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EP0322926A2
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Assays utilizing improved chemiluminescent esters, thioesters and amides
Assignee: Nichols Institute Diagnostics
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Filing: 1988-12-30
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Granted: —
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Expires: 2008-12-30
Abstract
Abstract In accordance with the present invention, specific binding assays are disclosed which utilize an chemiluminescent compound, i.e., moiety, which has increased stability in aqueous solution. The chemiluminescent moiety is an ester, thioester or amide in which the ester, thioester or amide linkage is between (1) a heterocyclic ring or ring system containing a carbon atom to which the linkage is attached, wherein the heteroatom within the heterocyclic ring or ring system is in an oxidation state which renders such carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiluminescence, and (2) an aryl ring or ring system. The aryl ring or ring system contains at least one substituted six-member ring. The substituted six-member ring has two or more substituent groups, where at least two of said two or more substituent groups sterically hinder hydrolysis of said linkage. One or more of the substituent groups which sterically hinder hydrolysis of said linkage may be an electron withdrawing group. The substituted six-member ring may have one or more additional substituent groups in addition to the substituent groups which sterically hinder hydrolysis of the linkage. Such additional substituent groups may also be an electron withdrawing group. The carbon atoms in the heterocyclic ring or ring system, to which the linkage is attached, may also have a secondary substituent of the formula R n X-, where X is selected from the group consisting of O, N, S and C, where R is any group, and where n is a number such that X has proper valency. Other chemiluminescent moieties are disclosed which are characterized by a heterocyclic ring or ring system and a secondary substituent of the formula RnX-, with the ester, thioester or amide linkage being between the heterocyclic ring or ring system and a leaving group. The disclosed chemiluminescent moieties can also include substituents at peri positions within the heterocyclic ring or ring system. Also in accordance with the present invention, compositions including and assay kits incorporating such chemiluminescent moieties are disclosed.
Inventors
- 👤 Frank Mccapra
- 👤 Iraj Beheshti
- 👤 Russell C. Hart
- 👤 Harlen Koelling
- 👤 Ashokkumar Patel
- 👤 Kastooriranganathan Ramakrishnan
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-27 18:35
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; C; C07; C07D; C07D215/00; C07D215/02; C07D215/16; C07D215/48; C07D215/50; C07D215/52; C07D219/00; C07D219/04; C07D219/06; C07D221/00; C07D221/02; C07D221/04; C07D221/06; C07D221/10; C07D221/12; C07D403/00; C07D403/02; C07D403/12; C07J; C07J43/00; C07J43/003; G01N33/531; G01N33/532; G01N33/533; G01N33/536; G01N33/542; G01N33/58; G01N33/582
Claims Analysis
Total Claims: 59
Claim 1
Independent Claim
291 words
In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent conjugate comprising a chemiluminescent moiety attached to a specific binding material and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products containing said chemiluminescent conjugate, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products containing said chemiluminescent conjugate; and measuring the chemiluminescence of (i) said one or more specific binding reaction products containing said chemiluminescent conjugate or (ii) the chemiluminescent conjugate not contained in said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between; a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiluminescence; and an aryl ring or ring system, said aryl ring or ring system containing at least one substituted six-member ring, said substituted six-member ring having two or more substituent groups, where at least two of said two or more substituent groups sterically hinder the hydrolysis of said linkage; and wherein said carbon atoms to which said linkage is attached has a secondary substituent of the formula R n X, where X is selected from the group consisting of O, N, S and C, where R is any group, and where n is a number such that X has proper valency.
Claim 2
Dependent Claim
291 words
In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent conjugate comprising a chemiluminescent moiety attached to a specific binding material and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products containing said chemiluminescent conjugate, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products containing said chemiluminescent conjugate; and measuring the chemiluminescence of (i) said one or more specific binding reaction products containing said chemiluminescent conjugate or (ii) the chemiluminescent conjugate not contained in said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between; a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiluminescence; and an aryl ring or ring system, said aryl ring or ring system containing at least one substituted six-member ring, said substituted six-member ring having two or more substituent groups, where at least two of said two or more substituent groups sterically hinder the hydrolysis of said linkage; and wherein said carbon atoms to which said linkage is attached has a secondary substituent of the formula R n X, where X is selected from the group consisting of O, N, S and C, where R is any group, and where n is a number such that X has proper valency.
Claim 3
Dependent Claim
285 words
In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent moiety and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products not containing said chemiluminescent moiety, and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said one or more specific binding reaction products, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products; and measuring the chemiluminescence of said chemiluminescent moiety caused by said formation of said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between:: a heterocyclic ring or ring system containing a carbon atom in which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiluminescence; and an aryl ring or ring system, said aryl ring or ring system containing at least one substituted six-member ring, said substituted six-member ring having two or more substituent groups, where at least two of said two or more substituent groups sterically hinder the hydrolysis of said linkage; and wherein said carbon atom to which said linkage is attached has a secondary substituent of the formula R n X-, where X is selected from the group consisting of O, N, S and C, where R is any group, and where n is a number such that X has proper valency.
Claim 4
Dependent Claim
285 words
In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent moiety and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products not containing said chemiluminescent moiety, and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said one or more specific binding reaction products, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products; and measuring the chemiluminescence of said chemiluminescent moiety caused by said formation of said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between:: a heterocyclic ring or ring system containing a carbon atom in which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiluminescence; and an aryl ring or ring system, said aryl ring or ring system containing at least one substituted six-member ring, said substituted six-member ring having two or more substituent groups, where at least two of said two or more substituent groups sterically hinder the hydrolysis of said linkage; and wherein said carbon atom to which said linkage is attached has a secondary substituent of the formula R n X-, where X is selected from the group consisting of O, N, S and C, where R is any group, and where n is a number such that X has proper valency.
Claim 5
Dependent Claim
40 words
The specific binding assay of claim 2 wherein said assay further utilizes a reactant capable of binding with said analyte to form an analyte-reactant complex and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said analyte-reactant complex.
Claim 6
Dependent Claim
51 words
The specific binding assay of any of claims 1 to 3 wherein said secondary substituent is selected from the group consisting of - CN, - OR, · NR₂, · NR₃⁺, · SR, - SR₂⁺, -S₂R,- NRCO₂R, · NHNR₂, · NRNR₂, · NHOR, · ONR₂, · CR(CN)₂, · CR(COR)₂, · CR₂NO₂, -C≡COR,
Claim 7
Dependent Claim
31 words
The specific binding assay of claim 4 wherein each of said substituent groups which sterically hinder hydrolysis of said linkage is selected from the group consisting of -CH₃, -OCH₃ and t-propyl.
Claim 8
Dependent Claim
27 words
The specific binding assay of claim 4 wherein at least one of said substituent groups which sterically hinder hydrolysis of said linkage is an electron withdrawing group.
Claim 9
Dependent Claim
45 words
The specific binding assay of claim 4 wherein said substituted six-member ring has at least one additional substituent group, in addition to said substituent groups which sterically hinder hydrolysis of said linkage, and said at least one additional substituent group is an electron withdrawing group.
Claim 10
Dependent Claim
30 words
The specific binding assay of claim 7 wherein said at least one additional substituent group is selected from the the group consisting of -NO₂, -SO₂Cl, -Br,-N(CH₃)₃⁺ and -H, preferably -H.
Claim 11
Dependent Claim
27 words
The specific binding assay of claim 4 wherein said aryl ring or ring system is selected from the group consisting of phenyl, naphthalene and anthracene, preferably phenyl.
Claim 12
Dependent Claim
35 words
The specific binding assay of claim 9 wherein the sum of the sigma p values for all substituent groups on said phenyl which are electron withdrawing groups is less than or equal to about 1.0.
Claim 13
Dependent Claim
22 words
The specific binding assay of any of claims 1 to 10 wherein said chemiluminescent moiety is selected from (2,6-dimethoxy-3-chlorosulfonyl)-phenyl-N-methyl-acridan-9-ethoxy-9-carboxylate, (2,6-dimethoxy-3-chloro-sulfonyl)phenyl-N-methyl-acridan-9-methoxy-9-carboxylate, and (2,6-dimethyl-S-chlorosulfonyl)phenyl-N-methyl-acridan-9-methoxy-9-carboxylate.
Claim 14
Dependent Claim
247 words
In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent conjugate comprising a chemiluminescent moiety attached to a specific binding material and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products containing said chemiluminescent conjugate, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products containing said chemiluminescent conjugate; and measuring the chemiluminescence of (i) said one or more specific binding reaction products containing said chemiluminescent conjugate or (ii) the chemiluminescent conjugate not contained in said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between: a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiluminescence; and a leaving group; and wherein said carbon atom to which said linkage is attached has a secondary substituent of the formula R n X-, where X is selected from the group consisting of O, N, S and C, where R is any group, and where n is a number such that X has proper valency.
Claim 15
Dependent Claim
247 words
In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent conjugate comprising a chemiluminescent moiety attached to a specific binding material and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products containing said chemiluminescent conjugate, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products containing said chemiluminescent conjugate; and measuring the chemiluminescence of (i) said one or more specific binding reaction products containing said chemiluminescent conjugate or (ii) the chemiluminescent conjugate not contained in said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between: a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiluminescence; and a leaving group; and wherein said carbon atom to which said linkage is attached has a secondary substituent of the formula R n X-, where X is selected from the group consisting of O, N, S and C, where R is any group, and where n is a number such that X has proper valency.
Claim 16
Dependent Claim
75 words
The specific binding assay of any of claims 1 to 12 wherein said specific binding material is capable of specifically binding with the analyte and said one or more specific binding reaction products is an analyte-chemiluminescent conjugate complex, said assay further comprising: allowing under suitable conditions substantial formation of said analyte-chemiluminescent conjugate complex; and measuring the chemiluminescence of (i) said analyte-chemiluminescent conjugate complex or (ii) the chemiluminescent conjugate not contained in said analyte-chemiluminescent conjugate complex.
Claim 17
Dependent Claim
91 words
The specific binding assay of any of claims 1 to 13 wherein said assay further utilizes a reactant which is capable of specifically binding (i) with said analyte to form an analyte-reactant complex and (ii) with said specific binding material to form a chemiluminescent conjugate-reactant complex, and wherein said one or more specific binding reactions products is said chemiluminescent conjugate-reactant complex, said assay further comprising: allowing under suitable conditions substantial formation of said analyte-reactant complex and said chemiluminescent conjugate-reactant complex; and measuring the chemiluminescence of (i) said chemiluminescent conjugate-chemiluminescent conjugate-reactant complex.
Claim 18
Dependent Claim
97 words
The specific binding assay of any of the claims 1 to 14 wherein said specific binding material is capable of specifically binding with said analyte and said assay further utilizes a reactant capable of specifically binding with said analyte to form a reactant-analyte-chemiluminescent conjugate complex, and wherein said one or more specific binding reaction products is said reactant-analyte-chemiluminescent conjugate complex, said assay further comprising: allowing under suitable conditions substantial formation of said reactant-analyte-chemiluminescent conjugate complex; and measuring the chemiluminescence of (i) said reactant-analyte-chemiluminescent conjugate complex or (ii) the chemiluminescent conjugate not contained in said reactant-analyte-chemiluminescent conjugate complex.
Claim 19
Dependent Claim
242 words
In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent moiety and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products not containing said chemiluminescent moiety, and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said one or more specific binding reaction products, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products; and measuring the chemiluminescence of said chemiluminescent moiety caused by said formation of said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between:: a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiluminescence; and a leaving group: and wherein said carbon atom to which said linkage is attached has a secondary substituent of the formula R n X-, where X is selected from the group consisting of O, N, S and C, where R is any group, and where n is a number such as that X has proper valency.
Claim 20
Dependent Claim
242 words
In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent moiety and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products not containing said chemiluminescent moiety, and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said one or more specific binding reaction products, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products; and measuring the chemiluminescence of said chemiluminescent moiety caused by said formation of said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between:: a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiluminescence; and a leaving group: and wherein said carbon atom to which said linkage is attached has a secondary substituent of the formula R n X-, where X is selected from the group consisting of O, N, S and C, where R is any group, and where n is a number such as that X has proper valency.
Claim 21
Dependent Claim
40 words
The specific binding assay of claim 16 wherein said assay further utilizes a reactant capable of binding with said analyte to form an analyte-reactant complex and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said analyte-reactant complex.
Claim 22
Dependent Claim
51 words
The specific binding assay of claim 12 or 16 wherein said secondary substituent is selected from the group consisting of - CN, - OR, - NR₂, · NR₃⁺, · SR, - SR₂⁺, - S₂R, - NRCO₂R, - NHNR₂, - NRNR₂, · NHOR, · ONR₂, · CR(CN)₂, · CR(COR)₂ · CR₂NO₂, -CECOR,
Claim 23
Dependent Claim
18 words
The specific binding assay of claim 18 wherein said leaving group is an aryl ring or ring system.
Claim 24
Dependent Claim
27 words
The specific binding assay of claim 19 wherein said aryl ring or ring system is selected from the group consisting of phenyl, naphthalene and anthracene, preferably phenyl.
Claim 25
Dependent Claim
13 words
The specific binding assay of claim 20 wherein said phenyl ring is unsubstituted.
Claim 26
Dependent Claim
99 words
The specific binding assay of claim 18 wherein said chemiluminescent moiety is an amide and said leaving group is a group of the formula NR′(SO₂-R˝-Y), where R′ and R˝ are selected from the group consisting of alkyl, alkylene, aryl, optionally substituted alkyl, optionally substituted alkylene, optionally substituted aryl, alkyloxy, aryloxy, halo, optionally protected amino, substituted aminohydroxy, protected hydroxy, oxo, thio, imino, optionally substituted mercapto, a heterocyclic ring, and a heteroalkyl group, and where Y is selected from the group consisting of a hydrogen, carboxyl, carbonyl halide, sulfonyl halide, carboalkoxy, carboxamido, carboaryloxy, cyano, carboximido, isocyanato, isothiocyanate, sulfo, N-succinimidylcarboxy and N-maleimide.
Claim 27
Dependent Claim
51 words
The specific binding assay of claim 18 wherein said chemiluminescent moiety is a thioester and said leaving group is a group of the formula V-R‴ where V is an aliphatic or aromatic group and where R‴ is a group which is useful for attaching said moiety to a specific binding partner.
Claim 28
Dependent Claim
17 words
The specific binding assay of claim 23 wherein said leaving group is a sulfonamido or sulfocarbonyl group.
Claim 29
Dependent Claim
68 words
The specific binding assay of any of the claims 4 to 18 wherein said heterocyclic ring or ring system is a reduced form of a member selected from the group consisting of acridinium, benz[a]acridinium, benz[b]acridinium, benz[c]acridinium, a 1,2,4-triazole cation, an isooxazole cation, an isothioazole cation, a 1,2-azole cation, an imidazole cation, a benzimidazole cation, quinolinium, isoquinolinium, quinolizinium, a cyclic substituted quinolinium, pyridinium pyrimidinium, pyridazinium, pyrazinium, phenanthridinium, and quinoxalinium.
Claim 30
Dependent Claim
23 words
The specific binding assay of claim 25 wherein said heterocyclic ring or ring system is a reduced form of acridinium, quinolinium or phenanthridinium.
Claim 31
Dependent Claim
21 words
The specific binding assay of claim 26 wherein said secondary substituent is selected from the group consisting of ·OCH₃ and ·OCH₂CH₃.
Claim 32
Dependent Claim
31 words
The specific binding assay of claim 25 wherein said heterocyclic ring or ring system has at least one peri substituent, peri to said carbon atom to which said linkage is attached.
Claim 33
Dependent Claim
21 words
The specific binding assay of claim 28 wherein said least one peri substituent is an alkyl or aryl group, preferably -CH₃.
Claim 34
Dependent Claim
13 words
The specific binding assay of claim 29 wherein said chemiluminescent moiety is phenyl-N-methyl-1,3-dimethyl-acridinium-9-methoxy-9-carboxylate.
Claim 35
Dependent Claim
238 words
In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent conjugate comprising a chemiluminescent moiety attached to a specific binding material and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products containing said chemiluminescent conjugate, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products containing said chemiluminescent conjugate; and measuring the chemiluminescence of (i) said one or more specific binding reaction products containing said chemiluminescent conjugate or (ii) the chemiluminescent conjugate not contained in said one or more specific binding reaction products; wherein the improvement comprises as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between: a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiluminescence; and an aryl ring or ring system, said aryl ring or ring system containing at least one substituted six-member ring, said substituted six-member ring having two or more substituent groups, where at least two of said two or more substituent groups sterically hinder the hydrolysis of said linkage.
Claim 36
Dependent Claim
238 words
In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent conjugate comprising a chemiluminescent moiety attached to a specific binding material and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products containing said chemiluminescent conjugate, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products containing said chemiluminescent conjugate; and measuring the chemiluminescence of (i) said one or more specific binding reaction products containing said chemiluminescent conjugate or (ii) the chemiluminescent conjugate not contained in said one or more specific binding reaction products; wherein the improvement comprises as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between: a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiluminescence; and an aryl ring or ring system, said aryl ring or ring system containing at least one substituted six-member ring, said substituted six-member ring having two or more substituent groups, where at least two of said two or more substituent groups sterically hinder the hydrolysis of said linkage.
Claim 37
Dependent Claim
71 words
The specific binding assay of claim 31 wherein said specific binding material is capable of specifically binding with the analyte and said one or more specific binding reaction products is an analyte-chemiluminescent conjugate complex, said assay further comprising: allowing under suitable conditions substantial formation of said analyte-chemiluminescent conjugate complex; and measuring the chemiluminescence of (i) said analyte-chemiluminescent conjugate complex or (ii) the chemiluminescent conjugate not contained in said analyte-chemiluminescent conjugate complex.
Claim 38
Dependent Claim
99 words
The specific binding assay of claim 31 wherein said assay further utilizes a reactant which is capable of specifically binding (i) with said analyte to form an analyte-reactant complex and (ii) with said specific binding material to form a chemiluminescent conjugate-reactant complex, and wherein said one or more specific binding reaction products is said chemiluminescent conjugate-reactant complex, said assay further comprising: allowing under suitable conditions a substantial formation of said analyte-reactant complex and said chemiluminescent conjugate-reactant complex; and measuring the chemiluminescence of (i) said chemiluminescent conjuate-reactant complex or (ii) the chemiluminescent conjugate not contained in said chemiluminescent conjugate-reactant complex.
Claim 39
Dependent Claim
92 words
The specific binding assay of claim 31 wherein said specific binding material is capable of specifically binding with said analyte and said assay further utilizes a reactant capable of specifically binding with said analyte to form a reactant-analyte-chemiluminescent conjugate complex, and wherein said one or more specific binding reaction products is said reactant-analyte-chemiluminescent conjugate complex, said assay further comprising: allowing under suitable conditions substantial formation of said reactant-analyte-chemiluminescent conjugate complex; and measuring the chemiluminescence of (i) said reactant-analyte-chemiluminescent conjugate complex or (ii) the chemiluminescent conjugate not contained in said reactant-analyte-chemiluminescent conjugate complex.
Claim 40
Dependent Claim
233 words
In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent moiety and (ii) the presence of analytic in said sample is proportional to the formation of one or more specific binding reaction products not containing said chemiluminescent moiety, and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said one or more specific binding reaction products, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products; and measuring the chemiluminescence of said chemiluminescent moiety caused by said formation of said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between:: a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiluminescence; and an aryl ring or ring system, said aryl ring or ring system containing at least one substituted six-member ring, said substituted six-member ring having two or more substituent groups, where at least two of said two or more substituent groups sterically hinder the hydrolysis of said linkage.
Claim 41
Dependent Claim
233 words
In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent moiety and (ii) the presence of analytic in said sample is proportional to the formation of one or more specific binding reaction products not containing said chemiluminescent moiety, and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said one or more specific binding reaction products, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products; and measuring the chemiluminescence of said chemiluminescent moiety caused by said formation of said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between:: a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiluminescence; and an aryl ring or ring system, said aryl ring or ring system containing at least one substituted six-member ring, said substituted six-member ring having two or more substituent groups, where at least two of said two or more substituent groups sterically hinder the hydrolysis of said linkage.
Claim 42
Dependent Claim
40 words
The specific binding assay of claim 35 wherein said assay further utilizes a reactant capable of binding with said analyte to form an analyte-reactant complex and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said analyte-reactant complex.
Claim 43
Dependent Claim
33 words
The specific binding assay of claim 31 or 35 wherein each of said substituent groups which sterically hinder hydrolysis of said linkage is selected from the group consisting of -CH₃, ·OCH₃ and i-propyl.
Claim 44
Dependent Claim
29 words
The specific binding assay of claim 31 to 35 wherein at least one of said substituent groups which sterically hinder hydrolysis of said linkage is an electron withdrawing group.
Claim 45
Dependent Claim
47 words
The specific binding assay of claim 31 to 35 wherein said substituted six-member ring has at least one additional substituent group, in addition to said substituent groups which sterically hinder hydrolysis of said linkage, and said at least one additional substituent group is an electron withdrawing group.
Claim 46
Dependent Claim
28 words
The specific binding assay of claim 39 wherein said at least one additional substituent group is selected from the group consisting of -NO₂, -SO₂Cl. -Br, -N(CH₃)₃+ and -H.
Claim 47
Dependent Claim
14 words
The specific binding assay of claim 40 wherein said electron withdrawing group is -H.
Claim 48
Dependent Claim
29 words
The specific binding assay of claim 31 or 35 wherein said aryl ring or ring system is selected from the group consisting of phenyl, naphthalene and anthracene, preferably phenyl.
Claim 49
Dependent Claim
35 words
The specific binding assay of claim 42 wherein the sum of the sigma p values for all substituent groups on said phenyl which are electron withdrawing groups is less than or equal to about 1.0.
Claim 50
Dependent Claim
19 words
The specific binding assay of claim 42 or 43 wherein said chemiluminescent moiety has one of the following formulae:
Claim 51
Dependent Claim
59 words
The specific binding assay of claim 31 or 35 wherein said heterocyclic ring or ring system is selected from the group consisting of acridinium, benz[a]acridinium, benz[b]acridinium, benz[c]acrydinium, a 1,2,4-triazole cation, an isooxazole cation, an isothioazole cation, a 1,2-azole cation, an imidazole cation, a benzimidazole cation, quinolinium, isoquinolinium, quinolizinium, a cyclic substituted quinolinium, pyridinium, pyrimidinium, pyridazinium, pyrazinium, phenanthridinium, and quinoxalinium.
Claim 52
Dependent Claim
27 words
The specific binding assay of claim 45 wherein said heterocyclic ring or ring system is selected from the group consisting of acridinium, quinolinium and phenanthridinium, preferably acridinium.
Claim 53
Dependent Claim
65 words
The specific binding assay of claim 31 or 35 wherein said heterocyclic ring or ring system is a reduced form of a member selected from the group consisting of acridinium, benz[a]acridinium, benz[b]acridinium, benz[c]acridinium, a 1,2,4-triazole cation, an isooxazole cation, an isothioazole cation, a 1,2-azole cation, an imidazole cation, a benzimidazole cation, quinolinium, isoquinolinium, quinolizinium, a cyclic substituted quinolinium, pyridinium, pyrimidinium, pyridazinium, pyrazinium, phenanthridinium, and quinoxalinium.
Claim 54
Dependent Claim
23 words
The specific binding assay of claim 47 wherein said heterocyclic ring or ring system is a reduced form of acridinium, quinolinium or phenanthridinium.
Claim 55
Dependent Claim
41 words
The specific binding assay of claim 47 wherein said carbon atom has a substituent selected from the group consisting of -H, -CN, a halogen, -OR, -NR₂, -NR₃⁺, -SR, -SR₂⁺, and -S₂R, wherein R= alkyl, aryl, an amino acid or a sugar.
Claim 56
Dependent Claim
13 words
The specific binding assay of claim 48 wherein said conjugate has the formula:
Claim 57
Dependent Claim
17 words
A composition comprising the chemiluminescent moiety of any of claims 1 to 11 or 31 to 50.
Claim 58
Dependent Claim
20 words
A composition comprising the chemiluminescent moiety of any of claims 1 to 50 and further comprising a specific binding partner.
Claim 59
Dependent Claim
23 words
As an article of manufacture, a specific binding assay kit comprising a vial containing at least the composition of claim 51 to 52.
References Cited (Backward Citations)
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