Optimization of the detection of inhibitory autoantibodies against the VWF-cleaving protease ADAMTS13 with an automated chemiluminescent ADAMTS13 activity immunoassay.

INTRODUCTION

Acquired thrombotic thrombocytopenic purpura is a rare disease associated with the production of autoantibodies against the VWF-cleaving protease ADAMTS13. The detection of these antibodies is made difficult by the instability of ADAMTS13 in citrated plasma and the time-consuming ADAMTS13 assays. The aim of our study was to evaluate the optimal conditions for detecting anti-ADAMTS13 inhibitory antibodies with the novel automated chemiluminescent immunoassay HemosIL AcuStar ADAMTS13 Activity assay.

METHODS

The parallelism between the AcuStar ADAMTS13 calibration curve and ADAMTS13 concentrations in serially diluted citrated plasma was evaluated after 2 hours incubation at 25°C, 37°C, or 37°C after addition of Ca to preserve the activity of the metalloprotease. Using Bethesda assays based on the 3 incubation procedures and the HemosIL AcuStar ADAMTS13 Activity assay, the inhibitor titers were determined in patients' samples with ADAMTS13 antibodies and compared with those determined using the Technozym ADAMTS13 activity ELISA.

RESULTS

The criterion of parallelism was respected for the 3 incubation methods over the range of ADAMTS13 concentrations relevant for the detection of ADAMTS13 inhibitor antibodies in a Bethesda assay. In agreement with this observation, all the incubation methods permitted the accurate detection and quantification of inhibitory anti-ADAMTS13 antibodies in the samples from patients with acquired thrombotic thrombocytopenic purpura.

CONCLUSION

Incubation of plasma samples with normal plasma at 25°C, 37°C, or 37°C after addition of Ca can be used in a Bethesda assay for quantifying the inhibitory activity of antibodies interfering with ADAMTS13 in the chemiluminescent HemosIL AcuStar ADAMTS13 Activity assay.