DiaPharma Group, Inc.

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

Reagents -- Composition, Packaging and Storage

1. Capture Antibody (Catalog No. TF-EIA-C): One yellow-capped vial containing 0.4 ml of monoclonal anti-TF antibody for coating plates.
2. Detecting Antibody (Catalog No. TF-EIA-D): One red-capped vial containing 0.4 ml of polyclonal anti-TF antibody for detection of captured Tissue Factor (TF).

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

Monoclonal antibody to Tissue Factor (TF) 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 biological fluids containing TF are applied.

The coated antibody will capture the TF in the sample. After washing the plate to remove unbound material, a peroxidase conjugated polyclonal antibody to TF is added to the plate to bind to the captured TF. 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 colour produced is quantified using a microplate reader. The colour generated is proportional to the concentration of TF 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 overnight at 4°C.
2. Blocking: Empty contents of plate and add 150 microliters of blocking buffer to every well and incubate for 90 minutes @ 22°C. Wash plate X 3 with wash buffer.
3. Preparation of Reference Standards and Test Samples: Reconstitute vial of recombinant apo-TF to 10 micrograms/milliliters in water (unused material may be frozen in aliquots and stored at -70°C). Further dilute stock TF 1/200 in sample diluent to achieve a final concentration of 50 nanograms/milliliters, then serial 1/2's down to 1/6400 (1.56 ng/ml). Test samples are diluted 1/4 and 1/8. All dilutions are made in sample diluent. Apply 100 microliters/well and incubate plate @ 22°C for 60 minutes. Wash plate X 3 with wash buffer.
4. Detecting Antibody: Dilute the detecting antibody 1/100 in conjugate diluent. Apply 100 microliters to each well. Incubate plate @ 22°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 color reaction with the addition of 50 microliters/well of 2.5 M H2SO4. The plate can be read at wavelength of 490 nm.