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US 4,275,149 AGranted
Macromolecular environment control in specific receptor assays
AssigneeSyva Company
InventorsDavid J. Litman, Zvi Harel, Edwin F. Ullman
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Abstract
Method and compositions are provided for performing protein binding assays involving a homologous pair consisting of ligand and receptor for the ligand. The method employs a label conjugated to a member of said homologous pair and a uniformly dispersed discontinuous phase of discrete particles in a continuous aqueous phase, where the discrete particles create microenvironments which allow for discrimination between the label associated with the particle--in a discontinuous phase--and the label in the continuous phase. Various conjugates and particles are provided which find use in the subject method.
Claims46
An assay method for determining the presence in a sample of an analyte which is a member of a specific binding pair consisting of ligand and its homologous receptor;
- said method employing:
- (a) a continuous aqueous medium;
- (b) discrete dispersable solid particles to which is conjugated one of the members of said pair to provide a particle conjugate, said particle conjugate defining an environment about said conjugated member of said pair different from said aqueous medium, wherein when a signal label is bound to said particle conjugate, the signal resulting from such signal label is different from the signal resulting from the signal label in said continuous aqueous medium;
- (c) a signal producing system capable of producing a measurable signal, the level of said signal being affected by the extent to which said signal producing system is within the environment of said particles;
- (d) at least one member of said signal producing system being conjugated to a pair member to provide a signal label conjugate,
- wherein the amount of signal label conjugate bound to said particle conjugate is related to the amount of analyte in said aqueous medium;
- said method comprising:
- combining in an aqueous assay medium,
- (a) said sample;
- (b) said particle conjugate substantially uniformly dispersed in said medium;
- (c) said signal label conjugate;
- (d) the homologous member of said specific binding pair, where the analyte, particle conjugate, and signal label conjugate are the same member of the specific binding pair;
- and
- (e) any remaining members of said signal producing system;
- with the proviso that when the analyte is a first receptor, said member of said pair conjugated to said particle can be the homologous ligand or receptor for said first receptor, and said member of said signal label conjugate can be receptor for said first receptor or the homologous ligand respectively;
- and
- determining the level of said signal in said aqueous assay medium as compared to an assay medium having a known amount of analyte.
14 more elements
An assay method for determining the presence in a sample of a ligand analyte, wherein the ligand with its homologous antiligand defines a specific binding pair,
- wherein said method employs (1) a medium comprised of an aqueous buffered continuous phase at a pH in the range of about 6.5 to 9.5 and a discontinuous solid phase of discrete porous dispersable particles to which is conjugated one of the members of said specific binding pair to provide a particle conjugate;
- and (2) a signal producing system capable of producing a measureable signal and having at least one member of said system conjugated to a member of said specific binding pair to provide a signal label conjugate, said particle conjugate defining an environment which affects the level of said measurable signal different from said aqueous medium, so that said measurable signal varies in relation to the partitioning of said signal producing system between said particles and said aqueous medium, said partitioning being related to the amount of analyte in said medium;
- said method comprising:
- combining in said aqueous medium,
- (a) said sample;
- (b) said particle conjugate substantially uniformly dispersed;
- (c) said signal label conjugate;
- (d) the homologous member of said specific binding pair, when said analyte, particle conjugate and signal label conjugate have the same member of said specific binding pair;
- and
- (e) any additional members of said signal producing system,
- whereby said signal label conjugate is partitioned between said aqueous medium and said particle conjugate to a degree dependent upon the amount of ligand in said sample;
- and
- determining the level of said signal in said aqueous medium as compared to an assay medium having a known amount of ligand.
10 more elements
An assay method for determining the presence in a sample of a ligand analyte, wherein the ligand with its homologous antiligand define a specific binding pair,
- wherein said method employs (1) a medium comprised of an aqueous buffered continuous phase at a pH in the range of about 6.5 to 9.5 and a discontinuous solid phase of dispersable porous discrete particles to which is conjugated one of the members of said specific binding pair and a first enzyme to provide a particle conjugate and (2) a signal producing system capable of producing a measurable signal and having a second enzyme conjugated to a member of said specific binding pair to provide a signal label conjugate, wherein said first and second enzymes with appropriate substrates and cofactors define said signal producing system and said first and second enzymes are related by the product of one being the substrate of the other, said particle conjugate defining an environment which affects the production of said signal differently from said aqueous medium, so that said measurable signal varies in relation to the partitioning of said signal label conjugate between said particles and said aqueous medium, said partitioning being related to the amount of analyte in said medium,
- said method comprising:
- combining in said aqueous medium,
- (a) said sample;
- (b) said particle conjugate substantially uniformly dispersed in said aqueous medium;
- (c) said signal label conjugate;
- (d) the homologous member of said specific binding pair, when said analyte, particle conjugate and signal label conjugate in said aqueous medium have the same member;
- and
- (e) any additional members of said signal producing system,
- whereby said signal label conjugate will be partitioned between said aqueous medium and said particle conjugate to a degree dependent upon the amount of ligand in said sample;
- and
- determining the level of said signal in said aqueous medium as compared to an assay medium having a known amount of ligand.
9 more elements
An assay method for determining the presence in a sample of a ligand analyte, wherein the ligand with its homologous antiligand defines a specific binding pair,
- wherein said method employs (1) a medium comprised of an aqueous buffered continuous phase at a pH in the range of about 6.5 to 9.5 and a discontinuous solid phase of discrete porous dispersable particles to which ligand is conjugated to provide a particle conjugate;
- and (2) a signal producing system capable of producing a measurable signal having a first enzyme conjugated to antiligand to provide a signal label conjugate, said particle conjugate defining an environment which affects the level of said measurable signal different from said aqueous medium, so that said measurable signal varies in relation to the partitioning of said signal producing system between said particles and said aqueous medium, said partitioning being related to the amount of analyte in said medium;
- said method comprising:
- combining in said aqueous medium,
- (a) said sample;
- (b) said particle conjugate substantially uniformly dispersed;
- (c) said signal label conjugate;
- (d) any additional members of said signal producing system,
- whereby said signal label conjugate is partitioned between said aqueous medium and said particle conjugate to a degree dependent upon the amount of ligand in said sample;
- and
- determining the level of said signal in said aqueous assay medium as compared to an assay medium having a known amount of ligand.
8 more elements
An assay method for determining the presence in a sample of an antiligand analyte, wherein the antiligand with its homologous ligand defines a specific binding pair
- wherein said method employs (1) a medium comprised of an aqueous buffered continuous phase at a pH in the range of about 6.5 to 9.5 and a discontinuous solid phase of discrete porous dispersable particles to which is conjugated one of the members of said specific binding pair to provide a particle conjugate;
- and (2) a signal producing system capable of producing a measurable signal and having at least one member of said system conjugated to a member of said specific binding pair to provide a signal label conjugate, said particle conjugate defining an environment which affects the level of said measurable signal different from said aqueous medium, so that said measurable signal varies in relation to the partitioning of said signal producing system between said particles and said aqueous medium, said partitioning being related to the amount of analyte in said medium;
- said method comprising:
- combining in said aqueous medium,
- (a) said sample;
- (b) said particle conjugate substantially uniformly dispersed;
- (c) said signal label conjugate;
- (d) the homologous member of said specific binding pair, when said analyte, particle conjugate and signal label conjugate have the same member of said specific binding pair;
- and
- (e) any additional members of said signal producing system,
- whereby said signal label conjugate is partitioned between said aqueous medium and said particle conjugate to a degree dependent upon the amount of antiligand in said sample;
- and
- determining the level of said signals in said assay medium as compared to an assay medium having a known amount of antiligand.
10 more elements
A composition comprising a discrete porous particle of a size in the range of about 500 nm to 100.mu. to which is conjugated a first enzyme and at least one molecule of a member of a specific binding pair consisting of ligand and antiligand;
- and a second enzyme which is a member of a signal producing system covalently bonded to a member of said specific binding pair, wherein said signal producing system member is bound to said particle through the intermediacy of the non-covalent binding of members of said specific binding pair and said first and second enzymes are characterized by the product of one of said enzymes being the substrate of the other of said enzymes.
Dependencies are inferred from the claim text.