Tropix, Inc.

Other

Applera Corporation

Pe Corporation

Life Technologies Corporation

Irena Y Bronstein

Brooks Edwards

John C Voyta

Alison Sparks

Christopher Martin

Rouh-Rong Juo

Arthur P Schaap

Zhixian Wang

Yu-xin Yan

John J Fortin

Chris S Martin

Larry J Kricka

Patricia McGrath

Michelle A J Palmer

Bonnie Tillotson

Anthony C Chiulli

Corinne Olesen

Michelle Palmer

Corinne E M Olesen

Alison A Laird

Chemiluminescent heteroaryl substituted benzothiazole 1,2-dioxetane compounds capable of producing light energy when decomposed are provided. These chemiluminescent compounds are represented by the general formula:The heteroaryl substituent Y can be, for example, a pyridyl group or a benzothiazolyl group. The heteroaryl substituted benzothiazole compounds are substantially stable at room temperature. Kits including the heteroaryl substituted dioxetane compounds as well as methods for using these compounds for detecting the presence of one or more analytes in a sample are also provided.

Heteroaryl substituted benzothiazole dioxetanes

Probes labeled with 1,2-dioxetane precursors can be employed in a variety of assays. The probes may be nucleic acid, peptide nucleic acid, proteins including enzyme, antibody or antigen, steroid, carbohydrate, drug or non-drug hapten. The probe is provided with a 1,2-dioxetane precursor bound thereto, generally either covalently, or a strong ligand bond. The dioxetane precursor moiety is converted to a bound 1,2-dioxetane by exposure to singlet oxygen. These dioxetane (labels) either spontaneously decompose, or are induced to decompose by an appropriate trigger to release light. The trigger may be a change in pH temperature, or an agent which removes a protective group. Assay formats in which these 1,2-dioxetane labeled probes and referents may be used to include hybridization assays, immuno assays, gel-based assays and Capillary Zone Electrophoresis.

Dioxetane labeled probes and detection assays employing the same

In an assay method in which a member of a specific binding pair is detected by means of an optically detectable reaction, the improvement wherein the optically detectable reaction includes the reaction, with an enzyme, of a dioxetane having the formulawhere T is a cycloalkyl or polycycloalkyl group bonded to the 4-membered ring portion of the dioxetane by a spiro linkage; Y is a fluorescent chromophore; X is H, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl, or enzyme-cleavable group; and Z is H or an enzyme-cleavable group, provided that at least one of X or Z must be an enzyme-cleavable group, so that the enzyme cleaves the enzyme-cleavable group from the dioxetane to form a negatively charged substituent bonded to the dioxetane, the negatively charged substituent causing the dioxetane to decompose to form a luminescent substance that includes group Y of said dioxetane.

Kit for conducting an assay to detect a substance using enzymatically-induced decomposition of dioxetanes

Chemiluminescent 1,2-dioxetane compounds capable of producing light energy when decomposed, substantially stable at room temperature, represented by the formulas I or II:wherein T is:

Benzothiazole dioxetanes

A competitive assay for the concentration of a cyclic nucleotide such as cyclic adenosine monophosphate combines, in a reaction chamber provided with a capture antibody, an antibody for the cAMP or other cyclic nucleotide, the sample to be assayed and a conjugate of cAMP and alkaline phosphatase. The mixture is incubated and washed, and after washing, a 1,2-dioxetane which is caused to decompose when contacted by the alkaline phosphatase of said conjugate is added. The light emission caused by decomposition is measured, with the strength of the signal being inversely related to the concentration of the cyclic nucleotide. Optionally, an enhancement agent in the form of a polymeric onium salt may be added, to further enhance the light emission.

Competitive chemiluminescent assay for cyclic nucleotide monophosphates

A method of measuring the activity of at least two reporter gene products in an aliquot of a sample extract is disclosed. The activities of a first and second reporter enzyme are quantified by measuring the light signal produced by degradation of a first substrate by the first reporter enzyme and the light signal produced by the degradation of a second substrate by a second reporter enzyme. Both quantifications are sequentially performed on the same aliquot of sample extract.

Multiple reporter gene assay

The present invention discloses multiple enzyme assays which measure the activity of at least one endogenous enzyme in a single aliquot and a method of measuring the activity of multiple enzymes in an aliquot of a cell extract, wherein at least one of the enzymes is an endogenous enzyme. In one embodiment of the invention the activity of a first enzyme is quantified by measuring the light signal produced by degradation of a first enzyme substrate by the first enzyme and the activity of the second enzyme is quantified by measuring the light signal produced by the degradation of a second substrate. In the method of the present invention, both quantifications are performed on the same aliquot of cell extract. Different embodiments of the present invention provide for the detection of more than one endogenous enzyme and for the detection of at least one reporter enzyme and at least one endogenous enzyme. The present invention also discloses kits for detecting the activity of multiple enzymes.

Multiple enzyme assays

Chemiluminescent substrates for neuraminidase, assays for detection of neuraminidase and kits therefor

A kit for detecting a first substance in a sample including a stabilized 1,2-dioxetane bearing an enzyme-labile substituent, which is destabilized and caused to decompose by contacting the 1,2-dioxetane with an enzyme under conditions which cause the enzyme to cleave the enzyme-labile substituent from the dioxetane, thereby yielding a negatively charged oxygen anion bonded to the 1,2-dioxetane, which causes the 1,2-dioxetane to decompose without input from an external excitation energy source, the decomposition being accompanied by chemiluminescence; and a second component selected from the group consisting of a specific affinity substance (e.g., an antigen, an antibody or a nucleic acid probe) and an enzyme which destabilizes said 1,2-dioxetane.

Synthesis of 1,2-dioxetanes and intermediates therefor

Chemiluminescent 1,2-dioxetane compounds capable of producing light energy when decomposed, substantially stable at room temperature, represented by the formulas I or II:

Spiroadamantyl dioxetanes bearing an alkoxy substituent, and an aromatic substituent of phenyl or naphthyl on the dioxetane ring can be activated to chemiluminesce if the aromatic substituent bears a moiety designated OX, wherein the X is cleaved by an enzyme with which the dioxetane is permitted to come in contact with. The T kinetics of the chemiluminescent reaction, as well as the signal intensity, or quantum yield of the chemiluminescent reaction, can be altered by selection of an electron-withdrawing or an electron-donating group Z, at positions on the aromatic substituent other than those adjacent the point of attachment to the dioxetane. Signal strength can further be enhanced by recognized chemiluminescent enhancers.

Growing community of inventors

Newton, MA, United States of America

Irena Y Bronstein