(416d) Purification of Factor V Leiden Molecule from Homozygous Patient's Plasma for Biosensor Development

Factor V (FV) is an important coagulant in blood plasma. FV is inactivated by activated protein C (APC) to prevent blood clotting inside blood vessels. FV is composed of a heavy chain (M= 105,000) and a light chain (M= 74,000), which are non-covalently associated. Factor V Leiden (FVL) is an abnormality by a single point gene mutation in position 506 of the FV gene in heavy chain that replaces an arginine to a glutamine residue. FVL have the same function as coagulant, but it cannot be deactivated by APC, increasing the risk of venous thrombosis, stroke, heart attack, or even death. FVL is the most common hereditary blood coagulation disorder that presents in 3~8 % of US and European populations. Current method of diagnosis is DNA analysis, which is costly and time consuming. To develop a biosensor to diagnose FVL, currently purifying FVL molecule is being purified from the homozygous patient plasma by immunoaffinity chromatography. Blood plasma was drawn from homozygous patient.

Currently, there is no commercially available antibody against FVL, because of the difficulties involved in the generation of specific antibodies against the molecules with the difference of a single amino acid.

Since the amino acid sequence of light chain of FV and FVL is identical and the single point mutation is in heavy chain, we hypothesized those commercially available antibodies against the light chain of FV molecule can be used for purifying FVL from homozygous patient. To select the best antibody for immunopurification, commercially available antibodies against light chain of FV were screened by ELISA. The one showed highest affinity was immobilized on Actigel-A and CNBr-Sepharose immuno affinity gel matrices. The affinity between the antibody and antigen was too high and FV was not eluted from these matrices (1 and 3 % purification yield). Using the lowest affinity antibody (from the ELISA result) immobilized on CNBr-Sepharose matrix, approximately 30 % yield was achieved using pure FV as a source material.

The plasma was pretreated with barium citrate and polyethylene glycol and was centrifuged after each step and the feasibility study for FV purification from plasma was performed. Approximately 20 % purification yield for FV purification from plasma was obtained.

Using developed protocol, FVL purification from homozygous patient plasma was performed. The samples were quantified and an overall yield of 5 % was achieved. The purity of sample was analyzed using SDS-PAGE. Purified FVL will be used as the standard for the sensor.

Authors acknowledge National Institutes of Health (5 R21EB003485-02) for the financial support and Dr. Sharma from the Oncology department at the University of Louisville for his clinical assistance to obtain the plasma from homozygous patient.