PB1057 - A novel, easy-to-use ADAMTS13 activity assay using a fiber-optic surface plasmon resonance biosensor for diagnosis of thrombotic thrombocytopenic purpura

Background: Determination of ADAMTS13 activity is crucial to differentiate thrombotic thrombocytopenic purpura (TTP) from other thrombotic microangiopathies. Current ADAMTS13 activity assays are complex and only performed in specialized laboratories, hampering fast diagnosis in non-specialized centers. Hence, there is an unmet need to develop easy-to-use ADAMTS13 activity assays to allow swift diagnosis of TTP in both reference and non-reference centers.

Aims: Develop an ADAMTS13 activity assay on the easy-to-use fiber-optic surface plasmon resonance (FO-SPR) biosensor.

Methods: Anti-GST antibody was immobilized to gold-coated optical fibers using EDC/NHS chemistry. After blocking, recombinant GST-VWF73 was added. Addition of plasma ADAMTS13 triggers the cleavage of the Tyr-Met bond within GST-VWF73 after which the resulting decapeptide (DREQAPNLVY) is detected by mouse anti-N10 antibody followed by signal amplification using goat anti-mouse IgG-labeled gold nanoparticles. Detection limit was determined by evaluating ADAMTS13 activity of heat-inactivated plasma. The assay was validated by measuring ADAMTS13 activity of healthy donor (HD), acute TTP, sepsis and hemolytic uremic syndrome (HUS) patient samples and compared to ADAMTS13 activity determined in the FRETS-VWF73 assay.

Results: By optimizing anti-GST antibody, GST-VWF73 and anti-N10 detection antibody concentrations and selecting optimal buffer conditions, an FO-SPR ADAMTS13 activity assay was developed. A calibration curve was established using pooled normal human plasma. The assay detection limit was 6.8% (%CV=9.51). ADAMTS13 activity was undetectable in all TTP samples and above 10% in HD (51.83%-81.04%), HUS (27.91%-107.24%) and sepsis (14.18%-44.25%) samples. Significant correlation (Pearson r=0.8544, p< 0.0001) was found between ADAMTS13 activity determined in both FO-SPR and FRETS-VWF73 assays.

Conclusion(s): An ADAMTS13 activity assay was successfully developed on the novel, easy-to-use FO-SPR biosensor, allowing TTP patient discrimination in about 3 hours. Previously, ADAMTS13 antigen, conformation and anti-ADAMTS13 autoantibody assays were developed on the FO-SPR platform. Hence, simultaneous testing of all ADAMTS13 parameters is now available on the FO-SPR biosensor which can ameliorate TTP diagnosis.