Patent ID: 8182988

Claim:
A homogenous bioassay method for use in measurement of biological activity, its modulation or analyte concentration of a sample, said bioassay comprising i) a first group comprising an acceptor, which acceptor is a short lifetime fluorescent compound capable of energy transfer, and ii) a second group comprising a quencher, which quencher is capable of energy transfer from an acceptor, and the first and second group are linked by at least a first linkage, and an increase, of fluorescence of said acceptor, due to the decrease, of energy transfer from said acceptor to said quencher resulting from cleavage of said first linkage, is measured, iii) a third group comprising a donor for energy transfer to said acceptor, which donor is an up-conversion fluorescent compound, a long-lifetime fluorescent compound or an electrogenerated luminescent compound; and wherein a conformational or terminal epitope is created on said first group through said cleavage of the linkage, and said third group comprises a binder with affinity for binding to said conformational or terminal epitope; and wherein fluorescence of said acceptor is brought about by exciting the donor resulting in energy being transferred from the donor to the acceptor, wherein said assay method includes the following steps a) bringing the sample, the first group, the second group and optionally the third group, in contact with each other to obtain an assay mixture, b) allowing the assay mixture to react, c) bringing the third group in contact with the assay mixture if it was not brought in contact with the assay mixture in step a), d) allowing the third group to react with the assay mixture if it was brought in contact with the assay mixture in step c), e) exciting the donor, and f) measuring the sensitized emission of the acceptor.