REAADS® vWF Antigen

The REAADS® vWF Antigen (vWF:Ag) test kit is an enzyme linked immunosorbent assay for determining von Willebrand Factor levels in citrated human plasma, expressed in relative percent (%) of normal. The assay is intended to be used as an aid in the diagnosis of von Willebrand Disease in patients with bleeding disorders, and to help distinguish between vWD and classic Hemophilia A. REAADS® vWF:Ag Test Kit will accurately detect antigen levels as low as 5% of normal.

Reagents

• 12 x 8 anti-human Von Willebrand Factor antibody coated microwells.
• 60 ml Sample Diluent (blue-green solution); contains sodium azide.
• 3 x 0.5 ml lyophilized Reference Plasma, with assay sheet.
• 12 ml anti-human VWF HRP Conjugate (red solution).
• 13 ml Substrate (TMB and H2O2).
• 15 ml Stopping Solution (0.36 N sulfuric acid).
• 30 ml Wash Concentrate (33X phosphate buffered saline with 0.01% Tween 20). Note: turbidity may appear in wash concentrate which will not affect component performance and should disappear when working dilution is prepared.

Materials required but not supplied

VWF:Act Control Plasma. Reconstitute Control Plasma selected for use following manufacturer’s instructions, and store as recommended.

• vWF:Ag Control Plasma. Follow manufacturer’s instructions, and store as recommended.
• Reagent grade water (1L) to prepare PBS/Tween 20 wash solution, to reconstitute Reference Plasma, and to zero or blank the plate reader during the final assay step
• Graduated cylinders
• Precision pipettors capable of delivering between 5 and 1000 µl, with appropriate tips
• Miscellaneous glassware appropriate for small volume handling 3
• Flask or bottle, 1 liter
• Wash bottles, preferably with the tip partially cut back to provide a wide stream, or an automated or semi-automated washing system
• Disposable gloves, powder-free recommended
• Plate reading spectrophotometer capable of reading absorbance at 450nm (with a 650nm reference if available)
• Multichannel pipettors capable of delivering to 8 wells simultaneously
• Microdilution tubes for patient sample preparation

Features

• Convenient ELISA procedure
• Objective, accurate and reproducible
• Reagent complete kit
• Total incubation time: 40 minutes

Measurement Principle

Principle
The REAADS® von Willebrand Factor Antigen (vWF:Ag) Test Kit is an enzyme linked immunosorbent assay for determining von Willebrand Factor levels in human plasma, expressed in relative percent (%) of normal. The assay is intended to be used as an aid in the diagnosis of von Willebrand Disease in patients with bleeding disorders, and to help distinguish between vWD and classic Hemophilia A. REAADS® vWF:Ag test kit will accurately detect antigen levels as low as 5% of normal.

Procedure
Diluted citrated patient plasma and controls are incubated in microwells coated with capture antibody specific for human vWF, allowing patient vWF to bind to the surface. Following an incubation period, the wells are washed, and a horseradish peroxidase (HRP) conjugated anti-human vWF detection antibody is added. After incubation, the wells are washed, substrate is added, and color development is measured in a spectrophotometer at 450nm following the addition of a stop solution. Patient vWF:Ag levels are determined from a six-point curve prepared from the reference plasma provided in the kit. Total incubation time is 40 minutes at room temperature.

Assay Procedure

1. Remove any microwell strips that will not be used from the frame and store them in the bag provided.
2. Assay each reference plasma dilution in duplicate. Duplicate determinations are also recommended for patient and control samples. One well should be run as a reagent blank; sample diluent without plasma is added to the well as explained in step 6 of this section. This well is treated the same as a patient sample in subsequent assay steps. A water blank well should be included with each plate; it is to remain empty until 200 μL of reagent grade water is added at the completion of the assay, immediately prior to reading the plate. The water blank well is to be used to zero the plate reader.
3. Using the Reference Plasma provided with the kit, prepare six reference plasma dilutions as described below:

4. Prepare a 1:26 dilution of each patient sample and control plasma selected for use in Sample Diluent (blue-green solution); e.g. 20 μl sample added to 500 μl Sample Diluent. Mix thoroughly.
5. Add 100 μl of the dilutions (reference plasmas x 6, patient samples and controls) to the appropriate microwells. 6. Add 100 μl of Sample Diluent to the reagent blank well. Leave the water blank well empty.
6. Incubate 15 minutes at room temperature. After the incubation is complete, carefully invert the microwells and dump the fluid. Do not allow samples to contaminate other microwells.
7. Wash 4 times with working wash solution (PBS/Tween 20). Each well should be filled with wash solution per wash. Wash solution in the empty well intended to serve as a water blank will not interfere with the procedure. Invert microwells between each wash to empty fluid. Use a snapping motion of the wrist to shake the liquid from the wells. The frame must be squeezed at the center on the top and bottom to retain microwell modules during washing. Blot on absorbent paper to remove residual wash fluid. Do not allow wells to dry out between steps.
8. Add 100 μl Conjugate (red) to each well (except the water blank well).
9. Incubate for 15 minutes at room temperature. After the incubation is complete, carefully invert the microwells and dump the conjugate solution.
10. Wash 4 times with working wash solution (PBS/Tween 20) as in step 8. Wash solution in the water blank well will not interfere with the procedure. Use a snapping motion to drain the liquid, and blot on absorbent paper after the final wash. Do not allow the wells to dry out.
11. Add 100 μl Substrate to each well (except for the water blank well) and incubate for 10 minutes at room temperature. Add the substrate to the wells at a steady rate. Blue color will develop in wells with positive samples.
12. Add 100 μl Stopping Solution (0.36 N sulfuric acid) to each well (except for the water blank well) to stop the enzyme reaction. Be sure to add Stopping Solution to the wells in the same order and at the same rate as the Substrate Solution was added. Blue substrate will turn yellow and colorless substrate will remain colorless. Do not add Stopping Solution to the water blank well. Instead, add 200 μl reagent grade water to the water blank well. Blank or zero the plate reader against the water blank well. Read the O.D. of each well at 450nm, against a 650nm reference filter (if available). For best results, the O.D. values should be measured within 30 minutes after the addition of Stopping Solution.

Performance

Clinical Performance
The clinical performance was determined by testing healthy blood donors and von Willebrand Disease plasma samples with REAADS® vWF:Ag Test Kit and with a well established, commercially available von Willebrand Factor Antigen Rocket EIA method. The results correlated well, and were shown to be statistically similar by single factor Anova.

Technical Performance
Intra-assay precision of REAADS® vWF:Ag test kit is 3.6%, while inter-assay precision is 5.0%. Linearity, expressed as the coefficient of determination (r2) is 0.996, with a mean accuracy of 103.6%.The REAADS® vWF:Ag test kit is a rapid, convenient, highly accurate and precise method for the quantitative determination of vWF:Ag levels.

Background

von Willebrand Factor (vWF), a procoagulant protein, plays two important roles in hemostasis:

1. mediates platelet adhesion to sites of vascular injury
2. protects factor VIII from proteolytic cleavage in circulation. In von Willebrand Disease (vWD), which is caused by either quantitative (Type I) or qualitative (Type II) deficiencies in vWF, abnormal bleeding may result due to impaired platelet function and clotting factor inhibition.

vWD is the most common inherited bleeding disorder, and is clinically characterized by easy bruising or prolonged bleeding from mucosal surfaces. While approximately 80% of vWD patients have Type I deficiency, both quantitative (antigenic) and qualitative (functional) assays may be required for a laboratory diagnosis of vWD.

von Willebrand Factor Antigen (vWF:Ag or Factor VIII-related protein) is a plasma protein found in circulation combined by non-covalent interactions with Factor VIII (FVIII:C), a pro-coagulant protein also known as the anti-hemophilic factor. These two proteins show distinct biochemical and functional properties as well as different antigenic determinants; their plasma levels may vary independently of each other. Deficiency of FVIII causes classic hemophilia while deficiency of vWF causes von Willebrand disease. Most of vWF:Ag is synthesized and stored by endothelial cells while 15-20% is synthesized by megakaryocytes and stored in circulating platelets. A vWF:Ag unit has a molecular weight of about 250 kD and tends to polymerize in circulation, with multimers ranging in size from 850kD to as large as 15×106 D.
vWF:Ag plays a very important role in hemostasis; it protects FVIII from proteolytic cleavage in circulation and helps platelets to aggregate or to adhere to sites of vascular damage. The in vivo half-life of FVIII:C without vWF:Ag is shortened from 10-12 hours to a few minutes. These two mechanisms prevent bleeding. Von Willebrand disease is characterized by an inherited deficiency of vWF. A decreased vWF activity in plasma can be the result of low concentrations (quantitative or type I defect) or a deficient function of vWF (qualitative or type II defect). von Willebrand disease is the most common inherited bleeding disorder characterized by easy bruising and prolonged bleeding from mucosal surfaces. The prevalence of von Willebrand disease has been estimated to be 1-3% of the general population. Approximately 80% of von Willebrand disease patients have a type I deficiency.
The laboratory diagnosis of von Willebrand disease may require both quantitative and qualitative (functional) determinations. Quantitative determinations are based on immunologic techniques such as radial immunodiffusion in gel and Laurell rocket immunoelectrophoresis. ELISA procedures9 applied to measure vWF:Ag are less labor intensive and offer several advantages including more objective, accurate and reproducible results. In addition, ELISA allows automation with commonly available laboratory instruments.

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