CA2033331C - Method of detecting a substance using enzymatically-induced decomposition of dioxetanes - Google Patents

Abstract

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 formula (see formula I) where 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 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 the enzyme cleaves the enzyme-cleavable group from the dioxetane to form a negatively charged substituent banded to the dioxetane, the negatively charged substituent causing the dioxetane to decompose to form a luminescent substance that includes group Y of said dioxetane.

Description

T1 L~ L~ rD T TAT T /1TT
Method of Detecting A Substance Using Enzymatically-Induced Decomposition of Dioxetanes Field Of The Invention The invention relates to the use of dioxetanes to detect a substance in a sample.
Background Of The Invention Dioxetanes are compounds having a 4-membered ring in which 2 of the members are adjacent oxygen atoms. Dioxetanes can be thermally or photochemically decomposed to form carbonyl products, e.g., esters, ketones or aldehydes. Release of energy in the form of light (i.e., luminescence) accompanies the decompositions.
Summary Of The Invention In general, the invention features in a first aspect an improvement in art assay method in which a member of a specific binding pair (i.e., two substances which bind specifically to each other) is detected by means of an optically detectable reaction. The improvement includes the reaction, with an enzyme, of a dioxetane having the formula ~X
T Y-Z
where T is substituted (i.e., containing one or more C1-C, alkyl groups or heteroatom groups, e.g., carbonyl groups) or unsubstituted cycloalkyl ring (having between 6 and 12 carbon atoms, inclusive, in the ring) or poly-cycloalkyl .group (having 2 or more fused rings, each ring independently having between 5 and 12 carbon atoms, inclusive), banded to the 4-membered dioxetane ring by a spiro linkage; Y is a fluorescent chromophore, (i.e., Y
is group capable of absorbing energy to form an excited, i.e., higher energy, state, from which it emits light to return to its original energy state): X is hydrogen, a l0 straight ar branched chain alkyl group (having between 1 and T carbon atoms, inclusive, e.g., methyl), a straight chain or branched heteroalkyl group (having between 1 and 7 carbon atoms, inclusive, e.g., methoxy, hydroxyethyl, or hydroxypropyl), an aryl group (having at least 1 ring, e.g., phenyl), a heteroaryl group (having at least 1 ring, e.g., pyrrolyl or pyrazolyl), a heteroalkyl group (having between 2 and 7 carbon atoms, inclusive, in the ring, s.g., dioxane), an aralkyl group (having at least 1 ring, e.g., benzyl), an alkaryl group (having at least 1 ring, e.g., tolyl), or an enzyme-cleavable group, i.e., a group having a bond which can be cleaved by an enzyme to yield an electron-rich moiety bonded to the dioxetane, e.g., phosphate, where a phosphorus-oxygen bond can be cleaved by an enzyme, e.g., acid phosphatase or alkaline phos-phatase, to yield a negatively charged oxygen bonded to the dioxetane; and Z is hydrogen, hydroxyl, or an enzyme-eleavmble group (as defined above), provided that at least one o! X or Z must be an enzyme-cleavable group, so that the enzyme cleaves the enzyme-cleavable group to form a negatively charged substituent (e. g., an oxygen anion) bonded to the dioxetane, the negatively charged substi-tuent causing the dioxetane to decompose to form a luminescent substance (i.e., a substance that Baits energy in the form o! light) that includes group Y. The lumines-cent substance is detected as an indication o! the pres-ence o! the first substance. By measuring the intensity 3 x_2033331 of luminescence, the concentration of the first substance can be determined.
In preferred embodiments, one or more of groups T, X, or Y further include a solubilizing substituent, e.g., carboxylic acid, sulfonic acid, or cjuaternary amino salt;
group T of the dioxetane is a polycycloalkyl group, preferably adamantyl; the enzyme-cleavable group includes phosphate; and the enzyme includes phosphatase.
The invention also features a kit for detecting a to first substance in a sample.
In a second aspect, the invention features a method of detecting an enzyme in a sample. The method involves contacting the sample with the above-described dioxetane in which group Z is capable of being cleaved by the enzyme being detected. The enzyme cleaves group Z to form a negatively charged substituent (e. g., an oxygen anion) bonded to the dioxetane. This substituent destabilises the dioxetane, thereby causing the dioxetane to decompose to form a luminescent substance that includes group Y of the dioxetane. The luminescent substance is detected as an indication of the presence of the enzyme. By measuring the intensity of luminescence, the concentration of the enzyme can also be determined.
The invention provides a simple, very sensitive method for detecting substances in samples, a.g., biologi cal samples, and is particularly useful for substances present in lore concentrations. Because dioxetane decom position serres as the excitation energy source for chromophore Y, an external excitation energy source, e.g., light, is not necessary. In addition, because the dioxe-tane molecules are already in the proper oxidation state for decomposition, it is not necessary to add external oxidants, e.g., Hi02 or OZ. Enzyme-activated decomposi-tion allows for high sensitivity because one enzyme molecule can cause many dioxetane molecules to luminesce, thus creating an amplification effect. Moreover, the wavelength (or energy) of emission and the quantum yields of luminescence can be varied according to the choice of the Y substituent of the dioxetane (as used herein, "quantum yield" refers to the number of photons emitted from the luminescent product per number of moles of dioxe-tane decomposed). In addition, through appropriate modi-fications of the T, X, and Y groups of the dioxetane, the solubility of the dioxetane and the kinetics of dioxetane decomposition can be varied. The dioxetanes can also be attached to a variety of molecules, e.g., proteins or haptens, or immobilization substrates, e.g., polymer membranes, or included as a side group in a homopolymer or copolymer.
Other features and advantages of the invention will be apparent from the following description of the pre ferred embodiments thereof, and from tho claims.
Description Of The Drawinq~
FIGURE 1 compares a solid state quantitative color-imetric assay for human chorionic gonadotropin (hCG) using p-nitrophenyl phosphate (PNPP) as chromogen with the quan-titative chemiluminescence assay of the invention using 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phos-phoryloxy)-phenyl-1,2-dioxetane, disodium salt (AMPPD) as the lumogen.
FIGURE 2 shows the results of tho solid state immuno assay for hCG of the invention using AMPPD as the lumoqen and using film exposure for detection o! the hCG.
FIGURE 3 is a standard curve for the quantitative estimation of the concentration of the enzyme alkaline phoaphatase by the AMPPD chemfluminescence assay of the 3o invention.
FIGURE 4 compares the quantitative estimation of the concentration of the enzyme alkaline phosphatase by the AMPPD chemiluminescence assay of the invention in the presence and absence of bovine serum albumin (BSA), fluor-escein (BS~r-Fluor.) poly[vinylbenzyl(benzyldimethyl ammonium chloride)] (BDMQ), and BDMQ-Fluor.

FIGURE S shows the results of a Herpes Simplex Virus I (HSVI) ONA probe assay using a specific alkaline phosphatase-labeled DNA probe in conjunction with the AMPPD chemiluminescence assay of the invention.
5 FIGURE 6 shows the time course of the AMPPD chemi-luminescence method of the invention applied to the hybridization-based detection of hepatitis H core antigen plasmid DNA (HBVe) with an alkaline phosphatase-DNA probe conjugate, using a film detection technique.
l0 FIGURE 7 shows the time course of the colorimetric detection of Hepatitis B core antigen plasmid DNA with an alkaline phosphatase-DNA probe conjuqate using nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) as substrates.
FIGURE 8 shows the quantitative application of the assay of Figure 6, wherein the film images were quantified by measuring reflection densities.
FIGURE 9 compares a solid state ELISA method for alpha feto protein (AFP) using PNPP as a colorimetric substrate and the AMPPD chemiluminescence method of the invention ror the quantitative estimation of AFP, wherein alkaline phosphatase is covalently linked to anti-AFP
antibody.
FIGURE 10 shows a solid state monoclonal antibody BLISA for thyroid stimulating hormone (TSH) using the Al~IPPD chemiluminescence method of the invention wherein monoclonal anti-~-TSH antibody conjugated to alkaline phosphatase was used was the detection antibody.
FIGURE 11 represents the assay of Figure 10 carried out both in the absence and presence of BSA.
FIGURE 12 shows the application of a solid state ELISA to the estimation of carcino-embryonic antigen (CEA), wherein a-CEA antibody-alkaline phosphatase was the detection antibody and the AMPPD chemiluminescence method of the invention was used to quantify the CEA.

FIGURE 13 is a diagram of the device used for the solid state immunoassay for human luteinizing hormone (hhH).
FIGURE 14 shows the assay images on film for a solid state immunoassay for hLH wherein monoclonal anti-hLH
antibody-alkaline phosphatase is the detection antibody and the AMPPD chemiluminescence assay of the invention was used to detect the hLH antigen.
FIGURE 15 shows a standard curve obtained for hLFi wherein the film images obtained by the method of Figure 14 ware quantified by reflection density det~rminations at each concentration of hLH.
FIGURE 16 shows a plot of chemiluminescance as a function of ~-galactosidase concentration in the chemi luminescence assay of the invention wherein the substrate for the enayme is 3-(2~-spiroadamantane)-4-methoxy-4-(3"-~B-D-galactopyranosyl) phenyl-1, 2-dioxetana (A?IPGD).
FIGURE 17 shows the pH dependence o1~8-galactosidase-activated chemiluminescence from AMPGD.
FIGURE 18 shows the production of light by p-galactosidase decomposition of AMPGD wherein the light intensity was measured after enzyme incubation at a pH of 7.3 and adjusting the pH to 12 with alkali.
FIGURE 19 shows a two-hour lumiautogram on X-ray film (A) and Polaroid instant black and white film (B) of DNA
fragments visualized by AMPPD chemiluminescence following electrophoretic separation of DNA fragments produced by the Sangar sequencing protocol.
~7gscr p~ion Qf The Pre,~rred Embodiments 3o The structure, synthesis, and use of preferred esbodiments of the invention will now be described.
Structure The invention employs dioxetanes having the structure recited in the Summary of the Invention above. The purpose o! group T is to stabilize the dioxetane, i.e., to .233331 prevent the dioxetane from decomposing before the enzyme-cleavable group Z is cleaved. Large, bulky, sterically hindered molecules, e.g., fused polycyclic molecules, are the most effective stabilizers. In addition, T preferably contains only C-C and C-H single bonds. The most preferred molecule is an adamantyl group consisting of 3 fused cyclohexyl rings. The adamantyl group is bonded to the 4-membered ring portion of the dioxetane through a spiro linkage.
Group Y is a fluorescent chromophore bonded to enzyme-cleavable group Z. Y becomes luminescent when an enzyme cleaves group Z, thereby creating an electron-rich moiety which destabilizes the dioxetane, causing the dioxetane to decompose. Decomposition produces two indi-vidual carbonyl compounds, one of which contains group T, and the other of which contains groups X, Y, and z; the energy released from dioxetane decomposition causes the Y
groups of the latter carbonyl compound to luminesce (if group X is hydrogen, an aldehyde is produced).
The excited state energy of chromophore Y (i.e., the energy chromophore Y must possess in order to emit light) .
is preferably less than the excited state energy of the ketone containing group T in order to confine luminescence to group Y. For example, when Y.is adamantyl, the excited state energy o! chromophore Y is preferably less than the excited state energy of spiroadamantane.
llny chromophore Y can be used according to the inven tion. In general, it is desirable to use a chromophore which aaximizes the quantum yield in order to increase sensitivity.
Examples of suitable chromophores include the following:
1) anthracene and anthracene derivatives, e.g " 9, io-diphenylanthracene, 9-methylanthracene, 9-anthracene carboxaldehyde, anthryl alcohols and 9-phenylanthracene:
2) rhodamine and rhodamine derivatives, e.g., rhodols, tetramethyl rhodamine, tetraethyl rhodamine, diphenyldimethyl rhodamine, diphenyldiethyl rhodamine, and dinaphthyl rhodamine:
3) fluorescein and fluorescein derivatives, e.g., 5-iodoacetamido fluorescein, 6-iodoacetamido fluorescein, and fluorescein-5-maleimide:
9) eosin and eosin derivatives, e.g., hydroxy eosins, eosin-5-iodoacetamide, and eosin-5-maleimide;
5) coumarin and coumarin derivatives, e.g., 7 dialkylamino-4-methylcoumarin, 4-bromomethyl-7-methoxy coumarin, and 4-bromomethyl-7-hydroxycoumarin:

6) erythrosin and erythrosin derivatives, e.g., hydroxy erythrosins, erythrosin-5-iodoacetamide and erythrosin-5-maleimide:

7) aciridine and aciridine derivatives, e.g., hydraxy aeiridines and 9-methyl aciridine:

8) pyrene and pyrene derivatives, e.g., N-(1-pyrene) iodoacetamide, hydroxy pyrenes, and 1-pyrenemethyl iodoacetate:

9) stilbone and stilbene derivatives, e.g. 6,6'-dibromostilbene and hydroxy stilbenes;

10) naphthalene and naphthalene derivatives, e.g., 5-dimethylaminonaphthalene-1-sulfonic acid and hydroxy naphthalene:

11) nitrobenzoxadiazoles and nitrobenzoxadiazole derivatives, e.g., hydroxy nitrobenzoxadiazoles, 4 chloro-7-nitrobenz-2-oxa-1,3-diazole, 2-(7-nitrobenz-2 oxa-1,3-diazol-4-yl-amino)hexanoic acid;

12) quinoline and quinoline derivatives, e.g., 6-hydroxyquinoline and 6-aminoquinoline:

13) acridine and acridine derlvatfves, e.q., N-methylacridine and N-phenylacridine;

14) acidoacridine and acidoacridine derivatives, e.g., 9-methylscfdoacridine and hydroxy-9-methylacido-acridine;

15) carbazola and carbazole derivatives, e.g., N-methylcarbazole and hydroxy-N-methylcarbazole:

9 = ?

16) fluorescent cyanines, e.g., DCM (a laser dye), .~ ' Q J J 3 3 hydroxy cyanines, 1,6-Biphenyl-1,3,5-hexatriene, 1-(4-dimethyl aminophenyl)6-phenylhexatriene, and the corresponding 1,3-butadienes.

17) carbocyanine and carbocyanine derivatives, e.g., phenylcarbocyanine and hydroxy carbocyanines;

18) pyridinium salts, e.g., 4(4-dialkyldiamino-styryl) N-methyl pyridinium iodate and hydroxy-substituted pyridinium salts;

19) oxonols: and 20) rasorofins and hydroxy resorofins.
The most preferred chromophores are hydroxy derivatives o! anthracene or naphthalene; the hydro~cy group facilitates bonding to group Z.
Group Z is bonded to chromophore Y through an enzyme-cleavable bond. Contact with the appropriate enzyme cleaves the enzyme-cleavable bond, yielding an electron-rich moiety bonded to a chromophore Y: this moiety initiates the decomposition of the .dioxetane into two individual carbonyl compounds e.g., into a ketone or an ester and an aldehyde if group X is hydrogen. Examples of electron-rich moieties include oxygen, sulfur, and amine or amino anions. The most preferred moiety is an oxygen anion. Examples of suitable Z groups, and the enzymes specific to these groups are given below in Table 1: an arrow denotes the enzyme-cleavable bond. The most preferred group is a phosphate ester, which is cleaved by alkaline or acid phosphatase enzymes.
Grouu Z
1) alkaline and acid O phosphatases AO_P ifl_Y
~D

phosphate ester esterases 2 0 3 3 3 3 1 o acetate ester decarboxylases Y-O-C-o9 carboxyl 4) phospholipase D
O
v o cxi o_c_ ( cxz > ~-cHa n t cH3- ( cHZ ) ~ c-o-cH o O
3-phospho-1,2-diacyl glycerides 5) ,A-xylosidass NoC~~ O-Y

No ~
p-D-xyloside _-ON
6) ~B-D-fucosidase CWf N of y h ~-D-fucoside a'"Z ~ ~ ~ thioglucosidase 1~ 0 1-thio-D-glucoside m Q-D-galactosidase ~ 2 0 3 3 3 ~ 1 CN
,B-D-galactoside a-D-galactosidase ~Y
Ch a-D-galactoside C.~,6N
S 10) a-D-glucosidase ~N
~+b ~Hb ~ Y
a-D-glucoside 11) CN,bN
p ~ p-D-glucosidase ~-D-qlucoside CI~~O~i 12) a-D-mannosidase a o~ ~
po b-Y
a-D-mannoside 13j ,B-D-mannosidase 2 Q 3 3 3 3 1 CHZOH
O O-Y
off off .-s Ho ~ ~
R-D-mannoside 14j HOCHZ O O-Y ~-D-fructofuranosidase OH ~-CHZOH
OH
~-D-fructofuranoside 15j ~-D-glucosiduronase 1' 0 0-Y
OH
HO H
~9-D-glucosfduronate 16 j ~0 0 ~e/VN~~~., ~ ~ ~ trypsln Q
~~l H
=NW
I
~1~~
p-toluenesulfonyl-L-arginine ester f 13 ,2033331 17) trypsin O o CH3 ~ ~' S-NH-CH-C NH-Y
ii ~ t O ~ iHZ) s N+i C=NN
NNs.
p-toluenesulfonyl-L-arginine amide Suitable X groups are described in the Summary of the Invention, above. Preferably, X contains one or more sol-ubilizing substituents, i.e., substituants'~which enhance the solubility of the dioxetane in aqueous'~solution.
Examples of solubilizing substituents include carboxylic acids, e.g., acetic acid; sulfonic acids, e.g., methane-sulfonic acid; and quaternary amino salts, e.g., ammonium bromide; the most preferred solubilizing substituent is methane-or athanesulfonic acid.
Preferably, the enzyme which cleaves group Z is co-valently bondod to a substance having a specific affinity for the substance being detected. Examples of specific affinity substances include antibodies, e.g., anti-hCG;
antigens, e.g., hCG, where the substance being detected is an antibody, e.g., anti-hCG; a probe capable of binding to all or a portion of a nucleic acid, e.g., DNA or RNA, being defeated; or an enzyme capable of cleaving the Y-Z
bond. Honding is preferably through an amide bond.
synthesis In general, the dioxetanes of the invention are synthesized in two steps. The first step involves syn-thesizing an appropriately substituted olefin having the formula -X
3d T Y-Z

wherein T, X, Y, and Z are as described above. These olefins are preferably synthesized using the Wittig reaction, in which a ketone containing the T group is reacted with a phosphorus ylide (preferably based on triphenylphosphine) containing the X, Y, and Z groups, as follows:
x x s= o t r.~
s Y-z The reaction is preferably carried out below about -70~C
in an ethereal solvent, e.g., tetrahydrofuran (TNF).
The phosphorus ylide is prepared by reacting triphenyl phosphine with a halogenated compound containing the X, Y, and Z groups in the presence of base. examples of preferred bases include n-butyllithium, sodium amide, sodium hydrid~, and sodium alkoxide: the most preferred base is n-butyllithium. The reaction sequence is as follows:
x z o p . di-x y-2 where Q is a halogen, e.g., C1, Hr, or I. The preferred halogen is Br. The reaction is preferably carried out below about -70'C in THF.
The olefin where T is adamantyl (Ad) , X is methoxy (oCIi~), Y is anthracene (An), and Z is phosphate (P04) can be synthesized as follows.
Hr-CH-OCH, is phosphorylated by treating it with the An-OH
product of phosphorus acid reacted in the presence of HgCh with N-methylimidazole: the net result is to replace the hydroxyl group of An with a phosphate group. The phosphorylated product is then reacted with triphenyl-phosphine below about -70'C in THF to form the phosphorus glide having the formula oe~, P
~An_P04 The reaction is conducted in a dry argon atmosphere, Spiroadamantanone (Ad = Oj is then added to the solution containing the glide, while maintaining the temperature below about -70'C, to form the olefin having the formula 10 ~OCHy Ad An-PO~
The olefin is then purified using conventional chromato-graphy methods.
The second step in the synthesis of the dioxetanes 15 involves converting the olefin described above to the dioxetane. Preferably, the conversion is effected photo chemically by treating by olefin with singlet oxygen ('02) in the presence o! light. ('OZ) adds across the double bond to form the dioxetane as follows:
_ X
O-O X
~+ X02 ~ ~.
T ~'-Z T \Y Z
The reaction is preferably carried out below about -70'C
in a halogenated solvent, e.g., methylene chloride. 'OZ is generated using a photosensitizer. Examples of photosen-sitizers include polymer-bound Rose Bengal (commercially known as Sansitox* Z and available from Hydron Laboratories, New Brunswick, N.J.), which ie preferred, and methylene blue (a well-known dye and pH indicator).
The synthesis of the dioxetane having the formula * Trade-mark o-o 'OCH 3 Ad ~ ~-~ a follows.
The olefin having the formula oCFis Ad An-PO~
is dissolved in methylene chloride, and the solution is placed in a 2-cm2 Pyrex tube equipped with a glass paddle;
the paddle is driven from above by an attached, glass enclosed, bar magnet. Ths solution is cooled to below about -70'C and lg of polymer-bound Rose Bengal is added with stirring. oxygen is then passed over the surface of the agitated solution while the reaction tube is exposed to light from a 500 W tungsten-halogen lamp (GE Q500 C1) equipped with a W-cut off filter (Corning 3060: trans-mission at 3s5 nm a 0.5!). Thin layer chromatography (tlc) is used to monitor the disappearance of the olefin and the concurrent appearance of the dioxetane. After the 2o reaction is complete (as indicated by tlc), the solvent is removed and the dioxetane is isolated.
A widt variety of assays exist which use visually detectable means to determine the presence or conaentra-tion of a particular substance in a sample. The above-described dioxetanes can be used in any of these assays.
Examples o! such assays include immunoassays to detect antibodies or antigens, e.g., a or ~-hCGt enzyme assays:
chemical assays to detect, e.g., potassium or sodium ions:
and nucleic acid assays to detect, e.g., viruses (e. g., HTLV IIZ or cytomegalovirus, or bacteria (e. g., ~. coli)).
When the detectable substance is an antibody, antigen, or nucleic acid, the enzyme capable of cleaving group Z of the dioxetane is preferably bonded to a substance having a specific affinity for the detectable substance (i.e., a substance that binds specifically to the detectable substance , e.g, an antigen, antibody, or nucleic acid probe, respectively. conventional methods, e.g., carbodiimide coupling, are used to bond the enzyme to the specific affinity substance; bonding is preferably through an amide linkage.
In general, assays are performed as follows. A
sample suspected of containing a detectable substance is contacted with a buffered solution containing an enzyme l0 bonded to a substance having a specific affinity for the detectable substance. The resulting solution is incubated to alloy the detectable substance to bind to the specific affinity portion of the specific affinity-enzyme compound.
Excess specific affinity-enzyme compound is then washed away, and a dioxetane having a group Z that is cleavable by the enzyme portion of the specific affinity enzyme compound is added. The enzyme cleaves group Z, causing the dioxetane to decompose into two carbonyl compounds (e. g., an ester or ketone when group X is other than 2o hydrogen and an aldehyde when group X is hydrogen)f chromophore y bonded to one of the ketones is thus excited and luminesces. Luminescence is detected using e.g., a cuvette or camera luminometer, as an indication of the presence of the detectable substance in the sample.
Luminescence intensity is measured to determine the concentration o! the substance.
When the detectable substance is an enzyme, a specific affinity substance is not necessary. Instead, a dioxetane having a z group that is cleavnble by the enzyme being detected is used. Therefore, an assay for the enzyme involves adding the dioxetane to the enzyme-containing sample, and detecting the resulting luninescance as an indication of the presence and the concentration of the enzyme.
Examples of specific assays follow.

i8 A. Assay for Human IaG
A 96-well microtiter plate is coated with sheep anti-human IgG (F(ab)2 fragment specific). A serum sample containing human IgG is then added to the wells, and the wells are incubated fox 1 hour at room temperature.
Following the incubation period, the serum sample is removed from the wells, and the vsIls are washed four times with an aqueous buffer solution containing 0.15 M
NaCl, 0.01 M phosphate, and 0.1% bovine serum albumin (pH
7.4).
Alkaline phosphatase bonded to anti-human IgG is added to each well, and the wells are incubated for 1 hr.
The wells are then washed four times with ttxa above buffer solution, and a buffer solution of a phosphate-containing dioxetane is added. The resulting luminescence caused by enzymatic degradation of the dioxetane is detected in a luminometer, or with photographic film in a camera luminometer.
B. Assa,~ for hCG
Rabbit anti-a-hCG is adsorbed onto a nylon-mesh membrane. A sample solution containing hCG, e.g., urine from a pregnant woman, is blotted through the membrane, after which the membrane is washed with 1 ml of a buffer solution containing 0.15 M NaCl, 0.01 h phosphate, and 0.1% bovine serum albumin (pH 7.4).
Alkaline phosphatase-labelled anti-~-hCG is added to the membrane, and the membrane is washed again with 2 ml of the above buffer solution. The membrane is then placed in the cuvette of a luminometer or into a camera luminometer, and contacted with a phosphate-containing dioxetane. The luainescencs resulting from enzymatic degradation of the dioxetane is than detected.
C. $~asa~, fgr ~,~erum A~~7ljne Phodphatase 2.7 ml of an aqueous buffer solution containing 0.8 M
2-methyl-2-aminopropanol is placed in a 12 x 75 mm pyrex test tube, and o.l ml of a serum sample containing alka-line phosphatase added. The solution is then equilibrated to 30'C. 0.2 ml of a phosphate-containing dioxetane is added, and the test tube immediately placed in a lumino-meter to record the resulting luminescence. The level of light emission will be proportional to the rate of alkaline phosphatase activity.
D. Nucleic Acid Hybridization Assav A sample of cerebrospinal fluid (CSF) suspected of containing cytomegalovirus is collected and placed on a nitrocellulose membrane. The sample is then chemically treated with urea or guanidinium isothiocyanate to break the cell walls and to degrade all cellular components except the viral DNA. The.strands of the viral DNA thus produced are separated and attached to the nitrocellulose filter: A DNA probe specific to the viral DNA and labelled with alkaline phosphatase is then applied to the filter: the probe hybridizes wig the. complementary viral DNA strands. After hybridization, the filter is washed with an aqueous buffer solution containing 0.2 M NaCl and 0.1 Tris-HC1 (pIi ~ 8.10) to remove excess probe molecules.
A phosphate-containing dioxetane is added and the resulting luminescence from the enzymatic degradation of the dioxetane is measured in a luminometer or detected with photographic film.
E. Assav for Galactosidase In the assays described above and in the Examples to follow dioxetanes containing a- or ~9- galactosidase-cleavabl4 a-D- or S-D-galactopyranoside groups, respect-ively, can be added, and the luminescence resulting from the enzymatic cleavage of the sugar moiety from the chromophors measured in a luminometer or detected with photographic film.

zo F. Electronhoresis Electrophoresis allows one to separate complex mixtures of proteins and nucleic acids according to their molecular size and structure on gel supports in an electrical field. This technique is also applicable to separate fragments of protein after proteolysis, or fragments of nucleic acids after scission by restriction endonucleases (as iri DNA sequencing). After electro-phoretic resolution of species in the gel, or after transfer of the separated species from a gel to a membrane, the bonds are probed with an enzyme bound to a ligand. For example, peptide fragments are probed with an antibody covalently linked to alkaline phosphatase. For another example, in DNA sequencing alkaline phosphatase -avidity binds to a biotinylated nucleotide base. There-after, AMPPD is added to the gel or membrane filter.
After short incubation, light is emitted as the result of enzymatic activation of the dioxetane to form the emitting species. The luminescence is detected by either X-ray or instant photographic film, or scanned by a luminometer.
Multichannel analysis further improves the process by allowing one to probe for more than one fragment simultaneously.
G. In solid state assays, it is desireabls to block nonspecific binding to the matrix by pretreatment of nonspaci!ic binding sites with nonspecific proteins such as bovine serum albumin (BSA) or gelatin. Applicant has determined that soma commercial preparations of BSA
contain small amounts of phosphatase activity that will produce undesirable background chemiluminescence from AMPPD. Applicant has discovered that certain watar-soluble synthetic macromolecular substances are efficient block.rs of nonspecific binding in solid state assays using dioxetanes. preferred among such substances are water-soluble polymeric quaternary ammonium salts such as poly(vinylbenzyltrimethyl ammonium chloride) (TMQ) or poly(vinylbenzyl(benzyldimethylammonium chlorid~)](BDMQ).
H. ss ~ for Nuc~gotidase An assay for the enzyme ATPase is performed in two steps. In the first step, the enzyme is reacted at its optimal pH (typically pH 7.4) with a substrate comprising ATP covalantly linked via a terminal phosphoester bond to a chromophore-substituted 1,2-dioxetane to produce a phos phorylchromophore substituted 1,2-dioxstane. In the l0 second step, the product of the first step is decomposed by the addition of acid to bring the pH to below 6, pref-erably to pN 2-4, and the resulting light measured in a luminometer or detected with chromatographic film. In a similar two-step procedure, ADPase is assayed using as the substrate an ADP derivative of a chromophore-substituted 1,2-dioxetane, and 5~-nucleotidase assayed using as the substrate an adenylic acid derivative of a chromophore-substitutsd 1,2-dioxetane. The second step can also be carried aut by adding the enzyme alkaline phosphatase to decompose the phosphoryl-chromophore-substituted 1,2-dioxetane.
I. Nucleic Acid g~vuencing DNA or atNA fragments, produced fn sequencing proto cols, can bs detected after electrophoretfc separation using the chemiluminescent 1,2-dioxetanes at this invention.
DNA sequencing can be performed by a dideoxy chain termination method [Sanger, F., g~ ~., Proc. Nat. Aced.
Sci. (USAl, 74:5463 (1977)). Briefly, for each of the 3o four sequencing reactions, single.stranded template DNA is mixed with dideoxynucleotides and biotinylated primer strand DNA. After annealing, Klenow enzyme and deoxy-adenosine triphosphate are incubated with each of the four sequencing reaction mixtures, then chase deoxynucleotide triphosphate is added and the incubation continued.

_2033331 Subsequently, DNA fragments in reaction mixtures are separated by polyacrylamide gel electrophoresis (PAGE).
The fragments are transferred to a membrane, preferably a nylon membrane, and the fragments cross-linked to the membrane by exposure to W light, preferably of short wave length.
After blocking non-specific binding sites with a polymer, e.g., heparin, casein or serum albumin, the DNA
fragments on the membrane are contacted with avidin or streptavidin covalently linked to an enzyme specific for the enzyme cleavable group of the 1,2-dioxetane substrates of this invention. As avidin or streptavidin bind avidly to biotin, biotinylated DNA fragments will now be tagged with an enzyme. For example, when the chemiluminescent substrate is 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phos-phoryloxy)phenyl-1,2-dioxetana salt (AMPPD), avidin or streptavidin will be conjugated to a phosphatase.
Similarly, when the chemiluminascant substrata is 3-(2'-spiroadamantaae)-4-methoxy-4-(3"-~-D-galactopyranosyl) phenyl-1,2-dioxetane (AMPGD), avidin or streptavidin are conjugated with p-galactosidase.
Following generation of luminescence by contacting the complex of DNA fragment biotinavidin (or strepta-vidin) enzyme with the appropriate 1,2-dioxetane at alkaline pH values, e.g., above about pH 8.5, DNA frag-ments era vfsualiaad on light-sensitive film, e.g, x-ray or instant film, or in a photoelectric luminometer instrument.
The detection method outlined above can also be applied to the genomic DNA sequencing protocol of Church g~ ~. [Church, G.M., g~ ate., Proc. Nat. Aced. Sci. (USA1, 81:1991 (1984)x. After transferring chemically cleaved and electrophoretically separated DNA [Maxam, A.M. g~ ~., Proc. Nat. Aced. Sci. (USA1, 74:560 (1977)] to a membrane, preferably a nylon membrane, and cross-linking the ladders to the membrane by light, specific DNA sequences may b~
detected by sequential addition of: biotinylated oligo-nucleotides as hybridization probes; avidin or strepta-vidin covalently linked to an enzyme specific for an enzyme.cleavable chemiluminescent 1,2-dioxetane of this invention; and, the appropriate 1,2-dfoxetane. Images of sequence ladders (produced by PAGE) may be obtained as described above.
Serial reprobing of sequence ladders can be accomplished by first stripping the hybridized probe and chemiluminescent material from a membrane by contacting the membrane with a heated solution of a detergent, e.g., from about 0.5 to about -5~ sodium dodecylsulfate (SDS) in water at from about 80'C to about 90°C , cooling to from about 50'C to about 70'C, hybridizing the now-naked DNA fragments with another biotinylated oligonucleotide probe to generate a different sequence, then generating an imaging chemiluminescence as described above.
Similar visualization methods can be applied to RNA
fragments generated by RNA sequencing methods.
Other embodiments are within the following claims.
For example, the enzyme-cleavable group Z can be bonded to group X of the dioxetane, instead of group Y.
The specific affinity substance can be bonded to the dioxetane through groups X, Y, or T (preferably group x), instead of the enzyme. In this case, the group to which the specific affinity substance is bonded is provided with, e.g., a carboxylic acid, amino, or maleimide subetituent to facilitate bonding.
Groups X, Y, or T of the dioxetane can be bonded to a polymerizable group, e.g., a vinyl group, which can be polymerized to form a homopolymer or copolymer.
Groups x, Y, or T of the dioxetane can be bonded to, e.g., membranes, films, beads, or polymers for use in immuno- or nucleic acid assays. The groups are provided with. e.g., carboxylic acid, amino, or maleimide substi-tuents to facilitate bonding.
Groups X, Y, or T of the dioxetane can contain substituents which enhance the kinetics of the dioxetane enzymatic degradation, e.g., electron-rich moieties (e. g., methoxy).
Groups Y and T of the dioxetane,. as well as group x, can contain solubilizing substituents.
Appropriately substituted dioxetanes can be synthe-sized chemically, as well as photochemically. For example, the olefin prepared from the Wittig reaction can be epoxidized using a peracid, e.g., p.nitroperbenzoic acid. The epoxidized olefin can then be converted to the dioxetane by treatment with an ammonium salt, e.g., tetra-methylammonium hydroxide.
Another example of a chemical synthesis involves converting the olefin prepared from the Wittig reaction to a 1, 2-hydroperoxide by reacting the olefin with HZOZ and dibromantin (1,3-dibromo-5,5-dimethyl hydantoin). Treat-ment of the 1,2-bromohydroperoxide with base, e.g., an alkali or alkaline earth methylhydroxide such as sodium hydroxide or a silver salt, e.g., silver bromide, forms the dioxetane.
2o Olefin precursors for the dioxetane can be synthe-sized by reacting a ketone with a ester in the presence of TiCl and lithium aluminum hydride (LAN). For example, to synthesize an olefin where T is adaman~yl (Adj, X is methoxy (OCHIj, Y is anthracene (Anj, and Z is phosphate (POD), the following reaction sequence is used:

Ad ~ 0 + An - C - 0 - CHI , T,~ Cl~j,B,)~ AD-"", s ~_~<
To phoephorylate chromophore Y, e.g., anthracene, a 3o hydraicyl derivative of the chromophore, e.g., hydroxy anthracene, can be reacted with a cyclic aryl phosphate having the following formula:

~O
CH3 CO ~ P _OCH3 The reaction product is then hydrolyzed with water to yield the phosphorylated chromophore. The cyclic acyl phosphate is prepared by reacting 2,2,2-trimethoxy-4,5-dimethyl-1,3-dioxaphospholene with phosgene at 0°C, 5 following by heating at 120°C for 2 hr.
The following examples are intended to illustrate the invention in detail, but they are in no way to be taken as limiting, and the present invention is intended to encom-pass modifications and variations of these examples within 10 the framework of their contents and the claims.
orioaic GQnad~ouhin (hCG) Assa In the following, an hCG assay method is described in 15 which 3-(2'spiroadamantane)-d-methoxy-4-(3"-phosphoryi-oxy)phenyl-1,2 dioxetane, disodium salt (AMPPD, synthe sized as described above), was used as a substrate of alkaline phosphatase. For comparison, a colorimetric assay was conducted using p-nitrophanylphosphoric acid 20 (PNPP) as a substrata.
1. Placed one bead which was previously coated with anti-hCG in each tube (12 x 75 mm) alter blotting excess buffer from bead.
Z. Added 1o0 dal of anti-hCG antibody-alkaline 25 phosphataea conjugate to each tube.
3. To each tube added i00 pl of sample. Separate tubes were prepared for each of the following:
a) Control Zaro Sample, (male serum or urine) b) 25 mIU/ml hCG standard (serum or urine) 3o c) 200 mIU/ml hCG standard (serum or urine) d) Patient sample (serum or urine) 4. After mixing, the tubes Were covered and incubated for 90 minutes at » 'C.
5. The reaction solution containing the conjugate and sample were aspirated to waste.

6. The beads were washed 5 times with 2.0 ml of phosphate buffered saline, pH 7.4, containing 0.1~ Tween 20.
For Colorimetric Assav ~hemfluminescence 7. N/A 7. Washed once with O.D5 M carbonate, 1 mM
MgClZ pH 9.5.
8. Added 200 ~1 1 mg/ml p- e. Added 250 ~1 of 0.4 nitrophenyl-phosphate mM AMPPD in O.oS M
(PNPP) in 0.1 m GLYCINE, carbonate, 1 mM MgClZ, 1 Mm MgClZ, pH 10.4 pH 9.5 9. Incubated for 30 minutes 9. Incubated for 20 at room temperature. minutes at 30~C.
10. Added 11.5 ml of 0.1 M 10. N/A
glycine, 10 mM of EDTA, pH 9.5, to stop color development.
11. Read absorbance at 405 11. Read 10 sec. integral nm in spectrophotometer of luminescence from each tube in Turner 2oE Luminometer 12. Plotted both sets of data as the signal at each concentration of hCG divided by the signal at zero hCG vs.
concentration of hCG. Typical data are plotted in Fig. 1, wherein PNPP represents the colorimetric assay and AMPPD
the chemiluminescence assay. The chemiluminescence assay was over ten times as sensitive as the colorimetric assay.
Example z Tandem Icon II hCG Assay (By Film Exposure) 3o Used a commercial Tandem ICON * II assay kit (Hybritech, Inc.j. Buffers and antibodies used were included in the kit and AMPPD was used as a substrate of alkaline phosphatass.
* Trade-mark Method 1. Prepared hCG standards at 0, 5, 10, 50 mIU/ml diluted in. control negative (male) urine for use as test samples.
2. Added 5 drops of the sample to the center of an ICON membrane device.
3. Added 3 drops of enzyme antibody conjugate to the center of each device. ' 4, Incubated for 1 minute.
5. Added 2 ml of Hybritech ICON wash solution to the device. Allowed to drain.
6. Added 500 ~C1 of 0.1% BSA inØl M Tris buffer, 1 mM MgCIZ, pH 9.8. Allowed to drain.
7. Added 200 ~l of 50 ~g/ml AMPPD in 0.1% BSA, 0.1 Tris buffer, pH 9.8, 1 mM MgCl2.
8. Transferred ICON membrane to a piece of Mylar polyester film and inserted into a black box to expose film. (Polaroid Type 612).
9. Exposed film for 30 seconds, The results of a typical assay are shown in Fig. 2. Intense chemilumines cence from positive samples occurred within a 30-second reaction time.
Example 3 Alkaline Phosnhatase Assav An assay for alkaline phosphatase was conducted in tha lollowinq manner.
ComDOnents 0.05 M carbonate, i mM MgClZ at pH 9.5.
&ubstrate:
3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD) at o.4 mM
concentration.

Alkali~pe Phosnhatase:
stock solution at 1.168 ~g/ml in the buffer.
Serial dilutions of alkaline phosphatase stock solu tions were made in tubes with final enzyme concentrations of 4.17 x10 ~~M 1.67 x 10~~5M

8.34 x10~~2M 8.34 x 10-~bM

1.67 x10~~ZM 4.17 x 10 ~bM

3.34 x10-~3M 2.09 x 10~~6!!

6.68 x10 ~~'M

1.34 x10~~~'M

3.34 x10 GSM

p_rp~e ur : ' Duplicate tubes at each of the above concentrations of alkaline phosphatase also containing 0.4 mM AMPPD were incubated at 30'C for 20 minutes.
After incubation, 30-second light integrals were measured in a Turner 20E Luminometer. The limits of detection of alkaline phosphatase is shown in Table II.
Data for the detection of alkaline phosphatase using 0.4 m2t AMPPD is shown in Figure 3. Light production was linear between 10'4 to 10'~~ M enzym~.
Concentration of Alkaline Minimum Detectable Phosphatase for 2X Conc. of Alkaline $$~qn Hackaround Phosnhatase None 1.0 x 10~~~ 1.67 x 10~~sM (1.12) 1. Buffer: 0.05 M sodium carbonate, 1 mM MgCl2, pH 9.5.
Temperature: 30'C. A?IPPD concentration was 0.4 mM.
a . The number in parentheses is the aultiple of back-qround at the indicated concentration.

Exay le 4 Alkaline P' osp~atase Assay in the Presence of Bovine Serum Alb~,~. BSA-Fluor. HDMO and BDMO-Fluor An assay for alkaline phosphatase was conducted in the following manner.
components:
B~fer: 0.05 M sodium carbonate, 1 mM MgClz, at pH 9.5.
S~strate: 3-(2'-spiroadamantane)-4-methoxy-4-(3"phos phoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD) at l0 0.4 mM concentration.
~, kalin~Phos ate: stock solution at 1.168 pg/ml in the buffer.
Conditions Tasted:
1. Buffer alone, control.
2. Buffer plus 0.1% bovine serum albumin (eSA).
3. Buffer plus 0.1% HSA-fluorescein (BSA to fluorexcein ratio 1 to 3).
4. Buffer plus 0.1% poly[vinylbenzyl(benzyl-dimethylammonium chloride)) (BDMQ).
5. Buffer plus 0.1% BDMQ and fluorescein (0.01 mg of fluorescein disodium salt mixed with 1 0l of HDMQ).
Serial dilutions of alkaline phosphatase stock solutions wars made in tubes at the final enzyme concentrations of:
4.17 x 10~~~M 1.67 x 10'~SM
8.34 x 10~~ZM 8.34 x 10 ~s!!
1.67 x 10 ~ZM 4.17 x 10-~~9 3.34 x 10 ~jM 2.09 x 10'~bll 6.68 x 10~"M 1.0 x 10'~aM
1.34 x 10 ~~M 5.0 x 10~~rM
3.34 x 10 ~sM 2.5 x 10~~TM

procedure:
Duplicate tubes with alkaline phosphatase at concen-trations described above also containing 0.4 mM AMPPD were incubated at 30'C under various conditions. Test tubes 5 were incubated for 20 minutes under conditions 1, 4 and 5, while incubated for 90 minutes under conditions 2 and 3.
After incubation, 30 second light integrals were measured in a Turner 20E Luminometer. The effect of BSA, BDMQ and fluorescein on the liaits of detection of to alkaline phosphatase is shown in Fig. 4 and Table III. In Fig. 4, - Q - corresponds to results under condition 1 above: ... . . condition 2: ... Q ... condition 3:
... ~ ..~ondition 4; and .. Q .:. condition 5, respectively.
15 fable III
Concentration of Alkaline Minimum Detectable Phosphatasefor 2X Cone, of alkaline gddition Backgro und Phosphat ase None 1. 0 x 10-~~ 1. 67 x 10 ( GSM 1.12 ) ~

20 0.1~ BSJ~ 9.5 x 10 ESN 8.34 x 10~1~!!(1.06) 0.1~ HSJi: 1.3 x 10~~sM 4.17 x 10-~6M (1.04) Fluorescein O.li HOtiQ 4.0 x 10~t~1 1.00 x 10~~6t~t(1.07) 0.1~ BDMQ: 3.4 x 10-~SM 2.09 x 10-~aM (1.06) 25 Fluorascein The number in parenthesis is the multiple of background at the indicated concentration.
ample 5 30 $SVZ DNA Probe Assay b;~eri s and Buffers:
liembrane: Game Screen Plus; Positively charged nylon membrane.
Buffers: Denaturation Buffer: 0.5 H NaOH
Neutralization Buffer: 0.4 M NaHiPO~ pH 2.0 * Trade-mark Loading Suffer, 1 part Denaturation Buffer, 1 part Neutralization Buffer Membrane Wetting Buffer: 0.4 M Tris buffer pH 7.5 Membrane Prehybridization Buffer:

Final Concentration 0.5 ml loo x Denhardt's 5%

solution 0.5 ml 10% SDS 0.5%

102.5 ml 20 x SSPE 5%

2.0 mg denatured, sonicated salmon sperm DNA
' 200 ~tg/ml 1510 ml Membrane Hybridization Buffer:

Final Concentration 0.5 ml 100 x Denhardt's 5%

20solution 0.5 ml 10% SDS 0.5%

2.5 ml 20 x SSPE 5%

2.0 mg salmon sperm DNA 200 ~g/ml 2.0 ml 50% Dextran sulfate 10%

ml ~aeh Buffer I:

1 x SSPE/0.1% SDS

ml 20 x SSPE

304 ml 10% SDS.

376 ml ddH20 400 ml Wash Buffet II: 0.1 x SSPE/0.1%DS preheated S to wash temperature.

352 ml 20 x SSPE

4 ml 10% SDS

ddHZO

?8951-11 400 ml (heated) Wash Buffer III:
0.1 x SSPE/0.1~ SDS
20 ml 20xSSPE
4 ml lOt SDS
394 ml ddH20 400 ml.
Wash Hufler IV:
3 mN Tris-HCl (pH 9.5) 0.6 ml IN Trizma Base 199.4 ml ddH20 200.0 nl.
100X Denhart's Solution:
Dissolved 2 g of polyvinylpyrrolidone mol. wt.
40K (PVP-40) and 2 g of Ficoll at temperatures greater than 65'C hut less than boiling. Cooled the solution to approximately 40'C, added 2 g of BSA and brought the final volume of 100 ml with ddH20. Aliquots were stored at -20'C.

2oX SSC (for 100 ml) ,3.0 N Sodium Chloride 17.48 0.3 N Sodium Citrate 8.8q Bring volume to 100 ml and filter through a 0.45~tm nitrocellulose filter. Store at room temperature.

2oX SSPE pH 7.4 (for 1 liter) 3.6N NaCl 210.248 200 mM Sodium phosphate 238 dibasic, 5.928 monobasic 20 mN EDTA 7.448 Dissolve, adjust pH to 7.4 with 5 N NaOH
Bring volume to 1 liter and filter through a 0.45~m nitrocellulose filter.

1X TE buffer 10 ~i Tris (pH 7.0) * Trade-mark 1 mM EDTA
Autoclave Method:
1. Prewetted membrane with Wetting Buffer for 15 min.
2. Inserted membrane into a vacuum manifold device.
3. Denatured the DNA sample by adding 50 ~ul of DNA
sample (with known number of copies of HSVI DNA) to 200 ~l of Denaturation Buffer. Incubated l0 min. at room temper l0 ature. Added 250 ml of ice cold Neutralization Buffer and kept denatured DNA on ice.
4. Added 200 ~l of Loading Buffer to~each well and aspirated through membrane.
5. Loaded denatured DNA samples to each well, and aspirated through membrane.
6. Repeated Step 4.
7. Dissembled manifold and removed membrane.
8. W-fixed DNA to membrane using a W
Transilluminator for 5 minutes.
9. Incubated the membrane in 0.1% (w/v) BDMQ in phosphate-buffered saline for 15 minutes.
10. Incubated membrane in Prehybridization Buffer at 70'C for 1 hour.
11. Added alkaline phosphatase-labeled SNAP probe specific for HSVI dissolved in Membrane Hybridization Buffer. Incubated for 3-5 hours at 70'C.
12. Removed membrane from Hybridization Buffer and incubated in 400 nl of wash Buffer i, while agitating at room temperature for 10 minutes.
13. Washed with 400 ml of Wash Buffer II at 50'C for 30 minutes.
14. Washed with 400 ml of Wash Buffer III at room temperature for 10 minutes.
15. Washed with 200 ml of Wash Buffer IV at room temperature for 10 minutes.

16. Added 2 ml of 300 ~g/ml AMPPD in 0.1 M Tris buffer, 1 mM MgClZ, pH 9,8 to the membrane.
17. Transferred the membranes to a piece of Mylar polyester film, and then to a black box containing Type 612 Polaroid film.
18. Exposed film for 30 minutes. Typical results are shown in Fig. 5, wherein Fig. 5A shows the results at 60 ~ag/ml AMPPD, Fig. 5B at 300 ~g/ml AMPPD, and Fig. 5C
after the first 30 min. o! reaction at 300 ~g/ml AMPPD.
Exa~ggle 6 Hepatitis B V rus DNA Hybridization Assay We compared the sensitivity of a chemiluminesceat substrate (AMPPD) and a chromogenic substrate (BCIP/NBT) for detection of an alkaline phosphate label in Hepatitis B Virus Core Antigen DNA HBV~ probe hybridization assay (SNAP~_, DuPont). Chemiluminescent signals obtained from AMPPD hydrolysis by said phosphatase was detected with Polaroid Instant Black and White Typs 612 film.
~,ethods and Materials:
1. Chemiluminescent Substrate: AMPPD
2. Protocol iQ~ Determining the Sensit~vit_y of SNAPe/Test for HBV~ ~(~gpatitis B "Core Antigen" DNA1 The levels of detection, or the sensitivity, of the SNAPS DNA probe test for Hepatitis 8 "Core Antigen~ DNA
were determined by performing the test using serially diluted HBV~ control plasmid DNA.
The assay protocol involved the following steps:
a. Preuaration of Positive HBV_ DNA Plasmic Controls A stock solution of HBV~ plasmfd was prepared by dissolving 100 ng (1.2 x 1010 copies) of the plasmid in 25 ul of sterile, deionized HZO and serially diluted with 0.3 N NaOH to produce plasmid samples in the concentra-tions range of 4.88 x 103 - 0.96 x l0a copiea/ul. The samples were allowed to denature for 15 minutes at room temperature.
b. Preparation of the Membranes. Immobilization of HBV_ Plasmid Control DN1, 5 Gene Screen"' Plus membranes were cut into 1 x B cm strips. 1 ul of each dilution of HBV~ plasmid sample was spotted on the dry membrane with a pipette tip in contact with the membrane surface to obtain very small, concen-trated spots. The membranes were then rinsed with 100 ul 10 of 2 M ammonium acetate per spot to neutralize the target immobilized nucleic acid. They were subsequently rinsed with 0.6 M sodium chloride, 0.08 M sodium citrate, pH 7.0 buffer.
c. probe Hybridization 15 (1) Prehybridization The membranes containing plasmid samples were placed in a heat-sealable pouch in 3 ml of Hybridization Buffer.
Prehybridization was carried out for 15 minutes at 55'C.
(ii) Hybridization 20 SN7~Pa alkaline phosphatase labeled probe was recon-stituted with 100 ul of the sterile deionized HzO. The hybridization solution was prepared using 2.5 ul alkaline phosphatase labeled probe solution dissolved in 0.5 ml Hybridization Buffer. Hybridization was performed in a 25 new, heat sealed pouch, with 0.5 ml hybridization solution, for 30 minutes at 55'C. lifter hybridization, the pouch was opened and the menbranas carefully removed and washed with the following buffers:
1. twice with 0.1 M sodium chloride, 0.02 K sodium 30 citrhte, pH 7.0, plus i0 g SDS butter, for 5 minutes at room temperature, 2. twice with 0.1 H sodium chloride, 0.02 Ii sodium citrate, pH 7.0, plus 10 ml Triton*X-10o (sigma chemical Co., St. Louis, MO), for 5 minutes at 55'C, * Trade-mark 3. twice with the above buffer for 5 minutes at room temperature, 4. twice with 0.1 M sodium chloride, 0.02 M sodium citrate, pH 7.0 buffer for 5 minutes at room temperature, 5. once with 0.1% BSA in 0.05 M carbonate buffer at pH 9.5.
Hybridization Hu~Ler was prepared by mixing 250 ml of 3 M sodium chloride, 0.4 M sodium citrate, pH 7,0, diluted to 800 ml with deionized HZO, with 5 g Bovine Serum l0 Albumin, 5 g polyvinylpyrrolidone (average MW 40,000) and g SDS, warmed and mixed to dissolve.
d. C~,~milw~inescent Detection of HBV Plasmid DNA with ppp ' The hybridized membrane strips were saturated with 100 ul of 1.6 mM AMPPD in 0.1% BSA in 0.05 M carbonate Buffer, 1.0 MgCl2 at pH 9.5. The membranes were then sealed in a plastic pouch and immediately placed in a camera luminometer where light emission was imaged on Polaroid Instant Hlack/White 20,000 ASA film.
e. detection w.~~h SNAPa Chromogenic S~strates ('[iitro Blue Tatrazoliym (NHT) 5-Bromo-4-Chlora-3-Indolyl phosgjiate l'~CIPLIPerformed According- to the Manu~r~,uer~s instructions) Hybridized membranes which were developed with the chromogonic substrates did not undergo wash step ~5.
Substrate solution was prepared by mixing 33 ul NBT and 25 ul of BLIP in 7.5 ml of alkaline phosphatase substrate buffer provided by the manufacturer. Washed hybridized membranes were transferred to a heat sealed pouch with the substrates containing buffers. Tha color was allowed to develop in the dark, as NBT is light sensitive.
f. Photaq~~hic Detection of AMPPD Signal The results of assays performed with AMPPD were imaged on Polaroid Instant Black and White Type 61Z photo-graphic film. The images were subsequently digitized using a black and white RBP Densitometer, Tobias Associates, Inc., Ivyland, PA.
Results:
Figure 6 shows a time course of the chemiluminescent for serially diluted Hepatitis B Virus "Core Antigen"
plasmid hybridized with alkaline phosphatase labeled probe and imaged onto photographic film. Each photograph corresponds to a 30 minute exposure on Polaroid Instant Black and White Type Copies of 612 film. A comparable set of serially diluted Hepatitis B Virus "Core Antigen"
plasmid DNA hybridized with alkaline phosphatasa labeled probe and detected BcIP/NBT substrate is shown in Figure 7. The chemiluminescent assay detected 1.18 x 106 copies of HBV~ DNA. The colorimetric test showed a detection of 1.07 x 10' copies. After a two hour incubation, the chemiluminescent assay detected 4.39 x l0~ copies of HBV~
DNA. The colorimetric test showed a detection of 1.07 x iD~ copies after the same incubation time. After a 4 incubation, the colorimetric assay detected 1.18 x 106 copies of HBV~ DNA.
Table IV sumvearizes the results of chemiluminescent detection limits of HBV~ using AMPPD and the colorimetric detection with HCIP/NBT substrates. Sensitivity of the SNAPS hybridization kit was improved over 100-fold using the chemiluminescent assay based upon A1~IPPD. The AMPPD-based assay detected as few as about 44,000 copies of HBV~
plasmid DNA, compared to the BCIP/NBT colorimetric assay which required 10,700,000 copies for detection. In addi-tion, AMPPD reduced the assay time from 4 hours to 30 minutes.

Table IV
Comcarison of Detection Limits for Hepatitis 8 "Core Anti-~en~ Plasmid DNA Using! Chemiluminescent and Chromoqeni_c Substrates in SNAP' HS~bridization Kit Chemiluminescent AMPPD Colorimetxic BCIP/
Copies of BHS~ Substrate Detection in NBT Substrates DNA Per Spot Minutes Detection in Minutes 9.8 x 10~ 30 30 3.2 x 10~ 30 60 1.07 x 10~ 30 120 3.56 x 106 30 180 1.18 x 106 30 240 3.95 x 105 60 no color 1.31 x 105 90 no color 4.39 x 10~ 120 no color Quantitative chemiluminescence results could be obtained by measuring reflection densities directly from the imaged Black and White Polaroid Type 612 instant photographic film strips using a Tobias RBP Hlack and White Reflection Densitometer, as shown in Figure 8. The results show that a dose response curve can be generated of tht reflection densities as a function of HBV~ plasmid concentration. This dose response curie can be subse-quently used as a calibration for the determination of HBV~
DNA levels in clinical specimens.
Example 7 Anti=AFP antibody coated beads and anti-AFP antibody:
alkaline phosphatase conjugates were obtained from a Hybri.tech Tandem Assay kit.
1. To each tube was added 20 ul of sample. Samples were 0, 25, 50, 100, and 200 mg/ml AFP.
2. Placed one bead in each tuba.
3. Added 200 ~1 of anti-AFP antibody alkaline phosphatase conjugate to each tube.
4. Shook rack to mix contents of tubes.

5. Covered tubes.
6, incubated for 2 flours at 37'C.
7. Aspirated off antibody and sample to waste.
e. Washed beads 3 times with c.Q ml of 0.1~ Tween 20 in ghosphate buffered saline, pH 9.4.
For Colorimetric Assav Chemilumine~ence 9. N/A 9. Washed 1 time with 0.5 H carbonate, 1 mM
MgClt pH 9.5.
10. Added 200 ~1 of 1 mq/ml 10. Added 250 ~ of 0.4 mM
p-nitrophenylphosphate AISPPD in 0.05 M in (PNPP) glycine 1 mM 0.1 carbonate, 1 mM
MgClZ pH 10.4 MgCl= pH 9.5.
11, Incubated for 30 minutes 11. Incubated for 20 at room temperature minutes at 30'C
12 . Added 1. 5 ml of 0 .1 M 12 , td/A
glycine, 10 mM EDTA, pH
9.5 to stop color devel-opment 13. Read in absorbance at 13. Read 10 sec. integrnl 410 nm in spectrophoto- of each tube in meter Turner luminometer 14. Plotted both sets of data as the signal at each concentration of AFP divided by the signal at zero AFP vs.
concentration o! AFP. As shown in Fig. 9, the results of the colorimetric assay are shown in the PHPP curve, and that o! the chemiluminescenca assay in the AtrIPPD curve.
It can be seen that the latter assay is about 10 times as sensitive as the former assay.
3o Hxamole 9 Assav for Thyroid Stimulating~"r~rmone fTSH1 Mouse monoclonal anti-TSH-~ antibody was used to coat 1/8 inch beads for analyta capture. Mouse monoclonal anti-TSH antibody was conjugated with alkaline phosphatase * Trade-mark c __ ____ __ __ and used as a detection antibody (antibody-enzyme conjugate).
TSH was obtained from Calbiochem, Catalog No. 609396, and BSA (type V - fatty acid free) was obtained from 5 Sigma, Catalog No. A6003.
The buffer solution~used for the analyte and antibody enzyme conjugate contained 0.1 M Tris-HC1, l mM MgCll, and 2~ by weight BSA (pM 7.5). The substrate buffer solution contained 0.1 H Tris, 0.1 mM MgCli, (pH 9.5), and the 10 substrate AriPPD (50 ~g/ml).
A TSH-containing analyte solution (15 ul) was mixed with 135 ~1 of antibody enzyme conjugate solution. Two 1/8 inch beads coated as described above were added to the solution and incubated for 2 hours at 23 ~ C. The beads 15 were then washed four times with 0.1 M Tris buffer (pH
7.5) and transferred to a reaction tube. 200 ~1 of the buffer solution containing the substrate described above was added to the tube. Following an incubation period of 20 minutes, light emission was recorded as ten second 20 counts using a Berthold Clinilumat Luminescence Analyzer.
Figure 10, which is a plot of the data in Table V
below, shows luminescence intensity for a given TSH
concentration. Linearity was achieved between 1 and 8 ~U/ml of TSH.
25 Table V
TSH Concentration fuU/mll jCounts/10 sec X 10~~1 1 0.25 2 0.49 30 4 1.1 An identical TSH assay was also performed in the absence of HSA for the sake of comparison. As shown in Fig. 11, the BSA- containing sample (Curve A) showed greater luminescence intensity for a given TSH concentra-35 tion than the sample without BSA (Curve B).
* Trade-mark Example 9 Assay for Carcinoe,~b~ryo~ic Ant:~,gen lCEA1 in the Head Format Anti-CEA coated beads and anti-CEA antibody: alkaline phosphatase conjugates were obtained from a Hybritech Tandem Assay kit.
1. To each tube were added 20 ul of sample.
Standards of 0, 2.5, 5, 10, 20, and 50 ng/ml CEA were used:
2. One bead was placed in each tube.
3. Added 200 ul of anti-CEA antibody enzyme conjugate to each tube.
4. Shook rack to mix contents of tubes.
5. Covered tubes.
6. Incubated for 2 hours at 37'C.
7. Aspirated off antibody and sample to waste.
8. Washed beads 3 times with 2.0 ml of 0.1~ Tween*
in phosphate buffered saline, pH 7.4.
9. Washed once with 0.05 % sodium carbonate, 1 mM
20 MgClt, pH 9.5.
10. Added 250 ul of 0.4 mM AlSPPD in 0.05 M sodium carbonate, 1 mM MgCl=, pH 9.5.
11. Incubated for 20 minutes at 30'C.
12. Read 10 sac. integral of luminescence frbm each tube in Turner 2oE Luminometer.
13. Plotted both sets of data as the signal at each concentration of hCG divided by the signal at zero CEA vs.
concentration of CEA. Typical data for a CEA assay using AMPPD are shown in Figure 12. Linearity was achieved between 0 and 20 ng/ml of CEA.
Exam l Assay for Numa~ Lvti,~niz ~ Hormone (hLH1 A nylon membrane, (Pall Immunodyne; 0.45 micron pore size), approximately 3mm in diameter was sensitized with 5 ul of a solution of 1 ~g/ml o! capture monoclonal anti LH antibodies for solid phase in phosphate buffered saline * Trade-mark (PBS), purchased from Medix, catalog #L-461-09. The membrane was subsequently blocked with 2% casein in phos-phate buffered saline at pii 7.3. The membrane was then enclosed in the device shown in Figure 13, which included blotting paper layers. In Fig. 13, A shows the prefilter cup; a plexiglass top: C Pall Immunodyne membrane (pore size 0.450 ; D polypropylene acetate fluffy layer: E
blotting paper: and F plexfglass.
The detection antibody used was mouse monoclonal anti-Lli, purchased from Medix, catalog $L-461-03. This antibody was derivatized with alkaline phosphatase, (purchased from Biozyme, catalog #ALPI-11G), using the glutaraldehyde coupling procedure (volley, A. et.al:, Bull. World 'Health Org;, 53, 55 (1976)].
Procedure:
The detection antibody conjugate (50 pl) was added to tubes containing 200 ul of hLli of the following concentrations:
Tube ~I ~o~c. l7LFi ~n_~g,~mi of PBs a 1 The content o! each tube was then added to four nylon membranes previously derivatized with capture antibodies (described above). After a five minute incubation period, the prefilter cup was removed and the membranes were washed with 400 pl of 0.051 Tween 20 in PBS. Subse quently, 100 ~Cl of 0.4 mM AMPPD, in 0.05M carbonate, 1 mM
MgClZ, 0.1% by weight BSA at pH 9.5 were added. The nylon membxanea were placed in a camera luminometer containing type 612 Polaroid Instant Black and White film, and exposed for one minute. The results of the assay imaged on film are shown in Figure 14.
Subsequently, the reflection densities of the images were measured using the Tobias RBP Portable Black and White Reflection Densitometer (manufactured by Tobias Associates, Inc., 50 industrial Drive., P.O. Box 2699, Ivyland, PA 18974-0347). The reflection densities were plotted versus concentration of LH to yield a standard curve for hLH, as shown in Figure 15.
~xamnle 11 5'.hg~miluminescent Decomposi~.ion of 3-(2'Soi~oada,,Mantanel-4-Methoxv-4- ( 3'~8-D-GalactopyranQsvl-Phenlrl ) -1. 2-Dioxg~,ane ( AMPGD f Reacents:
1. AMPGD synthesis as described above was made up in i:i MeOH/HZO at a concentration of l0 mg/ml.
2. 0.01 M. sodium phosphate buffer, pH 7.3, containing 0.1 M NaCl and 1 MgCl2.
3. ~-galactosidase (Sigma Chem. Co., catalog 65635, mol. wt. 500,000), 1 mg/ml in phosphate-salt buffer, pH
7.3, diluted 1:100 to yield a 2 x 10° M solution.
Protocol AMPGD solution (9.3 ~l) was diluted in 490 ul of a buffer solution of variable pH. Subsequent addition of 5 ~1 of the diluted galactosidase solution was followed by 1 hr. incubation at 37~C. The final concentration of reactants waa o.4 mM AMPGD and i x 10'3 moles galactosidase, at various pH values, as required by the experiment.
lifter incubation, the solutions were activated in a Turner 20E Luminometer by the addition of 100 ~ui of 1 N
NaOH. Tha instrument temperature was 29'C, that of the NaOH room temperature.
Thus, the assay consisted of a two-step process wherein the substrate-enzyme incubation was perfonaed at various pH values appropriate to efficient catalysis, e.g., at pH 7.3, and subsequently the pH was adjusted to about 12 with NaOH, and luminescence was read again.

Results In Figure 16 is shown the chemiluminescence of a fixed concentration of AMPGD as a function of ~-galacto sidase concentration, wherein the enzyme reaction was run at gH 7.3 and luminescence measured at pH 12. The use able, i.e., linear, portion of the standard curve was at enzyme concentrations between 10~~3 and 10-e M.
In Figure 17 is shown the effect of pH on the decomposition of AMPGD by ~B-galactosidase. The data show that the optimum pH for the enzyme with this substrate is about pH 6.5.
Figure 18 shows the production of light from AMPGD as a function of ~A-galactosidase concentration, using the ., two-step protocol described above. At all enzyme concen-trations, adjustment of the pH to 12 from 7.3 produced over a 100-fold increase in chemfluminescence.
Examgls 12 Detection of DNA FracLments by Cjyemi yminescence fter Electrorhoretic SeRaration ~~'_Fraaments DNA sequencing was performed using the dideoxy chain termination method of Singer et al. (1977) above.
Biotinylated pBR322 primer (40 ng) was annealed to 5 ~tg of denatured pBR322 glasmid. Klenow Fragment (DNA
polymerise I), 2 units, was then added (final volume was 17 pl). Subsequently, 2 ~1 of this template - primer solution was used for each of four base-specific reactions (G, A, T, C). To each reaction mixture, we added these specific amounts of deoxynueleotides, and dideoxynucleotides.
Reaction tures fN noaramsof Nueleotidesl M~, ~eoxvnuc~,gotides c~ ~ ~ _C

dGTP 1022.9 1077.4 1102.9 1102.9 dCTP 1015.9 992.4 1015.9 942.9 dTTP 1048.6 1048.6 972.5 1048.6 dATP 985.5 985.5 985.5 985.5 p'deo nucleotides ddGTP 123.0 ddCTP 29.7 ddTTP 466.0 5 ddATP 113.0 An aliquot of each reaction mixture (1 ~1) was loaded on a standard sequencing gel and electrophoresed. The DNA
was electrophoretically transferred to a Pall Biodyne A
nylon membrane and then W fixed to the membrane. The 10 membrane was then dried, blocked for i hour with o.2~
casein in PBS (casein-PBS), incubated with streptavidin:-alkaline phosphatase (1:5000 in casein-PBS) for 30 minutes, washed first with casein-PBS, then with 0.3~
Tween* 20 in PBS, and finally with 0.05 ti bicarbonate/-15 carbonate, pH 9.5, 1 mM MgClZ. Substrate, 0.4 m1K AMPPD in the final wash buffer, was incubated with the membrane for 5 minutes. After wrapping the membrane in plastic wrap, the membrane was placed in contact with Kodak XAR film and Polaroid Instant Black and White film for 2 hours. The 20 order of sequence lanes is C T A G in Figures 19A (X-ray film) and 19 B (instant film).
Example 13 Effect of liembrane Composition On Detect.~n Qj DNA
Fra en s jay Chemiluminescence 25 Various amounts of the SNAPS Hepatitis B core antigen oligonucleotide probe conjugated to alkaline phosphntase (Molecular 8losystems, Inc., San Diego, CA), as listed in the left column of Table VI, were spotted on three types of transfer membranes: Gene Screen Plus" (Nylon), 30 Schleicher and Schuell nitrocellulose, and Millipore PVDF.
The- .spots were incubated with an AKPPD solution, lumin-escence generated, and light detected on instant film, as in Example 6(C).
Tha data of Table VI show the earliest detection 35 times at each level o! oligonucleotide for each of the three membranes. Luminescence was greatly increased in * Trade-mark intensity by the use of nylon-based membranes, as compared to the other two types. For example, with a nylon membrane, the smallest amount of oligonucleotide tested, i.e., 0.01 ng, was detected within 60 seconds of film exposure. In contrast, it required at least 67 ng of oliqonucleotide to be detectable in 60 seconds on a nitrocellulose membrane; amounts of 0.82 ng or less were not detectable within 10 minutes. In further contrast, no amount of oligonueleotide was detectable in periods as long as 10 minutes.
Table VI
Earliest Detection Time. Sec.
Oligonucleotide, nq~ ~ Nvlon NitrocelluloseP_VDF

7.4 1 300 2.5 1 300 0.82 1 0.27 1 0.091 10 *

0.03 60 0.01 60 *Not detectable by 10 min. of exposure.

Claims (29)

CLAIMS:

1. An assay method of detecting a member of a specific binding pair in a sample, which comprises:
contacting the sample with an enzyme bonded to a substance having a specific affinity for the member, wherein the enzyme, the substance or the member to be detected is carried by solid matrices;
reacting the enzyme with a dioxetane of the formula:
(wherein 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 hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl, or an enzyme-cleavable group;
and Z is hydrogen or an enzyme-cleavable group, provided that at least one of X and Z is an enzyme-cleavable group), so that the enzyme cleaves the enzyme-cleavable group from the dioxetane to allow the formation of a negatively charged substituent bonded to the dioxetane, the negatively charged substituent causing the dioxetane to decompose to form a luminescent substance containing the group Y of the dioxetane, and detecting the luminescent substance as an indication of the member of the specific binding pair, wherein the solid matrices are pretreated with a polyvinyl quaternary ammonium salt) to block nonspecific binding to the solid matrices.

2. A method of detecting an enzyme in a sample, which comprises:
(a) providing a dioxetane having the formula:
(wherein 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 a hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, or cycloheteroalkyl group, or a group capable of being cleaved by the enzyme; and Z is hydrogen or a group capable of being cleaved by the enzyme, provided that at least one of X and Z is a group capable of being cleaved by the enzyme);
(b) providing solid matrices which have been pretreated with a poly(vinyl quaternary ammonium salt) to block nonspecific binding and by which the dioxetane or the enzyme is carried;
(c) contacting the dioxetane with the sample containing the enzyme; whereupon the enzyme cleaves the enzyme-cleavable group from the dioxetane to allow the formation of a negatively charged substituent bonded to the dioxetane, the negatively charged substituent causing the dioxetane to decompose to form a luminescent substance containing the group Y of the dioxetane; and 48a (d) detecting the luminescent substance as an indication of the presence of the enzyme.

3. In an assay method in which a member of a specific binding pair comprising a nucleic acid which is DNA, RNA, or a fragment thereof produced by sequencing protocol and a probe capable of binding to all or a portion of said nucleic acid is detected by means of an optically detectable reaction, the improvement being (a) having the optically detectable reaction include the reaction, with an enzyme, of a dioxetane of the formula wherein T is a cycloalkyl or a polycycloalkyl group bonded to the 4-membered ring portion of said dioxetane by a spiro linkage; Y is a fluorescent chromophore; X is hydro-gen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, hetero-aryl, cycloalkyl, cycloheteroalkyl, or an enzyme-cleavable group; and z is hydrogen or an enzyme-cleavable group, provided that at least one of X or Z is an enzyme-cleavable group, so that said enzyme cleaves said enzyme-cleavable group to allow the formation of a negatively-charged substituent bonded to said dioxetane, said negatively charged substituent causing said dioxetane to decompose to form a luminescent substance containing said group Y of said dioxetane, (b) contacting said nucleic acid with a labeled complementary oligonucleotide probe to form a hybridizing pair, (c) contacting the hybridized pair with a molecule capable of strong binding to the label of the nucleotide covalently conjugated with an enzyme capable of cleaving said dioxetane to release light energy, (d) adding said dioxetane, and (e) detecting the light produced by having the light produced come in contact with light sensitive film.

4. In an assay method in which a member of a specific binding pair comprising a nucleic acid which is DNA, RNA, or a fragment thereof produced by a ser;uencing protocol and a probe capable of binding to all or a portion of said nucleic acid is detected by means of an optically detectable reaction, the improvement being (a) having the optically detectable reaction include the reaction, with an enzyme, of a dioxetane of the formula wherein T is a cycloalkyl or a polycycloalkyl 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 an enzyme-cleavable group; and Z is hydrogen or an enzyme-cleavable group, provided that at leash one of X or Z is an enzyme-cleavable group, so that said enzyme cleaves said enzyme-cleavable group to allow the formation of a negatively-charged substituent bonded. to said dioxetane, said negatively charged substituent causing said dioxetane to decompose to form a luminescent substance containing said group Y of said dioxetane, (b) contacting said nucleic acid with a labeled complementary oligonucleotide probe to form a hybridizing pair, (c) contacting the hybridized pair with a molecule capable of strong binding to the label of the nucleotide covalently conjugated with an enzyme capable of cleaving said dioxetane to release light energy, (d) adding said dioxetane, and (e) detecting the light produced by having the light produced come in contact with a photoelectric cell.

5. In an assay method in which a member of a specific binding pair comprising a nucleic acid and a probe capable of binding to all or a portion of said nucleic acid is detected by means of an optically detectable reaction, the improvement being (a) having the optically detectable reaction include the reaction, with an enzyme, of a dioxetane of the formula:
wherein T is a cycloalkyl or polycyclaalkyl 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 an enzyme-cleavable group; and Z is hydrogen or an enzyme-cleavable group, provided that at least one of X or Z is an enzyme-cleavable group), so that the enzyme cleaves the enzyme-cleavable group from the dioxetane to allow the formation of a negatively charged substituent bonded to the dioxetane, the negatively charged substituent causing the dioxetane to decompose to form a luminescent substance containing the group Y of the dioxetane and (b) carrying out the binding of the probe to the nucleic acid on a nylon membrane.

6. The method of claim 5, wherein the probe is a labeled oligonucleotide complementary to the nucleic acid.

7. The method of claim 6, wherein the oligonucleotide probe is covalently labeled with the enzyme capable of decomposing the dioxetane to emit light.

8. The method of claim 6, wherein the label on the oligonucleotide probe comprises a covalently bound antigen that is imunochemically bound to an antibody-enzyme conjugate, wherein the antibody is directed to the antigen and the enzyme is capable of decomposing the dioxetane to emit light.

9. The method of claim 7 or 8, wherein the enzyme is an acid or alkaline phosphatase and the dioxetane is 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD).

10. The method of claim 7 or 8, wherein the enzyme is a galactosidase and the dioxetane is 3-(2'-spiroadamantane)-4-methoxy-4-(3"-.beta.-D-galactopyranosyl)phenyl-1,2-dioxetane (AMPGD).

11. The method of claim 7 or 8, wherein the enzyme is a carboxyl acid esterase and the dioxetane is 3-(2'-spiroadamantane)-4-methoxy-4-(3"-acetoxy)phenyl-1,2-dioxetane.

12. In an assay method in which a member of a specific binding pair comprising a nucleic acid which is DNA, RNA, or a fragment thereof produced by a sequencing protocol and a probe capable of binding to all or a portion of the nucleic acid is detected by means of an optically detectable reaction, the improvement being:
(a) having the optically detectable reaction include the reaction, with an enzyme, of a dioxetane of the formula:
(wherein T is a cycloalkyl or a polycycloalkyl group bonded to the 4-membered ring portion of the dioxetane by a spiro linkage; Y is a fluorescent chromophore; X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl, or an enzyme-cleavable group;
and Z is hydrogen or an enzyme-cleavable group, provided that at least one of X or Z is an enzyme-cleavable group), so that the enzyme cleaves the enzyme-cleavable group to allow the formation of a negatively-charged substituent bonded to the dioxetane, the negatively charged substituent causing the dioxetane to decompose to form a luminescent substance containing the group Y of the dioxetane, (b) contacting the nucleic acid with a labeled complementary oligonucleotide probe to form a hybridizing pair, (c) contacting the hybridized pair with a molecule capable of strong binding to the label of the nucleotide covalently conjugated with an enzyme capable of cleaving the dioxetane to release light energy, (d) adding the dioxetane, (e) detecting the light produced, and (f) the hybridizing between the nucleic acid and the labeled oligonucleotide probe is conducted on a nylon membrane.

13. The method of claim 12, wherein the oligonucleotide label is biotin or a biotin derivative.

14. The method of claim 12, wherein the molecule capable of strong interaction with the label of the oligonucleotide is avidin or streptavidin.

15. The method of claim 12, wherein the enzyme is a phosphatase and the dioxetane is 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD).

16. The method of claim 12, wherein the enzyme is a galactosidase and the dioxetane is 3-(2'-spiroadamantane)-4-methoxy-4-(3"-.beta.-D-galactopyranosyl)phenyl-1,2-dioxetane (AMPGD).

17. A kit for detecting a nucleic acid or fragment thereof in a sample by hybridization of the nucleic acid or fragment to a comlementary labeled oligonucleotide probe, comprising:
a 1,2-dioxetane capable of producing light energy when decomposed having the formula:
(wherein T is a cycloalkyl or polycycloalkyl group bonded to the 4-membered ring portion of said dioxetane by a spiro linkage, 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 the nucleic acid or fragment thereof is hybridized to the oligonucleotide probe.

18. A kit for detecting a protein in a sample, comprising:
(a) a 1,2-dioxetane capable of producing light energy when decomposed having the formula:
(wherein 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 hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl, or an enzyme-cleavable group;
and Z is hydrogen or an enzyme-cleavable group, provided that at least one of X and 2 is 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 containing the group Y of the dioxetane); and (b) a nylon membrane upon which protein-antibody binding is conducted.

19. A kit for detecting a nucleic acid or fragment thereof in a sample by hybridization of the nucleic acid or fragment to a complementary labeled oligonucleotide probe, the kit comprising:
(a) a 1,2-dioxetane capable of producing light energy when decomposed having the formula:
(wherein T is a cycloalkyl or polycycloalkyl group bonded to the 4-membered ring portion of the dioxetane by a spiro linkage; X is hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an enzyme-cleavable group; Y is a chromophore capable of fluorescence; and 2 is hydrogen or an enzyme-cleavable group, provided that at least one of X or Z is an enzyme-cleavable group);
(b) (1) a covalently enzyme-labeled oligonucleotide or (2) both a complementary oligonucleotide probe covalently labeled with biotin or a biotin derivative and an enzyme capable of decomposing a 1,2-dioxetane to emit light covalently bound to avidin or streptavidin;
(c) a water-soluble enhancing substance that increases specific light energy production above that produced in its absence, wherein the water-soluble enhancing substance comprises a positively charged polymeric quaternary ammonium salt and an energy acceptor capable of forming a ternary complex with the 1,2-dioxetane anion produced following enzyme-catalyzed decomposition of the 1,2-dioxetane, whereby energy transfer occurs between the 1,2-dioxetane anion and the energy acceptor and light is emitted by the energy acceptor; and (d) a nylon membrane upon which nucleic acid-oligonucleotide probe hybridization is conducted.

20. The kit of claim 19, wherein the polymeric quaternary ammonium salt is poly(vinylbenzyltrimethyl-ammonium chloride), poly[vinylbenzyl(benzyldimethyl-ammonium chloride)], or poly(vinylbenzyltributyl-ammonium chloride).

21. The kit of claim 19 or 20, wherein the energy acceptor is fluorescein.

22. A kit for detecting a protein in a sample, the kit comprising:
(a) a 1,2-dioxetane capable of producing light energy when decomposed having the formula:
(wherein T is a cycloalkyl or polycycloalkyl group bonded to the 4-membered ring portion of the dioxetane by a spiro linkage; 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 is an enzyme-cleavable group);

(b) an antibody directed to the protein covalently bound to an enzyme capable of decomposing the 1,2-dioxetane to emit light;
(c) a water-soluble enhancing substance that increases specific light energy production above that produced in its absence; and (d) a nylon membrane upon which protein-antibody binding is conducted.

23. The kit of claim 22, wherein the water-soluble enhancing substance comprises a positively charged polymeric quaternary ammonium salt and an energy acceptor capable of forming a ternary complex with the 1,2-dioxetane anion produced following enzyme-catalyzed decomposition of the 1,2-dioxetanes whereby energy transfer occurs between the 1,2-dioxetane anion and the energy acceptor and light is emitted by the energy acceptor.

24. The kit of claim 23, wherein the polymeric quaternary ammonium salt is poly(vinylbenzyltrimethyl-ammonium chloride), poly[vinylbenzyl(benzyldimethyl-ammonium chloride)] or poly(vinylbenzyltributyl-ammonium chloride).

25. The kit of claim 23 or 24, wherein the energy acceptor is fluorescein.

26. A kit for detecting a protein and a sample, the kit comprising:
(a) a 1,2-dioxetane capable of producing light energy when decomposed having the formula:
(wherein T is a cycloalkyl or polycycloalkyl group bonded to the 4-membered ring portion of the dioxetane by a spiro linkage; 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 one of X or Z is an enzyme-cleavable group);
(b) a first antibody directed to the protein;
(c) a second antibody directed to the first antibody covalently bound to an enzyme capable of decomposing the 1,2-dioxetane; and (d) a water-soluble enhancing substance that increases specific light energy production above that produced in its absence.

27. The kit of claim 26, wherein the water-soluble enhancing substance comprises a positively charged polymeric quaternary ammonium salt and an energy acceptor capable of forming a ternary complex with the 1,2-dioxetane anion produced following enzyme-catalyzed decomposition of the 1,2-dioxetane anion and the energy transfer occurs between the 1,2-dioxetane anion and the energy acceptor and light is emitted by said energy acceptor.

28. The kit of claim 27 wherein the polymeric quaternary ammonium salt is poly(vinylbenzyltrimethyl-ammonium chloride), poly[vinylbenzyl(benzyldimethyl-ammonium chloride)], or poly(vinylbenzyltributyl-ammonium chloride).

29. The kit of claim 27 or 28 wherein the energy acceptor is fluorescein.