Analysis of von Willebrand factor multimers using a commercially available enhanced chemiluminescence kit.

AUTOR(ES)

Cumming, A M

RESUMO

AIMS--To develop a rapid, sensitive, and safe method for the analysis of von Willebrand factor (vWf) multimers in plasma or platelet lysates. METHOD--Analysis of vWf multimers was carried out by sodium dodecyl sulphate-agarose discontinuous gel electrophoresis followed by protein transfer to nitrocellulose membranes by western blotting. Blots were probed using horseradish peroxidase (HRP) conjugated rabbit anti-vWf; visualisation of vWf multimers was achieved using a commercially available enhanced chemi-Luminescence (ECL) kit for detecting HRP labelled antibodies on western blots. RESULTS--Electrophoretic transfer of vWf multimers to nitrocellulose membranes, including the higher molecular weight forms, was achieved satisfactorily and there was good resolution of individual multimer bands and of the triplet sub-band structure. Type II vWD variants were readily identifiable. The use of ECL conferred a high degree of sensitivity to the method and the end result on autoradiography film provided a permanent record which did not fade and which was suitable for scanning densitometry. CONCLUSION--The method for vWf multimer analysis described here is sensitive, simple to carry out, uses minimal amounts of reagents, produces results within 48 hours, and does not require the use of potentially hazardous radioactive materials or carcinogenic enzyme substrates.

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