A rapid enzyme-linked assay for ADAMTS-13

J Thromb Haemost. 2006 Jan;4(1):129-36. doi: 10.1111/j.1538-7836.2005.01677.x.


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 approximately 60 min (coefficient of variation 5.8%) using 1 microL 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • ADAM Proteins / blood*
  • ADAM Proteins / metabolism
  • ADAMTS13 Protein
  • Binding Sites
  • Biotinylation
  • Clinical Enzyme Tests / methods*
  • Clinical Enzyme Tests / standards
  • Horseradish Peroxidase
  • Humans
  • Peptide Fragments
  • Photometry
  • Purpura, Thrombotic Thrombocytopenic / diagnosis*
  • Reproducibility of Results
  • Sensitivity and Specificity


  • Peptide Fragments
  • Horseradish Peroxidase
  • ADAM Proteins
  • ADAMTS13 Protein
  • ADAMTS13 protein, human