CA2212738A1 - Chemiluminescent energy transfer assays - Google Patents

Abstract

A chemiluminescent assays for the determination of the presence or amount of a biopolymer in bound assays using 1,2-dioxetanes in connection with AttoPhosTM as chemiluminescent substrates for enzymelabeled targets or probes is provided. Further disclosed is a kit for conducting a bioassay for the presence or concentration of a biopolymer comprising a) an enzyme complex; b) a 1,2-dioxetane; and c) AttoPhosTM.

Description

PCTnUS95101506 DescriPtion Chemiluminescent Ener~Y Transfer Assavs Technical Field This invention relates to the energy transfer chemiluminescent assays for the determination of the presence or amount of a biological substance in surface-bound assays using 1,2-dioxetanes in connection with hydrophobic fluorometric substrates such as AttoPhos~ as chemiluminescent substrates for enzyme-labeled fluorometric substrate targets or probes. The chemiluminescence of the dioxetane AttoPhos~
acceptor substrate pair can be enhanced by the addition of a polymeric enhancer. Further enhancement can be achieved by adding, in sequence, AttoPhos~ and then the 1,2-dioxetane.

Bac~qround Art Chemiluminescent assays for the detection of the presence or concentration of a biological substance have received increasing attention in recent years as a fast, sensitive and easily read method of conducting bioassays. In such assays, a chemiluminescent compound is used as a reporter molecule, the reporter molecule chemiluminescing in response to the presence or the absence of the suspected biopolymer.
A wide variety of chemiluminescent compounds have been identified for use as reporter molecules. One class of compounds receiving particular attention is the 1,2-dioxetanes. 1,2-dioxetanes can be stabilized by the addition of a stabilizing group to at least one of the carbon atoms of the dioxetane ring. An exemplary stabilizing group is spiro-bound adamantane. Such dioxetanes can be further substituted at the other carbon position with an aryl moiety, preferably phenyl or naphthyl, the aryl moiety being substituted by an oxygen which is, in turn, bound to an enzyme-labile group.
When contacted by an enzyme capable of cleaving the labile group, the oxyanion of the dioxetane is formed, leading to decomposition of the dioxetane and spontaneous W 096/2S667 PCTnUS95101S06 --2--chemilumine~cence. A wide variety of such dioxetan~s are disclosQd in U.S. Patent 5,112,960. That patent f~C~ C on dioxetane~ which bear a substituent on the ~A~ntyl-stabilizing group, such as halo substituents, alkyl ~LoU~, alkoxy groups and the like. Such dioxetanes represQnt an advance over earlier-recogniz~d dioxetane~, such as 3-(4-methoxyspiro [1,2-dioxetane-3,2'-tricyclo]-3.3.1.13~7 ]decan]-4-yl) phenylphosphate, and in particular, the Ai~o~l; um salt thereof, generally identified as AMPPD. The chlorine-substituted counterpart, which converts the stabilizing adamantyl group from a passive group which allows the decomposition reaction to go forward, to an active group which gives rise to enhanced chemil- ; n~S~~ signal due to faster ~s~mrocition of the dioxetane anion, greater signal-to-noisQ
values and better sensitivity, is referred to as CSPD. other dioxetanes, such as the phenyloxy-~-D-galactopyranoside (AMPGD) are also well-~nown, and can be used as reporter molecules. These dioxetanes, and their preparation, do not constitute an aspect of the invention herein, per se.
A~says employing these dioxetanes can include conventional assays, such as Southern, Northern and Western blot assays, DNA sequencing, ELISA, as well as other liquid phase and iY~ phase assays performed on membranes and beads.
In general, procedures are performed according to st~n~A~d, well-known protocols except for the detection step. In DNA
assays, the target biological substance is bound by a DNA
probe with an enzyme covalently or indirectly linked thereto, the probe being admixed with the sample immobilized on a membrane, to permit hybridization. Thereafter, pycpcc enzyme complex is removed, and dioxetane added to the hybridized sample. If hybridization has occurred, the dioxetane will be activated by the bound enzyme, leading to decomposition of the dioxetane, and chemiluminp-ccencp. In solution-phase assays, the enzyme is frequently conjugated to a nucleic acid probe or immune complexed with an antibody responsive to the target biological substance, unbound components being removed, and w~s6/2s~67 PC~S95/01506 the dioxetane added, chemilumine~-c~n~e being proA~ A by the decomposition of the dioxetane activated by the amount of enzyme present. In cases where the enzyme it~elf is the ~ target, the dioxetane need only be added to the sample.
Again, a wide variety of assay modalities has been developed, a~ disclosed in U.S. Patent 5,112,960, as well as U.S. Patent 4,978,614.
It has been well-known that light-~l~n~hin~ reactions will occur if the dioxetane decomposition ~ in a protic solvent, such as water. As the samples suspected of containing or lacking the analyte in question are generally biological samples, these assays generally take place in an aqueous environment. The light-~nc~in~ reactions therefor may substantially reduce the chemilu~;ne-c~n~e actually observed from the decomposition of the dioxetan. In assays involving low-level detections of particular analytes, such as nucleic acids, viral anti ho~ ies and other protein~, particularly those prepared in solution or in solution-solid phase systems, the r~u~e~ chemilumineF-e~e obsQrved, coupled with unavoidable back~ o~.~ signals, may reduce the sensitivity of the assay such that extremely low levels of biological substances cannot be detected. One method of addressing this problem is the addition of water soluble macromolecules, which may includQ both natural and synthetic molecules, as is disclosed in detail in U.S. Patent 5,145,772.
The disclosure of this patent is incorporated herein, by reference. To similar effect, U.S. Patent 4,978,614 addresses the addition of variou~ watQr-soluble ~nh~cement~ agents to the sample, although the patent speaks to the problem of suppressinq non-specific bi n~ inq reactions in solid state assays. In U.S. Patent 5,112,960, preferred water-soluble polymeric quaternary ammonium salts such as poly(vinylbenzyltrimethylammonium chloride) (TMQ) poly(vinyl-benzyltributylammonium chloride) (TBQ) and poly(vinylbenzyl-dimethylbenzylammonium chloride) (BDMQ) are identified as water-soluble polymeric quaternary ammonium salt~ which W O 96/2S667 PCTnUS~5101S06 -4~

~ ce chemilum;ne~ence and provide greater sensitivity by incrQasing the signal-to-noise ratio. Similar phosphonium and sulfonium polymeric salts are also disclosed.
This D~hA~cement is achieved, at least in part, through the formation of hydrophobic regions in which the dioxetane oxyanion i8 seque2~tered. Decomposition in these hydrophobic regions ~h~re~ chemilumine~~n~e, because water-basQd light 'n~h;Sl~J reactions are suppre~sed. Among the r~co~;7ed water-soluble quaternary polymer salts employed, T~Q provides ctedly superior ~h~ ment~ through this hydrophobic region-forming ~chAn;sm.
The chemiluminescent e~h~nc~ment achieved by the addition of water-soluble polymeric substAnc ~ such as ammonium, phosphonium and sulfonium polymeric salts can be further improved by the inclusion, in the aqueous sample, of an additive, which improves the ability of the quaternary polymeric salt to sequester the dioxetane oxyanion and the resulting excited state emitter reporting molecule in a hydrophobic region. Thus, the combination of the polymeric quaternary salt and the additive, together, produce an increase in ~hA~c~ment far beyond that pro~ e~ separately by the addition of the polymeric quaternary salt, or the additive, which, when a surfactant or water-soluble polymer itself, may enhance chemilumin~ccence to a limited degree.
The synergistic combination of the polymeric quaternary salt and additives gives enhancement effects making low-level, reliable detection possible even in aqueous samples through the use of 1,2-dioxetanes. The polymeric quaternary salts, coupled with the additives, are sufficiently powerful enhancers to show dramatic 4 and 5-fold increases at levels below 0.005 percent down to 0.001 percent. Increased signal, and improved signal/noise ratios are achieved by the addition of further amounts of the polymeric quaternary salt, the additive, or both, in amounts up to as large as 50 percent or more. In general, levels for both polymeric quaternary salt and additive can be preferably within the range of 0.01 - 25 W096~25667 ~CT~S95101506 percent, more preferably from 0.025 - 15 percent by weight.
The details of this improvement are disclosed in U.S.
Application Serial No. 08/031,471 which is incorporated herein ~ by reference.
U.S. Patent 5,208,148 describe~ a clas~ of fluorescent substrates for detection of cells producing the glycosidase enzyme. The substrate is a fluorescein diglycoside which is a non-fluorescent substrate until hydrolyzed by glycosidase enzyme inside a cell to yield a fluorescent detection product excitable between about 460 nm and 550 nm. The fluorescent enzymatic hydrolysis products are specifically formed and adequately retained inside living cells, and are non-toxic to the cells. The substrates can penetrate the cell membrane under physiological conditions. Therefore, the invention permits analysis, sorting and cloning of the cells and monitoring of cell development in vit~o and in vivo. However, these fluorescent products are detected in the single cells and within specific organelles of single cells only after the spectral properties of the substrates are excited by an argon la~er at its principle wavelengths.
Known fluorescent emitters have been used with dioxetanes in bioassays. U.S. patents 4,959,182 and 5,004,565 describe methods and compositions for energy transfer enhancement of chemilumir.e-c~n~e from l,2-dioxetanes. ThQse patents utilize a fluorescent micelle comprising a surfactant and a fluorescent co-surfactant which exists in the bulk phase of the buffer solution used. The fluorescent cosurfactant is present in a form capable of energy transfer-based fluor~--Dn~e at all times. In contact with a solid phase containing an enzyme-labeled ligand bin~ing pair, the fluorescent moiety tends to remain associated with the micelle in the bulk phase. If any fluorescent co-surfactant is deposited on the solid phase, this occurs indiscriminately, in areas containing the immobilized ligand binding pair, and in areas which do not contain said pair. Thus a problem results in that the fluorescent emitters never are, or do not remain W096/2~667 PCT~S9Sl01506 associated with the immobilized enzyme conjugate. Thus the close proximity nee~ for energy transfer from the dioxetane to the fluorescent emitter is not efficient. Further because the fluorescent emitters can ~e deposited anywhere on the solid phase matrix, this method does not allow for specificity when used in bound assay. The majority of the examples in the 1182 and 1565 patents are solution pha~e enzyme acsays or chemical triggering experiments not utilizing enzymes. These examplQ3 are better matched to the bulk phasQ co-micelle as a means to promote the proximity of the dioxetane anion product with the energy accepting fluorescent surfactant. The only example of a solid phase assay occurs at columns 29 and 30.
This ELISA assay shows that light is produced on a well surface over the range of 112 ng to 1.3 ng of S-antigen.
However, there are no control experiments showing light production from the same dose-response experiment, but using dioxetane and CTAB surfactant in the Ah-?nce of fluorescent co-surfactant. Thus one cannot determine how efficient the energy transfer at the solid surface actually is. Certainly, however, this fluo --~Pnt co s~ ~actant i8 not a non-fluorescent enzyme substrate such as AttoPhos. Thus the present invention, wherein a fluorescent energy acceptor i5 produced directly, and locally on a surface, by the same enzyme which catalytically decomro-~c the dioxetane energy donor, is not suggested by these art references.
$here are several basic problems which relate to fluorescent substrates used in surface or blotting experiments. One is that the excitation of the phoephorylated chromophore has to be performed with a laser or a lamp with a filter or a monochromator. These light sources are not only cumbersome, but increase the expense of the assay. This n~C~csAry and key excitation step which is accomplished with W/blue light results in a second problem which is auto fluorescence of the mem~rane or surf ace and other solid supports which ordinarily contain fluorescent brighteners and other excitable fluorophores, as well as W 096/25667 PCTnUS95101506 exciting chromophores contained in the biological sample (i.e., proteins and nucleic acids). Such fluorescent signal of the surface or membrane support and sources other than the dephosphorylated or activated substrate, contribute to unacceptable levels of bac~ground which substantially lower - the sensitivity and specificity of the assay so that cubstrates such as these cannot be used.
Known fluorescQnt emittQrS have been used with dioxetanes in nQnh~llnA a~says. HowevQr, a problem rQsults in that the fluor~ nt emitters don-It stay associated with the enzyme conjugate. Therefore, the close proximity needed for the energy transfer from the dioxetane to the fluorescent emitter is not possible. Further, because the fluorescent emitters don't stay associated with the enzyme coniugate, the emitters do not allow for specificity when used in bound assays.
Therefore, notwiths~n~i n~ the advances in chemilumineAcenc~ te~hnology addressed by the above assays, it remains a goal of the industry to provide chemiluminescent assays providing overall more intense signals, thus having greater sensitivity and specificity without the use of ~Yren~ ive, c~l~h~rsome lasers or lamps, to determine the presence, co~c~ntration or both of a biological substance in a sample. 1,2-dioxetane compoundc have already been developed which show excellent Fotential as reporter molecules for such chemilum;n~-cent assays. However, it i8 still n~cescAry to improve upon the sens:tivity and specificity of the chemiluminescence of the 1, 2-dioxetane molecules by providing an efficient fluorescent acceptor emitter which stays in close contact with the dioxetane to thereby allow for the neceC-~y energy transfer, and further, to allow for sensitive and specific determination of the target.

~isclosure of the Invention Therefore, it is an object of the present invention to provide a method for determining the presence or amount of a PCT~US~5101506 WO g6/25667 biological substance in biological surface-bound and solution-phase assays using 1,2-dioxetane donor molecules in combination with a fluorescent acceptor emitter, which s pro~ides increased sensitivity or signal-to-noise ratio without the use of any outside light sources for excitation.
The above objects have been met by the present invention which provides a method for determining the presenc~ or the amount of a bioloqical substance in a biological sample, wherein the method comprisQs the staps of: a) forming an enzyme conjugated binder (antibody or DNA probe) with the biological ligand from the sample; b) ~inq a hydrophobic fluorometric substrate such as AttoPhos~ and a 1,2-dioxetane to the bound enzyme conjugated binder; c) wherein the enzyme of the enzyme conjugated biopolymer cleaves an enzyme cleavable group such as a phosphate moiety from the AttoPhos~
and from the dioxetane causing the dioxetane to ~9C. ,--~
through an excited state emitter form such that energy transfer occurs from the excited state chemilumin~-c~nt emitter to the ~pho~phorylated AttoPhos~, causing this moiety to emit; and d) determining the pre~Q~s or amount of the biological substance as a function of the amount of fluore-ce~e.
The objects have further been met by the present invention which further provides a kit for conducting a bioassay for the presence or concentration of a biological substanca which is detected either bound to a surface or in a solution assay, said kit comprising: a) an enzyme complex which will stably bind to a surface-bound biological substance; b) a 1,2-dioxetane which when contacted by the enzyme complex will be caused to de~ompoce into a ~co~rosition product which is capable of transferring its energy; and c) AttoPhos~.

Brief DescriDtion of the ~rawincs Figure 1 is an illustration of the met~od of the present W 096/25667 ~CTnUS95101506 _g_ invention showing the energy transfer from CS-D to dephosphorylated Atto, thereby releasing energy in the form of ~luor~ nse.
Figure 2 (A) - (D) is a CCD image of Western blot analysis of rabbit IgG on Nitrocellulose Membrane. A detailed description of Figure 2 can be found in Example 1.
Figure 3 is a graph of a Western blot analysic of rabbit IqG on Nitrocellulose Membrane showing chemiluminescent intensity (average and maximum).
Figure 4 (A) - (D) is a CCD image of Western blot analysis of rabbit IgG on PVDF membrane. Figure 4 is specifically explained in Example l.
Figure 5 is a graph of a Western blot analysis of rabbit IgG on PVDF membrane showing chemiluminescent intensity (average and ~x;~-lm).
Figure 6 (A) - (B) are graphs of PSA (Prostate Specific Antigen), ng/mL versus RLU, S sec of chemilumine~c~nt detection of PSA comparison of CSPD to CSPD + AttoPhos~.
Figure 7 is a chemilum;n-~ent emission spectrum (intensity v. wavelength) obt~in-~ with 0.25 mM CSPD, 50%
AttoPhos~, and alkaline phosphatase, as described in Example 3.
Figu~e 8 is a chemilumine~Qnc~ spectrum (intensity v.
wavelength) obtained with 1.0 mM CSPD, 50~ AttoPhos~, and alkaline phosphatase, as described in Example 3.
Figure 9 is a chemilu~;~e~-ence spe_L~um (intensity v.
wavelength) obtAin~ with 0.1 m~ CSPD, S0% AttoPhos~, 20%
BDMQ, and alkaline phosphatase, as described in Example 3.
Figure 10 is a chemiluminescence spectrum (intensity v.
wavelength) obtained with 0.25 mM CSPD, 50~ AttoPhos~, 20 BDMQ, and alkaline phosphatase, as described in Example 3.
Figure 11 is a chemiluminesc~nce spectrum (intensity v.
wavelength) obtained with 0.5 mM CSPD, 50% AttoPhos~, 20%
BDMQ, and alkaline phosphatase, as described in Example 3.
Figure 12 is a chemil~l~ine~cenr~ spectrum (intensity v.
wavelength) obtained with 1.0 mM CSPD, 50% AttoPhos~, 20%

PCT~S95)01506 W096l25667 BDMQ, and alkaline phosphatase, as described in Example 3.
Figure 13 is a chemilumine~nce spectrum (intensity v.
wavelength) obt~i n~ with 1.0 mM CSPD, 50% AttoPhos~, 10%
BD~Q, and alkaline phosphatase, as described in Example 3.
Figure 14 is a chemilumin-~ence spectrum (intensity v.
wavelength) obtained with 1.0 m~ CSPD, 10~ AttoPhos~, 20%
BDMQ, and alkaline phosphatase, as deccribed in Example 3.
Figure 15 is a chemilum;n~ nt emission spectrum (intensity v. wavelenqth) obt~i n~~ using 1.0 mM CSPD, 50%
AttoPhos~, 2.0 mg/ml polyvinylbenzyltriphenyl phosphonium chloride-copolyvinylbenzylenzyldimethyl ammonium chloride (40 mole~ TPP/60 mole% BDMQ), and alkaline phosphatase as described ~n Example 3.
Figure 16 is a chemiluminescent emission spectrum (intensity vs. wavelength) obtAi n-~A using 1. 0 mM CSPD, S0%
AttoPhos~, 2.0 mg/ml polyvinylbenzyltriphenyl phosphonium chloride-copolyvinylbenzyltributyl ammonium chloride (45 mole%
TPP/55 mole% TBQ), and alkaline phosphatase as described in Example 3.
Figure 17 is a chemilumin~--snt emis~ion spectrum (intensity vs. wavelength) obtained using a 30 minute preincubation of alkaline phosphatase in 50% AttoPhos~, 20%
BDMQ, followed by the addition of CSPD (0.25 mM final concentration) at time zero as described in Example 3.
Figure 18 is a graph showing the ratio of emission at 545 nm/465 nm obtained from the data in Figures 7-14 and Figure 17.
Figure 19 is a graph showing the sum of emission at 465 nm and 545 nm, obtained from the data in Figures 7-14 and Figure 17.
Figure 20 is a graph showing the ratio of emission at 545 nm/465 nm obtained from the data in Figures 15 and 16.
Figure 21 is a graph showing the sum of emission at 465 ~ nm and 545 nm, obtained from the data in Figures 15 and 16.
Figure 22 is a CCD camera image detecting the presence of biotinylated DNA.

W096125667 PCT~S95101506 Rest Mn~e for ~rrying Out the Invention The present invention will now be described more fully hereinafter with references to the ar~ p~nying drawings, in which preferred embcdiments of the invention are shown. This ~ invention can, however, be embodied in many different forms and should not be construed a~ limited to the em~o~ments set forth herein; rather, Applicant provides these embodiments so that this disclosure will be thorough and completQ, and will fully convey the scope of the invention to those skilled in the art. It should be noted that the fluorometric substrate is not specifically limited, save for hydrophobicity, ~; FC~ below. Exemplary substrates are disclosed in u.s.
Patent 5,208,148 incorporated herein by reference.
This invention makes use of a hydrophobic fluorometric substrate. By this is int~n~e~ a compol~nA which upon activation by an enzyme can be ;~ ce~ to emit in response to energy transfer from an excited state dioxetane decomposition product donor. As the donor is hydrophobic, the substrate, when activated, must be sufficiently hydrophobic as to be sequestered in the same hydrophobic regions to which the donor migrate~, for energy and transfer to occur.
The present invention is described in terms of a method for determining the pre~enC~ or amount of a substance or determined in a solution-phase assay biological substance using l,2-dioxetanes using the hydrophobic fluorometric substrate AttoPhos~. The kit of the present invention also for determining the presence or amount of a substance, i~
described using a suitable enzyme conjugate, a 1,2-dioxetane and AttoPhos~. Other fluorometric substrates may be used.
The present inventors have found for the first time that l,2-dioxetane in connection with AttoPhos~ improves both the specificity and sensitivity of sur~ ace-bound assays.
Further, these assays using l,2-dioxetane in connection with AttoPhos~ alleviate the need for light sources necessary for excitation.

W O 96125667 PCTnUS95101506 Specifically, the present invention USQI; the high quantum yield of fluor~nce, affinity for sur~aces pos~--r~ by AttoPhos~, coupled with the enzyme activated chemilumine~c~n~
of 1,2-dioxetane as the excitation source for the dephosphorylated AttoPhos~. Thus, dephosphorylated AttoPhos~
is pro~ e~ at the surface and staya in close proximity with the enzyme environment throughout the assay, and the excitation of the acceptor--~rh~-phorylated AttoPhos~ can be performed without any external i~ ~mentation and without possible excitation of chromophores which are other than the dephosphorylated AttoPhos~.
The method can be used for determining the presence or the amount of a biological substance in a biological sample.
The method comprises the steps of: a) forming a enzyme conjugated binder (antibody or nucleic acid probe) complex with a biological substance from the biological sample; b) A~Ai~ AttoPhos~ and a 1,2-dioxetane to the bound enzyme conjugate biological substance complex c) wherein the enzyme of the enzyme conjugate cleaves a phosphate moiety from the AttoPhos~ and from the dioxetane, thereby causing the dioxetane to decompose through an excited state form such that an energy transfer occurs from the excited state donor of dioxetane to the dephosphorylated AttoPhos~ acceptor, causing it to luminesce; and d) dete~ i~;ng the pre-en~q or amount of the biological substance as a function of the amount of lumi n~~~n~.
The kit of the present invention is also for determining the pr~~nce or co~c~ntration of a biopolymer and comprises:
a) an enzyme complex which will bind to a biological substance upon admixture therewith; b) a 1, 2-dioxetane which when contacted by the enzyme of the enzyme complex will be caused to decompose into a decomposition product which is in an excited state; and c) AttoPhos~.
The assays and kits of this invention employ water-soluble chemilumin~s~nt 1,2-dioxetanes. As noted above, these dioxetanes are well established in the art, and their W 096/25667 PCTnUS9S101506 identity and preparation do not constitute a novel aspect of this invention, per se. In general, any chemiluminescent dioxetane which exhibits sufficient solubility and stability in aqueous buffers to conduct the assay, and which may be caused to decompose and chemilumin~rce by interaction with an enzyme, and cleavage, by the enzyme, of an enzyme labile group inducing the decomposition, can be used in connection with this invention.
Typically, the 1,2-dioxetanes useful in this invention will have the general formula:

O--O
ORl (I) ' ~ 2 z Z ~ H, Cl, other halogens, alkyl, carboxy, or alkoxy groups;
R1 is C~-C20 alkyl or Cl_l2 aryl or aralkyl;
Y is phenyl or naphthyl, unsubstituted or substituted with an electron donating or electron withdrawing group;
R2 is meta-substituted or non-conjugated on Y with respect to the dioxetane, and is OX, wherein;
X is an enzyme cleavable group which, when cleaved, leaves the dioxetane ph~oYy or naphthoxy anion.
Suitable dioxetanes are those disclosed in U.S. Patent Application 08/057,903, the entire disclosure of which is incorporated herein by reference. Preferred dioxetanes include dioxetanes in which X is a phosphate moiety.
Particularly preferred dioxetanes include AMPPD, and in particular, its disodium salt, as well as CSPD, and in particular, its disodium salt. Method~ of preparing these dioxetanes are disclosed in the afore-referenced, commonly-assigned patents, as well as, e.g., U.S. Patent 4,857,652, assigned to Wayne State University. The preparation, W O 9612~667 PCT~US9~01~06 -14-purification and isolation of the dioxetanes does not constitute a novel aspect of the invention disclosed and claimed herein per se.
AttoPhos~ is a highly sensitive fluorometric substrate for the detection of alkaline phosphatase. The chemical structure of AttoPhos~ is not known at the present time.
However, the chemical properties of AttoPhos~ are known.
AttoPhos~ was developed by JBL Scientific and can be obtained from the JBL-Scientific catalog (1993) at catalog number 167OA.
The chemical and physical properties of AttoPhos~ are as follows. AttoPhos~ is a pale, yellow crystalline solid having a molecular weight of approximat~ly 580 grams/mol. The turnover number for AttoPhos~ is 85,400 molecules of AttoPhos~
per minute per molecule of alkaline phosphatase in 2.40 M DEA
(d$ethanolamine) pH 9.O, O.23 mM MgC12 and O.005% NaN, by weight. The solubility of AttoPhos~ is > 10 mM in aqueous 2.4 M DEA buffer at a pH of 9.O. The optimum alkaline phosphatase turnover occur~ at a substrate conce~tration of O.5-1.5 mM AttoPhos~. AttoPhos~ has a Km value of O.030 mM
and a molar absorptivity of 31.412.
When contacted with alkaline phosphatase, AttoPhos~ is known to become a fluorescent emitter. The mol~c~l A~ weight of the fluorescent emitter is approximately 290 g/mole. This ~luorescent emitter has an excitation maximum in the visible range at 430-450 nm with fluorescence monitored at 550-570 nm, in a DEA bu~fer. Best conditions are at 440 nm for excitation with 550 nm emission. The fluorescent emitter also has an emission maximum at 560 nm, and a large Stokes Shift of 140 nm. The Water Raman emission occurs at 470 nm with an excitation at 413 nm. The fluorescent emitter has a maximum at 418 nm with an coefficient of 26,484 in 0.392 M Na2C03 and a pH of 11.0 and is fully ionized at a pH > 10Ø
The dioxetane is added to an enzyme complex which is bound to a biological binder (antibody or nucleic probe). The enzyme complex is also bound to the target biological W 096125667 PCTnUS95101~06 substance. The dioxetane i~ therefore the substrate for the enzyme, the enzyme-catalyzed cleavage of the labile ~L OU~ of the substratQ from the body of the dioxetanQ resulting in the formation of the un:table oxyanion, and ~h-equent decomposition of the dioxetane. The enzyme i8 usually complexed with a binder moiety, such as a DN~ probe in a hybridization step or suitable antibody in an in~hAtion step, so as to help bind to the biological substancQ.
The hybridization step can be carried out using s~ rd~
wellknown pro~n-l ~es and using a suitable probe.
As an alternative to a hybridization step, an inCllh~tion step can be carried out in the usual ~e~ using a suitable antibody.
The enzyme conjugate can be any enzyme conjugate capable of stably binding to the biological substance. Examples of the enzyme conjugate are any ligand-binder pair, probe with a covalently attached enzyme, or antibody labelQd directly with alkaline phosphata~e. Alternatively, the nucleic acid probes and an~; h~.l ie may be labelled indirectly with enzymeE~ ~ia a biotin-t-L~L}avidin or antigen-antibody (such as degoxigenin-antidigoxigenin, fluore8cein-antiflUoresCQin) and other type coupling. Derivatized ~1 ~A 1 i n~ phosphatase such as Streptavidin-alkaline phosphata~e alkaline rho~rh~tase l ~h~
ant; hoA; es and DNA probec~ are the preferred enzyme conjugateff useful in the present invention.
After the enzyme conjugate-biological substance complex is formed. AttoPhos~ and the 1,2-dioxetane are added ~o th~
bound enzyme conjugate complexed with biological substance either simult~eo~ly, or AttoPhos~ is added first, allowed to dephosphorylate, and subsequently, a 1,2-dioxetane is added.
It will be apparent to those of skill in the art that it is the process of enzyme cleavage which places the energy-donating dioxetane emitter fragment in close proximity to Atto~ which is also produced locally by the same enzyme.
AttoPhos~ itself, like other fluorometric enzyme substrates is non-fluorescent in the bulk phase. Thus, any non-enzymatic W O 96/2S667 ~CTnUS95101506 decomposition of the dioxetane, which would produce a noise signal, i~ not amplified by energy transfer in the bulk phase.
Thus it i8 an enzyme reaction which produce~ the hydrophobic, fluorescent form allowing immobilization on the surface used to perform the assay. It will also be apparent that other hyd.G~hobic, fluorimetric enzyme substrate~ can also be used in the invention. U.S. Patent 5,208,148, referred to above, de~cribQs fluorescein diglycosid~s which are specifically modified by the inclusion of a range of l~y~hobic moietieO
attached to the planar, fluorophore itself. Such hydrophobic substrates would be useful for performing the bioassays of the invention where the enzyme label utilized is a glycosidase such as beta-galactosidase and the dioxetane was of the general structure shown above where for example, Z=Cl, Rl~methyl, Y=phenylene, and X=beta-D-galactopyranoside. O~
course, the hydrophobic hydroxyfluore~-sinA shown in this patent as precursors to the diglycosides may inDtead by rhoc~horylated using known art to give hydrophobic fluo~ n mono- and diphosphate derivatives which are useful in the present invention.
The enzyme cleaves a phosphate moiety from both the 1,2-dioxetane and AttoPhos~. AB the 1,2-dioxetane becomes ~h~ phorylated by the enzyme, the formed oxyanion becomeD
the excited state donor, and its energy is transferred to the closely positioned acceptor--the ~erhocrhorylated AttoPhos~
emitter, causing it to emit. Figure 1 illustrates the energy transfer from the 1,2dioxetane (CS-D) to the dephosphorylated AttoPhos~, which in turn, releasing energy in the form of lumin~-c-nc~. The energy transfer efficiency is ~nhAnc~ as the ~pho~phorylated product of AttoPhos~--acceptor, is hydrophobic and is immobilized in the surface/biological substance sites and therefore is in very close proximity to the chemilumine-~nt dephosphorylated 1,2-dioxetane's excited state fragment which is the energy donor.
The 1,2-dioxetane is added to the bound enzyme conjugate complexed with biological substance in an amount of from 0.01 W096/25667 PCT~S95101506 to 2.5 mM, preferably 0.25 to 1 mM. Most preferably, the 1,2 -dioxetane is added in an amount of 0.25 mM.
AttoPhos~ in the 2.40 M diethanolamine (DEA) in water buffer is added to the enzyme or enzyme conjugated binder completed with biological substance in an amount Or from l-100%, prefQrably 25 to 75~ by volume. ~ost preferably, lO to 50% by volume AttoPhos~ i5 added.
As stated above, it i5 preferred that AttoPhos~ is added ~irst, allowed to ~pho~rho~ylate, and ~ uently, a 1,2-dioxetane is added. The time period between addition of AttoPhos~ and addition of a l,2-dioxetane is preferably lO to 60 minutes, more preferably 20 to 40 minutes, and most preferably 25 to 30 minutes.
The signal can be further ~h~nc~ by the addition of a water-soluble macromolecule along with AttoPhos~ or other hydlG~ic fluorometric enzyme substrate. Preferred water-soluble polymers useful in practicing the invention, are based, in general, on polymeric onium salts, particularly guaternary salts based on phosphonium, sulfonium and, preferably, ammonium moietiea. The polymerfi have the general formula I shown below:

CH2-CH ~
CH2 M~ ( I ) 1~R2 In this formula each of Rl, R2 and R3 can be a straight or br~nche~ chain unsubstituted alkyl group having from l to 20 carbon atoms, inclusive, e.g., methyl, ethyl, n-butyl, t-butyl, hexyl, or the like; a straight or branched chain alkyl group having from l to 20 carbon atoms, inclusive, substituted with one or more hydroxy, alkoxy, e.g., methoxy, ethoxy, W096/2~667 PCT~S95101506 benzyloxy or polyoxethylethoxy, aryloxy, e.g., phPnoYy, amino or substituted amino, e.g., methylamino, amido, e.g., acetamido or ureido, e.g., phenyl ureido; or fluoro~lkAn~ or fluoroaryl, e . g ., heptafluorobutyl, ~lOU~, an unsubstituted monocycloalkyl group having from 3 to 12 carbon ring carbon atoms, inclusive, e.g., cyclohexyl or cyclooctyl, a substituted monocycloalkyl group having from 3 to 12 ring carbon atoms, inclusive, substituted with one or more alkyl, alkoxy or fused benzo groups,, e-g-,, methoxycyclohexyl or 1,2,3,4-tetrahydronaphthyl, a polycycloalkyl group having 2 or more fused rings, each having from 5 to 12 carbon atoms, inclusive, unsubstituted or substituted with one or more alkyl, alkoxy or aryl groups, e.g., l-adamantyl or 3-phenyl-1-adamantyl, an aryl, alkaryl or aralkyl group having at least one ring and from 6 to 20 carbon atoms in toto, ~lnCllhctitutQd or substituted with one or more alkyl, aryl, fluorine or hydkox~ ~~ou~_,, e.g., phenyl, naphthyl, pentafluorophenyl, ethylphenyl, benzyl, hydroxybenzyl, phenylbenzyl or dehydroabietyl; at least two of Rl, R2 and R3, together with the quaternary nitrogen atom to which they are ho~ , can form a saturated or unsaturated, ~n--~h~tituted or substitutQd nitrogencont~ining, phosphorus-containing or sulfur-con~A i ni hg -ring having from 3 to 5 carbon atoms, inclusive, and 1 to 3 heteroatoms, inclusive, and which may be benzoannulated, e.g., 1-pyridinium, 1-(3-alkyl or aralkyl)imidazolium, morpholino, alkyl morpholinium, alkylpiperidinium, N-acylpiperidinium, piperidino or acylpiperidino, benzoxazolium, benzthiazolium or benzamidazolium.
The symbol X~ represents a counterion which can include, alone or in combination, moieties such as halide, i.e., fluoride, chloride, bromide or iodide, sulfate, alkylsulfonate, e.g., methylsulfonate, arylsulfonate, e.g., p-toluenesulfonate, substituted arylsulfonate, e.g., anilinonaphthylenesulfonate (various isomers), diphenylanthracenesul~onate, Perchlorate, alkanoate, e.g., acetate, arylcar~oxylate, e.g., fluorescein or fluorescein W 096125667 PCTn~S95101S06 -19-derivatives, benzoheterocyclic arylcarboxylate, e.g., 7-diethylamino-4-cyAnoconmarin-3-carboxylate, organic dianions such as p-terephthalate may also be represQnted by X~.
The symbol n represents a number such that the mol~c~ r weight of such poly(vinylbenzyl Quaternary salts) will range from about 800 to about 200,000 (weight average), and preferably from about 20,000 to about 70,000, as determined by intrinsic viscosity or LALLS techniques.
Methods for the preparation of these polymers, related copolymers and the related starting materials where M i~
nitrogen are disclosed in G. D. Jones et Al, Jo~l~nAl of pol~mer Science, ~, 201, 1958; in U.S. Patents 2,780,604;

3,178,396; 3,770,439; 4,308,335; 4,340,522; 4,424,326 and German Offenle~lncc~hrift 2,447,611.
The sym~ol M may also represent phosphorous or sulfur whereupon the co~Le~o.,ding sulfonium or phosphonium polymers have been described in the prior art: U.S. Patentc 3,236,820 and 3,065,272.
Methods of preparation of the two polymers of this invention are set forth in the referenced U.S. Patents, and do not constitute any aspect of this invention, per SQ.
Copolymers cont~in;ng 2 or more different pen~Ant onium groups may also be utilized in the invention described herein:

CH2-CH~ (CH2-CH ~
~ ~ (II) CH2 fH2 R~ X Rl M~ X
R2/\R3 R2'/R~' The symbols X, M~, Rl~, R2~, R3~ are as described above for X, M, Rl-R3. The symbols Y and Z represent the mole fraction of the individual monomers comprising the copolymer. The symbols Y and Z may thus individually vary from .01 to .99, with the W O 96125667 PCT~US95101S06 sum always equalling one.
As preferred moieties, M is N or P, and Rl-R3 are individually, ;n~epen~ntly~ alkyl, cycloalkyl, polycycloalkyl (e.g. adamantane) aralkyl or aryl, having 1 to 20 carbon atoms, unsubstituted or further sub~tituted with hydroxyl, amino, amido, ureido ~-ou~, or combine to form via a spiro linkage to the M atom a heterocyclic (aromatic, aliphatic or mixed, optionally including other N, S or O hetero atoms) onium moiety.
X is preferably selected to im~Lo~e solubility and to change ionic strength as desired, and is preferably halogen, a sulfate, a sulfonate. In copolymers, each of Rl-R3 may be the same as or different from the corresponding Rl-~3~. Examples of preferred polymers include the following:

W O 96/25667 P ~ nUS95101S0 ~' Cl- ' CH~P(CH~CHlCH2CH~)~
~oly~inyl~3nzyltributyl ~h~-3~h~ni um chlorido ~x [ ~Y

C~P[~CH2) 7C~3] 3 ~ Cl CH~Pt(CH~)3CH313 poly~inyIbenzyltrioctyl ph~3~ i chloride-co-Poly~inylbenzyltribut ~hoe~h~-~; chlorido ~_]x [~y ~ ~Nt(CH2)3CH3]~ ~ Cl C~I~P tC6~] 3 polyvinylbenzyltributyl a~30niUm chloride-Co-~olyVinYlbenZYltri ph ~3~h ~n i U13 chloridQ

~ Cl' CH~

polyvinylbenzylbonzyldimethyl : i chlorid~

~Cl CH~N~CH2CH~CH2CH3) 3 ~olyvinylbenzyltributyl~ ium chloride ~]X [~Y

CH~N [ ( CH 2 ) sCH3 ] ~ ~ Cl CH~N t (CH2 ) 3C}~3] 3 polyvinylbenzyltrihexyl ammonium chloride-co-polyvinylbenzyl tributyl A~m~nium chloride (THQ-TBQ) W 096/25667 ~CTnUS95101506 These vinylbenzyl quaternary ammonium salt polymers can be prepared by free radical polymerization of the appropriate precursor monomers or by exhaustive alkylation of the corresponding tertiary amines or phosphines with polyvinylbenzyl chl~ride, or copolymers cont~ining a rsn~t ~ benzyl chloride function. This same approach can be taken using other polymeric alkylating agents such as chloromethylated polyphenylene oxide or polyepichlorohydrin.
The same polymeric alkylating agents can be used aa initiators of oxazoline ring-opening polymerization, which, after hydrolysis, yields polyethyleneimine graft copolymers. Such copolymers can then be quaternized, preferably with aralkyl groups, to give the final polymer.
water soluble acetals of the polyvinylalcohol and a formylbenzyl quaternary salt, having the formula OHC X
~ \R (III) wherein each R4 is the same or a different aliphatic substituent and X1 is an anion, as disclosed and claimed in Bronstein-30nte et al U.S. Patent 4,124,388, can also be used in practicing this invention. And, the individual vinylbenzyl quaternary ammonium salt monomers used to prepare the poly(vinylbenzyl quaternary ammonium salts) of formula I above can also be copolymerized with other ethylenically unsaturated monomers having no quaternary ammonium functionality, to give polymers such as those disclosed and claimed in Land et al U.S. Patent 4,322,489; Bronstein-Bonte et al U.S. Patent 4,340,522; Land et al U.S. Patent 4,424,326; Bronstein-Bonte U.S. Patent 4,503,138; Bronstein-Bonte U.S. Patent 4,563,411;
and Cohen et al U.S. Patent 3,898,088, all of which polymers can also be used as ~nh~cer substances in practicing this invention. Preferably these quaternized polymers will have W 09612S667 PCTnUS95/01506 -2~-molec~ r weights within the ranges given above for the poly(vinylbenzyl quaternary ammonium salts) of Formula I.
As it will be apparent to one skilled in the art, the use of cationic microgels or crosslinked latices are more suitable for the direct formation of cast membranes, but can also be used for the overcoating of praformed membranes. Such materials are well known as photographic mordants and may be synthesized using a n~ -~ mixture which contains a cros81in~irlg moiety substituted with two ethylenically unsaturated groups. Quaternary ammonium or phosphonium salt containing latices can be prepared using methodologies described in C~mrhDll et al U.S. Patent 3,958,995.

~ CH2- CH)x ~CH2- CH)y (CH2_ CH

X-- CH2~-- CH2 11\ R~
~3 R2 Formula IV generally represents a useful subset of such water-soluble latex copolymers wherein the symbols X~, Rl, R2 and R3 are as described above. The symbols X, Y and Z are mole fractions which must add together to give unity.
Preferably, a polymeric enhancer such as BDMQ is added to the enzyme or enzyme conjugate biological substance sources in an amount of 0.01 to 26% (0.1 to 250 mg/ml), more preferably 0.025 to 15% (25 to 150 mg/ml). Most preferably, BDMQ is added in an amount of 0.1 to 0.2% (i to 2 mg/ml).
The emitted signal resulting from the dephosphorylated AttoPhos~ is by way of an energy transfer excitation from the excited state dioxetane dense fragment. The emitted signal can be captured on a green sensitive film or in a luminometer, CCD camera. The amount of emission detected will be responsive both to the prec~nc~ of the biopolymer, and to the amount of ~e surface-bound biopolymer. The amount of w096/25667 PCT~S95101506 biological sub~tance is a function of the intensity of the emission.
The m~thod~ and the kits of the present invention can be used to determine the pr~nc~ or con~ .Llation of any biological substance, including RNA, DNA, proteins and - haptens. Further, the methods and kits of the present invention can be used for detections performed on membranes such as Western, Southern, Northern blotting and DNA
sequencing, and can also be used for solution-phase assays.
In the solution-based a~say or when enhancing polymers are employed, they may require the ~ephocphorylated products of both AttoPhos~ and l,2-dioxetane substrates, and thereby increasing the proximity between the donor and acceptor moieties.

~X~
~X~hnPT.~ 1 ~-st~rn blottlng on nltrocolluloso an~ PVDF ~etoct~on of prot-~ns Ig~ on m mbran-s ~m~ge~ on ~hotometr~cs 8t~r 1 CCD
au~-r~).
Dilutions of rabbit IgG were electrophoresed on a 10%
polyacrylamide gel using st~ rd, known methods. The IgG
samples were 200, 66.7, 22.2, 7.4 and 2.4 ng per lane for nitrocellulose and lOO, 33.3, ll.l, 3.7 and l.2 ng per lane for PVDF. The protein was then transferred to the membrane as follows: the gel was equilibrated in transfer buffer (5 mM
MOPS, 2 mM sodium acetate, 20% methanol, pH 7.5) and then elecL~uLLansferred to nitrocellulose (Schleicher and Schuell BAS85) or PVDF (Tropix) at so volts for 1 hour at 4~C.
After transfer, the membranes were rinsed with phosphate buffered saline (PBS), blocked with 0.2% casein, 0.1% Tween-20 in PBS(blocking buffer), incubated for 30 minutes with a l-~ lO,OOO dilution of alkaline phosphatase conjugated goat anti-rabbit ~ntibody (GAR-AP) in blocking buffer, the PVDF
membranes were washed twice for 5 minutes in bloc~ing buffer, the nitrocellulose membranes were washed twice in O.1% Tween-W 096/25667 PCTnUS95101506 20 in PBS, all membranQs were washed twice for 5 minutes in 0.1 M diethanolamine, 1 mM MgC12, pH 10 (substrate buffer), nr~h~ted for 5 minutes in a 1-20 dilution of Nitro-Block (Tropix) in substrate buffer, washed twice for 5 minutes in substrate buffer, incllh~ted for 5 minutes in 0.25 mM CSPD in substrate buffer and AttoPhos~ under various conditions, ~e~ in a plastic report cover, in~llhAted for approximately 1 hour and imaged for 5 minutes with a Star I CCD camera (Photometrics).
Chemilumin~-c~nt images were obt~in~ by integration Or the chemilum;n~s~ent signal for 5 minutes with a Star 1 CCD
camera interfaced to an Apple Macintosh IIci computer using IPLab Spectrum software. The CCD images were transferred into the NIH Image software package, and average and maximum pixel intensities were measured for each band.
The cCD images, shown in Figure~ 2 and 4, are compo~ites of the Western blot images. Blot A was in~hAted in 0.25 mM
CSPD in substrate buffer. Blot B was ~nc~h~ted in 0.25 mM
CSPD and 50~ AttoPhos~ (50% AttoPhos~ buffer) simult~n~o~l~ly.
Blot C was incl~h~ted first in 50% AttoPhos~ (50% substratQ
buffer) for 30 minutes, the AttoPhos~ was removed, the membrane was washed twice for 5 minutes in substrate buffer, and 0.25 mM CSPD in substrate buffer was added. Blot D wa~
incubated for 30 minutes with undiluted AttoPhos~ st~nA~d, then the membrane was washed twice for 5 minutes in substrate buffer followed by 0.25 mM CSPD in substrate buffer. Images were obtained approximately 1 hour after the initial addition of CSPD. The average and maximum signal intensities were plotted for the top dilution for each of the conditions described above as shown in Figures 3 and 5.
The results shown in Figures 2-5 demonstrate that maximum intensity is ob~ine~ by the addition of AttoPhos~ followed by subsequent addition of the 1,2-dioxetane after a set period of time.

~ nPT~ 2 W096/25667 PCT~S95101506 PQA ~ "~~~~y t~Ybr~t--Ch Pro--tat-- 8~ G A~tlg--~ ~PBA)~.
The s~n~rds from a Hybritech Tandem-E PSA kit (catalog ~4823) were quantitated using the protocol and rQagQnts supplied by the manufacturQr, oxcapt for the detection step.
The a~say was performed as follows. An amount of 100 ~L of each s~nA~rd was aliquoted into 12 X 75 mm glass tubes (6 triplicates of the zero and tripliCatQs of the other s~A~ds). An amount of 100 ~L of th~ Al~Al~nq phosphatasQ
conjugated mouse anti-PSA was added to each tubQ followed by one bead with attached capture anti-PSA antibody. The tubQs were then incubated for 2 hours at room temperature on a chAk; ng platform at 170 RPM. The beads were washed three times with 2 mL of Hybritech wash solution and once with 0.1 M
diethanolamine, 1 mM MgCl2, pH 10 (substrate ~uffer).
Substrate was then added to each tube. The following three substrate compositions (200 ~L per tube) were te~ted: 0.25 mM
CSPD, 1 mg/mL BDMQ in substrate buffer added at time zero;
0.25 mM CSPD, 1 mg/mL BDMQ, 50~ AttoPhos~ in substrate buffer ~e~ at time zero; 50% AttoPhos~, 1 mg/mL BDMQ in ~ubstrate buffer for 30 minute~ followed by the addition of CSPD (final con~ntration 0.25 mM). The chemilumin~-~ent signal was measured 25 minutes after the addition of CSPD (or CSPD/AttoPhos~ mixture) with a Berthold 9S2T luminometer.
Figures 6(A) and (B) demonstrate that both the signal and signal/noise ratios are greater with CSPD and AttoPhos~ than with CSPD alone. Therefore, increased signal was the result of use of CSPD in connection with AttoPhos~.

~X ~ pT.~ 3 8O1ution onorgy transfer (onergy trans~er b-t~een th-dophosphorylated AttoPhos~ an~ tho dephosphoryl~to~ C8PD).
- The ~ollowing is a list of the samples used for the Spex emission spectra. For all samples, 0. 1 M diethanolamine, 1 mM Mc-12, pH 10 was used to adjust the final volume to 2 ml.
100% Sapphire is equivalent to 10.0 mg/ml BDMQ.
1. Fig. 7 0.25 mM CSPD, 50% AttoPhos~

WOs6/25667 PCT~S9SJ01506 2. Fig. 8 1.0 mM CSPD, 50~ AttoPhos~
3. Fig. 9 0.1 mM CSPD, 50~ AttoPhos~, 20~ BDMQ
4. Fig. 10 0.25 mM CSPD, 50~ AttoPhos~, 20% BDMQ

5. Fig. 11 0.5 mM CSPD, 50~ AttoPhos~, 20~ BDMQ

6. Fig. 12 1.0 mM CSPD, 50~ AttoPhos~, 20~ BDMQ

7. Fig. 13 1.0 mM CSPD, 50~ AttoPhos~, 10% BDMQ

8. Fig. 14 1.0 mM CSPD, lOS AttoPhos~, 20~ BDMQ

9. Fig. 15 1.0 mM CSPD, 50~ AttoPhos~, 2.0 mg/ml TPP(0.4)/BDMQ(0.6) 10. Fig. 16 1.0 mM CSPD, 50~ AttoPhos~, 2.0 mg/ml TPP(0.45)/TBQ(0.55) 11. Fig. 17 a 30 minute preincllh~tion of alkaline phosphatase in 50% AttoPhos~, 20~ BDMQ, followed by the addition of CSPD (o.2s mM final concentration) at time zero At time ~ 0, alkaline rho~rh~tasQ was added to each sample (f inal cQncentration, 1.12 X lo~11 M) and the ~v~e was inserted into the fluorimeter (Spex Fluorolog). Emission spectra were obtained with the monochrometer slits set at 10 mm and signal was integrated for 0.5 seconds per nm.
Spectra were recorded at 2, 10, 20, 30, 40, 50 and 60 minutes, in most cases.
The results are shown in Figures 7-21.
This set of experiments shows energy transfer from CSPD to AttoPhos~ in a buffer. Such solution-based assays are used with immuno~c~ys which are performed in buffers.
Figures 7-21 demonstrate that there is an energy transfer between the dephosphorylated emitter of CSPD and the dephosphorylated AttoPhos~. Figures 9-17 further show that this energy transfer is greatly improved by the presence of ncing polymers. Figures 7 and 8 demonstrate that an increase in the donor, dephosphorylated CSPD emitter increases the signal via energy transfer, i.e., the Attoemission. In this case, the blue emission (CSPD chemiluminescence) increases. This may be due to a population of the methyl-W096/2~667 PCT~S95101506 metaoxybenzoate anion (CSPD emitter) which is not within the energy transfer distance from the Atto~ acceptor. Figure 14 demonstrates that the green signal originates from Atto~, because when the conc~tration of AttoPhos~ is low, the energy transfer signal iB also very low. Figure 12 shows that the relative energy transfer signal when the substrates are added sequentially, i.e., first A~;nq AttoPhos~ which becomes o~phorylated creating the ground state emitter, followed by CSPD addition which upon ~r~ocphorylanon~ fragments and, generates the excited state donor which transfers its energy to the accumulated acceptor from the dephosphorylated AttoPhos~.

~A~rPT.~ 4 D-t-ct~on of b~ot~nyl~te~ D~a Biotinylated DNA was detected by bi ~A; n1 streptavidin alkaline phosphatase, and then ~lh-~uQntly in~llhAting with either CSPD 1,2-dioxetane substrate for alkaline phosphatase or mixtures of CSPD and the fluorescent alkaline phosphatase substrate AttoPhos~. Specifically, biotinylated 35mer wa~
spotted on to Pall Biodyne A nylon membrane, 210 pg in tho top spot followed by s~l~ce-~ive 1:3 dilutions. DNA was detected by performing the Tropix Southern-~ightTm procedure up to the substrate in~hAtion step. Esch membrane was then individually ;n~llhAted with a different substrate solution as follows:
1) 0.25 mM CSPD in assay buffer (O.lM DEA pH 10, 1 mH
Mgcl2) ~
2) 50% AttoPhos~ solution; 50% 1 mM CSPD in assay buffer, 3) 50% AttoPhos~ solution; 50% 0.25 mM CSPD in assay buffer, 4) 1 mM CSPD in AttoPhos~ solution, 5) membrane coated with dephosphorylated AttoPhos~ and then incubated with 0.25 mM CSPD in assay buffer, 6) AttoPhos~ solution.

W096/25667 PCT~S9~01506 The image was obtained using a Photometrics Star 1 CCD
Camera in a light-tight box without any external light source.
Figure 22 show~ an increa~Qd light signal from the sample~ of AttoPhos~ in combination with CSPD.
Applicants have endeavored to illustrate their invention by extensive emho~;ment of possiblQ combinations.
Nonetheless, it is recognized that the possible combinations are endles~, and cannot be exhaustively embodied. Given thQ
above ~ ing~ those of ordinary skill in the art will arrive at ~nh~n~ement agents and additives not specifically exemplified in the foregoing application. The examples are not inte~e~ to be limiting, and the identification of other combinations, given the foregoing disclosure, is well within the skill of those practicing this t~ch~ology without undue experimentation. Such combinations are intended to be within the scope of the invention, save a~ expressly limited or excluded by the claims set forth below.

Claims (15)

Claims

1. A method for determining the presence or the amount of a substance in a biological sample, said method comprising the steps of:
a) completing an enzyme or enzyme conjugate with said substance from said sample;
b) adding a hydrophobic fluorometric substrate and a 1,2-dioxetane of the following formula (I) to said bound enzyme complex:

Z = H, Cl, other halogens, alkyl, carboxy or alkoxy groups;
R1 is C1-C20 alkyl or C1-12 aryl or aralkyl;
Y is phenyl or naphthyl unsubstituted or substituted with an electron donating or electron withdrawing group;
R2 is meta-substituted or nonconjugated on Y with respect to the dioxetane, and is OX, wherein;
X is an enzyme cleavable moiety which dioxetane, when X
is cleaved, is hydrophobic;
c) wherein the enzyme of said enzyme-complexed substance cleaves an enzyme cleavable moiety from each of said hydrophobic fluorometric substrate and dioxetane, thereby causing said dioxetane to decompose to form an excited state donor such that an energy transfer occurs from said excited state emitter to said fluorometric substrate as acceptor, causing said acceptor to emit;
d) determining the presence or amount of said substrate as a function of the amount of emission.

2. The method of Claim 1, further comprising adding to said enzyme or enzyme conjugate complexed with said substance one or more enhancement polymeric salts selected from the group consisting of ammonium, phosphonium, and sulfonium polymeric salts.

3. The method of Claim 2, wherein the polymeric salts are selected from the group consisting of poly(vinylbenzyltrimethylammonium chloride) (TMQ), poly(vinylbenzyltributylammoniumchloride) (TBQ), and polyvinylbenzyldimethylbenzylammoniumchloride) (BDMQ).

4. The method of Claim 1, wherein said substance is selected from the group consisting of RNA, DNA, proteins and hapteas.

5. The method of Claim 1, wherein hydrophobic fluorometric substrate is added to enzyme or enzyme conjugate completed with said substance, and the 1,2-dioxetane is added subsequently, after a period of time sufficient to allow the enzyme to cleave an enzyme cleavable group from said fluorometric substrate.

6. The method of Claim 5, wherein the period of time is 25 to 30 minutes.

7. The method of Claim 1, wherein said 1,2-dioxetane is present in concentrations of 0.25 to 1.0 mM.

8. The method of Claim 1, wherein said fluorometric substrate is AttoPhos~ and is present in the form of a 2.40 M
diethanolamine (DEA) in water buffer in concentrations of 10 to 100 percent by volume.

9. The method of Claim 8, wherein poly(vinylbenzyldimethylbenzylammonium chloride) (BDMQ) is added at step (b) in an amount of 1 to 2 mg/ml in addition to AttoPhos~ and 1,2-dioxetane.

10. The method of Claim 1, wherein prior to step (b), a hybridization or an immunocomplexion step is performed.

11. The method of Claim 1, wherein said complexed enzyme is bound to a membrane or beads.

12. The method of Claim 1, wherein said hydrophobic fluorometric substrate is AttoPhos~.

13. A kit for conducting a bioassay for the presence of concentration of a substance in a biological sample comprising:
a) an enzyme or enzyme conjugate which will complex with a biological substance upon admixture therewith;
b) a 1,2-dioxetane which when contacted by the enzyme or said enzyme complex will be caused to decompose into a decomposition product forming a hydrophobic excited state donor; and c) a hydrophobic fluorometric substrate.

14. A method of determining the presence or amount of an enzyme in a biological sample, comprising:
a) adding to said sample a hydrophobic fluorometric substrate and a dioxetane of Formula I:

wherein Z = H, Cl, other halogens, alkyl, carboxy or alkoxy groups;
R1 is C1-20 alkyl or C1-12 aryl or aralkyl;
Y is phenyl or naphthyl unsubstituted or substituted with an electron donating or electron withdrawing group;
R2 is meta-substituted or nonconjugated on Y with respect to the dioxetane, and is OX, wherein;
X is an enzyme cleavable moiety which dioxetane, when X
is cleaved, is hydrophobic;
b) detecting the presence or amount of emission from said hydrophobic fluorescent substrate after admixture, wherein said enzyme cleaves an enzyme cleavable moiety from said hydrophobic fluorescent substrate and said dioxetane, such that an energy transfer occurs from an excited state decomposition product of said dioxetane to said hydrophobic fluorescent dioxetane.

14. A method of determining the presence or amount of an enzyme in a biological sample, comprising:
a) adding to said sample a hydrophobic fluorometric substrate and a dioxetane of Formula I:

wherein Z is H, Cl, halogens other than Cl, alkyl, carboxy or alkoxy; R1 is C1-20 alkyl or C1-12 aryl or aralkyl, Y
is phenyl or naphthyl unsubstituted or substituted with an electron donating or electron withdrawing group; R2 is meta-substituted or nonconjugated with respect to the dioxetane ring, and is OX, wherein X is an enzyme cleavable moiety, which dioxetane, when X is cleaved, is hydrophobic;
b) detecting the presence or amount of emission from said hydrophobic fluorescent substrate after admixture, wherein said enzyme cleaves an enzyme cleavable moiety from said hydrophobic fluorescent substrate and said dioxetane, such that an energy transfer occurs from an excited state decomposition product of said dioxetane to said hydrophobic fluorescent dioxetane.

15. A method for determining the presence or the amount of a substance in a biological sample, said method comprising the steps of:
a) complexing an enzyme or enzyme conjugate with said substance from said sample;
b) adding a hydrophobic fluorophore to said complex;
c) adding a 1,2-dioxetane of the following formula to said complex and fluorophore;

wherein Z = H, Cl, halogens other than Cl, alkyl, carboxy or alkoxy groups;
R1 is C1-C23 alkyl or C1-12 aryl or aralkyl, Y is phenyl or naphthyl unsubstituted or substituted with an electron donating or electron withdrawing group;
R2 is meta-substituted or nonconjugated on Y with respect to the dioxetane, and is OX, wherein X is enzyme cleavable moiety, which dioxetane, when X is cleaved, is hydrophobic;

wherein the enzyme of said complex cleaves an enzyme cleavable moiety from said dioxetane, thereby causing said dioxetane to decompose to form an excited-state donor such that energy transfer occurs from said excited-state donor to said fluorophore as acceptor, causing said acceptor to emit, and determining the presence or amount of said substrate as a function of the amount of emission.