DEVELOPMENT OF ADAMTS13 ACTIVITY ELISA KIT AND EVALUATION OF ITS PERFORMANCE

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  • ＡＤＡＭＴＳ１３活性測定用ＥＬＩＳＡキットの開発

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Abstract

ADAMTS13 specifically cleaves von Willebrand factor (VWF), which is a haemostatic factor, and hydrolyzes the peptide bond between Tyr1605-Met1606 in the VWF-A2 domain. The development of a mouse monoclonal antibody recognizing the C-terminal edge residue Tyr1605, the enzymatic cleavage site of the VWF-A2 domain, enabled the direct assay of the products cleaved by ADAMTS13. An ADAMTS13 activity ELISA kit based on this principle was developed, and its fundamental performance was studied.<br> The minimum detection limit was 0.4% of the normal detection limit. The intra- and inter- reproducibility study showed the results of CVs as 1.1-4.7% and 2.6-7.5%, respectively. The ideal linearity extrapolated to the original point was obtained in a dilution test. No interference with the ADAMTS13 activity assay was observed at the tested concentrations in the presence of various interferants such as hemoglobin, bilirubin, chylomicron, and rheumatoid factor. EDTA completely inhibited ADAMTS13 activity.<br> ADAMTS13 activities of 109 plasma samples were measured using this assay kit. The samples from Upshaw-Schulman syndrome (USS), Thrombotic Thrombocytopenic Purpura (TTP), USS carrier, and normal samples showed assay values 0.5-2.7%, 0.5-58.1%, 7.7-85.3%, and 54.7-134.4%, respectively. The test kit has been proven to have a sufficient clinical discriminate ability for diagnostic testing. We observed good correlation between this method and the SDS-Agarose gel electrophoresis method (r=0.931).<br> This assay kit can be used for routine laboratory test in blood transfusion medicine and clinical laboratory practice.<br>

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