A rapid enzyme-linked assay for ADAMTS-13.
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.


METHODS
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).


CONCLUSIONS
The HRP-linked substrate provides a rapid, sensitive, and reproducible way of determining the levels of ADAMTS-13 activity and inhibitory antibodies in plasma.
Wu
J-J
JJ
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Fujikawa
K
K
Lian
E C
EC
McMullen
B A
BA
Kulman
J D
JD
Chung
D W
DW
D051722:ADAM Proteins / Q000097:blood* / Q000378:metabolism; D000071120:ADAMTS13 Protein; D001665:Binding Sites; D019921:Biotinylation; D004796:Clinical Enzyme Tests / Q000379:methods* / Q000592:standards; D006735:Horseradish Peroxidase; D006801:Humans; D010446:Peptide Fragments; D010783:Photometry; D011697:Purpura, Thrombotic Thrombocytopenic / Q000175:diagnosis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D010446:Peptide Fragments; D006735:Horseradish Peroxidase; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human