Obelin mutants as reporters in bioluminescent dual-analyte binding assay
Two obelin mutants distinctly different in the color of bioluminescence were successfully applied to simultaneous detection of two analytes in a single well. The flash-type signals were triggered by single injection of Ca2+ and were discriminated by the spectral and time resolutions. The technique was developed in a high-throughput format, and applied to simultaneous immunoassay of two gonadotropic hormones – luteinizing (lutropin or hLH) and follicle stimulating (hFSH) – in clinical sera, and to simultaneous detection of two gene allelic variants at single nucleotide polymorphism genotyping of the human F5 gene encoding factor V Leiden polymorphism 1691 G→A (R506Q). A considerable number of samples were investigated and the results obtained were in good correlation with those obtained by using traditional techniques.