Preferential signal production on a surface in immunoassays

Claim 1

A method for detecting the presence of an analyte in a sample suspected of containing said analyte, where said analyte is a member of an immunological pair (mip) consisting of ligand and homologous antiligand; said method involving (1) the partitioning of an enzyme bound to a mip--enzyme-bound-mip--between a surface and a liquid phase, where said partitioning is through the intermediacy of ligand-antiligand binding to a mip-bound-surface in relation to the amount of analyte in said sample;, said surface being porous and permitting approach and binding of enzyme-bound-mip to mip of said mip-bound-surface, either directly or in combination with said ligand and (2) the change in intensity of a detectible signal from a surface resulting from the change in concentration of a signal generating compound associated with said surface, said change in intensity being related to the amount of reaction product produced by said enzyme bound to said surface; said method comprising: (a) combining in an aqueous assay medium; (1) said sample; (2) mip-bound-surface, substantially immersed in said medium wherein substantially all of said mip-bound-surface is uniformly contacted with said sample in said medium; (3) enzyme-bound-mip; and (4) the remaining members of the signal producing system, which system includes at least a second enzyme other than said enzyme-bound-mip, wherein the product of one enzyme is the substrate of the other enzyme, and a solute which is capable of undergoing a catalyzed reaction to produce a product which results in a change in amount of a signal generating compound associated with said surface and capable of producing a detectible signal, (b) waiting a sufficient time for enzyme-bound-mip to diffuse to said surface and at least a portion of said enzyme-bound-mip to bind to said surface through the intermediacy of ligand-antiligand binding and for a change in the amount of signal generating compound associated with said surface in relation to the amount of analyte in said sample; (c) determining the intensity of said detectible signal at said surface as a function of the amount of analyte in said sample.

Claim 2

A method for detecting the presence of an analyte in a sample suspected of containing said analyte, where said analyte is a member of an immunological pair (mip) consisting of ligand and homologous antiligand; said method involving (1) the partitioning of an enzyme bound to a mip--enzyme-bound-mip--between a surface and a liquid phase, where said partitioning is through the intermediacy of ligand-antiligand binding to a mip-bound-surface in relation to the amount of analyte in said sample;, said surface being porous and permitting approach and binding of enzyme-bound-mip to mip of said mip-bound-surface, either directly or in combination with said ligand and (2) the change in intensity of a detectible signal from a surface resulting from the change in concentration of a signal generating compound associated with said surface, said change in intensity being related to the amount of reaction product produced by said enzyme bound to said surface; said method comprising: (a) combining in an aqueous assay medium; (1) said sample; (2) mip-bound-surface, substantially immersed in said medium wherein substantially all of said mip-bound-surface is uniformly contacted with said sample in said medium; (3) enzyme-bound-mip; and (4) the remaining members of the signal producing system, which system includes at least a second enzyme other than said enzyme-bound-mip, wherein the product of one enzyme is the substrate of the other enzyme, and a solute which is capable of undergoing a catalyzed reaction to produce a product which results in a change in amount of a signal generating compound associated with said surface and capable of producing a detectible signal, (b) waiting a sufficient time for enzyme-bound-mip to diffuse to said surface and at least a portion of said enzyme-bound-mip to bind to said surface through the intermediacy of ligand-antiligand binding and for a change in the amount of signal generating compound associated with said surface in relation to the amount of analyte in said sample; (c) determining the intensity of said detectible signal at said surface as a function of the amount of analyte in said sample.

Claim 4

A method according to any one of claims 1 or 2, where said signal generating compound is an insoluble dye produced by desolubilization of a soluble precursor.

Claim 5

A method according to any one of claims 1 or 2, where said second enzyme is bound to said surface.

Claim 6

A method according to any one of claims 1 or 2, where a scavenger is included in the signal producing system which reacts with a component of said signal producing system to inhibit operation of said signal producing system in said liquid phase.

Claim 9

A method for detecting an haptenic ligand analyte in a sample suspected of containing said analyte, where said analyte is a member of an immunological pair (mip) consisting of haptenic ligand and homologous antiligand; said method involving (1) the partitioning of an enzyme catalyst bound to a haptenic ligand--enzyme-bound-haptenic ligand--between a surface and a liquid phase, where said partitioning is through the intermediacy of ligand-antiligand binding to an antiligand-bound-surface, said surface being porous and permitting approach and binding of enzyme-bound-haptenic ligand to antiligand of said antiligand-bound-surface, where said partitioning is in relation to the amount of haptenic ligand analyte in said sample; and (2) the change in concentration of a signal generating compound associated with said surface, where said signal generating compound is an insoluble dye produced as a product of said enzyme of said enzyme-bound-haptenic ligand, said change in concentration being in relation to the amount of insoluble dye produced by said enzyme bound to said surface; said method comprising, combining in an aqueous assay medium (1) said sample; (2) antiligand-bound-surface substantially immersed in said medium and wherein substantially all of said antiligand-bound-surface is uniformly contacted with said sample in said medium; (3) enzyme-bound-haptenic ligand; and (4) the remaining members of the signal producing system, which system includes at least a second enzyme other than said enzyme-bound-haptenic ligand, wherein the product of one enzyme is the substrate of the other enzyme, and one or more enzyme substrates which include a leuco dye or source of leuco dye which is capable of undergoing an enzyme catalyzed reaction to produce an insoluble dye which precipitates on said surface, (b) waiting a sufficient time for enzyme-bound-haptenic ligand to diffuse to said surface and said enzyme-bound-haptenic ligand to bind to said surface through the intermediacy of ligand-antiligand binding and for a change to occur in the amount of said insoluble dye associated with said surface in relation to the amount of haptenic ligand in said sample; and (c) determining the amount of detectible signal at said surface from said insoluble dye as a function of the amount of haptenic ligand in said sample.

Claim 10

A method for detecting an haptenic ligand analyte in a sample suspected of containing said analyte, where said analyte is a member of an immunological pair (mip) consisting of haptenic ligand and homologous antiligand; said method involving (1) the partitioning of an enzyme catalyst bound to a haptenic ligand--enzyme-bound-haptenic ligand--between a surface and a liquid phase, where said partitioning is through the intermediacy of ligand-antiligand binding to an antiligand-bound-surface, said surface being porous and permitting approach and binding of enzyme-bound-haptenic ligand to antiligand of said antiligand-bound-surface, where said partitioning is in relation to the amount of haptenic ligand analyte in said sample; and (2) the change in concentration of a signal generating compound associated with said surface, where said signal generating compound is an insoluble dye produced as a product of said enzyme of said enzyme-bound-haptenic ligand, said change in concentration being in relation to the amount of insoluble dye produced by said enzyme bound to said surface; said method comprising, combining in an aqueous assay medium (1) said sample; (2) antiligand-bound-surface substantially immersed in said medium and wherein substantially all of said antiligand-bound-surface is uniformly contacted with said sample in said medium; (3) enzyme-bound-haptenic ligand; and (4) the remaining members of the signal producing system, which system includes at least a second enzyme other than said enzyme-bound-haptenic ligand, wherein the product of one enzyme is the substrate of the other enzyme, and one or more enzyme substrates which include a leuco dye or source of leuco dye which is capable of undergoing an enzyme catalyzed reaction to produce an insoluble dye which precipitates on said surface, (b) waiting a sufficient time for enzyme-bound-haptenic ligand to diffuse to said surface and said enzyme-bound-haptenic ligand to bind to said surface through the intermediacy of ligand-antiligand binding and for a change to occur in the amount of said insoluble dye associated with said surface in relation to the amount of haptenic ligand in said sample; and (c) determining the amount of detectible signal at said surface from said insoluble dye as a function of the amount of haptenic ligand in said sample.

Claim 12

A method according to any of claims 8 or 9, wherein said haptenic ligand is a hormone.

Claim 13

A method according to any of claims 8 or 9, wherein said haptenic ligand is an analgesic.

Claim 14

A method according to any of claims 8 or 9, wherein said haptenic ligand is an aralkyl amine.

Claim 15

A method according to any of claims 8 to 9, wherein said haptenic ligand is a steroid.

Claim 16

A method according to any of claims 8 or 9, wherein said haptenic ligand is a heterocyclic drug.

Claim 17

A method according to any of claims 8 or 9, wherein said haptenic ligand is a thyroid hormone.

Claim 18

A method according to any of claims 8 or 9, wherein said haptenic ligand is an antibiotic.

Claim 19

A method according to any of claims 8 or 9, wherein said haptenic ligand is tetrahydrocannabinol or derivative thereof.

Claim 20

A method for detecting the presence of a polyepitopic antigenic analyte in a sample suspected of containing said analyte, where said antigenic analyte is a member of an immunological pair (mip) consisting of ligand and homologous antiligand; said method involving (1) the partitioning of an enzyme catalyst bound to a mip--enzyme-bound-mip--between a surface and a liquid phase, said surface being porous and permitting approach and binding of enzyme-bound-mip to mip of said mip-bound-surface, either directly or in combination with said ligand, where said partitioning is through the intermediacy of ligand-antiligand binding to a mip-bound-surface in relation to the amount of analyte in said sample; and (2) measuring the change in concentration of a signal generating compound associated with said surface, where said signal generating compound is an insoluble dye, said change in concentration being in proportion to the amount of insolable dye produced by said enzyme bound to said surface; said method comprising: (a) combining in an aqueous assay medium, (1) said sample; (2) mip-bound-surface substantially immersed in said medium, wherein substantially all of said mip-bound-surface is uniformly contacted with said sample; (3) enzyme-bound-mip, wherein one of the mips of said mip-bound-surface and enzyme-bound mip is antiligand; and (4) remaining members of said signal producing system, which system includes at least a second enzyme other than said enzyme-bound-mip, wherein the product of one enzyme is the substrate of the other enzyme, and wherein the enzyme catalyzed reaction of said enzyme-bound-mip results in the production of an insoluble dye as the signal generating compound, which precipitates onto said surface through the intermediacy of ligand-antiligand binding, (b) waiting a sufficient time for enzyme-bound-mip to diffuse to said surface and said enzyme-bound-mip to bind to said surface through the intermediacy of ligand-antiligand binding and for a change in the amount of signal generating compound associated with said surface in relation to the amount of analyte in said sample; and (c) determining the amount of detectible signal at said surface as a function of the amount of analyte in said sample.

Claim 21

A method for detecting the presence of a polyepitopic antigenic analyte in a sample suspected of containing said analyte, where said antigenic analyte is a member of an immunological pair (mip) consisting of ligand and homologous antiligand; said method involving (1) the partitioning of an enzyme catalyst bound to a mip--enzyme-bound-mip--between a surface and a liquid phase, said surface being porous and permitting approach and binding of enzyme-bound-mip to mip of said mip-bound-surface, either directly or in combination with said ligand, where said partitioning is through the intermediacy of ligand-antiligand binding to a mip-bound-surface in relation to the amount of analyte in said sample; and (2) measuring the change in concentration of a signal generating compound associated with said surface, where said signal generating compound is an insoluble dye, said change in concentration being in proportion to the amount of insolable dye produced by said enzyme bound to said surface; said method comprising: (a) combining in an aqueous assay medium, (1) said sample; (2) mip-bound-surface substantially immersed in said medium, wherein substantially all of said mip-bound-surface is uniformly contacted with said sample; (3) enzyme-bound-mip, wherein one of the mips of said mip-bound-surface and enzyme-bound mip is antiligand; and (4) remaining members of said signal producing system, which system includes at least a second enzyme other than said enzyme-bound-mip, wherein the product of one enzyme is the substrate of the other enzyme, and wherein the enzyme catalyzed reaction of said enzyme-bound-mip results in the production of an insoluble dye as the signal generating compound, which precipitates onto said surface through the intermediacy of ligand-antiligand binding, (b) waiting a sufficient time for enzyme-bound-mip to diffuse to said surface and said enzyme-bound-mip to bind to said surface through the intermediacy of ligand-antiligand binding and for a change in the amount of signal generating compound associated with said surface in relation to the amount of analyte in said sample; and (c) determining the amount of detectible signal at said surface as a function of the amount of analyte in said sample.

Claim 24

A method according to any of claims 18 or 19, wherein said antigenic ligand is an immunoglobulin.

Claim 25

A method according to any of claims 18 or 19, wherein said antigenic ligand is a surface antigen.

Claim 28

A method according to any of claims 18 or 19, wherein said antigenic ligand is a complement factor.

Claim 29

A method according to any of claims 18 or 19, wherein said antigenic ligand is a hormone.

Claim 33

A method for detecting the presence of a ligand analyte in a sample suspected of containing said analyte, where said ligand analyte is a member of an immunological pair (mip) consisting of ligand and homologous antiligand; said method involving (1) the partitioning of an enzyme bound to a mip--enzyme-bound-mip--between a surface and a liquid phase, said surface being porous and permitting approach and binding of enzyme-bound-mip to mip of said mip-bound-surface, either directly or in combination with said ligand, where said partitioning is through the intermediacy of ligand-antiligand binding to a mip-bound-surface in relation to the amount of analyte in said sample; and (2) the change in concentration of a signal generating compound associated with such surface, said change in concentration being in relation to the amount of reaction product produced by said enzyme bound to said surface; said method comprising (a) combining in an aqueous assay medium, (1) said sample; (2) mip-bound-surface substantially completely immersed in said sample, wherein substantially all of said mip-bound-surface is uniformly contacted with said sample; and (3) enzyme-bound-mip; (b) incubating said mixture for a time sufficient to allow enzyme-bound-mip to bind to said surface through the intermediacy of ligand-antiligand binding in an amount proportional to the amount of analyte in said sample; (c) after said sufficient time, and without separation of said surface from said aqueous assay medium, adding remaining components of said signal producing system, where components of said signal producing system include the enzyme of said enzyme-bound-mip and a second enzyme and a solute which is capable of undergoing a catalyzed reaction to produce a product which results in the formation of an insoluble dye associated with said surface, the amount of dye associated with said surface being related to the amount of enzyme-bound-mip bound to said surface; and (d) determining the amount of detectible signal from said insoluble dye at said surface as a function of the amount of analyte in said sample.

Claim 34

A method for detecting the presence of a ligand analyte in a sample suspected of containing said analyte, where said ligand analyte is a member of an immunological pair (mip) consisting of ligand and homologous antiligand; said method involving (1) the partitioning of an enzyme bound to a mip--enzyme-bound-mip--between a surface and a liquid phase, said surface being porous and permitting approach and binding of enzyme-bound-mip to mip of said mip-bound-surface, either directly or in combination with said ligand, where said partitioning is through the intermediacy of ligand-antiligand binding to a mip-bound-surface in relation to the amount of analyte in said sample; and (2) the change in concentration of a signal generating compound associated with such surface, said change in concentration being in relation to the amount of reaction product produced by said enzyme bound to said surface; said method comprising (a) combining in an aqueous assay medium, (1) said sample; (2) mip-bound-surface substantially completely immersed in said sample, wherein substantially all of said mip-bound-surface is uniformly contacted with said sample; and (3) enzyme-bound-mip; (b) incubating said mixture for a time sufficient to allow enzyme-bound-mip to bind to said surface through the intermediacy of ligand-antiligand binding in an amount proportional to the amount of analyte in said sample; (c) after said sufficient time, and without separation of said surface from said aqueous assay medium, adding remaining components of said signal producing system, where components of said signal producing system include the enzyme of said enzyme-bound-mip and a second enzyme and a solute which is capable of undergoing a catalyzed reaction to produce a product which results in the formation of an insoluble dye associated with said surface, the amount of dye associated with said surface being related to the amount of enzyme-bound-mip bound to said surface; and (d) determining the amount of detectible signal from said insoluble dye at said surface as a function of the amount of analyte in said sample.

Claim 37

A method according to any of claims 30, 31 or 32, wherein said analyte is a hapten, said mip of said enzyme bound mip is a ligand, and said mip of said mip-bound-surface is antiligand.

Claim 38

A method according to any of claims 30, 31, or 32, wherein said analyte is a polyepitopic antigen, said mip of said enzyme-bound-mip is antiligand and said mip of said mip-bound-surface is antiligand.

Claim 39

A method for detecting the presence of a ligand analyte in a sample suspected of containing said ligand where said ligand is a member of an immunological pair (mip) consisting of ligand and homologous antiligand; said method involving (1) the partitioning of an enzyme bound to a mip--enzyme-bound-mip--between a surface and a liquid phase, said surface being porous and permitting approach and binding of enzyme-bound-mip to mip of said mip-bound-surface, either directly or in combination with said ligand, where said partitioning is through the intermediacy of ligand-antiligand binding to a mip-bound-surface in relation to the amount of analyte in said sample; and (2) the change in concentration of a signal generating compound associated with said surface, where said signal generating compound is an insoluble dye, said change in concentration being in relation to the amount of insoluble dye produced by said enzyme bound to said surface said method comprising: (a) combining in an aqueous assay medium, (1) said sample; (2) mip-bound-surface, wherein said mip-bound-surface is immersed in said medium and is substantially uniformly contacted with said sample; (b) incubating for sufficient time for ligand analyte to bind to said mip-bound-surface; (c) after sufficient time for said incubation, adding substantially concurrently enzyme-bound-mip; and (d) remaining members of said signal producing system, which system includes at least said enzyme of said enzyme-bound-mip, a second enzyme and a solute which is capable of undergoing a catalyzed reaction to produce a product which results in the formation of a signal generating compound, which is an insoluble dye capable of producing a detectible signal, wherein the product of one enzyme is the substrate of the other enzyme (e) incubating for sufficient time for enzyme-bound-mip to diffuse to said surface and for said insoluble dye to precipitate on said surface in proportion to the amount of analyte in said sample; and (f) determining the amount of said detectible signal on said surface as a function of the amount of analyte in said sample.

Claim 40

A method for detecting the presence of a ligand analyte in a sample suspected of containing said ligand where said ligand is a member of an immunological pair (mip) consisting of ligand and homologous antiligand; said method involving (1) the partitioning of an enzyme bound to a mip--enzyme-bound-mip--between a surface and a liquid phase, said surface being porous and permitting approach and binding of enzyme-bound-mip to mip of said mip-bound-surface, either directly or in combination with said ligand, where said partitioning is through the intermediacy of ligand-antiligand binding to a mip-bound-surface in relation to the amount of analyte in said sample; and (2) the change in concentration of a signal generating compound associated with said surface, where said signal generating compound is an insoluble dye, said change in concentration being in relation to the amount of insoluble dye produced by said enzyme bound to said surface said method comprising: (a) combining in an aqueous assay medium, (1) said sample; (2) mip-bound-surface, wherein said mip-bound-surface is immersed in said medium and is substantially uniformly contacted with said sample; (b) incubating for sufficient time for ligand analyte to bind to said mip-bound-surface; (c) after sufficient time for said incubation, adding substantially concurrently enzyme-bound-mip; and (d) remaining members of said signal producing system, which system includes at least said enzyme of said enzyme-bound-mip, a second enzyme and a solute which is capable of undergoing a catalyzed reaction to produce a product which results in the formation of a signal generating compound, which is an insoluble dye capable of producing a detectible signal, wherein the product of one enzyme is the substrate of the other enzyme (e) incubating for sufficient time for enzyme-bound-mip to diffuse to said surface and for said insoluble dye to precipitate on said surface in proportion to the amount of analyte in said sample; and (f) determining the amount of said detectible signal on said surface as a function of the amount of analyte in said sample.