Antigenic Peptides Capable of Inducing Specific Antibodies for Detection of the Major Alterations Found in Type 2B Von Willebrand Disease

Abstract

Von Willebrand disease (VWD) is an inherited hemorrhagic disorder promoted by either quantitative or qualitative defects of the von Willebrand factor (VWF). The disease represents the most common human coagulopathy afflicting 1.3% of the population. Qualitative defects are subdivided into four subtypes and classified according to the molecular dysfunction of the VWF. The differential diagnosis of the VWD is a difficult task, relying on a panel of tests aimed to assess the plasma levels and function of the VWF. Here, we propose biochemical approaches for the identification of structural variants of the VWF. A bioinformatic analysis was conducted to design seven peptides among which three were representatives of specific amino acid sequences belonging to normal VWF and four encompassed sequences found in the most common VWD subtype 2B. These peptides were used to immunize mice, after which, peptide-specific immunoglobulins were purified. This resulted in four Ig preparations capable of detecting alterations in the subtype 2B VWD plus additional three antibody fractions targeting the normal VWF. The panel of antibodies could serve many applications among them (1) assessment of VWF: antigen interaction, (2) VWF multimer analysis, and (3) production of monoclonal antibodies against VWF for therapeutic purposes as in thrombotic thrombocytopenic purpura.

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.