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##                                                              
