VWF antigen (VWF:Ag) levels in the plasma samples obtained from the in vitro LVAD systems, were quantified by ELISA as described [30 (link),31 (link)]. Briefly, a 96-well microtiter plate was coated with 4.1 μg/mL of polyclonal anti-human VWF antibodies (Dako, Glostrup, DK), blocked with a 3% milk powder solution and a 1/80 dilution of human or bovine plasma was added and the samples were further diluted ½ in PBS containing 0.3% milk. Next, bound VWF was detected using polyclonal anti-human VWF antibodies labelled with horse radish peroxidase (HRP) (Dako). Colouring reaction was performed with orthophenylene diamine (OPD) and H2O2. The reaction was stopped with 4M sulfuric acid. Absorbance was measured at 490nm. A normal human plasma pool from 20 individuals (NHP) or a normal bovine plasma pool from 6 calves (NBP) was used to set up a calibration curve and undiluted plasma was set at 100%. The anti-VWF antibodies are known to cross react with bovine VWF.
Polyclonal anti human vwf antibody
Manufactured by Agilent Technologies
Sourced in Denmark, France
The Polyclonal anti-human VWF antibody is a laboratory reagent used for the detection and analysis of von Willebrand factor (VWF) in biological samples. It is a mixture of antibodies that recognize multiple epitopes on the VWF protein.
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3 protocols using polyclonal anti human vwf antibody
1
Quantification of VWF Antigen Levels
2
Quantification of Murine VWF Antigen
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VWF antigen (VWF:Ag) levels in plasma were determined using an in-house developed enzyme-linked immunosorbent assay as described42 (link). Briefly, a microtiter plate was coated with polyclonal anti-human VWF antibody (Dako, Glostrup, Denmark), known to cross-react with mVWF. Captured mVWF was detected using a mixture of in-house generated biotinylated anti-mVWF monoclonal antibodies and horseradish peroxidase-labeled streptavidin. Visualization was obtained with H2O2 and ortho-phenylenediamine. Pooled plasma from 38 Vwf+/+ mice was used as a normal murine plasma pool (NMP) reference and results were calculated and expressed as percentage of VWF level compared to the mean of the baseline samples (100%).
3
Equine Collagen Fibrillar Characterization
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Fibrillar collagen (equine type I) was obtained from Kordia (Leiden, The Netherlands). Apyrase (grade VII), bovine serum albumin (BSA), hexadecyltrimethylammonium bromide, rhodamine 6G, bovine thrombin and ferric chloride were obtained from Sigma (Saint-Quentin-Fallavier, France). d-Phe-Pro-Arg chloromethylketone dihydrochloride (PPACK) was purchased from Calbiochem-VWR (Fontenay-sous-Bois, France). Polyclonal anti-human VWF antibody was from Dako (Les Ulis, France). Immunoglobulins (IgG) anti-BSA were from MP Biomedicals (Illkirch, France). Mixture of purified rat monoclonal antibodies directed against mouse GPIbα were from Emfret Analytics (Eibelstadt, Germany). Peroxidase-labeled anti-human Fc or anti-HPC4 antibodies were from Sanquin (Amsterdam, the Netherlands) and Roche (Meylan, France), respectively.
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