Squarate dye conjugate, process for making it and its use in assay methods and kits

Claim 2

A compound for use in an assay for determining an analyte in a sample, said compound being a conjugate of a dye and a member of a specific binding pair (sbp) consisting of ligand and its complementary receptor, characterized in that a squarate dye is employed as the dye in said conjugate.

Claim 3

A compound according to Claim 1, of the formula - wherein: D and Y are the same or different and each comprises a chain of from 1 to 6 moieties selected from -CH= and -N=, said chain having alternating single and double bonds and terminating in a functionality selected from the group consisting of 0, S, and disubstituted nitrogen, wherein the sum of the number of said moieties and said terminal functionality in both D and Y is an even number, said chain, or part thereof including the terminal functionality, being aliphatic or part of one or more alicyclic or aromatic rings, or a combination thereof, said chain or rings, or functionalities having zero, one, or more substituents comprising from 1 to 30 atoms other than hydrogen, which atoms are selected from carbon, oxygen, nitrogen, sulfur, halogen of atomic numbers 9 to 53, arsenic, silicon, selenium and phosphorus wherein said substituents may be taken together to form one or more rings which may be alicylic or aromatic, wherein at least one of D or Y is linked to A; A is a group comprising at least one member of a specific binding pair (sbp) consisting of ligand and its complementary receptor; n is a number on the average of 1 up to the molecular weight of A divided by 100; and q is 1 or 2.

Claim 4

A compound according to Claim 2, selected from and wherein: A3 is a member of a specific binding pair; n 3 is a number on the average of 1 up to the molecular weight of A3 divided by 5000; t is 1 or 2; q 3 is 1 or 2 ; R 3 is independently selected from substituents having 1-10 carbon atoms, 0-6 oxygen atoms, and 0-3 nitrogen atoms, and may be taken together to form an alicylic or aromatic ring; R4 is independently selected from hydroxyl, hydrogen, phosphate, phosphonate, sulfonate, nitro, halogen, amino and R 3 ; and L replaces any hydrogen of R 3 and is a bond or a linking group to A3.

Claim 5

A compound of Claim 3 wherein at least one R 3 on nitrogen has a hydrogen replaced by L.

Claim 6

A compound of Claim 4 wherein every remaining R 3 on nitrogen is lower alkyl of 1 to 5 carbons.

Claim 7

A compound according to anyone of the Claims 3 to 5 wherein t is 1 and R 4 is hydroxyl.

Claim 8

A compound according to anyone of the Claims 3 to 6 wherein L is selected from non-oxo-carbonyl, carbamoyl, thiocarbamoyl, and amino.

Claim 9

A compound of anyone of the Claims 1 to 7, wherein said squarate dye has an absorption maximum greater than 600nm.

Claim 10

An assay for an analyte in a sample suspected of containing said analyte, which analyte is a member of a specific binding pair (sbp member), said method involving an sbp member conjugated to a dye, characterized in that a squarate dye is employed as the conjugated dye.

Claim 11

An assay for an analyte in a sample suspected of containing said analyte, which analyte is a member of a specific binding pair (sbp member), said method involving an sbp member conjugated to a dye, characterized in that a squarate dye is employed as the conjugated dye.

Claim 12

An assay according to Claim 9, which assay comprises - combining in an assay medium the sample, the sbp member squarate dye compound, and a third sbp member, wherein two of the three sbp members are complementary to the remaining member, and determining the effect of said sample on the fluorescence of the compound as related to the presence or amount of analyte in said sample.

Claim 13

An assay of Claim 10 wherein said third sbp member is conjugated to a compound capable of quenching the fluorescence of the compound when said third sbp member is bound to said compound.

Claim 14

An assay for a material of interest in a sample suspected of containing the material of interest wherein a fluorescent compound is employed to generate a signal in relation to the presence or amount of the material of interest in said sample and an energy source for excitation of said fluorescent compound is also employed, characterized in that a squarate dye is employed as the fluorescent compound and a helium/neon laser as the energy source.

Claim 15

An assay for a material of interest in a sample suspected of containing the material of interest wherein a fluorescent compound is employed to generate a signal in relation to the presence or amount of the material of interest in said sample and an energy source for excitation of said fluorescent compound is also employed, characterized in that a squarate dye is employed as the fluorescent compound and a helium/neon laser as the energy source.

Claim 16

An assay of Claim 12 wherein said squarate dye is water compatible.

Claim 17

An assay of Claim 12 or 13, wherein said squarate dye is not incorporated into a cell.

Claim 18

A kit for use in determining the presence or amount of an analyte in a sample suspected of containing said analyte, said analyte being a member of a specific binding pair (sbp), said kit comprising in packaged combination - - a first compound comprising a squarate dye conjugated to a second sbp member, a second compound comprising a third sbp member wherein two of the three sbp members are complementary to the remaining sbp member, and ancillary materials as required.

Claim 19

A kit for use in determining the presence or amount of an analyte in a sample suspected of containing said analyte, said analyte being a member of a specific binding pair (sbp), said kit comprising in packaged combination - - a first compound comprising a squarate dye conjugated to a second sbp member, a second compound comprising a third sbp member wherein two of the three sbp members are complementary to the remaining sbp member, and ancillary materials as required.

Claim 20

A process for preparing the compound of Claim 1, which process comprises conjugating the sbp member with the squarate dye.

Claim 21

A process which comprises the production of a compound for use in an assay for determining an analyte in a sample, said compound being a conjugate of a squarate dye and a member of a specific binding pair (sbp) consisting of ligand and its complementary receptor.

Claim 22

A process which comprises the production of a compound for use in an assay for determining an analyte in a sample, said compound being a conjugate of a squarate dye and a member of a specific binding pair (sbp) consisting of ligand and its complementary receptor.

Claim 23

A process according to claim 1 wherein the compound has the formula - wherein: D and Y are the same or different and each comprises a chain of from 1 to 6 moieties selected from -CH= and -N=, said chain having alternating single and double bonds and terminating in a functionality selected from the group consisting of 0, S, and disubstituted nitrogen, wherein the sum of the number of said moieties and said terminal functionality in both D and Y is an even number, said chain, or part thereof including the terminal functionality, being aliphatic or part of one or more alicyclic or aromatic rings, or a combination thereof, said chain or rings, or functionalities having zero, one, or more substituents comprising from 1 to 30 atoms other than hydrogen, which atoms are selected from carbon, oxygen, nitrogen, sulfur, halogen of atomic numbers 9 to 53, arsenic, silicon, selenium and phosphorus wherein said substituents may be taken together to form one or more rings which may be alicylic or aromatic, wherein at least one of D or Y is linked to A; A is a group comprising at least one member of a specific binding pair (sbp) consisting of ligand and its complementary receptor; n is a number on the average of 1 up to the molecular weight of A divided by 100; and q is 1 or 2.

Claim 24

A process according to claim 2 wherein the compound is selected from and wherein: A3 is a member of a specific binding pair; n 3 is a number on the average of 1 up to the molecular weight of A 3 divided by 5000; t is 1 or 2; q 3 is 1 or 2; R 3 is independently selected from substituents having 1-10 carbon atoms, 0-6 oxygen atoms, and 0-3 nitrogen atoms, and may be taken together to form an alicylic or aromatic ring; R 4 is independently selected from hydroxyl, hydrogen, phosphate, phosphonate, sulfonate, nitro, halogen, amino and R 3 ; and L replaces any hydrogen of R 3 and is a bond or a linking group to A3.

Claim 25

A process according to claim 3 wherein at least one R 3 on nitrogen has a hydrogen replaced by L.

Claim 26

A process according to claim 4 wherein every remaining R 3 on nitrogen is lower alkyl of 1 to 5 carbons.

Claim 27

A process according to anyone of the Claims 3 to 5 wherein t is 1 and R 4 is hydroxyl.

Claim 28

7 . A process according to anyone of the Claims 3 to 6 wherein L is selected from non-oxo-carbonyl, carbamoyl, thiocarbamoyl, and amino.

Claim 29

A process according to any one of the claims 1 to 7, wherein said squarate dye has an absorption maximum greater than 600nm.

Claim 30

An assay for an analyte in a sample suspected of containing said analyte, which analyte is a member of a specific binding pair (sbp member), said method involving an sbp member conjugated to a dye, characterized in that a squarate dye is employed as the conjugated dye.

Claim 31

An assay for an analyte in a sample suspected of containing said analyte, which analyte is a member of a specific binding pair (sbp member), said method involving an sbp member conjugated to a dye, characterized in that a squarate dye is employed as the conjugated dye.

Claim 32

An assay according to Claim 9, which assay- comprises - combining in an assay medium the sample, the sbp member squarate dye compound, and a third sbp member, wherein two of the three sbp members are complementary to the remaining member, and determining the effect of said sample on the fluorescence of the compound as related to the presence or amount of analyte in said sample.

Claim 33

An assay of Claim 10 wherein said third sbp member is conjugated to a compound capable of quenching the fluorescence of the compound when said third sbp member is bound to said compound.

Claim 34

An assay for a material of interest in a sample suspected of containing the material of interest wherein a fluorescent compound is employed to generate a signal in relation to the presence or amount of the material of interest in said sample and an energy source for excitation of said fluorescent compound is also employed, characterized in that a squarate dye is employed as the fluorescent compound and a helium/neon laser as the energy source.

Claim 35

An assay for a material of interest in a sample suspected of containing the material of interest wherein a fluorescent compound is employed to generate a signal in relation to the presence or amount of the material of interest in said sample and an energy source for excitation of said fluorescent compound is also employed, characterized in that a squarate dye is employed as the fluorescent compound and a helium/neon laser as the energy source.

Claim 36

An assay of Claim 12 wherein said squarate dye is water compatible.

Claim 37

An assay of Claim 12 or 13, wherein said squarate dye is not incorporated into a cell.

Claim 38

A kit for use in determining the presence or amount of an analyte in a sample suspected of containing said analyte, said analyte being a member of a specific binding pair (sbp), said kit comprising in packaged combination - a first compound comprising a squarate dye conjugated to a second sbp member, a second compound comprising a third sbp member wherein two of the three sbp members are complementary to the remaining sbp member, and ancillary materials as required.

Claim 39

A kit for use in determining the presence or amount of an analyte in a sample suspected of containing said analyte, said analyte being a member of a specific binding pair (sbp), said kit comprising in packaged combination - a first compound comprising a squarate dye conjugated to a second sbp member, a second compound comprising a third sbp member wherein two of the three sbp members are complementary to the remaining sbp member, and ancillary materials as required.

Claim 40

A process for preparing the compound of Claim 1, which process comprises conjugating the sbp member with the squarate dye.