U.S. patent application number 11/447839 was filed with the patent office on 2007-03-15 for chemiluminescence enhancer.
This patent application is currently assigned to FUJIREBIO INC.. Invention is credited to Yoshihiro Ashihara, Tatsuki Matsuno, Kazushige Moriyama, Tetsuji Tanimoto.
Application Number | 20070059786 11/447839 |
Document ID | / |
Family ID | 29996684 |
Filed Date | 2007-03-15 |
United States Patent
Application |
20070059786 |
Kind Code |
A1 |
Moriyama; Kazushige ; et
al. |
March 15, 2007 |
Chemiluminescence enhancer
Abstract
The present invention provides a chemiluminescence enhancer
treated to retain favorable dispersibility of fine solid carriers
and stably exert a chemiluminescence enhancing action. The
invention provides a chemiluminescence enhancer used for signal
detection in a solid phase immunoassay using antigen or/and
antibody immobilized onto fine solid carriers dispersible in a
liquid medium, consisting of a water soluble macromolecular
quaternary ammonium salt, a quaternary sulfonium salt or a
quaternary phosphonium salt in order to enhance emission of light
caused by an enzymatic reaction of a chemiluminescent substrate
having dioxetane, wherein the chemiluminescence enhancer is given
an aggregation inhibition treatment of the fine solid carriers by
the treatment with an oxidizing agent or a reducing agent, and a
chemiluminescence method and a kit using the chemiluminescence
enhancer.
Inventors: |
Moriyama; Kazushige; (Tokyo,
JP) ; Tanimoto; Tetsuji; (Tokyo, JP) ;
Matsuno; Tatsuki; (Tokyo, JP) ; Ashihara;
Yoshihiro; (Tokyo, JP) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
FUJIREBIO INC.
Tokyo
JP
|
Family ID: |
29996684 |
Appl. No.: |
11/447839 |
Filed: |
June 7, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10518586 |
Dec 21, 2004 |
|
|
|
PCT/JP03/07985 |
Jun 24, 2003 |
|
|
|
11447839 |
Jun 7, 2006 |
|
|
|
Current U.S.
Class: |
435/7.92 ;
436/524; 549/510 |
Current CPC
Class: |
G01N 21/76 20130101;
G01N 33/533 20130101; G01N 33/532 20130101 |
Class at
Publication: |
435/007.92 ;
436/524; 549/510 |
International
Class: |
G01N 33/551 20060101
G01N033/551; C07D 305/02 20060101 C07D305/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 24, 2002 |
JP |
2002-183720 |
Claims
1. A chemiluminescence enhancer, the enhancer comprising at least
one of a water soluble macromolecular quaternary ammonium salt, a
quaternary sulfonium salt or a quaternary phosphonium salt capable
of enhancing emission of light caused by enzymatic reaction of a
chemiluminescent substrate having dioxetane each salt having been
treated with an oxidizing agent or a reducing agent to provide
aggregation inhibition of an antigen and/or an antibody immobilized
on a solid fine carrier.
2. The chemiluminescence enhancer according to claim 1 wherein the
enhancer does not substantially comprise a component with a
molecular weight of more than 400,000 daltons in the molecular
weights separated by an ultrafiltration method.
3. The chemiluminescence enhancer according to claim 1 wherein the
chemiluminescent substrate is a substrate represented by the
general formula: ##STR4## wherein R.sub.2 is an aryl group
substituted with an X-oxy group, which forms a 1,2-dioxetane
compound that is an unstable oxide intermediate when X is
eliminated by an activator selected from acid, base, enzyme,
organic or inorganic catalyst and electron donor to induce a
reaction, which unstable 1,2-dioxetane compound is decomposed with
releasing electron energy to produce light and two
carbonyl-containing compounds of general formulae, ##STR5## and X
is a chemically easily reactive group that is eliminated by an
enzyme; R.sub.1 is selected from the group consisting of an alkyl
group, an alkoxy group, an aryloxy group, a dialkylamino group, a
trialkylsilyloxy group, an arylsilyloxy group, an aryl group and an
aryl group that is bound to an aryl group R.sub.2 to form a
polycyclic aryl group with X-oxy group substitution, which
spiro-binds to a 1,2-dioxetane ring; R.sub.3 and R.sub.4 are each
an alkyl group or a heteroalkyl group, or R.sub.3 and R.sub.4 may
be together bound to form a polycyclic alkylene group which
spiro-binds to the 1,2-dioxetane ring.
4. The chemiluminescence enhancer according to claim 1 wherein the
enhancer is prepared from a monomer selected from the group
consisting of a quaternary ammonium salt, a quaternary sulfonium
salt, a quaternary phosphonium salt and mixtures thereof.
5. The chemiluminescence enhancer according to claim 1 wherein the
enhancer is a polymerized quaternary ammonium salt, a polymerized
quaternary sulfonium salt, a polymerized quaternary phosphonium
salt or copolymers thereof.
6. The chemiluminescence enhancer according to claim 1 wherein the
enhancer is selected from the group consisting of
poly[vinylbenzyl(benzylmethyl ammonium chloride)],
poly(vinylbenzyltrimethyl ammonium chloride),
poly[vinylbenzyl(tributyl ammonium chloride)], benzylmethylcetyl
ammonium chloride, polymethacrylamidepropylenemethyl ammonium
chloride, poly[vinylbenzyl(triethyl ammonium chloride)],
poly[vinylbenzyl(2-benzyl-amino)ethyldimethyl ammonium chloride],
poly[vinylbenzyldimethyl(2-hydroxy)-ethyl ammonium chloride],
poly[vinylbenzyl(trimethylphosphonium chloride)],
poly[vinylbenzyl-(tributylphosphonium chloride and
poly[vinylbenzyl(trioctylphosphonium chloride)] and copolymers
thereof.
7. The chemiluminescence enhancer according to claim 1 wherein the
fine solid carrier is a particle.
8. The chemiluminescence enhancer according to claim 7 wherein the
particle is a magnetic particle.
9. The chemiluminescence enhancer according to claim 1 wherein the
reagent having an oxidation or reduction property is selected from
the group consisting of ammonium persulfate, sodium sulfite, sodium
hypochlorite, hydrogen peroxide, sodium metaperiodate, potassium
permanganate and potassium dichromate.
10-15. (canceled)
16. In combination, a chemiluminescent substrate, an antigen and/or
an antibody immobilized on a fine solid carrier, and a
chemiluminescence enhancer having dioxetane, the enhancer
comprising at least one of a water-soluble macromolecular
quaternary ammonium salt, a quaternary sulfonium salt or a
quaternary phosphonium salt capable of enhancing emission of light
caused by enzymatic reaction of the chemiluminescent substrate
having dioxetane, each salt having been treated with an oxidizing
agent or a reducing agent to provide aggregation inhibition of the
antigen and/or antibody immobilized on the fine solid carrier.
Description
TECHNICAL FIELD
[0001] The present invention relates to a chemiluminescence
enhancer used for enhancing chemiluminescence which is caused by
enzymatic action of a chemiluminescent substrate in signal
detection in a solid phase immunoassay using antigen or/and
antibody immobilized onto fine solid carriers dispersible in a
liquid medium.
BACKGROUND ART
[0002] In the field of measuring trace components, particularly in
the field of clinical diagnostics, measurement methods of applying
principles of immunology are utilized and immunoassays using fine
solid carriers as a solid phase are widely used. The fine solid
carriers include erythrocytes, gelatin particles, latex particles
and the like, and a quantitative analysis is performed by absorbing
antigen or/and antibody on the surface thereof and reacting
immunologically the antibody or/and the antigen against antigen
or/and antibody in a test sample. It is well-known that the
immunoassays using these fine solid carriers are also commonly used
even in the fields other than clinical diagnosis.
[0003] Chemiluminescence measurement utilizing chemiluminescence
reaction in which the chemiluminescence is caused by allowing an
enzyme such as alkaline phosphatase to act upon a chemiluminescent
substrate such as 1,2-dioxetanes can rapidly and sensitively
measure the presence or concentration of a measurement subject in a
specimen, and has been widely used to measure viruses such as HIV
and HCV, and other trace components in vivo (JP 96-507694).
[0004] It is well-known that a quenching reaction occurs in a
liquid medium, particularly in an aqueous medium with
chemiluminescence by decomposition of chemiluminescent substrate
having dioxetane. Many test samples are biological samples in
general, and thus the measurement of the samples by such a method
is generally performed in an aqueous medium. Therefore, the
quenching reaction sometimes reduces substantially the
chemiluminescence occurred by the decomposition of the
chemiluminescent substrate having dioxetane, which is actually
obsereved. In the measurement methods of certain test samples,
e.g., nucleic acids, a viral antibody and other proteins, in which
a detection at a low level is required, the chemiluminescence
reduced by the quenching reaction in combination with unavoidable
background signals reduces the sensitivity of the measurement
method, and thus in some cases, those at an extremely low level can
not be detected. In order to improve these quenching reactions, an
addition of a water soluble macromolecule including both naturally
occurring and synthetic molecules (see U.S. Pat. No. 5,145,722), an
addition of various water soluble enhancers to test samples (see
U.S. Pat. No. 4,978,614), or water soluble polymerized quaternary
ammonium salts such as poly(vinylbenzyltrimethyl ammonium chloride)
(TMQ), poly(vinylbenzyltributyl ammonium chloride) (TBQ) and
poly(vinylbenzyldimethylbenzyl ammonium chloride) (BDMQ) as the
water soluble polymerized quaternary ammonium salts has been used
(see U.S. Pat. No. 5,112,960 and JP 8-507694 T).
[0005] In the meanwhile, the chemiluminescence enhancers such as
TMQ, TBQ and BDMQ described above are polymers with high molecular
weight. In the case of being used for a signal detection in a solid
phase immunoassay using antigen or/and antibody immobilized onto a
fine solid carrier dispersible in a liquid medium, when the
carriers are once physically aggregated, for example, in order to
wash after an immune reaction, these polymers prevent the fine
solid carriers from dispersing thereafter, inhibit luminescence
caused by an enzymatic reaction of the chemiluminescent substrate
having dioxetane depending on the concentration of the subject
substance to be detected, and have sometimes had a problem in that
no precise measurement value can be obtained.
DISCLOSURE OF THE INVENTION
[0006] The aim of the invention is to provide a chemiluminescence
enhancer made by treating a known chemiluminessence enhancer to
retain favorable dispersibility of fine solid carriers in a
chemiluminescence reaction by a chemiluminescent substrate and
stably exert a chemiluminescence enhancing action.
[0007] As a result of intensive study, the inventors of the present
invention have found that a chemiluminescence enhancer such as
water soluble macromolecular quaternary ammonium salt, quaternary
sulfonium salt or quaternary phosphonium salt treated with a
reagent having an oxidation or reduction property more stably
enhances emission of light caused by an enzymatic reaction of a
chemiluminescent substrate having dioxetane in signal detection in
a solid phase immunoassay and provides more precise measurement
results, and have completed the invention.
[0008] That is, the invention provides a chemiluminescence enhancer
used for signal detection in a solid phase immunoassay using
antigen or/and antibody immobilized onto fine solid carriers
dispersible in a liquid medium, consisting of a water soluble
macromolecular quaternary ammonium salt, a quaternary sulfonium
salt or a quaternary phosphonium salt in order to enhance emission
of light caused by an enzymatic reaction of a chemiluminescent
substrate having dioxetane, wherein the chemiluminescence enhancer
is given an aggregation inhibition treatment of the fine solid
carriers by the treatment with an oxidizing agent or a reducing
agent, and a chemiluminescence method and a kit using the
chemiluminescence enhancer.
[0009] A preferable aspect of the enhancer is the chemiluminescence
enhancer which does not substantially contain a component of a
molecular weight more than about 400,000 daltons in the molecular
weight separated by an ultrafiltration method.
BEST MODE FOR CARRYING OUT THE INVENTION
[0010] As described above, the chemiluminescence enhancer of the
invention is water soluble macromolecular quaternary ammonium salt,
quaternary sulfonium salt or quaternary phosphonium salt, or the
like which is treated with a reagent having oxidation or reduction
property, and is represented by following general formula (I):
##STR1##
[0011] In general formula (I), each of R.sub.5, R.sub.6 and R.sub.7
may be a straight or branched, unsubstituted alkyl group having 1
to 20 carbon atoms (e.g., methyl group, ethyl group, n-butyl group,
t-butyl group or hexyl group or the like); a straight or branched
alkyl group having 1 to 20 carbon atoms substituted with one or
more of a hydroxyl group, an alkoxy group (e.g., methoxy, ethoxy,
benzyloxy or polyoxyethylethoxy group), an aryloxy group (e.g.,
phenoxy group), an amino group, a substituted amino group (e.g.,
methylamino), an amide group (e.g., acetamide group) or an ureide
group (e.g., phenylureide group); a fluoroalkane group; a
fluoroaryl group (e.g., heptafluorobutyl group); an unsubstituted
monocycloalkyl group having 3 to 12 ring carbon atoms (e.g.,
cyclohexyl or cyclooctyl group); a substituted monocycloalkyl group
having 3 to 12 ring carbon atoms substituted with one or more of an
alkyl group, an alkoxy group or a condensed benzo group (e.g.,
methoxycyclohexyl or 1,2,3,4-tetrahydronaphthyl group);
polycycloalkyl group having two or more condensed rings each having
5 to 12 carbon atoms, which is unsubstituted or substituted with
one or more of an alkyl group, an alkoxy group or an aryl group
(e.g., 1-adamantyl or 3-phenyl-1-adamantyl group); an aryl group,
an alkaryl group or an aralkyl group having at least one ring and
totally 6 to 20 carbon atoms, which is unsubstituted or substituted
with one or more of an alkyl group, an aryl group, fluorine or a
hydroxyl group (e.g., phenyl, naphthyl, pentafluorophenyl,
ethylphenyl, benzyl, hydroxybenzyl, phenylbenzyl or dehydroabiethyl
group). At least two of R.sub.5, R.sub.6 and R.sub.7 mean the
groups capable of forming together with a quaternary atom to which
they are bound a saturated or unsaturated, unsubstituted or
substituted ring having 3 to 5 carbon atoms and 1 to 3 heteroatoms
and containing nitrogen, phosphorus or sulfur, to which a benzene
ring may be condensed (e.g., 1-pyridinium, 1-(3-alkyl)imidazolium,
1-(3-aralkyl)imidazolium, morpholino, alkyl morpholinium, alkyl
piperidinium, N-acyl piperidinium, piperidino, acyl piperidino,
benzoxazolium, benzothiazolium or benzamidazolium).
[0012] A symbol Y in the general formula [I] alone or in
combination represents a counter ion capable of including moieties
such as a halogen ion, i.e., fluorine ion, chlorine ion, bromine
ion and iodine ion, sulfate ion, alkyl sulfonate ion (e.g., methyl
sulfonate ion), aryl sulfonate ion (e.g., p-toluene sulfonate ion),
substituted aryl sulfonate ion (e.g., anilinonaphthylene sulfonate
ion and various isomers thereof), diphenylantracene sulfonate ion,
perchlorate ion, alkanoate ion (e.g., acetate ion), aryl
carboxylate ion (e.g., fluorescein or fluorescein derivatives),
benzene heterocyclic aryl carboxylate ion (e.g.,
7-diethylamino-4-cyanocoumarin-3-carboxylate ion). Organic dianions
such as p-terephthalate ion may be represented by Y.
[0013] Furthermore, a symbol n represents a number over the range
of about 500 to about 500,000 (average molecular weight),
preferably about 20,000 to about 70,000 when the molecular weight
of such poly(vinylbenzyl quaternary salt) is measured using an
intrinsic viscosity or an LALLS method. Methods of preparing these
polymers where M is nitrogen, related copolymers and related
starting materials are disclosed in Jones, G. D., Journal of
Polymer Science, Vol. 25:201, 1958; U.S. Pat No. 2,780,604, U.S.
Pat. No. 3,178,396, U.S. Pat. No. 3,770,439, U.S. Pat. No.
4,308,335, U.S. Pat. No. 4,340,522, U.S. Pat. No. 4,424,326 and
German published Patent No. 2,447,611. A symbol M may be phosphorus
or sulfur, and here, corresponding phosphonium or sulfonium
polymers are described in prior art (U.S. Pat. No. 3,236,820 and
U.S. Pat. No. 3,065,272).
[0014] As the chemiluminescence enhancer having the structure
denoted by the general formula [I], those selected from
poly[vinylbenzyl(benzylmethyl ammonium chloride)] (BDMQ),
poly(vinylbenzyltrimethyl ammonium chloride) (TMQ),
poly[vinylbenzyl(tributyl ammonium chloride)] (TBQ),
benzylmethylcetyl ammonium chloride (BDMCAC),
polymethacrylamidepropylenemethyl ammonium chloride (poly MAPTAC),
poly[vinylbenzyl(triethyl ammonium chloride)] (TEQ),
poly[vinylbenzyl(2-benzylamino)ethyldimethyl ammonium chloride]
(BAEDM), poly[vinylbenzyldimethyl(2-hydroxy)ethyl ammonium
chloride] (DME(OH)B), poly[vinylbenzyl(trimethylphosphonium
chloride)] (TM), poly[vinylbenzyl(tributylphosphonium chloride)]
(TB) and poly[vinylbenzyl(trioctylphosphonium chloride)] (TO) and
copolymers thereof are suitable.
[0015] The chemiluminescence enhancers of general formula [I] which
is particularly preferable in the invention are those selected from
poly[vinylbenzyl(benzylmethyl ammonium chloride)] (BDMQ),
poly(vinylbenzyltrimethyl ammonium chloride) (TMQ),
poly(vinylbenzyl(tributyl ammonium chloride)] (TBQ),
poly[vinylbenzyl(triethyl ammonium chloride)] (TEQ),
poly[vinylbenzyl(trimethylphosphonium chloride)] (TM),
poly[vinylbenzyl(tributylphosphonium chloride)] (TB) and
poly[vinylbenzyl(trioctylphosphonium chloride)] (TO) and copolymers
thereof, more preferably those selected from
poly(vinylbenzyltrimethyl ammonium chloride) (TMQ),
poly[vinylbenzyl(tributyl ammonium chloride)] (TBQ) and
poly[vinylbenzyl(benzylmethyl ammonium chloride)] (BDMQ),
poly[vinylbenzyl(triethyl ammonium chloride)] (TEQ), and copolymers
thereof, and most preferably poly[vinylbenzyl(tributyl ammonium
chloride)] (TBQ) and poly[vinylbenzyl(benzylmethyl ammonium
chloride)] (BDMQ).
[0016] As described in the following general formula [II], a
copolymer having two or more different onium side chains can be
also utilized in the invention set forth here. The symbols Y, M',
R.sub.5', R.sub.6' and R.sub.7' are the same as defined above Y, M,
R.sub.5, R.sub.6 and R.sub.7. The symbols y and z represent a molar
fraction of an individual monomer which constitutes the copolymer.
Therefore, a sum of the symbols y and z is always equal to 1, and
each of them may vary from 0.01 to 0.99. As a suitable moiety, M is
nitrogen or phosphorus, and R.sub.5 to R.sub.7 are each
independently an alkyl, cycloalkyl, polycycloalkyl (e.g.,
adamantane group), aralkyl or aryl group having 1 to 20 carbon
atoms unsubstituted or further substituted with a hydroxyl, amino,
amide or ureide group, or together form a heterocyclic (in some
cases, aromatic or aliphatic or mixed including other heteroatoms
such as nitrogen, sulfur or oxygen) onium group via a spiro-bond
with an M atom. ##STR2##
[0017] The chemiluminescence reaction itself in which the
chemiluminescence enhancer of the invention is used is known.
Enzymes for performing the chemiluminescence reaction can include
acid phosphatase, alkali phosphatase, galactosidase, glucosidase,
glucuronidase or esterase, and preferable examples can include acid
phosphatase, alkali phosphatase, glucosidase, galactosidase and
esterase. The most preferable is alkali phosphatase. These enzymes
can be purified from animals, plants, bacteria and the like by
methods known publicly, and are also commercially available.
Commercially available articles can be also preferably used. These
enzymes may be in a free state or in a state bound to the other
substance such as an antigen, an antibody and a hapten.
[0018] The substrate of the chemiluminescence reaction used here
can include a dioxetane derivative represented by the following
general formula [III]: ##STR3## wherein R.sub.2 is an aryl group
substituted with an X-oxy group, which forms 1,2-dioxetane compound
which is an unstable oxide intermediate when X is eliminated by an
activator selected from acid, base, salt, enzyme, organic or
inorganic catalyst and electron donor to induce a reaction, and the
unstable 1,2-dioxetane compound is decomposed with releasing
electron energy to produce light and two carbonyl containing
compounds, further, X is a chemically easily reactive group which
is eliminated by an enzyme; R.sub.1 is selected from the group
consisting of an alkyl group, alkoxy group, aryloxy group,
dialkylamino group, trialkylsilyloxy group, arylsilyloxy group,
aryl group, and an aryl group which forms a polycyclic aryl group
of X-oxy substituent spiro-bound to a 1,2-dioxetane ring by binding
to an aryl group R.sub.2; R.sub.3 and R.sub.4 are each an alkyl or
heteroalkyl group and R.sub.3 and R.sub.4 may be bound one another
to form a polycyclic alkylene group spiro-bound to the
1,2-dioxetane ring.
[0019] In general formula [III], when R.sub.1 is not bound to
R.sub.2, this R.sub.1 is an alkyl, alkoxy, aryloxy, dialkylamino,
trialkylsilyloxy, arylsilyloxy or aryl group as described above,
and preferably a lower alkyl or alkoxy group having 1 to 8 carbon
atoms. R.sub.1 may be also an aryl, aryloxy or arylsilyloxy group
having 6 to 20 carbon atoms. When R.sub.1 is bound to R.sub.2 which
is an aryl group to form a polycyclic aryl group spiro-bound to the
1,2-dioxetane ring, it is preferable that the polycyclic aryl group
has carbon atoms up to 30. The polycyclic aryl group in this case
may be one where an oxygen atom is included in place of a carbon
atom as a xanthenyl group, and fluorenyl or xanthenyl where the
spiro-bound polycyclic aryl group is spiro-bound to the
1,2-dioxetane ring at position C9 of the group is preferable.
[0020] R.sub.2 is an aryl group substituted with X-oxy group (OX
group), and the group including the aryl group may be a phenyl,
biphenyl, bound phenyl group or other aryl group, include 6 to 30
carbon atoms, and include another substituent. X is a group
eliminated from dioxetane by the activator in order to decompose a
stable dioxetane structure to produce chemiluminescence (signal).
It is preferred that the OX group is selected from a hydroxyl,
alkylsilyloxy, arylsilyloxy group, an inorganic oxy acid salt
(particularly, phosphate salt or sulfate salt), pyranoside oxygen,
an arylcarboxylester or alkylcarboxylester group. When the OX group
is a hydroxy group, a hydrogen atom of the group is easily reactive
with an organic base such as potassium t-butoxide or an inorganic
base such as potassium hydroxide, and can be decomposed by the base
to produce the chemiluminescence. When the activator is an enzyme
typically frequently used as a label for an immunoassay or a
detection of a DNA probe, the OX group having X which is easily
reactive with the enzyme could be appropriately selected. For
example, when the activator is alkali phosphatase,
.beta.-galactosidase, aryl or acetylcholine esterase, or the like
commonly used as one detected by a colorimetric substrate or a
fluorescent substrate in the immunoassay or the detection of the
DNA probe, a phosphate salt, pyranoside oxygen or an acetate ester
group can be selected as the OX group.
[0021] R.sub.3 or R.sub.4 is each an alkyl group or a heteroalkyl
group, and may be bound one another to form a ring structure and
make a polycyclic alkylene group. The polycyclic alkylene group may
include 6 to 30 carbon atoms, and include heteroatoms (nitrogen,
oxygen, sulfur or phosphorus). The preferable polycyclic alkylene
group is an adamantyl group. R.sub.3 and R.sub.4 bring stability to
the dioxetane structure, and may have a substituent so long as the
substituent does not impair the stability.
[0022] In the compounds having the dioxetane structure as in the
above, the suitable chemiluminescent substrates are
3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.sup.3.7]decane]-4-yl)-
phenyl phosphoric acid and particularly disodium salt thereof
(AMPPD), and
3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.sup.3.7]de-
cane]-4-yl)phenyl phosphoric acid and particularly disodium salt
thereof (CSPD) (see U.S. Pat. No. 4,962,162, and Japanese Patent
No. 2552413).
[0023] The fine solid carriers used in the invention include animal
erythrocytes, gelatin particles, latex particles, magnetic
particles, and the like. The gelatin particle referred to here
indicates a particle composed of water soluble polysaccharides,
sodium metaphosphate and an aldehyde crosslinking agent and the
like in addition to gelatin (see JP 93-306113 A and JP 88-48021 B),
the latex particle indicates a particle composed of a synthetic
resin such as a polystyrene and acrylic resin which is an organic
macromolecule, and is also commercially available. It is also
possible to preferably use commercially available articles.
[0024] The fine solid carriers used in the invention include those
processed to have magnetism as a magnetic particle whose core is an
organic macromolecule and surface has a ferric oxide type ferrite
coating layer. The magnetic particles can efficiently perform B/F
separation utilizing magnetic force (see, Japanese Patents No.
3192149 and No. 2979414). In particular, the magnetic particles
preferably used include, for example, particles where a core is
magnetite and silane is coated (see JP 80-141670 A and JP 75-122997
A), particles where a core is a magnetic metal oxide and silane is
coated (see JP 85-1564 A), magnetic particles where a core is an
organic macromolecular compound, having a ferric oxide type ferrite
coating layer (see Japanese Patent No. 2979414), and further
particles having gelatin on a surface of magnetic particles where a
core is an organic macromolecular compound (see Japanese Patent No.
3192149).
[0025] The fine solid carriers are not particularly limited
thereto, and proper carriers are selected depending on a purpose of
each measurement and a constitution of a kit used. So long as it is
the carrier used in the solid phase immunoassay, the effects of the
invention are exerted by the chemiluminescence enhancer.
[0026] The chemiluminescence enhancer according to the invention
can be prepared by the treatment using the reagent having the
oxidation or reduction property. An oxidizing agent or reducing
agent used here includes ammonium persulfate, sodium periodate,
sodium sulfite, sodium hypochlorite, hydrogen peroxide, sodium
metaperiodate, potassium permanganate, potassium dichromate, and
the like. The particularly preferable oxidizing agent or reducing
agent can include ammonium persulfate, sodium sulfite, sodium
hypochlorite, and sodium metaperiodate, and more preferably can
include sodium sulfite and sodium hypochlorite.
[0027] When the chemiluminescence enhancer according to the
invention is prepared, a chemiluminescence enhancer of the above
general formula [I] or [II] manufactured by the known method or the
chemiluminescence enhancer of a commercially available article is
treated with the above oxidizing or reducing agent under an
appropriate condition. As the treatment condition, by considering
chemical species, molecular weight, concentration and the like of
the chemiluminescence enhancer, it is possible to appropriately
select the reagent concentration, the reaction time, the reaction
temperature, the solvent system used and the like of the oxidizing
agent or the reducing agent. The condition is not particularly
limited, and for example, when the chemiluminescence enhancer is at
an amount of about several g to some hundreds g, the
chemiluminescence enhancer according to the invention can be
obtained by the treatment with 5 mM sodium sulfite at room
temperature for one hour, or 0.1% sodium hypochlorite at room
temperature for two hours, or 1 mM ammonium persulfate and 1 mM
sodium metaperiodate at 60.degree. C. for two hours, or 15%
hydrogen peroxide at room temperature for 17 hours, or 1 mM
potassium permanganate and 1 mM potassium dichromate at 80.degree.
C. for two hours. It is known that sodium sulfite exhibits the
oxidation property or the reduction property depending on the
condition.
[0028] As exemplified in the following Examples, it is desirable
that the chemiluminescence enhancer of the invention is purified
using a dialysis membrane which cuts off ones with and less than
the molecular weight of about 14,000 after the treatment with
sodium hypochlorite. It has been shown that many of the untreated
chemiluminescence enhancers can not pass through a 300,000
molecular weight cut-off ultrafiltration filter whereas the
chemiluminescence enhancer of the invention can pass through it.
Furthermore, it has been found that even in the chemiluminescence
enhancer whose permeability through the filter is low depending on
the condition of oxidation/reduction, the permeability of the
chemiluminescence enhancer of the invention is enhanced by changing
a condition of a dialysis solution.
[0029] As exemplified in the following Examples, it has been shown
that when the chemiluminescence enhancer of the invention is added
to the fine solid carriers, i.e., for example, a dispersion system
of magnetic particles, dispersibility is improved, compared to the
addition of the untreated chemiluminescence enhancer. It is
believed that the favorable dispersibility of such fine solid
carriers brings enhancement and stabilization of luminescence
(signal) by the enzymatic reaction of the chemiluminescent
substrate having dioxetane.
EXAMPLES
[0030] Hereinafter, the invention is concretely explained based on
Examples. However, the invention is not limited to the following
Examples.
Example 1-1
Effect of Untreated TBQ and Oxidized/Reduced TBQ on
Chemiluminescence Measurement
[0031] To 2 ml of TBQ solution (35.2 mg/ml), 2 ml of 2 mM sodium
sulfite solution, 0.015% (effective chlorine concentration) sodium
hypochlorite solution, 2 mM sodium metaperiodate or 2 mM ammonium
persulfate was added, mixed, and subsequently treated at 60.degree.
C. for 4 hours. This treated TBQ was dispensed in a dialysis
membrane (molecular weight cut-off, 12,000 to 14,000, supplied from
Sanko Junyaku Co., Ltd.), and then dialyzed using MilliQ water
(ultrapure water) as an external solution to make the treated TBQ.
Next, 0.4 mg/ml AMPPD solution (0.2 M diethanolamine (DEA), 1 mM
magnesium chloride (MgCl.sub.2), 0.05% sodium azide (NaN.sub.3), pH
10.0) containing 0.8 mg/ml untreated TBQ or treated TBQ was
prepared (substrate solution). Magnetic particles (200 .mu.l)
binding 0.015% alkali phosphatase (ALP) was dispensed in a reaction
vessel, and the particles were attracted to a magnet by putting the
magnet close to the particles to eliminate a supernatant and wash.
To this reaction vessel, 200 .mu.l of the above substrate solution
was added and mixed, reacted at 37.degree. C. for 5 min,
subsequently emission of light(signal) was counted by a photon
counter (supplied from Hamamatsu Photonics K.K.), and an integrated
value for 2 seconds was calculated. The reaction of the above
magnetic particles with the substrate was performed five times.
This result is shown in Table 1. Compared to the untreated TBQ, in
the treated TBQ group, in all treatment conditions, the signal was
increased and repeatability (CV value) was also enhanced.
TABLE-US-00001 TABLE 1 Treated TBQ Ammonium Untreated TBQ Na
sulfite Na hypochlorite Na metaperiodate persulfate Count 1188601
1795546 1900953 1882699 1887294 1144091 1803082 1893218 1880734
1853619 1159564 1760605 1859213 1864943 1855904 1100897 1774889
1881518 1859709 1852409 1164963 1768074 1864585 1845497 1837743
Average 1151623 1780439 1879897 1866716 1857394 CV 2.8% 1.0% 1.0%
0.8% 1.0%
Example 1-2
Effect of Untreated TBQ and Oxidized/Reduced TBQ on Particle
Dispersion
[0032] DEA solution (0.1 M, pH 10.0) containing 0.8 mg/ml untreated
TBQ or treated TBQ was prepared. Then, 100 .mu.l of 0.03% magnetic
particles were dispensed in a reaction vessel, and the particles
were attracted to a magnet by putting the magnet close to the
particles and the supernatant was eliminated. The DEA solution (200
.mu.l) including the above TBQ was added and stirred for 30
seconds. Fifteen seconds after stirring, 150 .mu.l was taken from a
solution surface by a Pipetman (supplied from Gilson, a
micropipette), dispensed in a cell for a spectrophotometer, and
after 10 seconds, a turbidity (OD500) was measured by the
spectrophotometer (UV-1200, supplied from Shimadzu Corporation).
This result is shown in Table 2. Compared to the untreated TBQ, in
the treated TBQ group, the turbidity was higher and the dispersion
of particles was enhanced. TABLE-US-00002 TABLE 2 Treated TBQ
Ammonium Untreated TBQ Na sulfite Na hypochlorite Na metaperiodate
persulfate Turbidity 0.035 0.261 0.368 0.430 0.487
Example 2-1
Effect of Untreated TBQ and Oxidized/Reduced TBQ on
Chemiluminescence Measurement
[0033] To 2 ml of 35.2 mg/ml TBQ solution, 2 ml of a solution
including 0.005%, 0.05%, or 0.5% sodium hypochlorite as an
effective chlorine concentration was added, mixed, and subsequently
left at 25.degree. C. for 24 hours. Next, a mixture is dispensed in
a dialysis membrane (molecular weight cut-off, 12,000 to 14,000
supplied from Sanko Junyaku Co., Ltd.), and subsequently the
dialysis using MilliQ water as an external solution was performed
to make the treated TBQ. Then, 0.2 mg/ml AMPPD solution (0.1 M DEA,
1 mM MgCl.sub.2, 0.05% NaN.sub.3, pH 10.0) containing 0.8 mg/ml
untreated TBQ or treated TBQ was prepared (substrate solution).
Next, 200 .mu.l of 0.015% ALP-binding magnetic particles were
dispensed in a reaction vessel, and the particles were attracted to
a magnet by putting the magnet close to the particles, and the
supernatant was eliminate and washed. Then, 200 .mu.l of the above
substrate solution was added and mixed, reacted at 37.degree. C.
for 5 min, subsequently emission of light (signal) was counted by a
photon counter (supplied from Hamamatsu Photonics K.K.), and an
integrated value for 2 seconds was calculated. This result is shown
in Table 3. Compared to the untreated TBQ, in the treated TBQ
group, in all treatment conditions, the signal was increased and
repeatability (CV value) was also enhanced. TABLE-US-00003 TABLE 3
TBQ treated with sodium hypochlorite 0.005% 0.05% 0.5% Untreated
TBQ treatment treatment treatment Count 1243067 1939907 1874673
1742837 1138533 1951098 1904467 1747014 1185676 1958058 1896528
1742927 1213137 1958925 1928099 1749573 1046363 1944462 1889804
1753686 1198577 1931453 1879883 1738024 Average 1170892 1947317
1895576 1745677 CV 6.0% 0.6% 1.0% 0.3%
Example 2-2
Effect of Untreated TBQ and Oxidized/Reduced TBQ on Particle
Dispersion
[0034] A treated TBQ was obtained by the method described in
Example 2-1. A DEA solution (0.1 M, pH 10.0) containing 0.8 mg/ml
untreated TBQ or treated TBQ was prepared. Then, 100 .mu.l of 0.03%
magnetic particles were dispensed in a reaction vessel, and the
particles were attracted to a magnet by putting the magnet close to
the particles and the supernatant was eliminated. The DEA solution
(200 .mu.l) including the TBQ was added and stirred for 30 seconds.
Fifteen seconds after stirring, 150 .mu.l was taken from a solution
surface by a Pipetman (supplied from Gilson, a micropipette),
dispensed in a cell for a spectrophotometer, and after 10 seconds,
a turbidity (OD500) was measured by the spectrophotometer (UV-1200,
supplied from Shimadzu Corporation). This result is shown in Table
4. Compared to the untreated TBQ, in the treated TBQ group, the
turbidity was higher and the dispersion of particles was enhanced.
TABLE-US-00004 TABLE 4 TBQ treated with sodium hypochlorite 0.005%
0.05% 0.5% Untreated TBQ treatment treatment treatment Turbidity
0.035 0.314 0.385 0.510
Example 3
Ultrafiltration Filter Permeability of Untreated TBQ and
Oxidized/Reduced TBQ
[0035] The untreated TBQ and the treated TBQ were diluted to 1
mg/ml or less with 0.1 M or 1 M sodium chloride (NaCl). This
solution was ultrafiltrated through a 300,000 molecular weight
cut-off ultrafiltration filter (supplied from Millipore), an
absorbance (OD268) of a filtrate was measured, and a permeability
was calculated. The result is shown in Table 5. Compared to the
untreated TBQ, the filter permeability of the treated TBQ was
increased. For TBQ treated with 0.005% sodium hypochlorite whose
permeability was low in 0.1 M NaCl solution, when dissolved in 1 M
NaCl solution, its filter permeability was enhanced. TABLE-US-00005
TABLE 5 TBQ treated with sodium hypochlorite 0.005% 0.05% 0.5%
Untreated TBQ treatment treatment treatment Filter 0.1 M NaCl
solution 3% 31% 94% 95% permeability 1 M NaCl solution 7% 96%
Example 4
Effect of Untreated TBQ and Oxidized/Reduced TBQ on
Chemiluminescence Immunoassay (1)
[0036] To 2 ml of 35.2 mg/ml TBQ, 2 ml of 2 M sodium sulfite
solution was added, mixed, and then treated at 60.degree. C. for 4
hours. Next, this was dispensed in a dialysis membrane (molecular
weight cut-off, 12,000 to 14,000, supplied from Sanko Junyaku Co.,
Ltd.), and the dialysis was performed using MilliQ water as an
external solution to make the treated TBQ. Next, 0.02 mg/ml AMPPD
solution (0.1 M DEA, 1 mM MgCl.sub.2, 0.05% NaN.sub.3, pH 10.0)
containing 0.8 mg/ml untreated TBQ or treated TBQ was prepared
(substrate solution). Test samples containing 0, 10, 100, 800 and
2000 ng/ml of a fetoprotein (AFP) were diluted to 20 times with a
BSA solution. Each measurement sample (20 .mu.l) was added into a
reaction vessel in which 50 .mu.l of 0.03% anti-AFP
antibody-binding magnetic particles were placed, mixed, and reacted
at 37.degree. C. for 8 min. Subsequently, the particles were
attracted to a magnet by putting the magnet close to the reaction
vessel to eliminate a supernatant and wash. Then, 50 .mu.l of 0.1
.mu.g/ml ALP-conjugating anti-AFP antibody solution was added,
mixed, and reacted at 37.degree. C. for 8 min. After the reaction,
the particles were attracted to a magnet by putting the magnet
close to the reaction vessel, the supernatant was eliminated and
washed. The above substrate solution (200 .mu.l) was added to these
particles, mixed, and reacted at 37.degree. C. for 4 min.
Subsequently, emission of light (signal) was counted by a photon
counter (supplied from Hamamatsu Photonics K.K.), and an integrated
value for two seconds was calculated. The result is shown in Table
6. Compared to the cases of the untreated TBQ, repeatability (CV
value) using the TBQ treated with sodium sulfite was enhanced.
[0037] [Table 6] TABLE-US-00006 TABLE 6 AFP concentration (ng/ml) 0
10 100 800 2000 Untreated TBQ Count 737 22413 190157 1204985
2201469 689 25275 205970 874704 1396455 822 30756 199645 932275
2335765 824 30605 205809 816211 2214843 859 33443 189985 946657
1430978 853 22297 196053 1064297 1374801 Average 797 27465 197937
973188 1825719 CV 8.6% 17.3% 3.6% 14.4% 25.6% TBQ treated with
sodium sulfite Count 354 17298 165651 1230392 2391362 380 17595
166482 1236687 2514891 370 17634 167006 1245664 2397218 385 17515
167636 1192276 2515427 381 17557 166714 1191075 2442702 371 16806
164900 1216407 2540877 Average 374 17401 166398 1218750 2467080 CV
3.0% 1.8% 0.6% 1.9% 2.6%
Example 5
Effect of untreated TBQ and Oxidized/Reduced TBQ on
Chemiluminescence Immunoassay (2)
[0038] To 300 ml of 35.2 mg/ml TBQ solution, 15 ml of 1 M sodium
sulfite-HCl solution (pH 6.0) was added, mixed and reacted at
25.degree. C. for 24 hours. Next, this was dispensed in a dialysis
membrane (molecular weight cut-off, 12,000 to 14,000, supplied from
Sanko Junyaku Co., Ltd.), and the dialysis was performed using
MilliQ water as an external solution to make the treated TBQ. Next,
0.2 mg/ml AMPPD solution (0.1 M DEA, 1 mM MgCl.sub.2, 0.05%
NaN.sub.3, pH 10.0) containing 0.8 mg/ml untreated TBQ or treated
TBQ was prepared (substrate solution). As with the above Example 4,
using .alpha.-fetoprotein as a test sample, an immunoassay was
performed, and chemiluminescence (signal) was counted to perform a
measurement. The result is shown in Table 7. Compared to the cases
of the untreated TBQ, repeatability (CV value) using the TBQ
treated with sodium sulfite was enhanced. TABLE-US-00007 TABLE 7
AFP concentration (ng/ml) 0 10 100 800 2000 Untreated TBQ Count 549
37542 188792 1064676 1714369 560 38510 219614 908606 1985371 609
41285 212975 958981 1866508 551 38169 207767 1051242 1464529 505
41844 232717 983756 1498278 472 27779 211984 1054501 1684947
Average 541 37522 212308 1003627 1702334 CV 8.8% 13.5% 6.8% 6.3%
11.9% TBQ treated with sodium sulfite Count 374 27079 234281
1140508 2163595 380 25361 233081 1160725 1925163 362 27141 220616
1153611 2031717 363 25705 228368 1117495 2060136 426 27389 221167
1104866 1978776 399 24851 227564 1130180 2036848 Average 384 26254
227513 1134564 2032706 CV 6.4% 4.1% 2.5% 1.9% 4.0%
Example 6
Effect of untreated BDMQ and Oxidized/Reduced BDMQ on
Chemiluminescence Immunoassay
[0039] To 10 ml of BDMQ solution (25.4 mg/ml), 0.5 ml of 1 M sodium
sulfite-HCl solution (pH 6.0) was added, mixed and reacted at
25.degree. C. for 24 hours. Next, this was dispensed in a dialysis
membrane (molecular weight cut-off, 12,000 to 14,000, supplied from
Sanko Junyaku Co., Ltd.), and the dialysis was performed using
MilliQ water as an external solution to make a treated BDMQ.
[0040] Next, 0.2 mg/ml AMPPD solution [0.1 M diethanolamine (DEA),
1 mM magnesium chloride (MgCl.sub.2), 0.05% sodium azide
(NaN.sub.3), pH 10.0] containing 0.4 mg/ml untreated BDMQ or
treated BDMQ was prepared (substrate solution).
[0041] Test samples containing 0, 10, 100, 800 and 2000 ng/ml of
.alpha. fetoprotein (AFP) were diluted to 10 times with a BSA
solution. Each measurement sample (20 .mu.l) was added into a
reaction vessel in which 250 .mu.l of 0.015% anti-AFP
antibody-binding magnetic particles were placed, mixed, and reacted
at 37.degree. C. for 10 min. Subsequently, the particles were
attracted to a magnet by putting the magnet close to the reaction
vessel to eliminate a supernatant and wash. Then, 250 .mu.l of 0.1
.mu.g/ml ALP-conjugating anti-AFP antibody was added, mixed, and
reacted at 37.degree. C. for 10 min. After the reaction, the
particles were attracted to a magnet by putting the magnet close to
the reaction vessel, the supernatant was eliminated and washed. The
above substrate solution (200 .mu.l) was added to these particles,
mixed, and reacted at 37.degree. C. for 5 min. Subsequently,
chemiluminescence (signal) was counted by a photon counter
(supplied from Hamamatsu Photonics K.K.), and an integrated value
for two seconds was calculated. The result is shown in Table 8.
Compared to the cases of the untreated BDMQ, repeatability (CV
value) using the BDMQ treated with sodium sulfite was enhanced.
TABLE-US-00008 TABLE 8 AFP concentration (ng/ml) 0 10 100 800 2000
Untreated BDMQ Count 235 29915 200489 860407 1600060 242 30126
203080 904037 1735060 222 29949 198255 891405 1759976 242 29466
208122 935003 1512594 Average 235 29864 202487 897713 1651923 CV
4.00% 0.90% 2.10% 3.40% 7.10% BDMQ treated with sodium sulfite
Count 201 24969 198765 1212549 2354980 212 25586 195978 1210283
2371754 207 25043 196434 1213840 2339963 184 25086 195111 1219413
2371016 Average 201 29864 202487 897713 1651923 CV 6.10% 0.90%
0.80% 0.40% 0.90%
Example 7
Effects of Untreated BDMQ and Oxidized/Reduced BDMQ on Particle
Dispersion
[0042] To 10 ml of BDSMQ solution (25.4 mg/ml), 0.5 ml of 1 M
sodium sulfite-HCl solution (pH 6.0) was added, mixed and reacted
at 25.degree. C. for 24 hours. Next, this was dispensed in a
dialysis membrane (molecular weight cut-off, 12,000 to 14,000,
supplied from Sanko Junyaku Co., Ltd.), and the dialysis was
performed using MilliQ water as an external solution to make the
treated BDMQ.
[0043] Next, 0.1 M DEA (pH 10.0) containing 0.4 mg/ml untreated
BDMQ or treated BDMQ was prepared. Then, 100 .mu.l of 0.03%
magnetic particles were dispensed to a reaction vessel, and the
particles were attracted to a magnet by putting the magnet close to
the particles, and the supernatant was eliminated. The DEA solution
(200 .mu.l) including the above BDMQ was added and stirred for 30
seconds. Fifteen seconds after stirring, 150 .mu.l was taken from a
solution surface by a Pipetman (supplied from Gilson, a
micropipette), dispensed in a cell for a spectrophotometer, and
after 10 seconds, a turbidity (OD500) was measured by the
spectrophotometer (UV-1200, supplied from Shimadzu Corporation.
This result is shown in Table 9. Compared to the untreated BDMQ, in
the treated BDMQ group, the turbidity was higher and the dispersion
of particles was enhanced. TABLE-US-00009 TABLE 9 Untreated BDMQ
Treated BDMQ Turbidity 0.045 0.339
EFFECTS OF THE INVENTION
[0044] As in the above, by treating with the oxidizing agent or the
reducing agent, the chemiluminescence enhancer has been provided,
which is used for the signal detection in the solid immunoassay
using the antigen or/and the antibody immobilized onto fine solid
carriers dispersible in the liquid medium, improves the
dispersibility of the fine solid carriers compared to conventional
chemiluminescence enhancers, and is excellent in enhancement of
chemiluminescence by the enzymatic reaction of the chemiluminescent
substrate having dioxetane. Also, the chemiluminescence method and
the kit using the chemiluminescence enhancer have been provided.
When using the chemiluminescence enhancer of the invention, the
within-run reproducibility of the measurement values is enhanced
and more precise quantification becomes possible.
* * * * *