Rporation (Paisley, UK). Production of cleaved C1-INH C1-Inhibitor was cleaved by incubation with trypsin Sepharose (30 mg/ml) for six h. The Sepharose was removed by centrifugation for three two min at 1000 g. Residual trypsin activityNIH-PA Author ManuscriptInnate Immun. Author manuscript; available in PMC 2011 January 1.Thorgersen et al.Pagein the C1-INH containing supernatant was checked inside a chromogenic assay with all the chromogenic substrate S2222 (1 mM). Residual trypsin activity was 0.1 .NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSerum experiments Sera from 3 distinctive pigs plus a serum pool of 20 humans have been pre-incubated for 5 min with C1-INH, iC1-INH, SPICE (pig experiments), compstatin (human experiments) or HSA. Escherichia coli was added to a final concentration of 108 bacteria/ml serum and incubated for 30 min at 37 . Complement activation was stopped by adding ethylenediaminetetraacetic acid (EDTA) to a final concentration of 20 mM. The samples were kept on ice then centrifuged for ten min at 1400 g (4), for removal of E. coli in the samples. The baseline sample (T0) was processed immediately. The sera have been stored at -70 until analysed for complement activation. Whole-blood experiments Blood was collected from seven diverse pigs (Sus scrofa domesticus, Landrace, out-bred stock) and six different humans. Each porcine and human blood was anticoagulated with lepirudin (50 g/ml) as described previously,17 and promptly split in 1.8-ml or four.5 mlNunc tubes (Roskilde, Denmark) for incubation. The blood was applied for complement, cytokine or flow cytometric analyses. For complement and cytokine analyses, blood was pre-incubated at 37 for 5 min with C1INH, iC1-INH, a complement inhibitor (SPICE in porcine experiments and compstatin in human experiments), or HSA. Escherichia coli was then added to a final concentration of 108 bacteria/ml complete blood in the porcine experiments and 107 bacteria/ml whole blood in the human experiments. The samples were incubated for two h and four h. The baseline sample (T0) was processed immediately. Complement activation was stopped by adding EDTA to a final concentration of 20 mM immediately following incubation. The blood samples have been centrifuged for ten min at 1400 g (4) and the plasma collected and stored at -70 till analysed. For the flow cytometry experiments, blood was pre-incubated at 37 for five min with C1INH, iC1-INH, a complement inhibitor (SPICE or compstatin), or HSA. Escherichia coli was then added to a final concentration of 108 bacteria/ml whole blood in both the porcine and human experiments and also the samples have been incubated for ten min at 37 ahead of evaluation. Enzyme immunoassays The soluble terminal C5b-9 complement complicated (TCC) was measured in an enzyme immunoassay (EIA), as described previously,21 and later modified.22 Briefly, the monoclonal IL-23 review antibody (mAb) aE11 reacting having a neoepitope exposed in C9 immediately after incorporation inside the C5b-9 HSV site complex was applied as capture antibody at a final concentration of three g/ml. A biotinylated anti-C6 mAb (Quidel Corporation, San Diego, CA, USA) was made use of as detection antibody at a final concentration of four g/ml. Each antibodies cross-react with pig along with the assay can be applied to detect porcine TCC.22 The regular was regular human serum activated with zymosan and defined to include 1000 arbitrary units (AU)/ml. Zymosan-activated porcine serum was applied as a positive handle. The buffer applied as a diluent for the requirements and samples and.