Chemiluminographic detection of von Willebrand factor multimeric composition.
Diagnosis of von Willebrand's disease (vWD) requires quantitation of von Willebrand factor (vWF) in plasma plus qualitative assessment of the vWF multimers according to molecular size ranges. Characterization of vWF multimeric size distributions is typically done using sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) followed by immunoblotting in the gel with radiolabeled antibody against vWF and autoradiographic exposure. We applied a western blot technique to vWF multimeric analysis. It included SDS-AGE, electroblotting onto a membrane, and chemiluminescent detection using rabbit anti-human vWF as primary antibody and goat anti-rabbit IgG as secondary antibody conjugated with horseradish 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 detection was at least 4-fold enhanced by chemiluminescence compared to radiolabel. Specificity of the assay was confirmed by analysis of plasma samples known to be deficient to different degrees in the larger vWF multimers. The chemiluminographic assay for vWF multimers is superior to the autoradiographic one because it is more sensitive, avoids use of radioactivity, and has shorter total assay time (under 2 days versus five radiolabel).
Wen
L T
LT
Scripps Immunology Reference Laboratory, Scripps Research Institute, La Jolla, California.
Smolec
J M
JM
Coughlin
J
J
McPherson
R A
RA
D000818:Animals; D001345:Autoradiography; D001797:Blood Protein Electrophoresis / Q000379:methods / Q000706:statistics & numerical data; D004587:Electrophoresis, Agar Gel / Q000379:methods / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D006801:Humans; D007124:Immunoenzyme Techniques / Q000706:statistics & numerical data; D008163:Luminescent Measurements; D011487:Protein Conformation; D011817:Rabbits; D012680:Sensitivity and Specificity; D014842:von Willebrand Diseases / Q000097:blood* / Q000175:diagnosis*; D014841:von Willebrand Factor / Q000032:analysis / Q000737:chemistry*
D014841:von Willebrand Factor