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 <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.
Department of Medicine, Thrombosis Research Section, Baylor College of Medicine, Houston, TX, USA.
J L
JL
Whitelock
L
L
Nolasco
A
A
Bernardo
J
J
Moake
J-F
JF
Dong
M A
MA
Cruz
D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; D001665:Binding Sites; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism*
D011994:Recombinant Proteins; D014841:von Willebrand Factor; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human