Anti mpo monoclonal antibody clone 4a4

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).

Citrated plasma samples were collected at the third visit of RS-I (1997–1999) and the baseline examination of RS-II (2000–2001), and stored at −80°C. We determined circulating levels of NETs by measuring myeloperoxidase (MPO)-DNA complexes with a capture ELISA as reported earlier (4 (link)). Briefly, we adjusted the commercial human cell death ELISA kit (Cell death detection ELISAPLUS, Cat. No 11-774-425-002; Roche Diagnostics Nederland B.V., Almere, the Netherlands) in which we used anti-MPO monoclonal antibody (clone 4A4, ABD Serotec, # 0400-002) as the capturing antibody. Patient plasma was added together with the peroxidase-labeled anti-DNA monoclonal antibody (component No.2 of the commercial cell death detection ELISA kit; Roche, #11-774-425-002). The absorbance at 405 nm wavelength was measured using BioTek Synergy HT plate reader with a reference filter of 490 nm. Values are expressed as milli arbitrary units (mAU/ml). The reference line to define the mAU was composed after isolation and induction of neutrophils from a healthy donor. NET formation was induced by adding phorbol 12-myristaat 13-acetaat (PMA).