US 5,512,451 AGranted

Enhancement of chemiluminescent reactions

AssigneeBritish Technology Group Limited

InventorLarry J. Kricka

Life of this patent

Estimating the term…

Abstract

In enhanced chemiluminescent (ECL) reactions of a fused aromatic diacyl cyclic hydrazide such as luminol, a peroxidase enzyme catalyst, an oxidant such as hydrogen peroxide and an enhancer, it has been found advantageous to use a combination of an organoboron enhancer such as 4-biphenylboronic acid with a non boron-containing enhancer, especially a phenolic or aromatic amine enhancer, particularly 4-iodophenol. ECL reactions are useful in diagnostic assay.

Claims30

Dependent claims

A method of enhancing a chemiluminescent reaction between a fused aromatic diacyl cyclic hydrazide (FADCH), a peroxidase enzyme and an oxidant, said enhancing increasing the signal to background ratio thereof, "signal" being light output in the presence of the peroxidase enzyme, "background" in its absence, which method comprises the step of carrying out said chemiluminescent reaction in the presence of an organoboron enhancer together with a non-boron-containing organic enhancer, said organoboron cnhancer being selected from the group consisting of the compounds of formula (I) ##STR10## in which the R groups are the same and each is selected from the group consisting of hydrogen, n-butyl, 4'-chlorophenyl and 3',5'-dicholorophenyl;

or the R's together are 0,0-propylene thereby forming with the boron atom, a cyclic ether,
W is selected from the group consisting of hydrogen, methyl, methoxy, hydroxy and chloro;
X is selected from the group consisting of hydrogen, chloro, amino and nitro;
Y is selected from the group consisting of hydrogen, methyl, carboxy, chloro, bromo, iodo, phenyl, phenoxy, 4'-chloroanilino, 4'-boronylphenyl, 4'-bromophenyl, 2'-carboxyethenyl and trimethylsilyl;
Z is selected from the group consisting of hydrogen, 5-chloro, 5-bromo, 5-(3'-trifluoromethyl)phenylazo and 6-chloro;
or
W and X together may represent a fused benzene ring and X and Y together may represent a fused benzene ring substituted by hydroxy in the 6-position of the naphthalene ring numbering, provided that
(1) when each R is hydrogen;
(a) W, X, Y, Z are each hydrogen;
or
(b) W, X and Z are each hydrogen and Y is selected from the group consisting of iodo, bromo, chloro, trimethylsilyl, phenoxy, phenyl, 4'-chloroanilino, methyl, 4'-boronylphenyl and 2'-carboxyethenyl;
or
(c) W and Z are each hydrogen and
(i) X and Y together represent a fused benzene ring substituted by hydroxy in the 6-position of the naphthalene ring numbering:
or
(ii) X is either nitro or chloro and Y is chloro;
or
(iii) X is nitro and Y is carboxy;
(d) W, Y and Z are each hydrogen and X is amino, chloro or nitro;
or
(e) W and X together represent a fused benzene ring and Y and Z are each hydrogen;
(f) X and Y are each hydrogen:
(i) W is methoxy and Z is 5-bromo;
or
(ii) W is hydroxy and Z is 5-(3'-trifluoromethyl)phenylazo;
or
(iii) W is methyl and Z is hydrogen;
(g) W is chloro, X is chloro and Y and Z are each hydrogen;
or
(h) W and Y are each chloro, X is amino and Z is 6-chloro;
(2) when each R is n-butyl, W, X and Z are each hydrogen and Y is bromo or 4'-bromophenyl;
(3) when each R is 4'-chlorophenyl W, X and Z are each hydrogen and Y is chloro;
(4) when each R is 3',5'-dichlorophenyl W and Y are each hydrogen, X is chloro and Z is 5-chloro;
and
(5) when the R's together represent 0,0-propylene, X, Y and Z are each hydrogen;
and the compounds bis(catechol)borate, boroglycine, pentaerythritol borate, 4-(3'-borono-4'-hydroxy-phenylazo benzoic acid, diphenylisobutoxyborane, diphenylboronic anhydride and dimethylphenylboronic acids, said non-boron-containing organic enhancer being selected from the group consisting of phenolic enhancers and amine enhancers, said organoboron enhancer and said non-boron-containing enhancer being present in concentrations effective to increase the signal or signal to background ratio or to decrease the background, compared with carrying out said chemiluminescent reaction in the presence of each of said organoboron enhancer and said non-boron-containing organic enhancer alone.

33 more elements

A method of increasing the light, output or signal to background ratio of light output from a chemiluminescent reaction between a dihydrophthalazinedione (DPD), a peroxidase enzyme and an oxidant, "signal" being in the presence of the peroxidase enzyme, "background" in its absence, which method comprises the step of carrying out said chemiluminescent reaction in the presence of an organoboron enhancer together with a phenolic enhancer, said organoboron enhancer being selected from the group consisting of the compounds of formula (I) ##STR23## in which the R groups are the same and each is selected from the group consisting of hydrogen, n-butyl, 4'-chlorophenyl and 3',5'-dichlorophenyl;

or the R's together are 0,0-propylene thereby forming with the boron atom, a cyclic ether,
W is selected from the group consisting of hydrogen, methyl, methoxy, hydroxy and chloro;
X is selected from the group consisting of hydrogen, chloro, amino and nitro;
Y is selected from the group consisting of hydrogen, methyl, carboxy, chloro, bromo, iodo, phenyl, phenoxy, 4'-chloroanilino, 4'-boronylphenyl, 4'-bromophenyl, 2'-carboxyethenyl and trimethylsilyl;
Z is selected from the group consisting of hydrogen, 5-chloro, 5-bromo, 5-(3'-trifluoromethyl)phenylazo and 6-chloro;
or
W and X together may represent a fused benzene ring and X and Y together may represent a fused benzene ring substituted by hydroxy in the 6-position of the naphthalene ring numbering, provided that
(1) when each R is hydrogen;
(a) W, X, Y, Z are each hydrogen;
or
(b) W, X and Z are each hydrogen and Y is selected from the group consisting of iodo, bromo, chloro, trimethylsilyl, phenoxy, phenyl, 4'-chloroanilino, methyl, 4'-boronylphenyl and 2'-carboxyethenyl;
or
(c) W and Z are each hydrogen and
(i) X and Y together represent a fused benzene ring substituted by hydroxy in the 6-position of the naphthalene ring numbering;
or
(ii) X is either nitro or chloro and Y is chloro;
or
(iii) X is nitro and Y is carboxy;
(d) W, Y and Z are each hydrogen and X is amino, chloro or nitro;
or
(e) W and X together represent a fused benzene ring and Y and Z are each hydrogen;
(f) X and Y are each hydrogen:
(i) W is methoxy and Z is 5-bromo;
or
(ii) W is hydroxy and Z is 5-(3'-trifluoromethyl)phenylazo;
or
(iii) W is methyl and Z is hydrogen;
(g) W is chloro, X is chloro and Y and Z are each hydrogen;
or
(h) W and Y are each chloro, X is amino and Z is 6-chloro;
(2) when each R is n-butyl, W, X and Z are each hydrogen and Y is bromo or 4'-bromophenyl;
(3) when each R is 4'-chlorophenyl W, X and Z are each hydrogen and Y is chloro;
(4) when each R is 3',5'-dichlorophenyl W and Y are each hydrogen, X is chloro and Z is 5-chloro;
and
(5) when the R's together represent 0,0-propylene, X, Y and Z are each hydrogen;
and the compounds bis(catechol)borate, boroglycine, pentaerythritol borate, 4-(3'-borono-4'-hydroxy-phenylazo benzoic acid, diphenylisobutoxyborane, diphenylboronic anhydride and dimethylphenylboronic acids.

33 more elements

A method of diagnostic assay of a substance, the presence or amount of which is relatable to the presence or amount of a reaction partner selected from the group consisting of a dihydrophthalazincdionc (DPD), a peroxidase enzyme and an oxidant which together are reactable in a chemiluminescent reaction, which method comprises the steps of:

carrying out said chemiluminescent reaction in the presence of an organoboron enhancer together with a phenolic enhancer, said organoboron enhancer being selected from the group consisting of the compounds of formula (I) ##STR24## in which the R groups are the same and each is selected from the group consisting of hydrogen, n-butyl, 4'-chlorophenyl and 3',5'-dichlorophenyl;
or the R's together are 0,0-propylene thereby forming with the boron atom, a cyclic ether,
W is selected from the group consisting of hydrogen, methyl, methoxy, hydroxy and chloro;
X is selected from the group consisting of hydrogen, chloro, amino and nitro;
Y is selected from the group consisting of hydrogen, methyl, carboxy, chloro, bromo, iodo, phenyl, phenoxy, 4'-chloroanilino, 4'-boronylphenyl, 4'-bromophenyl, 2'-carboxyethenyl and trimethylsilyl;
Z is selected from the group consisting of hydrogen, 5-chloro, 5-bromo, 5-(3'-trifluormethyl)phenylazo and 6-chloro;
or
W and X together may represent a fused benzene ring and X and Y together may represent a fused benzene ring substituted by hydroxy in the 6-position of the naphthalene ring numbering, provided that
(1) when each R is hydrogen;
(a) W, X, Y, Z are each hydrogen;
or
(b) W, X and Z are each hydrogen and Y is selected from the group consisting of iodo, bromo, chloro, trimethylsilyl, phenoxy, phenyl, 4'-chloroanilino, methyl, 4'-boronylphenyl and 2'-carboxyethenyl;
or
(c) W and Z are each hydrogen and
(i) X and Y together represent a fused benzene ring substituted by hydroxy in the 6-position of the naphthalene ring numbering;
or
(ii) X is either nitro or chloro and Y is chloro;
or
(iii) X is nitro and Y is carboxy;
(d) W, Y and Z are each hydrogen and X is amino, chloro or nitro;
or
(e) W and X together represent a fused benzene ring and Y and Z are each hydrogen;.
(f) X and Y are each hydrogen:
(i) W is methoxy and Z is 5-bromo;
or
(ii) W is hydroxy and Z is 5-(3'-trifluoromethyl)phenylazo;
or
(iii) W is methyl and Z is hydrogen;
(g) W is chloro, X is chloro and Y and Z are each hydrogen;
or
(h) W and Y are each chloro, X is amino and Z is 6-chloro;
(2) when each R is n-butyl, W, X and Z are each hydrogen and Y is bromo or 4'-bromophenyl;
(3) when each R is 4'-chlorophenyl W, X and Z are each hydrogen and Y is chloro;
(4) when each R is 3',5'-dichlorophenyl W and Y are each hydrogen, X is chloro and Z is 5-chloro;
and
(5) when the R's together represent 0,0-propylene, X, Y and Z are each hydrogen;
and the compounds bis(catechol)borate, boroglycine, pentaerythritol borate, 4-(3'-borono-4'-hydroxy-phenylazo benzoic acid, diphenylisobutoxyborane, diphenylboronic anhydride and dimethylphenylboronic acids,
detecting or measuring the light output of the chemiluminescent reaction, and
relating the presence or amount of the substance to be assayed to said light output.

36 more elements

A kit for use in a diagnostic assay, comprising in separate containers first and second enhancers which increase the signal or signal to background ratio of light output, in a chemiluminescent reaction of a fused aromatic diacyl cyclic hydrazide (FADCH), a peroxidase enzyme and an oxidant, "signal" being light output in the presence of the peroxidase enzyme, "background" in its absence, said first enhancer being an organoboron compound and the second enhancer a non-boron-containing organic compound selected from the group consisting of phenolic enhancers and amine enhancers, said organoboron compound being selected from the group consisting of the compounds of formula (I) ##STR25## in which the R groups are the same and each is selected from the group consisting of hydrogen, n-butyl, 4'-chlorophenyl and 3',5'-dichlorophenyl;

or the R's together are 0,0-propylene thereby forming with the boron atom, a cyclic ether;
W is selected from the group consisting of hydrogen, methyl, methoxy, hydroxy and chloro;
X is selected from the group consisting of hydrogen, chloro, amino and nitro;
Y is selected from the group consisting of hydrogen, methyl, carboxy, chloro, bromo, iodo, phenyl, phenoxy, 4'-chloroanilino, 4'-boronylphenyl, 4'-bromophenyl, 2'-carboxyethenyl and trimethylsilyl;
Z is selected from the group consisting of hydrogen, 5-chloro, 5-bromo, 5-(3'-trifluormethyl)phenylazo and 6-chloro;
or
W and X together may represent a fused benzene ring and X and Y together may represent a fused benzene ring substituted by hydroxy in the 6-position of the naphthalene ring numbering, provided that
(1) when each R is hydrogen;
(a) W, X, Y, Z are each hydrogen;
or
(b) W, X and Z are each hydrogen and Y is selected from the group consisting of iodo, bromo, chloro, trimethylsilyl, phenoxy, phenyl, 4'-chloroanilino, methyl, 4'-boronylphenyl and 2'-carboxyethenyl;
or
(c) W and Z are each hydrogen and
(i) X and Y together represent a fused benzene ring substituted by hydroxy in the 6-position of the naphthalene ring numbering;
or
(ii) X is either nitro or chloro and Y is chloro;
or
(iii) X is nitro and Y is carboxy;
(d) W, Y and Z are each hydrogen and X is amino, chloro or nitro;
or
(e) W and X together represent a fused benzene ring and Y and Z are each hydrogen;
(f) X and Y are each hydrogen:
(i) W is methoxy and Z is 5-bromo;
or
(ii) W is hydroxy and Z is 5-(3'-trifluoromethyl)phenylazo;
or
(iii) W is methyl and Z is hydrogen;
(g) W is chloro, X is chloro and Y and Z are each hydrogen;
or
(h) W and Y are each chloro, X is amino and Z is 6-chloro;
(2) when each R is n-butyl, W, X and Z are each hydrogen and Y is bromo or 4'-bromophenyl;
(3) when each R is 4'-chlorophenyl W, X and Z are each hydrogen and Y is chloro;
(4) when each R is 3',5'-dichlorophenyl W and Y are each hydrogen, X is chloro and Z is 5-chloro;
and
(5) when the R's together represent 0,0-propylene, X, Y and Z are each hydrogen;
and the compounds bis(catechol)borate, boroglycine, pentaerythritol borate, 4-(3'-borono-4'-hydroxy-phenylazo benzoic acid, diphenylisobutoxyborane, diphenylboronic anhydride and dimethylphenylboronic acids.

33 more elements

A kit for use in a diagnostic assay, comprising in separate containers first and second enhancers which increase the signal or signal to background ratio of light output in a chemiluminescent reaction of a dihydrothalazinedione (DPD), a peroxidase enzyme and an oxidant, "signal" being light output in the presence of the peroxidase enzyme, "background" in its absence, the first enhancer being an organoboron compound and the second a phenolic enhancer, said organoboron compound being selected from the group consisting of the compounds of formula (I) ##STR32## in which the R groups are the same and each is selected from the group consisting of hydrogen, n-butyl, 4'-chlorophenyl and 3',5'-dichlorophenyl;

or the R's together are 0,0-propylene thereby forming with the boron atom, a cyclic ether;
W is selected from the group consisting of hydrogen, methyl, methoxy, hydroxy and chloro;
X is selected from the group consisting of hydrogen, chloro, amino and nitro;
Y is selected from the group consisting of hydrogen, methyl, carboxy, chloro, bromo, iodo, phenyl, phenoxy, 4'-chloroanilino, 4'-boronylphenyl, 4'-bromophenyl, 2'-carboxyethenyl and trimethylsilyl;
Z is selected from the group consisting of hydrogen, 5-chloro, 5-bromo, 5-(3'-trifluoromethyl)phenylazo and 6-chloro;
or
W and X together may represent a fused benzene ring and X and Y together may represent a fused benzene ring substituted by hydroxy in the 6-position of the naphthalene ring numbering, provided that
(1) when each R is hydrogen:
(a) W, X, Y, Z are each hydrogen;
or
(b) W, X and Z are each hydrogen and Y is selected from the group consisting of iodo, bromo, chloro, trimethylsilyl, phenoxy, phenyl, 4'-chloroanilino, methyl, 4'-boronylphenyl and 2'-carboxyethenyl;
or
(c) W and Z are each hydrogen and
(i) X and Y together represent a fused benzene ring substituted by hydroxy in the 6-position of the naphthalene ring numbering;
or
(ii) X is either nitro or chloro and Y is chloro;
or
(iii) X is nitro and Y is carboxy;
(d) W, Y and Z are each hydrogen and X is amino, chloro or nitro;
or
(e) W and X together represent a fused benzene ring and Y and Z are each hydrogen;
(f) X and Y are each hydrogen:
(i) W is methoxy and Z is 5-bromo;
or
(ii) W is hydroxy and Z is 5-(3'-trifluoromethyl)phenylazo;
or
(iii) W is methyl and Z is hydrogen;
(g) W is chloro, X is chloro and Y and Z are each hydrogen;
or
(h) W and Y are each chloro, X is amino and Z is 6-chloro;
(2) when each R is n-butyl, W, X and Z are each hydrogen and Y is bromo or 4'-bromophenyl;
(3) when each R is 4'-chlorophenyl W, X and Z are each hydrogen and Y is chloro;
(4) when each R is 3',5'-dichlorophenyl W and Y are each hydrogen, X is chloro and Z is 5-chloro;
and
(5) when the R's together represent 0,0-propylene, X, Y and Z are each hydrogen;
and the compounds bis(catechol)borate, boroglycine, pentaerythritol borate, 4-(3'-borono-4'-hydroxy-phenylazo benzoic acid, diphenylisobutoxyborane, diphenylboronic anhydride and dimethylphenylboronic acids.

33 more elements

Dependencies are inferred from the claim text.

Facts

Application: 08/217,845Filed 25 Mar 1994 · continuation in part
Priority: GB 93068881 Apr 1993
Granted: 30 Apr 1996
Term ends: Not estimatedThe source records don’t support an estimate.

Classification4 codes

C12Q 1/28
Involving oxidoreductase: involving peroxidase
Y10S 435/968
Chemistry: molecular biology and microbiology: high energy substrates, e.g. fluorescent, chemiluminescent, radioactive
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms
Y10S
Technical subjects covered by former uspc cross-reference art collections [xracs] and digests

Family

Serial41,233Parent of this continuation-in-part · filed 1 Apr 1993
US 5,512,451 AThis patent

From the US filing history. Foreign family members aren’t listed.

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Abstract

or the R's together are 0,0-propylene thereby forming with the boron atom, a cyclic ether,
W is selected from the group consisting of hydrogen, methyl, methoxy, hydroxy and chloro;
X is selected from the group consisting of hydrogen, chloro, amino and nitro;
Y is selected from the group consisting of hydrogen, methyl, carboxy, chloro, bromo, iodo, phenyl, phenoxy, 4'-chloroanilino, 4'-boronylphenyl, 4'-bromophenyl, 2'-carboxyethenyl and trimethylsilyl;
Z is selected from the group consisting of hydrogen, 5-chloro, 5-bromo, 5-(3'-trifluoromethyl)phenylazo and 6-chloro;
or
W and X together may represent a fused benzene ring and X and Y together may represent a fused benzene ring substituted by hydroxy in the 6-position of the naphthalene ring numbering, provided that
(1) when each R is hydrogen;
(a) W, X, Y, Z are each hydrogen;
or
(b) W, X and Z are each hydrogen and Y is selected from the group consisting of iodo, bromo, chloro, trimethylsilyl, phenoxy, phenyl, 4'-chloroanilino, methyl, 4'-boronylphenyl and 2'-carboxyethenyl;
or
(c) W and Z are each hydrogen and
(i) X and Y together represent a fused benzene ring substituted by hydroxy in the 6-position of the naphthalene ring numbering:
or
(ii) X is either nitro or chloro and Y is chloro;
or
(iii) X is nitro and Y is carboxy;
(d) W, Y and Z are each hydrogen and X is amino, chloro or nitro;
or
(e) W and X together represent a fused benzene ring and Y and Z are each hydrogen;
(f) X and Y are each hydrogen:
(i) W is methoxy and Z is 5-bromo;
or
(ii) W is hydroxy and Z is 5-(3'-trifluoromethyl)phenylazo;
or
(iii) W is methyl and Z is hydrogen;
(g) W is chloro, X is chloro and Y and Z are each hydrogen;
or
(h) W and Y are each chloro, X is amino and Z is 6-chloro;
(2) when each R is n-butyl, W, X and Z are each hydrogen and Y is bromo or 4'-bromophenyl;
(3) when each R is 4'-chlorophenyl W, X and Z are each hydrogen and Y is chloro;
(4) when each R is 3',5'-dichlorophenyl W and Y are each hydrogen, X is chloro and Z is 5-chloro;
and
(5) when the R's together represent 0,0-propylene, X, Y and Z are each hydrogen;
and the compounds bis(catechol)borate, boroglycine, pentaerythritol borate, 4-(3'-borono-4'-hydroxy-phenylazo benzoic acid, diphenylisobutoxyborane, diphenylboronic anhydride and dimethylphenylboronic acids, said non-boron-containing organic enhancer being selected from the group consisting of phenolic enhancers and amine enhancers, said organoboron enhancer and said non-boron-containing enhancer being present in concentrations effective to increase the signal or signal to background ratio or to decrease the background, compared with carrying out said chemiluminescent reaction in the presence of each of said organoboron enhancer and said non-boron-containing organic enhancer alone.

33 more elements

Facts

Application

08/217,845Filed 25 Mar 1994 · continuation in part

Priority

GB 93068881 Apr 1993

Granted

30 Apr 1996

Term ends

Not estimatedThe source records don’t support an estimate.

Classification4 codes

C12Q 1/28
Involving oxidoreductase: involving peroxidase
Y10S 435/968
Chemistry: molecular biology and microbiology: high energy substrates, e.g. fluorescent, chemiluminescent, radioactive
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms
Y10S
Technical subjects covered by former uspc cross-reference art collections [xracs] and digests