight. The plates were blocked with 1% BSA/PBS at 37°C for 1 hour, and THP was added with concentration varied from 0.3125 to10 μg/mL at 37°C for 1 hour. For CL‐11 and ficolins binding assay, THP was diluted in VBS buffer with 0.5 mmol/L MgCl₂ 0.15 mmol/L CaCl₂. The CaCl₂ concentration was 1 mmol/L for MBL binding assay. Subsequently, wells were incubated with rabbit anti‐human uromodulin polyclonal antibody (Biomedical Technologies Inc, USA) and mouse anti‐rabbit IgG alkaline phosphatase (Sigma‐Aldrich, USA) both at 37°C for 1 hour. The OD at 405 nm was measured using a microplate reader (iMark, BIO‐RAD).