Near real-time immuno-optical sensor for diagnosing single point mutation: a model system: sensor for factor V Leiden diagnosis.
Factor V leiden (FVL) is an abnormality of factor V (FV), a blood coagulation factor. It is a hereditary blood coagulation disorder with a high frequency (3-7% of general population). The most common type of FVL is caused by a single amino acid mutation and, therefore, its diagnosis is currently done only by DNA analysis, which takes a long time and is expensive. We have developed a rapid, accurate, and cost-effective, sandwich immuno-optical sensing method. To produce monoclonal antibodies against FV or FVL, having minimal cross-reactivity with the other molecule, a 20 amino acid sequence (20-mer) of FV or FVL at around the mutation site was utilized. The antibodies were screened first with the 20-mers and then the ones showing no cross-affinity were reacted with native FV or FVL molecules and they showed some cross-reactivity. Using two antibodies having strongest affinity to either FV or FVL molecule, a FV and a FVL preferred sensors, were produced. After verifying that the levels of the antibody affinity to the two different molecules remained constant with changes in analyte concentration, a two-sensor system is developed to quantify FV and FVL in plasma samples. The system quantified the levels of FV and FVL at the maximum error of 0.5 microg/ml-plasma, in their physiological concentration range of 0-12 microg/ml-plasma. The levels of both molecules may provide us whether the patient has FVL or not but also the seriousness level of the disease (homozygous and different level of heterozygous).
Kang
Kyung A
KA
Department of Chemical Engineering, University of Louisville, Louisville, KY 40292, USA. Kyung.Kang@louisville.edu
Ren
Yongjie
Y
Sharma
Vivek R
VR
Peiper
Stephen C
SC