US4978614A — Method of detecting a substance using enzymatical…

Claim 1

Independent Claim 146 words

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 said optically detectable reaction includes the reaction, with, an enzyme, of a dioxetane having the formula ##STR31## where T is a cycloalkyl or olycycloalkyl group bonded to the 4-membered ring portion of said dioxetane by a spiro linkage; Y is a fluorescent chromophore; X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl, or enzyme-cleavable group; and Z is hydrogen or an enzyme-cleavable group, provided that at least one of X or Z must be an enzyme-cleavable group, so that said enzyme cleaves said enzyme-cleavable group from said dioxetane to form a negatively charged substituent bonded to said dioxetane, said negatively charged substituent causing said dioxetane to decompose to form a luminescent substance comprising said group Y of said dioxetane.

Claim 2

Dependent Claim 146 words

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 said optically detectable reaction includes the reaction, with, an enzyme, of a dioxetane having the formula ##STR31## where T is a cycloalkyl or olycycloalkyl group bonded to the 4-membered ring portion of said dioxetane by a spiro linkage; Y is a fluorescent chromophore; X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl, or enzyme-cleavable group; and Z is hydrogen or an enzyme-cleavable group, provided that at least one of X or Z must be an enzyme-cleavable group, so that said enzyme cleaves said enzyme-cleavable group from said dioxetane to form a negatively charged substituent bonded to said dioxetane, said negatively charged substituent causing said dioxetane to decompose to form a luminescent substance comprising said group Y of said dioxetane.

Claim 3

Dependent Claim 18 words

The method of claim 1 wherein said groups T, X, or Y, independently, further comprise a solubilizing substituent.

Claim 4

Dependent Claim 16 words

The method of claim 1 wherein said specific binding pair comprises an antigen and an antibody.

Claim 5

Dependent Claim 29 words

The method of claim 1 wherein said specific binding pair comprises a nucleic acid and a probe capable of binding to all or a portion of said nucleic acid.

Claim 6

Dependent Claim 16 words

The method of claim 1 wherein said group T of said dioxetane is a polycycloalkyl group.

Claim 7

Dependent Claim 13 words

The method of claim 1 wherein said group T is an adamantyl group.

Claim 8

Dependent Claim 17 words

The method of claim 1 wherein said enzyme cleavable group comprises phosphate, and said enzyme comprises phosphatase.

Claim 9

Dependent Claim 23 words

The method of claim 1 wherein said specific binding pair comprises an enzyme and a dioxetane containing a group cleavable by said enzyme.

Claim 10

Dependent Claim 18 words

The method of claim 1 wherein said enzyme-cleavable group comprises a galactopyranoside, and said enzyme comprises a galactosidase.

Claim 11

Dependent Claim 20 words

The method of claim 1 wherein said enzyme-cleavable group comprises a carboxylic acid ester, and said enzyme comprises an esterase.

Claim 12

Dependent Claim 30 words

The method of claim 1 further comprising, when applied to solid state methods, blocking nonspecific binding to solid matrices by pretreating said solid matrices with a poly(vinyl quaternary ammonium salt).

Claim 13

Dependent Claim 15 words

The method of claim 4 wherein the nucleic acid is DNA, RNA or fragment thereof.

Claim 14

Dependent Claim 17 words

The method of claim 4 wherein the probe is a labeled oligonucleotide complementary to the nucleic acid.

Claim 15

Dependent Claim 11 words

The method of claim 13 wherein the oligonucleotide probe is biotinylated.

Claim 16

Dependent Claim 18 words

The method of claim 12 wherein the DNA RNA or fragment thereof is produced by a sequencing protocol.

Claim 17

Dependent Claim 74 words

The method of claim 15 further comprising the steps of (a) contacting the DNA, RNA or fragment thereof with a labeled complementary oligonucleotide probe to form a hybridizing pair, (b) contacting the hybridized pair with a molecule capable of strong binding to the label of the oligonucleotide covalently conjugated with an enzyme capable of cleaving an enzyme-cleavable 1,2-dioxetane to release light energy, (c) adding such a 1,2-dioxetane substrate, and (d) detecting the light produced.

Claim 18

Dependent Claim 15 words

The method of claim 16 wherein the oligonucleotide label is biotin or a biotin derivative.

Claim 19

Dependent Claim 22 words

The method of claim 16 wherein the molecule capable of strong interaction with the label of the oligonucleotide is avidin or streptavidin.

Claim 20

Dependent Claim 16 words

The method of claim 16 wherein the enzyme is a phosphatase and the 1,2-dioxetane is AMPPD.

Claim 21

Dependent Claim 16 words

The method of claim 16 wherein the enzyme is a galactosidase and the 1,2-dioxetane is AMPGD.

Claim 22

Dependent Claim 12 words

The method of claim 16 wherein light is detected by light-sensitive film.

Claim 23

Dependent Claim 13 words

The method of claim 16 wherein light is detected by a photoelectric cell.

Claim 24

Dependent Claim 23 words

The method of claim 13, wherein said oligonucleotide probe is covalently labeled with an enzyme capable of decomposing said 1,2-dioxetane to emit light.

Claim 25

Dependent Claim 45 words

The method of claim 13, wherein said label on said oligonucleotide probe comprises a covalently bound antigen that is immunochemically bound to an antibody-enzyme conjugate, wherein said antibody is directed to said antigen and said enzyme is capable of decomposing said 1,2-dioxetane to emit light.

Claim 26

Dependent Claim 22 words

The method of either claim 23 or 24, wherein said enzyme is an acid or alkaline phosphatase and said 1,2-dioxetane is AMPPD.

Claim 27

Dependent Claim 19 words

The method of either claim 23 or 14, wherein said enzyme is a galactosidase and said 1,2-dioxetane is AMPGD.

Claim 28

Dependent Claim 21 words

The method of either claim 23 or 24, wherein said enzyme is a carboxyl acid esterase and said 1,2-dioxetane is 3-(2'-spiroadamantane)-4-methoxy-4-(3"-acetoxy)phenyl-1,2-dioxetane.

Claim 29

Independent Claim 29 words

The method of any one of claims 4, 13 or 23-27, inclusive, wherein the binding of said probe to said nuclei acid is carried out on a nylon membrane.

Claim 30

Dependent Claim 18 words

The method of claim 16, wherein said enzyme is a carboxylic acid esterase and said 1,2-dioxetane is 3-(2'-spiroadamantane)-4-methoxy-4-(3"-acetoxy)phenyl-1,2-dioxetane.

Claim 31

Independent Claim 30 words

The method of any one of claims 16-20, inclusive, wherein the hybridizing between said DNA, RNA or fragment thereof and said labeled oligonucleotide probe is conducted on a nylon membrane.

Claim 32

Dependent Claim 92 words

A kit for detecting a first substance in a sample comprising a dioxetane having the formula ##STR32## where T is a cycloalkyl group bonded to the 4-membered ring portion of said dioxetane by a spiro linkage; Y is a chromophore capable of fluorescence; X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cyclo heteroalkyl, or enzyme-cleavable group; and Z is hydrogen or an enzyme-cleavable group, provided that at least one of X or Z must be an enzyme-cleavable group; and an enzyme capable of cleaving said enzyme-cleavable group of said dioxetane.

Claim 33

Dependent Claim 92 words

A kit for detecting a first substance in a sample comprising a dioxetane having the formula ##STR32## where T is a cycloalkyl group bonded to the 4-membered ring portion of said dioxetane by a spiro linkage; Y is a chromophore capable of fluorescence; X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cyclo heteroalkyl, or enzyme-cleavable group; and Z is hydrogen or an enzyme-cleavable group, provided that at least one of X or Z must be an enzyme-cleavable group; and an enzyme capable of cleaving said enzyme-cleavable group of said dioxetane.

Claim 34

Dependent Claim 117 words

A kit for detecting a nucleic acid or fragment thereof in a sample by hybridization of said nucleic acid or fragment to a complementary labeled oligonucleotide probe, comprising a 1,2-dioxetane capable of producing light energy when decomposed having the formula ##STR33## wherein T is a stabilizing cycloalkyl group spiro bonded to the 4-membered dioxetane ring, X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an enzyme-cleavable group, Y is a chromophore capable of fluorescence, and Z is hydrogen or an enzyme-cleavable group provided that at least one of X or Z must be an enzyme-cleavable group; a covalently enzyme-labeled oligonucleotide probe; and, a nylon membrane upon which said nucleic acid-oligonucleotide probe hybridization is conducted.

Claim 35

Dependent Claim 117 words

A kit for detecting a nucleic acid or fragment thereof in a sample by hybridization of said nucleic acid or fragment to a complementary labeled oligonucleotide probe, comprising a 1,2-dioxetane capable of producing light energy when decomposed having the formula ##STR33## wherein T is a stabilizing cycloalkyl group spiro bonded to the 4-membered dioxetane ring, X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an enzyme-cleavable group, Y is a chromophore capable of fluorescence, and Z is hydrogen or an enzyme-cleavable group provided that at least one of X or Z must be an enzyme-cleavable group; a covalently enzyme-labeled oligonucleotide probe; and, a nylon membrane upon which said nucleic acid-oligonucleotide probe hybridization is conducted.

Claim 36

Dependent Claim 18 words

The kit of claim 32, wherein said 1,2-dioxetane is AMPPD and said enzyme is acid or alkaline phosphatase.

Claim 37

Dependent Claim 15 words

The kit of claim 32, wherein said 1,2-dioxetane is AMPGD and said enzyme is galactosidase.

Claim 38

Dependent Claim 18 words

The kit of claim 32, wherein said enzyme is a carboxylic acid esterase and said 1,2-dioxetane is 3-(2'-spiroadamantane)-4-methoxy-4(3"-acetoxy)phenyl-1,2-dioxetane.

Claim 39

Dependent Claim 18 words

The kit of any one of claims 27-30, inclusive, further comprising image-reproducing means for detecting said light energy.

Claim 40

Dependent Claim 12 words

The kit of claim 36, wherein said image-producing means is photographic film.

Claim 41

Dependent Claim 145 words

A kit for detecting a nuclei acid or fragment thereof in a sample by hybridization of said nuclei acid or fragment to a complementary labeled oligonucleotide probe, comprising a 1,2-dioxetane capable of producing light energy when decomposed having the formula ##STR34## wherein T is a stabilizing cycloalkyl group spiro bonded to the 4-membered dioxetane ring, X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an enzyme-cleavable group, Y is a chromophore capable of fluorescence, and Z is hydrogen or an enzyme-cleavable group, provided that at least one of X or Z must be an enzyme-cleavable group; a complementary oligonucleotide probe covalently labeled with biotin or a biotin derivative; avidin or streptavidin covalently bound to an enzyme capable of decomposing a 1,2-dioxetane to emit light; and, a nylon membrane upon which said nuclei acid or fragment thereof is hybridized to said oligonucleotide probe.

Claim 42

Dependent Claim 145 words

A kit for detecting a nuclei acid or fragment thereof in a sample by hybridization of said nuclei acid or fragment to a complementary labeled oligonucleotide probe, comprising a 1,2-dioxetane capable of producing light energy when decomposed having the formula ##STR34## wherein T is a stabilizing cycloalkyl group spiro bonded to the 4-membered dioxetane ring, X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an enzyme-cleavable group, Y is a chromophore capable of fluorescence, and Z is hydrogen or an enzyme-cleavable group, provided that at least one of X or Z must be an enzyme-cleavable group; a complementary oligonucleotide probe covalently labeled with biotin or a biotin derivative; avidin or streptavidin covalently bound to an enzyme capable of decomposing a 1,2-dioxetane to emit light; and, a nylon membrane upon which said nuclei acid or fragment thereof is hybridized to said oligonucleotide probe.

Claim 43

Dependent Claim 18 words

The kit of claim 38, wherein said 1,2-dioxetane is AMPPD and said enzyme is acid or alkaline phosphatase.

Claim 44

Dependent Claim 16 words

The kit of claim 38, wherein said 1,2-dioxetane is AMPGD and said enzyme is a galactosidase.

Claim 45

Dependent Claim 18 words

The kit of claim 38, wherein said 1,2-dioxetane is 3-(2'-spiroadamantane)-4-methoxy-4-(3"-acetoxy)phenyl-1,2-dioxetane and said enzyme is a carboxylic acid esterase.

Claim 46

Dependent Claim 18 words

The kit of any one of claims 38-41, inclusive, further comprising image-reproducing means for detecting said light energy.

Claim 47

Dependent Claim 12 words

The kit of claim 42, wherein said image-reproducing means is photographic film.

Claim 48

Dependent Claim 145 words

A kit for detecting a nucleic acid or fragment thereof in a sample by hybridization of said nuclei acid or fragment to a complementary labeled oligonucleotide probe, comprising a 1,2-dioxetane capable of producing light energy when decomposed having the formula ##STR35## wherein T is a stabilizing cycloalkyl group spiro bonded to the 4-membered dioxetane ring, X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an enzyme-cleavable group, Y is a chromophore capable of fluorescence, and Z is hydrogen or an enzyme-cleavable group, provided that at least one of X or Z must be an enzyme-cleavable group; a complementary oligonucleotide probe covalently labeled with an antigen; an antibody directed to said antigen covalently bound to an enzyme capable of decomposing a 1,2-dioxetane to emit light; and, a nylon membrane upon which said nucleic acid or fragment thereof is hybridized to said oligonucleotide probe.

Claim 49

Dependent Claim 145 words

A kit for detecting a nucleic acid or fragment thereof in a sample by hybridization of said nuclei acid or fragment to a complementary labeled oligonucleotide probe, comprising a 1,2-dioxetane capable of producing light energy when decomposed having the formula ##STR35## wherein T is a stabilizing cycloalkyl group spiro bonded to the 4-membered dioxetane ring, X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an enzyme-cleavable group, Y is a chromophore capable of fluorescence, and Z is hydrogen or an enzyme-cleavable group, provided that at least one of X or Z must be an enzyme-cleavable group; a complementary oligonucleotide probe covalently labeled with an antigen; an antibody directed to said antigen covalently bound to an enzyme capable of decomposing a 1,2-dioxetane to emit light; and, a nylon membrane upon which said nucleic acid or fragment thereof is hybridized to said oligonucleotide probe.

Claim 50

Dependent Claim 19 words

The kit of claim 44, wherein said enzyme is an acid or alkaline phosphatase and said 1,2-dioxetane is AMPPD.

Claim 51

Dependent Claim 16 words

The kit of claim 44, wherein said enzyme is a galactosidase and said 1,2-dioxetane is AMPGD.

Claim 52

Dependent Claim 18 words

The kit of claim 44, wherein said enzyme is a carboxyl acid esterase and said 1,2-dioxetane is 3-(2'-spirodamantane)-4-methoxy-4-(3"-acetoxy)phenyl-1,2-dioxetane.

Claim 53

Dependent Claim 19 words

The kit of any one of the claims 44-47, inclusive, further comprising image-reproducing means for detecting said light energy.

Claim 54

Dependent Claim 12 words

The kit of claim 48, wherein said image-reproducing means is photographic film.

Claim 55

Dependent Claim 110 words

A kit for detecting a protein in a sample, comprising a 1,2-dioxetane capable of producing light energy when decomposed having the formula ##STR36## wherein T is a stabilizing cycloalkyl group spiro bonded to the 4-membered dioxetane ring, X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an enzyme-cleavable group, Y is a chromophore capable of fluorescence, and Z is hydrogen or an enzyme-cleavable group, provided that at least one of X or Z must be an enzyme-cleavable group; an antibody directed to said protein covalently bound to an enzyme capable of decomposing said 1,2-dioxetane derivatives to emit light; and, a membrane upon which protein-antibody binding is conducted.

Claim 56

Dependent Claim 110 words

A kit for detecting a protein in a sample, comprising a 1,2-dioxetane capable of producing light energy when decomposed having the formula ##STR36## wherein T is a stabilizing cycloalkyl group spiro bonded to the 4-membered dioxetane ring, X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an enzyme-cleavable group, Y is a chromophore capable of fluorescence, and Z is hydrogen or an enzyme-cleavable group, provided that at least one of X or Z must be an enzyme-cleavable group; an antibody directed to said protein covalently bound to an enzyme capable of decomposing said 1,2-dioxetane derivatives to emit light; and, a membrane upon which protein-antibody binding is conducted.

Claim 57

Dependent Claim 14 words

The kit of claim 50, wherein said membrane is a nylon or nitrocellulose membrane.

Claim 58

Dependent Claim 18 words

The kit of claim 50, wherein said 1,2-dioxetane is AMPPD and said enzyme is acid or alkaline phosphatase.

Claim 59

Dependent Claim 16 words

The kit of claim 50, wherein said 1,2-dioxetane is AMPGD and said enzyme is a galactosidase.

Claim 60

Dependent Claim 18 words

The kit of claim 50, wherein said 1,2-dioxetane is 3-(2'-spiroadamantane)-4-methoxy-4-(3"-acetoxy)phenyl-1,2-dioxetane and said enzyme is a carboxylic acid esterase.

Claim 61

Dependent Claim 18 words

The kit of any one of claims 50-54, inclusive, further comprising image-reproducing means for detecting said light energy.

Claim 62

Dependent Claim 13 words

The kit of claim 55, wherein said image reproducing means is photographic film.

Claim 63

Dependent Claim 108 words

A kit for detecting a protein in a sample comprising a 1,2-dioxetane capable of producing light energy when decomposed having the formula ##STR37## wherein T is a stabilizing cycloalkyl group spiro bonded to the 4-membered dioxetane ring, X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an enzyme-cleavable group, Y is a chromophore capable of fluorescence, and Z is hydrogen or an enzyme-cleavable group, provided at at least one of X or Z must be an enzyme-cleavable group; a first antibody directed to said protein; and, a second antibody directed to said first antibody covalently bound to an enzyme capable of decomposing said 1,2-dioxetane derivative.

Claim 64

Dependent Claim 108 words

A kit for detecting a protein in a sample comprising a 1,2-dioxetane capable of producing light energy when decomposed having the formula ##STR37## wherein T is a stabilizing cycloalkyl group spiro bonded to the 4-membered dioxetane ring, X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an enzyme-cleavable group, Y is a chromophore capable of fluorescence, and Z is hydrogen or an enzyme-cleavable group, provided at at least one of X or Z must be an enzyme-cleavable group; a first antibody directed to said protein; and, a second antibody directed to said first antibody covalently bound to an enzyme capable of decomposing said 1,2-dioxetane derivative.

Claim 65

Dependent Claim 18 words

The kit of claim 57, wherein said 1,2-dioxetane is AMPPD and said enzyme is acid or alkaline phosphatase.

Claim 66

Dependent Claim 16 words

The kit of claim 57, wherein said 1,2-dioxetane is AMPGD and said enzyme is a galactosidase.

Claim 67

Dependent Claim 18 words

The kit of claim 57, wherein said 1,2-dioxetane is 3-(2'-spiroadamantane)-4-methoxy-4-(3"-acetoxy)phenyl-1,2-dioxetane and said enzyme is a carboxylic acid esterase.

Claim 68

Dependent Claim 30 words

The kit of any one of claims 37, 43, 55 or 57, further comprising a water-soluble enhancing substance that increases specific light energy production above that produced in its absence.

Claim 69

Dependent Claim 13 words

The kit of claim 61, wherein said water-soluble enhancing substance is serum albumin.

Claim 70

Dependent Claim 15 words

The kit of claim 61, wherein said enhancing substance is a polymeric quaternary ammonium salt.

Claim 71

Dependent Claim 17 words

The kit of claim 63 wherein said polymeric quaternary ammonium salt is poly(vinylbenzyltrimethylammonium chloride) or poly[vinylbenzyl(benzyldimethylammonium chloride)].

Claim 72

Dependent Claim 52 words

The kit of claim 61, wherein said enhancing substance comprises a positively charged polymeric quaternary ammonium salt and fluorescein capable of forming a ternary complex with the 1,2-dioxetane anion produced following enzyme-catalyzed decomposition of said 1,2-dioxetane, whereby energy transfer occurs between said 1,2-dioxetane anion and fluorescein and light is emitted by fluorescein.

Claim 73

Dependent Claim 17 words

The kit of claim 65 wherein said polymeric quaternary ammonium salt is poly(vinylbenzyltrimethylammonium chloride) or poly[vinylbenzyl(benzyldimethylammonium chloride)].

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1993-12-17	1994-06-18	1994-12-18	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1997-12-17	1998-06-18	1998-12-18	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2001-12-17	2002-06-18	2002-12-18	$7700.00	Window Closed / Lapsed

Method of detecting a substance using enzymatically-induced decomposition of dioxetanes

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