METHOD OF DETECTING A SUBSTANCE USING ENZYMATICALLY-INDUCED DECOMPOSITION OF DIOXETANES

METHODE POUR DECELER UNE SUBSTANCE GRACE A LA DECOMPOSITION DE DIOXETANES INDUITE PAR VOIE ENZYMATIQUE

English Abstract

In an assay method in which a member of a specific
binding pair is detected by means of an optically detectable
reaction, the improvement wherein the optically
detectable reaction includes the reaction, with an enzyme,
of a dioxetane having the formula
(see formula I)
where T is a cycloalkyl or polycycloalkyl group bonded to
the 4-membered ring portion of the dioxetane by a spiro
linkage; Y is a fluorescent chromophore; X is hydrogen,
alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl,
cycloalkyl, cycloheteroalkyl, or enzyme-cleavable group;
and Z is hydrogen or an enzyme-cleavable group, provided
that at least one of X or Z must be an enzyme-cleavable
group, so that the enzyme cleaves the enzyme-cleavable
group from the dioxetane to form a negatively charged
substituent banded to the dioxetane, the negatively
charged substituent causing the dioxetane to decompose to
form a luminescent substance that includes group Y of said
dioxetane.

Claims

47
CLAIMS:
1. An assay method of detecting a member of a
specific binding pair in a sample, which comprises:
contacting the sample with an enzyme bonded to a
substance having a specific affinity for the member, wherein
the enzyme, the substance or the member to be detected is
carried by solid matrices;
reacting the enzyme with a dioxetane of the
formula:
<IMG>
(wherein T is a cycloalkyl or polycycloalkyl group bonded to
the 4-membered ring portion of the dioxetane by a spiro
linkage; Y is a fluorescent chromophore; X is hydrogen,
alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl,
cycloalkyl, cycloheteroalkyl, or an enzyme-cleavable group;
and Z is hydrogen or an enzyme-cleavable group, provided
that at least one of X and Z is an enzyme-cleavable group),
so that the enzyme cleaves the enzyme-cleavable group from
the dioxetane to allow the formation of a negatively charged
substituent bonded to the dioxetane, the negatively charged
substituent causing the dioxetane to decompose to form a
luminescent substance containing the group Y of the
dioxetane, and
detecting the luminescent substance as an
indication of the member of the specific binding pair,

48
wherein the solid matrices are pretreated with a
polyvinyl quaternary ammonium salt) to block nonspecific
binding to the solid matrices.
2. A method of detecting an enzyme in a sample, which
comprises:
(a) providing a dioxetane having the formula:
<IMG>
(wherein T is a cycloalkyl or polycycloalkyl group bonded to
the 4-membered ring portion of the dioxetane by a spiro
linkage; Y is a fluorescent chromophore; X is a hydrogen,
alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl,
cycloalkyl, or cycloheteroalkyl group, or a group capable of
being cleaved by the enzyme; and Z is hydrogen or a group
capable of being cleaved by the enzyme, provided that at
least one of X and Z is a group capable of being cleaved by
the enzyme);
(b) providing solid matrices which have been
pretreated with a poly(vinyl quaternary ammonium salt) to
block nonspecific binding and by which the dioxetane or the
enzyme is carried;
(c) contacting the dioxetane with the sample
containing the enzyme; whereupon the enzyme cleaves the
enzyme-cleavable group from the dioxetane to allow the
formation of a negatively charged substituent bonded to the
dioxetane, the negatively charged substituent causing the
dioxetane to decompose to form a luminescent substance
containing the group Y of the dioxetane; and

48a
(d) detecting the luminescent substance as an
indication of the presence of the enzyme.
3. In an assay method in which a member of a specific
binding pair comprising a nucleic acid which is DNA, RNA, or
a fragment thereof produced by sequencing protocol and a
probe capable of binding to all or a portion of said nucleic
acid is detected by means of an optically detectable
reaction, the improvement being (a) having the optically
detectable reaction include the reaction, with an enzyme, of
a dioxetane of the formula
<IMG>
wherein T is a cycloalkyl or a polycycloalkyl group bonded
to the 4-membered ring portion of said dioxetane by a

49
spiro linkage; Y is a fluorescent chromophore; X is hydro-
gen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, hetero-
aryl, cycloalkyl, cycloheteroalkyl, or an enzyme-cleavable
group; and z is hydrogen or an enzyme-cleavable group,
provided that at least one of X or Z is an enzyme-
cleavable group, so that said enzyme cleaves said enzyme-
cleavable group to allow the formation of a negatively-
charged substituent bonded to said dioxetane, said
negatively charged substituent causing said dioxetane to
decompose to form a luminescent substance containing said
group Y of said dioxetane, (b) contacting said nucleic
acid with a labeled complementary oligonucleotide probe to
form a hybridizing pair, (c) contacting the hybridized
pair with a molecule capable of strong binding to the
label of the nucleotide covalently conjugated with an
enzyme capable of cleaving said dioxetane to release light
energy, (d) adding said dioxetane, and (e) detecting the
light produced by having the light produced come in
contact with light sensitive film.
4. In an assay method in which a member of a
specific binding pair comprising a nucleic acid which is
DNA, RNA, or a fragment thereof produced by a ser;uencing
protocol and a probe capable of binding to all or a
portion of said nucleic acid is detected by means of an
optically detectable reaction, the improvement being (a)
having the optically detectable reaction include the
reaction, with an enzyme, of a dioxetane of the formula
<IMG>
wherein T is a cycloalkyl or a polycycloalkyl group bonded
to the 4-membered ring portion of said dioxetane by a
spiro linkage: Y is a fluorescent chromophore: X is
hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl,

50
heteroaryl, cycloalkyl, cycloheteroalkyl, or an enzyme-
cleavable group; and Z is hydrogen or an enzyme-cleavable
group, provided that at leash one of X or Z is an enzyme-
cleavable group, so that said enzyme cleaves said enzyme-
cleavable group to allow the formation of a negatively-
charged substituent bonded. to said dioxetane, said
negatively charged substituent causing said dioxetane to
decompose to form a luminescent substance containing said
group Y of said dioxetane, (b) contacting said nucleic
acid with a labeled complementary oligonucleotide probe to
form a hybridizing pair, (c) contacting the hybridized
pair with a molecule capable of strong binding to the
label of the nucleotide covalently conjugated with an
enzyme capable of cleaving said dioxetane to release light
energy, (d) adding said dioxetane, and (e) detecting the
light produced by having the light produced come in
contact with a photoelectric cell.
5. In an assay method in which a member of a
specific binding pair comprising a nucleic acid and a
probe capable of binding to all or a portion of said
nucleic acid is detected by means of an optically
detectable reaction, the improvement being (a) having the
optically detectable reaction include the reaction, with
an enzyme, of a dioxetane of the formula:
<IMG>
wherein T is a cycloalkyl or polycyclaalkyl group bonded
to the 4-membered ring portion of said dioxetane by a
spiro linkage; Y is a fluorescent chromophore; X is
hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl,
heteroaryl, cycloalkyl, cycloheteroalkyl, or an enzyme-
cleavable group; and Z is hydrogen or an enzyme-cleavable
group, provided that at least one of X or Z is an enzyme-

51
cleavable group), so that the enzyme cleaves the enzyme-
cleavable group from the dioxetane to allow the formation of
a negatively charged substituent bonded to the dioxetane,
the negatively charged substituent causing the dioxetane to
decompose to form a luminescent substance containing the
group Y of the dioxetane and (b) carrying out the binding of
the probe to the nucleic acid on a nylon membrane.
6. The method of claim 5, wherein the probe is a
labeled oligonucleotide complementary to the nucleic acid.
7. The method of claim 6, wherein the oligonucleotide
probe is covalently labeled with the enzyme capable of
decomposing the dioxetane to emit light.
8. The method of claim 6, wherein the label on the
oligonucleotide probe comprises a covalently bound antigen
that is imunochemically bound to an antibody-enzyme
conjugate, wherein the antibody is directed to the antigen
and the enzyme is capable of decomposing the dioxetane to
emit light.
9. The method of claim 7 or 8, wherein the enzyme is
an acid or alkaline phosphatase and the dioxetane is 3-(2'-
spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-
dioxetane (AMPPD).
10. The method of claim 7 or 8, wherein the enzyme is
a galactosidase and the dioxetane is 3-(2'-spiroadamantane)-
4-methoxy-4-(3"-.beta.-D-galactopyranosyl)phenyl-1,2-dioxetane
(AMPGD).
11. The method of claim 7 or 8, wherein the enzyme is
a carboxyl acid esterase and the dioxetane is 3-(2'-
spiroadamantane)-4-methoxy-4-(3"-acetoxy)phenyl-1,2-
dioxetane.

52
12. In an assay method in which a member of a specific
binding pair comprising a nucleic acid which is DNA, RNA, or
a fragment thereof produced by a sequencing protocol and a
probe capable of binding to all or a portion of the nucleic
acid is detected by means of an optically detectable
reaction, the improvement being:
(a) having the optically detectable reaction
include the reaction, with an enzyme, of a dioxetane of the
formula:
<IMG>
(wherein T is a cycloalkyl or a polycycloalkyl group bonded
to the 4-membered ring portion of the dioxetane by a spiro
linkage; Y is a fluorescent chromophore; X is hydrogen,
alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl,
cycloalkyl, cycloheteroalkyl, or an enzyme-cleavable group;
and Z is hydrogen or an enzyme-cleavable group, provided
that at least one of X or Z is an enzyme-cleavable group),
so that the enzyme cleaves the enzyme-cleavable group to
allow the formation of a negatively-charged substituent
bonded to the dioxetane, the negatively charged substituent
causing the dioxetane to decompose to form a luminescent
substance containing the group Y of the dioxetane,
(b) contacting the nucleic acid with a labeled
complementary oligonucleotide probe to form a hybridizing
pair,
(c) contacting the hybridized pair with a molecule
capable of strong binding to the label of the nucleotide
covalently conjugated with an enzyme capable of cleaving the
dioxetane to release light energy,

53
(d) adding the dioxetane,
(e) detecting the light produced, and
(f) the hybridizing between the nucleic acid and
the labeled oligonucleotide probe is conducted on a nylon
membrane.
13. The method of claim 12, wherein the
oligonucleotide label is biotin or a biotin derivative.
14. The method of claim 12, wherein the molecule
capable of strong interaction with the label of the
oligonucleotide is avidin or streptavidin.
15. The method of claim 12, wherein the enzyme is a
phosphatase and the dioxetane is 3-(2'-spiroadamantane)-4-
methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD).
16. The method of claim 12, wherein the enzyme is a
galactosidase and the dioxetane is 3-(2'-spiroadamantane)-4-
methoxy-4-(3"-.beta.-D-galactopyranosyl)phenyl-1,2-dioxetane
(AMPGD).
17. A kit for detecting a nucleic acid or fragment
thereof in a sample by hybridization of the nucleic acid or
fragment to a comlementary labeled oligonucleotide probe,
comprising:
a 1,2-dioxetane capable of producing light energy
when decomposed having the formula:
<IMG>
(wherein T is a cycloalkyl or polycycloalkyl group bonded to
the 4-membered ring portion of said dioxetane by a spiro

54
linkage, X is hydrogen, alkyl, aryl, aralkyl, alkaryl,
heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an
enzyme-cleavable group, Y is a chromophore capable of
fluorescence, and Z is hydrogen or an enzyme-cleavable
group, provided that at least one of X or Z must be an
enzyme-cleavable group);
a complementary oligonucleotide probe covalently
labeled with biotin or a biotin derivative;
avidin or streptavidin covalently bound to an
enzyme capable of decomposing a 1,2-dioxetane to emit light;
and,
a nylon membrane upon which the nucleic acid or
fragment thereof is hybridized to the oligonucleotide probe.
18. A kit for detecting a protein in a sample,
comprising:
(a) a 1,2-dioxetane capable of producing light
energy when decomposed having the formula:
<IMG>
(wherein T is a cycloalkyl or polycycloalkyl group bonded to
the 4-membered ring portion of the dioxetane by a spiro
linkage; Y is a fluorescent chromophore; X is hydrogen,
alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl,
cycloalkyl, cycloheteroalkyl, or an enzyme-cleavable group;
and Z is hydrogen or an enzyme-cleavable group, provided
that at least one of X and 2 is an enzyme-cleavable group,
so that the enzyme cleaves the enzyme-cleavable group from
the dioxetane to form a negatively charged substituent
bonded to the dioxetane, the negatively charged substituent

55
causing the dioxetane to decompose to form a luminescent
substance containing the group Y of the dioxetane); and
(b) a nylon membrane upon which protein-antibody
binding is conducted.
19. A kit for detecting a nucleic acid or fragment
thereof in a sample by hybridization of the nucleic acid or
fragment to a complementary labeled oligonucleotide probe,
the kit comprising:
(a) a 1,2-dioxetane capable of producing light
energy when decomposed having the formula:
<IMG>
(wherein T is a cycloalkyl or polycycloalkyl group bonded to
the 4-membered ring portion of the dioxetane by a spiro
linkage; X is hydrogen, alkyl, aryl, aralkyl, alkaryl,
heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an
enzyme-cleavable group; Y is a chromophore capable of
fluorescence; and 2 is hydrogen or an enzyme-cleavable
group, provided that at least one of X or Z is an enzyme-
cleavable group);
(b) (1) a covalently enzyme-labeled oligonucleotide
or (2) both a complementary oligonucleotide probe covalently
labeled with biotin or a biotin derivative and an enzyme
capable of decomposing a 1,2-dioxetane to emit light
covalently bound to avidin or streptavidin;
(c) a water-soluble enhancing substance that
increases specific light energy production above that
produced in its absence, wherein the water-soluble enhancing
substance comprises a positively charged polymeric

56
quaternary ammonium salt and an energy acceptor capable of
forming a ternary complex with the 1,2-dioxetane anion
produced following enzyme-catalyzed decomposition of the
1,2-dioxetane, whereby energy transfer occurs between the
1,2-dioxetane anion and the energy acceptor and light is
emitted by the energy acceptor; and
(d) a nylon membrane upon which nucleic acid-
oligonucleotide probe hybridization is conducted.
20. The kit of claim 19, wherein the polymeric
quaternary ammonium salt is poly(vinylbenzyltrimethyl-
ammonium chloride), poly[vinylbenzyl(benzyldimethyl-ammonium
chloride)], or poly(vinylbenzyltributyl-ammonium chloride).
21. The kit of claim 19 or 20, wherein the energy
acceptor is fluorescein.
22. A kit for detecting a protein in a sample, the kit
comprising:
(a) a 1,2-dioxetane capable of producing light
energy when decomposed having the formula:
<IMG>
(wherein T is a cycloalkyl or polycycloalkyl group bonded to
the 4-membered ring portion of the dioxetane by a spiro
linkage; X is hydrogen, alkyl, aryl, aralkyl, alkaryl,
heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an
enzyme-cleavable group; Y is a chromophore capable of
fluorescence; and Z is hydrogen or an enzyme-cleavable
group, provided that at least one of X or Z is an enzyme-
cleavable group);

57
(b) an antibody directed to the protein covalently
bound to an enzyme capable of decomposing the 1,2-dioxetane
to emit light;
(c) a water-soluble enhancing substance that
increases specific light energy production above that
produced in its absence; and
(d) a nylon membrane upon which protein-antibody
binding is conducted.
23. The kit of claim 22, wherein the water-soluble
enhancing substance comprises a positively charged polymeric
quaternary ammonium salt and an energy acceptor capable of
forming a ternary complex with the 1,2-dioxetane anion
produced following enzyme-catalyzed decomposition of the
1,2-dioxetanes whereby energy transfer occurs between the
1,2-dioxetane anion and the energy acceptor and light is
emitted by the energy acceptor.
24. The kit of claim 23, wherein the polymeric
quaternary ammonium salt is poly(vinylbenzyltrimethyl-
ammonium chloride), poly[vinylbenzyl(benzyldimethyl-ammonium
chloride)] or poly(vinylbenzyltributyl-ammonium chloride).
25. The kit of claim 23 or 24, wherein the energy
acceptor is fluorescein.
26. A kit for detecting a protein and a sample, the
kit comprising:
(a) a 1,2-dioxetane capable of producing light
energy when decomposed having the formula:
<IMG>

58
(wherein T is a cycloalkyl or polycycloalkyl group bonded to
the 4-membered ring portion of the dioxetane by a spiro
linkage; X is hydrogen, alkyl, aryl, aralkyl, alkaryl,
heteroalkyl, heteroaryl, cycloalkyl, cycloheteroalkyl or an
enzyme-cleavable group; Y is a chromophore capable of
fluorescence; and Z is hydrogen or an enzyme-cleavable
group, provided that one of X or Z is an enzyme-cleavable
group);
(b) a first antibody directed to the protein;
(c) a second antibody directed to the first
antibody covalently bound to an enzyme capable of
decomposing the 1,2-dioxetane; and
(d) a water-soluble enhancing substance that
increases specific light energy production above that
produced in its absence.
27. The kit of claim 26, wherein the water-soluble
enhancing substance comprises a positively charged polymeric
quaternary ammonium salt and an energy acceptor capable of
forming a ternary complex with the 1,2-dioxetane anion
produced following enzyme-catalyzed decomposition of the
1,2-dioxetane anion and the energy transfer occurs between
the 1,2-dioxetane anion and the energy acceptor and light is
emitted by said energy acceptor.
28. The kit of claim 27 wherein the polymeric
quaternary ammonium salt is poly(vinylbenzyltrimethyl-
ammonium chloride), poly[vinylbenzyl(benzyldimethyl-ammonium
chloride)], or poly(vinylbenzyltributyl-ammonium chloride).
29. The kit of claim 27 or 28 wherein the energy
acceptor is fluorescein.

Description

CA 02033331 2001-O1-10
78951-11
1
T1 L~ L~ rD T TAT T /1TT
Method of Detecting A Substance Using Enzymatically-Induced
Decomposition of Dioxetanes
Field Of The Invention
The invention relates to the use of dioxetanes to
detect a substance in a sample.
Background Of The Invention
Dioxetanes are compounds having a 4-membered ring in
which 2 of the members are adjacent oxygen atoms. Dioxetanes
can be thermally or photochemically decomposed to form carbonyl
products, e.g., esters, ketones or aldehydes. Release of
energy in the form of light (i.e., luminescence) accompanies
the decompositions.
Summary Of The Invention
In general, the invention features in a first aspect
an improvement in art assay method in which a member of a
specific binding pair (i.e., two substances which bind
specifically to each other) is detected by means of an
optically detectable reaction. The improvement includes the
reaction, with an enzyme, of a dioxetane having the formula
~X
T Y-Z
where T is substituted (i.e., containing one or more C1-C, alkyl
groups or heteroatom groups, e.g., carbonyl

2033331
2
groups) or unsubstituted cycloalkyl ring (having between
6 and 12 carbon atoms, inclusive, in the ring) or poly-
cycloalkyl .group (having 2 or more fused rings, each ring
independently having between 5 and 12 carbon atoms,
inclusive), banded to the 4-membered dioxetane ring by a
spiro linkage; Y is a fluorescent chromophore, (i.e., Y
is group capable of absorbing energy to form an excited,
i.e., higher energy, state, from which it emits light to
return to its original energy state): X is hydrogen, a
l0 straight ar branched chain alkyl group (having between 1
and T carbon atoms, inclusive, e.g., methyl), a straight
chain or branched heteroalkyl group (having between 1 and
7 carbon atoms, inclusive, e.g., methoxy, hydroxyethyl, or
hydroxypropyl), an aryl group (having at least 1 ring,
e.g., phenyl), a heteroaryl group (having at least 1 ring,
e.g., pyrrolyl or pyrazolyl), a heteroalkyl group (having
between 2 and 7 carbon atoms, inclusive, in the ring,
s.g., dioxane), an aralkyl group (having at least 1 ring,
e.g., benzyl), an alkaryl group (having at least 1 ring,
e.g., tolyl), or an enzyme-cleavable group, i.e., a group
having a bond which can be cleaved by an enzyme to yield
an electron-rich moiety bonded to the dioxetane, e.g.,
phosphate, where a phosphorus-oxygen bond can be cleaved
by an enzyme, e.g., acid phosphatase or alkaline phos-
phatase, to yield a negatively charged oxygen bonded to
the dioxetane; and Z is hydrogen, hydroxyl, or an enzyme-
eleavmble group (as defined above), provided that at least
one o! X or Z must be an enzyme-cleavable group, so that
the enzyme cleaves the enzyme-cleavable group to form a
negatively charged substituent (e. g., an oxygen anion)
bonded to the dioxetane, the negatively charged substi-
tuent causing the dioxetane to decompose to form a
luminescent substance (i.e., a substance that Baits energy
in the form o! light) that includes group Y. The lumines-
cent substance is detected as an indication o! the pres-
ence o! the first substance. By measuring the intensity

3 x_2033331
of luminescence, the concentration of the first substance
can be determined.
In preferred embodiments, one or more of groups T, X,
or Y further include a solubilizing substituent, e.g.,
carboxylic acid, sulfonic acid, or cjuaternary amino salt;
group T of the dioxetane is a polycycloalkyl group,
preferably adamantyl; the enzyme-cleavable group includes
phosphate; and the enzyme includes phosphatase.
The invention also features a kit for detecting a
to first substance in a sample.
In a second aspect, the invention features a method
of detecting an enzyme in a sample. The method involves
contacting the sample with the above-described dioxetane
in which group Z is capable of being cleaved by the enzyme
being detected. The enzyme cleaves group Z to form a
negatively charged substituent (e. g., an oxygen anion)
bonded to the dioxetane. This substituent destabilises
the dioxetane, thereby causing the dioxetane to decompose
to form a luminescent substance that includes group Y of
the dioxetane. The luminescent substance is detected as
an indication of the presence of the enzyme. By measuring
the intensity of luminescence, the concentration of the
enzyme can also be determined.
The invention provides a simple, very sensitive
method for detecting substances in samples, a.g., biologi
cal samples, and is particularly useful for substances
present in lore concentrations. Because dioxetane decom
position serres as the excitation energy source for
chromophore Y, an external excitation energy source, e.g.,
light, is not necessary. In addition, because the dioxe-
tane molecules are already in the proper oxidation state
for decomposition, it is not necessary to add external
oxidants, e.g., Hi02 or OZ. Enzyme-activated decomposi-
tion allows for high sensitivity because one enzyme
molecule can cause many dioxetane molecules to luminesce,
thus creating an amplification effect. Moreover, the
wavelength (or energy) of emission and the quantum yields

20 3333 1
of luminescence can be varied according to the choice of
the Y substituent of the dioxetane (as used herein,
"quantum yield" refers to the number of photons emitted
from the luminescent product per number of moles of dioxe-
tane decomposed). In addition, through appropriate modi-
fications of the T, X, and Y groups of the dioxetane, the
solubility of the dioxetane and the kinetics of dioxetane
decomposition can be varied. The dioxetanes can also be
attached to a variety of molecules, e.g., proteins or
haptens, or immobilization substrates, e.g., polymer
membranes, or included as a side group in a homopolymer or
copolymer.
Other features and advantages of the invention will
be apparent from the following description of the pre
ferred embodiments thereof, and from tho claims.
Description Of The Drawinq~
FIGURE 1 compares a solid state quantitative color-
imetric assay for human chorionic gonadotropin (hCG) using
p-nitrophenyl phosphate (PNPP) as chromogen with the quan-
titative chemiluminescence assay of the invention using 3-
(2'-spiroadamantane)-4-methoxy-4-(3"-phos-phoryloxy)-
phenyl-1,2-dioxetane, disodium salt (AMPPD) as the
lumogen.
FIGURE 2 shows the results of tho solid state immuno
assay for hCG of the invention using AMPPD as the lumoqen
and using film exposure for detection o! the hCG.
FIGURE 3 is a standard curve for the quantitative
estimation of the concentration of the enzyme alkaline
phoaphatase by the AMPPD chemfluminescence assay of the
3o invention.
FIGURE 4 compares the quantitative estimation of the
concentration of the enzyme alkaline phosphatase by the
AMPPD chemiluminescence assay of the invention in the
presence and absence of bovine serum albumin (BSA), fluor-
escein (BS~r-Fluor.) poly[vinylbenzyl(benzyldimethyl
ammonium chloride)] (BDMQ), and BDMQ-Fluor.

2033331
FIGURE S shows the results of a Herpes Simplex
Virus I (HSVI) ONA probe assay using a specific alkaline
phosphatase-labeled DNA probe in conjunction with the
AMPPD chemiluminescence assay of the invention.
5 FIGURE 6 shows the time course of the AMPPD chemi-
luminescence method of the invention applied to the
hybridization-based detection of hepatitis H core antigen
plasmid DNA (HBVe) with an alkaline phosphatase-DNA probe
conjugate, using a film detection technique.
l0 FIGURE 7 shows the time course of the colorimetric
detection of Hepatitis B core antigen plasmid DNA with an
alkaline phosphatase-DNA probe conjuqate using nitroblue
tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate
(BCIP) as substrates.
FIGURE 8 shows the quantitative application of the
assay of Figure 6, wherein the film images were quantified
by measuring reflection densities.
FIGURE 9 compares a solid state ELISA method for
alpha feto protein (AFP) using PNPP as a colorimetric
substrate and the AMPPD chemiluminescence method of the
invention ror the quantitative estimation of AFP, wherein
alkaline phosphatase is covalently linked to anti-AFP
antibody.
FIGURE 10 shows a solid state monoclonal antibody
BLISA for thyroid stimulating hormone (TSH) using the
Al~IPPD chemiluminescence method of the invention wherein
monoclonal anti-~-TSH antibody conjugated to alkaline
phosphatase was used was the detection antibody.
FIGURE 11 represents the assay of Figure 10 carried
out both in the absence and presence of BSA.
FIGURE 12 shows the application of a solid state
ELISA to the estimation of carcino-embryonic antigen
(CEA), wherein a-CEA antibody-alkaline phosphatase was the
detection antibody and the AMPPD chemiluminescence method
of the invention was used to quantify the CEA.

6
FIGURE 13 is a diagram of the device used for the
solid state immunoassay for human luteinizing hormone
(hhH).
FIGURE 14 shows the assay images on film for a solid
state immunoassay for hLH wherein monoclonal anti-hLH
antibody-alkaline phosphatase is the detection antibody
and the AMPPD chemiluminescence assay of the invention was
used to detect the hLH antigen.
FIGURE 15 shows a standard curve obtained for hLFi
wherein the film images obtained by the method of Figure
14 ware quantified by reflection density det~rminations at
each concentration of hLH.
FIGURE 16 shows a plot of chemiluminescance as a
function of ~-galactosidase concentration in the chemi
luminescence assay of the invention wherein the substrate
for the enayme is 3-(2~-spiroadamantane)-4-methoxy-4-(3"-
~B-D-galactopyranosyl) phenyl-1, 2-dioxetana (A?IPGD).
FIGURE 17 shows the pH dependence o1~8-galactosidase-
activated chemiluminescence from AMPGD.
FIGURE 18 shows the production of light by
p-galactosidase decomposition of AMPGD wherein the light
intensity was measured after enzyme incubation at a pH of
7.3 and adjusting the pH to 12 with alkali.
FIGURE 19 shows a two-hour lumiautogram on X-ray film
(A) and Polaroid instant black and white film (B) of DNA
fragments visualized by AMPPD chemiluminescence following
electrophoretic separation of DNA fragments produced by
the Sangar sequencing protocol.
~7gscr p~ion Qf The Pre,~rred Embodiments
3o The structure, synthesis, and use of preferred
esbodiments of the invention will now be described.
Structure
The invention employs dioxetanes having the structure
recited in the Summary of the Invention above. The
purpose o! group T is to stabilize the dioxetane, i.e., to
2033331

.233331
prevent the dioxetane from decomposing before the enzyme-
cleavable group Z is cleaved. Large, bulky, sterically
hindered molecules, e.g., fused polycyclic molecules, are
the most effective stabilizers. In addition, T preferably
contains only C-C and C-H single bonds. The most preferred
molecule is an adamantyl group consisting of 3 fused
cyclohexyl rings. The adamantyl group is bonded to the
4-membered ring portion of the dioxetane through a spiro
linkage.
Group Y is a fluorescent chromophore bonded to
enzyme-cleavable group Z. Y becomes luminescent when an
enzyme cleaves group Z, thereby creating an electron-rich
moiety which destabilizes the dioxetane, causing the
dioxetane to decompose. Decomposition produces two indi-
vidual carbonyl compounds, one of which contains group T,
and the other of which contains groups X, Y, and z; the
energy released from dioxetane decomposition causes the Y
groups of the latter carbonyl compound to luminesce (if
group X is hydrogen, an aldehyde is produced).
The excited state energy of chromophore Y (i.e., the
energy chromophore Y must possess in order to emit light) .
is preferably less than the excited state energy of the
ketone containing group T in order to confine luminescence
to group Y. For example, when Y.is adamantyl, the excited
state energy o! chromophore Y is preferably less than the
excited state energy of spiroadamantane.
llny chromophore Y can be used according to the inven
tion. In general, it is desirable to use a chromophore
which aaximizes the quantum yield in order to increase
sensitivity.
Examples of suitable chromophores include the
following:
1) anthracene and anthracene derivatives, e.g " 9,
io-diphenylanthracene, 9-methylanthracene, 9-anthracene
carboxaldehyde, anthryl alcohols and 9-phenylanthracene:
2) rhodamine and rhodamine derivatives, e.g.,
rhodols, tetramethyl rhodamine, tetraethyl rhodamine,

2033331
diphenyldimethyl rhodamine, diphenyldiethyl rhodamine, and
dinaphthyl rhodamine:
3) fluorescein and fluorescein derivatives, e.g.,
5-iodoacetamido fluorescein, 6-iodoacetamido fluorescein,
and fluorescein-5-maleimide:
9) eosin and eosin derivatives, e.g., hydroxy
eosins, eosin-5-iodoacetamide, and eosin-5-maleimide;
5) coumarin and coumarin derivatives, e.g., 7
dialkylamino-4-methylcoumarin, 4-bromomethyl-7-methoxy
coumarin, and 4-bromomethyl-7-hydroxycoumarin:
6) erythrosin and erythrosin derivatives, e.g.,
hydroxy erythrosins, erythrosin-5-iodoacetamide and
erythrosin-5-maleimide:
7) aciridine and aciridine derivatives, e.g.,
hydraxy aeiridines and 9-methyl aciridine:
8) pyrene and pyrene derivatives, e.g., N-(1-
pyrene) iodoacetamide, hydroxy pyrenes, and 1-pyrenemethyl
iodoacetate:
9) stilbone and stilbene derivatives, e.g. 6,6'-
dibromostilbene and hydroxy stilbenes;
10) naphthalene and naphthalene derivatives, e.g.,
5-dimethylaminonaphthalene-1-sulfonic acid and hydroxy
naphthalene:
11) nitrobenzoxadiazoles and nitrobenzoxadiazole
derivatives, e.g., hydroxy nitrobenzoxadiazoles, 4
chloro-7-nitrobenz-2-oxa-1,3-diazole, 2-(7-nitrobenz-2
oxa-1,3-diazol-4-yl-amino)hexanoic acid;
12) quinoline and quinoline derivatives, e.g., 6-
hydroxyquinoline and 6-aminoquinoline:
13) acridine and acridine derlvatfves, e.q., N-
methylacridine and N-phenylacridine;
14) acidoacridine and acidoacridine derivatives,
e.g., 9-methylscfdoacridine and hydroxy-9-methylacido-
acridine;
15) carbazola and carbazole derivatives, e.g., N-
methylcarbazole and hydroxy-N-methylcarbazole:

9 = ?
16) fluorescent cyanines, e.g., DCM (a laser dye), .~ ' Q J J 3 3
hydroxy cyanines, 1,6-Biphenyl-1,3,5-hexatriene, 1-(4-
dimethyl aminophenyl)6-phenylhexatriene, and the
corresponding 1,3-butadienes.
17) carbocyanine and carbocyanine derivatives, e.g.,
phenylcarbocyanine and hydroxy carbocyanines;
18) pyridinium salts, e.g., 4(4-dialkyldiamino-
styryl) N-methyl pyridinium iodate and hydroxy-substituted
pyridinium salts;
19) oxonols: and
20) rasorofins and hydroxy resorofins.
The most preferred chromophores are hydroxy
derivatives o! anthracene or naphthalene; the hydro~cy
group facilitates bonding to group Z.
Group Z is bonded to chromophore Y through an enzyme-
cleavable bond. Contact with the appropriate enzyme
cleaves the enzyme-cleavable bond, yielding an electron-
rich moiety bonded to a chromophore Y: this moiety
initiates the decomposition of the .dioxetane into two
individual carbonyl compounds e.g., into a ketone or an
ester and an aldehyde if group X is hydrogen. Examples
of electron-rich moieties include oxygen, sulfur, and
amine or amino anions. The most preferred moiety is an
oxygen anion. Examples of suitable Z groups, and the
enzymes specific to these groups are given below in
Table 1: an arrow denotes the enzyme-cleavable bond. The
most preferred group is a phosphate ester, which is
cleaved by alkaline or acid phosphatase enzymes.
Grouu Z
1) alkaline and acid
O phosphatases
AO_P ifl_Y
~D
0
phosphate ester

10
esterases 2 0 3 3 3 3 1
o
CH3 C O-Y
acetate ester
decarboxylases
Y-O-C-o9
carboxyl
4) phospholipase D
O
v
o cxi o_c_ ( cxz > ~-cHa
n t
cH3- ( cHZ ) ~ c-o-cH o
CHZ-0-P-O-Y
O
3-phospho-1,2-diacyl glycerides
5) ,A-xylosidass
NoC~~ O-Y
0
No ~
p-D-xyloside _-
ON
6) ~B-D-fucosidase
CWf
N of y
h
~-D-fucoside
a'"Z ~ ~ ~ thioglucosidase
1
1~ 0
1-thio-D-glucoside

m
Q-D-galactosidase ~ 2 0 3 3 3 ~ 1
CN
,B-D-galactoside
a-D-galactosidase
~Y
Ch
a-D-galactoside
C.~,6N
S 10) a-D-glucosidase
~N
~+b ~Hb ~ Y
a-D-glucoside
11) CN,bN
p ~ p-D-glucosidase
~-D-qlucoside
CI~~O~i
12) a-D-mannosidase
a o~ ~
po b-Y
a-D-mannoside

12
13j ,B-D-mannosidase 2 Q 3 3 3 3 1
CHZOH
O O-Y
off off .-
s Ho ~ ~
R-D-mannoside
14j
HOCHZ O O-Y ~-D-fructofuranosidase
OH ~-
CHZOH
OH
~-D-fructofuranoside
15j
~-D-glucosiduronase
0008
1' 0 0-Y
OH
HO H
~9-D-glucosfduronate
16 j ~0 0
~e/VN~~~., ~ ~ ~ trypsln
Q
~~l
H
=NW
I
~1~~
p-toluenesulfonyl-L-arginine
ester
f

13 ,2033331
17) trypsin
O o
CH3 ~ ~' S-NH-CH-C NH-Y
ii ~ t
O ~ iHZ) s
N+i
C=NN
NNs.
p-toluenesulfonyl-L-
arginine amide
Suitable X groups are described in the Summary of the
Invention, above. Preferably, X contains one or more sol-
ubilizing substituents, i.e., substituants'~which enhance
the solubility of the dioxetane in aqueous'~solution.
Examples of solubilizing substituents include carboxylic
acids, e.g., acetic acid; sulfonic acids, e.g., methane-
sulfonic acid; and quaternary amino salts, e.g., ammonium
bromide; the most preferred solubilizing substituent is
methane-or athanesulfonic acid.
Preferably, the enzyme which cleaves group Z is co-
valently bondod to a substance having a specific affinity
for the substance being detected. Examples of specific
affinity substances include antibodies, e.g., anti-hCG;
antigens, e.g., hCG, where the substance being detected is
an antibody, e.g., anti-hCG; a probe capable of binding to
all or a portion of a nucleic acid, e.g., DNA or RNA,
being defeated; or an enzyme capable of cleaving the Y-Z
bond. Honding is preferably through an amide bond.
synthesis
In general, the dioxetanes of the invention are
synthesized in two steps. The first step involves syn-
thesizing an appropriately substituted olefin having the
formula
-X
3d T Y-Z

24 3333 1
14
wherein T, X, Y, and Z are as described above. These
olefins are preferably synthesized using the Wittig
reaction, in which a ketone containing the T group is
reacted with a phosphorus ylide (preferably based on
triphenylphosphine) containing the X, Y, and Z groups, as
follows:
x x
s= o t r.~
s Y-z
The reaction is preferably carried out below about -70~C
in an ethereal solvent, e.g., tetrahydrofuran (TNF).
The phosphorus ylide is prepared by reacting
triphenyl phosphine with a halogenated compound containing
the X, Y, and Z groups in the presence of base. examples
of preferred bases include n-butyllithium, sodium amide,
sodium hydrid~, and sodium alkoxide: the most preferred
base is n-butyllithium. The reaction sequence is as
follows:
x
z o p . di-x
y-2
where Q is a halogen, e.g., C1, Hr, or I. The preferred
halogen is Br. The reaction is preferably carried out
below about -70'C in THF.
The olefin where T is adamantyl (Ad) , X is methoxy
(oCIi~), Y is anthracene (An), and Z is phosphate (P04) can
be synthesized as follows.
Hr-CH-OCH, is phosphorylated by treating it with the
An-OH
product of phosphorus acid reacted in the presence of
HgCh with N-methylimidazole: the net result is to replace
the hydroxyl group of An with a phosphate group. The
phosphorylated product is then reacted with triphenyl-

CA 02033331 2001-O1-10
78951-11
phosphine below about -70'C in THF to form the phosphorus
glide having the formula
oe~,
P
~An_P04
5
The reaction is conducted in a dry argon atmosphere,
Spiroadamantanone (Ad = Oj is then added to the solution
containing the glide, while maintaining the temperature
below about -70'C, to form the olefin having the formula
10 ~OCHy
Ad An-PO~
The olefin is then purified using conventional chromato-
graphy methods.
The second step in the synthesis of the dioxetanes
15 involves converting the olefin described above to the
dioxetane. Preferably, the conversion is effected photo
chemically by treating by olefin with singlet oxygen ('02)
in the presence o! light. ('OZ) adds across the double
bond to form the dioxetane as follows:
_ X
O-O X
~+ X02 ~ ~.
T ~'-Z T \Y Z
The reaction is preferably carried out below about -70'C
in a halogenated solvent, e.g., methylene chloride. 'OZ is
generated using a photosensitizer. Examples of photosen-
sitizers include polymer-bound Rose Bengal (commercially
known as Sansitox* Z and available from Hydron
Laboratories, New Brunswick, N.J.), which ie preferred,
and methylene blue (a well-known dye and pH indicator).
The synthesis of the dioxetane having the formula
* Trade-mark

CA 02033331 2001-O1-10
78951-11
16
o-o 'OCH 3
Ad ~ ~-~ a
follows.
The olefin having the formula
oCFis
Ad An-PO~
is dissolved in methylene chloride, and the solution is
placed in a 2-cm2 Pyrex tube equipped with a glass paddle;
the paddle is driven from above by an attached, glass
enclosed, bar magnet. Ths solution is cooled to below
about -70'C and lg of polymer-bound Rose Bengal is added
with stirring. oxygen is then passed over the surface of
the agitated solution while the reaction tube is exposed
to light from a 500 W tungsten-halogen lamp (GE Q500 C1)
equipped with a W-cut off filter (Corning 3060: trans-
mission at 3s5 nm a 0.5!). Thin layer chromatography
(tlc) is used to monitor the disappearance of the olefin
and the concurrent appearance of the dioxetane. After the
2o reaction is complete (as indicated by tlc), the solvent
is removed and the dioxetane is isolated.
A widt variety of assays exist which use visually
detectable means to determine the presence or conaentra-
tion of a particular substance in a sample. The above-
described dioxetanes can be used in any of these assays.
Examples o! such assays include immunoassays to detect
antibodies or antigens, e.g., a or ~-hCGt enzyme assays:
chemical assays to detect, e.g., potassium or sodium ions:
and nucleic acid assays to detect, e.g., viruses (e. g.,
HTLV IIZ or cytomegalovirus, or bacteria (e. g., ~. coli)).
When the detectable substance is an antibody,
antigen, or nucleic acid, the enzyme capable of cleaving
group Z of the dioxetane is preferably bonded to a
substance having a specific affinity for the detectable

CA 02033331 2001-O1-10
78951-11
17
substance (i.e., a substance that binds specifically to
the detectable substance , e.g, an antigen, antibody, or
nucleic acid probe, respectively. conventional methods,
e.g., carbodiimide coupling, are used to bond the enzyme
to the specific affinity substance; bonding is preferably
through an amide linkage.
In general, assays are performed as follows. A
sample suspected of containing a detectable substance is
contacted with a buffered solution containing an enzyme
l0 bonded to a substance having a specific affinity for the
detectable substance. The resulting solution is incubated
to alloy the detectable substance to bind to the specific
affinity portion of the specific affinity-enzyme compound.
Excess specific affinity-enzyme compound is then washed
away, and a dioxetane having a group Z that is cleavable
by the enzyme portion of the specific affinity enzyme
compound is added. The enzyme cleaves group Z, causing
the dioxetane to decompose into two carbonyl compounds
(e. g., an ester or ketone when group X is other than
2o hydrogen and an aldehyde when group X is hydrogen)f
chromophore y bonded to one of the ketones is thus excited
and luminesces. Luminescence is detected using e.g., a
cuvette or camera luminometer, as an indication of the
presence of the detectable substance in the sample.
Luminescence intensity is measured to determine the
concentration o! the substance.
When the detectable substance is an enzyme, a
specific affinity substance is not necessary. Instead, a
dioxetane having a z group that is cleavnble by the enzyme
being detected is used. Therefore, an assay for the
enzyme involves adding the dioxetane to the enzyme-
containing sample, and detecting the resulting
luninescance as an indication of the presence and the
concentration of the enzyme.
Examples of specific assays follow.

2033331
i8
A. Assay for Human IaG
A 96-well microtiter plate is coated with sheep anti-
human IgG (F(ab)2 fragment specific). A serum sample
containing human IgG is then added to the wells, and the
wells are incubated fox 1 hour at room temperature.
Following the incubation period, the serum sample is
removed from the wells, and the vsIls are washed four
times with an aqueous buffer solution containing 0.15 M
NaCl, 0.01 M phosphate, and 0.1% bovine serum albumin (pH
7.4).
Alkaline phosphatase bonded to anti-human IgG is
added to each well, and the wells are incubated for 1 hr.
The wells are then washed four times with ttxa above buffer
solution, and a buffer solution of a phosphate-containing
dioxetane is added. The resulting luminescence caused by
enzymatic degradation of the dioxetane is detected in a
luminometer, or with photographic film in a camera
luminometer.
B. Assa,~ for hCG
Rabbit anti-a-hCG is adsorbed onto a nylon-mesh
membrane. A sample solution containing hCG, e.g., urine
from a pregnant woman, is blotted through the membrane,
after which the membrane is washed with 1 ml of a buffer
solution containing 0.15 M NaCl, 0.01 h phosphate, and
0.1% bovine serum albumin (pH 7.4).
Alkaline phosphatase-labelled anti-~-hCG is added to
the membrane, and the membrane is washed again with 2 ml
of the above buffer solution. The membrane is then placed
in the cuvette of a luminometer or into a camera
luminometer, and contacted with a phosphate-containing
dioxetane. The luainescencs resulting from enzymatic
degradation of the dioxetane is than detected.
C. $~asa~, fgr ~,~erum A~~7ljne Phodphatase
2.7 ml of an aqueous buffer solution containing 0.8 M
2-methyl-2-aminopropanol is placed in a 12 x 75 mm pyrex

2033331
19
test tube, and o.l ml of a serum sample containing alka-
line phosphatase added. The solution is then equilibrated
to 30'C. 0.2 ml of a phosphate-containing dioxetane is
added, and the test tube immediately placed in a lumino-
meter to record the resulting luminescence. The level of
light emission will be proportional to the rate of
alkaline phosphatase activity.
D. Nucleic Acid Hybridization Assav
A sample of cerebrospinal fluid (CSF) suspected of
containing cytomegalovirus is collected and placed on a
nitrocellulose membrane. The sample is then chemically
treated with urea or guanidinium isothiocyanate to break
the cell walls and to degrade all cellular components
except the viral DNA. The.strands of the viral DNA thus
produced are separated and attached to the nitrocellulose
filter: A DNA probe specific to the viral DNA and
labelled with alkaline phosphatase is then applied to the
filter: the probe hybridizes wig the. complementary viral
DNA strands. After hybridization, the filter is washed
with an aqueous buffer solution containing 0.2 M NaCl and
0.1 Tris-HC1 (pIi ~ 8.10) to remove excess probe molecules.
A phosphate-containing dioxetane is added and the
resulting luminescence from the enzymatic degradation of
the dioxetane is measured in a luminometer or detected
with photographic film.
E. Assav for Galactosidase
In the assays described above and in the Examples to
follow dioxetanes containing a- or ~9- galactosidase-
cleavabl4 a-D- or S-D-galactopyranoside groups, respect-
ively, can be added, and the luminescence resulting from
the enzymatic cleavage of the sugar moiety from the
chromophors measured in a luminometer or detected with
photographic film.

2033331
zo
F. Electronhoresis
Electrophoresis allows one to separate complex
mixtures of proteins and nucleic acids according to their
molecular size and structure on gel supports in an
electrical field. This technique is also applicable to
separate fragments of protein after proteolysis, or
fragments of nucleic acids after scission by restriction
endonucleases (as iri DNA sequencing). After electro-
phoretic resolution of species in the gel, or after
transfer of the separated species from a gel to a
membrane, the bonds are probed with an enzyme bound to a
ligand. For example, peptide fragments are probed with an
antibody covalently linked to alkaline phosphatase. For
another example, in DNA sequencing alkaline phosphatase -
avidity binds to a biotinylated nucleotide base. There-
after, AMPPD is added to the gel or membrane filter.
After short incubation, light is emitted as the result of
enzymatic activation of the dioxetane to form the emitting
species. The luminescence is detected by either X-ray or
instant photographic film, or scanned by a luminometer.
Multichannel analysis further improves the process by
allowing one to probe for more than one fragment
simultaneously.
G. In solid state assays, it is desireabls to block
nonspecific binding to the matrix by pretreatment of
nonspaci!ic binding sites with nonspecific proteins such
as bovine serum albumin (BSA) or gelatin. Applicant has
determined that soma commercial preparations of BSA
contain small amounts of phosphatase activity that will
produce undesirable background chemiluminescence from
AMPPD. Applicant has discovered that certain watar-
soluble synthetic macromolecular substances are efficient
block.rs of nonspecific binding in solid state assays
using dioxetanes. preferred among such substances are
water-soluble polymeric quaternary ammonium salts such as

2033331
21
poly(vinylbenzyltrimethyl ammonium chloride) (TMQ) or
poly(vinylbenzyl(benzyldimethylammonium chlorid~)](BDMQ).
H. ss ~ for Nuc~gotidase
An assay for the enzyme ATPase is performed in two
steps. In the first step, the enzyme is reacted at its
optimal pH (typically pH 7.4) with a substrate comprising
ATP covalantly linked via a terminal phosphoester bond to
a chromophore-substituted 1,2-dioxetane to produce a phos
phorylchromophore substituted 1,2-dioxstane. In the
l0 second step, the product of the first step is decomposed
by the addition of acid to bring the pH to below 6, pref-
erably to pN 2-4, and the resulting light measured in a
luminometer or detected with chromatographic film. In a
similar two-step procedure, ADPase is assayed using as the
substrate an ADP derivative of a chromophore-substituted
1,2-dioxetane, and 5~-nucleotidase assayed using as the
substrate an adenylic acid derivative of a chromophore-
substitutsd 1,2-dioxetane. The second step can also be
carried aut by adding the enzyme alkaline phosphatase to
decompose the phosphoryl-chromophore-substituted 1,2-
dioxetane.
I. Nucleic Acid g~vuencing
DNA or atNA fragments, produced fn sequencing proto
cols, can bs detected after electrophoretfc separation
using the chemiluminescent 1,2-dioxetanes at this
invention.
DNA sequencing can be performed by a dideoxy chain
termination method [Sanger, F., g~ ~., Proc. Nat. Aced.
Sci. (USAl, 74:5463 (1977)). Briefly, for each of the
3o four sequencing reactions, single.stranded template DNA is
mixed with dideoxynucleotides and biotinylated primer
strand DNA. After annealing, Klenow enzyme and deoxy-
adenosine triphosphate are incubated with each of the four
sequencing reaction mixtures, then chase deoxynucleotide
triphosphate is added and the incubation continued.

_2033331
22
Subsequently, DNA fragments in reaction mixtures are
separated by polyacrylamide gel electrophoresis (PAGE).
The fragments are transferred to a membrane, preferably a
nylon membrane, and the fragments cross-linked to the
membrane by exposure to W light, preferably of short wave
length.
After blocking non-specific binding sites with a
polymer, e.g., heparin, casein or serum albumin, the DNA
fragments on the membrane are contacted with avidin or
streptavidin covalently linked to an enzyme specific for
the enzyme cleavable group of the 1,2-dioxetane substrates
of this invention. As avidin or streptavidin bind avidly
to biotin, biotinylated DNA fragments will now be tagged
with an enzyme. For example, when the chemiluminescent
substrate is 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phos-
phoryloxy)phenyl-1,2-dioxetana salt (AMPPD), avidin or
streptavidin will be conjugated to a phosphatase.
Similarly, when the chemiluminascant substrata is 3-(2'-
spiroadamantaae)-4-methoxy-4-(3"-~-D-galactopyranosyl)
phenyl-1,2-dioxetane (AMPGD), avidin or streptavidin are
conjugated with p-galactosidase.
Following generation of luminescence by contacting
the complex of DNA fragment biotinavidin (or strepta-
vidin) enzyme with the appropriate 1,2-dioxetane at
alkaline pH values, e.g., above about pH 8.5, DNA frag-
ments era vfsualiaad on light-sensitive film, e.g, x-ray
or instant film, or in a photoelectric luminometer
instrument.
The detection method outlined above can also be
applied to the genomic DNA sequencing protocol of Church
g~ ~. [Church, G.M., g~ ate., Proc. Nat. Aced. Sci. (USA1,
81:1991 (1984)x. After transferring chemically cleaved
and electrophoretically separated DNA [Maxam, A.M. g~ ~.,
Proc. Nat. Aced. Sci. (USA1, 74:560 (1977)] to a membrane,
preferably a nylon membrane, and cross-linking the ladders
to the membrane by light, specific DNA sequences may b~
detected by sequential addition of: biotinylated oligo-

CA 02033331 2001-O1-10
78951-11
23
nucleotides as hybridization probes; avidin or strepta-
vidin covalently linked to an enzyme specific for an
enzyme.cleavable chemiluminescent 1,2-dioxetane of this
invention; and, the appropriate 1,2-dfoxetane. Images of
sequence ladders (produced by PAGE) may be obtained as
described above.
Serial reprobing of sequence ladders can be
accomplished by first stripping the hybridized probe and
chemiluminescent material from a membrane by contacting
the membrane with a heated solution of a detergent, e.g.,
from about 0.5 to about -5~ sodium dodecylsulfate (SDS)
in water at from about 80'C to about 90°C , cooling to
from about 50'C to about 70'C, hybridizing the now-naked
DNA fragments with another biotinylated oligonucleotide
probe to generate a different sequence, then generating an
imaging chemiluminescence as described above.
Similar visualization methods can be applied to RNA
fragments generated by RNA sequencing methods.
Other embodiments are within the following claims.
For example, the enzyme-cleavable group Z can be
bonded to group X of the dioxetane, instead of group Y.
The specific affinity substance can be bonded to the
dioxetane through groups X, Y, or T (preferably group x),
instead of the enzyme. In this case, the group to which
the specific affinity substance is bonded is provided
with, e.g., a carboxylic acid, amino, or maleimide
subetituent to facilitate bonding.
Groups X, Y, or T of the dioxetane can be bonded to
a polymerizable group, e.g., a vinyl group, which can be
polymerized to form a homopolymer or copolymer.
Groups x, Y, or T of the dioxetane can be bonded to,
e.g., membranes, films, beads, or polymers for use in
immuno- or nucleic acid assays. The groups are provided
with. e.g., carboxylic acid, amino, or maleimide substi-
tuents to facilitate bonding.
Groups X, Y, or T of the dioxetane can contain
substituents which enhance the kinetics of the dioxetane

CA 02033331 2001-O1-10
78951-11
24
enzymatic degradation, e.g., electron-rich moieties (e. g.,
methoxy).
Groups Y and T of the dioxetane,. as well as group x,
can contain solubilizing substituents.
Appropriately substituted dioxetanes can be synthe-
sized chemically, as well as photochemically. For
example, the olefin prepared from the Wittig reaction can
be epoxidized using a peracid, e.g., p.nitroperbenzoic
acid. The epoxidized olefin can then be converted to the
dioxetane by treatment with an ammonium salt, e.g., tetra-
methylammonium hydroxide.
Another example of a chemical synthesis involves
converting the olefin prepared from the Wittig reaction to
a 1, 2-hydroperoxide by reacting the olefin with HZOZ and
dibromantin (1,3-dibromo-5,5-dimethyl hydantoin). Treat-
ment of the 1,2-bromohydroperoxide with base, e.g., an
alkali or alkaline earth methylhydroxide such as sodium
hydroxide or a silver salt, e.g., silver bromide, forms
the dioxetane.
2o Olefin precursors for the dioxetane can be synthe-
sized by reacting a ketone with a ester in the presence of
TiCl and lithium aluminum hydride (LAN). For example, to
synthesize an olefin where T is adaman~yl (Adj, X is
methoxy (OCHIj, Y is anthracene (Anj, and Z is phosphate
(POD), the following reaction sequence is used:
0 OCHI
Ad ~ 0 + An - C - 0 - CHI , T,~ Cl~j,B,)~ AD-"",
s ~_~<
To phoephorylate chromophore Y, e.g., anthracene, a
3o hydraicyl derivative of the chromophore, e.g., hydroxy
anthracene, can be reacted with a cyclic aryl phosphate
having the following formula:
CH3 O
~O
CH3 CO ~ P _OCH3

2033331
The reaction product is then hydrolyzed with water to
yield the phosphorylated chromophore. The cyclic acyl
phosphate is prepared by reacting 2,2,2-trimethoxy-4,5-
dimethyl-1,3-dioxaphospholene with phosgene at 0°C,
5 following by heating at 120°C for 2 hr.
The following examples are intended to illustrate the
invention in detail, but they are in no way to be taken as
limiting, and the present invention is intended to encom-
pass modifications and variations of these examples within
10 the framework of their contents and the claims.
orioaic GQnad~ouhin (hCG) Assa
In the following, an hCG assay method is described in
15 which 3-(2'spiroadamantane)-d-methoxy-4-(3"-phosphoryi-
oxy)phenyl-1,2 dioxetane, disodium salt (AMPPD, synthe
sized as described above), was used as a substrate of
alkaline phosphatase. For comparison, a colorimetric
assay was conducted using p-nitrophanylphosphoric acid
20 (PNPP) as a substrata.
1. Placed one bead which was previously coated with
anti-hCG in each tube (12 x 75 mm) alter blotting excess
buffer from bead.
Z. Added 1o0 dal of anti-hCG antibody-alkaline
25 phosphataea conjugate to each tube.
3. To each tube added i00 pl of sample. Separate
tubes were prepared for each of the following:
a) Control Zaro Sample, (male serum or urine)
b) 25 mIU/ml hCG standard (serum or urine)
3o c) 200 mIU/ml hCG standard (serum or urine)
d) Patient sample (serum or urine)
4. After mixing, the tubes Were covered and
incubated for 90 minutes at » 'C.
5. The reaction solution containing the conjugate
and sample were aspirated to waste.

CA 02033331 2001-O1-10
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26
6. The beads were washed 5 times with 2.0 ml of
phosphate buffered saline, pH 7.4, containing 0.1~ Tween
20.
For Colorimetric Assav ~hemfluminescence
7. N/A 7. Washed once with O.D5
M carbonate, 1 mM
MgClZ pH 9.5.
8. Added 200 ~1 1 mg/ml p- e. Added 250 ~1 of 0.4
nitrophenyl-phosphate mM AMPPD in O.oS M
(PNPP) in 0.1 m GLYCINE, carbonate, 1 mM MgClZ,
1 Mm MgClZ, pH 10.4 pH 9.5
9. Incubated for 30 minutes 9. Incubated for 20
at room temperature. minutes at 30~C.
10. Added 11.5 ml of 0.1 M 10. N/A
glycine, 10 mM of EDTA,
pH 9.5, to stop color
development.
11. Read absorbance at 405 11. Read 10 sec. integral
nm in spectrophotometer of luminescence from
each tube in Turner
2oE Luminometer
12. Plotted both sets of data as the signal at each
concentration of hCG divided by the signal at zero hCG vs.
concentration of hCG. Typical data are plotted in Fig. 1,
wherein PNPP represents the colorimetric assay and AMPPD
the chemiluminescence assay. The chemiluminescence assay
was over ten times as sensitive as the colorimetric assay.
Example z
Tandem Icon II hCG Assay (By Film Exposure)
3o Used a commercial Tandem ICON * II assay kit
(Hybritech, Inc.j. Buffers and antibodies used were
included in the kit and AMPPD was used as a substrate of
alkaline phosphatass.
* Trade-mark

20 33331
27
Method
1. Prepared hCG standards at 0, 5, 10, 50 mIU/ml
diluted in. control negative (male) urine for use as test
samples.
2. Added 5 drops of the sample to the center of an
ICON membrane device.
3. Added 3 drops of enzyme antibody conjugate to
the center of each device. '
4, Incubated for 1 minute.
5. Added 2 ml of Hybritech ICON wash solution to
the device. Allowed to drain.
6. Added 500 ~C1 of 0.1% BSA inØl M Tris buffer,
1 mM MgCIZ, pH 9.8. Allowed to drain.
7. Added 200 ~l of 50 ~g/ml AMPPD in 0.1% BSA, 0.1
Tris buffer, pH 9.8, 1 mM MgCl2.
8. Transferred ICON membrane to a piece of Mylar
polyester film and inserted into a black box to expose
film. (Polaroid Type 612).
9. Exposed film for 30 seconds, The results of a
typical assay are shown in Fig. 2. Intense chemilumines
cence from positive samples occurred within a 30-second
reaction time.
Example 3
Alkaline Phosnhatase Assav
An assay for alkaline phosphatase was conducted in
tha lollowinq manner.
ComDOnents
0.05 M carbonate, i mM MgClZ at pH 9.5.
&ubstrate:
3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)
phenyl-1,2-dioxetane disodium salt (AMPPD) at o.4 mM
concentration.

20 3333 1
28
Alkali~pe Phosnhatase:
stock solution at 1.168 ~g/ml in the buffer.
Serial dilutions of alkaline phosphatase stock solu
tions were made in tubes with final enzyme concentrations
of
4.17 x10 ~~M 1.67 x 10~~5M
8.34 x10~~2M 8.34 x 10-~bM
1.67 x10~~ZM 4.17 x 10
~bM
3.34 x10-~3M 2.09 x 10~~6!!
6.68 x10 ~~'M
1.34 x10~~~'M
3.34 x10 GSM
p_rp~e ur : '
Duplicate tubes at each of the above concentrations
of alkaline phosphatase also containing 0.4 mM AMPPD were
incubated at 30'C for 20 minutes.
After incubation, 30-second light integrals were
measured in a Turner 20E Luminometer. The limits of
detection of alkaline phosphatase is shown in Table II.
Data for the detection of alkaline phosphatase using
0.4 m2t AMPPD is shown in Figure 3. Light production was
linear between 10'4 to 10'~~ M enzym~.
Concentration of Alkaline Minimum Detectable
Phosphatase for 2X Conc. of Alkaline
$$~qn Hackaround Phosnhatase
None 1.0 x 10~~~ 1.67 x 10~~sM (1.12)
1. Buffer: 0.05 M sodium carbonate, 1 mM MgCl2, pH 9.5.
Temperature: 30'C. A?IPPD concentration was 0.4 mM.
a . The number in parentheses is the aultiple of back-
qround at the indicated concentration.

2033331
29
Exay le 4
Alkaline P' osp~atase Assay in the Presence of Bovine Serum
Alb~,~. BSA-Fluor. HDMO and BDMO-Fluor
An assay for alkaline phosphatase was conducted in
the following manner.
components:
B~fer: 0.05 M sodium carbonate, 1 mM MgClz, at pH 9.5.
S~strate: 3-(2'-spiroadamantane)-4-methoxy-4-(3"phos
phoryloxy)phenyl-1,2-dioxetane disodium salt (AMPPD) at
l0 0.4 mM concentration.
~, kalin~Phos ate: stock solution at 1.168 pg/ml in the
buffer.
Conditions Tasted:
1. Buffer alone, control.
2. Buffer plus 0.1% bovine serum albumin (eSA).
3. Buffer plus 0.1% HSA-fluorescein (BSA to
fluorexcein ratio 1 to 3).
4. Buffer plus 0.1% poly[vinylbenzyl(benzyl-
dimethylammonium chloride)) (BDMQ).
5. Buffer plus 0.1% BDMQ and fluorescein (0.01 mg
of fluorescein disodium salt mixed with 1 0l of HDMQ).
Serial dilutions of alkaline phosphatase stock
solutions wars made in tubes at the final enzyme
concentrations of:
4.17 x 10~~~M 1.67 x 10'~SM
8.34 x 10~~ZM 8.34 x 10 ~s!!
1.67 x 10 ~ZM 4.17 x 10-~~9
3.34 x 10 ~jM 2.09 x 10'~bll
6.68 x 10~"M 1.0 x 10'~aM
1.34 x 10 ~~M 5.0 x 10~~rM
3.34 x 10 ~sM 2.5 x 10~~TM

CA 02033331 2001-O1-10
78951-11
procedure:
Duplicate tubes with alkaline phosphatase at concen-
trations described above also containing 0.4 mM AMPPD were
incubated at 30'C under various conditions. Test tubes
5 were incubated for 20 minutes under conditions 1, 4 and 5,
while incubated for 90 minutes under conditions 2 and 3.
After incubation, 30 second light integrals were
measured in a Turner 20E Luminometer. The effect of BSA,
BDMQ and fluorescein on the liaits of detection of
to alkaline phosphatase is shown in Fig. 4 and Table III. In
Fig. 4, - Q - corresponds to results under condition 1
above: ... . . condition 2: ... Q ... condition 3:
... ~ ..~ondition 4; and .. Q .:. condition 5,
respectively.
15 fable III
Concentration of Alkaline Minimum Detectable
Phosphatasefor 2X Cone, of alkaline
gddition Backgro und Phosphat ase
None 1. 0 x 10-~~ 1. 67 x 10 (
GSM 1.12
)
~
20 0.1~ BSJ~ 9.5 x 10 ESN 8.34 x 10~1~!!(1.06)
0.1~ HSJi: 1.3 x 10~~sM 4.17 x 10-~6M (1.04)
Fluorescein
O.li HOtiQ 4.0 x 10~t~1 1.00 x 10~~6t~t(1.07)
0.1~ BDMQ: 3.4 x 10-~SM 2.09 x 10-~aM (1.06)
25 Fluorascein
The number in parenthesis is the multiple of background
at the indicated concentration.
ample 5
30 $SVZ DNA Probe Assay
b;~eri s and Buffers:
liembrane: Game Screen Plus; Positively charged nylon
membrane.
Buffers: Denaturation Buffer: 0.5 H NaOH
Neutralization Buffer: 0.4 M NaHiPO~ pH 2.0
* Trade-mark

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Loading Suffer, 1 part Denaturation
Buffer, 1 part
Neutralization Buffer
Membrane Wetting Buffer: 0.4
M Tris buffer pH 7.5
Membrane Prehybridization Buffer:
Final
Concentration
0.5 ml loo x Denhardt's 5%
solution
0.5 ml 10% SDS 0.5%
102.5 ml 20 x SSPE 5%
2.0 mg denatured,
sonicated salmon
sperm DNA
' 200 ~tg/ml
1510 ml
Membrane Hybridization Buffer:
Final
Concentration
0.5 ml 100 x Denhardt's 5%
20solution
0.5 ml 10% SDS 0.5%
2.5 ml 20 x SSPE 5%
2.0 mg salmon sperm DNA 200 ~g/ml
2.0 ml 50% Dextran sulfate 10%
25
ml
~aeh Buffer I:
1 x SSPE/0.1% SDS
ml 20 x SSPE
304 ml 10% SDS.
376 ml ddH20
400 ml
Wash Buffet II: 0.1 x SSPE/0.1%DS preheated
S to
wash temperature.
352 ml 20 x SSPE
4 ml 10% SDS
ddHZO

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32
400 ml (heated)
Wash Buffer III:
0.1 x SSPE/0.1~ SDS
20 ml 20xSSPE
4 ml lOt SDS
394 ml ddH20
400 ml.
Wash Hufler IV:
3 mN Tris-HCl (pH 9.5)
0.6 ml IN Trizma Base
199.4 ml ddH20
200.0 nl.
100X Denhart's Solution:
Dissolved 2 g of polyvinylpyrrolidone mol. wt.
40K (PVP-40) and 2 g of Ficoll at temperatures
greater than 65'C hut less than boiling. Cooled
the solution to approximately 40'C, added 2 g of
BSA and brought the final volume of 100 ml with
ddH20. Aliquots were stored at -20'C.
20X SSC
2oX SSC (for 100 ml)
,3.0 N Sodium Chloride 17.48
0.3 N Sodium Citrate 8.8q
Bring volume to 100 ml and filter through a
0.45~tm nitrocellulose filter. Store at room
temperature.
20X SSPE
2oX SSPE pH 7.4 (for 1 liter)
3.6N NaCl 210.248
200 mM Sodium phosphate 238 dibasic, 5.928
monobasic
20 mN EDTA 7.448
Dissolve, adjust pH to 7.4 with 5 N NaOH
Bring volume to 1 liter and filter through a
0.45~m nitrocellulose filter.
1X TE
1X TE buffer 10 ~i Tris (pH 7.0)
* Trade-mark

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33
1 mM EDTA
Autoclave
Method:
1. Prewetted membrane with Wetting Buffer for 15
min.
2. Inserted membrane into a vacuum manifold device.
3. Denatured the DNA sample by adding 50 ~ul of DNA
sample (with known number of copies of HSVI DNA) to 200 ~l
of Denaturation Buffer. Incubated l0 min. at room temper
l0 ature. Added 250 ml of ice cold Neutralization Buffer and
kept denatured DNA on ice.
4. Added 200 ~l of Loading Buffer to~each well and
aspirated through membrane.
5. Loaded denatured DNA samples to each well, and
aspirated through membrane.
6. Repeated Step 4.
7. Dissembled manifold and removed membrane.
8. W-fixed DNA to membrane using a W
Transilluminator for 5 minutes.
9. Incubated the membrane in 0.1% (w/v) BDMQ in
phosphate-buffered saline for 15 minutes.
10. Incubated membrane in Prehybridization Buffer at
70'C for 1 hour.
11. Added alkaline phosphatase-labeled SNAP probe
specific for HSVI dissolved in Membrane Hybridization
Buffer. Incubated for 3-5 hours at 70'C.
12. Removed membrane from Hybridization Buffer and
incubated in 400 nl of wash Buffer i, while agitating at
room temperature for 10 minutes.
13. Washed with 400 ml of Wash Buffer II at 50'C for
30 minutes.
14. Washed with 400 ml of Wash Buffer III at room
temperature for 10 minutes.
15. Washed with 200 ml of Wash Buffer IV at room
temperature for 10 minutes.

2033331
34
16. Added 2 ml of 300 ~g/ml AMPPD in 0.1 M Tris
buffer, 1 mM MgClZ, pH 9,8 to the membrane.
17. Transferred the membranes to a piece of Mylar
polyester film, and then to a black box containing Type
612 Polaroid film.
18. Exposed film for 30 minutes. Typical results
are shown in Fig. 5, wherein Fig. 5A shows the results at
60 ~ag/ml AMPPD, Fig. 5B at 300 ~g/ml AMPPD, and Fig. 5C
after the first 30 min. o! reaction at 300 ~g/ml AMPPD.
Exa~ggle 6
Hepatitis B V rus DNA Hybridization Assay
We compared the sensitivity of a chemiluminesceat
substrate (AMPPD) and a chromogenic substrate (BCIP/NBT)
for detection of an alkaline phosphate label in Hepatitis
B Virus Core Antigen DNA HBV~ probe hybridization assay
(SNAP~_, DuPont). Chemiluminescent signals obtained from
AMPPD hydrolysis by said phosphatase was detected with
Polaroid Instant Black and White Typs 612 film.
~,ethods and Materials:
1. Chemiluminescent Substrate: AMPPD
2. Protocol iQ~ Determining the Sensit~vit_y of
SNAPe/Test for HBV~ ~(~gpatitis B "Core Antigen" DNA1
The levels of detection, or the sensitivity, of the
SNAPS DNA probe test for Hepatitis 8 "Core Antigen~ DNA
were determined by performing the test using serially
diluted HBV~ control plasmid DNA.
The assay protocol involved the following steps:
a. Preuaration of Positive HBV_ DNA Plasmic Controls
A stock solution of HBV~ plasmfd was prepared by
dissolving 100 ng (1.2 x 1010 copies) of the plasmid in 25
ul of sterile, deionized HZO and serially diluted with
0.3 N NaOH to produce plasmid samples in the concentra-
tions range of 4.88 x 103 - 0.96 x l0a copiea/ul. The

CA 02033331 2001-O1-10
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samples were allowed to denature for 15 minutes at room
temperature.
b. Preparation of the Membranes. Immobilization of HBV_
Plasmid Control DN1,
5 Gene Screen"' Plus membranes were cut into 1 x B cm
strips. 1 ul of each dilution of HBV~ plasmid sample was
spotted on the dry membrane with a pipette tip in contact
with the membrane surface to obtain very small, concen-
trated spots. The membranes were then rinsed with 100 ul
10 of 2 M ammonium acetate per spot to neutralize the target
immobilized nucleic acid. They were subsequently rinsed
with 0.6 M sodium chloride, 0.08 M sodium citrate, pH 7.0
buffer.
c. probe Hybridization
15 (1) Prehybridization
The membranes containing plasmid samples were placed
in a heat-sealable pouch in 3 ml of Hybridization Buffer.
Prehybridization was carried out for 15 minutes at 55'C.
(ii) Hybridization
20 SN7~Pa alkaline phosphatase labeled probe was recon-
stituted with 100 ul of the sterile deionized HzO. The
hybridization solution was prepared using 2.5 ul alkaline
phosphatase labeled probe solution dissolved in 0.5 ml
Hybridization Buffer. Hybridization was performed in a
25 new, heat sealed pouch, with 0.5 ml hybridization
solution, for 30 minutes at 55'C. lifter hybridization,
the pouch was opened and the menbranas carefully removed
and washed with the following buffers:
1. twice with 0.1 M sodium chloride, 0.02 K sodium
30 citrhte, pH 7.0, plus i0 g SDS butter, for 5 minutes at
room temperature,
2. twice with 0.1 H sodium chloride, 0.02 Ii sodium
citrate, pH 7.0, plus 10 ml Triton*X-10o (sigma chemical
Co., St. Louis, MO), for 5 minutes at 55'C,
* Trade-mark

2033331
36
3. twice with the above buffer for 5 minutes at
room temperature,
4. twice with 0.1 M sodium chloride, 0.02 M sodium
citrate, pH 7.0 buffer for 5 minutes at room temperature,
5. once with 0.1% BSA in 0.05 M carbonate buffer at
pH 9.5.
Hybridization Hu~Ler was prepared by mixing 250 ml of
3 M sodium chloride, 0.4 M sodium citrate, pH 7,0, diluted
to 800 ml with deionized HZO, with 5 g Bovine Serum
l0 Albumin, 5 g polyvinylpyrrolidone (average MW 40,000) and
g SDS, warmed and mixed to dissolve.
d. C~,~milw~inescent Detection of HBV Plasmid DNA with
ppp '
The hybridized membrane strips were saturated with
100 ul of 1.6 mM AMPPD in 0.1% BSA in 0.05 M carbonate
Buffer, 1.0 MgCl2 at pH 9.5. The membranes were then
sealed in a plastic pouch and immediately placed in a
camera luminometer where light emission was imaged on
Polaroid Instant Hlack/White 20,000 ASA film.
e. detection w.~~h SNAPa Chromogenic S~strates ('[iitro
Blue Tatrazoliym (NHT) 5-Bromo-4-Chlora-3-Indolyl
phosgjiate l'~CIPLIPerformed According- to the
Manu~r~,uer~s instructions)
Hybridized membranes which were developed with the
chromogonic substrates did not undergo wash step ~5.
Substrate solution was prepared by mixing 33 ul NBT and 25
ul of BLIP in 7.5 ml of alkaline phosphatase substrate
buffer provided by the manufacturer. Washed hybridized
membranes were transferred to a heat sealed pouch with the
substrates containing buffers. Tha color was allowed to
develop in the dark, as NBT is light sensitive.
f. Photaq~~hic Detection of AMPPD Signal
The results of assays performed with AMPPD were
imaged on Polaroid Instant Black and White Type 61Z photo-

2Q33331
37
graphic film. The images were subsequently digitized
using a black and white RBP Densitometer, Tobias
Associates, Inc., Ivyland, PA.
Results:
Figure 6 shows a time course of the chemiluminescent
for serially diluted Hepatitis B Virus "Core Antigen"
plasmid hybridized with alkaline phosphatase labeled probe
and imaged onto photographic film. Each photograph
corresponds to a 30 minute exposure on Polaroid Instant
Black and White Type Copies of 612 film. A comparable set
of serially diluted Hepatitis B Virus "Core Antigen"
plasmid DNA hybridized with alkaline phosphatasa labeled
probe and detected BcIP/NBT substrate is shown in Figure
7. The chemiluminescent assay detected 1.18 x 106 copies
of HBV~ DNA. The colorimetric test showed a detection of
1.07 x 10' copies. After a two hour incubation, the
chemiluminescent assay detected 4.39 x l0~ copies of HBV~
DNA. The colorimetric test showed a detection of 1.07 x
iD~ copies after the same incubation time. After a 4
incubation, the colorimetric assay detected 1.18 x 106
copies of HBV~ DNA.
Table IV sumvearizes the results of chemiluminescent
detection limits of HBV~ using AMPPD and the colorimetric
detection with HCIP/NBT substrates. Sensitivity of the
SNAPS hybridization kit was improved over 100-fold using
the chemiluminescent assay based upon A1~IPPD. The AMPPD-
based assay detected as few as about 44,000 copies of HBV~
plasmid DNA, compared to the BCIP/NBT colorimetric assay
which required 10,700,000 copies for detection. In addi-
tion, AMPPD reduced the assay time from 4 hours to 30
minutes.

CA 02033331 2001-O1-10
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38
Table IV
Comcarison of Detection Limits for Hepatitis 8 "Core Anti-
~en~ Plasmid DNA Using! Chemiluminescent and Chromoqeni_c
Substrates in SNAP' HS~bridization Kit
Chemiluminescent AMPPD Colorimetxic BCIP/
Copies of BHS~ Substrate Detection in NBT Substrates
DNA Per Spot Minutes Detection in Minutes
9.8 x 10~ 30 30
3.2 x 10~ 30 60
1.07 x 10~ 30 120
3.56 x 106 30 180
1.18 x 106 30 240
3.95 x 105 60 no color
1.31 x 105 90 no color
4.39 x 10~ 120 no color
Quantitative chemiluminescence results could be
obtained by measuring reflection densities directly from
the imaged Black and White Polaroid Type 612 instant
photographic film strips using a Tobias RBP Hlack and
White Reflection Densitometer, as shown in Figure 8. The
results show that a dose response curve can be generated
of tht reflection densities as a function of HBV~ plasmid
concentration. This dose response curie can be subse-
quently used as a calibration for the determination of HBV~
DNA levels in clinical specimens.
Example 7
Anti=AFP antibody coated beads and anti-AFP antibody:
alkaline phosphatase conjugates were obtained from a
Hybri.tech Tandem Assay kit.
1. To each tube was added 20 ul of sample. Samples
were 0, 25, 50, 100, and 200 mg/ml AFP.
2. Placed one bead in each tuba.
3. Added 200 ~1 of anti-AFP antibody alkaline
phosphatase conjugate to each tube.
4. Shook rack to mix contents of tubes.

CA 02033331 2001-O1-10
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39
5. Covered tubes.
6, incubated for 2 flours at 37'C.
7. Aspirated off antibody and sample to waste.
e. Washed beads 3 times with c.Q ml of 0.1~ Tween
20 in ghosphate buffered saline, pH 9.4.
For Colorimetric Assav Chemilumine~ence
9. N/A 9. Washed 1 time with
0.5 H carbonate, 1 mM
MgClt pH 9.5.
10. Added 200 ~1 of 1 mq/ml 10. Added 250 ~ of 0.4 mM
p-nitrophenylphosphate AISPPD in 0.05 M in
(PNPP) glycine 1 mM 0.1 carbonate, 1 mM
MgClZ pH 10.4 MgCl= pH 9.5.
11, Incubated for 30 minutes 11. Incubated for 20
at room temperature minutes at 30'C
12 . Added 1. 5 ml of 0 .1 M 12 , td/A
glycine, 10 mM EDTA, pH
9.5 to stop color devel-
opment
13. Read in absorbance at 13. Read 10 sec. integrnl
410 nm in spectrophoto- of each tube in
meter Turner luminometer
14. Plotted both sets of data as the signal at each
concentration of AFP divided by the signal at zero AFP vs.
concentration o! AFP. As shown in Fig. 9, the results of
the colorimetric assay are shown in the PHPP curve, and
that o! the chemiluminescenca assay in the AtrIPPD curve.
It can be seen that the latter assay is about 10 times as
sensitive as the former assay.
3o Hxamole 9
Assav for Thyroid Stimulating~"r~rmone fTSH1
Mouse monoclonal anti-TSH-~ antibody was used to coat
1/8 inch beads for analyta capture. Mouse monoclonal
anti-TSH antibody was conjugated with alkaline phosphatase
* Trade-mark

CA 02033331 2001-O1-10
c __ ____ __ __
78951-11
and used as a detection antibody (antibody-enzyme
conjugate).
TSH was obtained from Calbiochem, Catalog No. 609396,
and BSA (type V - fatty acid free) was obtained from
5 Sigma, Catalog No. A6003.
The buffer solution~used for the analyte and antibody
enzyme conjugate contained 0.1 M Tris-HC1, l mM MgCll, and
2~ by weight BSA (pM 7.5). The substrate buffer solution
contained 0.1 H Tris, 0.1 mM MgCli, (pH 9.5), and the
10 substrate AriPPD (50 ~g/ml).
A TSH-containing analyte solution (15 ul) was mixed
with 135 ~1 of antibody enzyme conjugate solution. Two
1/8 inch beads coated as described above were added to the
solution and incubated for 2 hours at 23 ~ C. The beads
15 were then washed four times with 0.1 M Tris buffer (pH
7.5) and transferred to a reaction tube. 200 ~1 of the
buffer solution containing the substrate described above
was added to the tube. Following an incubation period of
20 minutes, light emission was recorded as ten second
20 counts using a Berthold Clinilumat Luminescence Analyzer.
Figure 10, which is a plot of the data in Table V
below, shows luminescence intensity for a given TSH
concentration. Linearity was achieved between 1 and 8
~U/ml of TSH.
25 Table V
TSH Concentration
fuU/mll jCounts/10 sec X 10~~1
1 0.25
2 0.49
30 4 1.1
An identical TSH assay was also performed in the
absence of HSA for the sake of comparison. As shown in
Fig. 11, the BSA- containing sample (Curve A) showed
greater luminescence intensity for a given TSH concentra-
35 tion than the sample without BSA (Curve B).
* Trade-mark

CA 02033331 2001-O1-10
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41
Example 9
Assay for Carcinoe,~b~ryo~ic Ant:~,gen lCEA1 in the Head
Format
Anti-CEA coated beads and anti-CEA antibody: alkaline
phosphatase conjugates were obtained from a Hybritech
Tandem Assay kit.
1. To each tube were added 20 ul of sample.
Standards of 0, 2.5, 5, 10, 20, and 50 ng/ml CEA were
used:
2. One bead was placed in each tube.
3. Added 200 ul of anti-CEA antibody enzyme
conjugate to each tube.
4. Shook rack to mix contents of tubes.
5. Covered tubes.
6. Incubated for 2 hours at 37'C.
7. Aspirated off antibody and sample to waste.
8. Washed beads 3 times with 2.0 ml of 0.1~ Tween*
in phosphate buffered saline, pH 7.4.
9. Washed once with 0.05 % sodium carbonate, 1 mM
20 MgClt, pH 9.5.
10. Added 250 ul of 0.4 mM AlSPPD in 0.05 M sodium
carbonate, 1 mM MgCl=, pH 9.5.
11. Incubated for 20 minutes at 30'C.
12. Read 10 sac. integral of luminescence frbm each
tube in Turner 2oE Luminometer.
13. Plotted both sets of data as the signal at each
concentration of hCG divided by the signal at zero CEA vs.
concentration of CEA. Typical data for a CEA assay using
AMPPD are shown in Figure 12. Linearity was achieved
between 0 and 20 ng/ml of CEA.
Exam l
Assay for Numa~ Lvti,~niz ~ Hormone (hLH1
A nylon membrane, (Pall Immunodyne; 0.45 micron pore
size), approximately 3mm in diameter was sensitized with
5 ul of a solution of 1 ~g/ml o! capture monoclonal anti
LH antibodies for solid phase in phosphate buffered saline
* Trade-mark

20 3333 1
42
(PBS), purchased from Medix, catalog #L-461-09. The
membrane was subsequently blocked with 2% casein in phos-
phate buffered saline at pii 7.3. The membrane was then
enclosed in the device shown in Figure 13, which included
blotting paper layers. In Fig. 13, A shows the prefilter
cup; a plexiglass top: C Pall Immunodyne membrane (pore
size 0.450 ; D polypropylene acetate fluffy layer: E
blotting paper: and F plexfglass.
The detection antibody used was mouse monoclonal
anti-Lli, purchased from Medix, catalog $L-461-03. This
antibody was derivatized with alkaline phosphatase,
(purchased from Biozyme, catalog #ALPI-11G), using the
glutaraldehyde coupling procedure (volley, A. et.al:,
Bull. World 'Health Org;, 53, 55 (1976)].
Procedure:
The detection antibody conjugate (50 pl) was added to
tubes containing 200 ul of hLli of the following
concentrations:
Tube ~I ~o~c. l7LFi ~n_~g,~mi of PBs
1 0
a 1
3 10
4 100
The content o! each tube was then added to four nylon
membranes previously derivatized with capture antibodies
(described above). After a five minute incubation period,
the prefilter cup was removed and the membranes were
washed with 400 pl of 0.051 Tween 20 in PBS. Subse
quently, 100 ~Cl of 0.4 mM AMPPD, in 0.05M carbonate, 1 mM
MgClZ, 0.1% by weight BSA at pH 9.5 were added. The nylon
membxanea were placed in a camera luminometer containing
type 612 Polaroid Instant Black and White film, and
exposed for one minute. The results of the assay imaged
on film are shown in Figure 14.
Subsequently, the reflection densities of the images
were measured using the Tobias RBP Portable Black and

2033331
43
White Reflection Densitometer (manufactured by Tobias
Associates, Inc., 50 industrial Drive., P.O. Box 2699,
Ivyland, PA 18974-0347). The reflection densities were
plotted versus concentration of LH to yield a standard
curve for hLH, as shown in Figure 15.
~xamnle 11
5'.hg~miluminescent Decomposi~.ion of 3-(2'Soi~oada,,Mantanel-
4-Methoxv-4- ( 3'~8-D-GalactopyranQsvl-Phenlrl ) -1. 2-Dioxg~,ane
( AMPGD f
Reacents:
1. AMPGD synthesis as described above was made up
in i:i MeOH/HZO at a concentration of l0 mg/ml.
2. 0.01 M. sodium phosphate buffer, pH 7.3,
containing 0.1 M NaCl and 1 MgCl2.
3. ~-galactosidase (Sigma Chem. Co., catalog 65635,
mol. wt. 500,000), 1 mg/ml in phosphate-salt buffer, pH
7.3, diluted 1:100 to yield a 2 x 10° M solution.
Protocol
AMPGD solution (9.3 ~l) was diluted in 490 ul of a
buffer solution of variable pH. Subsequent addition of 5
~1 of the diluted galactosidase solution was followed by
1 hr. incubation at 37~C. The final concentration of
reactants waa o.4 mM AMPGD and i x 10'3 moles
galactosidase, at various pH values, as required by the
experiment.
lifter incubation, the solutions were activated in a
Turner 20E Luminometer by the addition of 100 ~ui of 1 N
NaOH. Tha instrument temperature was 29'C, that of the
NaOH room temperature.
Thus, the assay consisted of a two-step process
wherein the substrate-enzyme incubation was perfonaed at
various pH values appropriate to efficient catalysis,
e.g., at pH 7.3, and subsequently the pH was adjusted to
about 12 with NaOH, and luminescence was read again.

24 33331
44
Results
In Figure 16 is shown the chemiluminescence of a
fixed concentration of AMPGD as a function of ~-galacto
sidase concentration, wherein the enzyme reaction was run
at gH 7.3 and luminescence measured at pH 12. The use
able, i.e., linear, portion of the standard curve was at
enzyme concentrations between 10~~3 and 10-e M.
In Figure 17 is shown the effect of pH on the
decomposition of AMPGD by ~B-galactosidase. The data show
that the optimum pH for the enzyme with this substrate is
about pH 6.5.
Figure 18 shows the production of light from AMPGD as
a function of ~A-galactosidase concentration, using the .,
two-step protocol described above. At all enzyme concen-
trations, adjustment of the pH to 12 from 7.3 produced
over a 100-fold increase in chemfluminescence.
Examgls 12
Detection of DNA FracLments by Cjyemi yminescence fter
Electrorhoretic SeRaration ~~'_Fraaments
DNA sequencing was performed using the dideoxy chain
termination method of Singer et al. (1977) above.
Biotinylated pBR322 primer (40 ng) was annealed to
5 ~tg of denatured pBR322 glasmid. Klenow Fragment (DNA
polymerise I), 2 units, was then added (final volume was
17 pl). Subsequently, 2 ~1 of this template - primer
solution was used for each of four base-specific reactions
(G, A, T, C). To each reaction mixture, we added these
specific amounts of deoxynueleotides, and
dideoxynucleotides.
Reaction tures fN noaramsof Nueleotidesl
M~,
~eoxvnuc~,gotides c~ ~ ~ _C
dGTP 1022.9 1077.4 1102.9 1102.9
dCTP 1015.9 992.4 1015.9 942.9
dTTP 1048.6 1048.6 972.5 1048.6
dATP 985.5 985.5 985.5 985.5

CA 02033331 2001-O1-10
78951-11
p'deo nucleotides
ddGTP 123.0
ddCTP 29.7
ddTTP 466.0
5 ddATP 113.0
An aliquot of each reaction mixture (1 ~1) was loaded
on a standard sequencing gel and electrophoresed. The DNA
was electrophoretically transferred to a Pall Biodyne A
nylon membrane and then W fixed to the membrane. The
10 membrane was then dried, blocked for i hour with o.2~
casein in PBS (casein-PBS), incubated with streptavidin:-
alkaline phosphatase (1:5000 in casein-PBS) for 30
minutes, washed first with casein-PBS, then with 0.3~
Tween* 20 in PBS, and finally with 0.05 ti bicarbonate/-
15 carbonate, pH 9.5, 1 mM MgClZ. Substrate, 0.4 m1K AMPPD in
the final wash buffer, was incubated with the membrane for
5 minutes. After wrapping the membrane in plastic wrap,
the membrane was placed in contact with Kodak XAR film and
Polaroid Instant Black and White film for 2 hours. The
20 order of sequence lanes is C T A G in Figures 19A (X-ray
film) and 19 B (instant film).
Example 13
Effect of liembrane Composition On Detect.~n Qj DNA
Fra en s jay Chemiluminescence
25 Various amounts of the SNAPS Hepatitis B core antigen
oligonucleotide probe conjugated to alkaline phosphntase
(Molecular 8losystems, Inc., San Diego, CA), as listed in
the left column of Table VI, were spotted on three types
of transfer membranes: Gene Screen Plus" (Nylon),
30 Schleicher and Schuell nitrocellulose, and Millipore PVDF.
The- .spots were incubated with an AKPPD solution, lumin-
escence generated, and light detected on instant film, as
in Example 6(C).
Tha data of Table VI show the earliest detection
35 times at each level o! oligonucleotide for each of the
three membranes. Luminescence was greatly increased in
* Trade-mark

2033331
46
intensity by the use of nylon-based membranes, as compared
to the other two types. For example, with a nylon
membrane, the smallest amount of oligonucleotide tested,
i.e., 0.01 ng, was detected within 60 seconds of film
exposure. In contrast, it required at least 67 ng of
oliqonucleotide to be detectable in 60 seconds on a
nitrocellulose membrane; amounts of 0.82 ng or less were
not detectable within 10 minutes. In further contrast, no
amount of oligonueleotide was detectable in periods as
long as 10 minutes.
Table VI
Earliest Detection Time. Sec.
Oligonucleotide,
nq~ ~ Nvlon NitrocelluloseP_VDF
200 1 60
67 1 60
22 1 300
7.4 1 300
2.5 1 300
0.82 1
0.27 1
0.091 10 *
0.03 60
0.01 60
*Not detectable by 10 min. of exposure.

Representative Drawing