Immunoassays utilizing covalent conjugates of polymerized enzyme and antibody

Claim 2

A process for producing a polymeric-enzyme/antibody conjugate, which comprises (1) covalently coupling at least two enzyme molecules to produce a prepolymerized enzyme: and (2) coupling covalently the prepolymerized enzyme to an antibody or fragment thereof.

Claim 3

A process according to claim 1 wherein the enzyme is selected from beta-D-glactosidase, glucose oxidase, horseradish peroxidase, alkaline phosphatase, beta-lactamase, glucose-6-phosphate dehydrogenase, urease, uricase, superoxide dismutase, luciferase, pyruvate kinase, lactate dehydrogenase, galactose oxidase, acetylcholine-sterase, enterokinase, tyrosinase and xanthine oxidase.

Claim 4

A process according to claim 1 or 2 wherein the coupling step (1) and/or the coupling step (2) is carried out using a crosslinking reagent.

Claim 5

A process according to claim 3 wherein the reagent is a homobifunctional cross-linking reagent.

Claim 6

A process according to claim 4 wherein the homobifunctional cross-linking reagent is selected from o-phenylenedimaleimide, 3,3'-dithiobis(sulfosuccinimidyl propionate), bis(sulfosuccinimidyl)suberate, bis(maleimido) methyl ester, dimethyl suberimidate.2HCl, dimethyl pimelimidate, dimethyl adipimidate and dithiobis(succinimidyl propionate).

Claim 7

A process according to claim 4 wherein the crosslinking reagent is a heterobifunctional cross-linking reagent selected from the group consisting of m-maleimidobenzoyl N-hydroxysuccinimide ester, N-succinimidyl 3-(2-pyridyldithio)propionate, succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate, succinimidyl-4-(p-maleimidophenyl)butyrate, N-succinimidyl (4-iodoacetyl)aminobenzoate, maleimidohexanoyl-N-hydroxysuccinimide ester, m-maleimidobenzoyl sulfosuccinimide ester, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate, sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate, N-5-azido-2-nilrobenzoyloxysuccinimide, N-hydroxysuccinimidyl-4-azidobenzoate, sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate.

Claim 8

A process according to any one of the preceding claims wherein the antibody or fragment thereof is an IgG molecule, Fab fragment, Fab' fragment, F(ab') 2 fragment or half-molecule.

Claim 9

A process according to claim 7 wherein the Fab' fragment is an Fab'-SH fragment.

Claim 10

A process according to claim 1 wherein the antibody is an Fab'-SH fragment, the enzyme is beta-D-galactosidase, and the coupling steps (1) and (2) are carried out using o-phenylenedimaleimide.

Claim 11

An immunoassay for the determination of an analyte in a liquid sample, said immunoassay comprising: (1) contacting the sample with, as an enzyme-labeled anti-analyte antibody, a polymeric-enzyme/antibody conjugate as defined in any one of claims 1 to 9 to form (i) a complex comprising enzyme-labeled anti-analyte antibody and analyte and (ii) free enzyme-labeled anti-analyte antibody; (2) optionally separating the complex from the free enzyme-labeled anti-analyte antibody; (3) measuring the enzymatic activity of either the complex or the free enzyme-labeled anti-analyte antibody; and (4) relating the enzymatic activity to the amount of analyte initially present in the sample.

Claim 12

An immunoassay according to claim 10 wherein the analyte is digoxin and the covalent conjugate comprises an Fab' fragment and prepolymerized beta-D-galactosidase of molecular weight from 20,000,000 to 35,000,000, the molar ratio of Fab' fragment to prepolymerized beta-D-galactosidase being from 20:1 to 40:1.