title: Evaluation of a disintegrin-like and metalloprotease with thrombospondin type 1 repeat motifs 13 (ADAMTS13) activity enzyme-linked immunosorbent assay for measuring plasma ADAMTS13 activity in dogs.
abstract: A disintegrin-like and metalloprotease with thrombospondin type 1 repeat motifs 13 (ADAMTS13) is a von Willebrand factor (vWF)-cleaving protease. Deficiencies in ADAMTS13 activity are known to cause thrombotic diseases in human beings. The present study evaluated whether the human ADAMTS13 activity enzyme-linked immunosorbent assay (ELISA) kit containing human vWF73 (a minimal substrate) and anti-N10 antibody (which specifically recognizes the decapeptide of the C-terminal edge of cleaved vWF by human ADAMTS13) is applicable to the measurement of canine plasma ADAMTS13 activity. Human vWF73 fused with a GST-tag and a His-tag (GST-hvWF73-His) was reacted with recombinant canine (rc)ADAMTS13, canine plasma, and human plasma, and then used in Western blotting using anti-N10 antibody. Linearity and intra- and interassay reproducibility of the human ADAMTS13 activity ELISA kit in canine plasma were further evaluated. Finally, plasma ADAMTS13 activity was measured in 13 healthy dogs and 6 dogs with bacteremia using the human ADAMTS13 activity ELISA kit. Cleaved products with a 28-kDa GST-hvWF73-His were detected specifically in rcADAMTS13 as well as in human ADAMTS13, and also in canine plasma by anti-N10 antibody, showing excellent linearity. Intra-assay coefficient of variation (CV) was 3.0-12.4%, and interassay CV was 11.5-12.5%. The ADAMTS13 activity was significantly lower in dogs with bacteremia than in healthy dogs (P = 0.0025). The current study revealed that the human ADAMTS13 activity ELISA kit is applicable for measurement of canine plasma ADAMTS13 activity to elucidate the pathology of thrombotic diseases in dogs.
authors.affiliation: 1Haruhiko Maruyama, Laboratory of Veterinary Pathobiology, Department of Veterinary Medicine, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan. maruyama.haruhiko@nihon-u.ac.jp.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Maruyama
authors.forename: Michiko
authors.initials: M
authors.lastname: Kaneko
authors.forename: Taiga
authors.initials: T
authors.lastname: Otake
authors.forename: Rui
authors.initials: R
authors.lastname: Kano
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Yamaya
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Watari
authors.forename: Atsuhiko
authors.initials: A
authors.lastname: Hasegawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kamata
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016470:Bacteremia; D015153:Blotting, Western / Q000662:veterinary; D004285:Dogs / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D021122:Protein Subunits; D015203:Reproducibility of Results; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D014841:von Willebrand Factor; D051722:ADAM Proteins
keywords: Anti-N10 antibody; disintegrin-like and metalloprotease with thrombospondin type 1 repeat motifs 13; dogs; von Willebrand factor 73