Biologically active reagent, analytical element and methods for use of the reagent

Claim 2

A biologically active reagent comprising: (I) a water-insoluble, nonporous particle, and (II) a biologically active substance covalently attached to the particle through the reactive groups, the reagent characterized wherein the particle is composed of, at least on its surface, a copolymer having recurring units derived from: (a) at least 0.5 mole % of one or more ethylenically unsaturated polymerizable monomers having reactive groups which are capable of reaction with free amine or sulfhydryl groups of biologically active substances, (b) from 0.1 to 20 mole % of one or more ethylenically unsaturated polymerizable monomers having polyoxyalkylene side chains, each side chain having a molecular weight of at least 88, and (c) up to 99.4 mole % of one or more ethylenically unsaturated monomers which provide hydrophobicity to the copolymer.

Claim 3

The reagent as claimed in claim 1 wherein the biologically active substance is an amine, enzyme, amino acid, peptide, polypeptide, protein, lipoprotein, glycoprotein, hormone, drug, steroid, vitamin, polysaccharide, glycolipid, alkaloid, microorganism, virus, protozoan, fungus, parasite, rickettsia, mold, blood component, tissue and organ component, pharmaceutical, hapten, lectin, toxin, nucleic acid, antigenic material, biotin or derivative thereof or a component thereof.

Claim 4

The reagent as claimed in either of claims 1 or 2 wherein the biologically active substance is an antibody.

Claim 5

An analytical element comprising a fluid-permeable substrate having one or more reaction zones therein, and containing in at least one of the zones, a biologically active reagent, the element characterized wherein the reagent comprises: (I) a water-insoluble, nonporous particle composed of, at least on its surface, a copolymer having recurring units derived from: (a) at least 0.5 mole % of one or more ethylenically unsaturated polymerizable monomers having reactive groups which are capable of reaction with free amine or sulfhydryl groups of biologically active substances, (b) from 0.1 to 20 mole % of one or more ethylenically unsaturated polymerizable monomers having polyoxyalkylene side chains, each side chain having a molecular weight of at least 88, and (c) up to 99.4 mole % of one or more ethylenically unsaturated monomers which provide hydrophobicity to the copolymer, and (II) a biologically active substance covalently attached to the particle through the reactive groups

Claim 6

An analytical element comprising a fluid-permeable substrate having one or more reaction zones therein, and containing in at least one of the zones, a biologically active reagent, the element characterized wherein the reagent comprises: (I) a water-insoluble, nonporous particle composed of, at least on its surface, a copolymer having recurring units derived from: (a) at least 0.5 mole % of one or more ethylenically unsaturated polymerizable monomers having reactive groups which are capable of reaction with free amine or sulfhydryl groups of biologically active substances, (b) from 0.1 to 20 mole % of one or more ethylenically unsaturated polymerizable monomers having polyoxyalkylene side chains, each side chain having a molecular weight of at least 88, and (c) up to 99.4 mole % of one or more ethylenically unsaturated monomers which provide hydrophobicity to the copolymer, and (II) a biologically active substance covalently attached to the particle through the reactive groups

Claim 7

The element as claimed in claim 4 wherein the biologically active substance is an antibody to phenobarbital, thyroxine, carbamazepine, C-reactive protein, phenytoin or digoxin.

Claim 8

The analytical element as claimed in either of claims 4 or 5 which comprises a nonporous support, having imposed thereon, and in order and in fluid contact, a reagent layer containing one or more reagents, and a porous spreading layer containing the reagent.

Claim 11

The method as claimed in claim 7 wherein the ligand is a protein, carbohydrate, hapten, hormone, or drug, and the receptor is an antibody for the protein, carbohydrate, hapten, hormone or drug.

Claim 12

The method as claimed in claim 7 wherein the ligand is a single-stranded nucleic acid, and the receptor is a second single-stranded oligonucleotide which is substantially complementary to the nucleic acid.

Claim 13

A competitive binding assay for the determination of a specific binding ligand comprising: A. contacting a specimen containing a water-soluble specific binding ligand with a water-soluble receptor therefor, and with a reagent comprising: (I) a water-insoluble, nonporous particle composed of, at least on its surface, a copolymer having recurring units derived from: (a) at least 0.5 mole % of one or more ethylenically unsaturated polymerizable monomers having reactive groups which are, directly or indirectly, capable of reaction with free amine or sulfhydryl groups of biologically active substances, (b) from 0.1 to 20 mole % of one or more ethylenically unsaturated polymerizeable monomers having polyoxyalkylene side chains, each side chain having a molecular weight of at least 88, and (c) up to 99.4 mole % of one or more ethylenically unsaturated monomers which provide hydrophobicity to the copolymer, and (II) molecules of the ligand covalently attached to the particle through the reactive groups, to form specific binding complex (a) between the receptor and the ligand, and specific binding complex (b) between the receptor and the water-insoluble reagent, and B. after separating said complexes (a) and (b), detecting the presence of either complex as an indication of the presence or amount of the ligand in the specimen.

Claim 14

A competitive binding assay for the determination of a specific binding ligand comprising: A. contacting a specimen containing a water-soluble specific binding ligand with a water-soluble receptor therefor, and with a reagent comprising: (I) a water-insoluble, nonporous particle composed of, at least on its surface, a copolymer having recurring units derived from: (a) at least 0.5 mole % of one or more ethylenically unsaturated polymerizable monomers having reactive groups which are, directly or indirectly, capable of reaction with free amine or sulfhydryl groups of biologically active substances, (b) from 0.1 to 20 mole % of one or more ethylenically unsaturated polymerizeable monomers having polyoxyalkylene side chains, each side chain having a molecular weight of at least 88, and (c) up to 99.4 mole % of one or more ethylenically unsaturated monomers which provide hydrophobicity to the copolymer, and (II) molecules of the ligand covalently attached to the particle through the reactive groups, to form specific binding complex (a) between the receptor and the ligand, and specific binding complex (b) between the receptor and the water-insoluble reagent, and B. after separating said complexes (a) and (b), detecting the presence of either complex as an indication of the presence or amount of the ligand in the specimen.

Claim 17

The method as claimed in claim 11 wherein the uncomplexed materials are separated from the immunological complex by filtration with a microporous membrane.

Claim 19

An assay for the determination of a specific binding ligand comprising: detecting a water-insoluble specific binding complex formed between a ligand of interest and a receptor therefor, the receptor provided as a component of a reagent comprising: (I) a water-insoluble, nonporous particle composed of, at least on its surface, a copolymer having recurring units derived from: (a) at least 0.5 mole % of one or more ethylenically unsaturated polymerizable monomers having reactive groups which are capable of reaction with free amine or sulfhydryl groups of biologically active substances, (b) from 0.1 to 20 mole % of one or more ethylenically unsaturated polymerizeable monomers having polyoxyalkylene side chains, each side chain having a molecular weight of at least 88, and (c) up to 99.4 mole % of one or more ethylenically unsaturated monomers which provide hydrophobicity to the copolymer, and (II) the receptor for the ligand being covalently attached to the particle through the reactive groups.

Claim 20

An assay for the determination of a specific binding ligand comprising: detecting a water-insoluble specific binding complex formed between a ligand of interest and a receptor therefor, the receptor provided as a component of a reagent comprising: (I) a water-insoluble, nonporous particle composed of, at least on its surface, a copolymer having recurring units derived from: (a) at least 0.5 mole % of one or more ethylenically unsaturated polymerizable monomers having reactive groups which are capable of reaction with free amine or sulfhydryl groups of biologically active substances, (b) from 0.1 to 20 mole % of one or more ethylenically unsaturated polymerizeable monomers having polyoxyalkylene side chains, each side chain having a molecular weight of at least 88, and (c) up to 99.4 mole % of one or more ethylenically unsaturated monomers which provide hydrophobicity to the copolymer, and (II) the receptor for the ligand being covalently attached to the particle through the reactive groups.

Claim 21

An analytical separation method comprising: A. passing a specimen containing a mixture of biologically active substances over an affinity chromatography reagent comprising: (I) a water-insoluble, nonporous particle composed of, at least on its surface, a copolymer having recurring units derived from: (a) at least 0.5 mole % of one or more ethylenically unsaturated polymerizable monomers having reactive groups which are capable of reaction with free amine or sulfhydryl groups of biologically active substances, (b) at least 0.1 mole % one or more ethylenically unsaturated polymerizable monomers having polyoxyalkylene side chains, each side chain having a molecular weight of at least 88, and (c) up to 99.4 mole % of one or more ethylenically unsaturated monomers which provide hydrophobicity to the copolymer, and (II) a specific binding substance covalently attached to the particle through the reactive groups, the specific binding substance being specific to one or more predetermined biologically active substances in the specimen mixture of biologically active substances to form a complex of the reagent with the predetermined substances, and B. collecting either the one or more complexed predetermined substances or one or more substances remaining in the eluent.

Claim 22

An analytical separation method comprising: A. passing a specimen containing a mixture of biologically active substances over an affinity chromatography reagent comprising: (I) a water-insoluble, nonporous particle composed of, at least on its surface, a copolymer having recurring units derived from: (a) at least 0.5 mole % of one or more ethylenically unsaturated polymerizable monomers having reactive groups which are capable of reaction with free amine or sulfhydryl groups of biologically active substances, (b) at least 0.1 mole % one or more ethylenically unsaturated polymerizable monomers having polyoxyalkylene side chains, each side chain having a molecular weight of at least 88, and (c) up to 99.4 mole % of one or more ethylenically unsaturated monomers which provide hydrophobicity to the copolymer, and (II) a specific binding substance covalently attached to the particle through the reactive groups, the specific binding substance being specific to one or more predetermined biologically active substances in the specimen mixture of biologically active substances to form a complex of the reagent with the predetermined substances, and B. collecting either the one or more complexed predetermined substances or one or more substances remaining in the eluent.

Claim 25

The method as claimed claim 16 wherein the nucleic acid of interest is amplified using polymerase chain reaction prior to contact with the reagent.

Claim 26

A kit for a hybridization assay for a nucleic acid of interest comprising: a. a reagent comprising: (I) a water-insoluble particle, and (II) an oligonucleotide covalently attached to the particle through the reactive groups, the oligonucleotide being substantially complementary to a nucleic acid of interest, and b. separately packaged, one or more additional reagents, solutions or equipment needed for the assay, the kit characterized wherein the particle is composed of, at least on its surface, a copolymer having recurring units derived from: (a) at least 0.5 mole % of one or more ethylenically unsaturated polymerizable monomers having reactive groups which are capable of reaction with free amine or sulfhydryl groups of biologically active substances, (b) at least 0.1 mole % one or more ethylenically unsaturated polymerizable monomers having polyoxyalkylene side chains, each side chain having a molecular weight of at least 88, and (c) up to 99.4 mole % of one or more ethylenically unsaturated monomers which provide hydrophobicity to the copolymer.

Claim 27

A kit for a hybridization assay for a nucleic acid of interest comprising: a. a reagent comprising: (I) a water-insoluble particle, and (II) an oligonucleotide covalently attached to the particle through the reactive groups, the oligonucleotide being substantially complementary to a nucleic acid of interest, and b. separately packaged, one or more additional reagents, solutions or equipment needed for the assay, the kit characterized wherein the particle is composed of, at least on its surface, a copolymer having recurring units derived from: (a) at least 0.5 mole % of one or more ethylenically unsaturated polymerizable monomers having reactive groups which are capable of reaction with free amine or sulfhydryl groups of biologically active substances, (b) at least 0.1 mole % one or more ethylenically unsaturated polymerizable monomers having polyoxyalkylene side chains, each side chain having a molecular weight of at least 88, and (c) up to 99.4 mole % of one or more ethylenically unsaturated monomers which provide hydrophobicity to the copolymer.

Claim 28

A kit for a specific binding assay for the determination of a ligand of interest comprising: a. a reagent comprising: (I) a water-insoluble particle, and (II) a biologically active substance covalently attached to the particle through the reactive groups, said substance being specifically reactive with either the ligand of interest or a receptor therefor, and b. separately packaged, one or more additional reagents, solutions or articles needed for the assay, the kit characterized wherein the particle is composed of, at least on its surface, a copolymer having recurring units derived from: (a) at least 0.5 mole % of one or more ethylenically unsaturated polymerizable monomers having reactive groups which are capable of reaction with free amine or sulfhydryl groups of biologically active substances, (b) at least 0.1 mole % one or more ethylenically unsaturated polymerizable monomers having polyoxyalkylene side chains, each side chain having a molecular weight of at least about 88, and (c) up to 99.4 mole % of one or more ethylenically unsaturated monomers which provide hydrophobicity to the copolymer.

Claim 29

A kit for a specific binding assay for the determination of a ligand of interest comprising: a. a reagent comprising: (I) a water-insoluble particle, and (II) a biologically active substance covalently attached to the particle through the reactive groups, said substance being specifically reactive with either the ligand of interest or a receptor therefor, and b. separately packaged, one or more additional reagents, solutions or articles needed for the assay, the kit characterized wherein the particle is composed of, at least on its surface, a copolymer having recurring units derived from: (a) at least 0.5 mole % of one or more ethylenically unsaturated polymerizable monomers having reactive groups which are capable of reaction with free amine or sulfhydryl groups of biologically active substances, (b) at least 0.1 mole % one or more ethylenically unsaturated polymerizable monomers having polyoxyalkylene side chains, each side chain having a molecular weight of at least about 88, and (c) up to 99.4 mole % of one or more ethylenically unsaturated monomers which provide hydrophobicity to the copolymer.

Claim 30

The invention as claimed in any of claims 1 to 19 wherein the monomers of (b) are represented by the structure: wherein R is hydrogen or methyl, R 1 is alkylene having 2 to 4 carbon atoms, X is hydrogen or acyl and n is 2 to 30.

Claim 31

The reagent as claimed in claim 20 wherein R 1 is alkylene having 2 or 3 carbon atoms and n is 2 to 20.