[Development of a novel ADAMTS13 activity assay for diagnosis of TMA and criteria for judgment of platelet transfusion].
ADAMTS13 specifically cleaves unusually large von Willebrand factor (VWF) multimers, which induce platelet thrombi formation under high shear stress. The determination of plasma levels of ADAMTS13 activity is prerequisite for a differential diagnosis of thrombotic microangiopathies (TMAs). Here, we describe a unique and highly sensitive enzyme immunoassay of ADAMTS13 activity for routine laboratory use. ADAMTS13 hydrolyses the peptide bond between Y1605 and M1606 of VWF. In this enzyme immunoassay, a recombinant fusion protein (GST-VWF73-His) is used as a substrate. We have produced a panel of mouse monoclonal antibodies (anti-N10 mAb) that specifically recognizes the peptide containing Y1605 which is the C-terminal edge residue newly generated by the enzymatic cleavage. Using horseradish peroxidase-conjugated anti N10 mAb, a standard enzyme activity assay was established by ELISA. This assay was highly sensitive, and the detection limit was 0.5% of the normal. Further, an inhibitor of ADAMTS13 was measured to a level of 0.1 Bethesda U/ml. The activity measured by our novel assay and by the classic VWF multimer assay showed high correlation. We have also established ADAMST13 antigen ELISA assay using two clones of anti-ADAMST13 monoclonal antibody. The plasma antigen level of ADAMTS13 has generally good correlate to the plasma activity, but the several samples from acquired TTP patients with below 3% activity showed apparently high antigen level from 5 to 30%. Here, we have established a convenient and highly sensitive enzyme immunoassay for ADAMTS13 activity. This assay will be introduced for routine laboratory work in transfusion medicine.
Hiura
Hisahide
H
Japan Clinical Laboratories, Inc. Kyoto.
D051722:ADAM Proteins / Q000097:blood*; D000071120:ADAMTS13 Protein; D015415:Biomarkers / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D017713:Platelet Transfusion; D011697:Purpura, Thrombotic Thrombocytopenic / Q000097:blood / Q000175:diagnosis* / Q000628:therapy; D014841:von Willebrand Factor
D015415:Biomarkers; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human