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

We describe an enzyme-linked immunosorbent assay (ELISA) based primer exten sion method for the detection of the factor V Leiden (FVL) mutation, The wi ldtype nucleotide at position 1691 or the mutant nucleotide at the compleme ntary position on the antisense strand were detected by the incorporation o f biotinylated complementary bases onto fluorescein isothiocyanate (FITC) l abelled mini-sequence primers with specificity for the sense and antisense gene segments downstream from the bases adjacent to position 1691. The reac tions took place in pairs of tubes containing the complementary bases to ei ther the wild-type or mutant nucleotide respectively. Primer extension prod ucts from each reaction tube pair which have incorporated biotinylated base s were then captured in streptavidin-coated microtitre plate wells and dete cted colourimetrically using an ELISA procedure. 200 patient samples were t ested to validate the assay and there was complete genotypic agreement betw een the ELISA method and restriction site analysis using Mn1 I (137 wild ty pe, 55 FVL heterozygotes and eight homozygotes). The method utilizes non-ra dioactive reagents and does not require electrophoretic techniques. It is t herefore a safe, simple and rapid assay which lends itself to automation.