All P-values <5% were considered significant. Wistar rats were immunized with a complex consisting of the 15 C-terminal amino acid residues of MASP-1

(i.e. a sequence not shared by MASP-3 and MAp44) coupled to keyhole limpet haemocyanin (KLH). The sera were tested for reactivity to rCCP1-CCP2-SP coated in microtitre wells. The specificity of the preferred serum was examined further by application to blots of MBL/MASP complexes purified from human serum. A Western blot is shown in Fig. 1a, lane 1, where reaction with protein can be seen at a position corresponding to the previously observed Palbociclib ic50 mobility of pro-enzyme MASP-1 (Mr ∼75 kDa). We saw no reactivity with material at the positions of MASP-3 (Mr ∼105 kDa), MASP-2 (Mr ∼70 kDa), MAp44 (Mr ∼44 kDa) or MAp19 (Mr ∼19 kDa), which were selleck compound revealed by incubating parallel strips with the relevant antibodies (not shown), as described previously in detail [10,21]. No reactivity of a normal rat serum is seen (Fig. 1a, lane 2). The anti-MASP-1 serum was tested further on Western blots of rCCP1-CCP2-SP. A reactivity corresponding to ∼45 kDa was seen, which is the expected size of the construct (calculated at 45 073 g/mol) (Fig. 1a, lane 4). No reactivity was

seen by a normal rat serum (Fig. 2a, lane 3). The reaction of the rat anti-MASP-1 anti-serum was also evaluated by TRIFMA, as described below. Preliminary attempts to construct a sandwich-type assay were non-productive and we thus turned towards an inhibition assay based on inhibition of the binding of anti-MASP-1 antibody to a surface coat of rCCP1-CCP2-SP. Accordingly, decreasing signals were seen when increasing

concentration of plasma were applied, as illustrated by the standard curve in Fig. 1b, which was generated by applying serial dilutions of our standard plasma pool. The value for MASP-1 content of this pool was estimated at 5·7 µg/ml by comparison with a preparation of pure rCCP1-CCP2-SP. Figure 1b shows a dilution curve of this reagent next to the dilution curve of the standard serum. A number of dilution buffers were assessed. The most consistent results for plasma and serum were obtained with the complex assay buffer composition detailed in Materials and methods. This is Rutecarpine a high-ionic-strength calcium-containing buffer (the high ionic strength lowers the background in the assay but also prevents coagulation if diluting, e.g. EDTA or citrate plasma in a calcium-containing buffer) with proteins added to reduce background signals. We found a 60-fold dilution to be suitable for plasma samples to be assayed for MASP-1. For routine analyses, three internal controls were added to each assay plate. The means and interassay coefficients of variation (CVs), determined from 10 individual assays for the three internal controls, were: 15·5 µg/ml, 7·68 µg/ml, 3·72 µg/ml and 11%, 13% and 8%, respectively. The sensitivity of the assay, i.e.