Quantification of ADAMTS13 Antigen Levels in Healthy Donors and Patients with Thrombotic Microangiopathies by a Newly Developed Sandwich ELISA.
Authors: Rieger Manfred , Ferrari Silvia , Herzog Andrea , Konetschny Christian , Dockal Michael , Plaimauer Barbara , Scheiflinger Friedrich 
Published: October 2019

Abstract
The prolonged presence of unusually large VWF multimers due to reduced or absent proteolytic degradation by ADAMTS13 has been recognized as the main cause for thrombotic thrombocytopenic purpura (TTP). Congenital TTP is associated with gene defects whereas acquired TTP, is caused by autoantibodies to ADAMTS13. Such antibodies might block ADAMTS13 enzymatic activity or enhance its clearance from the circulation. There is an unknown correlation between ADAMTS13 activity and ADAMTS13 antigen level. Here we report for the first time the development of an ELISA to quantify ADAMTS13 antigen levels in human plasma derived from healthy controls, and patients diagnosed with thrombotic microangiopathies (TMAs) like TTP or the haemolytic uraemic syndrome (HUS). To set up the ELISA system, we used an affinity purified polyclonal rabbit anti-human ADAMTS13 IgG fraction to capture the human plasmatic ADAMTS13 and the same antibody preparation, although horseradish-peroxidase-labeled, as detection antibody. Quantification of bound ADAMTS13 was performed using ADAMTS13 depleted human plasma as matrix and recombinant human ADAMTS13 as standard. An interference of ADAMTS13 autoantibodies with the ELISA system was excluded by spiking experiments. In total we analyzed 94 individuals, 25 healthy donors and 69 patients with the clinical diagnosis of TMA. In the TMA group 17 patients were suffering from hereditary TTP/HUS, 40 patients had acquired TTP and 12 patients had HUS. In healthy donors the ADAMTS13 activity range was 0.46-1.25U/ml and the normal range obtained for ADAMTS13 antigen levels was 680–1350ng/ml (mean 1010ng/ml). Patients with diagnosis of hereditary TTP/HUS had low or undetectable ADAMTS13 activities and antigen levels <100ng/ml, except for a single patient where we found an ADAMTS13 antigen of 940ng/ml with an activity of 35%, suggesting a missense mutation not interfering with ADAMTS13 secretion, but compromising ADAMTS13 activity. Patients with acquired TTP (n=40) due to autoantibodies had variable ADAMTS13 antigen levels. Some (n=13) patients had very low or undetectable antigen levels (<100ng/ml) in correlation with undetectable ADAMTS13 activities, indicating an enhancing effect of ADAMTS13 clearance caused by the antibodies. A group of 21 patients with undetectable ADAMTS13 activity presented antigen levels ranging from 110ng/ml to 1040ng/ml, suggesting the presence of antibodies blocking ADAMTS13 activity, without enhancing its clearance. Four patients had ADAMTS13 antigen levels of 150–1250ng/ml and ADAMTS13 activities ranging from 13%–56%. A group of 2 patients with acute acquired TTP but without detectable autoantibodies showed ADAMTS13 antigen levels of 450 and 690ng/ml and ADAMTS13 activities of 30% and 49%, respectively. HUS patients (n=12, no anti-ADAMTS13 antibodies) had ADAMTS13 antigen levels close to normal or borderline normal (620–1430ng/ml) and ADAMTS13 activities of 30–60% compared to healthy individuals. In summary, this new developed diagnostic assay allows a rapid and very sensitive determination of ADAMTS13 antigen in human plasma, independent of anti-ADAMTS13 antibodies.