(657a) Quantification of Fv and Fvl in Plasma by Using Fiber-Optic Sensing System

The human coagulation factor - Factor V (FV) is a single-chain glycoprotein (MW = 330,000). Factor V Leiden (FVL), an abnormality with a single mutation on heavy chain of FV, is the most common hereditary blood coagulation disorder in the United States, with the frequency 3~7% of general population and 20~40% of individuals with thrombosis. For heterozygotes, the risk of venous thrombosis increases 3~8 folds, and for homozygotes, 30-140 folds. The current FVL diagnosis is performed by DNA analysis, taking a long assay time with high cost and technical difficulty. Early diagnosis of FVL and accurate quantification of the abnormality can minimize traumatic complications by allowing immediate treatments. A portable, accurate and inexpensive dual-FV/FVL sensing system is being developed by our research group for near real-time FVL diagnosis, by providing the amount of both normal FV and FVL in the plasma.

The dual-FV/FVL sensing system performs fiber-optic immuno-biosensing. The system performs a fluorophore mediated sandwich immunoassay within the evanescent wave field on the surface of two optical fibers. There is no commercially available antibody against FVL molecule without cross-reacting with FV molecule and it is extremely difficult to develop antibodies against FVL molecules only. A 20mer – polypeptide sequence including the mutation region was used to stimulate the production of antibodies targeted against antigen FV or FVL only, with a minimal cross-reactivity.

Due to the similarity of the amino acid sequence of FV/FVL molecule, the resulting monoclonal antibodies generated were not specific to only one antigen and the cross-reactivity between antibodies to FV and antibodies to FVL are not neglected in immunoreactions. Therefore, the concentration of the antigens cannot be obtained from single measurement. By solving the linear equations with two variables, which is acquired from multiple measurements, the amount of antigens (FV/FVL) could be calculated. The total measurement will be completed in 10 minutes within a multiple channels chip.

We acknowledge the National Institutes of Health (5R21EB003485-02) for the financial support and Dr. Sharma from the Oncology department at the University of Louisville for providing plasma from homozygous patient.