Electroblot and Immunoperoxidase Staining for Rapid Screening of the Abnormalities of the Multimeric Structure of von Willebrand Factor in von Willebrand’s Disease

Summary

A new electrophoretic method is described for rapid screening of abnormalities of the multimeric structure of von Willebrand factor in von Willebrand’s disease. The method is based on the transfer of the separated proteins from agarose gels onto nitrocellulose foils followed by immunoperoxidase staining. It has the advantage of not requiring radio-iodinated antibodies and reduces the working time for the entire procedure from 5-6 days to 3 days. Electroblotting followed by immunoperoxidase staining differentiates patients with intact multimeric structure from those without intermediate and/or large multimers. The more subtle defects of the inner structure of the smallest multimers found in patients with type II von Willebrand’s disease can also be identified. A potential disadvantage of electroblotting and immunoperoxidase staining is the lesser sensitivity of this technique, which results in the detection of a smaller number of multimers (11-12 bands) than by autoradiography without transfer onto nitrocellulose (16-17 bands).

Key words

Electroblots - von Willebrand Factor - von Willebrand’s Disease - Immunoperoxidase

• References

• 1 Zimmerman TS, Ruggeri ZM. von Willebrand’s disease. Clin Haematol 1983; 12: 175-200
• 2 Ruggeri ZM, Zimmerman TS. Variant von Willebrand’s disease. Characterization of two subtypes by analysis of multimeric composition of factor VIII/von Willebrand factor in plasma and in platelets. J Clin Invest 1980; 65: 1318-1325
• 3 Hoyer LW, Shainoff JR. Factor VIII related protein circulates in normal human plasma as high molecular weight multimers. Blood 1980; 55: 1056-1059
• 4 Ruggeri ZM, Zimmerman TS. The complex multimeric compositionof factor VIII/von Willebrand factor. Blood 1981; 57: 1140-1143
• 5 Ruggeri ZM, Nilsson IM, Lombardi R, Holmberg L, Zimmerman TS. Aberrant multimeric structure of von Willebrand factor in a new variant of von Willebrand’s disease (Type II C). J Clin Invest 1982; 70: 1124-1127
• 6 Kinoshita S, Harrison J, Lazerson J, Abildgaard CF. A new variant of dominant type II von Willebrand’s disease with aberrant multimeric pattern of factor VIII-related antigen (type II D). Blood 1984; 63: 1369-1371
• 7 Mannucci PM, Abildgaard CF, Gralnick HR, Hill FGH, Hoyer LW, Lombardi R, Nilsson IM, Tuddenham E, Meyer D. Multicenter comparison of von Willebrand factor multimer sizing techniques. Thromb Haemostas 1985; 54: 873-877
• 8 Ruggeri ZM, Mannucci PM, Jeffcoate SC, Ingram GIC. Immunoradiometric assay of factor VIII related antigen, with observations in 32 patients with von Willebrand’s disease. Br J Haematol 1976; 33: 221-230
• 9 Mannucci PM, Lombardi R, Bader R, Vianello L, Federici AB, Solinas S, Mazzucconi MG, Mariani G. Heterogeneity of type I von Willebrand disease: evidence for a subgroup with an abnormal von Willebrand factor. Blood 1985; 66: 796-802
• 10 Hoyer LW, Rizza CR, Tuddenham EGD, Carta CA, Armitage H, Rotblat F. von Willebrand factor multimer patterns in von Willebrand’s disease. Br J Haematol 1983; 55: 493-507
• 11 Furlong BL, Peake IR. An electroblotting technique for the detection of factor VIII/von Willebrand factor multimers in plasma. Br J Haematol 1983; 53: 641-643
• 12 Brosstad F, Kjønniksen I, Rønning B, Stormorken H. SDS-agarose pattern of vWF-multimers visualized after immunoblotting with peroxidase-conjugated secondary antibodies: a new approach. Thromb Haemostas 1985; 54: 74
• 13 Chow R, Savidge GF. Enzyme linked antibody systems for the visualization of factor VIII multimers. Thromb Haemostas 1985; 54: 75
• 14 Millar DS, Mibashan RS, Rodeck CH, Nicolaides KH. Detection of factor VIII/vWF multimers in fetal, adult and variant vWD plasmas using a peroxidase anti-peroxidase system. Thromb Haemostas 1985; 54: 76
• 15 Miller MA, Palascak JE, Thompson MR, Martelo OJ. A modified SDS agarose gel method for determining factor VIII von Willebrand factor multimers using commercially available reagents. Thromb Res 1985; 39: 777-780