Assay method and reagent therefor

Claim 2

In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) the presence of said ligand or receptor in said solution causes a conjugate in a solution to become matrix bound, said conjugate comprising a ligand or receptor labelled with an enzyme; (b) the matrix bound conjugate is separated from unbound conjugate, and (c) the matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) contacting the conjugate to be assayed with a precursor for a modulator for a cyclic chemical reaction whereby said modulator is produced as a result of reaction of label and precursor for the modulator, and (e) contacting the thus produced modulator with the components of a cyclic chemical reaction which is activated by the modulator, which cyclic chemical reaction interconverts said modulator and its reduced or oxidized form and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.

Claim 3

The improvement according to claim 1 wherein the modulator is a cofactor which the cyclic chemical reaction interconverts with its reduced form.

Claim 4

The improvement according to claim 2 wherein the cofactor is a nicotinamide adenine dinucleotide.

Claim 5

The improvement according to claim 2 wherein NAD is converted to NADH by a dehydrogenase.

Claim 6

The improvement according to claim 4 wherein the dehydrogenase is an alcohol dehydrogenase.

Claim 7

The improvement according to claim 4 wherein the conversion of NADH to NAD accompanies reduction of a tetrazolium compound whereby a colour change is produced.

Claim 8

The improvement according to claim 4 wherein the cyclic chemical reaction comprises two enzymatic reactions.

Claim 9

The improvement acccording to claim 1 wherein the ligand is an antibody or antigen.

Claim 10

The improvement according to claim 1 wherein the ligand in solution and conjugate are added sequentially.

Claim 11

The improvement according to claim 9 wherein the ligand in solution is added before the conjugate.

Claim 12

In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) a matric to which is bound a ligand or receptor is contacted with a conjugate and with the, said conjugate comprising a ligand or receptor labelled with an enzyme; (b) the matrix bound conjugate is separated from unbound conjugate, and (c) the matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) contacting the conjugate to be assayed with a precursor for a modulator for a cyclic chemical reaction whereby said modulator is produced as a result of reaction of label and precursor for the modulator, and (e) contacting the thus produced modulator with the components of a cyclic chemical reaction which is activated by the modulator, which cyclic chemical reaction interconverts said modulator and its reduced or oxidized form and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.

Claim 13

In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) a matric to which is bound a ligand or receptor is contacted with a conjugate and with the, said conjugate comprising a ligand or receptor labelled with an enzyme; (b) the matrix bound conjugate is separated from unbound conjugate, and (c) the matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) contacting the conjugate to be assayed with a precursor for a modulator for a cyclic chemical reaction whereby said modulator is produced as a result of reaction of label and precursor for the modulator, and (e) contacting the thus produced modulator with the components of a cyclic chemical reaction which is activated by the modulator, which cyclic chemical reaction interconverts said modulator and its reduced or oxidized form and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.

Claim 14

The improvement according to claim 11 wherein the modulator is a cofactor which the cyclic chemical reaction interconverts with its reduced form.

Claim 15

The improvement according to claim 12 wherein the cofactor is a nicotinamide adenine dinucleotide.

Claim 16

The improvement according to claim 12 wherein the NAD is converted to NADH by a dehydrogenase.

Claim 17

The improvement according to claim 14 wherein the dehydrogenase is an alcohol dehydrogenase.

Claim 18

The improvement according to claim 14 wherein the conversion of NADH to NAD accompanies reduction of tetrazolium compound whereby a colour change is produced.

Claim 19

The improvement according to claim 14 wherein the cyclic chemical reaction comprises two enzymatic reactions.

Claim 20

The improvement according to claim 11 wherein the ligand is an antibody or antigen.

Claim 21

In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) the presence of said ligand or receptor in said solution causes a conjugate in a solution to become matrix bound, said conjugate comprising a ligand or receptor labelled with an enzyme; (b) the matrix bound conjugate is separated from unbound conjugate, and (c) the matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) contacting the conjugate to be assayed with a precursor for a modulator for a cyclic chemical reactor whereby said modulator is produced as a result of reaction of label and precursor for the modulator, and (e) contacting the thus produced modulator with the components of a cyclic chemical reaction interconverts said modulator and a modified modulator which has more or fewer phosphate groups attached thereto, and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.

Claim 22

In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) the presence of said ligand or receptor in said solution causes a conjugate in a solution to become matrix bound, said conjugate comprising a ligand or receptor labelled with an enzyme; (b) the matrix bound conjugate is separated from unbound conjugate, and (c) the matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) contacting the conjugate to be assayed with a precursor for a modulator for a cyclic chemical reactor whereby said modulator is produced as a result of reaction of label and precursor for the modulator, and (e) contacting the thus produced modulator with the components of a cyclic chemical reaction interconverts said modulator and a modified modulator which has more or fewer phosphate groups attached thereto, and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.

Claim 23

The improvement according to claim 19 wherein the modulator has one less phosphate group than the modified modulator.

Claim 24

The improvement according to claim 20 wherein the modulator is fructose-6-phosphate and the modified modulator is fructose-1,6-diphosphate.

Claim 25

The improvement according to claim 19 wherein the modulator has one more phosphate group than the modified modulator.

Claim 26

The improvement according to claim 22 wherein the modulator is ATP and the modified modulator is ADP.

Claim 27

The improvement according to claim 19 wherein the determinable product is inorganic phosphate.

Claim 28

The improvement according to claim 19 wherein the ligand is an antibody.

Claim 29

The improvement according to claim 19 wherein the ligand is an antigen.

Claim 30

In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) a matrix to which is bound a receptor or ligand is contacted with a conjugate and with the solution, said conjugate comprising a receptor or ligand labelled with an enzyme; (b) the matrix bound conjugate is separated from unbound conjugate, and (c) the matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) contacting the conjugate to be assayed with a precursor for a modulator for a cyclic chemical reaction whereby said modulator is produced as a result of reaction of label and precursor for the modulator, and (e) contacting the thus produced modulator with the components of a cyclic chemical reaction which is activated by the modulator, which cyclic chemical reaction interconverts said modulator and a modified modulator which has more or fewer phosphate groups attached thereto, and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.

Claim 31

In a method of determining a ligand or receptor in solution which comprises an assay wherein: (a) a matrix to which is bound a receptor or ligand is contacted with a conjugate and with the solution, said conjugate comprising a receptor or ligand labelled with an enzyme; (b) the matrix bound conjugate is separated from unbound conjugate, and (c) the matrix bound or the unbound conjugate is assayed, the improvement which comprises: (d) contacting the conjugate to be assayed with a precursor for a modulator for a cyclic chemical reaction whereby said modulator is produced as a result of reaction of label and precursor for the modulator, and (e) contacting the thus produced modulator with the components of a cyclic chemical reaction which is activated by the modulator, which cyclic chemical reaction interconverts said modulator and a modified modulator which has more or fewer phosphate groups attached thereto, and which cyclic chemical reaction thereby regenerates the modulator produced by the conjugate and produces a determinable change, said components of the cyclic chemical reaction being present in sufficient quantity to maintain the cyclic chemical reaction and produce determinable product as the cyclic chemical reaction proceeds, whereby amplification occurs.

Claim 32

The improvement according to claim 27 wherein the modulator has one less phosphate group than the modified modulator.

Claim 33

The improvement according to claim 28 wherein the modulator is fructose-6-phosphate and the modified modulator is fructose-1,6-diphosphate.

Claim 34

The improvement according to claim 27 wherein the modulator has one or more phosphate groups than the modified modulator.

Claim 35

The improvement according to claim 30 wherein the modulator is ATP and the modified modulator is ADP.

Claim 36

The improvement according to claim 27 wherein the determinable product is inorganic phosphate.

Claim 37

The improvement according to claim 27 wherein the ligand is an antibody.

Claim 38

The improvement according to claim 27 wherein the ligand is an antigen.

Claim 39

The improvement according to claim 27 wherein the ligand in solution and conjugate are added sequentially.

Claim 40

The improvement according to claim 27 wherein the ligand in solution is added before the conjugate.