CHEMILUMINOGRAPHIC DETECTION OF VON-WILLEBRAND-FACTOR MULTIMERIC COMPOSITION

Diagnosis of von Willebrand's disease (vWD) requires quantitation of v on Willebrand factor (vWF) in plasma plus qualitative assessment of th e vWF multimers according to molecular size ranges. Characterization o f vWF multimeric size distributions is typically done using sodium dod ecyl sulfate-agarose gel electrophoresis (SDS-AGE) followed by immunob lotting in the gel with radiolabeled antibody against vWF and autoradi ographic exposure. We applied a western blot technique to vWF multimer ic analysis. It included SDS-AGE, electroblotting onto a membrane, and chemiluminescent detection using rabbit anti-human vWF as primary ant ibody and goat anti-rabbit IgG as secondary antibody conjugated with h orseradish peroxidase. Using this method, 18 to 20 vWF multimers were regularly resolved in normal plasma with exposure times of 2 to 4 sec compared to 4 hr or longer by autoradiography. Sensitivity of detectio n was at least 4-fold enhanced by chemiluminescence compared to radiol abel. Specificity of the assay was confirmed by analysis of plasma sam ples known to be deficient to different degrees in the larger vWF mult imers. The chemiluminographic assay for vWF multimers is superior to t he autoradiographic one because it is more sensitive, avoids use of ra dioactivity, and has shorter total assay time (under 2 days versus fiv e radiolabel). (C) 1993 Wiley-Liss, Inc.