ADAMTS-13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant VWF-A2 domain as substrate
| Autor: | Joel F. Moake, Aubrey Bernardo, Leticia Nolasco, Jody L. Whitelock, Jing-fei Dong, Miguel A. Cruz |
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| Rok vydání: | 2004 |
| Předmět: |
medicine.drug_class
ADAMTS13 Protein Peptide Enzyme-Linked Immunosorbent Assay Monoclonal antibody Epitope law.invention Substrate Specificity Von Willebrand factor law von Willebrand Factor medicine Peptide bond Humans chemistry.chemical_classification Binding Sites biology Chemistry ADAMTS Metalloendopeptidases Hematology Molecular biology Enzyme assay Recombinant Proteins ADAM Proteins Kinetics Biochemistry biology.protein Recombinant DNA |
| Zdroj: | Journal of thrombosis and haemostasis : JTH. 2(3) |
| ISSN: | 1538-7933 |
| Popis: | 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 |
| Databáze: | OpenAIRE |
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