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Docling/tests/data/uspto/pftaps057006474.txt
Cesar Berrospi Ramis 4e087504cc
feat: create a backend to parse USPTO patents into DoclingDocument (#606) 
* feat: add PATENT_USPTO as input format

Signed-off-by: Cesar Berrospi Ramis <ceb@zurich.ibm.com>

* feat: add USPTO backend parser

Add a backend implementation to parse patent applications and
grants from the United States Patent Office (USPTO).

Signed-off-by: Cesar Berrospi Ramis <75900930+ceberam@users.noreply.github.com>

* refactor: change the name of the USPTO input format

Change the name of the patent USPTO input format to show the typical format (XML).

Signed-off-by: Cesar Berrospi Ramis <75900930+ceberam@users.noreply.github.com>

* refactor: address several input formats with same mime type

Signed-off-by: Cesar Berrospi Ramis <75900930+ceberam@users.noreply.github.com>

* refactor: group XML backend parsers in a subfolder

Signed-off-by: Cesar Berrospi Ramis <75900930+ceberam@users.noreply.github.com>

* chore: add safe initialization of PatentUsptoDocumentBackend

Signed-off-by: Cesar Berrospi Ramis <75900930+ceberam@users.noreply.github.com>

---------

Signed-off-by: Cesar Berrospi Ramis <ceb@zurich.ibm.com>
Signed-off-by: Cesar Berrospi Ramis <75900930+ceberam@users.noreply.github.com>
2024-12-17 16:35:23 +01:00

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PATN
WKU 057006474
SRC 8
APN 5686806
APT 1
ART 189
APD 19951207
TTL Carbocation containing cyanine-type dye
ISD 19971223
NCL 20
ECL 1
EXP Houtteman; Scott W.
NDR 1
NFG 1
INVT
NAM Miyazaki; Takeshi
CTY Ebina
CNT JPX
INVT
NAM Tanaka; Kazumi
CTY Yokohama
CNT JPX
INVT
NAM Santo; Tsuyoshi
CTY Yokohama
CNT JPX
INVT
NAM Ohnishi; Toshikazu
CTY Machida
CNT JPX
INVT
NAM Fukui; Tetsuro
CTY Kawasaki
CNT JPX
INVT
NAM Okamoto; Tadashi
CTY Yokohama
CNT JPX
ASSG
NAM Canon Kabushiki Kaisha
CTY Tokyo
CNT JPX
COD 03
PRIR
CNT JPX
APD 19910621
APN 3-150428
PRIR
CNT JPX
APD 19911028
APN 3-281645
PRIR
CNT JPX
APD 19920610
APN 4-150665
RLAP
COD 74
APN 900302
APD 19920618
PSC 01
PNO 5512446
CLAS
OCL 435 6
XCL 436139
XCL 430 93
XCL 585 16
XCL 585406
XCL 260350
EDF 6
ICL C12Q 168
FSC 435
FSS 6
FSC 436
FSS 139;63;56
FSC 430
FSS 93
FSC 585
FSS 16;406
FSC 260
FSS 350
UREF
PNO 3770383
ISD 19731100
NAM Price
OCL 436509
UREF
PNO 3789116
ISD 19740100
NAM Kay
OCL 436800
UREF
PNO 4738908
ISD 19880400
NAM Oguchi et al.
OCL 430 20
UREF
PNO 5112960
ISD 19920500
NAM Bronstein et al.
OCL 536 18.1
FREF
PNO 2191674
ISD 19900700
CNT JPX
OREF
PAL K. Sauda et al., "Determination of Protein in Human Serum by
High-Performance Liquid Chromatography with Semiconductor Laser
Fluorometric Detection," Analytical Chemistry, vol. 58, No. 13, Nov. 1986,
pp. 2649-2653.
PAL Derwent Abstract Accession No. 91-068399/10 (1991).
PAL Smith, et al., Nature, vol. 321 (1986), pp. 674-679.
PAL Wingrove, Organic Chemistry, pub. by Harper & Row, New York, pp. 163-166,
1981.
PAL Mujumdar et al., Cytometry 10:11-19, 1989.
LREP
FRM Fitzpatrick, Cella, Harper & Scinto
ABST
PAL To provide a reagent with excellent stability under storage, which can
detect a subject compound to be measured with higher specificity and
sensitibity.
PAL Complexes of a compound represented by the general formula (IV):
##STR1##
PARN
PAR This application is a division of application Ser. No. 07/900,302 filed
Jun. 18, 1992, now U.S. Pat. No. 5,512,446.
BSUM
PAC BACKGROUND OF THE INVENTION
PAR 1. Field of the Invention
PAR The present invention relates to a labeled complex for microassay using
near-infrared radiation. More specifically, the present invention relates
to a labeled complex capable of specifically detecting a certain
particular component in a complex mixture with a higher sensitivity.
PAR 2. Related Background Art
PAR On irradiating a laser beam on a trace substance labeled with dyes and the
like, information due to the substance is generated such as scattered
light, absorption light, fluorescent light and furthermore light
acoustics. It is widely known in the field of analysis using lasers, to
detect such information so as to practice microassays rapidly with a
higher precision.
PAR A gas laser represented by an argon laser and a helium laser has
conventionally been used exclusively as a laser source. In recent years,
however, a semi-conductor laser has been developed, and based on the
characteristic features thereof such as inexpensive cost, small scale and
easy output control, it is now desired to use the semiconductor laser as a
light source.
PAR If diagnostically useful substances from living organisms are assayed by
means of the wave-length in ultraviolet and visible regions as has
conventionally been used, the background (blank) via the intrinsic
fluorescence of naturally occurring products, such as flavin, pyridine
coenzyme and serum proteins, which are generally contained in samples, is
likely to increase. Only if a light source in a near-infrared region can
be used, such background from naturally occurring products can be
eliminated so that the sensitivity to substances to be measured might be
enhanced, consequently.
PAR However, the oscillation wavelength of a semiconductor laser is generally
in red and near-infrared regions (670 to 830 nm), where not too many dyes
generate fluorescence via absorption or excitation. A representative
example of such dyes is polymethine-type dye having a longer conjugated
chain. Examples of labeling substances from living organisms with a
polymethine-type dye and using the labeled substances for microanalysis
are reported by K. Sauda, T. Imasaka, et al. in the report in Anal. Chem.,
58, 2649-2653 (1986), such that plasma protein is labeled with a cyanine
dye having a sulfonate group (for example, Indocyanine Green) for the
analysis by high-performance liquid chromatography.
PAR Japanese Patent Application Laid-open No. 2-191674 discloses that various
cyanine dyes having sulfonic acid groups or sulfonate groups are used for
labeling substances from living organisms and for detecting the
fluorescence.
PAR However, these known cyanine dyes emitting fluorescence via absorption or
excitation in the near-infrared region are generally not particularly
stable under light or heat.
PAR If the dyes are used as labeling agents and bonded to substances from
living organisms such as antibodies for preparing complexes, the complexes
are likely to be oxidized easily by environmental factors such as light,
heat, moisture, atmospheric oxygen and the like or to be subjected to
modification such as generating cross-links. Particularly in water, a
modification such as hydrolysis is further accelerated, disadvantageously.
Therefore, the practical use of these complexes as detecting reagents in
carrying out the microassay of the components of living organisms has
encountered difficulties because of their poor stability under storage.
PAC SUMMARY OF THE INVENTION
PAR The present inventors have made various investigations so as to solve the
above problems, and have found that a dye of a particular structure, more
specifically a particular polymethine dye, and among others, a dye having
an azulene skelton, are extremely stable even after the immobilization
thereof as a labeling agent onto substances from living organisms. Thus,
the inventors have achieved the present invention. It is an object of the
present invention to provide a labeled complex with excellent storage
stability which can overcome the above problems.
PAR According to an aspect of the present invention, there is provided a
labeled complex for detecting a subject compound to be analyzed by means
of optical means using near-infrared radiation which complex comprises a
substance from a living organism and a labeling agent fixed onto the
substance and is bonded to the subject compound to be analyzed, wherein
the labeling agent comprises a compound represented by the general formula
(I), (II) or (III):
##STR2##
wherein R.sub.1 through R.sub.7 are independently selected from the group
consisting of hydrogen atom, halogen atom, alkyl group, aryl group,
aralkyl group, sulfonate group, amino group, styryl group, nitro group,
hydroxyl group, carboxyl group, cyano group, or arylazo group; R.sub.1
through R.sub.7 may be bonded to each other to form a substituted or an
unsubstituted condensed ring; R.sub.1 represents a divalent organic
residue; and X.sub.1.sup..crclbar. represents an anion;
##STR3##
wherein R.sub.8 through R14 are independently selected from the group
consisting of hydrogen atom, halogen atom, alkyl group, aryl group,
aralkyl group, sulfonate group, amino group, styryl group, nitro group,
hydroxyl group, carboxyl group, cyano group, or arylazo group; R.sub.8
through R14 may be bonded to each other to form a substituted or an
unsubstituted condensed ring; and R.sub.A represents a divalent organic
residue;
##STR4##
wherein R.sub.15 through R.sub.21 are independently selected from the
group consisting of hydrogen atom, halogen atom, alkyl group, aryl group,
a substituted or an unsubstituted aralkyl group, a substituted or an
unsubstituted amino group, a substituted or an unsubstituted styryl group,
nitro group, sulfonate group, hydroxyl group, carboxyl group, cyano group,
or arylazo group; R.sub.15 through R.sub.21 may or may not be bonded to
each other to form a substituted or an unsubstituted condensed ring;
R.sub.B represents a divalent organic residue; and X.sub.1.sup..crclbar.
represents an anion.
PAR According to another aspect of the present invention, there is provided a
labeled complex for detecting a subject compound to be analyzed by means
of optical means using near-infrared radiation which complex comprises a
substance from a living organism and a labeling agent fixed onto the
substance and is bonded to the subject compound to be analyzed, wherein
the labeling agent comprises a compound represented by the general formula
(IV):
##STR5##
wherein A, B, D and E are independently selected from the group consisting
of hydrogen atom, a substituted or an unsubstituted alkyl group having two
or more carbon atoms, alkenyl group, aralkyl group, aryl group, styryl
group and heterocyclic group; r.sub.1 ' and r.sub.2 ' are individually
selected from the group consisting of hydrogen atom, a substituted or an
unsubstituted alkyl group, cyclic alkyl group, alkenyl group, aralkyl
group and aryl group; k is 0 or 1; 1 is 0, 1 or 2; and
X.sub.2.sup..crclbar. represents an anion.
PAR According to another aspect of the present invention, there is provided a
method of detecting a subject compound to be analyzed by means of optical
means which method comprises using a labeled complex comprised of a
substance from a living organism and a labeling agent fixed onto the
substance and bonding the complex to the subject compound to be analyzed,
wherein the labeling agent comprises a compound represented by the general
formula (I), (II) or (III).
PAR According to still another aspect of the present invention, there is
provided a method of detecting a subject compound to be analyzed by means
of optical means which method comprises using a labeled complex comprised
of a substance from a living organism and a labeling agent fixed onto the
substance and bonding the complex to the subject compound to be analyzed,
wherein the labeling agent comprises a compound represented by the general
formula (iv).
DRWD
PAC BRIEF DESCRIPTION OF THE DRAWINGS
PAR FIG. 1 depicts one example of fluorescence emitting wave form of a labeling
agent.
DETD
PAC DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
PAR The present invention will now be explained in detail hereinbelow.
PAR In accordance with the present invention, the compound of the general
formula (I), (II) or (III) is employed as a labeling agent, wherein
R.sub.1 to R.sub.21 individually represent hydrogen atom, halogen atom
(chlorine atom, bromine atom, and iodine atom) or a monovalent organic
residue, and other such functional groups described above. The monovalent
organic residue can be selected from a wide variety of such residues.
PAR The alkyl group is preferably in straight chain or branched chain, having a
carbon number of 1 to 12, such as for example methyl group, ethyl group,
n-propyl group, iso-propyl group, n-butyl group, sec-butyl group,
iso-butyl group, t-butyl group, n-amyl group, t-amyl group, n-hexyl group,
n-octyl group, t-octyl group and the like.
PAR The aryl group preferably has a carbon number of 6 to 20, such as for
example phenyl group, naphthyl group, methoxyphenyl group,
diethylaminophenyl group, dimethylaminophenyl group and the like.
PAR The substituted aralkyl group preferably has a carbon number of 7 to 19,
such as for example carboxybenzyl group, sulfobenzyl group, hydroxybenzyl
group and the like.
PAR The unsubstituted aralkyl group preferably has a carbon number of 7 to 19,
such as for example benzyl group, phenethyl group, .alpha.-naphthylmethyl
group, .beta.-naphthylmethyl group and the like.
PAR The substituted or unsubstituted amino group preferably has a carbon number
of 10 or less, such as for example amino group, dimethylamino group,
diethylamino group, dipropylamino group, acetylamino group, benzoylamino
group and the like.
PAR The substituted or unsubstituted styryl group preferably has a carbon
number of 8 to 14, such as for example styryl group, dimethylaminostyryl
group, diethylaminostyryl group, dipropylaminostyryl group, methoxystyryl
group, ethoxystyryl group, methylstyryl group and the like.
PAR The aryl azo group preferably has a carbon number of 6 to 14, such as for
example phenylazo group, .alpha.-naphthylazo group, .beta.-naphthylazo
group, dimethylaminophenylazo group, chlorophenylazo group, nitrophenylazo
group, methoxyphenylazo group and the like.
PAR Of the combinations of R.sub.1 and R.sub.2, R.sub.2 and R.sub.3, R.sub.3
and R.sub.4, R.sub.4 and R.sub.5, R.sub.5 and R.sub.6, and R.sub.6 and
R.sub.7 of the general formula (I), at least one combination may form a
substituted or an unsubstituted condensed ring. The condensed ring may be
five, six or seven membered, including aromatic ring (benzene,
naphthalene, chlorobenzene, bromobenzene, methyl benzene, ethyl benzene,
methoxybenzene, ethoxybenzene and the like); heterocyclic ring (furan
ring, benzofuran ring, pyrrole ring, thiophene ring, pyridine ring,
quinoline ring, thiazole ring and the like); and aliphatic ring
(dimethylene, trimethylene, tetramethylene and the like). This is the case
with the general formulas (II) and (III).
PAR For the general formula (II), at least one combination among the
combinations of R.sub.8 and R.sub.9, R.sub.9 and R.sub.10, R.sub.10 and
R.sub.11, R.sub.11 and R.sub.12, R.sub.12 and R.sub.13, and R.sub.13 and
R.sub.14, may form a substituted or an unsubstituted condensed ring.
PAR Also for the general formula (III), at least one combination of the
combinations of R.sub.15 and R.sub.16, R.sub.16 and R.sub.17, R.sub.17 and
R.sub.18, R.sub.18 and R.sub.19, R.sub.19 and R.sub.20, and R.sub.20 and
R.sub.21, may form a substituted or an unsubstituted condensed ring.
PAR In the general formulas (I) to (IV) described above, the general formula
(I) is specifically preferable; preference is also given individually to
hydrogen atom, alkyl group and sulfonate group in the case of R.sub.1 to
R.sub.7 ; hydrogen atom, alkyl group and sulfonate group in the case of
R.sub.8 to R.sub.14 ; hydrogen atom, alkyl group and sulfonate group in
the case of R.sub.15 to R.sub.21 ; alkyl group and aryl group in the case
of A, B, D and E; hydrogen atom and alkyl group in the case Of r.sub.1 '
to r.sub.2 '.
PAR In the general formula (I), R represents a divalent organic residue bonded
via a double bond. Specific examples of a compound containing such R to be
used in the present invention, include those represented by the following
general formulas (1) to (12), wherein Q.sup..sym. represents the following
azulenium salt nucleus and the right side excluding Q.sup..sym.
represents R.
##STR6##
wherein the relation between the azulenium salt nucleus represented by
Q.sup..crclbar. and the azulene salt nucleus on the right side in the
formula (3) may be symmetric or asymmetric.
##STR7##
In the above formulas (1) to (12) as in the case of R.sub.1 to R.sub.7,
R.sub.1 ' to R.sub.7 ' and R.sub.1 " to R.sub.7 " independently represent
hydrogen atom, halogen atom, alkyl group, aryl group, aralkyl group, amino
group, styryl group, nitro group, hydroxyl group, carboxyl group, cyano
group or aryl azo group, while R.sub.1 ' to R.sub.7 ' and R.sub.1 " to
R.sub.7 " independently may form a substituted or an unsubstituted
condensed ring; n is 0, 1 or 2; r is an integer of 1 to 8; S represents 0
or 1; and t represents 1 or 2.
PAR M.sub.2 represents a non-metallic atom group required for the completion of
a nitrogen-containing heterocyclic ring.
PAR Specific examples of M.sub.2 are atom groups required for the completion of
a nitrogen-containing heterocyclic ring, including pyridine, thiazole,
benzothiazole, naphthothiazole, oxazole, benzoxazole, naphthoxazole,
imidazole, benzimidazole, naphthoimidazole, 2-quinoline, 4-quinoline,
isoquinoline or indole, and may be substituted by halogen atom (chlorine
atom, bromine atom, iodine atom and the like), alkyl group (methyl, ethyl,
propyl, butyl and the like), aryl group (phenyl, tolyl, xylyl and the
like), and aralkyl (benzene, p-trimethyl, and the like).
PAR R.sub.22 represents hydrogen atom, nitro group, sulfonate group, cyano
group, alkyl group (methyl, ethyl, propyl, butyl and the like), or aryl
group (phenyl, tolyl, xylyl and the like). R.sub.23 represents alkyl group
(methyl, ethyl, propyl, butyl and the like), a substituted alkyl group
(2-hydroxyethyl, 2-methoxyethyl, 2-ethoxyethyl, 3-hydroxypropyl,
3-methoxypropyl, 3-ethoxypropyl, 3-chloropropyl, 3-bromopropyl,
3-carboxylpropyl and the like ), a cyclic alkyl group (cyclohexyl,
cyclopropyl), aryl aralkyl group (benzene, 2-phenylethyl, 3-phenylpropyl,
3-phenylbutyl, 4-phenylbutyl, .alpha.-naphthylmethyl,
.beta.-naphthylmethyl), a substituted aralkyl group (methylbenzyl,
ethylbenzyl, dimethylbenzyl, trimethylbenzyl, chlorobenzyl, bromobenzyl
and the like), aryl group (phenyl, tolyl, xylyl, .alpha.-naphtyl,
.beta.-naphthyl) or a substituted aryl group (chlorophenyl,
dichlorophenyl, trichlorophenyl, ethylphenyl, methoxydiphenyl,
dimethoxyphenyl, aminophenyl, sulfonate phenyl, nitrophenyl, hydroxyphenyl
and the like).
PAR R.sub.24 represents a substituted or an unsubstituted aryl group or the
cation group thereof, specifically including a substituted or an
unsubstituted aryl group (phenyl, tolyl, xylyl, biphenyl, aminophenyl,
.alpha.-naphthyl, .beta.-napthyl, anthranyl, pyrenyl, methoxyphenyl,
dimethoxyphenyl, trimethoxyphenyl, ethoxyphenyl, diethoxyphenyl,
chlorophenyl, dichlorophenyl, trichlorophenyl, bromophenyl, dibromophenyl,
tribromophenyl, ethylphenyl, diethylphenyl, nitrophenyl, aminophenyl,
dimethylaminophenyl, diethylaminophenyl, dibenzylaminophenyl,
dipropylaminophenyl, morpholinophenyl, piperidinylphenyl,
piperidinophenyl, diphenylaminophenyl, acetylaminophenyl,
benzoylaminophenyl, acetylphenyl, benzoylphenyl, cyanophenyl, sulfonate
phenyl, carboxylate phenyl and the like).
PAR R.sub.25 represents a heterocyclic ring or the cation group thereof,
specifically including a monovalent heterocyclic ring derived from cyclic
rings, such as furan, thiophene, benzofuran, thionaphthene, dibenzofuran,
carbazole, phenothiazine phenoxazine, pyridine and the like.
PAR R.sub.26 represents hydrogen atom, alkyl group (methyl, ethyl, propyl,
butyl and the like), or a substituted or an unsubstituted aryl group
(phenyl, tolyl, xylyl, biphenyl, ethylphenyl, chlorophenyl, methoxyphenyl,
ethoxyphenyl, nitrophenyl, aminophenyl, dimethylaminophenyl,
diethylaminophenyl, acetylaminophenyl, .alpha.-naphthyl, .beta.-naphthyl,
anthraryl, pyrenyl, sulfonate phenyl, carboxylate phenyl and the like. In
the formula, Z.sub.7 represents an atom group required for the completion
of pyran, thiapyran, selenapyran, telluropyran, benzopyran,
benzothiapyran, benzoselenapyran, benzotelluropyran, naphthopyran,
naphthothiapyran, or naphthoselenapyran, or naphthotelluropyran.
PAR L.sub.7 represents sulfur atom, oxygen atom or selenium atom or tellurium
atom.
PAR R.sub.27 and R.sub.28 individually represent hydrogen atom, alkoxy group, a
substituted or an unsubstituted aryl group, alkenyl group and a
heterocyclic group,
PAR More specifically, R.sub.27 and R.sub.28 individually represent hydrogen
atom, alkyl group (methyl, ethyl, propyl, butyl and the like), alkyl
sulfonate group, alkoxyl group (methoxy, ethoxy, propoxy, ethoxyethyl,
methoxyethyl and the like), aryl group (phenyl, tolyl, xylyl, sulfonate
phenyl, chlorophenyl, biphenyl, methoxyphenyl and the like), a substituted
or an unsubstituted styryl group (styryl, p-methylstyryl, o-chlorostyryl,
p-methoxystyryl and the like), a substituted or an unsubstituted 4-phenyl,
1,3-butadienyl group (r-phenyl, 1,3-butadienyl, 4-(p-methylphenyl),
1,3-butadienyl and the like), or a substituted or an unsubstituted
heterocyclic group (quinolyl, pyridyl, carbazoyl, furyl and the like).
PAR As in the case of R, the same is true with R.sub.A and R.sub.B of the
general formulas (II) and (III), respectively.
PAR Then, in R, the symbols R.sub.8 ' to R.sub.14 ' individually correspond to
R.sub.1 ' to R.sub.7 '; R.sub.8 " to R.sub.14 " individually correspond to
R.sub.1 " to R.sub.7 "; in R.sub.B, R.sub.14 ' to R.sub.21 " individually
correspond to R.sub.1 ' to R.sub.7 '; R.sub.14 " to R.sub.21 "
individually correspond to R.sub.1 " to R.sub.7 ".
PAR In the azulenium nucleus of the (1) to (12), described above, those
represented by the formulas (3), (9) and (10) are more preferably used;
and particularly, the formula (3) is preferable.
PAR R.sub.1 to R.sub.28, R.sub.1 ' to R.sub.21 ' and R.sub.1 " to R.sub.21 "
preferably contain one or more well-known polar groups in order to impart
water solubility to a compound (labeling agent) represented by the general
formula (I), (II) or (III). The polar groups include, for example,
hydroxyl group, alkylhydroxyl group, sulfonate group, alkylsulfonate
group, carboxylate group, alkylcarboxylate group, tetra-ammonium base and
the like. R.sub.1 to R.sub.28, R.sub.1 ' to R.sub.21 ', and R.sub.1 " to
R.sub.21 " preferably contain one or more well-known reactive groups in
order that the compound of the general formula (I) can form a covalent
bond with a substance from a living organism.
PAR The reactive groups include the reactive sites of isocyanate,
isothiocyanate, succinimide ester, sulfosuccinimide ester, imide ester,
hydrazine, nitroaryl halide, piperidine disulfide, maleimide,
thiophthalimide, acid halide, sulfonyl halide, aziridine, azide
nitrophenyl, azide amino, 3-(2-pyridyldithio) propionamide and the like.
In these reactive sites, the following spacer groups
##STR8##
(n=0, 1 to 6) may be interposed in order to prevent steric hindrance
during on the bonding of a labeling agent and a substance from a living
organism.
PAR Preferable such reactive groups include isothiocyanate, sulfosuccinimide
ester, succinimide ester maleimide and the like X.sub.1.sup..sym.
represents an anion, including chloride ion, bromide ion, iodide ion,
perchlorate ion, benzenesulfonate ion, p-toluene sulfonate ion,
methylsulfate ion, ethylsulfate ion, propylsulfate ion, tetrafluoroborate
ion, tetraphenylborate ion, hexafluorophosphate ion, benzenesulfinic acid
salt ion, acetate ion, trifluoroacetate ion, propionate ion, benzoate ion,
oxalate ion, succinate ion, malonate ion, oleate ion, stearate ion,
citrate ion, monohydrogen diphosphate ion, dihydrogen monophosphate ion,
pentachlorostannate ion, chlorosulfonate ion, fluorosulfonate ion,
trifluoromethane sulfonate ion, hexafluoroantimonate ion, molybdate ion,
tungstate ion, titanate ion, zirconate ion and the like.
PAR Specific examples of these labeling agents are illustrated in Tables 1, 2
and 3, but are not limited thereto.
PAR The synthetic method of these azulene dyes is described in U.S. Pat. No.
4,738,908.
TBL3 TABLE 1
- No. G R X.sub.1 R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 R.sub.6
R.sub.7
1 (I) (3) R'.sub.1 = R'.sub.3 = R'.sub.5 = R'.sub.6 = HR'.sub.2 =
R'.sub.7 = CH.sub.3R'.sub.4 = CH(CH.sub.3).sub.2, R'.sub.22 = H, n = 2
BF.sub.4 H H H
##STR9##
H H CH.sub.3
2 (I) (3) R'.sub.1 = R'.sub.2 = R'.sub.4 = R'.sub.6 = H ClO.sub.4 H H
CH.sub.3 H OCH.sub.3 H CH.sub.3
R'.sub.3 = R'.sub.7 =
CH.sub.3 R'.sub.5 =
OCH.sub.3, R'.sub.22 = H, n =
2 3 (I) (3) R'.sub.1 =
R'.sub.2 = R'.sub.3 = R'.sub.7 = R'.sub.4 = R'.sub.6 = H I H H CH.sub.3
H CH.sub.2
CH.sub.2 H CH.sub.3
R'.sub.5 = CH.sub.2 CH.sub.2 CH.sub.2 COONa CH.sub.2 CH.sub.3
R'.sub.22 = H n =
2 4 (I) (3) R'.sub.1 =
R'.sub.2 = R'.sub.3 = R'.sub.4 = R'.sub.6 = R'.sub.7 = H ClO.sub.4 H H'
H H C(CH.sub.2).sub.3 H H
R'.sub.5 =
C(CH.sub.2).sub.3 R'.sub.22
= H, n =
2
5 (I) (3) R'.sub.1 = R'.sub.5 = R'.sub.6 = HR'.sub.2 and R'.sub.3
are cyclizedwith (CH.sub.2).sub.2R'.sub.4 = R'.sub.7 = CH.sub.3,
R'.sub.22 = H, n = 2 BF.sub.4 H SO.sub.3.sup..crclbar. Na.sup..sym. H
##STR10##
H H CH.sub.3
6 (I) (3)
##STR11##
BF.sub.4 H H H H C(CH.sub.2).sub.3 H H
7 (I) (3) R'.sub.1 = R'.sub.2 = R'.sub.4 = R'.sub.6 = HR'.sub.3 =
R'.sub.5 = R'.sub.7 = CH.sub.3R'.sub.22 = H, n =
2
##STR12##
H H CH.sub.3 H CH.sub.3 H CH.sub.3
8 (I) (9)
##STR13##
BF.sub.4 H CH.sub.3 H R.sub.4 and R.sub.5 arecombined to formSCHC(CH.sub
.3) H CH.sub.3
9 (I) (10)
##STR14##
ClO.sub.4 H SO.sub.3.sup..crclbar. Na.sup..sym. H CH(CH.sub.3).sub.2
##STR15##
H CH.sub.3
10 (I) (11)
##STR16##
BF.sub.4 H CH.sub.3 H CH(CH.sub.3).sub.2 H H CH.sub.3
11 (I) (12)
##STR17##
##STR18##
H CH.sub.3 H CH(CH.sub.3).sub.2 H H CH.sub.3
*G: General Formula
TBL TABLE 2
__________________________________________________________________________
No.
G R.sub.A R.sub.8
R.sub.9
R.sub.10
R.sub.11
R.sub.12 R.sub.13
R.sub.14
__________________________________________________________________________
12 (II)
(1)
R'.sub.8 = R'.sub.10 = R'.sub.12 = R'.sub.13 = H
H SO.sub.3.sup..crclbar. Na.sup..sym.
H CH(CH.sub.3).sub.2
H H CH.sub.3
R'.sub.9 = R'.sub.14 = CH.sub.3
R'.sub.11 = CH(CH.sub.3).sub.2
13 (II)
(1)
R'.sub.8 = R'.sub.12 = R'.sub.13 = R'.sub.14 = H
H CH.sub.3
SCHC(CH.sub.3)
H H H
R'.sub.10 &lt; R'.sub.11CSCHC(CH.sub.3)
14 (II)
(2)
R'.sub.8 = R'.sub.10 = R'.sub.12 = R'.sub.13 = H
H SO.sub.3.sup..crclbar. Na.sup..sym.
H CH(CH.sub.3).sub.2
H H CH.sub.3
R'.sub.9 = R'.sub.14 = CH.sub.3
R'.sub.11 = CH(CH.sub.3).sub.2
15 (II)
(2)
R'.sub.8 = R'.sub.9 = R'.sub.11 = R'.sub.13 = H R'.sub.10 =
R'.sub.14 = CH.sub.3 R'.sub.12 = OC.sub.2 H.sub.5
H H CH.sub.3
H
##STR19## H CH.sub.3
__________________________________________________________________________
*G: General Formula
TBL3 TABLE 3
- No. G R.sub.B X.sub.1 R.sub.15 R.sub.16 R.sub.17 R.sub.18 R.sub.19
R.sub.20 R.sub.21
16 (III) (10)
##STR20##
BF.sub.4 H CH.sub.3 H formation ofSCHC H CH.sub.3
1
7 (III) (4) R'.sub.15 = R'.sub.16 = R'.sub.18 = R'.sub.20 = H I H
SO.sub.3.sup..crclbar.
Na.sup..sym. H H CH.sub.3 H CH.sub.3 R'.sub.17 =
R'.sub.19 = R'.sub.21 =
CH.sub.3 r =
1
18 (III) (10)
R'.sub.15 = R'.sub.18 = R'.sub.20 = HR'.sub.16 = NO.sub.2R'.sub.17 =
R'.sub.19 = R'.sub.21 = CH.sub.3r =
3
##STR21##
H NO.sub.2 CH.sub.3 H CH.sub.3 H CH.sub.3
19 (III) (5) R'.sub.15 = R'.sub.16 = R'.sub.17 = R'.sub.18 = HR'.sub.19
= R'.sub.20 = R'.sub.21 = HR".sub.15 = R".sub.17 = R".sub.18 = R".sub.19
=R".sub.20 = R".sub.21 = H ClO.sub.4 H SO.sub.3.sup..crclbar.
Na.sup..sym. H H
##STR22##
H H
20 (III) (8)
##STR23##
##STR24##
H CH.sub.3 H CH(CH.sub.3).sub.2 H H CH.sub.3
21 (III) (9)
##STR25##
BF.sub.4 H SO.sub.3.sup..crclbar. Na.sup..sym. H H n-C.sub.8 H.sub.17 H
H
22 (III) (10)
##STR26##
##STR27##
H SO.sub.3.sup..crclbar. Na.sup..sym. H CH(CH.sub.3).sub.2 H H CH.sub.3
23 (III) (12)
##STR28##
I H CH.sub.3 H CH(CH.sub.3).sub.2
##STR29##
H CH.sub.3
No. G R X.sub.1 R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.5 R.sub.6
R.sub.7
24 I (6)
##STR30##
I H H H
##STR31##
H H CH.sub.3
25 I (7)
##STR32##
BF.sub.4 H H H CH(CH.sub.3).sub.2 H H CH.sub.3
26 I (3) R'.sub.1 = R'.sub.3 = R'.sub.5 = R'.sub.6 = HR'.sub.2 =
SO.sub.3.sup..crclbar. Na.sup..sym.R'.sub.7 = CH.sub.3 R'.sub.22 =
HR'.sub.4 = CH(CH.sub.3).sub.2n =
2 I H H CH.sub.3
##STR33##
H H CH.sub.3
27 I (3) R'.sub.1 = R'.sub.3 = R'.sub.4 = R'.sub.6 = R'.sub.7 =
HR'.sub.2 = SO.sub.3.sup..crclbar. Na.sup..sym.R'.sub.5 =
(CH.sub.2).sub.3COO.sup..crclbar.
Na.sup..sym. BF.sub.4 H SO.sub.3.sup..crclbar.
Na.sym. H H
##STR34##
H H
*G: General Formula
PAR These illustrated labeling agents absorb light in a near-infrared
wavelength region of 670 to 900 nm, and the molar absorption coefficient
.epsilon. is in the region of 50,000 to 300,000 1/mol.cm. The illustrated
labeling agents include those generating strong fluorescence.
PAR Table 4 shows the maximum absorption wavelength (.lambda.max) and maximum
fluorescence wavelength of (.lambda.em) each of the labeling agents
generating fluorescence in the region of the semiconductor laser
wavelength (medium: ethanol/dichloromethane=1/4).
TBL TABLE 4
______________________________________
Maximum absorption
Maximum fluorescence
No. wavelength (.lambda. max)
wavelength (.lambda. em)
______________________________________
2 828 863
3 833 871
4 825 857
6 825 851
7 830 871
16 790 828
27 826 870
______________________________________
PAR FIG. 1 shows the fluorescence emitting wave form on the incidence of
semiconductor laser beam (10 mW) of 830 nm into a labeling agent No. 3.
The apparatus for measurement is IMUC-7000 manufactured by Otsuka Electron
Co., Ltd.
PAR In FIG. 1, curve A shows incident wave form of semiconductor laser beam.
Curve B shows A fluorescence emitting wave form of a labeling agent No. 3.
PAR Alternatively, the labeling agent to be used in the present invention is a
compound of the general formula (IV), wherein A, B, D and E individually
represent hydrogen atom or alkyl group (for example, ethyl group, n-propyl
group, iso-propyl group, n-butyl group, sec-butyl group, iso-butyl group,
t-butyl group, n-amyl group, t-amyl group, n-hexyl group, n-octyl group,
t-octyl group and the like); and additionally, other alkyl groups such as
for example a substituted alkyl group (for example, 2-hydroxyethyl group,
3-hydroxypropyl group, 4-hydroxybutyl group, 2-acetoxyethyl group,
carboxymethyl group, 2-carboxyethyl group, 3-carboxypropyl group,
2-sulfoethyl group, 3-sulfopropyl group, 4-sulfobutyl group, 3-sulfate
propyl group, 4-sulfate butyl group, N-(methylsulfonyl)-carbamylmethyl
group, 3-(acetyl-sulfamyl)propyl group, 4-(acetylsulfamyl)butyl group and
the like); cyclic alkyl groups (for example cyclohexyl group), allyl group
(CH.sub.2 .dbd.CH--CH.sub.2 --), alkenyl group (vinyl group, propenyl
group, butenyl group, pentenyl group, hexenyl group, heptenyl group,
octenyl group, dodecyl group, prenyl group and the like), aralkyl group
(for example, benzyl group, phenethyl group, .alpha.-naphthylmethyl group,
.beta.-naphthylmethyl group and the like), a substituted aralkyl group
(for example, carboxybenzyl group, sulfobenzyl group, hydroxybenzyl group
and the like), a substituted or an unsubstituted aryl group (for example,
phenyl group, aminophenyl group, naphthyl group, tolyl group, xylyl group,
methoxyphenyl group, dimethoxyphenyl group, trimethoxyphenyl group,
ethoxyphenyl group, dimethylaminophenyl group, diethylaminophenyl group,
dipropylaminophenyl group, dibenzylaminophenyl group, diphenylaminophenyl
group, sulfonate phenyl group, carboxylate phenyl group and the like), a
substituted or an unsubstituted heterocyclic group (for example, pyridyl
group, quinolyl group, lepidyl group, methylpyridyl group, furyl group,
phenyl group, indolyl group, pyrrolle group, carbazolyl group,
N-ethylcarbazolyl group and the like), or a substituted or an
unsubstituted styryl group (for example, styryl group, methoxystyryl
group, dimethoxystyryl group, trimethoxystyryl group, ethoxystyryl group,
aminostyryl group, dimethylaminostyryl group, diethylaminostyryl group,
dipropylaminostyryl group, dibenzylaminostyryl group, diphenylaminostyryl
group, 2,2-diphenylvinyl group, 2-phenyl-2-methylvinyl group,
2-(dimethylamino-phenyl)-2-phenylvinyl group,
2-(diethylaminophenyl)-2-phenylvinyl group,
2-(dibenzylaminophenyl)-2-phenylvinyl group, 2,2-di(diethylaminophenyl
)vinyl group, 2,2-di(methoxyphenyl)vinyl group, 2,2-di(ethoxylphenyl)vinyl
group, 2-(dimethylaminophenyl)-2-methylvinyl group,
2-(diethylaminophenyl)-2-ethylvinyl group, and the like).
PAR r.sub.1 ' and r.sub.2 ' individually represent hydrogen atom or alkyl group
(for example, methyl group, ethyl group, n-propyl group, iso-propyl group,
n-butyl group, sec-butyl group, iso-butyl group, t-butyl group, n-amyl
group, t-amyl group, n-hexyl group, n-octyl group, t-octyl group and the
like); and additionally, other alkyl groups such as for example a
substituted alkyl group (for example, 2-hydroxyethyl group,
3-hydroxypropyl group, 4-hydroxybutyl group, 2-acetoxyethyl group,
carboxymethyl group, 2-carboxyethyl group, 3-carboxypropyl group,
2-sulfoethyl group, 3-sulfopropyl group, 4-sulfobutyl group, 3-sulfate
propyl group, 4-sulfate butyl group, N-(methylsulfonyl)-carbamylmethyl
group, 3-(acetylsulfamyl)propyl group, 4-(acetylsulfamyl)butyl group and
the like); cyclic alkyl group (for example, cyclohexyl group), allyl group
(CH.sub.2 .dbd.CH--CH.sub.2 --), alkenyl group (vinyl group, propenyl
group, butenyl group, pentenyl group, hexenyl group, heptenyl group,
octenyl group, dodecyl group, prenyl group and the like), aralkyl group
(for example, benzyl group, phenethyl group, .alpha.-naphthylmethyl group,
.beta.-naphthylmethyl group and the like), and a substituted aralkyl group
(for example, carboxybenzyl group, sulfobenzyl group, hydroxybenzyl group
and the like).
PAR A, B, D, r.sub.1 ' and r.sub.2 ' preferably contain one or more well-known
polar groups in order to impart water solubility to the labeling agent
(dye) of the general formula (IV). The reactive group includes for example
hydroxyl group, alkylhydroxyl group, sulfone group, alkyl sulfone group,
carboxyl group, alkylcarboxyl group, tetra-ammonium base and the like. A,
B, D, r.sub.1 ' and r.sub.2 ' preferably contain one or more well-known
reactive groups in order that the labeling agent of the general formula
(IV) can form a covalent bond with a substance from a living organism.
PAR The reactive group includes the reactive sites of isocyanate,
isothiocyanate, succinimide ester, sulfosuccinimide ester, imide ester,
hydrazine, nitroaryl halide, piperidine disulfide, maleimide, thiophthal
imide, acid halide, sulfonyl halide, aziridine, azide nitrophenyl, azide
amino, 3-(2-pyridyldithio) propionamide and the like. In these reactive
sites, the following spacer groups
##STR35##
(n=0, 1 to 16) may be interposed in order to prevent the steric hindrance
on the bonding of a labeling agent and a substance from a living organism.
PAR Preferable such reactive groups include isothiocyanate, sulfosuccinimide
ester, succinimide ester, maleimide and the like.
PAR The k in the general formula (IV) is 0 or 1 and 1 is 1 or 2.
PAR X.sub.2.sup..crclbar. represents an anion including chlorine ion, bromine
ion, iodine ion, perchlorate ion, benzenesulfonate ion, p-toluene
sulfonate ion, methylsulfate ion, ethylsulfate ion, propylsulfate ion,
tetrafluoroborate ion, tetraphenylborate ion, hexafluorophosphate ion,
benzenesulfinic acid ion, acetate ion, trifluoroacetate ion, propionate
ion, benzoate ion, oxalate ion, succinate ion, malonate ion, oleate ion,
stearate ion, citrate ion, monohydrogen diphosphate ion, dihydrogen
monophosphate ion, pentachlorostannate a ion, chlorosulfonate ion,
fluorosulfonate ion, trifluoromethane sulfonate ion, hexafluoroantimonate
ion, molybdate ion, tungstate ion, titanate ion, zirconate ion and the
like.
PAR Specific examples of these labeling agents are illustrated in Table 5, but
are not limited thereto.
TBL3 TABLE 5
- No A B D E r.sub.1 ' r.sub.2
' X.sub.2
28 ph .rarw. .rarw.
##STR36##
-- -- BF.sub.4 k = 0,l =
1
29
##STR37##
.rarw. .rarw.
##STR38##
-- -- BF.sub.4 k = 0,l =
1
30
##STR39##
##STR40##
(CH.sub.3).sub.2N
##STR41##
-- -- I k = 0,l =
1gleaming
31
##STR42##
##STR43##
(C.sub.2 H.sub.5).sub.2
N
##STR44##
H CH.sub.3 AsF.sub.6 k = 1,l =
0
32
##STR45##
.rarw. .rarw.
##STR46##
-- -- FSO.sub.3 k = 0,l =
1
33
##STR47##
##STR48##
##STR49##
##STR50##
-- --
##STR51##
k = 0,l =
1
34
##STR52##
Ph
##STR53##
##STR54##
-- --
##STR55##
k = 0,l =
1
35
##STR56##
.rarw. .rarw. .rarw. H
##STR57##
BF.sub.4 k = 1,l =
0
36
##STR58##
H
##STR59##
.rarw. -- -- AsF.sub.6 k = 0l =
0
37
##STR60##
.rarw. .rarw. .rarw. H
##STR61##
BF.sub.4 k = 1,l =
0
38
##STR62##
.rarw.
##STR63##
.rarw. -- -- FSO.sub.3 k = 0,l =
1
39
##STR64##
.rarw. .rarw. .rarw. H C.sub.2 H.sub.5 BF.sub.4 k = 1,l =
0
40
##STR65##
.rarw. .rarw.
##STR66##
-- -- BF.sub.4 k = 0,l =
1
41
##STR67##
.rarw. .rarw. .rarw. H
##STR68##
BF.sub.4 k = 1,l =
0
42
##STR69##
CH.sub.3
##STR70##
CH.sub.3 H CH.sub.3 FSO.sub.3 k = 1,l =
0
43
##STR71##
C.sub.3
H.sub.7
##STR72##
C.sub.3
H.sub.7 H
##STR73##
BF.sub.4 k = 1,l =
0
44
##STR74##
##STR75##
##STR76##
##STR77##
-- --
##STR78##
k = 0,l =
1
45
##STR79##
##STR80##
##STR81##
##STR82##
-- -- AsF.sub.6 k = 0,l =
1
46
##STR83##
##STR84##
##STR85##
##STR86##
--
##STR87##
I k = 0,l =
1
47
##STR88##
##STR89##
##STR90##
##STR91##
--
##STR92##
I k = 1,l =
0
48
##STR93##
##STR94##
##STR95##
##STR96##
-- -- BF.sub.4 k = 0,l =
1
PAR These illustrated labeling agents absorb light in a near-infrared
wavelength region of 670 to 900 nm, and the molar absorption coefficient e
is in the region of 50,000 to 300,000 1/mol.cm. Some of the illustrated
labeling agents generate intense fluorescence.
PAR The dye No. 30 illustrated in Table 5 exhibits the maximum absorption at a
wavelength of 819 nm in a near-infrared region and emits fluorescence. The
maximum fluorescence wavelength (.lambda.em) is 864 nm (medium;
dichloromethane).
PAR In accordance with the present invention, the labeling agents described
above are immobilized onto a substance from a living organism, but the
substance from a living organism to be immobilized is selectively
determined based on a substance to be analyzed or a subject sample. That
is, if a substance is selected from a living organism having a biological
specificity to a subject sample, the substance to be analyzed can be
detected with specificity. By the term "substance from a living organism"
is meant naturally occurring or synthetic peptides, proteins, enzymes,
sugars, rectins, viruses, bacteria, nucleic acids, DNA, RNA, antigens
(including for example recombinant antigens), antibodies and the like. The
substances specifically useful in terms of clinical pathology include the
following; immunoglobulin such as IgG, IgM, IgE and the like; plasma
proteins and antibodies thereof, such as compliments, CRP, ferritin,
.alpha.1-microglobulin, .beta.2-microglobulin, and the like; tumor markers
and antibodies thereof, such as .alpha.-fetoprotein, carcinoembryonic
antigen (CEA), prostate acid phosphatase (PAP), CA19-9, CA-125 and the
like; hormones and antibodies thereof such as luteinizing hormone (LH),
follicle stimulating hormone (FSH), human chorionic gonadotropin (hCG),
estrogen, insulin and the like; substances in relation with virus
infection and antibodies thereof, such as HBV-related antigens (HBs, HBe,
HBc), HIV, ATL and the like; bacteria and antibodies thereof, such as
Corynebacterium diphteriae, Clostridium botulinum, mycoplasma, Treponema
pallidum and the like; protozoae and antibodies thereof such as Toxoplasma
gondii, Trichomonas, Leishmania, Tripanozoma, malaria protozoa and the
like; pharmaceutical agents and antibodies thereof, such as antileptic
agents including phenytoin, phenobarbital and the like, cardiovascular
agents including quinidine and digoxin, antasthmatic agents including
theophylline, antibiotics including chloramphenicol and gentamycin; as
well as, enzymes, enterotoxin (streptolysin O) and the antibodies thereof.
Depending on the type of sample, a substance which can incur the
antigen-antibody reaction with a substance to be measured in a sample is
appropriately selected for use.
PAR In accordance with the present invention, the following known method can be
utilized in order to immobilize a labeling agent onto a substance from a
living organism such as a physiological active substance.
PAR There are illustrated for example i) ion bonding method, ii) physical
absorption method, iii) covalent bonding method and the like.
PAR The ion bonding method comprises electrostatically bonding a labelling
agent having principally a positive charge to a substance from a living
organism such as proteins, DNA, RNA and the like.
PAR The physical absorption method comprises utilizing the hydrophobic bond
between the lipophilic part of a labeling agent and the lipophilic part of
a protein.
PAR The reaction process of bonding is simple in accordance with the ion
bonding method and physical absorption method, but the bonding strength of
a labeling agent and a substance from a living organism is weak.
PAR On contrast, the covalent bonding method comprises bonding a highly
reactive functional group to at least one of a labeling agent and a
substance from a living organism, and covalently bonding the two through
the functional group whereby a highly intense bonding strength can be
generated. In bonding a labeling agent with a substance from a living
organism such as physiological active substances via covalent bonds, the
functional groups being present in the substance from a living organism
and which can be involved in the bonding, include free amino group,
hydroxyl group, phosphate group, carboxyl group, the sulfhydryl group of
cysteine, the imidazole group of histidine, phenol group of tyrosine, the
hydroxyl group of serine and threonine, and the like.
PAR These functional groups react with a variety of diazonium salts, acid
amides, isocyanate, active-type halogenated alkyl groups, active-type
ester groups and the like. Therefore, by a variety of methods, dyes can be
immobilized onto a substance from a living organism by introducing these
functional groups into a labeling agent. Alternatively, the conformation
of a substance from a living organism, specifically that of proteins, is
readily damaged because it is retained through relatively weak bonds such
as hydrogen bond, hydrophobic bond, ion bond and the like. Thus, the
immobilization with a labeling agent preferably should be carried out
under mild conditions, without processing by means of high temperatures,
strong acids and strong alkalis.
PAR One method of carrying out the immobilization under mild conditions
includes the use of bifunctional cross-linking agents which react with a
labeling agent and with the functional groups of a substance from a living
organism. The bifunctional cross-linking agents include, for example,
carbodiimide represented by the general formula R--N.dbd.C.dbd.N--R',
dialdehyde represented by the general formula CHO--R--CHO, diisocyanate
represented by O.dbd.C.dbd.N--R--N.dbd.C.dbd.O (wherein R and R' represent
individually the same or a different substituted or unsubstituted alkyl
group, aryl group alkylaryl group or aryl alkyl group), and the like.
PAR The analysis of a certain particular objective substance is conducted by
using the resulting labeled complex in which a labeling agent is
immobilized onto a substance from a living organism.
PAR If a target (analytical subject) is one species of cell, the labeled
complex is bonded to a specific substance on the cell complimentary to the
substance from a living organism bonded to the labeled complex via a
specific bonding such as an antigen-antibody reaction or the hydrogen
bonding between nucleic acids. Then, the amount of such antigen, antibody
or nucleic acids can be measured based on the fluorescence or absorbance
of the system.
PAR If the analysis is effected of a target in relation with an antigen and an
antibody, a complex bonded through the labeling agent to an antigen (or an
antibody) and an antibody (an antigen if a labeling agent is immobilized
onto the antibody) to be measured are subjected to antigen-antibody
reaction. The complex (B; bonded type) bonded to the antibody (antigen) is
then separated from the complex (F; free type) which is not bonded to the
antibody (antigen) (B/F separation). Thereafter, the amount of the complex
(B) is determined based on the fluorescence or absorbance. The technique
utilizing the antigen-antibody reaction described above is described in
details in "Examination and Technology", Vol. 16, No. 7 (1988).
PAR In terms of detection sensitivity, furthermore, it is preferable that two
or more, preferably 10 or more labeling agents are bonded to one molecule
of a substance from a living organism. In terms of synthesis and
sensitivity, preferably 10 to 100, more preferably 20 to 50 such agents
may be bonded to one molecule thereof.
PAR The present invention will now be explained with reference to examples.
PAC EXAMPLE 1
PAR Anti-human CRP sheep serum (IgG fraction; manufactured by Cooper Biomedical
Inc.) was diluted with phosphate buffer, pH 8.0, to a concentration of 0.5
mg/ml, to prepare an antibody solution. To 8 ml of the antibody solution
were added 0.2 mg of a labeling agent No. 3 of Table 1 (.lambda.max=833
nm) and 0.09 g of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (referred to as WSC hereinafter) (manufactured by Dojin
Chemicals, Co. Ltd.) for reaction at room temperature for three hours, to
generate a labeling agent-antibody complex. The labeling agent-antibody
complex was separated and purified from unreacted substances by gel
filtration chromatography on a column packed with Sepharose 6B. The
bonding molar ratio of the labeling agent and the antibody in the complex
thus obtained was 2.1:1. By using a spectrophotometer Shimadzu UV-3100S,
the absorbance of the complex was measured at wavelengths .lambda.=833 nm
and .lambda.=280 nm, separately, to calculate the molar ratio of the
labeling agent and the antibody.
PAC EXAMPLE 2
PAR Rectin. Concanavalin A (manufactured by E. Y. Laboratories Co. Ltd.) was
diluted with phosphate buffer, pH 8.2, to a concentration of 0.2 mg/ml, to
prepare a rectin solution.
PAR With 10 ml of the rectin solution was reacted 0.2 mg of a labeling agent
No. 6 of Table 1 (.lambda.max=825 nm) at room temperature for three hours.
The labeling agent-rectin complex was separated and purified on a gel
filtration chromatocolumn packed with Sepharose 6B. The molar ratio of the
labeling agent and the rectin in the complex obtained was 3.7:1. The
absorbances at wavelengths .lambda.=825 and .lambda.=280 nm were measured
by a spectrophotometer Shimadzu UV-3100S, to calculate the molar ratio of
the labeling agent and the rectin.
PAC EXAMPLE 3
PAR Anti-human HCG monoclonal antibody (manufactured by ZyMED Lab. Inc.) was
diluted with phosphate buffered physiological saline (PBS), pH 7.2, to a
concentration of 0.2 mg/ml, to prepare a monoclonal antibody solution.
PAR To 8 ml of the antibody solution was added 0.3 mg of a labeling agent No.
12 of Table 1 (.lambda.max=705 nm) for agitation at room temperature for
three hours. The labeling agent-antibody complex was separated and
purified by gel filtration chromatography on a column packed with
Sepharose 6B.
PAR The molar ratio of the labeling agent and the antibody in the labeling
agent-antibody complex thus obtained was 1.7:1. By using a
spectrophotometer 1 Shimadzu UV-3100S, the absorbance of the complex was
measured at wavelengths .lambda.=705 nm and .lambda.=280 nm, separately,
to calculate the molar ratio of the labeling agent and the antibody.
PAC EXAMPLE 4
PAR M13mp18 single-strand DNA (7249 bases) (manufactured by TAKARA Liquor KK.)
(0.1 mg) was diluted with 5 mmol phosphate buffer, pH 6, to prepare a DNA
solution. A labeling agent No. 5 (0.1 mg) shown in Table 1
(.lambda.max=796 nm) was dissolved in 5 ml of distilled water, and
subsequently, 5 ml of the DNA solution was gradually added dropwise to the
resulting dye solution. Agitation was further effected at room temperature
for 2 hours, to produce a DNA-labeling agent complex.
PAR To the solution of the DNA-labeling agent complex described above was added
further 40 ml of ethanol, to precipitate the DNA-labeling agent complex.
The DNA-labeling agent complex was separated on a filter, followed by
washing with ethanol. The DNA-labeling agent complex after the washing was
again dissolved in 2 ml of the phosphate buffer, pH 6. The amount of the
labeling agent bonded to that of the DNA was 0.5 .mu.g per .mu.g.DNA. The
absorbance of the complex was measured at wavelengths .lambda.=705 nm and
.lambda.=280 nm, separately, to calculate the concentrations of the
labeling agent and the DNA.
PAC EXAMPLE 5
PAR A 20-mer oligonucleotide having a base sequence partially complimentary to
the base sequence of a model target nucleic acid M13mp18 ss DNA was
synthesized by a DNA synthesizer 381 A, manufactured by ABI Co. Ltd. Then,
a primary amine was introduced into the 5' terminus of the oligonucleotide
by using a N-MMT-hexanol amine linker manufactured by Milligen Co. Ltd.,
instead of general amidide reagents. A predetermined protocol was followed
to perform cutting out from the CPG-support, deprotection (including the
deprotection of monomethoxytrityl group as a protective group of the
primary amine), and the purification by high-performance liquid
chromatography.
PAR After mixing together 200 .mu.g of the oligonucleotide, 100 .mu.l of 1M
sodium carbonate buffer, pH 9.0, and 700 .mu.l of water, 2 mg of a
labeling agent No. 27 (.lambda.max=826 nm) shown in Table 1, which had
preliminarily been dissolved in 200 .mu.l of dimethyl formamide, was
gradually added under agitation. After the reaction at room temperature
for 24 hours, the peak of the nucleic acid was decreased on a
high-performance liquid chromatogram, whereas a new peak having the
absorbances of the nucleic acid and the labeling agent developed. Thus,
the reaction solution was nearly purified on a gel filtration column,
NAP-50, manufactured by Pharmacia, which was then purified by HPLC to
obtain 175 .mu.g of the nucleic acid-labeling agent complex.
PAC COMPARATIVE EXAMPLE 1
PAR The chemical structure of a well-known cyanine-type near-infrared
absorption dye NK-1967 (manufactured by Nippon Photosensitive Dye Research
Institute) is depicted hereinbelow.
##STR97##
PAR To 5 ml of the antibody solution prepared in Example 1 was added 0.3 mg of
the cyanine dye, and agitated at room temperature for 3 hours, to generate
a labeling agent-antibody complex.
PAR The labeling agent-antibody complex was separated and purified by gel
filtration chromatography on a column packed with Sepharose 6B.
PAR The molar ratio of the labeling agent and the antibody was 1.7:1. The
absorbances at wavelength p=747 nm and p=280 nm were measured by a
spectrophotometer Shimadzu UV-3100S to calculate the molar ratio of the
labeling agent and the antibody. Complex stability under storage
PAR In order to examine the complex stability under storage, the following
experiments were carried out.
PAR The labeled complexes prepared in Examples 1 to 5 and Comparative Example 1
were prepared to predetermined concentrations with 10 mmol phosphate
buffer, pH 7.2. The solutions of the labeled complexes were kept in dark
at 7.degree. C. for three days. At the initiation and termination of the
test of complex stability under storage, the absorbance was measured at
predetermined wavelengths to calculate the ratio of the absorbance at the
termination, provided that the absorbance at the initiation was designated
as 100.
PAR For the complexes exhibiting fluorescence, the ratio of the fluorescence
intensity at the termination was calculated, provided that the
fluorescence intensity at the initiation was designated as 100.
PAR The results are shown in Table 6.
TBL TABLE 6
______________________________________
Stability under storage of labeled complexes
Change in
Change in fluorescence
absorbance*
intensity**
(wavelength
(wavelength
Concentration
in nm) in nm)
______________________________________
Example
1 0.4 g/ml 93.4 (833)
94 (875)
2 0.4 g/ml 91.9 (825)
90 (870)
3 0.5 g/ml 94.2 (705)
--
4 0.4 g/ml 95.1 (796)
--
5 0.5 g/ml 96.1 (826)
93 (870)
Comparative
Example
1 0.5 g/ml 71.2 (747)
63 (820)
______________________________________
*The initial absorbance was designated as 100.
**The initial fluorescence was designated as 100.
PAR As is shown in Table 6, the labeled complexes of the present invention
showed lower change of the absorbance or fluorescence intensity in water
than those of Comparative Example.
PAC EXAMPLE 6
PAR Anti-human CRP sheep serum (IgG fraction; manufactured by Cooper Biomedical
Inc.) was diluted with PBS, pH 7.2, to a concentration of 0.5 mg/ml, to
prepare an antibody solution. To 8 ml of the antibody solution were added
0.2 mg of a labeling agent No. 29 of Table 5 (.lambda.max=819 nm) and 0.09
g of WSC for reaction at room temperature for three hours, to generate a
dye-antibody complex. The dye-antibody complex was separated and purified
from unreacted substances by gel filtration chromatography on a column
packed with Sepharose 6B. The molar ratio of the dye and the antibody in
the complex thus obtained was 2.5:1. By using a spectrophotometer,
Shimadzu UV-3100S, the absorbance of the complex was measured at
wavelength .lambda.=819 nm and .lambda.=280 nm, separately, to calculate
the molar ratio of the dye and the antibody.
PAC EXAMPLE 7
PAR Anti-human HCG monoclonal antibody (manufactured by ZyMED Lab, Inc.) was
diluted with PBS to a concentration of 0.4 mg/ml, to prepare a monoclonal
antibody solution. To 2 ml of the monoclonal antibody solution were added
0.3 mg of a dye No. 32 of Table 5 (.lambda.max=825 nm) and 0.10 g of
Woodward reagent (manufactured by Tokyo Chemicals, Co. Ltd.) for reaction
at room temperature for three hours. The dye-antibody complex was
separated and purified by gel filtration chromatography on a column packed
with Sepharose 6B. The molar ratio of the dye and the antibody in the
dye-antibody complex thus obtained was 3.1:1. By using a spectrophotometer
Shimadzu UV-3100S, the absorbance of the complex was measured at
wavelengths .lambda.=825 nm and .lambda.=280 nm, separately, to calculate
the molar ratio of the dye and the antibody.
PAC EXAMPLE 8
PAR Rectin.Concanavalin A (manufactured by E. Y. Laboratories Co. Ltd.) was
diluted with PBS to a concentration of 0.2 mg/ml, to prepare a rectin
solution. With 10 ml of the rectin solution were added 0.2 mg of a dye No.
40 of Table 5 (.lambda.max=805 nm) and 10 ml of 0.05 M sodium borate
buffer, pH 8.0 containing 1% glutaraldehyde at room temperature for one
hour. The dye-rectin complex was separated and purified on a gel
filtration chromatocolumn packed with Sepharose 6B. The molar ratio of the
dye and the rectin in the complex obtained was 1.7:1. The absorbances at
wavelengths .lambda.=805 and .lambda.=280 nm were measured by a
spectrophotometer Shimadzu UV-3100S, to calculate the molar ratio of the
dye and the rectin.
PAC EXAMPLE 9
PAR M13mp18 single-strand DNA (7249 bases) (manufactured by TAKARA Liquor KK.)
(0.1 mg) was diluted with 5 mmol phosphate buffer, pH 6, to prepare a DNA
solution. A dye No. 35 (0.1 mg) shown in Table 5 (.lambda.max=780 nm) was
dissolved in 2 ml of ethanol, followed by gradual dropwise addition of 5
ml of the DNA solution to the resulting dye solution under stirring.
Agitation was further effected at room temperature for 2 hours, to produce
a DNA-dye complex.
PAR To the solution of the DNA-dye complex described above was added further 40
ml of ethanol, to precipitate the DNA-dye complex. The DNA-dye complex was
separated on a filter, followed by washing several times with ethanol. The
DNA-dye complex after the washing was again dissolved in 2 ml of the
phosphate buffer, pH 6. The amount of the dye bonded to that of the DNA
was 0.5 .mu.g per .mu.g.DNA. The absorbance of the complex was measured at
wavelengths .lambda.=780 nm and .lambda.=260 nm, separately, to calculate
the concentrations of the dye and the DNA.
PAC EXAMPLE 10
PAR A 20-mer oligonucleotide having a base sequence partially complimentary to
the base sequence of a model target nucleic acid M13mp18 ss DNA was
synthesized by a DNA synthesizer 381 A, manufactured by ABI Co. Ltd. Then,
a primary amine was introduced into the 5' terminus of the oligonucleotide
by using a N-MMT-hexanol amine linker manufactured by Milligen Co. Ltd.,
instead of general amidide reagents. A predetermined protocol was followed
to perform cutting out from the CPG-support, deprotection (including the
deprotection of monomethoxytrityl group as a protective group of the
primary amine), and the purification by high-performance liquid
chromatography.
PAR After mixing together 200 .mu.g of the oligonucleotide, 100 .mu.l of 1M
sodium carbonate buffer, pH 9.0, and 700 .mu.l of water, 2 mg of a dye No.
46 (.lambda.max=810 nm) shown in Table 5, which had preliminarily been
dissolved in 200 N1 of dimethyl formamide, was gradually added under
agitation. After the reaction at room temperature for 24 hours, the peak
of the nucelic acid was decreased on a high-performance liquid
chromatogram, whereas a new peak having the absorbances of the nucleic
acid and the dye developed. Thus, the reaction solution was nearly
purified on a gel filtration column, NAP-50, manufactured by Pharmacia,
which was then purified by HPLC to obtain 175 .mu.g of the nucleic
acid-dye complex. Complex stability under storage
PAR In order to examine the stability under storage of dye complexes, the
following experiments were carried out.
PAR The labeled dye complexes prepared in Examples 6 to 10 were prepared to
predetermined concentrations with 10 mmol phosphate buffer, pH 7.2. The
solutions of the labeled complexes were kept in dark at 7.degree. C. for
three days. At the initiation and termination of the test of complex
stability under storage, the absorbance was measured at predetermined
wavelengths. Then, the ratio of the absorbance at the termination was
calculated, provided that the absorbance at the initiation was designated
as 100.
PAR The results are shown in Table 7.
TBL TABLE 7
______________________________________
Stability under storage of labeled dye complexes
Change in absorbance
Wavelength
(initial absorbance
for was designated
Example
Concentration
measurement
as 100)
______________________________________
6 0.5 g/ml 819 95.1
7 0.5 g/ml 825 94.5
8 0.5 g/ml 805 91.3
9 0.4 g/ml 780 95.7
10 0.4 g/ml 810 93.9
______________________________________
PAR As is shown in Table 7, the labeled dye complexes of the present invention
showed lower change of the absorbance in water than those of Comparative
Example.
PAC EXAMPLE 11
PAR A 20-mer oligonucleotide having a base sequence partially complimentary to
the base sequence of a model target nucleic acid M13mp18 ss DNA was
synthesized by a DNA synthesizer 381 A, manufactured by ABI Co. Ltd. Then,
by using a deoxyuridylic acid derivative monomer:
##STR98##
with an amino group introduced, instead of general amidide reagents, 20
such deoxyuridylic acid derivatives each having a primary amine group were
added to the 5' terminus of the oligonucleotide. Routine method was
followed to perform cutting out from the CPG-support, deprotection
(including the deprotection of trifluoroacetyl group as a protective group
of the primary amine), and the purification by high-performance liquid
chromatography.
PAR After mixing together 200 .mu.g of the oligonucleotide bonding the primary
amines, 100 .mu.l of 1M sodium carbonate buffer, pH 9.0, and 700 .mu.l of
water, 5 mg of a dye No. 27 (.lambda.max=826 nm) shown in Table 1, which
had preliminarily been dissolved in 200 .mu.l of dimethyl formamide, was
gradually added under agitation. After the reaction at 40.degree. C. for
24 hours, the peak of the nucleic acid was decreased on a high-performance
liquid chromatogram, whereas a new peak having the absorbances of the
nucleic acid and the labeling agent developed. Thus, the reaction solution
was nearly purified on a gel filtration column, NAP-50, manufactured by
Pharmacia, which was then purified by HPLC to obtain 350 .mu.g of the
nucleic acid-labeling agent complex. The absorbance of the nucleic
acid-labeling agent complex at 826 nm had the intensity about 20-fold that
of the nucleic acid-labeling agent shown in Example 5.
PAR In accordance with the present invention, a stable complex can be formed
with less decomposition of dyes, and hence with less change in absorbance
or with less change in fluorescence, by bonding a labeling agent of a
particular structure to a substance from a living organism.
PAR Therefore, the complex of the present invention can provide a reagent with
excellent stability under storage for the application to microanalysis.
CLMS
STM What is claimed is:
NUM 1.
PAR 1. A labeled complex for detecting a subject compound to be analyzed by
means of optical means using near-infrared radiation which complex
comprises a substance from a living organism and a labeling agent fixed
onto the substance, the substance capable of specifically binding to the
subject compound, wherein the labeling agent comprises a compound
represented by the general formula (IV):
##STR99##
wherein A, B, D and E are independently selected from the group consisting
of hydrogen atom, a substituted or an unsubstituted alkyl group having two
or more carbon atoms, alkenyl group, aralkyl group, aryl group, styryl
group and heterocyclic group, and at least one of A and B is a substituted
or unsubstituted aryl group, and at least one of D and E is a substituted
or unsubstituted aryl group;
PA1 r.sub.1 ' and r.sub.2 ' are individually selected from the group consisting
of hydrogen atom, a substituted or an unsubstituted alkyl group, cyclic
alkyl group, alkenyl group, aralkyl group and aryl group; k is 0 or 1; is
0, 1 or 2; and X.sub.2.sup..crclbar. represents an anion.
NUM 2.
PAR 2. The labeled complex according to claim 1, wherein the substance from a
living organism is an antibody or an antigen.
NUM 3.
PAR 3. The labeled complex according to claim 1, wherein the substance from a
living organism is a nucleic acid.
NUM 4.
PAR 4. The labeled complex according to claim 1, wherein the substituted aryl
group constituting at least one of A and B is phenyl group substituted by
dialkylamino group.
NUM 5.
PAR 5. The labeled complex according to claim 1, wherein the substituted aryl
group constituting at least one of D and E is phenyl group substituted by
dialkylamino group.
NUM 6.
PAR 6. The labeled complex according to claim 4 or 5, wherein the dialkylamino
group is a diethylamino group.
NUM 7.
PAR 7. The labeled complex according to claim 1, wherein each of A, B and D is
dimethylaminophenyl group, E is aminophenyl group, k is 0 and l is 1.
NUM 8.
PAR 8. The labeled complex according to claim 1, wherein each of A, B and D is
diethylaminophenyl group, E is phenyl group substituted by carboxyl group,
k is 0 and l is 1.
NUM 9.
PAR 9. The labeled complex according to claim 1, wherein each of A, B, D and E
is diethylaminophenyl group, k is 1 and l is 0.
NUM 10.
PAR 10. The labeled complex according to claim 1, wherein each of A, B, and D
is diethylaminophenyl group, E is aminophenyl group, K is 0 and l is 1.
NUM 11.
PAR 11. The labeled complex according to claim 1, wherein A is
dimethylaminophenyl group, each of B and E is ethoxyphenyl group, k is 0,
1 is l and D is represented by the following formula:
##STR100##
NUM 12.
PAR 12. A method of detecting a subject compound to be analyzed in a sample
comprising the steps of:
PA1 providing a labeled complex comprising a substance from a living organisms
and a labeling agent fixed onto the substance, the substance being capable
of specifically binding to the subject compound;
PA1 binding the labeled complex to the subject compound; and
PA1 detecting the labeled complex to which the subject compound is bonded by
means of optical means, wherein the labeling agent comprises a compound
represented by the general formula (IV):
##STR101##
wherein A, B, D and E are independently selected from the group consisting
of hydrogen atom, a substituted or an unsubstituted alkyl group having two
or more carbon atoms, alkenyl group, aralkyl group, aryl group, styryl
group and heterocyclic group, and at least one of A and B is a substituted
or unsubstituted aryl group, and at least one of D and E is a substituted
or unsubstituted aryl group;
PA1 r.sub.1 ' and r.sub.2 ' are individually selected from the group consisting
of hydrogen atom, a substituted or an unsubstituted alkyl group, cyclic
alkyl group, alkenyl group, aralkyl group and aryl group; k is 0 or 1; is
0, 1 or 2; and X.sub.2.sup..crclbar. represents an anion.
NUM 13.
PAR 13. The method according to claim 12, wherein the substance from a living
organism is an antibody or an antigen.
NUM 14.
PAR 14. The method according to claim 12, wherein the substance from a living
organism is a nucleic acid.
NUM 15.
PAR 15. The analyzing method according to any one of claims 12, 13 and 14,
wherein the optical means is an optical means using near-infrared ray.
NUM 16.
PAR 16. The method according to claim 12, wherein each of A, B and D is
dimethylaminophenyl group, E is aminophenyl group, k is 0 and l is 1.
NUM 17.
PAR 17. The method according to claim 12, wherein each of A, B and D is
diethylaminophenyl group, E is phenyl group substituted by carboxyl group,
k is 0 and l is 1.
NUM 18.
PAR 18. The method according to claim 12, wherein each of A, B, D and E is
diethylaminophenyl group, k is 1 and l is 0.
NUM 19.
PAR 19. The method according to claim 12, wherein each of A, B and D is
diethylaminophenyl group, E is aminophenyl group, k is 0 and l is 1.
NUM 20.
PAR 20. The method according to claim 12, wherein A is dimethylaminophenyl
group, each of B and E is ethoxyphenyl group, k is 0, l is 1 and D is
represented by the following formula:
##STR102##
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