Method and compositions are provided for chemical analysis of an analyte which is a member of a specific binding pair of organic substances consisting of ligand and ligand receptor (antiligand). The method involves bringing together the following reagents with the analyte in an aqueous assay medium under mild conditions.
The first reagent is a conjugate of a member of the specific binding pair with a chemical entity which provides a means for chemically changing the concentration of a compound which acts as a signal mediator. The second reagent is the signal mediator precursor. The third reagent is a conjugate of a member of the specific binding pair with a component of a signal producing system of which system the signal mediator is a member.
The amount of signal which can be detected is affected by the local concentration of the signal mediator. By bringing the reagents together in the presence of analyte, where the signal mediator concentration changing means is brought together in a microenvironment with the conjugated signal producing system component, localized concentrations of the signal mediator can be created which differ from the gross concentration of the signal mediator in the assay medium. The degree to which the signal mediator concentration changing means is in close proximity to the signal producing means in a microenvironment will affect the observed signal. By appropriate choice of the two conjugates in conjunction with the analyte, the observed signal can be related to the amount of analyte in the medium.
Novel conjugates are provided, as well as combinations of conjugates in specific proportions to substantially optimize the assay sensitivity. The combinations are provided as kits, where ancillary reagents can also be included, so as to simplify the combination of reagents, as well as provide for more accurate measurements and relative proportions of reagents.