Aihara M, Sawada Y, Ueno K, Morimoto S, Yoshida Y, de Serres M, Cooper H A, Wagner R H
Thromb Haemost. 1986 Apr 30;55(2):263-7.
A technique for the detection of von Willebrand factor multimers separated by discontinuous SDS agarose electrophoresis has been developed using non-radioactive compounds. The multimeric patterns were visualized by monospecific anti-human vWF:Ag followed by incubation with biotinylated antibody. After addition of avidin-biotin-peroxidase complex, the peroxidase activity was detected by 4-chloro-1-naphthol, giving sharp bands with a clear background. By this method, the differences of vWF:Ag multimers could be easily observed between normal plasma and the plasmas from variant type vWD (IIA, IIB, platelet-type). Large and intermediate multimers were absent in the plasma with vWD type IIA, while only large multimers were absent in the plasma with vWD IIB and platelet-type. The absence of large multimers was also observed in two commercial FVIII preparations having the ratio of vWF/vWF:Ag 0.18 and 0.63. The preparation with the ratio of 0.63 showed the presence of larger intermediate multimers. Electrophoresis in SDS 1.5% agarose gel revealed triplet structure of each small multimer, and a relative increase of the smallest subband was observed in vWD IIA plasma, platelet-type vWD plasma and commercial FVIII preparations. The procedures described are easy and safe to perform and are useful for screening or classifying cases with vWD in general laboratories.
已开发出一种使用非放射性化合物通过不连续SDS琼脂糖电泳检测血管性血友病因子多聚体的技术。通过单特异性抗人vWF:Ag，随后与生物素化抗体孵育来观察多聚体模式。加入抗生物素蛋白-生物素-过氧化物酶复合物后，用4-氯-1-萘酚检测过氧化物酶活性，可得到背景清晰的清晰条带。通过这种方法，可以很容易地观察到正常血浆与变异型vWD（IIA、IIB、血小板型）血浆之间vWF:Ag多聚体的差异。IIA型vWD血浆中缺乏大的和中等大小的多聚体，而IIB型和血小板型vWD血浆中仅缺乏大的多聚体。在两种vWF/vWF:Ag比率分别为0.18和0.63的市售FVIII制剂中也观察到大的多聚体缺失。比率为0.63的制剂显示存在较大的中等大小的多聚体。在1.5%琼脂糖SDS凝胶中进行电泳显示每个小多聚体的三重结构，并且在IIA型vWD血浆、血小板型vWD血浆和市售FVIII制剂中观察到最小亚带的相对增加。所述程序操作简便且安全，对一般实验室筛查或分类vWD病例很有用。
