Visualization of von Willebrand factor multimers by immunoenzymatic stain using avidin-biotin peroxidase complex.
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.
D001360:Avidin; D001710:Biotin; D055598:Chemical Phenomena; D002621:Chemistry; D004586:Electrophoresis / Q000379:methods*; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005169:Factor VIII / Q000032:analysis; D006801:Humans; D007124:Immunoenzyme Techniques; D009284:Naphthols; D013379:Substrate Specificity; D014842:von Willebrand Diseases / Q000175:diagnosis / Q000235:genetics; D014841:von Willebrand Factor / Q000032:analysis*
D009284:Naphthols; D014841:von Willebrand Factor; D001360:Avidin; C025917:4-chloro-1-naphthol; D001710:Biotin; D005169:Factor VIII