Novel monoclonal antibody-based enzyme immunoassay for determining plasma levels of ADAMTS13 activity.
BACKGROUND
ADAMTS13 specifically cleaves unusually large von Willebrand factor (VWF) multimers, which induce platelet thrombi formation under high shear stress. ADAMTS13 activity is deficient in patients with thrombotic thrombocytopenic purpura (TTP). The determination of plasma levels of ADAMTS13 activity is a prerequisite for a differential diagnosis of thrombotic microangiopathies. Here, a unique and highly sensitive enzyme immunoassay (EIA) of ADAMTS13 activity is described.


METHODS
ADAMTS13 hydrolyzes the peptide bond between Y1605 and M1606 of VWF. In this assay, a recombinant fusion protein (GST-VWF73-His) is used as a substrate. A panel of mouse monoclonal antibodies (MoAbs) that specifically recognizes Y1605, which is the C-terminal edge residue of the VWF-A2 domain and is generated by the enzymatic cleavage, has been produced. These antibodies were prepared with a synthetic decapeptide, termed N-10 (1596-DREQAPNLVY-1605), as the immunogen. Twenty-six clones specific to N10 were obtained, and one anti-N10 MoAb was used in this study.


RESULTS
With horseradish peroxidase-conjugated anti-N10 MoAb, a standard enzyme assay was established. This assay was highly sensitive, and the detection limit was 0.5 percent of the normal. Further, an inhibitor of ADAMTS13 was measured to a level of 0.1 Bethesda units per mL. ADAMTS13 activity was measured in 20 patients with Upshaw-Schulman syndrome, a congenital TTP, and 61 acquired TTP patients. The activity measured by this assay and by the classic VWF multimer assay showed high correlation.


CONCLUSIONS
A convenient and highly sensitive EIA for ADAMTS13 activity has been established. This assay can be introduced for routine laboratory work in transfusion medicine.
Kato
Seiji
S
Department of Blood Transfusion Medicine, Nara Medical University, Nara, Japan.
Matsumoto
Masanori
M
Matsuyama
Tomomi
T
Isonishi
Ayami
A
Hiura
Hisahide
H
Fujimura
Yoshihiro
Y
D051722:ADAM Proteins / Q000737:chemistry* / Q000276:immunology; D000071120:ADAMTS13 Protein; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011697:Purpura, Thrombotic Thrombocytopenic / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014841:von Willebrand Factor / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human