Reagents and method employing channeling

Claim 1

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.

Claim 2

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.

Claim 27

A method for determining the presence of a ligand in a sample suspected of containing said ligand, said ligand 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 first enzyme conjugated to a member of said specific binding pair, wherein said enzyme reacts with a signal mediator precursor to produce a signal mediator; (B) signal producing label conjugate, which consists of a second enzyme, which is a component of a detectable signal producing system, said second enzyme being 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; and (C) signal mediator precursor, which is an unconjugated compound which reacts with said first enzyme to produce, said signal mediator, which reacts with a product of said second enzyme or said second enzyme to produce said detectable signal; wherein the spatial proximity in said assay medium of said reactant label conjugate and said signal producing conjugate is affected by the concentration of ligand in said medium and the bringing together of said reactant label conjugate and said signal producing label conjugate by means of the binding together of said ligand or ligand analog and antiligand, brings said reactant label and said signal producing label into close spatial proximity in 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; (D) said signal mediator precursor (E) any auxiliary reagents necessary for the reaction of said first and second enzymes; (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.

Claim 28

A method for determining the presence of a ligand in a sample suspected of containing said ligand, said ligand 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 first enzyme conjugated to a member of said specific binding pair, wherein said enzyme reacts with a signal mediator precursor to produce a signal mediator; (B) signal producing label conjugate, which consists of a second enzyme, which is a component of a detectable signal producing system, said second enzyme being 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; and (C) signal mediator precursor, which is an unconjugated compound which reacts with said first enzyme to produce, said signal mediator, which reacts with a product of said second enzyme or said second enzyme to produce said detectable signal; wherein the spatial proximity in said assay medium of said reactant label conjugate and said signal producing conjugate is affected by the concentration of ligand in said medium and the bringing together of said reactant label conjugate and said signal producing label conjugate by means of the binding together of said ligand or ligand analog and antiligand, brings said reactant label and said signal producing label into close spatial proximity in 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; (D) said signal mediator precursor (E) any auxiliary reagents necessary for the reaction of said first and second enzymes; (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.

Claim 49

A method for determining the presence of a ligand which is human γ-globulin, wherein said human γ-globulin is a member of a specific binding pair consisting of human γ-globulin and anti(human γ-globulin), wherein the amount of a detectible signal is a function of the amount of human γ-globulin in an assay medium, said detectible signal being produced by having three primary reagents: (a) (human γ-globulin)-galactose oxidase conjugate; (b) anti(human γ-globulin)-horse radish peroxidase conjugate; and (c) galactose, which undergoes a catalyzed reaction by galactose oxidase to produce hydrogen peroxide; wherein the spatial proximity in said assay medium of said human (γ-globulin)-galactose oxidase conjugate and said anti(human γ-globulin)-horse radish peroxidase conjugate is affected by the concentration of human γ-globulin in said medium, wherein when said (human γ-globulin)-galactose oxidase conjugate binds to said anti(human γ-globulin)-horse radish peroxidase conjugate, there results an enhanced concentration of hydrogen peroxide in the presence of said horse radish peroxidase differing from the concentration of hydrogen peroxide in the mass of said assay medium; said method comprising: combining in an aqueous assay medium: (a) said sample; (b) said (human γ-globulin)-galactose oxidase conjugate; (c) said anti(human γ-globulin)-horse radish peroxidase conjugate; (d) said galactose; and (e) luminol; and determining the light emitted from said assay medium as compared to an assay medium having a known amount of human γ-globulin.

Claim 50

A method for determining the presence of a ligand which is human γ-globulin, wherein said human γ-globulin is a member of a specific binding pair consisting of human γ-globulin and anti(human γ-globulin), wherein the amount of a detectible signal is a function of the amount of human γ-globulin in an assay medium, said detectible signal being produced by having three primary reagents: (a) (human γ-globulin)-galactose oxidase conjugate; (b) anti(human γ-globulin)-horse radish peroxidase conjugate; and (c) galactose, which undergoes a catalyzed reaction by galactose oxidase to produce hydrogen peroxide; wherein the spatial proximity in said assay medium of said human (γ-globulin)-galactose oxidase conjugate and said anti(human γ-globulin)-horse radish peroxidase conjugate is affected by the concentration of human γ-globulin in said medium, wherein when said (human γ-globulin)-galactose oxidase conjugate binds to said anti(human γ-globulin)-horse radish peroxidase conjugate, there results an enhanced concentration of hydrogen peroxide in the presence of said horse radish peroxidase differing from the concentration of hydrogen peroxide in the mass of said assay medium; said method comprising: combining in an aqueous assay medium: (a) said sample; (b) said (human γ-globulin)-galactose oxidase conjugate; (c) said anti(human γ-globulin)-horse radish peroxidase conjugate; (d) said galactose; and (e) luminol; and determining the light emitted from said assay medium as compared to an assay medium having a known amount of human γ-globulin.