Abstract: An assay for the presence of luciferase in a biological sample offers heightened sensitivity, signal intensity and persistence. The assay is sensitive down to 50 fg luciferase. The biological sample is combined with essential ingredients luciferin, ADP, myokinase and Mg++. The myokinase converts ADP to ATP, necessary for the luciferase reaction, and AMP, which retards the reaction kinetics. The resulting assay exhibits a persistent glow emission which makes it adaptable to automation.

Abstract: Dioxetanes that can be triggered by bases are provided which can be used to detect bases in organic solvents, aqueous preparations and detect gaseous bases in the atmosphere. The dioxetanes can be used to detect the release of bases from various labels, as a means to detect the presence of a base released by physical or natural processes, to calibrate light measuring apparatus and to determine the amount of reducing or oxidizing agent present in the base.

Abstract: Chemiluminescent bioassays for the presence or concentration of an analyte in a sample use 1,2-dioxetanes as substrates for the enzyme of an enzyme complex that bind to the analyte. The chemiluminescence obtained from the decomposition of the dioxetane triggered by the enzyme through the formation of the corresponding 1,2-dioxetane oxyanion of the enzyme complex is enhanced by the addition of TBQ as an enhancement agent. Other polymeric quaternary onium salts can be used as enhancement agents in conjunction with enhancement additives which improve the ability of the enhancement agent to form hydrophobic regions in the aqueous sample, in which regions the 1,2-dioxetane oxyanion and its chemiluminescent decomposition products can be sequestered. A kit for performing such assays is also provided.

Abstract: 1,2-dioxetane compounds bearing a proteolytic enzyme specific amino acid or peptide are provided, which amino acid or peptide can be removed by action of the corresponding protease. When the amino acid or peptide is removed, the 1,2-dioxetane decomposes with chemiluminescence, the generation of light providing a rapid, ultra-sensitive and convenient means for detecting the presence of the protease in the sample being inspected. The amount of light generated, or degree of chemiluminescence, can be correlated with the amount of protease present. Immunoassays, as well as DNA hybridization and DNA probe assays are provided.

Abstract: 1, 2-dioxetane compounds bearing a proteolytic enzyme specific amino acid or peptide are provided, which amino acid or peptide can be removed by action of the corresponding protease. When the amino acid or peptide is removed, the 1,2-dioxetane decomposes with chemiluminescence, the generation of light providing a rapid, ultra-sensitive and convenient means for detecting the presence of the protease in the sample being inspected. The amount of light generated, or degree of chemiluminescence, can be correlated with the amount of protease present. Immunoassays, as well as DNA hybridization and DNA probe assays are provided.

Abstract: Dioxetanes that can be triggered by bases are provided which can be used to detect bases in organic solvents, aqueous preparations and detect gaseous bases in the atmosphere. The dioxetanes can be used to detect the release of bases from various labels, as a means to detect the presence of a base released by physical or natural processes, to calibrate light measuring apparatus and to determine the amount of reducing or oxidizing agent present in the base.

Abstract: Chemiluminescent bioassays for the presence or concentration of an analyte in a sample use 1,2-dioxetanes as substrates for the enzyme of an enzyme complex that bind to the analyte. The chemiluminescence obtained from the decomposition of the dioxetane triggered by the enzyme through the formation of the corresponding 1,2-dioxetane oxyanion of the enzyme complex is enhanced by the addition of TBQ as an enhancement agent. Other polymeric quaternary onium salts can be used as enhancement agents in conjunction with enhancement additives which improve the ability of the enhancement agent to form hydrophobic regions in the aqueous sample, in which regions the 1,2-dioxetane oxyanion and its chemiluminescent decomposition products can be sequestered. A kit for performing such assays is also provided.

Abstract: 1,2-dioxetane compounds bearing a proteolytic enzyme specific amino acid or peptide are provided, which amino acid or peptide can be removed by action of the corresponding protease. When the amino acid or peptide is removed, the 1,2-dioxetane decomposes with chemiluminescence, the generation of light providing a rapid, ultra-sensitive and convenient means for detecting the presence of the protease in the sample being inspected. The amount of light generated, or degree of chemiluminescence, can be correlated with the amount of protease present. Immunoassays, as well as DNA hybridization and DNA probe assays are provided.

Abstract: Chemiluminescent bioassays for the presence or concentration of an analyte in a sample use 1,2-dioxetanes as substrates for the enzyme of an enzyme complex that bind to the analyte. The chemiluminescence obtained from the decomposition of the dioxetane triggered by the enzyme through the formation of the corresponding 1,2-dioxetane oxyanion of the enzyme complex is enhanced by the addition of TBQ as an enhancement agent. Other polymeric quaternary onium salts can be used as enhancement agents in conjunction with enhancement additives which improve the ability of the enhancement agent to form hydrophobic regions in the aqueous sample, in which regions the 1,2-dioxetane oxyanion and its chemiluminescent decomposition products can be sequestered. A kit for performing such assays is also provided.

Abstract: Methods are disclosed for purifying chemiluminescent water-soluble 1,2-dioxetane derivatives suitable for use as reporter molecules in a variety of biological analytical systems, including enzyme-linked immunoassays, nucleic acid probe techniques, and structural determinations. The methods are based upon high pressure, medium pressure or low pressure liquid chromatography using as the stationary phase alkaline pH-stable compositions with the chromatographic characteristics of reversed-phase adsorbents, at alkaline pH values, and in the absence of acid-forming compounds or compounds with an unshared pair of electrons. Desalting of substantially pure water soluble 1,2-dioxetane derivatives may be accomplished by the same chromatographic systems or by molecular sieve chromatography systems, but in the absence of salt buffers. Under some circumstances, purification and desalting may be combined in a single chromatographic step.

Abstract: Methods are disclosed for purifying chemiluminescent water-soluble 1,2-dioxetane derivatives suitable for use as reporter molecules in a variety of biological analytical systems, including enzyme-linked immunoassays, nucleic acid probe techniques, and structural determinations. The methods are based upon high pressure, medium pressure or low pressure liquid chromatography using as the stationary phase alkaline pH-stable compositions with the chromatographic characteristics of reversed-phase adsorbents, at alkaline pH values, and in the absence of acid-forming compounds or compounds with an unshared pair of electrons. Desalting of substantially pure water soluble 1,2-dioxetane derivatives may be accomplished by the same chromatographic systems or by molecular sieve chromatography systems, but in the absence of salt buffers. Under some circumstances, purification and desalting may be combined in a single chromatographic step.

Abstract: Water soluble naturally-occurring and synthetic enhancer substances, generally macromolecular in nature, for example globular proteins that include hydrophobic regions such as bovine serum albumin, and polymeric quaternary ammonium salts such as poly(vinylbenzyltrimethylammonium chloride), which have the ability to inhibit light-emitting fluorophores resulting from the decomposition of chemiluminescent compounds from releasing energy through non-light emitting pathways, are disclosed as permitting the stabilization, and hence increasing the light intensity, of such light-emitting fluorophores in aqueous media as compared to the intensity of the light emitted by the same quantities of such fluorophores in aqueous media in the absence of such enhancer substances. Any chemiluminescent enzymatically cleavable 1,2-dioxetane, for example 3-(2'-spiroadamantane)-4-methoxy-(3"-phosphoryloxy)phenyl-1,2-dioxetane disodium salt, can be used.

Abstract: Apparati and methods of use for static and flow cytometry of cells and other particulate matter that eliminate the need for a laser or other external energy source to separate and/or detect differences in cells and other particulate matter wherein components of cells and other particulate matter interact with added thermally, chemically, electrochemically, photochemically or enzymatically decomposible chemiluminescent compounds, to produce light energy emissions optically detectable by said apparati.

Abstract: Chemiluminescent 1,2-dioxetane compounds are disclosed in which the molecule is stabilized at the 3-position on the dioxetane ring against decomposition prior to the molecule's coming in contact with a labile group-removing substance (e.g., an enzyme that will cleave the labile group to cause the molecule to decompose to form at least one light-emitting fluorophore) and substituted at the 4-position on the dioxetane ring with a fused polycyclic ring-containing fluorophore moiety bearing a labile ring substituent whose point of attachment to the fused polycyclic ring, in relation to this ring's point(s) of attachment to the dioxetane ring, is such that the total number of ring atoms separating these points of attachment, including the ring atoms at the points of attachment, is an odd whole number. These odd pattern substituted compounds decompose to emit light of greater intensity and of a different wavelength than that emitted by the corresponding even pattern substituted isomers.

Abstract: Processes are disclosed in which light of different wavelengths is simultaneously released from two or more enzymatically decomposable chemiluminescent 1,2-dioxetane compounds, said compounds being configured, by means of the inclusion of a different light emitting fluorophore in each of them, to each emit light of said different wavelengths, by decomposing each of said compounds by means of a different enzyme. Such processes can be used in multi-channel assays--immunoassays, chemical assays and nucleic acid probe assays--to detect the presence or determine the concentration of chemical or biological substances, and in multi-channel chemical/physical probe procedures for studying the microstructures of macromolecules.

Abstract: Methods are disclosed for purifying chemiluminescent water-soluble 1,2-dioxetane derivatives suitable for use as reporter molecules in a variety of biological analytical systems, including enzyme-linked immunoassays, nucleic acid probe techniques, and structural determinations. The methods are based upon high pressure, medium pressure or low pressure liquid chromatography using as the stationary phase alkaline pH-stable compositions with the chromatographic characteristics of reversed-phase adsorbents, at alkaline pH values, and in the absence of acid-forming compounds or compounds with an unshared pair of electrons. Desalting of substantially pure water soluble 1,2-dioxetane derivatives may be accomplished by the same chromatographic systems or by molecular sieve chromatography systems, but in the absence of salt buffers. Under some circumstances, purification and desalting may be combined in a single chromatographic step.