Acl 300

ADAMTS13 activity was measured using the fluorescence resonance energy transfer substrate VWF73 (FRETS-VWF73) as previously described [18 (link)]. VWF antigen (VWF:Ag) levels were determined with an in-house enzyme-linked immunosorbent assay, using polyclonal rabbit anti-human VWF antibodies (DakoCytomation, Glostrup, Denmark) for catching and tagging. Fibrinogen levels were derived from the clotting curve of the prothrombin time assay using Thromborel S as a reagent on an automated coagulation laboratory (ACL 300, Instrumentation Laboratory). In a subset of 1208 participants of RS-I-3, an extended panel of inflammatory and immunology markers was measured as previously described, including complement, immunoglobulins, and cytokines [19 (link)].

Fibrinogen levels were derived from the clotting curve of the prothrombin time assay, using Thromborel S (Behringwerke, Marburg, Germany) on the ACL 300 coagulation analyzer (Instrumentation Laboratory). vWF:Ag levels were measured with an in-house ELISA using polyclonal rabbit antihuman VWF antibodies and horseradish-peroxidase-conjugated antihuman VWF antibodies (DakoCytomation, Glostrup, Denmark) to catch and tag vWF. ADAMTS13 activity was measured in a kinetic assay using Fluorescence Resonance Energy Transfer Substrate VWF 73 (FRETS-VWF73), as is thoroughly described in the previous articles [16 (link), 17 (link)].
We determined NET levels by measuring MPO–DNA complexes with an ELISA as reported earlier [18 (link)]. We adjusted the commercial human cell death ELISA kit (Cell death detection ELISAPLUS, Roche Diagnostics Nederland B.V., Almere, The Netherlands). Briefly, as the capturing antibody, we used anti-MPO monoclonal antibody (clone 4A4, ABD Serotec). Patient plasma was added in combination with the peroxidase-labeled anti-DNA monoclonal antibody (from cell death detection ELISA kit; Roche). The absorbance at 405 nm wavelength was measured using Biotek Synergy HT plate reader with a reference filter of 490 nm. The values are expressed as milli-arbitrary units (mAU/mL).