ADAMTS13 activity was quantified using a standardized fluorescent-labeled substrate, FRETS-VWF73 (AnaSpec: AS-63728-01). ADAMTS13 (2.5 nM) as wild-type (R&D Systems: 6156-AD) or as ADAMTS13-BirA* was reacted with FRETS-VWF73 (1 µM) in FRETS buffer (20 mM Tris–HCl, 25 mM CaCl2, 0.05% Tween-20, pH 7.4) and the activity was measured using SpectraMax M3 (Molecular Devices) fluorescence mode at ex = 340 nm and em = 450 nm. The proteolysis rate was calculated by linear regression of the initial slope of the measured activity using SoftMax Pro (v5.4). ADAMTS13 specific activity rate was calculated by linear regression of the slope of the change in proteolysis rate over the change in concentration of ADAMTS13 and plotted using GraphPad Prism (v. 6). The concentration of ADAMTS13 utilized ranged from 0.5 to 10 nM, with a replicate number of 2.
Frets vwf73
Manufactured by AnaSpec
Sourced in United States
FRETS-VWF73 is an engineered fluorogenic substrate designed for the quantitative measurement of ADAMTS13 activity. It consists of a short peptide with a fluorescent label and a quencher. The substrate is designed to be cleaved by ADAMTS13, resulting in a measurable fluorescence signal.
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4 protocols using frets vwf73
1
Quantifying ADAMTS13 Enzymatic Activity
2
Sepsis Biomarker Measurement Protocol
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ADAMTS13 activity was measured using FRETS-VWF73 (Anaspec Inc; Cat. #AS-63728-01; Fremont, USA). Total ADAMTS13 antigen in plasma was quantified using human ADAMTS13 Quantikine ELSIA kit (R&D Systems; Cat. #DADT130; Minneapolis, USA). VWF antigen was quantified using human VWF-A2 DuoSet ELISA (R&D Systems; Cat. #DY2764-05). Protein C antigen was quantified by ELISA (Affinity Biologicals Inc; Cat. #PC-EIA; Ancaster, Canada). MPO levels were quantified using human Myeloperoxidase Quantikine ELISA kit (R&D Systems; Cat. #DMYE00B). All commercial assays were conducted according to manufacturer’s protocols. ADAMTS13 activity, ADAMTS13 antigen, VWF antigen, PC antigen, and MPO values were expressed relative to healthy control plasma. All parameters were measured on Day 1 and Day Last (non-survivors septic: 10.6±8.8 days, survivors septic: 5.7±3.0 days, and non-septic: 5.0±2.2 days).
3
Quantifying VWF and ADAMTS13 in Plasma
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Three milliliters of fasting venous blood were drawn from all subjects and anticoagulated with sodium citrate. Within 20 min of collection, the samples were centrifuged (2000 g, 5 min) and the resulting plasma samples were stored at −80 °C. The concentration of VWF in plasma was measured using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit purchased from Abcam, following the manufacturer's guidelines. Fluorescence resonance energy transfer (FRET) was used to determine ADAMTS13 activity in participants' plasma [43]. FRETS-VWF73 (ADAMTS13 fluorescent substrate) was purchased from AnaSpec, USA. A standard plasma sample, a mixture of plasma from 20 healthy individuals provided by the Physical Examination Center of the Fifth People's Hospital of Shanghai, was used to set the ADAMTS13 activity level at 100 %. The percentage of ADAMTS13 enzyme activity was expressed in the assay.
4
Quantifying Pulmonary Inflammation and Coagulation
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Levels of lung MPO were quantified using a mouse MPO DuoSet (R&D Systems, Minneapolis, Minnesota, USA). Plasma levels of Interleukin (IL)-6, IL-10, and monocyte chemoattract protein (MCP-1)/CCL2 were measured using the mouse IL-6 Duoset, the mouse IL-10 quantikine ELISA, and the Mouse CCL2/JE/MCP-1 DuoSet respectively, (R&D Systems, Minneapolis, Minnesota, USA). Plasma levels of thrombin antithrombin (TAT) complexes were quantified using the matched-pair antibody set (Affinity Biologicals, Ancaster, Ontario, Canada) according to the manufacturer’s protocol. CFDNA was quantified as per the manufacturer’s instructions using their Quant-iT™ PicoGreen™ assay (ThermoFisher Scientific, Waltham, Massachusetts, USA). ADAMTS13 activity was measured using FRETS-VWF73 (Anaspec Inc, Fremont, California, USA).
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