A novel ELISA-based primer extension assay for the detection of the factor V Leiden mutation

We describe an enzyme-linked immunosorbent assay (ELISA) based primer extension method for the detection of the factor V Leiden (FVL) mutation. The wild-type nucleotide at position 1691 or the mutant nucleotide at the complementary position on the antisense strand were detected by the incorporation of biotinylated complementary bases onto fluorescein isothiocyanate (FITC) labelled mini-sequence primers with specificity for the sense and antisense gene segments downstream from the bases adjacent to position 1691. The reactions took place in pairs of tubes containing the complementary bases to either the wild-type or mutant nucleotide respectively. Primer extension products from each reaction tube pair which have incorporated biotinylated bases were then captured in streptavidin-coated microtitre plate wells and detected colourimetrically using an ELISA procedure. 200 patient samples were tested to validate the assay and there was complete genotypic agreement between the ELISA method and restriction site analysis using Mnl I (137 wild type, 55 FVL heterozygotes and eight homozygotes). The method utilizes non-radioactive reagents and does not require electrophoretic techniques. It is therefore a safe, simple and rapid assay which lends itself to automation.