Lt. Wen et al., CHEMILUMINOGRAPHIC DETECTION OF VON-WILLEBRAND-FACTOR MULTIMERIC COMPOSITION, Journal of clinical laboratory analysis, 7(6), 1993, pp. 317-323
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.