title: An ELISA for the quantitation of von Willebrand factor: osteoprotegerin complexes in plasma.
abstract: BACKGROUND
  Von Willebrand factor (VWF) is pivotal in arterial thrombosis, and osteoprotegerin (OPG) is besides being a bone protein also related to cardiovascular diseases. OPG can bind VWF, but the significance of this interaction is not known.
  
  
  OBJECTIVE
  The aim was to develop an assay for measurement of von Willebrand factor-osteoprotegerin complex (VWF:OPG) in human plasma. Furthermore, the significance of VWF:OPG complex as a marker of cardiovascular disease (CVD) was evaluated.
  
  
  METHODS
  A sandwich ELISA for quantification of VWF:OPG was developed using a polyclonal rabbit anti-human VWF capturing antibody and a monoclonal anti-human OPG detecting antibody. Samples were quantified relative to a standard curve obtained from dilutions of a plasma pool from healthy individuals. The assay was evaluated in two groups of patients with CVD and two groups of asymptomatic individuals with and without documented coronary calcification (total n=118).
  
  
  CONCLUSIONS
  The assay detected VWF:OPG complexes in human plasma, while no significant signal was observed when testing solutions containing VWF or recombinant OPG alone. Importantly, the ELISA assay was able to detect in vitro formed complexes between human VWF and recombinant OPG in a dose-dependent manner. There was a large inter-individual variation in plasma VWF:OPG levels, but we found no significant differences in the level of VWF:OPG complexes between the four groups. Thus, we conclude that increasing OPG plasma levels in atherosclerotic CVD are not derived from increased levels of complexed form of VWF and OPG, but are more likely due to increased amounts of OPG secreted into the circulation.
authors.affiliation: Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, DK-5000 Odense C, Denmark. pernille.vinholt@ouh.regionsyddanmark.dk
authors.forename: Pernille J
authors.initials: PJ
authors.lastname: Vinholt
authors.forename: Martin
authors.initials: M
authors.lastname: Overgaard
authors.forename: Axel C P
authors.initials: AC
authors.lastname: Diederichsen
authors.forename: Hans
authors.initials: H
authors.lastname: Mickley
authors.forename: Tina S
authors.initials: TS
authors.lastname: Poulsen
authors.forename: Niels Peter R
authors.initials: NP
authors.lastname: Sand
authors.forename: Mads
authors.initials: M
authors.lastname: Nybo
authors.forename: Lars M
authors.initials: LM
authors.lastname: Rasmussen
mesh_terms: D000368:Aged; D002318:Cardiovascular Diseases / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D053244:Osteoprotegerin / Q000032:analysis / Q000097:blood*; D014842:von Willebrand Diseases / Q000097:blood; D014841:von Willebrand Factor / Q000032:analysis / Q000378:metabolism*
chemical_list: D053244:Osteoprotegerin; D014841:von Willebrand Factor