A rapid enzyme-linked assay for ADAMTS-13

Abstract

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 plasmawere 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 lL of test plasma. Amino acid sequencing of the cleavage product con.rmed 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. © 2006 International Society on Thrombosis and Haemostasis.