Characterization of conformation-sensitive antibodies to ADAMTS13, the von Willebrand cleavage protease

Zuben E Sauna, Chinyere Okunji, Ryan C Hunt, Tanvi Gupta, Courtni E Allen, Elizabeth Plum, Adam Blaisdell, Vahan Grigoryan, S Geetha, Robert Fathke, Kenji Soejima, Chava Kimchi-Sarfaty
PloS One 2009 August 5, 4 (8): e6506

BACKGROUND: The zinc metalloprotease ADAMTS13 is a multidomain protein that cleaves von Willebrand Factor (VWF) and is implicated in Thrombotic Thrombocytopenic Purpura (TTP) pathogenesis. Understanding the mechanism of this protein is an important goal. Conformation sensitive antibodies have been used to monitor protein conformation and to decipher the molecular mechanism of proteins as well as to distinguish functional and non-functional mutants.

METHODOLOGY/PRINCIPAL FINDINGS: We have characterized several antibodies against ADAMTS13, both monoclonal and polyclonal. We have used flow cytometry to estimate the binding of these antibodies to ADAMTS13 and demonstrate that antibodies raised against the TSP and disintegrin domains detect conformation changes in the ADAMTS13. Thus for example, increased binding of these antibodies was detected in the presence of the substrate (VWF), mainly at 37 degrees C and not at 4 degrees C. These antibodies could also detect differences between wild-type ADAMTS13 and the catalytically deficient mutant (P475S). The flow cytometry approach also allows us to estimate the reactivity of the antibody as well as its apparent affinity.

CONCLUSIONS/SIGNIFICANCE: Our results suggest that these antibodies may serve as useful reagents to distinguish functional and non-functional ADAMTS13 and analyze conformational transitions to understand the catalytic mechanism.

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