A rapid ADAMTS13 activity assay using a portable readout device.

Thrombotic Thrombocytopenic Purpura (TTP) is a severe type of thrombotic microangiopathy (TMA)with a high mortality rate. Detection of plasma ADAMTS13 activity levels <0.1 IU/mL confirm the diagnosis of TTP, whereas anti-ADAMTS13 antibodies detection allow differentiation between congenital (cTTP) or acquired (iTTP) phenotype. Unfortunately, turn-around-times of ADAMTS13 assays are often long, hampering proper treatment decisions. This study describes the design and validation of a microfluidic luminescent-based ADAMTS13 assay (ADAMTS13 lum ) to quantify ADAMTS13 activity levels and detect ADAMTS13 antibodies in plasma samples using a portable device. The platform consists of a reusable portable processor and a disposable microfluidic cartridge. The cartridge contains an immobilized chimera of the VWF-A2 domain in tandem with luciferase (Nter-Nluc-VWFA2-Cys-Cter). Upon cleavage by ADAMTS13, the liberated luciferase of the chimera is transported to the detection area that contains furimazine, the substrate for luciferase. The portable processor contains miniaturized photon sensors to quantify the luminescent signals. The signal output of the ADAMTS13 lum assay is linear to the ADAMTS13 activity levels in plasma samples. ADAMTS13 levels measured with the ADAMTS13 lum assay are in good agreement with commercially available ADAMTS13 activity assays. Finally, inhibition of rADAMTS13 printed in a parallel line detects ADAMTS13 antibodies, allowing for simultaneous quantification of ADAMTS13 activity levels as well as detecting ADAMTS13 antibodies. This technology enables rapid quantification of ADAMTS13 activity levels and detection of ADAMTS13 antibodies using a portable device.