Method for detecting enzymatic activity using particle agglutination

Claim 2

An agglutination based method for detecting an enzyme in a liquid test sample comprising: (1) forming an agglutination system comprising: (i) highly refractive particles having a ligand disposed thereon. (ii) a binding partner specific for the ligand and capable of binding to at least two ligands, the binding partner and ligand being present at concentrations which permit agglutination, and (iii) a substrate for the enzyme, the enzyme, substrate, ligand and binding partner being such that a reaction between the enzyme and substrate produces a product which competes with the ligand for the binding partner; (2) contacting the agglutination system with the test sample to produce the product which competes with the ligand for the binding partner; (3) measuring a physical property of the agglutination system, which property is a function of agglutination; and (4) relating the measurement to the amount of enzyme initially present in the test sample.

Claim 3

An agglutination based assay for detecting an analyte in a liquid test sample, comprising: (1) contacting the sample with a conjugate comprising an enzyme and a first binding partner capable of binding to the analyte, whereby a fraction of the conjugate binds to the analyte to form a complex and a fraction of the conjugate remains free; (2) separating the free conjugate from the complex; (3) contacting either the free conjugate or the complex with an agglutination system comprising: (i) highly refractive particles having a ligand disposed thereon, (ii) a second binding partner capable of binding to at least two ligands, the second binding partner and particle bound ligand being present at concentrations which permit agglutination, and (iii) a substrate for the enzyme, the enzyme, substrate, ligand and second binding partner being such that a reaction between the enzyme and substrate produces a product which competes with the ligand for the second binding partner; (4) measuring a physical property of the agglutination system, which property is a function of agglutination; and (5) relating the measurement to the amount of analyte initially present in the liquid test sample.

Claim 4

An agglutination based assay for detecting an analyte in a liquid test sample, comprising: (1) contacting the sample with a conjugate comprising an enzyme and a first binding partner capable of binding to the analyte, whereby a fraction of the conjugate binds to the analyte to form a complex and a fraction of the conjugate remains free; (2) separating the free conjugate from the complex; (3) contacting either the free conjugate or the complex with an agglutination system comprising: (i) highly refractive particles having a ligand disposed thereon, (ii) a second binding partner capable of binding to at least two ligands, the second binding partner and particle bound ligand being present at concentrations which permit agglutination, and (iii) a substrate for the enzyme, the enzyme, substrate, ligand and second binding partner being such that a reaction between the enzyme and substrate produces a product which competes with the ligand for the second binding partner; (4) measuring a physical property of the agglutination system, which property is a function of agglutination; and (5) relating the measurement to the amount of analyte initially present in the liquid test sample.

Claim 5

An agglutination-based method for detecting an enzyme in a liquid test sample, comprising: (1) forming an agglutination system comprising: (i) highly refractive particles having coated thereon a substrate capable of being converted into a ligand upon reaction with the enzyme; and (ii) a binding partner capable of binding to at least two ligands, but incapable of binding to the substrate; (2) contacting the agglutination system with the sample to convert the substrate into the ligand; (3) measuring a physical property of the agglutination system which is a function of agglutination; and (4) relating the measurement to the amount of enzyme initially present in the test sample.

Claim 6

An agglutination-based method for detecting an enzyme in a liquid test sample, comprising: (1) forming an agglutination system comprising: (i) highly refractive particles having coated thereon a substrate capable of being converted into a ligand upon reaction with the enzyme; and (ii) a binding partner capable of binding to at least two ligands, but incapable of binding to the substrate; (2) contacting the agglutination system with the sample to convert the substrate into the ligand; (3) measuring a physical property of the agglutination system which is a function of agglutination; and (4) relating the measurement to the amount of enzyme initially present in the test sample.

Claim 7

An agglutination-based method for detecting an analyte in a liquid test sample, comprising: (1) contacting the sample with a conjugate comprising an enzyme and a first binding partner capable of binding to the analyte, whereby a fraction of the conjugate binds to the analyte to form a complex and a fraction of the conjugate remains free; (2) separating the free conjugate from the complex; (3) contacting either the free conjugate or the complex with an agglutination system comprising: (i) highly refractive particles having coated thereon a substrate capable of being converted into a ligand upon reaction with the enzyme; and (ii) a binding partner capable of binding to at least two ligands, but incapable of binding to the substrate; and (4) measuring a physical property of the agglutination system which is a function of agglutination; and (5) relating the measurement to the amount of enzyme initially present in the test sample.

Claim 8

An agglutination-based method for detecting an analyte in a liquid test sample, comprising: (1) contacting the sample with a conjugate comprising an enzyme and a first binding partner capable of binding to the analyte, whereby a fraction of the conjugate binds to the analyte to form a complex and a fraction of the conjugate remains free; (2) separating the free conjugate from the complex; (3) contacting either the free conjugate or the complex with an agglutination system comprising: (i) highly refractive particles having coated thereon a substrate capable of being converted into a ligand upon reaction with the enzyme; and (ii) a binding partner capable of binding to at least two ligands, but incapable of binding to the substrate; and (4) measuring a physical property of the agglutination system which is a function of agglutination; and (5) relating the measurement to the amount of enzyme initially present in the test sample.

Claim 9

A method according to claim 1 or 3 wherein the binding partner is an antibody having at least two ligand binding sites.

Claim 10

A method according to claim 1 or 2 wherein the enzyme is P-galactosidase, the substrate is ortho-nitrophenyl-β- galactopyranoside, the ligand is 3-nitro-4-hydroxybenzoic acid or ortho-nitrophenol, and the binding agent is an anti-3-nitro-4-hydroxybenzoic acid antibody.

Claim 11

A method according to claim 1 or 2 wherein the enzyme is alkaline phosphatase, the substrate is para-nitrophenyl phosphate, the ligand is para-nitrophenol, and the binding agent is an anti-para-nitrophenol antibody.

Claim 12

A method according to claim 2 or 4 wherein the first binding partner is an anti-analyte antibody.

Claim 13

A method according to claim 2,4 or 8 wherein the second binding partner is an antibody having at least two ligand binding sites.

Claim 14

A method according to claim 3 or 4 wherein the enzyme is β-galactosidase, the substrate is 2-nitro-4-0-(-P-D-galactopyranosyl)benzoic acid and the binding partner is an anti-(3-nitro)-4-hydroxybenzoic acid antibody.

Claim 17

A method according to claim 12 wherein the essentially spherical particles have a diameter of 30 nm to 100,000 nm.

Claim 18

A method according to claim 13 wherein the diameter is 30 nm to 100 nm.