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ADAMTS‐13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant VWF‐A2 domain as substrate
Author(s) -
Whitelock J. L.,
Nolasco L.,
Bernardo A.,
Moake J.,
Dong JF.,
Cruz M. A.
Publication year - 2004
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.1538-7836.2004.00601.x
Subject(s) - recombinant dna , peptide , von willebrand factor , epitope , chemistry , monoclonal antibody , microtiter plate , thrombotic thrombocytopenic purpura , microbiology and biotechnology , adamts13 , cleavage (geology) , epitope mapping , antibody , biochemistry , chromatography , biology , immunology , platelet , paleontology , fracture (geology) , gene
Summary. The metalloprotease ADAMTS‐13 cleaves von Willebrand factor (VWF) at the Y842/M843 peptide bond located in the A2 domain. Measurement of ADAMTS‐13 activity is a clinical utility for thrombotic diseases, but the current assays used for diagnostic and clinical research are non‐physiological and time consuming. We have expressed in bacteria a recombinant VWF‐A2 peptide (aa 718–905) that contains both a 6xHis tag at the N‐terminal end and a Tag‐100 epitope at the C‐terminal end. Diluted plasma was mixed with the VWF‐A2 peptide and digestion was allowed to proceed in a Ni 2+ ‐coated microtiter well plate for 2 h. The immobilized Ni 2+ captures the VWF‐A2 peptide by its 6xHis tag and cleavage of the A2 peptide is measured by the removal of the C‐terminus fragment of the A2 peptide that contains the Tag‐100. The cleavage activity for this assay was defined by the low detection of A2 peptide containing the Tag‐100 epitope by the antiTag‐100 monoclonal antibody. The assay was completed in <5 h. We then used the assay to analyze ADAMTS‐13 activity in plasma from 39 healthy donors and 16 samples from patients diagnosed as thrombotic thrombocytopenic purpura. The average of enzyme activity ± SEM for normal plasmas diluted 1 : 50 was 40 ± 4.2% while the value obtained for the patients was 2.4 ± 0.7%. These results were validated by a traditional long incubation assay (24 h). Our assay provides significant advantages over currently used assays because it is quicker, reproducible, cost effective and measures ADAMTS‐13 activity under physiological and non‐denaturing conditions. This assay is clinically useful and significant in measuring ADAMTS‐13 activity in plasma.