Anti human vwf antibody

Von Willebrand factor (VWF) antigen (VWF:Ag) was measured by an ELISA technique. A 96-well DynexImmulon 4HBX plate (Fisher Scientific, Loughborough, UK) was coated with a capture antibody consisting of an antihuman VWF antibody from Dako (Ely, UK) and incubated at 4^oC overnight. The plate was then blocked using a solution of 2% polyvinyl pyrrolidine. After washing the plate three times (wash buffer consisting of phosphate buffered saline and 0.05% Tween 20 v/v), 100 μl of test plasma and control plasma (CryoCheck Abnormal 1 and Abnormal 2 control plasma, Precision Biologic, Dartmouth, Nova Scotia, Canada) were added to the appropriate number of wells. A standard curve was constructed using Technoclone reference plasma from Pathway Diagnostics, UK. The plate was then covered and incubated for 2 h at room temperature. The plate was then washed three times and a rabbit anti-VWF horse-radish peroxidase immunoconjugate antibody (Dako) was added to each well. The plate was then covered and incubated for 1 h at room temperature before washing a further three times. Hundred microlitres of 3,3′,5,5′-tetramethylbenzidine liquid reagent (Skybio Ltd, Wyboston, Bedfordshire, UK) was added to each well and after 7 min 50 μl of Red-Stop reagent (Skybio Ltd) was added. Light absorbance was read at 405 nm on a plate reader (BioTek).

Vascular function was measured in 129 subjects with type 2 diabetes. The clinical characteristics of the population are shown in Table 1. Major clinical risk factors were categorized as follows: hypercholesterolemia (total plasma cholesterol >4.8 mM or use of cholesterol-lowering medication); smoking (current or within last 6 mo); diabetes (fasting glucose >5.5 mM or current treatment with insulin or oral hypoglycemic agents); hypertension (blood pressure >140/90 mmHg or current treatment with antihypertensive agents), and overweight and obesity (body mass index >25 kg/m²). Some of these subjects have been described in a prior publication (24 (link)). Flow-mediated dilatation (FMD) was used as a measure of endothelial function, as described in detail elsewhere (25 (link)). Images were analyzed by Vascular Tools 5 software by 2 independent blinded observers. Von Willebrand factor (vWF) was measured using sandwich immunoassay utilizing an anti-human vWF antibody (Dako) and expressed as percentage of reference sample (25 (link)). Serum RANTES levels were determined using a quantitative ELISA (CCL5/RANTES Quantikine ELISA kit; R&D Systems). The Jagiellonian University ethics committee approved all human studies. All subjects provided written informed consent before the study.