The study was approved by local Ethics Committees and informed consent was obtained from the donors. In the following sections references

are given to papers which have used the same samples for other purposes. A recombinant fragment of MASP-1 comprising the CCP1-CCP2-SP domains (rCCP1-CCP2-SP) was produced in Escherichia coli, and refolded and purified as described previously [13]. A synthetic peptide representing the 15 C-terminal amino acid residues of human MASP-1 (CHHNKDWIQRVTGVR) was coupled to keyhole limpet haemocyanin. Three Wistar rats were immunized four times subcutaneously with 10 µg of this conjugate, emulsified first in complete Freund’s adjuvant and then in incomplete Freund’s adjuvant for boosts. Sera from the animals were tested for reactivity towards Protease Inhibitor Library order rCCP1-CCP2-SP coated onto microtitre wells. All rats responded and the rat with the highest titre was selected. IgG was purified from the anti-serum by affinity chromatography on Protein G-coupled beads. The serum was diluted 1/1 in phosphate-buffered saline (PBS; 137 mM NaCl, 2·7 mM KCl, 1·5 mM KH2PO4, 8·1 mM Na2HPO4, pH 7·4) selleck compound containing 10 mM EDTA (PBS/EDTA), and passed through the beads. After washing, the

bound IgG was eluted with 0·1 M glycine, pH 2·4. The immunoglobulin concentration was determined by spectroscopy at 280 nm. The purified IgG was biotinylated by standard procedure [26] using 167 µg biotin-N-hydroxysuccinimide ester (Sigma-Aldrich, Vildagliptin St Louis, MO, USA) per mg antibody. The anti-MASP-1 anti-serum was tested by Western blotting. MBL/MASP complexes were purified from serum by affinity chromatography on mannan coupled to Sepharose beads, as described previously [8]. The complexes, as well as a preparation of rCCP1-CCP2-SP, were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in non-reducing conditions followed by blotting onto a membrane. For the preparation of Western blotting strips, MBL/MASP

complexes corresponding to 30 µg MBL were loaded onto a single-well XT-Criterion pre-cast 4–12% gradient Bis-Tris polyacrylamide gel (Bio-Rad, Copenhagen, Denmark) and cut into 2·5-mm-wide strips after blotting, resulting in approximately 1 µg MBL (+ associated proteins) per strip. The proteins were blotted onto a nitrocellulose membrane (Hybond-ECN; GE Healthcare, Hilleroed, Denmark) in transfer buffer (25 mM Tris, 0·192 M glycine, 20% v/v ethanol, 0·1% w/v SDS, pH 8·3) for 500 volt-hours. The membrane was blocked in 0·1% Tween 20 in Tris-buffered saline (TBS) (10 mM Tris–HCl, 140 mM NaCl, 1·5 mM NaN3, pH 7·4) before being cut into strips. The strips were incubated with primary antibodies (normal rat IgG or rat anti-MASP-1 antibody) diluted in primary buffer (TBS with 0·05% Tween 20 (TBS/Tw), 1 mM EDTA, with 1 mg human serum albumin (HAS) and 100 µg normal human IgG (hIgG) added per ml) in eight-well trays (Octaline, Pateof, Denmark) for 2·5 h on a rocking table.