Abstract: Stable, enzymatically triggered chemiluminescent 1,2-dioxetanes with improved water solubility and storage stability are provided as well as synthetic processes and intermediates used in their preparation. Dioxetanes further substituted with two or more water-solubilizing groups disposed on the dioxetane structure and an additional fluorine atom or lower alkyl group provide superior performance by eliminating the problem of reagent carryover when used in assays performed on capsule chemistry analytical systems. These dioxetanes display substantially improved stability on storage. Compositions comprising these dioxetanes, a non-polymeric cationic surfactant enhancer and optionally a fluorescer, for providing enhanced chemiluminescence are also provided.

Abstract: Methods of synthesizing polynucleotides are disclosed involving the simultaneous ligation of a set of oligomer 5'-phosphates onto a template-bound primer. The set of these oligomers can be preselected to contain oligomers which are complementary to the template strand or the oligomers can be supplied as a library and allowed to self select. The synthesis by ligation can proceed unidirectionally or bidirectionally from the primer and can be used to synthesize both strands simultaneously by the use of two primers. The ligation is preferably performed with a ligase enzyme. The methods of synthesis are useful in a variety of applications, including cloning, amplification, labeling, diagnostic assays, mutation analysis and screening, gene expression monitoring and sequence analysis.

Abstract: Methods of generating chemiluminescence rapidly without the use of enzymes are provided. The methods utilize chemiluminescent compounds comprising an acridan ring bearing an exocyclic double bond in a reaction with an acid, an oxidant and a base. The chemiluminescent methods are useful in assays to detect the amount of an analyte in a sample. The chemiluminescent compounds may be supplied as a label on an analyte or a specific binding partner or may be encapsulated within a liposome or latex particle.

Abstract: Methods of synthesizing polynucleotides are disclosed involving the simultaneous ligation of a set of oligomer 5'-phosphates onto a template-bound primer. The set of these oligomers can be preselected to contain oligomers which are complementary to the template strand or the oligomers can be supplied as a library and allowed to self select. The synthesis by ligation can proceed unidirectionally or bidirectionally from the primer and can be used to synthesize both strands simultaneously by the use of two primers. The ligation is preferably performed with a ligase enzyme. The methods of synthesis are useful in a variety of applications, including cloning, amplification, labeling, diagnostic assays, mutation analysis and screening, gene expression monitoring and sequence analysis.

Abstract: Methods of synthesizing polynucleotides are disclosed involving the simultaneous ligation of a set of oligomer 5'-phosphates onto a template-bound primer. The set of these oligomers can be preselected to contain oligomers which are complementary to the template strand or the oligomers can be supplied as a library and allowed to self select. The synthesis by ligation can proceed unidirectionally or bidirectionally from the primer and can be used to synthesize both strands simultaneously by the use of two primers. The ligation is preferably performed with a ligase enzyme. The methods of synthesis are useful in a variety of applications, including cloning, amplification, labeling, diagnostic assays, mutation analysis and screening, gene expression monitoring and sequence analysis.

Abstract: Methods of synthesizing polynucleotides are disclosed involving the simultaneous ligation of a set of oligomer 5'-phosphates onto a template-bound primer. The set of these oligomers can be preselected to contain oligomers which are complementary to the template strand or the oligomers can be supplied as a library and allowed to self select. The synthesis by ligation can proceed unidirectionally or bidirectionally from the primer and can be used to synthesize both strands simultaneously by the use of two primers. The ligation is preferably performed with a ligase enzyme. The methods of synthesis are useful in a variety of applications, including cloning, amplification, labeling, diagnostic assays, mutation analysis and screening, gene expression monitoring and sequence analysis.

Abstract: Compounds which generate red chemiluminescence on reaction with a phosphatase enzyme are provided as well as intermediates useful for their preparation. The chemiluminescent compounds comprise a luciferin ring system and an exocyclic enol phosphate group where the position on the luciferin ring system adjacent to the double bond is disubstituted. The chemiluminescent compounds are useful alone or within compositions containing a cationic aromatic compound in methods for producing chemiluminescence. The chemiluminescent reactions can be applied in assays for phosphatase enzymes and in assays employing enzyme-labeled specific binding pairs.

Abstract: A process for producing a stable triggerable dioxetane comprising;(a) reacting a vinyl sulfide compound containing a sulfur-substituent SR.sub.4, wherein R.sub.4 is an organic group containing 1 to 20 carbon atoms and optionally heteroatoms, with oxygen and light in the presence of a photosensitizer to form an intermediate sulfur-substituted dioxetane compound; and(b) reacting the sulfur-substituted dioxetane compound with an electrophilic compound E--Y and a hydroxylic compound R.sub.5 OH selected from the group consisting of alcohols, phenols and carboxylic acids or their salts and containing an OR.sub.5 group to replace the SR.sub.4 group of the dioxetane with the OR.sub.5 group.

Abstract: Methods and compositions for generating chemiluminescence on reaction with a peroxidase enzyme are provided as well as compounds to be used in the methods and compositions. The compounds have a C--C double bond substituted at one carbon with two oxygen or sulfur atom-containing groups. The compositions contain the double bond-containing compound and a peroxide in an aqueous solution. Additional components which can be included in the compositions are peroxidase activity enhancers, nonionic surfactants or cationic surfactants to improve detection sensitivity of the peroxidase. The chemiluminescent methods and compositions are useful in assays for peroxidase enzymes and in assays employing enzyme-labeled specific binding pairs.

Abstract: A chemiluminescent assay method and compositions are described which use a dialkyl-substituted dioxetane which is deprotected to trigger a chemiluminescent reaction. Chemiluminescent 1,2-dioxetane compounds substituted on the dioxetane ring with two nonspirofused alkyl groups which can be triggered by a reagent to generate light are disclosed. Dialkyl-substituted dioxetanes are useful for the detection of triggering agents including enzymes. The enzyme may be present alone or linked to a member of a specific binding pair in an immunoassay, DNA probe assay or other assay where the enzyme is bound to a reporter molecule.

Abstract: Vinyl sulfides bearing protected arlyoxy substituents and a process for their preparation are provided. In the process a vinyl sulfide is formed by coupling a ketone compound and a thioester compound with a low valent titanium reagent to form both carbon-carbon bonds in the same step. The vinyl sulfide compounds are useful as intermediates which are convertible into triggerable sulfur-substituted dioxetanes by addition of oxygen to the double bond. The sulfur-substituted dioxetanes additionally are useful for producing chemiluminescence or for further conversion into triggerable alkoxy, alkenyloxy, alkynloxy, aryloxy, aralkyloxy, or acyloxy-substituted dioxetanes. Triggerable dioxetanes are useful for producing chemiluminescence and in detecting activating agents in assays such as immunoassays and nucleic acid hybridization assays.

Abstract: Stable, enzymatically triggered chemiluminescent 1,2-dioxetanes with improved water solubility and storage stability are provided. Dioxetanes further substituted with two or more water-solubilizing groups disposed on the dioxetane structure and an additional fluorine atom or lower alkyl group provide superior performance by eliminating the problem of reagent carryover when used in assays performed on capsule chemistry analytical systems. These dioxetanes display substantially improved stability on storage. Compositions comprising these dioxetanes, a non-polymeric cationic surfactant enhancer and optionally a fluorescer, for providing enhanced chemiluminescence are also provided.

Abstract: Methods of detecting analytes or target species using two enzyme-labeled specific binding partners where the two enzymes function in concert to produce a detectable chemiluminescent signal are disclosed. The methods use a specific binding partner labeled with a hydrolytic enzyme to produce a phenolic enhancer in close proximity to a peroxidase-labeled second specific binding partner. The method is useful to detect and quantitate with improved specificity various biological molecules including antigens and antibodies by the technique of immunoassay, proteins by Western blotting, DNA by Southern blotting, RNA by Northern blotting. The method may also be used to detect DNA mutations and juxtaposed gene segments in chromosomal translocations and particularly to unambiguously identify heterozygous genotypes in a single test.

Abstract: Novel methods and compositions which generate chemiluminescence are provided. The compositions comprise a heterocyclic enol phosphate compound and a dihydroxyaromatic compound in which the two hydroxy groups are separated by an even number of ring carbon atoms. Novel methods and compositions for generating chemiluminescence by reaction with a hydrolytic enzyme are provided as well. The compositions comprise a heterocyclic enol phosphate compound and a protected dihydroxyaromatic compound in which one of the hydroxy groups of the dihydroxyaromatic compound is protected with an enzyme-cleavable group. The novel chemiluminescent compositions are useful in methods for producing chemiluminescence for use in assays of hydrolytic enzymes and enzyme inhibitors and in assays employing labeled specific binding pairs.

Abstract: Stable, enzymatically triggered chemiluminescent 1,2-dioxetanes with improved water solubility are provided. Dioxetanes further substituted with two or more water-solubilizing groups disposed on the dioxetane structure provide superior performance by eliminating the problem of reagent carryover when used in assays performed on capsule chemistry analytical systems. Compositions comprising a dioxetane with two or more water-solubilizing groups, a non-polymeric cationic surfactant enhancer and optionally a fluorescer, for providing enhanced chemiluminescence are also provided.

Abstract: Stable, enzymatically triggered chemiluminescent 1,2-dioxetanes with improved water solubility are provided. Dioxetanes further instituted with two or more water-solubilizing groups disposed on the dioxetane structure provide superior performance by eliminating the problem of reagent carryover when used in assays performed on capsule chemistry analytical systems. Compositions comprising a dioxetane with two or more water-solubilizing groups, a non-polymeric cationic surfactant enhancer and optionally a fluorescer, for providing enhanced chemiluminescence are also provided.

Abstract: Novel methods and compositions which generate chemiluminescence are provided. The compositions comprise a heterocyclic enol phosphate compound and a dihydroxyaromatic compound in which the two hydroxy groups are separated by an even number of ring carbon atoms.Novel methods and compositions for generating chemiluminescence by reaction with a hydrolytic enzyme are provided as well. The compositions comprise a heterocyclic enol phosphate compound and a protected dihydroxyaromatic compound in which one of the hydroxy groups of the dihydroxyaromatic compound is protected with an enzyme-cleavable group.The novel chemiluminescent compositions are useful in methods for producing chemiluminescence for use in assays of hydrolytic enzymes and enzyme inhibitors and in assays employing labeled specific binding pairs.

Abstract: A chemiluminescent assay method, compositions, kits and chemiluminescent acridan compounds are described which use a two-step chemiluminescent reaction process. The reaction involves an acridan compound, preferably a derivative of an N-alkylacridan-9-carboxylic acid, which undergoes a reaction with a peroxide compound, a peroxidase enzyme and an enhancer under conditions of time, temperature and pH which permit the accumulation of an intermediate compound, which is subsequently induced to produce a burst of light by raising the pH. The result is generation of very high intensity light from the reaction. The peroxidase 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. The method is particularly amenable to automated assays because of the separation of the incubation and light generating steps.

Abstract: Stable, enzymatically triggered chemiluminescent 1,2-dioxetanes with improved water solubility and storage stability are provided. Dioxetanes further substituted with two or more water-solubilizing groups disposed on the dioxetane structure and an additional fluorine atom or lower alkyl group provide superior performance by eliminating the problem of reagent carryover when used in assays performed on capsule chemistry analytical systems. These dioxetanes display substantially improved stability on storage. Compositions comprising these dioxetanes, a non-polymeric cationic surfactant enhancer and optionally a fluorescer, for providing enhanced chemiluminescence are also provided.

Abstract: A chemiluminescent assay method, compositions, kits and chemiluminescent acridan compounds are described which use a two-step chemiluminescent reaction process. The reaction involves an acridan compound, preferably a derivative of an N-alkylacridan-9-carboxylic acid, which undergoes a reaction with a peroxide compound, a peroxidase enzyme and an enhancer under conditions of time, temperature and pH which permit the accumulation of an intermediate compound, which is subsequently induced to produce a burst of light by raising the pH. The result is generation of very high intensity light from the reaction. The peroxidase 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. The method is particularly amenable to automated assays because of the separation of the incubation and light generating steps.