ADAMTS‐13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant VWF‐A2 domain as substrate

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 Ni2+‐coated microtiter well plate for 2 h. The immobilized Ni2+ 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