Department of Clinical Chemistry, Karolinska University Hospital, Institution of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden.
Thromb Res. 2011 Jan;127(1):47-50. doi: 10.1016/j.thromres.2010.10.024. Epub 2010 Nov 20.
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.
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.
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.
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.
需要更可靠的方法来测量血浆样本中凝血因子 VIII（FVIII）与血管性血友病因子（VWF）的结合，以便在临床常规中使用。我们已经开发出一种利用 ELISA 系统测量血浆中 FVIII 结合的方法。
微量滴定板用一种单克隆抗体（ESH-8）包被，该抗体与 FVIII 的 C2 结构域反应。然后用重组 FVIII（Kogenate Bayer®）处理孔。洗涤后，加入稀释的血浆样本并孵育 1 小时。然后加入 HRP 缀合的针对 VWF 的抗体，并用于定量结合的 VWF。
获得了与 VWF 浓度反应的强信号。来自不同类型血管性血友病患者的血浆经常给出减弱的反应。然而，通过计算 FVIII 结合/VWF 抗原比，对 VWF 抗原水平进行校正后，只有已知的血管性血友病 2N 型（n = 4）患者的结果明显异常。40 名健康个体的 FVIII 结合被确定为 1.08 ± 0.48 U/mL（SD）。校正 VWF 抗原水平后，结果为 0.94 ± 0.15。因此，通过这种校正，SD 显著下降。系列内 CV 和系列间 CV 分别为 6.8%和 11.3%。
我们已经建立了一种简单可靠的方法来检测血浆样本中 FVIII 与血管性血友病因子结合的减少。该方法可方便地用于研究大人群，以及发现患者中较小的结合缺陷。
