Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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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
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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
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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
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~ 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
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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
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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
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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
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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
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_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%
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W096l25667
--10--
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.
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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.
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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
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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,
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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
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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
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-16-
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
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-17-
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,
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-18-
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
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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
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-20-
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:
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-21-
~'
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
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--22--
~ 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)
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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
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-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
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-25-
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-
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-26-
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
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W096/25667 PCT~S95101506
-27-
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~
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-28-
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-
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-29-
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.
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-30-
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.
