DiaPharma Group, Inc.

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

Reagents -- Composition, Packaging and Storage

1. Capture Antibody (FIIZ-EIA-C): One yellow-capped vial containing 0.5 ml of polyclonal affinity purified antiprothrombin fragment 2 antibody for coating plates.
2. Detecting Antibody (FIIZ-EIA-D): Five neutral-capped vials, each containing 10 ml of pre-diluted peroxidase conjugated polyclonal anti-thrombin antibody for detection of intact prothrombin.

Store reagents at 2-8°C.

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 μl of blocking buffer to every well and incubate for 90 minutes @ 22°C. Wash plate X 3 with wash buffer.
3. Samples: Reference plasma is diluted 1/5,000 (100%) then serial 1/2's down to 1/160,000 (3.13%). Sample plasmas are diluted 1/10,000, 1/20,000 & 1/40,000. All dilutions are made in HBS-BSA-T20 sample diluent. Apply 100 microliters/well and incubate plate @ 22°C for 60 minutes. Wash plate X 3 with wash buffer.
4. Detecting Antibody: Apply the pre-diluted detecting antibody, 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 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:

• SAFII-IG - Sheep anti-human FII, IgG from antiserum
• SAFII-AP - Sheep anti-human FII, affinity-purified IgG
• SAFII-HRP - Sheep anti-human FII, IgG-peroxidase conjugate
• FII-EIA - Paired antibody set for ELISA of FII, 5 x 96 well
• FII-DP - Human plasma deficient in FII, immune depleted