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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

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.

대표청구항 ▼

A method for determining the presence of an analyte in a sample suspected of containing said analyte, said analyte being a member of a specific binding pair consisting of ligand and antiligand, wherein the amount of a detectable signal is a function of the amount of analyte in an assay medium, said

A method for determining the presence of an analyte in a sample suspected of containing said analyte, said analyte being a member of a specific binding pair consisting of ligand and antiligand, wherein the amount of a detectable signal is a function of the amount of analyte in an assay medium, said detectable signal being produced by having three primary reagents: (A) reactant label conjugate consisting of a reactive organic compound conjugated to a member of said specific binding pair which interacts with a signal mediator precursor to produce a signal mediator; (B) signal producing label conjugate, which consists of a compound which is a component of a detectable signal producing system conjugated to a member of said specific binding pair, wherein the amount of signal produced is affected by the localized concentration of said signal mediator, with the proviso that said signal producing label and said reactant label may be bonded to the same molecular member of said specific binding pair to produce a single reagent which is a reactant label-signal producing label conjugate; and (C) signal mediator precursor, which is an unconjugated compound which interacts with said reactant label to produce said signal mediator which is said precursor in an electronically excited state or a reaction product of said signal mediator precursor and said reactant label, with the proviso that when said reactant label and said signal producing label are both the same enzyme, said signal mediator precursor has a plurality of substituents having bonds cleavable by said enzyme and said signal mediator is at an intermediate level of substitution by said substituents. wherein the average spatial proximity in said assay medium of said reactant label and said signal producing label is affected by the concentration of analyte in said medium and the bringing together of said reactant label conjugates and said signal producing label conjugates or a plurality of reactant label-signal producing label conjugates by means of the binding together of said ligand or ligand analog and antiligand brings said reactant labels and said signal producing labels into a microenvironment, so as to create a localized concentration of signal mediator differing from the concentration of signal mediator in the mass of said assay medium; said method comprising: combining in an aqueous assay medium: (A) said sample; (B) said reactant label conjugate; (C) said signal producing label conjugate; with the proviso that both said reactant label conjugate and signal producing label conjugate may be replaced with reactant label-signal producing label conjugate; (D) said signal mediator precursor with the proviso that when said reactant label and said signal producing label are both the same enzyme, said signal mediator precursor is employed having at least two substituents having covalent bonds cleaved by said enzyme; (E) any auxiliary reagents necessary for the reaction of said reactant label and said signal producing label; and (F) poly(ligand analog), when said ligand is monoepitopic and monolabel-monoligand is employed as one of said conjugates; and determining the resulting detectable signal as compared to the detectable signal from an assay medium having a known amount of analyte.