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US4806488A
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Ligand-receptor assays employing squarate dye compositions
Assignee: Siemens Healthcare Diagnostics GmbH Germany
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Filing: 1985-09-06
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Granted: 1989-02-21
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Expires: 2005-09-06
Abstract
Abstract Novel assays for ligands and receptors employing novel compounds that are conjugates of squarates dyes and members of a specific binding pair (sbp) are disclosed. The sbp members are selected from the group consisting of ligand and its complementary receptor. The sbp member is covalently or non-covalently bound to the squarate dye, which usually has an absorption maximum greater than 600 nanometers. The novel conjugates are employed in assays for determining the presence or amount of an sbp member analyte in a sample suspected of containing such analyte. Kits comprising such novel conjugates are also disclosed.
Inventors
- 👤 Donald E. Berger, Jr.
- 👤 Thomas L. Tarnowski
- 👤 Edwin F. Ullman
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-07 15:36
CPC Classifications
C; C09; C09B; C09B57/00; C09B57/007; C09B23/00; C09B23/0066; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/531; G01N33/532; G01N33/533; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10S436/00; Y10S436/80; Y10S436/815
Claims Analysis
Total Claims: 93
Claim 1
Independent Claim
247 words
A conjugate of a squaraine dye covalently or non-covalently bonded to a member of a specific binding pair, said conjugate being of the formula ##STR13## wherein: D and Y are the same or different and each consists of a chain of from 1 to 6 moieties selected from the group consisting of --CH═ and --N═, said chain having alternating single and double bonds and terminating in a double or single bonded atom selected from the group consisting of O, S, and disubstituted nitrogen, wherein the sum of the number of said moieties and said terminal atoms in both D and Y is an even number, said chain, or part thereof including the terminal atom, being aliphatic or part of one or more alicyclic or aromatic rings, or a combination thereof, said chain or rings, or atoms having zero, one, or more substituents consisting of from 1 to 30 atoms other than hydrogen, which atoms are selected from the group consisting of carbon, oxygen, nitrogen, sulfur, halogen, arsenic, silicon, selenium and phosphorus wherein said substituents may be taken together to form one or more rings which may be alicyclic or aromatic, wherein at least one of D or Y is bonded to A; A is a group consisting of at least one member of a specific binding pair (sbp) consisting of ligand and its complementary receptor; n is a number from 1 up to the molecular weight of A divided by 100; and q is 1 or 2.
Claim 2
Dependent Claim
247 words
A conjugate of a squaraine dye covalently or non-covalently bonded to a member of a specific binding pair, said conjugate being of the formula ##STR13## wherein: D and Y are the same or different and each consists of a chain of from 1 to 6 moieties selected from the group consisting of --CH═ and --N═, said chain having alternating single and double bonds and terminating in a double or single bonded atom selected from the group consisting of O, S, and disubstituted nitrogen, wherein the sum of the number of said moieties and said terminal atoms in both D and Y is an even number, said chain, or part thereof including the terminal atom, being aliphatic or part of one or more alicyclic or aromatic rings, or a combination thereof, said chain or rings, or atoms having zero, one, or more substituents consisting of from 1 to 30 atoms other than hydrogen, which atoms are selected from the group consisting of carbon, oxygen, nitrogen, sulfur, halogen, arsenic, silicon, selenium and phosphorus wherein said substituents may be taken together to form one or more rings which may be alicyclic or aromatic, wherein at least one of D or Y is bonded to A; A is a group consisting of at least one member of a specific binding pair (sbp) consisting of ligand and its complementary receptor; n is a number from 1 up to the molecular weight of A divided by 100; and q is 1 or 2.
Claim 3
Dependent Claim
10 words
The conjugate of claim 1 wherein A is an antigen.
Claim 4
Dependent Claim
10 words
The conjugate of claim 1 wherein A is an antibody.
Claim 5
Dependent Claim
14 words
The conjugate of claim 1 wherein the sbp member is bound to a polymer.
Claim 6
Dependent Claim
212 words
A conjugate of a squaraine dye covalently or non-covalnetly bonded to a member of a specific binding pair, said conjugate being of the formula ##STR14## wherein: m is 1 to 4; k is 1 to 4; m+k is an even number; X is O, S, or N(R 1 ) 2 ; p is (-) when X is O or S and (+) when X is N(R 1 ) 2 ; R 1 is independently selected from a group consisting of hydrogen and substituents having from 1 to 30 atoms other than hydrogen selected from the group consisting of carbon, oxygen, nitrogen, sulfur, halogen, arsenic, silicon, selenium, and phosphorus, wherein R 1 may be taken together with one or more other R 1 s to form one or more rings, and wherein a hydrogen atom on at least one R 1 is replaced by a bond to A 1 ; n 1 is a number from 1 up to the molecular weight of A 1 divided by 100; A 1 is a group consisting of at least one member of a specific binding pair (sbp) consisting of ligand and its complementary receptor, wherein A 1 is bonded to R 1 , x, or x p ; and q 1 is 1 or 2.
Claim 7
Dependent Claim
212 words
A conjugate of a squaraine dye covalently or non-covalnetly bonded to a member of a specific binding pair, said conjugate being of the formula ##STR14## wherein: m is 1 to 4; k is 1 to 4; m+k is an even number; X is O, S, or N(R 1 ) 2 ; p is (-) when X is O or S and (+) when X is N(R 1 ) 2 ; R 1 is independently selected from a group consisting of hydrogen and substituents having from 1 to 30 atoms other than hydrogen selected from the group consisting of carbon, oxygen, nitrogen, sulfur, halogen, arsenic, silicon, selenium, and phosphorus, wherein R 1 may be taken together with one or more other R 1 s to form one or more rings, and wherein a hydrogen atom on at least one R 1 is replaced by a bond to A 1 ; n 1 is a number from 1 up to the molecular weight of A 1 divided by 100; A 1 is a group consisting of at least one member of a specific binding pair (sbp) consisting of ligand and its complementary receptor, wherein A 1 is bonded to R 1 , x, or x p ; and q 1 is 1 or 2.
Claim 8
Dependent Claim
38 words
The conjugate of claim 5 wherein m and k are each 4 and R 1 on carbon 1 and R 1 on carbon 4 are taken together to form an ethylene group as part of a benzene ring.
Claim 9
Dependent Claim
13 words
The conjugate of claim 6 wherein X is N(R 1 ) 2 .
Claim 10
Dependent Claim
9 words
The conjugate of claim 6 wherein X is oxygen.
Claim 11
Dependent Claim
48 words
The conjugate of claim 5 wherein m and k are each 2 and X is N(R 1 ) 2 and one R 1 of N(R 1 ) 2 is taken together with R 1 on the carbon bearing N(R 1 ) 2 to form a five membered ring.
Claim 12
Dependent Claim
16 words
The conjugate of claim 9 wherein a benzene ring is fused to the five membered ring.
Claim 13
Dependent Claim
11 words
The conjugate of claim 5 wherein A 1 is an antigen.
Claim 14
Dependent Claim
11 words
The conjugate of claim 5 wherein A 1 is an antibody.
Claim 15
Dependent Claim
14 words
The conjugate of claim 5 wherein the sbp member is bound to a polymer.
Claim 16
Dependent Claim
198 words
A composition of the formula ##STR15## wherein: q 2 is 1 or 2; A 2 is a group consisting of at lest one member of a specific binding pair (sbp) consisting of ligand and its complementary receptor; n 2 is a number from 1 up to the molecular weight of A divided by 100; D 1 is independently selected from the group consisting of ##STR16## Y 1 is independently selected from the group consisting of ##STR17## Z in D 1 and Z in Y 1 are indpendently selected from the group consisting of carbon, nitrogen, oxygen, sulfur, and selenium; R 2 is independently selected from group consisting of hydrogen and substituents having from 1 to 4 atoms other than hydrogen selected from the group consisting of carbon, oxygen, nitrogen, suflur, halogen, arsenic, silicon, selenium, and phosphorus, wherein R 2 may be taken together with one or more other R 2 s to form one or more rings, and wherein a hydrogen atom on at least one R 2 is replaced by a bond to A 2 ; s is 2 when Z is carbon, 1 when Z is nitrogen, and 0 when Z is oxygen, sulfur, or selenium.
Claim 17
Dependent Claim
198 words
A composition of the formula ##STR15## wherein: q 2 is 1 or 2; A 2 is a group consisting of at lest one member of a specific binding pair (sbp) consisting of ligand and its complementary receptor; n 2 is a number from 1 up to the molecular weight of A divided by 100; D 1 is independently selected from the group consisting of ##STR16## Y 1 is independently selected from the group consisting of ##STR17## Z in D 1 and Z in Y 1 are indpendently selected from the group consisting of carbon, nitrogen, oxygen, sulfur, and selenium; R 2 is independently selected from group consisting of hydrogen and substituents having from 1 to 4 atoms other than hydrogen selected from the group consisting of carbon, oxygen, nitrogen, suflur, halogen, arsenic, silicon, selenium, and phosphorus, wherein R 2 may be taken together with one or more other R 2 s to form one or more rings, and wherein a hydrogen atom on at least one R 2 is replaced by a bond to A 2 ; s is 2 when Z is carbon, 1 when Z is nitrogen, and 0 when Z is oxygen, sulfur, or selenium.
Claim 18
Dependent Claim
10 words
The conjugate of claim 14 wherein D 1 is ##STR18##
Claim 19
Dependent Claim
56 words
The conjugate of claim 14, wherein R 2 bound to nitrogen is independently selected from the group consisting of methyl and a group imparting water solubility and R 2 bound to carbon is hydrogen, wherein a hydrogen atom of at least one R 2 is replaced by a bond or linking group to A 2 .
Claim 20
Dependent Claim
23 words
The conjugate of claim 16 wherein the group imparting water solubility includes a moiety selected from the group consisting of sulfonates and phosphates.
Claim 21
Dependent Claim
10 words
The conjugate of claim 14 wherein D 1 is ##STR19##
Claim 22
Dependent Claim
12 words
The conjugate of claim 18 wherein Z(R 2 ) s is isopropylidene.
Claim 23
Dependent Claim
53 words
The conjugate of claim 19 wherein up to two of R 2 other than R 2 bound to Z are groups imparting water solubility and are otherwise hydrogen with the proviso that a hydrogen atom of at least one R 2 is replaced by a bond or linking group to A 2 .
Claim 24
Dependent Claim
10 words
The conjugate of claim 14 wherein Y 1 is ##STR20##
Claim 25
Dependent Claim
28 words
The conjugate of claim 21 wherein R 2 bound to nitrogen is selected independently from the group consisting of methyl and a linking group to A 2 .
Claim 26
Dependent Claim
10 words
The conjugate of claim 14 wherein Y 1 is ##STR21##
Claim 27
Dependent Claim
12 words
The conjugate of claim 23 wherein Z(R 2 ) s is isopropylidene.
Claim 28
Dependent Claim
53 words
The conjugate of claim 24 wherein up to two of R 2 other than R 2 bound to Z are groups imparting water solubility and are otherwise hydrogen with the proviso that a hydrogen atom of at least one R 2 is replaced by a bond or linking group to A 2 .
Claim 29
Dependent Claim
14 words
The conjugate of claim 14 wherein the sbp member is bound to a polymer.
Claim 30
Dependent Claim
11 words
The conjugate of claim 14 wherein A 2 is an antigen.
Claim 31
Dependent Claim
11 words
The conjugate of claim 14 wherein A 2 is an antibody.
Claim 32
Dependent Claim
125 words
A composition selected from the group consisting of ##STR22## wherein: A 3 is a member of a specific binding pair; n 3 is a number from 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 indpendently selected from the group consisting of substituents having 1-10 carbon atoms, 0-6 oxygen atoms, and 0-3 nitroen atoms, and may be taken together to form an alicyclic or aromatic ring; R 4 is indpendently selected from the group consisting of 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 A 3 .
Claim 33
Dependent Claim
125 words
A composition selected from the group consisting of ##STR22## wherein: A 3 is a member of a specific binding pair; n 3 is a number from 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 indpendently selected from the group consisting of substituents having 1-10 carbon atoms, 0-6 oxygen atoms, and 0-3 nitroen atoms, and may be taken together to form an alicyclic or aromatic ring; R 4 is indpendently selected from the group consisting of 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 A 3 .
Claim 34
Dependent Claim
19 words
The conjugate of claim 29 wherein at least one R 3 on nitrogen has a hydrogen replaced by L.
Claim 35
Dependent Claim
20 words
The conjugate of claim 30 wherein every remaining R 3 on nitrogen is lower alkyl of 1 to 5 carbons.
Claim 36
Dependent Claim
14 words
The conjugate of claim 29 wherein t is 1 and R 4 is hydroxyl.
Claim 37
Dependent Claim
19 words
The conjugate of claim 29 wherein L is selected from the group consisting of non-oxo-carbonyl, carbamoyl, thiocarbamoyl and amino.
Claim 38
Dependent Claim
14 words
The conjugate of claim 29 wherein the sbp member is bound to a polymer.
Claim 39
Dependent Claim
13 words
The conjugate of claim 29 wherein A 3 is an antigen or hapten.
Claim 40
Dependent Claim
10 words
The conjugate of claim 29 wherein A 3 is digoxin.
Claim 41
Dependent Claim
11 words
The conjugate of claim 29 wherein A 3 is an antibody.
Claim 42
Dependent Claim
27 words
A composition of the formula ##STR23## wherein: W is selected from the group consisting of antibody for digoxin and a digoxin analog capable of binding to anti-digoxin.
Claim 43
Dependent Claim
27 words
A composition of the formula ##STR23## wherein: W is selected from the group consisting of antibody for digoxin and a digoxin analog capable of binding to anti-digoxin.
Claim 44
Dependent Claim
53 words
In a conjugate 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, the improvement which comprises employing a squarate dye as the dye in said conjugate.
Claim 45
Dependent Claim
53 words
In a conjugate 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, the improvement which comprises employing a squarate dye as the dye in said conjugate.
Claim 46
Dependent Claim
15 words
The conjugate of claim 39 wherein said sbp member is an anitgen or an antibody.
Claim 47
Dependent Claim
12 words
The conjugate of claim 39 wherein said sbp member is an antibody.
Claim 48
Dependent Claim
17 words
The conjugate of claim 39 wherein said squarate dye has an absorption maximum greater than 600 nm.
Claim 49
Dependent Claim
14 words
The conjugate of claim 41 wherein the sbp member is bound to a polymer.
Claim 50
Dependent Claim
86 words
In an assay for an analyte in a sample suspected to containing said analyte, which analyte is a member of a specific binding pair (sbp member), said method involving the steps of (1) combining in an assay medium said sample and an sbp member conjugated to a dye and (2) determining the effect of said sample on the fluorescene of said dye, the effect thereof being related to the presence of analyte in said sample, the improvement which comprises employing as said dye a squarate dye.
Claim 51
Dependent Claim
86 words
In an assay for an analyte in a sample suspected to containing said analyte, which analyte is a member of a specific binding pair (sbp member), said method involving the steps of (1) combining in an assay medium said sample and an sbp member conjugated to a dye and (2) determining the effect of said sample on the fluorescene of said dye, the effect thereof being related to the presence of analyte in said sample, the improvement which comprises employing as said dye a squarate dye.
Claim 52
Dependent Claim
16 words
The assay of claim 44 wherein said conjugated sbp member is an antigen or a hapten.
Claim 53
Dependent Claim
13 words
The assay of claim 44 wherein said conjuated sbp member is an antibody.
Claim 54
Dependent Claim
17 words
The assay of claim 44 wherein said squarate dye has an absorption maximum greater than 600 nm.
Claim 55
Dependent Claim
103 words
A method for detecting the presence or amount of analyte in a sample suspected of containing said analyte, wherein said analyte is a member of a specific binding pair consisting of ligand and its complementary receptor, which method comprises combining in an assay medium the sample, the sbp member containing conjugate of claim 1, and a third sbp member, wherein two of the three spb members are complementary to the remaining member, and determining the effect of said sample on the fluorescence of the conjugate of claim 1, the effect thereof being related to the presence or amount of analyte in said sample.
Claim 56
Dependent Claim
39 words
The method of claim 48 wherein said third sbp member is conjugated to a compound capable of quenching the fluorescence of the compound of claim 1 when said third sbp member is bound to said conjugate of claim 1.
Claim 57
Dependent Claim
16 words
The method of claim 48 wherein said third sbp member is an antibody for the analyte.
Claim 58
Dependent Claim
19 words
The method of claim 48 wherein said third sbp member is bound to a particle or to a surface.
Claim 59
Dependent Claim
26 words
The method of claim 48 wherein said third sbp member and the sbp member of the conjugate of claim 1 are both antibodies for said analyte.
Claim 60
Dependent Claim
104 words
A method for detecting the presence or amount of analyte in a sample suspected of containing said analyte, said analyte being a member of a specific binding pair (sbp) consisting of ligand and its complementary receptor, which method comprises combining in an assay medium the sample, the sbp member containing conjugate of claim 5, and a third sbp member where two of the three spb members are complementary to the remaining sbp member, and determining the effect of said sample on the fluorescence of the conjugate of claim 5, the effect thereof being related to the presence or amount of analyte in said sample.
Claim 61
Dependent Claim
39 words
The method fo claim 53 wherein said third sbp member is conjugated to a compound capable of quenching the fluorescence of the compound of claim 7 when said third sbp member is bound to the conjugate of claim 5.
Claim 62
Dependent Claim
16 words
The method of claim 53 wherein said third sbp member is an antibody for the analyte.
Claim 63
Dependent Claim
18 words
The method of claim 53 wherein said third sbp member is conjugated to a particle or a surface.
Claim 64
Dependent Claim
26 words
The method of claim 53 wherein said third sbp member and the sbp member of the conjugate of claim 5 are both antibodies for said analyte.
Claim 65
Dependent Claim
104 words
A method for detecting the presence or amount of analyte in a sample suspected of containing said analyte, said analyte being a member of a specific binding pair (sbp) consisting of ligand and its complementary receptor, which method comprises combining in an assay medium the sample, the sbp member containing conjugate of claim 14, and a third sbp member where two of the three sbp members are complementary to the remaining sbp member, and determining the effect of said sample on the fluorescence of the conjugate of claim 14, the effect thereof being related to the presence or amount of analyte in said sample.
Claim 66
Dependent Claim
39 words
The method of claim 58 wherein said third sbp member is conjugated to a compound capable of quenching the fluorescence of the compound of claim 14 when said third sbp member is bound to the compound of claim 14.
Claim 67
Dependent Claim
16 words
The method of claim 58 wherein said third sbp member is an antibody for the analyte.
Claim 68
Dependent Claim
18 words
The method of claim 58 wherein said their sbp member is bound to a particle or a surface.
Claim 69
Dependent Claim
26 words
The method of claim 58 wherein said third sbp member and the sbp member of the conjugate of claim 14 are both antibodies for said analyte.
Claim 70
Dependent Claim
105 words
A method for detecting 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) consisting of ligand and its complementary receptor, which method comprises combining in an assay medium the sample, the sbp member containing conjugate of claim 29, and a third sbp member wherein two of the three sbp members are complementary to the remaining sbp member, and determining the effect of said sample on the fluorescence of the conjugate of claim 29, the effect thereof being related to the presence or amount of analyte in said sample.
Claim 71
Dependent Claim
39 words
The method of claim 63 wherein said third sbp member is conjugated to a compound capable of quenching the fluorescence of the conjugate of claim 29 when said third sbp member is bound to the conjugate of claim 29.
Claim 72
Dependent Claim
16 words
The method of claim 63 wherein the said third member is an antibody for the analyte.
Claim 73
Dependent Claim
18 words
The method of claim 63 wherein said third sbp member is bound to a particle or a surface.
Claim 74
Dependent Claim
26 words
The method of claim 63 wherein said third sbp member and the sbp member of the compound of claim 29 are both antibodies for said analyte.
Claim 75
Dependent Claim
63 words
A method for determining digoxin in a sample suspected of containing digoxin, which method comprises combining in an assay medium the sample, the conjugate of claim 38 wherein W is a digoxin analog, and an antibody for digoxin, and determining the effect of the sample on the fluorescence of said medium, the effect thereof being related to the presence digoxin in said sample.
Claim 76
Dependent Claim
14 words
The method of claim 68 wherein the quencher is gallocyanine conjugated to said antibody.
Claim 77
Dependent Claim
73 words
A method for determining digoxin in a sample suspected of containing digoxin, which method comprises combining in an assay medium the sample, the conjugate of claim 38 wherein W is antibody for digoxin, and a second conjugate comprising a digoxin analog conjugated to particles, and determining the effect of said sample on the flourescence of said medium or said particles, the effect thereof being related to the presence of digoxin in said sample.
Claim 78
Dependent Claim
92 words
In an assay for a material of interest in a sample suspected of containing the material of interest wherein said assay comprises the steps of (1) combining in an assay medium said sample with a fluorescent compound to generate a signal in relation to the presence or amount of the material of interest in said sample and (2) irradiating said fluorescent compound with an energy source for excitation of said fluorescent compound, the improvement which comprises employing a squarate dye as the flourescent compound and a helium/neon laser as the energy source.
Claim 79
Dependent Claim
92 words
In an assay for a material of interest in a sample suspected of containing the material of interest wherein said assay comprises the steps of (1) combining in an assay medium said sample with a fluorescent compound to generate a signal in relation to the presence or amount of the material of interest in said sample and (2) irradiating said fluorescent compound with an energy source for excitation of said fluorescent compound, the improvement which comprises employing a squarate dye as the flourescent compound and a helium/neon laser as the energy source.
Claim 80
Dependent Claim
12 words
The assay of claim 71 wherein said squarate dye is water compatible.
Claim 81
Dependent Claim
138 words
In a fluorescent assay employing as a reagent a fluorescent compound conjugated to a member of a specific binding pair wherein said pair consists of ligand and its complementary receptor and said assay is for the determination of an analyte which is also a member of a specific binding pair, the binding of said conjugate to said analyte, or a specific binding pair (sbp) member complex of said analyte, being indicative of the presence of said analyte, said assay comprising the steps of (1) combining a sample suspected of containing said analyte with said reagent and (2) determining the binding of said reagent to said analyte or an sbp member complex of said analyte, the improvement which comprises employing in said assay a reagent that is a squarate dye conjugated to a member of a specific binding pair.
Claim 82
Dependent Claim
138 words
In a fluorescent assay employing as a reagent a fluorescent compound conjugated to a member of a specific binding pair wherein said pair consists of ligand and its complementary receptor and said assay is for the determination of an analyte which is also a member of a specific binding pair, the binding of said conjugate to said analyte, or a specific binding pair (sbp) member complex of said analyte, being indicative of the presence of said analyte, said assay comprising the steps of (1) combining a sample suspected of containing said analyte with said reagent and (2) determining the binding of said reagent to said analyte or an sbp member complex of said analyte, the improvement which comprises employing in said assay a reagent that is a squarate dye conjugated to a member of a specific binding pair.
Claim 83
Dependent Claim
146 words
In a method for detecting the presence of a determinant site or a receptor by employing a fluorescent reagent having a fluorescent compound bound to a member of a specific binding pair, wherein the binding of said fluorescent reagent to said determinant site or said receptor, or a specific binding pair (sbp) member complex of said determinant site or said receptor, is determined as indicative of the presence of said determinant site or said receptor, said assay comprising the steps of (1) combining said determinant site or said receptor with said fluorescent reagent and (2) determining the extent of binding of said reagent to said determinant site or said receptor, or an sbp member complex of said determinant site or said receptor, the improvement which comprises employing a fluorescent reagent that is a conjugate of a squarate dye and a member of a specific binding pair.
Claim 84
Dependent Claim
146 words
In a method for detecting the presence of a determinant site or a receptor by employing a fluorescent reagent having a fluorescent compound bound to a member of a specific binding pair, wherein the binding of said fluorescent reagent to said determinant site or said receptor, or a specific binding pair (sbp) member complex of said determinant site or said receptor, is determined as indicative of the presence of said determinant site or said receptor, said assay comprising the steps of (1) combining said determinant site or said receptor with said fluorescent reagent and (2) determining the extent of binding of said reagent to said determinant site or said receptor, or an sbp member complex of said determinant site or said receptor, the improvement which comprises employing a fluorescent reagent that is a conjugate of a squarate dye and a member of a specific binding pair.
Claim 85
Dependent Claim
18 words
The method of claim 74 wherein said determinant site or said receptor is on the surface a cell.
Claim 86
Dependent Claim
78 words
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 87
Dependent Claim
78 words
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 88
Dependent Claim
17 words
The kit of claim 76 wherein said squarate dye has an absorption maximum greater than 600 nm.
Claim 89
Dependent Claim
21 words
The kit of claim 76 wherein said sbp members are selected from the group consisting of antigens or haptens and antibodies.
Claim 90
Dependent Claim
24 words
The kit of claim 76 wherein the sbp members in said first compound and in said second compound are both antibodies for said analyte.
Claim 91
Dependent Claim
14 words
The kit of claim 76 wherein said second compound is antibody for the analyte.
Claim 92
Dependent Claim
10 words
The kit of claim 76 wherein the analyte is digoxin.
Claim 93
Dependent Claim
31 words
The kit of claim 76 wherein the sbp member in said first compound is an antibody for a hapten and said second compound comprises a complementary hapten conjugated to a particle.
References Cited (Backward Citations)
US4166105A
Cited
EP0176252A2
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1992-02-21 | 1992-08-22 | 1993-02-21 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-02-21 | 1996-08-22 | 1997-02-21 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-02-21 | 2000-08-22 | 2001-02-21 | $7700.00 | Window Closed / Lapsed |