Chemiluminescent detection of hydrolytic enzymes using an acridan
AssigneeLumigen, Inc.
InventorsHashem Akhavan-Tafti, Zahra Arghavani, Renuka DeSilva
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Abstract
A chemiluminescent assay method, compositions and kits are described which use a protected phenolic enhancer compound which is deprotected by a hydrolytic enzyme and then enhances a chemiluminescent reaction. The reaction involves an acridan compound, preferably a derivative of an N-alkylacridan-9-carboxylic acid, which is activated to produce light by a peroxide compound and a peroxidase enzyme in the presence of the deprotected enhancer. The result is enhanced generation of light from the reaction. The hydrolytic enzyme is present alone or linked to a member of a specific binding pair in an immunoassay, DNA probe assay or other assay where the hydrolytic enzyme is bound to a reporter molecule.
Claims34
A method for producing chemiluminescence comprising reacting a hydrolytic enzyme with a protected enhancer, a peroxide compound, a peroxidase enzyme and an acridan compound to produce chemiluminescence;
- wherein the acridan has the formula:
- ##STR4## wherein R is an alkyl, heteroalkyl or aralkyl group, wherein R.sub.1 to R.sub.8 are selected independently from groups which allow the production of chemiluminescence and wherein Y is a leaving group;
- and wherein the protected enhancer compound has the formula ArOX wherein X is a leaving group and Ar is selected from the group consisting of a substituted phenyl, a napthyl and a substituted napthyl group, wherein the hydrolytic enzyme reacts with the protected enhancer compound to remove X, thus enhancing the level of chemiluminescence produced by the acridan compound upon reaction with the peroxidase and the peroxide as compared to the level of chemiluminescence produced in the absence of the enhancer compound.
A method of detecting the presence or amount of a hydrolytic enzyme in a sample wherein the hydrolytic enzyme may be present in the sample in free form or as a conjugate to a biological molecule comprising:
- (a) reacting the hydrolytic enzyme in the sample with a protected enhancer, a peroxide compound, a peroxidase enzyme and an acridan compound to produce chemiluminescence;
- wherein the acridan has the formula:
- ##STR5## wherein R is an alkyl, heteroalkyl or aralkyl group, wherein R.sub.1 to R.sub.8 are selected independently from groups which allow the production of chemiluminescence and wherein Y is a leaving group;
- and wherein the protected enhancer compound has the formula ArOX wherein X is a leaving group and Ar is selected from the group consisting of a substituted phenyl, a napthyl and a substituted napthyl group, wherein the hydrolytic enzyme reacts with the protected enhancer compound to remove X, thus enhancing the level of chemiluminescence produced by the acridan compound upon reaction with the peroxidase and the peroxide as compared to the level of chemiluminescence produced in the absence of the enhancer compound;
- and
- (b) measuring the amount of chemiluminescence produced and relating it to the presence or amount of the hydrolytic enzyme in the sample.
A kit for detecting the presence or amount of a hydrolytic enzyme in a sample wherein the hydrolytic enzyme may be present in the sample in free form or as a conjugate to a biological molecule comprising providing in one or more containers:
- (a) an acridan compound having the formula:
- ##STR6## wherein R is an alkyl, heteroalkyl or aralkyl group, wherein R.sub.1 to R.sub.8 are selected independently from groups which allow the production of chemiluminescence and wherein Y is a leaving group;
- (b) a peroxide compound;
- (c) a protected enhancer compound of the formula ArOX, wherein X is a leaving group and Ar is selected from the group consisting of a substituted phenyl, a napthyl and a substituted napthyl group, wherein the hydrolytic enzyme reacts with the protected enhancer compound to remove X, thus enhancing the level of chemiluminescence produced by the acridan compound upon reaction with the peroxidase and the peroxide as compared to the level of chemiluminescence produced in the absence of the enhancer compound;
- and
- (d) a peroxidase enzyme.
A kit for detecting the presence or amount of a hydrolytic enzyme in a sample wherein the hydrolytic enzyme may be present in the sample in free form or as a conjugate to a biological molecule comprising:
- a reagent composition which generates chemiluminescence in the presence of a hydrolytic enzyme, said reagent composition comprising, in an aqueous solution, a protected enhancer, a peroxide compound, a peroxidase enzyme and an acridan compound which generates chemiluminescence upon reaction with the peroxide compound and the peroxidase enzyme, wherein the acridan has the formula:
- ##STR7## wherein R is an alkyl, heteroalkyl or aralkyl group, wherein R.sub.1 to R.sub.8 are selected independently from groups which allow the production of chemiluminescence and wherein Y is a leaving group;
- and wherein the protected enhancer compound has the formula ArOX wherein X is a leaving group and Ar is selected from the group consisting of a substituted phenyl, a napthyl and a substituted napthyl group, wherein the hydrolytic enzyme reacts with the protected enhancer compound to remove X, thus enhancing the level of chemiluminescence produced by the acridan compound upon reaction with the peroxidase and the peroxide as compared to the level of chemiluminescence produced in the absence of the enhancer compound.
A composition which generates chemiluminescence in the presence of a hydrolytic enzyme which comprises in an aqueous solution:
- (a) a peroxide compound;
- (b) a peroxidase enzyme;
- (c) an acridan compound which generates light chemiluminescence upon reaction with the peroxide compound and the peroxidase enzyme, wherein the acridan has the formula:
- ##STR8## wherein R is an alkyl, heteroalkyl or aralkyl group, wherein R.sub.1 to R.sub.8 are selected independently from groups which allow the production of chemiluminescence and wherein Y is a leaving group;
- and
- (d) a protected enhancer compound of the formula ArOX, wherein X is a leaving group and Ar is selected from the group consisting of a substituted phenyl, a napthyl and a substituted napthyl group, wherein the hydrolytic enzyme reacts with the protected enhancer compound to remove X, thus enhancing the level of chemiluminescence produced by the acridan compound upon reaction with the peroxidase and the peroxide as compared to the level of chemiluminescence produced in the absence of the enhancer compound.
Dependencies are inferred from the claim text.