DiaPharma Group, Inc.

For Research Use Only. Not for use in diagnostic procedures.

Reagents -- Composition, Packaging and Storage

1. Capture Antibody (CFVIII-EIA-C): One yellow-capped vial containing 0.4 ml of polyclonal purified anti-canine FVIII antibody for coating plates.
2. Detecting Antibody (CFVIII-EIA-D): Four neutral-capped tubes each containing 10 ml of pre-diluted peroxidase conjugated polyclonal anti-canine FVIII antibody for detection of captured cFVIII.
3. Sample Diluent (CFVIII-EIA-SD): 100 ml bottle containing a green-colored diluent optimised for dilution of samples.

Store reagents at 2-8°C

Principle of Sandwich-style ELISA

Purified antibody to canine FVIII is coated onto the wells of a microtitre plate. The plate is washed and plasma or other fluids containing cFVIII are applied. The coated antibody will capture the cFVIII in the sample. After washing the plate to remove unbound material, a peroxidase conjugated second antibody to cFVIII is added to the plate to bind to the captured cFVIII. 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 cFVIII 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 at 22 degrees C or overnight at 2-8 degrees C.
2. Blocking: Blocking is not required under the conditions described. Washing the plate with PBS-Tween is sufficient to block nonspecific interactions. Wash plate X 3 with wash buffer.
3. Samples: To prepare a reference curve normal canine plasma is diluted 1/5 (100%) then serial 1/2's down to 1/160 (3.13%). Sample plasmas are diluted 1/10, 1/20 & 1/40. All dilutions are made in the provided green sample diluent. Apply 100 microliters/well and incubate plate at 22 degrees C for 120 minutes. Wash plate X 3 with wash buffer.
4. Detecting Antibody: Apply the pre-diluted detecting antibody, 100 microliters to each well. Incubate plate at 22 degrees C for 60 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 5-10 minutes then stop colour reaction with the addition of 50 microliters/well of 2.5 M H2SO4. The plate can be read at wavelength of 490 nm.