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


RESULTS
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
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.
Sauna
Zuben E
ZE
Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, United States of America. Zuben.sauna@fda.hhs.gov
Okunji
Chinyere
C
Hunt
Ryan C
RC
Gupta
Tanvi
T
Allen
Courtni E
CE
Plum
Elizabeth
E
Blaisdell
Adam
A
Grigoryan
Vahan
V
Geetha
S
S
Fathke
Robert
R
Soejima
Kenji
K
Kimchi-Sarfaty
Chava
C
D051722:ADAM Proteins / Q000276:immunology*; D000071120:ADAMTS13 Protein; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D005434:Flow Cytometry; D006801:Humans; D006868:Hydrolysis; D011487:Protein Conformation; D013696:Temperature
D000911:Antibodies, Monoclonal; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human