Assays for Plasma ADAMTS13 Activity and Inhibitors Using Recombinant FRETS-VWF73 Peptide

Abstract

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy caused by severe deficiency of plasma ADAMTS13 activity. ADAMTS13 cleaves ultra-large von Willebrand factor (ULVWF) multimers, thus preventing the pathological accumulation of platelet-VWF aggregates at sites of vascular injury. The deficiency of ADAMTS13 can be congenital or more commonly immune-mediated, involving the formation of autoantibodies that bind and inactivate plasma ADAMTS13 activity. A simple, rapid, and cost-effective assay is crucial for the diagnosis of TTP and for the conduction of translational research relevant to ADAMTS13 function. Here, we describe a FRETS-rVWF73 assay for ADAMTS13 activity and inhibition, starting from the peptide substrate expression, purification, labeling, detailed assay protocol, and potential pitfalls. This assay can be used to quantify ADAMTS13 enzymatic activity and inhibition in various biological conditions for translational research.

1. Introduction

Thrombotic thrombocytopenic purpura (TTP) is a rare, life-threatening thrombotic microangiopathy, characterized by microvascular thrombosis resulting in thrombocytopenia, hemolytic anemia, and multi-organ dysfunction [1-4]. The central pathogenic mechanism in TTP involves a marked deficiency of plasma ADAMTS13, a metalloprotease responsible for cleaving ultra-large von Willebrand factor (ULVWF) multimers into smaller, less adhesive fragments [5]. In the absence of plasma ADAMTS13 activity, ULVWF accumulates on the endothelial surface and in circulation, leading to excessive platelet-VWF aggregation and microvascular thrombosis, the characteristic pathological feature of TTP [3]. ADAMTS13 deficiency may result from inherited ADAMTS13 mutations as seen in congenital TTP [6] or, more commonly, from acquired autoantibodies that inhibit ADAMTS13 enzymatic activity and/or promote accelerated clearance of the protease as seen in immune-mediated TTP [4, 7-11].

Timely and accurate assessment of plasma ADAMTS13 activity is essential for confirming the diagnosis of TTP and for distinguishing TTP from other thrombotic microangiopathies [12], such as atypical hemolytic uremic syndrome (aHUS) [13]. When plasma ADAMTS13 activity level is below 10 IU/dL, the diagnosis of TTP is confirmed in the appropriate clinical context; when plasma ADAMTS13 level is above 20 IU/dL, this generally rules out TTP. If a value falls between 10 and 20 IU/dL, it requires careful interpretation in the context of the patient’s clinical presentation. Other causes of low ADAMTS13 activity should be considered, including sepsis, surgery, and disseminated intravascular coagulation (DIC). Furthermore, a detection of plasma anti-ADAMTS13 autoantibodies or inhibitors is crucial for differentiating immune-mediated TTP from hereditary TTP and informing therapeutic strategies, including plasma exchange and immunosuppressive treatment [14, 15] or simple ADAMTS13 replacement therapy [16].

Historically, plasma ADAMTS13 activity assays used a full-length multimeric von Willebrand factor as a substrate [17, 18]. This assay took days to yield a result and was technically challenging. Modern approaches rely on the use of a VWF fragment or a synthetic peptide to improve assay sensitivity, specificity, and reproducibility [19]. Among these, the FRETS-hVWF73 assay is widely utilized for its speed and quantitative capabilities [20-22]. This assay uses a fluorogenic peptide substrate derived from the A2 domain of VWF, designed with a fluorophore–quencher or fluorophore-fluorophone homoquecher pair to enable real-time monitoring of protease activity. When cleaved at the Tyr1605-Met1606 bond by ADAMTS13, fluorescence is restored, providing a direct readout of enzymatic function (Fig. 1). Both recombinant and synthetic versions of the FRETS-VWF73 substrate are available and designed to mimic the native cleavage site, offering robust performance for clinical and research applications.

Fig. 1.

Structure and mechanism of the FRETS-VWF73 assay. (a) This 73-amino-acid peptide corresponds to the D1596–R1668 region of von Willebrand factor (VWF). (b) The Q1599 and N1610 residues were replaced with cysteine residues, which were subsequently labeled with 5′-fluorescein-maleimide. Due to their close proximity, homo fluorophore-fluorophore fluorescence quenching occurs. Cleavage of the peptide bond between the fluorescent labels restores fluorescence

In addition to assessing plasma ADAMTS13 activity, the FRETS-rVWF73 assay can be adapted to assess the presence and potency of inhibitory autoantibodies in patients with immune-mediated TTP [11, 23, 24]. This is achieved by pre-incubating recombinant or plasma-derived ADAMTS13 with patient plasma or serum, followed by substrate addition. A reduction in fluorescence relative to control samples indicates inhibition of ADAMTS13 activity. Serial dilutions of the patient sample allow for the generation of inhibition curves and calculation of IC₅₀ values, providing a quantitative measure of antibody-mediated inhibition. As such, this assay serves not only as a tool for measuring ADAMTS13 activity but also for evaluating immune-mediated inhibition—making it an essential and versatile method for comprehensive laboratory evaluation of TTP and other disorders involving ADAMTS13 dysfunction.

This chapter provides comprehensive protocols and practical guidance for implementing the recombinant fluorescent resonance energy transfer (FRETS-VWF73) assay, including detailed steps for reagent preparation, assay setup, data analysis, and quality control. In addition to measuring ADAMTS13 activity, the assay can be adapted to quantify the inhibitory potency of anti-ADAMTS13 autoantibodies. By preparing serial dilutions of patient plasma or serum, inhibition curves can be generated, allowing calculation of IC₅₀ values—a quantitative measure of antibody-mediated inhibition. Together, these applications make the recombinant FRETS-VWF73 assay a powerful and versatile tool for the laboratory evaluation of TTP and other disease conditions involving ADAMTS13 dysfunction. This assay is an excellent tool for research purposes requiring assessing plasma or tissue ADAMTS13 activity.

2. Materials

Prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a sensitivity of 18 MΩ-cm at 25 °C) and analytical grade reagents.

2.1. Buffers and Solutions

1. Cell suspension buffer: 50 mM sodium phosphate, pH 8.0, 500 mM NaCl, 1% igepal, 20% glycerol, 1 mM PMSF, 10 mM benzamidine-HCl, and 0.1 mM dithiothreitol (DTT).

2. Cell lysis buffer: Add 50 mg/mL of lysozyme to the cell suspension buffer to make the cell lysis buffer.

3. Equilibration Buffer A: 50 mM sodium phosphate, pH 8.0, 500 mM NaCl, and 20 mM imidazole.

4. Elution Buffer B: 50 mM sodium phosphate, pH 8.0, 500 mM NaCl, and 250 mM imidazole.

5. FRETS buffer: 5 mM Bis–Tris, 25 mM CaCl2, 0.005% Tween 20, pH 6.0. Store at 4 °C with light protection. Warm it to room temperature before use.

2.2. Consumables and Laboratory Equipment

White 96-well plate, plate sealer or lid, plastic reagent reservoirs, serological pipettes, 1.5–15-mL centrifuge tubes, multichannel pipette, chromatography columns, PD-10 desalting columns, Amicon Ultra-1 centrifugal filter, Ultracel 3 K, pH meter, hotplate stirrer, glass flasks, shaking bacterial incubator, 37 °C incubator, high speed centrifuge/ultracentrifuge, NanoDrop spectrophotometer, sonicator, and SpectraMax Gemini XPS microplate reader (Molecular Devices).

2.3. Cells and Chemical Reagents

E. coli strain BL21 transformed with pQE100-hVWF73 plasmid that expresses recombinant VWF3 peptide, Luria–Bertani (LB) broth, ampicillin, isopropyl β-D-1-thiogalactopyranoside (IPTG), lysozyme from chicken egg white, fluorescein-5-maleimide, pooled normal plasma (NHP), chelating Sepharose Fast Flow resin, 0.1% nickel sulfate hexahydrate, 2 mol/L imidazole stock solution, bond breaker TCEP solution, 1 mol/L Tris–HCl buffer, pH 7.9, DTT (dithiothreitol), sodium chloride crystals, sodium phosphate monobasic anhydrous, sodium phosphate dibasic heptahydrate, 1 mol/L monobasic sodium phosphate (NaH₂PO₄), 0.5 M dibasic sodium phosphate (Na₂HPO₄), 0.5 mol/L EDTA, pH 8.0, 400 mM Pefabloc SC, glycerol, ultra ≥99% (GC grade), IGEPAL CA-630, 1 mol/L phenylmethanesulfonyl fluoride (PMSF), 1 mol/L benzamidine hydrochloride hydrate, fluorescein-5-maleimide, 0.1% trifluoroacetic acid (TFA), 100% dimethyl sulfoxide (DMSO), 0.5 M NaOH solution, and 0.1% trifluoroacetic acid.

3. Methods

3.1. E. coli Expression of FRETS-rVWF73 Peptide

1. Inoculation of starter culture (day 1): Add 100 μL of sterile-filtered ampicillin (100 mg/mL) to 100 mL of LB broth in a 250-mL flask. Retrieve E. coli BL21 containing the pQE100-hVWF73 plasmid from the freezer and inoculate the prepared LB broth using a sterile pipette tip to scrape a small frozen portion. Incubate the culture at 37 °C in a shaker set to 225 rpm for approximately 16 h overnight.

2. Large-scale culture and induction (day 2): Add 1 mL of sterile-filtered ampicillin (100 mg/mL) to each of two 1-L LB broth cultures in 2-L flasks. Collect 1 mL of each LB/ampicillin mixture to use as a blank in optical density (OD)600 measurements. Inoculate each 1-L flask with 50 mL of the overnight culture. Incubate the cultures at 37 °C and 225 rpm for approximately 15 min, then begin measuring OD600 using a NanoDrop 9000. Continue measurements every 15 min until the OD reaches between 0.6 and 0.8. When the OD600 reaches approximately 0.4, remove IPTG (Isopropylβ-D-1-thiogalactopyranoside) from the freezer to allow it to thaw. Once the OD600 reaches the target range, induce protein expression by adding 1 mL of IPTG (1.0 M stock) to each 1-L culture. Reduce the temperature to 30 °C and continue shaking at 225 rpm for 4–6 h, or until the OD600 reaches approximately 3.0.

3. Collection of cell pellet (day 3): Prechill an ultracentrifuge to 4 °C. Transfer cultures into 1-L centrifuge bottles, ensuring that the caps are fitted with rubber gaskets to prevent leakage. Centrifuge the cultures at 3500 rpm at 4 °C for 20 min. If necessary, decant the supernatant, pool any remaining culture, and repeat the centrifugation for another 20 min. Decant the final supernatant and freeze the cell pellets at −80 °C until further use.

3.2. Purification and Fluorescein Labeling of FRETS-rVWF73 Peptide

1. Cell lysis (day 3): Remove frozen cell pellets from −80 °C freezer and allow them to thaw at room temperature. Resuspend the cells in a cold cell lysis buffer (minimum 30 mL per 1.0 L of culture) using a serological pipette. Transfer the suspension to a 50-mL centrifuge tube and place on ice. Prepare the lysis buffer by adding lysozyme to a final concentration of 1.0 mg/mL (~0.025 g per 25 mL). Sonicate the suspension on ice for 10 s, repeating the pulse ten times. Check the sample under a microscope for sufficient lysis, and repeat sonication if necessary. Pre-cool the centrifuge to 4 °C, then transfer the lysate to 30-mL thick-walled centrifuge tubes and centrifuge at 12,000 rpm for 15 min at 4 °C. Transfer the resulting supernatant to a fresh 50-mL tube, discarding the pellet. Add 20 mM imidazole to the clarified lysate and adjust the pH to 8.0 using either 1.0 M Tris or HCl at room temperature.

2. Prepare a Ni-sepharose affinity column (day 3): Prepare a 5-mL Ni-column by rinsing the yellow casing with deionized water, drying the exterior with a paper towel, and ensuring both end plugs are in place. Fill a beaker with 200 mL of deionized water and position it above bench height. Prime the column tubing with a syringe and clamp it shut with a binder clip. Fill the column with 5 mL of deionized water, mark the fill level, and discard the water. Add Chelating Sepharose Fast Flow beads up to the fill line and allow them to settle. Add more beads if needed. Wash the beads with 10 column volumes of deionized water using gravity flow. Close the stopcock, remove the tubing, and drain the column to just above the bead bed (see Note 1). Add 7 mL of 0.1 M NiSO₄ to the beads until they turn teal in color. Gently shake the column to ensure even coating. Wash the column with 10 column volumes of deionized water, followed by 10 column volumes of Equilibration/Wash Buffer A (20 mM imidazole). If air bubbles interrupt the flow, detach and reattach the tubing. Do not let the column dry.

3. Affinity purification and in-column fluorescein labeling of FRETS-VWF73 peptide: Collect 20 μL of the E. coli cell lysate for SDS-PAGE analysis, then load the remaining lysate onto the Ni-column. Reload the flow-through three times to maximize protein binding. Wash the column sequentially with 10 column volumes each of Wash Buffer A containing 20 mM imidazole and Wash Buffer B containing 40 mM imidazole using gravity flow. Stop the flow and drain the buffer until just above the beads. Seal the column and close the stopcock.

   To reduce disulfide bonds of the substrate, add 20 mM bond breaker (~600 μL) to the column, invert gently to mix, and incubate horizontally on a shaker for 15 min at room temperature. Wash with 10 column volumes of Equilibration/Wash Buffer A (20 mM imidazole). Prepare 2.0 mg of fluorescein-5-maleimide in 1.0 mL of Equilibration Buffer and add it to the column. Invert to mix, protect from light, and incubate for 15 min on a shaker. Wash again with 10 column volumes of Equilibration/Wash Buffer A followed by 10 column volumes of Wash Buffer B. Continue washing until the green-yellow dye is no longer visible and the flow-through is clear. Prepare at least twenty 1.5-mL microcentrifuge tubes for elution. Elute the bound protein using the Elution Buffer containing 250 mM imidazole and collect fractions until the dye is no longer visible. Measure OD at 280 nm (protein) and 495 nm (fluorescent) using a NanoDrop 9000. Pool fractions based on the OD280 peak and store the pooled eluate at 4 °C protected from light overnight (see Note 2).

4. Concentration and cleanup (day 4): Concentrate the purified protein using a Millipore 3 K centrifugal filter at 3000–3500 rpm at 4 °C in 15–30-min intervals. Retain the flow-through in a 15-mL centrifuge tube and continue centrifuging until the final protein volume is less than 2.5 mL. Wash the filter membrane by adding an equal volume of flow-through and combine it with the concentrated sample. Prepare a PD-10 desalting column by removing the storage solution, cutting the bottom notch, and equilibrating with ~25 mL of 0.1% trifluoroacetic acid (TFA) buffer. Load 2.5 mL of the concentrated protein onto the column and discard the flow-through. Elute the protein by adding 3.5 mL of 0.1% TFA buffer and collect the eluate into a 15-mL centrifuge tube. Further concentrate the protein using the 3 K filter under the same conditions. Measure OD at 280 nm and 495 nm on a NanoDrop 9000 using 0.1% TFA as the blank.

5. Calculate protein concentration using the following formula:

   $$\text{FRETS}-\text{rVWF}73(\mu \mathrm{M})=\frac{(10\times (\text{OD}280)-10\times (\text{OD}495)\times 0.11)\times 0.94}{0.0079(\text{rVWF}73\text{molecularweight})}(see\mathbf{Note}3).$$

   (see Note3)

6. Continue concentrating until the protein concentration exceeds 200 μM. Aliquot 50-μL samples into microcentrifuge tubes and store at −80 °C until use.

3.3. Assaying ADAMTS13 Activity by FRETS-rVWF73 Peptide

1. Plate reader settings and configuration: Begin by ensuring the FRETS plate reader is properly connected to the computer and powered on before sample preparation. Confirm the following settings: set the temperature to 25 °C; under kinetic parameters, set time to 1:00:00, interval to 2:00, and total reads to 31. Configure the fluorescence settings with excitation at 485 nm, cutoff at 530 nm, and emission at 538 nm. For well definition, assign concentrations for the standard curve wells and define all sample wells, ensuring each sample is run in duplicate. Note that the software will automatically calculate sample activity based on the standard curve.

2. Samples and FRETS-hVWF73 substrate preparation: To begin sample preparation, calculate the total volume of FRETS buffer needed based on the number of samples, the standard curve, and the number of controls (see example Fig. 2a). Allow the buffer to reach room temperature and then add Pefabloc to a final concentration of 1 mM. For example, if testing three samples in duplicate, calculate as follows: (a) volume for standard curve and controls: 45 μL + 46 μL + 47 μL + 48 μL + 49 μL + 50 μL + 47.5 μL + 44.5 μL = 377 μL; (b) volume for experimental samples: 45 μL × 3 (samples) × 2 (duplicates) = 270 μL; (c) add 20% extra volume to account for pipetting error: (377 + 270) μL × 1.2 = ~800 μL; (d) to achieve 1 mM final concentration, add 2 μL of 400 mM Pefabloc stock solution to 800 μL of FRETS buffer; (e) final preparation: 800 μL FRETS buffer +2 μL Pefabloc = ready-to-use working solution.

3. Standard curve: To prepare the standard curve, dilute normal human plasma (NHP) in the FRETS buffer as shown in Table 1.

4. Positive and negative controls: NHP as a positive and NHP with ethylenediaminetetraacetic acid (EDTA) as a negative control, as shown in Table 2.

5. Sample preparation: For experimental samples, dilute 5 μL of sample plasma with 45 μL of FRETS buffer to reach a final volume of 50 μL per well.

6. FRETS-rVWF73 substrate preparation: dilute the stock (400 μM) to double the desired final concentration for a 100 μL well volume. For example, if preparing substrate for 14 wells, calculate as follows: (a) 50 μL × 14 wells × 1.2 (pipetting error) = 840 μL; (b) volume of 400 μM r-hVWF73 needs = 840 μL × (2 μM/400 μM) = 4.2 μL; (c) final preparation: mix 4.2 μL of 400 μM r-hVWF73 stock with 835.8 μL of FRETS buffer to prepare 840 μL of 2 μM of working substrate.

7. Kinetic cleavage of FRETS-rVWF73: Add 50 μL of each diluted sample or control to the appropriate wells of a white 96-well plate. Using a multichannel pipette, immediately add 50 μL of the prepared FRETS-rVWF73 substrate to each well to reach a final reaction volume of 100 μL. Promptly transfer the plate to the plate reader and initiate the run (see Note 4).

Fig. 2.

Layout and representative results from the 96-well assay plate. (a) The first column contains the ADAMTS13 standard curve (STD1–STD6), prepared using normal human plasma (NHP) at defined activity levels of 50, 40, 30, 20, 10, and 0 mU/mL. Below the standards are the assay controls: the negative control (25 mU/mL ADAMTS13 + EDTA) and the positive control (25 mU/mL ADAMTS13 without EDTA), used to verify inhibition and assay performance. The second column includes three sample groups, each tested in duplicate. (b) Based on the standard curve, the interpolated ADAMTS13 activities for the samples are as follows: Sample 1 (A2/B2), average value is 1.225, estimated activity: 35 mU/mL; Sample 2 (C2/D2), average value is 0.59, estimated activity: 12 mU/mL; and Sample 3 (E2/F2), average is 0.115, estimated activity ≈ 1 mU/mL, near the negative control level

Table 1.

Standard curve setup and corresponding value of ADAMTS13 activity

STD #	Activity concentration (mU/mL)	NHP (μL)	FRETS buffer (μL)
1	50	5	45
2	40	4	46
3	30	3	47
4	20	2	48
5	10	1	49
6	0	0	50

Table 2.

Positive and negative control setup in each assay

	Plasma	FRETS buffer (μL)
Positive control	2.5 μL NHP	47.5
Negative control	2.5 μL NHP μL + 3 μL 0.5 M EDTA	44.5

Configure the device with the following settings: Temperature to 25 °C, the time to 1:00:00, interval to 2:00, and number of reads to 31. Under fluorescence settings, select excitation at 485 nm, cutoff at 530 nm, and emission at 538 nm. Define the layout of the wells by assigning concentrations for the standard curve and designating the sample wells, making sure that each sample is run in duplicate (see Note 5).

After completing the run, ensure that the standard curve achieves an R² value greater than 0.95 before proceeding with data interpretation. The software will then use this validated curve to calculate ADAMTS13 activity for each sample (see example in Fig. 2b).

3.4. Determining Anti-ADAMTS13 Inhibitors by FRETS-rVWF73

1. Samples and FRETS-rVWF73 substrate preparation: Based on the number of standard curve points, controls, and experimental samples, calculate the total required FRETS buffer volume. Bring the buffer to room temperature and add Pefabloc to a final concentration of 1 mM. For example (Fig. 3a), if you are testing eight serial dilutions of an autoantibody, the volume calculation proceeds as follows:

   1. Volume for standard curve and controls: 45 μL + 46 μL + 47 μL + 48 μL + 49 μL + 50 μL + 47.5 μL + 44.5 μL = 377 μL.
   2. Volume for experimental sample wells: (32.5 μL FRETS buffer +15 μL autoantibody dilution) × 8 (serial dilutions) = 380 μL.
   3. Account for pipetting error (20% excess): (377 μL + 380 μL) × 1.2 = ~910 μL.
   4. Pefabloc addition: 910 μL × (1 mM/400 mM) = 2.3 μL of 400 mM Pefabloc stock.
   5. Final preparation: Mix 910 μL of FRETS buffer with 2.3 μL of 400 mM Pefabloc to prepare your working solution.

2. Preparation for standard curve: Prepare a six-point standard curve by diluting normal human plasma (NHP) in FRETS buffer as shown in Table 1.

3. Positive and negative controls: Positive and negative controls are set as in Table 2.

4. Experimental sample preparation: To test inhibition by autoantibodies, prepare each experimental sample by mixing 5 μL of NHP with 32.5 μL of FRETS buffer and 12.5 μL of serially diluted autoantibody, for a final volume of 50 μL, as shown in Table 3.

5. Serial dilution of plasma containing inhibitors: To test a series starting from 200 nM with a 2× dilution factor, begin by determining the highest working concentration needed for the 50 μL well volume (see sample in Fig. 3a). Since 12.5 μL of antibody makes up one-fourth of the total reaction, and considering an additional two-fold dilution when mixed with substrate, the initial antibody concentration required is: 200 nM × 4 (volume correction) × 2 (final well dilution) = 1.6 μM.

   Prepare 30 μL of 1.6 μM autoantibody using a 10 μM stock, mix: 4.8 μL of 10 μM stock +25.2 μL of FRETS buffer.

   Perform two-fold serial dilutions by transferring 15 μL of the 1.6 μM solution into 15 μL of FRETS buffer, and repeat this step seven times to obtain eight concentrations. Mix 37.5 μL of plasma in FRETS buffer with 12.5 μL of each antibody dilution to create the final 50 μL sample mixtures.

   Transfer 50 μL of each sample, standard, and control to a white 96-well plate. Cover with foil and incubate at 37 °C for 15 min.

   FRETS-rVWF73 substrate preparation: Dilute FRETS-rVWF73 (400 μM, stock) in FRETS buffer to double the final required concentration (2 μM) for a total volume of 100 μL per well. For 16 wells: (a) total buffer volume: 50 μL × 16 wells × 1.2 = 960 μL, (b) volume of FRETS-rVWF73 stock required: 960 μL × (2 μM / 400 μM) = 4.8 μL, (c) final preparation: Mix 4.8 μL of 400 μM r-hVWF73 with 955.2 μL of FRETS buffer to make 960 μL working solution.

6. Assay execution: Using a multichannel pipette, add 50 μL of diluted FRETS-rVWF73 substrate to each well (already containing 50 μL of sample/control). Immediately transfer the plate to the reader and start the program (see Note 6).

7. Data acquisition and analysis: Ensure that the standard curve R² value is ≥0.98 before interpreting results (see example in Fig. 3b). Calculate ADAMTS13 activity as a percentage using the formula:

   $$\begin{gathered} \%\text{Activity}=(\text{NHP}\text{with}\text{antibody}∕\text{NHP}\text{without} \\ \text{antibody})\times 100 \end{gathered}$$

   .

Fig. 3.

Dose–response curve and IC₅₀ determination of an autoantibody inhibiting ADAMTS13 activity. (a) ADAMTS13 activity was measured in a 96-well plate format with a serial dilution of an autoantibody (final concentrations: 0–200 nM). (b) The values were measured and converted to activity values (mU/mL) using a standard curve based on normal human plasma (STD1–STD6: 50–0 mU/mL, value range 1.8–0.1). Negative (EDTA-treated) and positive (25 mU/mL untreated) controls were included. (c) Activity values for each antibody concentration were calculated via interpolation: 0 nM (29.66 mU/mL), 3.125 nM (26.99 mU/mL), 6.25 nM (22.54 mU/mL), 12.5 nM (18.09 mU/mL), 25 nM (10.67 mU/mL), 50 nM (4.73 mU/mL), 100 nM (0.28 mU/mL), and 200 nM (0 mU/mL). A dose–response curve was plotted using the interpolated activity values, and the data were fit using a four-parameter logistic regression model. The half-maximal inhibitory concentration (IC₅₀) of the autoantibody was determined to be approximately 12.5 nM

Table 3.

Preparation for inhibition assay

Sample#	Concentration (nM)	Autoantibody (μL)	NHP (μL)	FRETS buffer (μL)
1	400	12.5	5	32.5
2	133	12.5	5	32.5
3	44	12.5	5	32.5
4	14.8	12.5	5	32.5
5	4.94	12.5	5	32.5
6	1.65	12.5	5	32.5
7	0.55	12.5	5	32.5
8	0	12.5	5	45.0

4. Notes

1. When preparing the gel column, do not allow it to dry at any point.

2. Column regeneration: To regenerate the column, begin by washing with 10 column volumes of deionized water, followed by 5 column volumes of 0.5 M NaOH to remove residual proteins. Next, rinse with 10 column volumes of 0.2 M EDTA (pH 8.0) to chelate any remaining metal ions, then wash again with 10 volumes of deionized water. Finally, clean the column with 5 column volumes of 20% ethanol in deionized water. Store the regenerated column at 4 °C until its next use.

3. The value 0.0079 in the formula corresponds to the molecular weight of rVWF73 (in mg/μmol).

4. The enzymatic reaction initiates immediately upon mixing rVWF73 with plasma; therefore, precise timing is essential.

5. Sample activity is automatically calculated by the software based on the generated standard curve.

6. The enzymatic reaction initiates immediately upon the addition of rVWF73 to the wells containing plasma.