For Research Use Only. Not For Use In Diagnostic Procedures.

Reagents -- Composition, Packaging and Storage

1. Capture Antibody (VWF-EIA-C): One yellow-capped vial containing 0.5 ml of polyclonal purified anti-vWF antibody for coating plates.
2. Detecting Antibody (VWF-EIA-D): One red-capped vial containing 0.5 ml of peroxidase conjugated polyclonal antivWF antibody for detection of captured vWF.

Note: Antibodies are supplied in a 50% (v/v) glycerol solution for storage at -10 to -20°C. Keep vials tightly capped. Do not store in frost-free freezers.

Principle of Sandwich-style ELISA

Affinity-purified antibody to vWF is coated onto the wells of a microtitre plate. Any remaining binding sites on the plastic wells are blocked with an excess of bovine serum albumin. The plates are washed and plasma or other fluids containing vWF are applied. The coated antibody will capture the vWF in the sample. After washing the plate to remove unbound material, a peroxidase conjugated second antibody to vWF is added to the plate to bind to the captured vWF. After washing the plate to remove unbound conjugated antibody, the peroxidase activity is expressed by incubation with o-phenylenediamine (OPD). After a fixed development time the reaction is quenched with the addition of H2SO4 and the color produced is quantified using a microplate reader. The color generated is proportional to the concentration of vWF present in the sample.

Assay Procedure:

1. Coating of plates: Dilute the capture antibody 1/100 in coating buffer (preferably in a polypropylene tube) and immediately add 100 microliters to every well in the plate. Incubate 2 hrs @ 22°C or overnight at 2-8°C.
2. Blocking: Empty contents of plate and add 150 microliters of blocking buffer to every well and incubate for 60 minutes @ 22°C. Wash plate X 3 with wash buffer.
3. Samples: Reference plasma is diluted 1/100 (100%) then serial 1/2's down to 1/3200 (3.13%). Sample plasmas are diluted 1/200, 1/400 & 1/800. All dilutions are made in HBS-BSAT20 sample diluent. Apply 100 microliters/well and incubate plate @ 22°C for 90 minutes. Wash plate X 3 with wash buffer.
4. Detecting Antibody: Dilute the detecting antibody 1/100 in HBS-BSA-T20 sample diluent and apply 100 microliters to each well. Incubate plate @ 22°C for 90 minutes. Wash plate X 3 with wash buffer.
5. OPD Substrate: Apply 100 microliters of freshly prepared OPD substrate to every well. Allow color to develop for 10-15 minutes then stop color reaction with the addition of 50 microliters/well of 2.5 M H2SO4. The plate can be read at a wavelength of 490 nm.

Related Products:

• GAVWF-IG - Goat anti-human vWF, whole IgG from antiserum
• GAVWF-AP - Goat anti-human vWF, affinity purified IgG
• GAVWF-HRP - Goat anti-human vWF, IgG-peroxidase
• VWF-DP - Plasma deficient in vWF/F.VIII, immune depleted