Antigenic Peptides Capable of Inducing Specific Antibodies for Detection of the Major Alterations Found in Type 2B Von Willebrand Disease
Figure 1
SDS-PAGE and western blotting approaches featuring the major steps involved in the production of a panel of anti-(KLH-peptide) antibodies for detection of VWD subtype 2B. (a) Lane 1, 10% SDS-PAGE profile of pooled human plasma after enrichment for normal VWF using ethanol precipitation; Lane 2, western blotting for detection of normal VWF, at approximately 250 kDa, using the commercially available antibody against VWF/FVIII—note the presence of additional protein bands being recognized, particularly at lower mass; Lane 3, a representative Western blotting reaction obtained with the generated panel of anti-(KLH-peptide) antibodies, targeting both normal and altered VWF. (b) Detection of normal VWf by western blotting following the subtractive affinity chromatography; Lane 4, detection of VWF prior to depletion of antibodies against the normal factor; Lanes 5 and 6, detection of VWF using the nonretained fractions from the first and sixth chromatographic steps, respectively—note that 6 column passages proved sufficient for complete removal of anti-(KLH-peptide) antibodies. (c) Lane 7, 10% SDS-PAGE profile representative of the eluates obtained after affinity purification of antibodies targeting specifically altered VWf peptides—note the presence of IgG heavy and light chains at approximately 50 and 25 kDa, respectively; Lanes 8, 9, and 10, 10% SDS-PAGE profile of nonderivatized BSA, BSA-(RPSELRR) and BSA-(SQKRIRVA), respectively; Lanes 8′, 9′, and 10′, corresponding western blotting reactions obtained using control sera, anti-(KLH-RPSELRR) and anti-(KLH-SQKRIRVA), respectively.
