TechnoZym® ADAMTS-13 activity assay for determination of inhibitory antibodies against ADAMTS-13
L. Wagner, F. Nasufi, M. Kafka, N. B. Binder (Vienna, Austria)
Time: 17:15 - 18:15
Objective: Acquired TTP is frequently caused by inhibitory auto-antibodies against ADAMTS-13. Determination of ADAMTS-13 inhibitors therefore provides a valuable tool for diagnosis and patient follow-up in TTP. ADAMTS-13 antibodies can be detected in vitro either as IgG by ELISA, or functionally in a Bethesda based assay due to their inhibitory effect on ADAMTS-13 activity. Aim of this study was to evaluate the usability of TECHNOZYM® ADAMTS-13 activity ELISA in combination with a Bethesda based assay for detection of inhibitory antibodies against ADAMTS-13.
Methods: We determined ADAMTS-13 activity in plasma samples from patients clinically diagnosed with TTP and in normal plasma as negative control. The inhibitory antibodies against ADAMTS-13 were determined by a Bethesda-type method similar to the one used to analyze inhibitory FVIII antibodies. Patient plasma was heated treated at 56°C for 30 min to eliminate endogenous ADAMTS-13 activity. Then, a serial dilution was made before mixing 1:1 with normal human plasma (NHP). A 1:1 mixture of NHP with buffer was used as control mix. ADAMTS-13 activity of all dilutions was determined with TECHNOZYM® ADAMTS-13 activity ELISA. Residual activity in % was calculated from the results obtained in IU/mL for all patient plasma dilutions and the control mixture. The residual activity of patient plasma between 25% and 75% was used to further calculate the Bethesda Units (BU), where one BU is defined as the amount necessary to reduce ADAMTS13 activity to 50% of control mixture. The results for inhibitory auto-antibodies were considered to be positive if the result of patient plasma was found to be >0.5BU.
Results: All normal plasma samples were found to be below the 0.5BU limit. The inhibitor titer in TTP samples ranged from 0.64 BU/mL to 8.24 BU/mL. When 2 dilutions had a residual activity between 25% and 75% the calculated BU/mL between dilutions correlated very well.
Conclusion: In this study we demonstrate that combining TECHNOZYM® ADAMTS-13 activity ELSIA with a Bethesda based assay setting provides a good method for measurement of functional inhibitors. That allows differentiation of different forms of TTP and may enable close monitoring of inhibitor titer changes in the course of the disease and in response to treatment.