Premium
A rapid enzyme‐linked assay for ADAMTS‐13
Author(s) -
WU J.J,
FUJIKAWA K.,
LIAN E. C.,
McMULLEN B. A.,
KULMAN J. D.,
CHUNG D. W.
Publication year - 2006
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/j.1538-7836.2005.01677.x
Subject(s) - chemistry , adamts , antibody , horseradish peroxidase , thrombotic thrombocytopenic purpura , cleavage (geology) , peptide , biochemistry , metalloproteinase , substrate (aquarium) , microbiology and biotechnology , enzyme , chromatography , thrombospondin , biology , immunology , paleontology , ecology , platelet , fracture (geology)
Summary. Background: A deficiency in the plasma metalloprotease ADAMTS‐13 is associated with deposition of microvascular thrombi that cause thrombotic thrombocytopenic purpura. Current assays for ADAMTS‐13 are technically complex and time‐consuming. The objective of this study is to devise a rapid and sensitive assay for ADAMTS‐13 activity in plasma and verify the site of cleavage. Method: A new enzyme‐linked substrate, which contains a core ADAMTS‐13‐specific peptide conjugated to horseradish peroxidase (HRP) at the N‐terminus, and labeled with biotin at the C‐terminus, was constructed. After cleavage of this substrate by plasma ADAMTS‐13 and removal of uncleaved substrate by adsorption with streptavidin–agarose, ADAMTS‐13 activity was quantitated by determining the unadsorbed HRP activity remaining in solution. Levels of inhibitory antibodies in test plasma were also determined by measuring the residual ADAMTS‐13 activity after varying amounts of test plasma were incubated with a known amount of ADAMTS‐13. Results: Plasma ADAMTS‐13 activity was readily determined in ∼60 min (coefficient of variation 5.8%) using 1 μ L of test plasma. Amino acid sequencing of the cleavage product confirmed that cleavage occurred at the Tyr1605‐Met1606 bond in the substrate. ADAMTS‐13 activities in the plasma of five TTP patients were below 2%. Inhibitory antibody titers in these samples varied from undetectable to 81 BU mL −1 . Conclusion: The HRP‐linked substrate provides a rapid, sensitive, and reproducible way of determining the levels of ADAMTS‐13 activity and inhibitory antibodies in plasma.