PB0317 - Novel luminescent-based ADAMTS13 activity assay

Background: The activity of Von Willebrand Factor (VWF) is modulated by ADAMTS13, a metalloprotease that cleaves VWF in the A2 domain. Deficiency in ADAMTS13 results in the disorder thrombotic thrombocytopenia purpura (TTP). ADAMTS13 activity analysis for initial diagnosis of TTP is the first-tier assay recommended by the ISTH in 2020. A point-of-care ADAMTS13 test could aid in the therapeutic decision-making process.

Aims: To develop a sensitive luminescent ADAMTS13 activity assay for a point-of-care platform.

Methods: A chimeric protein construct was expressed in BL21 E. coli containing the VWFA2 domain (with the ADAMTS13 cleavage site Y1605-M1606) in frame with N-terminal NanoLuc® Luciferase, a C-terminal His-tag, and a free cysteine at the C-terminus to enable purification and immobilization, respectively. This construct (N-NLuc-VWFA2-C) was purified and shown to be cleaved by ADAMTS13 by SDS-PAGE in the expected fragments. N-NLuc-VWFA2-C coupled via the free cysteine to commercially available maleimide-coated plates was also cleaved by ADAMTS13 as shown by the released luciferase activity in the supernatant transferred to wells with furimazine substrate. Titration of recombinant ADAMTS13 (TAK-755, Takeda) in ADAMTS13-deficient plasma was performed to verify sensitivity towards ADAMTS13 activity levels in plasma. The luminescent ADAMTS13 activity assay (ADAMTS13lum) was compared to the TECHNOZYM® ADAMTS13 Activity ELISA from Technoclone, using samples from ECAT surveys.

Results: Titration of rADAMTS13 in deficient plasma showed excellent concentration dependent luminescence of the ADAMTS13lum assay over the range of 0-2 IU/ml (R2 0.997). Estimation of ADAMTS13 activity levels of 4 ECAT samples with a range between 0.07 to 0.82 IU/ml with the ADAMTS13lum assay showed high level of agreement (deviance < 6%) with the levels estimated by TECHNOZYM® ADAMTS13 Activity ELISA.

Conclusion(s): A luminescent-based assay specific for ADAMTS13 activity was developed. Because of the high sensitivity of this technique, it is suitable for further miniaturization to a point-of-care platform and small plasma volumes.