title: A new method measuring the interaction between von Willebrand factor and coagulation factor VIII.
abstract: BACKGROUND
  There is a need for more reliable methods measuring the binding of coagulation factor VIII (FVIII) to von Willebrand factor (VWF) in plasma samples, for use in the clinical routine. We have developed such a method measuring FVIII binding in plasma, utilizing an ELISA system.
  
  
  METHODS
  Microtiter plates were coated with a monoclonal antibody (ESH-8), reacting with the C2 domain in FVIII. Thereafter the wells were treated with recombinant FVIII (Kogenate Bayer®). After washing, diluted plasma samples were added and incubated for 1h. Then HRP-conjugated antibodies against VWF were added and used for quantification of bound VWF.
  
  
  RESULTS
  A strong signal to VWF concentration response was obtained. Plasma from patients with different types of von Willebrand disease gave frequently diminished responses. However, after correction for the VWF antigen levels, by calculation of FVIII binding/VWF antigen ratio, only the patients with known von Willebrand disease type 2N (n = 4) had clearly abnormal results. The FVIII binding in 40 healthy individuals was determined as 1.08 ± 0.48 U/mL (SD). After correction for the VWF antigen levels the result was 0.94 ± 0.15. Thus, the SD declined substantially by this correction. The within-series CV and between-series CV were determined as 6.8 and 11.3%, respectively.
  
  
  CONCLUSIONS
  We have established a simple and reliable method to detect decreased binding of FVIII to von Willebrand factor in plasma samples. The method can conveniently be used to study large populations, as well as finding minor binding defects in patients.
authors.affiliation: Department of Clinical Chemistry, Karolinska University Hospital, Institution of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden. mattias.karlman@gmail.com
authors.forename: Mattias
authors.initials: M
authors.lastname: Karlman
authors.forename: Margareta
authors.initials: M
authors.lastname: Holmström
authors.forename: Björn
authors.initials: B
authors.lastname: Wiman
mesh_terms: D000911:Antibodies, Monoclonal; D015415:Biomarkers / Q000097:blood; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D011237:Predictive Value of Tests; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D020349:Surface Plasmon Resonance; D014842:von Willebrand Diseases / Q000097:blood / Q000175:diagnosis*; D014841:von Willebrand Factor / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D015415:Biomarkers; D011994:Recombinant Proteins; D014841:von Willebrand Factor; C078147:F8 protein, human; D005169:Factor VIII