Method for performing fluorescent protein binding assay employing novel alkyl substituted fluorescent compounds and conjugates

Claim 2

In a method for performing a fluorescent protein binding assay, which comprises combining in an assay medium a sample suspected of containing an analyte and members of a signal producing system wherein one of said members comprises a conjugate of a member of a specific binding pair either ligand or receptor and a spectroscopically active compound, and measuring the level of said signal in said assay medium, the improvement which comprises employing as said conjugate a compound of the formula ##STR9## wherein: α is a member of a specific binding pair either ligand or receptor covalently bonded to one of ψ or ψ'; ρ is an aliphatic hydrocarbon group of from about 1 to 12 carbon atoms. ψ' is hydrogen or non-oxocarbonyl; L is a bond or divalent radical; ψ is a linking group bonded to α or a group terminating in a heteroatom containing functionality when not bonded to α; and ν is a number between 1 and the molecular weight of α divided by 500.

Claim 3

In a method for performing a fluorescent protein binding assay, which comprises combining in an assay medium a sample suspected of containing an analyte, and members of a signal producing system wherein one of said members comprises a conjugate of a member of a specific binding pair either ligand or receptor and a spectroscopically active compound, the improvement which comprises employing as said conjugate a compound of the formula ##STR10## wherein: R is alkyl or carboxyalkyl of from 1 to 4 carbon atoms: Z is carboxy; A is a ligand or receptor; W is a bond or divalent radical of from 0 to 8 carbon atoms; Y is methylene or a heteroatom containing linking functionality; m is 0 to 3; n is a number between 1 and the molecular weight of A divided by 500; and said portion in the brackets of said formula having from 0 to 6 chloro groups at other than the 1,8-position of the xanthene portion of the molecule.

Claim 4

In a method for performing a fluorescent protein binding assay, which comprises combining in an assay medium a sample suspected of containing an analyte, and members of a signal producing system wherein one of said members comprises a conjugate of a member of a specific binding pair either ligand or receptor and a spectroscopically active compound, the improvement which comprises employing as said conjugate a compound of the formula ##STR10## wherein: R is alkyl or carboxyalkyl of from 1 to 4 carbon atoms: Z is carboxy; A is a ligand or receptor; W is a bond or divalent radical of from 0 to 8 carbon atoms; Y is methylene or a heteroatom containing linking functionality; m is 0 to 3; n is a number between 1 and the molecular weight of A divided by 500; and said portion in the brackets of said formula having from 0 to 6 chloro groups at other than the 1,8-position of the xanthene portion of the molecule.

Claim 5

In a method for performing a fluorescent protein binding assay, which comprises combining in an assay medium a sample suspected of containing an analyte, and members of a signal producing system wherein one of said members comprises a conjugate of a member of a specific binding pair either ligand or receptor and a spectroscopically active compound, the improvement which comprises employing as said conjugate a compound of the formula ##STR11## wherein: E b is hydrogen or chloro; E 2 is chloro; Z 2 is carboxy; R 2 is alkylene of from 1 to 3 carbon atoms; D 2 is hydrogen or carboxy; W 2 is a bond, alkylene, carboxamidoalkylene, or poly(carboxamidoalkylene), wherein alkylene is of from 1 to 2 carbon atoms; Y 2 is non-oxo-carbonyl, carbamoyl, thiocarbamoyl, amino, methylene, oxy or thio; A 2 is a ligand or receptor; m 2 is 2 to 3; X 2 is 0 to 4; and n 2 is a number between 1 and the molecular weight of A 2 divided by 500.

Claim 6

In a method for performing a fluorescent protein binding assay, which comprises combining in an assay medium a sample suspected of containing an analyte, and members of a signal producing system wherein one of said members comprises a conjugate of a member of a specific binding pair either ligand or receptor and a spectroscopically active compound, the improvement which comprises employing as said conjugate a compound of the formula ##STR11## wherein: E b is hydrogen or chloro; E 2 is chloro; Z 2 is carboxy; R 2 is alkylene of from 1 to 3 carbon atoms; D 2 is hydrogen or carboxy; W 2 is a bond, alkylene, carboxamidoalkylene, or poly(carboxamidoalkylene), wherein alkylene is of from 1 to 2 carbon atoms; Y 2 is non-oxo-carbonyl, carbamoyl, thiocarbamoyl, amino, methylene, oxy or thio; A 2 is a ligand or receptor; m 2 is 2 to 3; X 2 is 0 to 4; and n 2 is a number between 1 and the molecular weight of A 2 divided by 500.

Claim 7

In a method for performing a fluorescent protein binding assay, which comprises combining in an assay medium a sample suspected of containing an analyte, and members of a signal producing system wherein one of said members comprises a conjugate of a member of a specific binding pair either ligand or receptor and a spectroscopically active compound, which conjugate is further bound to a support, and measuring the level of said signal in said assay medium, the improvement which comprises employing as said conjugate bound to a support a compound of the formula ##STR12## wherein: n 3 is a number between 1 and the molecular weight of A 2 divided by 500, with the proviso that when q is 0, n 3 is 1; q is 0 to 1; p is at least 1 and up to the molecular weight of Support divided by 500; Support is a molecule of at least about 10,000 molecular weight having a plurality of functionalities for linking; the group in the brackets is bonded by any convenient functionality by a bond or linking group to the Support; E b is hydrogen or chloro; E 2 is chloro; Z 2 is carboxy; R 2 is alkylene of from 1 to 3 carbon atoms; D 2 is hydrogen or carboxy; W 2 is a bond, alkylene, carboxyamidoalkylene or poly(carboxyamidoalkylene), wherein alkylene is of from 1 to 2 carbon atoms; Y 2 is non-oxo-carbonyl, carbamoyl, thiocarbamoyl, amino, methylene, oxy or thio; A 2 is a ligand or receptor bonded to Y 2 ; m 2 is 0 to 3; and x 2 is 0 to 4.

Claim 8

In a method for performing a fluorescent protein binding assay, which comprises combining in an assay medium a sample suspected of containing an analyte, and members of a signal producing system wherein one of said members comprises a conjugate of a member of a specific binding pair either ligand or receptor and a spectroscopically active compound, which conjugate is further bound to a support, and measuring the level of said signal in said assay medium, the improvement which comprises employing as said conjugate bound to a support a compound of the formula ##STR12## wherein: n 3 is a number between 1 and the molecular weight of A 2 divided by 500, with the proviso that when q is 0, n 3 is 1; q is 0 to 1; p is at least 1 and up to the molecular weight of Support divided by 500; Support is a molecule of at least about 10,000 molecular weight having a plurality of functionalities for linking; the group in the brackets is bonded by any convenient functionality by a bond or linking group to the Support; E b is hydrogen or chloro; E 2 is chloro; Z 2 is carboxy; R 2 is alkylene of from 1 to 3 carbon atoms; D 2 is hydrogen or carboxy; W 2 is a bond, alkylene, carboxyamidoalkylene or poly(carboxyamidoalkylene), wherein alkylene is of from 1 to 2 carbon atoms; Y 2 is non-oxo-carbonyl, carbamoyl, thiocarbamoyl, amino, methylene, oxy or thio; A 2 is a ligand or receptor bonded to Y 2 ; m 2 is 0 to 3; and x 2 is 0 to 4.

Claim 9

In a method for performing a fluorescent protein binding assay, which comprises combining in an assay medium a sample suspected of containing an analyte, and members of a signal producing system wherein one of said members comprises a conjugate of a member of a specific binding pair either ligand or receptor and a spectroscopically active compound, the improvement which comprises employing as said conjugate a compound of the formula ##STR13## wherein: E b is hydrogen or chloro; R 2 is alkylene of from 1 to 2 carbon atoms; D 2 is hydrogen or carboxy; m 2 is 1 to 3; x 2 is 1 to 3; a 2 is 0 to 1; A 2 is a ligand or a receptor, said ligand and receptor being linked by amide linkages; n 2 is 1 when A 2 is hydroxyl or an electrophilic ester activating group and is otherwise a number between 1 and the molecular weight of A 2 divided by 500.

Claim 10

In a method for performing a fluorescent protein binding assay, which comprises combining in an assay medium a sample suspected of containing an analyte, and members of a signal producing system wherein one of said members comprises a conjugate of a member of a specific binding pair either ligand or receptor and a spectroscopically active compound, the improvement which comprises employing as said conjugate a compound of the formula ##STR13## wherein: E b is hydrogen or chloro; R 2 is alkylene of from 1 to 2 carbon atoms; D 2 is hydrogen or carboxy; m 2 is 1 to 3; x 2 is 1 to 3; a 2 is 0 to 1; A 2 is a ligand or a receptor, said ligand and receptor being linked by amide linkages; n 2 is 1 when A 2 is hydroxyl or an electrophilic ester activating group and is otherwise a number between 1 and the molecular weight of A 2 divided by 500.