Polypyridine Fluorescent labels for immunoassay

Claim 2

A fluorescent chelate of Eu -3 and a chelating agent having the structure ##STR30## wherein R represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, aryl, aryloxy, a heterocyclic group, an enzyme, antigen, or antibody; R 1 represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, substituted or unsubstituted aryl or a heterocyclic group; R 2 represents carboxy, hydroxy, carbonyliminodiacetic acid, methyleneiminodiacetic acid, hydrazinylylidenediacetic acid or a salt of such acids; n is 0 to 4; and m is 0 to 1 provided that m can be 1 only when n is 0.

Claim 3

The chelate of claim 1 wherein the chelating agent has the structure ##STR31## wherein R represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, aryl, aryloxy, a heterocyclic group, an enzyme, antigen, or antibody; R 1 represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, substituted or unsubstituted aryl or a heterocyclic group; R 2 represents carboxy, hydroxy, carbonyliminodiacetic acid, methyleneiminodiacetic acid, hydrazinylylidenediacetic acid or a salt of such acids; and n is 1 to 3.

Claim 4

The chelate of claim 1 wherein the chelating agent has the structure ##STR32## wherein R 1 represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, substituted or unsubstituted aryl or a heterocyclic group; and R 2 represents carboxy, hydroxy, carbonyliminodiacetic acid, methyleneimino diacetic acid, hydrazinylylidene diacetic acid or a salt of such acids.

Claim 5

The chelate of claim 2 wherein R represents hydrogen, --SCH 3 , --OCH 3 , --CN, --COOCH 3 or --CH 3 ; R 1 represents hydrogen, phenyl, methoxyphenyl, tolyl, cyanophenyl, p-methylthiophenyl, methoxycarbonylphenyl, p-(5,5-diphenylimidazolidine-2,4-dion-3-yl)methylphenyl; and R 2 represents carboxy, hydroxy, carbonyliminodiacetic acid, methyleneimino diacetic acid, hydrazinylylidene diacetic acid or a salt of such acids.

Claim 6

The chelate of claim 3 wherein R 1 , in the chelating agent, represents hydrogen; and R 2 represents carboxy, hydroxy, carbonyliminodiacetic acid, methyleneimino diacetic acid, hydrazinylylidene diacetic acid or a salt of such acids.

Claim 7

The chelate of claim 2 wherein the chelating agent has the structure ##STR33## wherein R 4 represents --H, --SCH 3 , --OCH 3 , --CN, --NO 2 , --CO 2 CH 3 , --CH 3 ; ##STR34## wherein R 5 represents --CH 3 , --NO 2 , or --OCH 3 ; ##STR35## wherein R 11 represents --CH 3 , --NO 2 , --OCH 3 ; or ##STR36##

Claim 8

The chelate of claim 1, 2, 3, 4, 5 or 6 wherein the mole ratio of Eu -3 to the chelating agent is from 1:1 to 2:1.

Claim 9

A fluorescently labeled specific binding reagent comprising a physiologically active material adsorbed or bound to a fluorescent label comprising a fluorescent chelate of a Eu -3 and a chelating agent having the structure ##STR37## wherein R represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, aryl, aryloxy, a heterocyclic group, an enzyme, antigen, or antibody; R 1 represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, substituted or unsubstituted aryl or a heterocyclic group; R 2 represents carboxy, hydroxy, carbonyliminodiacetic acid, methyleneimino diacetic acid, hydrazinylylidene diacetic acid or a salt of such acids; n is 0 to 4; and m is 0 to 1 provided that m can be 1 only when n is 0.

Claim 10

A fluorescently labeled specific binding reagent comprising a physiologically active material adsorbed or bound to a fluorescent label comprising a fluorescent chelate of a Eu -3 and a chelating agent having the structure ##STR37## wherein R represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, aryl, aryloxy, a heterocyclic group, an enzyme, antigen, or antibody; R 1 represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, substituted or unsubstituted aryl or a heterocyclic group; R 2 represents carboxy, hydroxy, carbonyliminodiacetic acid, methyleneimino diacetic acid, hydrazinylylidene diacetic acid or a salt of such acids; n is 0 to 4; and m is 0 to 1 provided that m can be 1 only when n is 0.

Claim 11

The binding reagent of claim 8 wherein the chelating agent has the structure ##STR38## wherein R 1 represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, substituted or unsubstituted aryl or a heterocyclic group; R 2 represents carboxy, hydroxy, carbonyliminodiacetic acid, methyleneimino diacetic acid, hydrazinylylidene diacetic acid or a salt of such acids; and n is 1 to 3.

Claim 12

The binding reagent of claim 8 wherein the chelating agent has the structure ##STR39## wherein R 1 represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, substituted or unsubstituted aryl or a heterocyclic group; and R 2 represents carboxy, hydroxy, carbonyliminodiacetic acid, methyleneimino diacetic acid, hydrazinylylidene diacetic acid or a salt of such acids.

Claim 13

The binding reagent of claim 9 wherein R 1 represents hydrogen, phenyl, methoxyphenyl, tolyl, cyanophenyl, p-methylthiophenyl, methoxycarbonylphenyl or p-(5,5-diphenylimidazolidine-2,4-dion-3-yl)methylphenyl; and R 2 represents carboxy, hydroxy, carbonyliminodiacetic acid, methyleneimino diacetic acid, hydrazinylylidene diacetic acid or a salt of such acids.

Claim 14

The binding reagent of claim 9 wherein the chelating agent has the structure ##STR40## wherein R 4 represents --H, --SCH 3 , --OCH 3 , --CN, --NO 2 , --CO 2 CH 2 , --CH 3 ; ##STR41## wherein R 5 represents --CH 3 , --NO 2 , or --OCH 3 ; ##STR42## wherein R 11 represents --CH 3 , --NO 2 , --OCH 3 ; or ##STR43##

Claim 15

The labeled specific binding reagent of claim 8, 9, 10, 11 or 12 wherein the physiologically active material is selected from the group consisting of antibodies, antigens, haptens, enzymes, enzyme substrates, metabolites, vitamins, hormones, hormone receptors, cell surface receptors and lectins.

Claim 16

The labeled specific binding reagent of claim 8, 9, 10, 11, or 12 wherein the mole ratio of Eu +3 to the chelating agent is from 1:1 to 2:1.

Claim 19

In a method for performing an immunoassay comprising the steps of (a) fluorescently labeling a ligand of interest, a derivative of said ligand or some other molecular entity, to produce a fluorescently labeled immunologically binding ligand analog of said ligand of interest; (b) adding a known amount of said fluorescently labeled ligand analog and an unknown amount of ligand to be assayed to a receptor which is capable of binding with said fluorescently labeled ligand analog and also is capable of binding with said ligand to be assayed; and (c) determining the unknown amount of said ligand to be assayed by measuring the fluorescence of said fluorescently labeled ligand analog bound to said receptor or by measuring the fluorescence of said fluorescently labeled ligand analog remaining unbound, the improvement wherein the labeled ligand analog comprises ligand adsorbed or covalently bound to a fluorescent label comprising a fluorescent chelate of a lanthanide metal and a chelating agent having the structure ##STR45## wherein R represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, aryl, aryloxy or a heterocyclic group; R 1 represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, substituted or unsubstituted aryl or a heterocyclic group; R 2 represents carboxy, hydroxy, carbonyliminodiacetic acid, methyleneimino diacetic acid, hydrazinylylidene diacetic acid or a salt of such acids; n is 0 to 4; and m is 0 to 1 provided that m can be 1 only when n is 0.

Claim 20

In a method for performing an immunoassay comprising the steps of (a) fluorescently labeling a ligand of interest, a derivative of said ligand or some other molecular entity, to produce a fluorescently labeled immunologically binding ligand analog of said ligand of interest; (b) adding a known amount of said fluorescently labeled ligand analog and an unknown amount of ligand to be assayed to a receptor which is capable of binding with said fluorescently labeled ligand analog and also is capable of binding with said ligand to be assayed; and (c) determining the unknown amount of said ligand to be assayed by measuring the fluorescence of said fluorescently labeled ligand analog bound to said receptor or by measuring the fluorescence of said fluorescently labeled ligand analog remaining unbound, the improvement wherein the labeled ligand analog comprises ligand adsorbed or covalently bound to a fluorescent label comprising a fluorescent chelate of a lanthanide metal and a chelating agent having the structure ##STR45## wherein R represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, aryl, aryloxy or a heterocyclic group; R 1 represents hydrogen, alkyl, alkoxy, alkylthio, alkylamino, substituted or unsubstituted aryl or a heterocyclic group; R 2 represents carboxy, hydroxy, carbonyliminodiacetic acid, methyleneimino diacetic acid, hydrazinylylidene diacetic acid or a salt of such acids; n is 0 to 4; and m is 0 to 1 provided that m can be 1 only when n is 0.