Method of using synthesis of 1,2-dioxetanes and kits therefore
AssigneeTropix, Inc.
InventorsIrena Y. Bronstein, Brooks Edwards
Life of this patent
Estimating the term…
Abstract
Kits comprising 1,2-dioxetanes which can be cause to chemiluminesce by contact with an enzyme, and the enzyme, are provided for use in optically detectable assays. The assay calls for binding the enzyme to the substance to be detected in a sample, removing any unbound enzyme, and then combining the treated sample with the dioxetane. If the substance to be detected is present, enzyme bound thereto will cleave the protecting group of the dioxetane, causing the dioxetane to decompose and chemiluminescence. The intensity of luminescence is indicative of the concentration of the substance in the sample. The substance to be detected may be an enzyme, in which case no binding group is necessary.
Claims11
A kit for the generation of light in a optically detectable assay, comprising:
- (a) a 1,2-dioxetane of the formula:
- ##STR41## wherein n-0-19, wherein Z is a moiety selected from the group consisting of a phosphoryloxy, oxacarboxylate, 1-phospho-2,3-diacyglyceride, D-xyloside, D-fucoside, 1-thio-D-glucoside, adenosine triphosphate, adenosine diphosphate adenosine monophosphate, adenosine, .alpha.-D-glucoside, .beta.-D-glucoside, .alpha.-D-mannoside, .beta.-D-mannoside, .beta.-D-fructofuranoside, .beta.-D-glucosiduronate, p-toluenesulfonyl-L-arginine ester, and p-toluenesulfonyl-1-arginine amide, and
- Q is selected from the group consisting of a perfluoroalkyl group of 1-7 carbon atoms, methylsufonyl, a halogen, cyano, nitro, alkoxy carbamoyl of 1-7 carbon atoms, alkanoyl of 1-7 carbon atoms, branched or straight chain alkyl of 1-7 carbon atom and alkoxy of 1-7 carbon atoms, and
- (b) an enzyme which, when brought into contact with said 1,2-dioxetane, cleaves said moiety Z.
A method of optically detecting the presence or concentration of a substance in a sample, comprising contacting said sample with an enzyme bound to a moiety having a specific affinity for said substance,
- removing all said enzyme-specific affinity complex not bound to any substance in said sample, and
- contacting said sample with a 1,2-dioxetane of the formula ##STR42## wherein n=0-19, wherein Z is a moiety selected from the group consisting of a phyosphoryloxy, oxacarboxylate, 1-phospho-2,3-diacyglyceride, D-xyloside, D-fucoside, 1-thio-D-glucoside, adenosine triphosphate, adenosine diphosphate adenosine monophosphate, adenosine, .alpha.-D-glucoside, .beta.-D-glucoside, .alpha.-D-mannoside, .beta.-D-mannoside, .beta.-D-fructofuranoside, .beta.-D-glucosiduronate, p-toluenesulfonyl-L-arginine ester, and p-toluenesulfonyl-1-arginine amide, and
- Q is selected from the group consisting of a perfluoroalkyl group of 1-7 carbon atoms, methylsufonyl, a halogen, cyano, nitro, alkoxy carbamoyl of 1-7 carbon atoms, alkanoyl of 1-7 carbon atoms, branched or straight chain alkyl of 1-7 carbon atom and alkoxy of 1-7 carbon atoms, wherein Z is selected so as to cleavable by said enzyme, and
- detecting any luminescence generated as a result of said contacting step,
- wherein luminescence is indicative of the presence of said substance.
A method of detecting an enzyme in a sample, comprising combining said sample with a 1,2-dioxetane of the formula ##STR43## wherein n=0-19, wherein Z is a moiety selected from the group consisting of a phyosphoryloxy, oxacarboxylate, 1-phospho-2,3-diacyglyceride, D-xyloside, D-fucoside, 1-thio-D-glucoside, adenosine triphosphate, adenosine diphosphate adenosine monophosphate, adenosine, .alpha.-D-glucoside, .beta.-D-glucoside, .alpha.-D-mannoside, .beta.-D-mannoside, .beta.-D-fructofuranoside, .beta.-D-glucosiduronate, p-tolenesulfonyl-L-arginine ester, and p-toluenesulfonyl-1-arginine amide, and
- Q is selected from the group consisting of a perfluoroalkyl group of 1-7 carbon atoms, methylsufonyl, a halogen, cyano, nitro, alkoxy carbamoyl of 1-7 carbon atoms, alkanoyl of 1-7 carbon atoms, branched or straight chain alkyl of 1-7 carbon atom and alkoxy of 1-7 carbon atoms,
- wherein Z is selected so as to be cleaved by said enzyme whose detection is sought, and
- observing said sample to detect luminescence, wherein detected luminescence is indicative of the presence of said enzyme.
Dependencies are inferred from the claim text.