An electroblotting technique for the detection of factor VIII/von Willebrand factor multimers in plasma

Abstract

A new, relatively simple technique was developed in order to study the multimers of factor VIII/von Willebrand factor (VIII/vWF). It involves electrophoresis on SDS [sodium dodecyl sulfate] agarose gels and electrophoretic transfer (electroblotting) of the separated protein bands onto nitrocellulose membranes, to which they are non-covalently bound. VIII/vWF multimers are then detected by 125I-labeled antibodies to VIII/vWF and autoradiography. Optimum electrophoretic transfer occurred at 0.5 A, for 18 h, on 0.8% agarose gels, thus enabling detection of the multimeric profile of VIII/vWF in 5 .mu.l of normal plasma. The multimeric profile for hemophilia A patients was identical to that for normal plasma. In plasma from patients with von Willebrand''s disease (vWd), various patterns were seen, with a preponderance of smaller multimers in type II (atypical) vWd, similar to those seen in cryosupernatant. Heterogeneity within a particular type of vWd was also evident. Investigation of commercial factor VIII concentrates showed the presence of doublets of VIII/vWF. Unlike other reported techniques, the rapid transfer and fixing of the protein bands to the nitrocellulose, minimizes loss of resolution, and handling of the paper is easier. It is possible to cut a sample electrophoresis strip into several areas, for incubation with different antibodies. Preliminary experiments also suggest that double antibody techniques are possible, or even removal of a 1st radiolabeled antibody by low pH, and then incubation of the separated proteins with a 2nd, unrelated antibody.