Amplified signal in binding assays
View Patent ↗Methods are disclosed for determining minute quantities of an analyte in a medium suspected of containing the analyte. One method comprises treating a medium suspected of containing an analyte under conditions such that the analyte, if present, causes a substrate having an oxidant cleavable linker and a photosensitizer to come into close proximity. The photosensitizer generates singlet oxygen which oxidatively cleaves the linker to form a product which can be detected in a sandwich detection assay such as LOCI. The amount of product detected is related to the amount of analyte in the medium. Compositions, kits, and compounds are also disclosed.
1. A method for determining the presence or concentration of an analyte in a medium, said method comprising:
providing a reaction mixture comprising in combination:
a medium suspected of containing an analyte;
a first specific binding pair member bound to a water-insoluble solid support;
a second specific binding pair member bound to a sensitizer, said sensitizer capable in its excited state of generating a reactive oxygen species, wherein the proximity of the first specific binding pair member with the second specific binding pair member is modulated by the presence of the analyte; and
digoxigenin-linked biotin linked to the solid support through a reactive oxygen cleavable linker;
incubating the reaction mixture;
exciting the sensitizer, said excitation of the sensitizer causing the formation of reactive oxygen, which cleaves the cleavable linker and releases digoxigenin-linked biotin from the solid support; and
detecting the released digoxigenin-linked biotin, the amount thereof being related to the amount of analyte in said medium.
2. The method of claim 1 wherein:
the proximity of the first and second specific binding pair members to one another results from the binding of the first and second specific binding pair members to the analyte;
the sensitizer is a photosensitizer;
the reactive oxygen species is singlet oxygen; and
the excitation step comprises irradiation of the photosensitizer with light.
3. The method of claim 1 wherein:
the step of detecting the released digoxigenin-linked biotin is carried out by a detection method employing, as a third specific binding pair member, avidin bound to a member of a signal producing system or anti-digoxigenin antibodies bound to a member of a signal producing system or both.