Quantification of ADAMTS-13 Activity and Antigen from the Same Sample by a Newly Developed Combi-Actibind Assay (TECHNOZYM® ADAMTS-13).

Defects in activity and/or antigen levels of ADAMTS-13, the von Willebrand Factor (vWF) cleaving protease, are viewed as the main cause inducing the microvascular thrombotic disorder TTP (thrombotic thrombocytopenic purpura) that is in more than 90% of cases fatal if not treated early and appropriately. Malfunction of ADAMTS-13 with respect to cleave multimeric vWF can be caused by auto-antibodies directed against ADAMTS-13, by decreased presence of ADAMTS-13 in the circulation or by defective activity of ADAMTS-13. Therefore rapid and reliable diagnosis of ADAMTS-13 parameters is a clinical need. We present here a new assay for quantification of ADAMTS-13 activity and antigen levels based on ELISA format and thus suitable for fast and routine measurements.

The method is based on the Actibind® technology, using a monoclonal antibody to capture ADAMTS-13 from plasma to a microtiter plate. Activity is then assayed by a fluorescent substrate. After removal of the substrate solution, antigen can be assayed by standard ELISA technology using a polyclonal antibody followed by a peroxidase labelled secondary antibody. Thus both parameters can be measured from the same sample consequently in the same assay allowing calculation of specific activities of ADAMTS-13.

Dilutions of normal plasma (pool of 100 normal donors) were used as calibrators. The standard curve for the activity assay comprises a range from 3.7% to 100 % of normal plasma activity, thus samples of severe TTP (definition: activity below 5%) can be detected with sufficient sensitivity. 42 samples from TTP patients were tested in this new activity/antigen assay as well as in the TECHNOZYM ADAMTS-13 INH assay that allows determination of anti ADAMTS-13 auto antibodies. Using these two assays a significant negative correlation was found between ADAMTS-13 activity and the presence of autoantibodies, 82% of the samples with extremely low ADAMTS-13 activity (<5%) had high levels of autoantibodies. A less stringent positive correlation was found between ADAMTS-13 antigen and ADAMTS-13 activity. Out of 30 samples with low ADAMTS-13 activity (< 25%) 15 samples had antigen levels less than 50% of normal. Out of the 42 samples from TTP patients, 3 samples were found without auto antibodies but still low activity. 2 of these samples also had low antigen levels. Moreover, the TTP samples had significantly higher levels of vWF:CBA activity (p<0.05) consistent with the presence of high molecular weight vWF.

This combined new assay clearly adds to the diagnostic possibilities to differentiate patients with TTP with respect to defects in ADAMTS-13 function and antigen concentration. In addition, the results of this assay correlate with the clinical picture and the decreased functional activity of ADAMTS-13 leading to increased levels of vWF multimers and in turn platelet consumption.