JP5189096B2 - Diagnostic agent and measuring method using the same - Google Patents

Description

The present invention relates to a diagnostic agent and a measurement method using the same, and more particularly to a diagnostic agent using an antibody labeled with a fluorescent dye and a measurement method using the same.

For the diagnosis of cancer, infectious diseases, etc., an immunoassay utilizing the specific recognition ability of an antibody is used (for example, Patent Document 1). The immunoassay is a method for detecting a target antigen using a labeled antibody, and an enzyme immunoassay (ELISA method) using an enzyme as a labeling substance or a fluorescent immunoassay (FIA method) using a fluorescent dye as a labeling substance is used. . In the ELISA method, the final detection is performed by detecting and quantifying various signals (color development, luminescence, chemiluminescence, etc.) generated by the reaction of the enzyme as a labeling substance. On the other hand, the FIA method is performed by irradiating a fluorescent dye, which is a labeling substance, with excitation light, and detecting and quantifying the resulting fluorescence.
International Publication No. 2003/010542 Pamphlet

  Since the FIA method uses a fluorescent dye, it has a clear contrast and excellent quantification, and has features that it can be detected in a shorter time and is easier to operate than the ELISA method. However, the conventional fluorescent dye has a problem that the labeling rate is low. For example, about 200-fold mol of the fluorescent dye is used with respect to the antibody, and the labeling rate is about 50-60% even under this condition. For this reason, it is necessary to use a large amount of fluorescent dye, so that the detection cost becomes high, and there is a problem that a processing step for removing the unreacted fluorescent dye is required and it takes a long time for detection. In addition, there is a problem that a fluorescent dye having a higher fluorescence intensity is required to perform high-sensitivity detection.

  As a result of diligent efforts to solve the above problems, the present inventor has used, as a fluorescent dye for labeling an antibody, a fluorescent dye having a coloring part composed of an organic EL dye and a binding part that binds to the antibody, The present invention has been completed by finding that the labeling rate for antibodies can be greatly improved. That is, the diagnostic agent of the present invention is a diagnostic agent containing at least an antibody and a fluorescent dye that labels the antibody, and the fluorescent dye comprises a coloring part composed of an organic EL dye and a binding part that binds to the antibody. It is characterized by having.

  Here, as the organic EL dye, a condensed polycyclic compound composed of a 5-membered ring compound containing one or more hetero atoms, selenium atoms, or boron atoms and a 6-membered ring compound having a conjugated system can be used.

  Further, as the condensed polycyclic compound, an azole derivative represented by any one of the following general formulas (1), (2) or (3) can be used.

Here, in the formula, R ₁ , R ₂ , R ₃ and R ₄ are each independently a hydrogen atom, a halogen atom, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkyl ester group, phosphoric acid as a substituent. An aromatic hydrocarbon group, an aliphatic hydrocarbon group or a heterocyclic group which may have an ester group, a sulfate ester group, a nitrile group, a hydroxyl group, a cyano group, a sulfonyl group, an aromatic hydrocarbon group or a heterocyclic group; X represents an optionally substituted nitrogen atom, sulfur atom, oxygen atom, selenium atom or boron atom, and R ′ represents an aliphatic hydrocarbon such as an alkyl group or an alkenyl group which may contain an aromatic ring. group or an aromatic hydrocarbon group, an ^- is a halide ^{_{ion, CF 3 SO 3 -, BF}} 4 - or PF ₆ ^- shows a.

In addition, as R ₂ and R ₃ , one selected from the group consisting of thiophene derivatives, furan derivatives, pyrrole derivatives, imidazole derivatives, oxazole derivatives, thiazole derivatives, pyrazole derivatives, and pyridine derivatives can be used.

In addition, a phenyl group having a sulfonyl group can be used for the above R ₂ and R ₃ .

  Moreover, the imidazole derivative shown by the following general formula (4), (5), (6), (7) or (8) can also be used for the said condensed polycyclic compound.

Here, in the formula, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ are each independently a hydrogen atom, a halogen atom, a substituent as an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkyl ester group. , Phosphoric acid ester group, sulfuric acid ester group, nitrile group, hydroxyl group, cyano group, sulfonyl group, aromatic hydrocarbon group or aliphatic hydrocarbon group or heterocyclic group which may have an aromatic hydrocarbon group or heterocyclic group R ₁ , R ₂ , R ₃ , R ₄ and R ₅ may be the same or different, and R ′ and R ″ may be an aliphatic group such as an alkyl group or an alkenyl group which may contain an aromatic ring. A hydrocarbon group or an aromatic hydrocarbon group, An ⁻ represents a halide ion, CF ₃ SO ₃ ⁻ , BF ₄ ⁻ or PF ₆ ⁻ .

In addition, as R ₂ and R ₃ , one selected from the group consisting of thiophene derivatives, furan derivatives, pyrrole derivatives, imidazole derivatives, oxazole derivatives, thiazole derivatives, pyrazole derivatives, and pyridine derivatives can be used.

In addition, a phenyl group having a sulfonyl group can be used for the above R ₂ and R ₃ .

  Moreover, the imidazole derivative shown by the following general formula can also be used for a color development part.

Here, in the formula, R ₁ , R ₄ and R ₅ are each independently a hydrogen atom, a halogen atom, a substituent as an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkyl ester group, a phosphate ester group, An aromatic hydrocarbon group, an aliphatic hydrocarbon group or a heterocyclic group which may have a sulfate ester group, a nitrile group, a hydroxyl group, a cyano group, a sulfonyl group, an aromatic hydrocarbon group or a heterocyclic group, and R ₁ , R ₄ and R ₅ may be the same or different.

  In addition, in the fluorescent dye, any one selected from a carboxylic acid group, an isocyanate group, an isothiocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group, a maleimide group, and an active esterified carbonyl group at the bonding portion. One type of reactive group can be used.

  Moreover, what has a spacer part which connects a color development part and a coupling | bond part can be used for a fluorescent dye.

The spacer part of the fluorescent dye includes -CH _2- , -NHCOO-, -CONH-, -COO-, -SO ₂ NH-, -HN-C (= NH) -NH-, -O-, -S- , —NR— (wherein R is an alkyl group), — (CH ₂ —CH ₂ —O) n— (n is an integer of 1 to 10), —CH═CH—, —C≡C—, —Ar— and — Those containing at least one functional group selected from the group consisting of CO—Ar—NR— can be used.

Here, what is represented by the following general formula (I) can be used for the said spacer part.
-(CHR ') pX- (CHR ") q- (I)
In the formula, X is a direct bond or -NHCOO-, -CONH-, -COO-, -SO ₂ NH-, -HN-C (= NH) -NH-, -O-, -S-, -NR- , —CH═CH—, —C≡C—, —Ar—, and —CO—Ar—NR—, each representing at least one functional group, wherein R ′ and R ″ are independently hydrogen An aliphatic hydrocarbon group such as an alkyl group or an alkenyl group which may contain an atom or an aromatic ring, or an aromatic hydrocarbon group, if necessary, a group consisting of a sulfonyl group, a hydroxyl group, a quaternary amine group and a carboxyl group And Ar represents an aryl group, p and q each independently represents an integer of 0 to 20, and p + q ≧ 1.

  Moreover, a peptide linker which consists of an amino acid or 2-20 amino acids can be used for a spacer part.

  Moreover, an amino acid can be used for a spacer part and a natural amino acid or a synthetic amino acid can be used for the amino acid.

  In addition, one amino acid selected from the group consisting of cysteic acid, 2-amino-3-sulfosulfanylpropanoic acid, 2-amino-3-sulfoxypropanoic acid, tyrosine, threonine, homoserine and serine is used as the amino acid. Can do.

  Alternatively, a peptide linker may be used for the spacer portion, and the peptide linker having at least one charged group selected from the group consisting of a sulfonyl group, a hydroxyl group, a quaternary amine group and a carboxyl group can be used.

  Further, the peptide linker may be at least one selected from the group consisting of cysteic acid, 2-amino-3-sulfosulfanylpropanoic acid, 2-amino-3-sulfoxypropanoic acid, tyrosine, threonine, homoserine and serine. Those containing amino acids can also be used.

  Moreover, the azole derivative shown by any one of the following general formula (9), (10) or (11) can also be used for the said condensed polycyclic compound.

Here, in the formula (9) and (11), R ₁ is represented, and in (10) _one of R ₁ and R ₄ is represented by the general formula LM, and M may have a substituent. Pyridinium group, secondary amino group, tertiary amino group, quaternary amino group, piperidinium group, piperazinium group, imidazolium group, thiazolium group, oxazolium group, quinolium group, benzoimidazolium group, benzothiazolium group or benzooxa represents nitrogen cation-containing group is a Zoriumu group, L is a direct bond _{or, - (CH 2) n- (} n is an integer of 1 to 4), - NHCOO -, - CONH -, - COO -, - SO ₂ NH-, -HN-C (= NH) -NH-, -O-, -S-, -NR- (R is an alkyl group), -Ar- (Ar is an aromatic hydrocarbon group) and -CO- A linker comprising one or more functional groups selected from the group consisting of Ar-NR-, linking M and the color-developing part, the remainder of R ₁ and R _{4 in} (10), and R ₂ and R ₃ are: Each independently a hydrogen atom, halo Having an alkyl group, alkoxy group, alkyl ester group, phosphate ester group, sulfate ester group, nitrile group, hydroxyl group, cyano group, sulfonyl group, aromatic hydrocarbon group or heterocyclic group as a substituent. X represents an optionally substituted aromatic hydrocarbon group, aliphatic hydrocarbon group or heterocyclic group, and X represents an optionally substituted carbon atom, nitrogen atom, sulfur atom, oxygen atom, selenium atom or boron atom. , R ′ represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group comprising an alkyl group which may contain an aromatic ring, and An ⁻ represents a halide ion, CF ₃ SO ₃ ⁻ , BF ₄ ⁻ or PF ₆ ⁻ .

In the above azole derivatives, R ₂ and R ₃ are each independently a group consisting of a thiophene derivative, a furan derivative, a pyrrole derivative, an imidazole derivative, an oxazole derivative, a thiadiazole derivative, a pyrazole derivative, a pyridine derivative, and a quinoline derivative. 1 type selected from can be used.

In the azole derivative, an aryl group having a sulfonyl group can be used for R ₂ and R ₃ .

In the azole derivative,-(CH ₂ ) n- (n is an integer of 1 to 4) can be used for the linker L.

  Moreover, the imidazole derivative shown by any 1 type of the following general formula (12), (13), (14), (15) or (16) can also be used for the said condensed polycyclic compound.

Here, in the formula, _one of R ₁ and R ₄ in (12), (14) and (15), and any one of R ₁ , R ₄ and R ₅ in (13) and (16) is It is represented by the general formula LM, and M is an optionally substituted pyridinium group, secondary amino group, tertiary amino group, quaternary amino group, piperidinium group, piperazinium group, imidazolium group, thiazolium group, oxazolium. A nitrogen cation-containing group which is a group, a quinolium group, a benzimidazolium group, a benzothiazolium group or a benzoxazolium group, and L is a direct bond or — (CH ₂ ) n— (n is 1 to 4) Integer)), -NHCOO-, -CONH-, -COO-, -SO ₂ NH-, -HN-C (= NH) -NH-, -O-, -S-, -NR- (R is an alkyl group) ), -Ar- (Ar is an aromatic hydrocarbon group) and -CO-Ar-NR-, one or more functional groups selected from the group consisting of, and a linker for linking M and the coloring part, (12 ), (14) and R ₁ and the balance R ₄ in (15), (13) and R _1, R ₄ and the remainder of R ₅ in (16) and R _2, R ₃ are each independently, a hydrogen atom, a halogen atom, an alkyl group as a substituent, an alkoxy group, an alkyl ester group, a phosphoric acid ester group A sulfuric ester group, a nitrile group, a hydroxyl group, a cyano group, a sulfonyl group, an aromatic hydrocarbon group or an aromatic hydrocarbon group or a heterocyclic group which may have an aromatic hydrocarbon group or a heterocyclic group; R ′ and R ″ are aliphatic hydrocarbon groups or aromatic hydrocarbon groups comprising an alkyl group which may contain an aromatic ring, An ⁻ represents a halide ion, CF ₃ SO ₃ ⁻ , BF ₄ ⁻ or PF ₆ ⁻ . Show.

In the imidazole derivative, R ₂ and R ₃ are each independently a group consisting of a thiophene derivative, a furan derivative, a pyrrole derivative, an imidazole derivative, an oxazole derivative, a thiadiazole derivative, a pyrazole derivative, a pyridine derivative, and a quinoline derivative. 1 type selected from can be used.

In the imidazole derivative, an aryl group having a sulfonyl group can be used for R ₂ and R ₃ .

In the imidazole derivative,-(CH ₂ ) n- (n is an integer of 1 to 4) can be used for the linker L.

The measuring method of the present invention is a measuring method of detecting the antigen in the subject, and a color portion comprising an organic EL-dye, an antibody labeled with a fluorescent dye having a coupling portion for coupling an antibody antigen And the fluorescence from the fluorescent dye is measured.

  Further, the method for producing a diagnostic agent of the present invention is a method for producing a diagnostic agent comprising at least an antibody and a fluorescent dye that labels the antibody, wherein the fluorescent dye comprises an organic EL dye and an antibody. At least one selected from the group consisting of an amide bond, an imide bond, a urethane bond, a thiourethane bond, an ester bond, and a guanidine bond between the organic EL dye and the antibody. The method includes the step of forming a bond.

  Since the diagnostic agent of the present invention uses a fluorescent dye having a coloring part composed of an organic EL dye and a binding part that binds to the antibody, the labeling ratio for the antibody is improved as compared with the conventional technique, and the fluorescence intensity is high. Antigen can be detected with higher sensitivity. Furthermore, since the organic EL dye has a high quantum yield in a solid state (including solid and semi-solid), it gives a high fluorescence intensity even in a dry state on a substrate such as a microarray or on a bead. In addition, since organic EL dyes are less expensive than conventional fluorescent dyes Cy3 and Cy5, antigens can be detected at a lower cost. In addition, since the excitation wavelength and emission wavelength can be changed by changing the substituent of the organic EL dye, the degree of freedom in selecting the fluorescence wavelength increases, and many fluorescence wavelengths such as orange, yellow, green, and blue are used. be able to. This makes it possible to use two or more fluorescent dyes having a large Stokes shift (the difference between the excitation wavelength and the fluorescence wavelength is large) and to simultaneously detect a plurality of antigens contained in one sample. . In addition, while Cy3 and Cy5 need to be stored frozen, organic EL dyes are chemically stable and can withstand long-term storage at room temperature, making them easy to handle.

In the detection method of this invention, it is a schematic diagram which shows an example of the preparation method of Fab 'fragment of IgG antibody. In the detection method of the present invention, it is a schematic diagram showing an example of a method for introducing an organic EL dye into an Fab ′ fragment of an IgG antibody. It is a graph which shows the result of an activity test in Example 1 of this invention. It is an example of the HPLC spectrum of BSA labeled with EL-OSu in Example 2 of the present invention. It is an example of the HPLC spectrum of BSA labeled with EL-OSu-Sp in Example 2 of the present invention. It is an example of the TOF MS spectrum of BSA after labeling with EL-OSu in Example 2 of the present invention. It is an example of the TOF MS spectrum of BSA before labeling in Example 2 of the present invention. It is a fluorescence micrograph which shows the result of Example 5 of this invention.

Hereinafter, embodiments of the present invention will be described in detail.
In the diagnostic agent of the present invention, a fluorescent dye having a coloring part composed of an organic EL dye and a binding part that binds to the antibody is used as a fluorescent dye for labeling the antibody.

(antibody)
The antibody used in the present invention is not particularly limited, and an antibody that specifically binds to at least one target antigen selected from plasma protein, lipoprotein, glycoprotein, polypeptide, lipid, polysaccharide, lipopolysaccharide and nucleic acid. Can be used. More preferably, the antigen is plasma protein, tumor marker, viral antigen, protein or the like. Examples of antibodies for tumor markers include monoclonal antibodies. Examples of antibodies for viral antigens include IgG antibodies and IgM antibodies.

  The antibodies used in the present invention include whole antibodies, antibody fragments, antibody derivatives, and modified antibodies labeled with biotin and the like. The antibody can be used by purifying an antiserum or ascites fluid obtained by administering an antigen to a mammal such as a horse, cow, sheep, or rat and immunizing it. In addition, a monoclonal antibody obtained by immunizing a suitable animal with an antigen, preparing a hybridoma between the collected antibody-producing cells and mouse myeloma cells, and screening for one having a specific amino acid sequence can also be used. Monoclonal antibodies can also be used as antibody fragments such as Fab, Fab ′, and F (ab ′) 2 after treatment with enzymes such as trypsin, papain, and pepsin. Examples of the subject containing the antibody include pathogens such as bacteria and viruses, blood separated from a living body, saliva, tissue lesions, feces and urine. Moreover, when performing prenatal diagnosis, fetal cells present in amniotic fluid can also be used. These specimens are concentrated as sediments by centrifugation or the like, and then used after disrupting cells by enzyme treatment, heat treatment, ultrasonic treatment or the like.

(Fluorescent dye)
The organic EL dye used for the fluorescent dye is sandwiched in a solid state between a pair of anode and cathode, and can emit light by the energy when the holes injected from the anode and the electrons injected from the cathode are recombined. If it is a pigment | dye, it will not specifically limit. For example, polycyclic aromatic compounds such as tetraphenylbutadiene and perylene, cyclopentadiene derivatives, distyrylpyrazine derivatives, acridone derivatives, quinacdrine derivatives, stilbene derivatives, phenothiazine derivatives, pyrazinopyridine derivatives, azole derivatives, imidazole derivatives, carbazole derivatives and tetraphenyl A thiophene derivative or the like can be used. Furthermore, it is preferable that it is a pigment | dye which has a carboxylic acid group in a molecule | numerator, or can introduce | transduce a carboxylic acid group. This is because a reactive group for binding to an antibody can be easily introduced as described below.

  The binding moiety used for the fluorescent dye has a reactive group that binds to the protein constituting the antibody, and the reactive group includes a substituent or a nucleophilic group that forms a covalent bond or an ionic bond with the antibody. Reagents or electrophilic reagents can be used. When forming a covalent bond, the reactive group is preferably a functional group capable of reacting with the amino group, imino group, thiol group or hydroxyl group of the antibody. As the covalent bond between the organic EL dye and the protein, it is preferable to form an amide bond, an imide bond, a urethane bond, a thiourethane bond, an ester bond, or a guanidine bond. The functional group includes, for example, isothiocyanate group, isocyanate group, epoxy group, sulfonyl halide group, acyl chloride group, halogenated alkyl group, glyoxal group, aldehyde group, triazine group, carbodiimide group, maleimide group and active esterification. The carbonyl group etc. which were made can be used. Preferably, any one selected from an isothiocyanate group, an isocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group, and an active esterified carbonyl group is preferably used. More preferably, any one selected from an isocyanate group, an isothiocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group, and an active esterified carbonyl group is preferably used. This is because it can form an amide bond with an amino group of a protein and can directly bond to an imino group of a protein. More preferred are a triazine group, a carbodiimide group, or an active esterified carbonyl group. In addition, when these organic EL dyes have a carboxylic acid group, it is possible to directly modify the amino group and imino group present in the protein in the presence of a carbodiimide derivative or a triazine derivative. An anionic group such as a sulfonyl group or a carboxyl group can be used as the reactive group that forms an ionic bond. These anionic groups are ionically bonded to a cationic group of the protein, such as an amino group.

In addition, the fluorescent dye can be provided with a spacer portion for connecting the color forming portion and the reactive group. The spacer part is a part containing a covalent bond or an atomic chain, and is —CH ₂ —, —NHCOO—, —CONH—, —COO—, —SO ₂ NH—, —HN—C (═NH) —NH—, — O -, - S -, - NR- (R is an alkyl _{group), - (CH 2 -CH 2} -O) n- (n is an integer of from 1 to 10), - CH = CH -, - C≡C- , One containing at least one functional group selected from the group consisting of —Ar— and —CO—Ar—NR— can be used. That is, the spacer portion may be composed of only one type of functional group selected from the above group or may be composed of two or more types of functional groups. Moreover, it can also be set as the structure containing 2 or more of the selected one functional groups.

Here, it is preferable to use what is represented by the following general formula (I) for a spacer part.
-(CHR ') pX- (CHR ") q- (I)
In the formula, X is a direct bond or -NHCOO-, -CONH-, -COO-, -SO ₂ NH-, -HN-C (= NH) -NH-, -O-, -S-, -NR- , —CH═CH—, —C≡C—, —Ar—, and —CO—Ar—NR— can be used, and at least one functional group selected from the group consisting of —COO—, — CONH-, -O-, -CH = CH-, -C≡C- or -Ar-, more preferably -COO-, -CONH-, -O- or -Ar- can be used. R ′ and R ″ are each independently a hydrogen atom, an aliphatic hydrocarbon group such as an alkyl group or an alkenyl group which may contain an aromatic ring, or an aromatic hydrocarbon group, and if necessary, a sulfonyl group, A group substituted with any one charged group selected from the group consisting of a hydroxyl group, a quaternary amine group and a carboxyl group can be used, and Ar is an aryl group, preferably a phenylene group or naphthylene. A group substituted with a sulfonyl group as necessary, p and q are each independently an integer of 0 to 20, preferably an integer of 0 to 10, more preferably an integer of 0 to 5; Yes, p + q ≧ 1.
Specific examples of the spacer part include-(CH ₂ ) p-CONH- (CH ₂ ) q-,-(CH ₂ ) p-COO- (CH ₂ ) q-,
-(CH ₂ ) p-CH (-R'-SO ₃ H)-(CH ₂ ) q-,-(CH ₂ ) p-CH (-R'-N ⁺ H ₃ )-(CH ₂ ) q- ,-(CH ₂ ) p-CH (-R'-COOH)-(CH ₂ ) q-,-(CH ₂ ) p-CH (-R'-OH)-(CH ₂ ) q-,-(CH ₂ ) p- (O-CH-) n- (CH ₂ ) q-,-(CH ₂ ) p-CONH (-R'-SO ₃ H)-(CH ₂ ) q-,-(CH ₂ ) p -CONH (-R'-SO ₃ H)-(CH ₂ ) q-,-(CH ₂ ) p-CONH (-R'-N ⁺ H ₃ )-(CH ₂ ) q-,-(CH ₂ ) p-CONH (-R'-OH)-(CH ₂ ) q-,-(CH ₂ ) p-CONH (-R'-COOH)-(CH ₂ ) q-,-(CH ₂ ) p-COO- R '(-SO ₃ H)-(CH ₂ ) q-,-(CH ₂ ) p-COO-R' (-OH)-(CH ₂ ) q-,-(CH ₂ ) p-COO-R ' (-N ⁺ H ₃ )-(CH ₂ ) q-,-(CH ₂ ) p-COO-R '(-COOH)-(CH ₂ ) q-,-(CH ₂ ) p-Ar- (CH ₂ ) q-,-(CH ₂ ) p- (Ar-COO)-(CH ₂ ) q-,-(CH ₂ ) p- (Ar-SO ₃ H)-(CH ₂ ) q-,-(CH ₂ ) p- (Ar-N ⁺ H ₃ )-(CH ₂ ) q-,-(CH ₂ ) p- (Ar-OH)-(CH ₂ ) q-,-(CH ₂ ) p- (Ar-COOH) )-(CH ₂ ) q-,-(CH ₂ ) pC≡C- (CH ₂ ) q-,-(CH ₂ ) pC = C- (CH ₂ ) q-,-(CH ₂ ) p-NR- (CH ₂ ) q-,-(CH ₂ ) pO- (CH ₂ ) q-,-(CH ₂ ) pS- (CH ₂ ) q-,-(CH ₂ ) p-HN-C (= NH)- NH- (CH ₂ ) q-,-(CH ₂ ) p-CO-Ar-NR- (CH ₂ ) q- and the like can be mentioned.

  Moreover, the peptide linker which consists of an amino acid or 2-20 amino acids can also be used for a spacer part. Natural or synthetic amino acids can be used as amino acids. Here, natural amino acids include glycine, alanine, valine, leucine, isoleucine, serine, homoserine, threonine, aspartic acid, glutamic acid, asparagine, glutamine, lysine, hydroxylysine, arginine, cysteine, cysteic acid, 2-amino-3 -Sulfosulfanylpropanoic acid, 2-amino-3-sulfoxypropanoic acid, cystine, methionine, phenylalanine, tyrosine, tryptophan, histidine, proline, 4-hydroxyproline and the like.

Synthetic amino acids include D-forms of the above natural amino acids and modified amino acids having at least an amino group and a carboxyl group in the molecule.
The modified amino acid can be represented by the general formula: HN (R ₁ )-(R ₂ —CO) —OH. Here, R ₁ and R ₂ are each independently a sulfonyl group, a hydroxyl group, a quaternary amine group, and a carboxyl group, with or without an ester, ether, thioester, thioether, amide, carbamide, or thiocarbamide. An aliphatic hydrocarbon group, an aromatic group, or a heterocyclic group substituted with any one charged group selected from the group consisting of: Further, the aliphatic hydrocarbon group, aromatic group or heterocyclic group may be substituted with at least one of a halogen atom, an alkyl group, an alkenyl group, an alkynyl group or an alkoxy group, respectively.

  More preferred amino acids for use in the spacer portion of the present invention are amino acids having a sulfonyl group, such as cysteic acid, 2-amino-3-sulfosulfanylpropanoic acid, 2-amino-3-sulfoxypropanoic acid, and a hydroxyl group. It is any one selected from the group consisting of tyrosine, threonine, and serine. This is because the water solubility of the fluorescent dye can be improved. More preferred is cysteic acid, homoserine or serine.

Peptide linkers include -C (-R ₁ ) -CONH-C (-R ₂ )-, -C (-R ₁ ) -CONH-C (-R ₂ ) -CONH-C (-R ₃ ), respectively. -, -C (-R ₁ ) -CONH-C (-R ₂ ) -CONH-C (-R ₃ ) -CONH-C (-R ₄ )- It is preferable. Here, R ₁ , R ₂ , R ₃ and R ₄ are a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alcohol group, an indole group, a hydroxyphenyl group, a benzyl group, a guanidine group, a thioether group, and an alkylthiol group. Represents a substituent such as an imidazole group or an alkylamine group. These peptides may be homo or hetero peptides. As specific examples, Ala-Ser, Glu-Ala, Glu-Ala-Leu, Gly-Pro, Gly-Pro-Asn, Ile-Val, Ile-Val-Met and the like can be used.

  Moreover, what has at least 1 sort (s) of charged group selected from the group which consists of a sulfonyl group, a hydroxyl group, a quaternary amine group, and a carboxyl group can be used for a part of peptide linker if needed. For example, a peptide linker containing one or more amino acids having any one of these charged groups can be used. Thereby, the water solubility of the fluorescent dye can be improved. For example, selected from the group comprising cysteic acid having a sulfonyl group, 2-amino-3-sulfosulfanylpropanoic acid, 2-amino-3-sulfoxypropanoic acid, tyrosine having a hydroxyl group, threonine, homoserine, serine Peptide linkers containing at least one amino acid can be used.

  By changing the length of the spacer part and its structure, it is possible to change the distance between the coloring part and the labeling site of the antibody molecule, thereby suppressing steric hindrance between the antibody molecule and the fluorescent dye. In other words, the structure of the fluorescent dye can be designed so as to suppress steric hindrance in accordance with the three-dimensional structure of proteins, peptides, etc. having a complicated structure, so that the labeling rate can be improved. Alternatively, by introducing a functional group that gives rigidity to the spacer part, for example, —CH═CH—, —C≡C—, —Ar—, and —CO—Ar—NR—, a specific labeling site, such as It is also possible to increase the steric hindrance with respect to the deeply labeled site. This allows selective labeling of only the marked part of the steric hindrance, for example, the shallow part, while distinguishing the deep part and the shallow part of the labeling part by labeling the deeply labeled part with another fluorescent dye having little steric hindrance. It is also possible to do.

When a reactive group is introduced into the fluorescent dye of the present invention, for example, the reaction shown in Scheme 1 can be used. Reaction formula (I) shows an example in which an active esterified carbonyl group is used as a reactive group, and -COO- is used as a functional group of the spacer portion bonded to the reactive group. For the active esterified carbonyl group, N-hydroxy-succinimide ester or maleimide ester can be used. By using N-hydroxy-succinimide and using DCC as the condensing agent, the organic EL dye and the antibody are bound by an amide bond via the N-hydroxy-succinimide ester.
In addition, the reaction formula (II) shows an example in which a triazine derivative is used for the active esterified carbonyl group, and -COO- is used for the functional group of the spacer portion bonded to the reactive group.
Further, the reaction formula (III) shows an example in which a carbodiimide group is used as a reactive group and -COO- is used as a functional group of a spacer portion bonded to the reactive group. As the carbodiimide group, a carbodiimide reagent such as N, N′-dicyclohexylcarbodiimide (DCC) or 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide can be used. An organic EL dye and an antibody can be bound by an amide bond via a carbodiimide body.
Reaction formula (IV) shows an example in which a carbodiimide group or a triazine group is introduced in advance into the spacer portion, that is, an example in which the functional group of the spacer portion bonded to the reactive group also serves as the reactive group. Thus, the fluorescent dye can be directly bonded to the amino group and imino group of the antibody without separately introducing a reactive group into the fluorescent dye.

スキーム１．

Scheme 1.

Preferred organic EL dyes used for fluorescent dyes include compounds containing a conjugated 5-membered ring compound, wherein the 5-membered ring compound contains one or more heteroatoms, selenium atoms, or boron atoms. it can. More specifically, a monocyclic compound composed of a 5-membered ring compound having a conjugated system and a condensed polycyclic compound composed of a 6-membered ring compound having a conjugated system with the 5-membered ring compound can be exemplified. This is because even in the solid state, the quantum yield is large and strong fluorescence is exhibited. The 5-membered ring compound is preferably an azole derivative or an imidazole derivative. Furthermore, the azole derivative or imidazole derivative preferably has one or more quaternary ammonium groups. It is because water solubility can be improved.
Below, the specific example of a condensed polycyclic compound is demonstrated.

(Monoazole derivative 1)

Here, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ are each independently a hydrogen atom, a halogen atom, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkyl ester group, phosphoric acid as a substituent. An aromatic hydrocarbon group, an aliphatic hydrocarbon group or a heterocyclic group which may have an ester group, a sulfate ester group, a nitrile group, a hydroxyl group, a cyano group, a sulfonyl group, an aromatic hydrocarbon group or a heterocyclic group; R ₁ , R ₂ , R ₃ , R ₄ , R ₅ may be the same or different. The alkyl group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. The alkenyl group is preferably a vinyl group, an allyl group, a crotyl group, a tiglyl group, or a prenyl group. The alkynyl group is preferably an ethynyl group or a propargyl group. The alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group or a phenoxy group. The aromatic hydrocarbon group includes a monocyclic or polycyclic ring, preferably a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a phenyl group. The heterocyclic group is preferably a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group or a quinoline group, more preferably a furan group, an imidazole group or a thiophene. It is a group.
R ′ represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group such as an alkyl group or an alkenyl group which may contain an aromatic ring. Here, as the alkyl group and the alkenyl group aromatic hydrocarbon group, those similar to the above can be used.
An ⁻ represents a halide ion such as Cl ⁻ , Br ⁻ or I ⁻ , CF ₃ SO ₃ ⁻ , BF ₄ ⁻ or PF ₆ ⁻ . The same applies to the following general formulas unless otherwise specified.

(Monoazole derivative 2)

Here, in the formula, R ₈ and R ₉ are each independently a hydrogen atom, a halogen atom, a substituent as an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkyl ester group, a phosphate ester group, a sulfate ester group. , A nitrile group, a hydroxyl group, a cyano group, a sulfonyl group, an aromatic hydrocarbon group which may have an aromatic hydrocarbon group or a heterocyclic group, an aliphatic hydrocarbon group or a heterocyclic group, R ₈ , R ₉ may be the same or different. The alkyl group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. The alkenyl group is preferably a vinyl group, an allyl group, a crotyl group, a tiglyl group, or a prenyl group. The alkynyl group is preferably an ethynyl group or a propargyl group. The alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group or a phenoxy group. The aromatic hydrocarbon group includes a monocyclic or polycyclic ring, preferably a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a phenyl group. The heterocyclic group is preferably a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group or a quinoline group, more preferably a furan group, an imidazole group or a thiophene. It is a group.
The same applies to the following general formulas unless otherwise specified. N is an integer of 1 or more, preferably 1 to 5, and the same applies to the following general formulas.

(Diazole derivative 1)

(Diazole derivative 2)

(Diazole derivative 3)

Here, in the formula, R ₁ , R ₂ , R ₃ and R ₄ are each independently a hydrogen atom, a halogen atom,
Each independently a hydrogen atom, a halogen atom, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkyl ester group, a phosphate ester group, a sulfate ester group, a nitrile group, a hydroxyl group, a cyano group, a sulfonyl group, An aromatic hydrocarbon group, an aliphatic hydrocarbon group or a heterocyclic group which may have an aromatic hydrocarbon group or a heterocyclic group, and R ₁ , R ₂ , R ₃ , R ₄ , R ₆ , R ₇ May be the same or different. R ₂ and R ₃ may be an aromatic hydrocarbon group which may have a substituent, preferably a phenyl group, and the substituent may be an alkyl group having 1 to 4 carbon atoms, an alkoxy group, or bromine. It is preferable to use atoms. Furthermore, it is preferable to use a methyl group for the alkyl group and a methoxy group for the alkoxy group. X is a nitrogen atom, sulfur atom, oxygen atom, selenium atom or boron atom which may have a substituent, and the same applies to the following general formulas unless otherwise specified.

(Diazole derivative 4)

ここで、N→Oは、窒素原子が酸素原子に配位結合している状態を示す。(Diazole derivative 5)

Here, N → O indicates a state in which a nitrogen atom is coordinated to an oxygen atom.

(Diazole derivative 6)

(Diazole derivative 7)

(Diazole derivative 8)

Here, in the formula, R ₁₀ and R ₁₁ are each independently a hydrogen atom, a halogen atom, a substituent such as an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkyl ester group, a phosphate ester group, a sulfate ester group. , A nitrile group, a hydroxyl group, a cyano group, a sulfonyl group, an aromatic hydrocarbon group which may have an aromatic hydrocarbon group or a heterocyclic group, an aliphatic hydrocarbon group or a heterocyclic group, R ₁₀ , R ₁₁ may be the same or different. The alkyl group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. The alkenyl group is preferably a vinyl group, an allyl group, a crotyl group, a tiglyl group, or a prenyl group. The alkynyl group is preferably an ethynyl group or a propargyl group. The alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group or a phenoxy group. The aromatic hydrocarbon group includes a monocyclic or polycyclic ring, preferably a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a phenyl group. The heterocyclic group is preferably a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group or a quinoline group, more preferably a furan group, an imidazole group or a thiophene. It is a group. R ₁₂ is an olefin group or a paraffin group which may have a substituent, and n is an integer of 1 to 3, preferably 1. The same applies to the following general formulas unless otherwise specified.

(Diazole derivative 9)

(Triazole derivative 1)

(Triazole derivative 2)

As the 5-membered ring compound, the following derivatives containing a thiophene group can also be used.
(Thiophene derivative 1)

(Thiophene derivative 2)

(Thiophene derivative 3)
In the case of a thiophene derivative, it is a non-condensed compound, and a 2,3,4,5-tetraphenylthiophene derivative represented by the following general formula can also be used.

Here, in the formula, R ₁₃ , R ₁₄ and R ₁₅ are each independently a hydrogen atom or a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted aryl group, preferably phenyl. Represents a group, a tolyl group, a xylyl group or a naphthyl group, or a substituted or unsubstituted aralkyl group, preferably a benzyl group or a phenethyl group, Ar ₁ and Ar ₂ are substituted or unsubstituted aryl groups, preferably a phenyl group Represents a tolyl group, a xylyl group or a naphthyl group, and Ar ₁ and Ar ₂ may form a nitrogen-containing heterocycle together with the nitrogen atom to which Ar ₁ and Ar ₂ are bonded. Y ₁ and Y ₂ are a hydrogen atom, a halogen atom, a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms, or a linear, branched or cyclic alkoxy group, preferably a methoxy group, an ethoxy group, Propoxy group, isopropoxy group, butoxy group, pentyloxy group or phenoxy group, or substituted or unsubstituted aryl group, preferably phenyl group, tolyl group, xylyl group or naphthyl group, or substituted or unsubstituted aralkyl group, preferably A benzyl group or a phenethyl group, or a substituted or unsubstituted amino group is represented.

(Thiophene derivative 4)
In addition, 2,3,4,5-tetraphenylthiophene derivatives represented by the following general formula can also be used.

Here, in the formula, Ar _{1 to} Ar ₆ each independently represents a substituted or unsubstituted aryl group, preferably a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and Ar ₁ and Ar ₂ , Ar ₃ And Ar ₄ and Ar ₅ and Ar ₆ may form a nitrogen-containing heterocycle together with the nitrogen atom to which they are bonded.

  Further, imidazole can be used as the five-membered ring compound and an imidazole derivative represented by the following general formula can be used. Here, the imidazole group constituting the imidazole derivative preferably has a quaternary ammonium group. It is because water solubility can be improved. Further, when a pyridino group is included, the pyridino group may also have a quaternary ammonium group in order to further improve water solubility. In the general formula below, R ″ represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group such as an alkyl group or an alkenyl group which may contain an aromatic ring.

(Imidazole derivative 1)

(Imidazole derivative 2)

(Imidazole derivative 3)

(Imidazole derivative 4)

Here, the imidazole skeleton may have a plurality of units bonded to any position of the central benzene ring R ₈ , R ₉ , R ₁₀ , R ₁₁ . R ₁₂ is an olefin group or a paraffin group which may have a substituent, and n is an integer of 1 to 3, preferably 1.

(Carbazole derivative)
Moreover, the carbazole derivative shown with the following general formula can also be used.

  Moreover, it is a 5-membered ring compound which has a conjugated system, and the monocyclic compound containing 1 or more types of hetero atoms, a selenium atom, or a boron atom can also be used. Although not particularly limited, for example, an imidazole derivative represented by the following general formula can be used.

Here, in the formula, R ₁ , R ₄ and R ₅ are each independently a hydrogen atom, a halogen atom, an alkyl group, alkenyl group, alkynyl group, alkoxy group, hydroxyl group, cyano which may have a substituent. Group, a sulfonyl group, a heterocyclic group which may have a substituent, an aromatic hydrocarbon group which may have a substituent such as a heterocyclic group, and R ₁ , R ₄ and R ₅ are the same or different It may be. The alkyl group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. The alkenyl group is preferably a vinyl group, an allyl group, a crotyl group, a tiglyl group, or a prenyl group. The alkynyl group is preferably an ethynyl group or a propargyl group. The alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group or a phenoxy group. The aromatic hydrocarbon group includes a monocyclic or polycyclic ring, preferably a biphenyl group, a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a biphenyl group or a phenyl group. The heterocyclic group is preferably a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group or a quinoline group, more preferably a furan group, an imidazole group or a thiophene. It is a group.

  The organic EL dye used in the fluorescent dye of the present invention is not particularly limited as long as it is the condensed polycyclic compound and monocyclic compound described above, but a diazole derivative or imidazole derivative represented by the following general formula is preferably used. be able to.

  Furthermore, among the above diazole derivatives and imidazole derivatives, diazolopyridine derivatives or imidazolopyridine derivatives can be suitably used.

  Particularly preferred fluorescent dyes of the present invention contain the above-mentioned diazolopyridine derivative or imidazolopyridine derivative in the coloring portion, and can be represented by the following general formula.

-(CHR ') pX- (CHR ") q- represents a spacer portion. Z represents the reactive group described above.
Here, it is preferable to use an aromatic hydrocarbon group or an aliphatic hydrocarbon group which may have a substituent for R ₂ and R ₃ described above. A green fluorescent dye corresponding to Cy3 can be obtained. The aromatic hydrocarbon group is a phenyl group, a tolyl group, a xylyl group or a naphthyl group, more preferably a phenyl group or a tolyl group. Furthermore, as a substituent, a sulfonium group is preferable. It is because water solubility can be improved.

Alternatively, R ₂ and R ₃ are each selected from the group consisting of an optionally substituted thiophene group, furan group, pyrrole group, imidazole group, oxazole group, thiazole group, pyrazole group and pyridine group. Seeds, more preferably thiophene groups, imidazole groups or furan groups can also be used. A red fluorescent dye corresponding to Cy5 can be obtained.

The fluorescent dye can be synthesized by various methods by combining the reactive group and the spacer portion. For example, when an active esterified carbonyl group is used as a reactive group, an active ester form of a diazolopyridine derivative or an imidazolopyridine derivative is synthesized in advance, and a spacer compound (for example, glycine, alanine, etc.) is synthesized on the active ester form. , 4-aminobutanoic acid, cysteic acid, serine, etc.) are reacted to obtain a carboxylic acid form, and this carboxylic acid form is reacted with N-hydroxy-succinimide to obtain an active ester form in which a spacer is introduced. be able to. For example, when glycine is used as the spacer compound, a spacer portion having —CONH— and — (CH ₂ ) — can be obtained. Further, when β-alanine is used, a spacer portion having —CONH— and — (CH ₂ ) ₂ — can be obtained. In addition, when 4-aminobutanoic acid is used, a spacer portion having —CONH— and — (CH ₂ ) ₃ — can be obtained. In addition, when cysteic acid is used, a spacer portion having —CONH— and —SO ₃ ^— can be obtained. In addition, when serine is used, a spacer portion having -CONH- and -OH can be obtained. By using cysteic acid and serine, a sulfonium group and a hydroxyl group can be introduced into the spacer portion, respectively, and the water solubility of the fluorescent dye can be improved.

  As another embodiment of the present invention, an azole derivative or imidazole derivative having a nitrogen cation-containing group can also be used. As the azole derivative, those represented by the following general formula (9), (10) or (11) can also be used.

Here, R ₁ in (9) and (11), and _one of R ₁ and R _{4 in} (10) is represented by the general formula LM, where M is a pyridinium group, a secondary amino group, a tertiary amino group. A nitrogen cation-containing group which is a group, quaternary amino group, piperidinium group, piperazinium group, imidazolium group, thiazolium group, oxazolium group, quinolium group, benzoimidazolium group, benzothiazolium group or benzoxazolium group , L is a direct bond _{or, - (CH 2) n- (} n is an integer of 1 to 4), - NHCOO -, - CONH -, - COO -, - SO 2 NH -, - HN-C (= NH ) -NH-, -O-, -S-, -NR- (R is an alkyl group), -Ar- (Ar is an aromatic hydrocarbon group) and -CO-Ar-NR- A linker composed of one or more functional groups and linking M and the coloring portion is shown. It is preferable to use a linker. The linker relaxes the steric hindrance between the coloring part and the biomolecule to be labeled, and facilitates the binding between the binding part and the labeling part of the biomolecule, so it can give a higher labeling rate. is there.

In addition, the remainder of R ₁ and R ₄ of (10) and R ₂ and R ₃ are each independently a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, an alkyl ester group, a phosphate ester group, a sulfuric acid group as a substituent. An aromatic hydrocarbon group, an aliphatic hydrocarbon group or a heterocyclic group which may have an ester group, a nitrile group, a hydroxyl group, a cyano group, a sulfonyl group, an aromatic hydrocarbon group or a heterocyclic group is shown. The alkyl group is, for example, a linear or branched alkyl group having 1 to 6 carbon atoms. Moreover, said alkoxy group is a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group, or a phenoxy group, for example. The alkyl ester group is a linear or branched alkyl ester having 1 to 6 carbon atoms. The aromatic hydrocarbon group is a monocyclic or polycyclic aryl group, specifically a phenyl group, a tolyl group, a xylyl group or a naphthyl group, more preferably a phenyl group. The heterocyclic group is, for example, a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group, or a quinoline group, and more preferably a furan group, an imidazole group, or a thiophene group. It is. The aliphatic hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. Preferably, R ₂ and R ₃ are each independently one selected from the group consisting of thiophene derivatives, furan derivatives, pyrrole derivatives, imidazole derivatives, oxazole derivatives, thiazole derivatives, pyrazole derivatives, pyridine derivatives and quinoline derivatives. is there. Alternatively, preferably, R ₂ and R ₃ are aryl groups having a sulfonyl group.

  X represents a carbon atom, nitrogen atom, sulfur atom, oxygen atom, selenium atom or boron atom which may have a substituent.

  R ′ represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group composed of an alkyl group which may contain an aromatic ring. Here, the aliphatic hydrocarbon group or the aromatic hydrocarbon group may be the same as described above.

An ⁻ represents a halide ion such as Cl ⁻ , Br ⁻ or I ⁻ , CF ₃ SO ₃ ⁻ , BF ₄ ⁻ or PF ₆ ⁻ .

  Although the example of the diazole derivative was shown as an azole derivative which has a nitrogen cation containing group, the triazole derivative represented by the following general formula can also be used. Even if a triazole derivative is used, the same effect as in the case of a diazole derivative can be obtained.

Here, R ₁ in (17) and (19), and _one of R ₁ and R _{7 in} (18) is represented by the general formula LM, where M is a pyridinium group, a secondary amino group, a tertiary amino group. A nitrogen cation-containing group which is a group, quaternary amino group, piperidinium group, piperazinium group, imidazolium group, thiazolium group, oxazolium group, quinolium group, benzoimidazolium group, benzothiazolium group or benzoxazolium group , L is a direct bond _{or, - (CH 2) n- (} n is an integer of 1 to 4), - NHCOO -, - CONH -, - COO -, - SO 2 NH -, - HN-C (= NH ) -NH-, -O-, -S-, -NR- (R is an alkyl group), -Ar- (Ar is an aromatic hydrocarbon group) and -CO-Ar-NR- A linker composed of one or more functional groups and linking M and the coloring portion is shown. It is preferable to use a linker.

Further, the remainder of R ₁ and R ₇ in (18) and R ₂ and R ₃ are each independently a hydrogen atom, a halogen atom, a substituent as an alkyl group, an alkoxy group, an alkyl ester group, a phosphate ester group, a sulfuric acid group An aromatic hydrocarbon group, an aliphatic hydrocarbon group or a heterocyclic group which may have an ester group, a nitrile group, a hydroxyl group, a cyano group, a sulfonyl group, an aromatic hydrocarbon group or a heterocyclic group is shown. The alkyl group is, for example, a linear or branched alkyl group having 1 to 6 carbon atoms. Moreover, said alkoxy group is a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group, or a phenoxy group, for example. The alkyl ester group is a linear or branched alkyl ester having 1 to 6 carbon atoms. The aromatic hydrocarbon group is a monocyclic or polycyclic aryl group, specifically a phenyl group, a tolyl group, a xylyl group or a naphthyl group, more preferably a phenyl group. The heterocyclic group is, for example, a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group, or a quinoline group, and more preferably a furan group, an imidazole group, or a thiophene group. It is. The aliphatic hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. Preferably, R ₂ and R ₃ are each independently one selected from the group consisting of thiophene derivatives, furan derivatives, pyrrole derivatives, imidazole derivatives, oxazole derivatives, thiazole derivatives, pyrazole derivatives, pyridine derivatives and quinoline derivatives. is there. Alternatively, preferably, R ₂ and R ₃ are aryl groups having a sulfonyl group directly or across a carbon chain.

  R ′ represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group composed of an alkyl group which may contain an aromatic ring. Here, the aliphatic hydrocarbon group or the aromatic hydrocarbon group may be the same as described above.

An ⁻ represents a halide ion such as Cl ⁻ , Br ⁻ or I ⁻ , CF ₃ SO ₃ ⁻ , BF ₄ ⁻ or PF ₆ ⁻ .

  The imidazole derivative having a nitrogen cation-containing group can be represented by the following general formula.

Here, _one of R ₁ and R ₄ in (12), (14) and (15), and any one of R ₁ , R ₄ and R ₅ in (13) and (16) is represented by the general formula LM M is a pyridinium group, secondary amino group, tertiary amino group, quaternary amino group, piperidinium group, piperazinium group, imidazolium group, thiazolium group, oxazolium group, quinolium group, benzoimidazolium group, benzoimidazole group A nitrogen cation-containing group which is a thiazolium group or a benzoxazolium group, and L is a direct bond or — (CH ₂ ) n— (n is an integer of 1 to 4), —NHCOO—, —CONH— , -COO-, -SO ₂ NH-, -HN-C (= NH) -NH-, -O-, -S-, -NR- (R is an alkyl group), -Ar- (Ar is an aromatic carbonization) A linker that comprises one or more functional groups selected from the group consisting of (hydrogen group) and -CO-Ar-NR-, and connects M and the coloring portion. It is preferable to use a linker.

Further, (12), (14) and the remainder of R ₁ and R ₄ in (15), (13) and R _1, the remainder of R ₄ and R ₅ in (16) and R _2, R _3, are each Independently, a hydrogen atom, a halogen atom, a substituent such as an alkyl group, an alkoxy group, an alkyl ester group, a phosphate ester group, a sulfate ester group, a nitrile group, a hydroxyl group, a cyano group, a sulfonyl group, an aromatic hydrocarbon group or a complex An aromatic hydrocarbon group, an aliphatic hydrocarbon group or a heterocyclic group which may have a cyclic group is shown. The alkyl group is, for example, a linear or branched alkyl group having 1 to 6 carbon atoms. Moreover, said alkoxy group is a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group, or a phenoxy group, for example. The alkyl ester group is a linear or branched alkyl ester having 1 to 6 carbon atoms. The aromatic hydrocarbon group is a monocyclic or polycyclic aryl group, specifically a phenyl group, a tolyl group, a xylyl group or a naphthyl group, more preferably a phenyl group. The heterocyclic group is, for example, a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group, or a quinoline group, and more preferably a furan group, an imidazole group, or a thiophene group. It is. The aliphatic hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. Preferably, R ₂ and R ₃ are each independently one selected from the group consisting of thiophene derivatives, furan derivatives, pyrrole derivatives, imidazole derivatives, oxazole derivatives, thiazole derivatives, pyrazole derivatives, pyridine derivatives and quinoline derivatives. is there. Alternatively, preferably, R ₂ and R ₃ are aryl groups having a sulfonyl group.

  R ′ and R ″ each represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group composed of an alkyl group that may contain an aromatic ring. Here, the aliphatic hydrocarbon group or the aromatic hydrocarbon group includes: The thing similar to the above can be used.

An ⁻ represents a halide ion such as Cl ⁻ , Br ⁻ or I ⁻ , CF ₃ SO ₃ ⁻ , BF ₄ ⁻ or PF ₆ ⁻ .

In any of the azole derivatives and imidazole derivatives having the aforementioned nitrogen cation-containing group, _{linker, - (CH 2) n- (} n is an integer of 1 to 4), - NHCOO -, - CONH -, - COO-, -SO ₂ NH-, -HN-C (= NH) -NH-, -O-, -S-, -NR- (R is an alkyl group), -Ar- (Ar is an aromatic hydrocarbon group) and- It may be composed of only one kind of functional group selected from the group consisting of CO—Ar—NR—, or may be composed of two or more kinds of functional groups. Moreover, it can also be set as the structure containing 2 or more of the selected one functional groups.

For example, in the case of constituting only one kind of functional group, —CONH—, —O—, —COO— or — (CH ₂ ) _n —, more preferably —COO— or — (CH ₂ ) _n —, Preferably,-(CH ₂ ) _n- . Here, _{n in} — (CH ₂ ) _n — is preferably 1 or 2.

Moreover, when comprised by 2 or more types of functional groups, it can be set as the following aspects.
(1) When composed of two types of functional groups
_{-CONH-COO -, - CH 2} -O- or -CH ₂ -NR- is preferred.
(2) When comprised by 3 or more types of functional groups (i) What is represented by the following general formula (I) can be used.
-(CHR ') pX- (CHR ") q- (I)
In the formula, X is a direct bond or -NHCOO-, -CONH-, -COO-, -SO ₂ NH-, -HN-C (= NH) -NH-, -O-, -S-, -NR- , -Ar- and -CO-Ar-NR- can be used, preferably at least one functional group selected from the group consisting of -COO-, -CONH-, -O- or -Ar- Preferably, -COO-, -CONH-, -O- or -Ar- can be used. R ′ and R ″ are each independently an aliphatic hydrocarbon group or an aromatic hydrocarbon group comprising a hydrogen atom or an alkyl group which may contain an aromatic ring, and if necessary, a sulfonyl group, a hydroxyl group, 4 A group substituted with any one charged group selected from the group consisting of a primary amino group and a carboxyl group can be used, and Ar is an aryl group, preferably a phenylene group or a naphthylene group. And p and q are each independently an integer of 0 to 20, preferably an integer of 0 to 10, more preferably an integer of 0 to 5, and p + q ≧ 1.
Specific examples of the linker include-(CH ₂ ) p-CONH- (CH ₂ ) q-,-(CH ₂ ) p-COO- (CH ₂ ) q-,
-(CH ₂ ) p-CH (-R'-SO ₃ H)-(CH ₂ ) q-,-(CH ₂ ) p-CH (-R'-N ⁺ H ₃ )-(CH ₂ ) q- ,-(CH ₂ ) p-CH (-R'-COOH)-(CH ₂ ) q-,-(CH ₂ ) p-CH (-R'-OH)-(CH ₂ ) q-,-(CH ₂ ) p- (O-CH-) n- (CH ₂ ) q-,-(CH ₂ ) p-CONH (-R'-SO ₃ H)-(CH ₂ ) q-,-(CH ₂ ) p -CONH (-R'-SO ₃ H)-(CH ₂ ) q-,-(CH ₂ ) p-CONH (-R'-N ⁺ H ₃ )-(CH ₂ ) q-,-(CH ₂ ) p-CONH (-R'-OH)-(CH ₂ ) q-,-(CH ₂ ) p-CONH (-R'-COOH)-(CH ₂ ) q-,-(CH ₂ ) p-COO- R '(-SO ₃ H)-(CH ₂ ) q-,-(CH ₂ ) p-COO-R' (-OH)-(CH ₂ ) q-,-(CH ₂ ) p-COO-R ' (-N ⁺ H ₃ )-(CH ₂ ) q-,-(CH ₂ ) p-COO-R '(-COOH)-(CH ₂ ) q-,-(CH ₂ ) p-Ar- (CH ₂ ) q-,-(CH ₂ ) p- (Ar-COO)-(CH ₂ ) q-,-(CH ₂ ) p- (Ar-SO ₃ H)-(CH ₂ ) q-,-(CH ₂ ) p- (Ar-N ⁺ H ₃ )-(CH ₂ ) q-,-(CH ₂ ) p- (Ar-OH)-(CH ₂ ) q-,-(CH ₂ ) p- (Ar-COOH) )-(CH ₂ ) q-,-(CH ₂ ) p-NR- (CH ₂ ) q-,-(CH ₂ ) pO- (CH ₂ ) q-,-(CH ₂ ) pS- (CH ₂ ) q-,-(CH ₂ ) p-HN-C (= NH) -NH- (CH ₂ ) q-,-(CH ₂ ) p-CO-Ar-NR- (CH ₂ ) q- Can do. More _{preferably, - (CH 2) p-} CONH- (CH 2) q- or _{- (CH 2) p-COO-} (CH 2) a q-.
(ii) Moreover, what is represented by the following general formula (II) can also be used.
-Y- (CHR ₃ ) rZ- (II)
Here, Y and Z are each independently, -NHCOO -, - CONH -, - COO -, - SO 2 NH -, - HN-C (= NH) -NH -, - O -, - S-, One functional group selected from the group consisting of —NR—, —Ar— and —CO—Ar—NR—, and preferably Y and Z are —CONH—, —COO— and —COO—, respectively. And -COO-, -COO- and -NR-, more preferably -COO- and -COO-, and -COO- and -NR-. R3 is a hydrogen atom or an aliphatic hydrocarbon group or an aromatic hydrocarbon group comprising an alkyl group which may contain an aromatic ring, and if necessary, a sulfonyl group, a hydroxyl group, a secondary amino group, a tertiary amino group A group substituted with any one charged group selected from the group consisting of a group, a quaternary amino group and a carboxyl group can be used. Ar is an aryl group, preferably a phenylene group or a naphthylene group, and those substituted with a sulfonyl group as necessary can be used. r is an integer of 0 to 20, preferably an integer of 0 to 10, more preferably an integer of 0 to 5. Specific examples of the spacer portion include -CONH- (CH ₂ ) r-COO-, -CONH-CH (-R ₃ -OH) -COO-, -CONH-CH (-R ₃ -COOH) -COO -, -CONH-CH (R ₃ -SO ₃ H) -COO-, -COO- (CH ₂ ) r-COO- and the like.

Moreover, a peptide linker can also be used for a linker. Peptide linkers include -C (-R ₁ ) -CONH-C (-R ₂ )-, -C (-R ₁ ) -CONH-C (-R ₂ ) -CONH-C (-R ₃ ), respectively. -, -C (-R ₁ ) -CONH-C (-R ₂ ) -CONH-C (-R ₃ ) -CONH-C (-R ₄ )- It is preferable. Here, R ₁ , R ₂ , R ₃ and R ₄ are a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alcohol group, an indole group, a hydroxyphenyl group, a benzyl group, a guanidine group, a thioether group, and an alkylthiol group. Represents a substituent such as an imidazole group or an alkylamino group. These peptides may be homo or hetero peptides. As specific examples, Ala-Ser, Glu-Ala, Glu-Ala-Leu, Gly-Pro, Gly-Pro-Asn, Ile-Val, Ile-Val-Met and the like can be used.

  The fluorescent dye having a nitrogen cation-containing group of the present invention can improve the labeling rate for biomolecules due to its high water solubility, and can detect biomolecules with high sensitivity. As a result, the amount of the labeling dye to be used can be greatly reduced, so that the cost for detecting the target molecule can be greatly reduced.

  Moreover, a coupling | bond part can be provided in a nitrogen cation containing group. As the bonding portion, a reactive group that binds to a biomolecule to form a covalent bond or an ionic bond can be used.

  As the covalent bond, for example, when an amide bond, an imide bond, a urethane bond, a thiourethane bond, an ester bond, or a guanidine bond is formed, the reactive group includes an amino group, an imino group, a thiol group, and a carboxyl group of a biomolecule. Or the functional group which can react with a hydroxyl group is preferable. The functional group includes, for example, isothiocyanate group, isocyanate group, epoxy group, sulfonyl halide group, acyl chloride group, halogenated alkyl group, glyoxal group, aldehyde group, triazine group, carbodiimide group, maleimide group and active esterification. The carbonyl group etc. which were made can be used. Preferably, any one selected from an isothiocyanate group, an isocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group, and an active esterified carbonyl group is preferably used. More preferably, any one selected from an isothiocyanate group, an isocyanate group, an epoxy group, a halogenated alkyl group, a triazine group, a carbodiimide group, and an active esterified carbonyl group is preferably used. More preferred are a triazine group, a carbodiimide group, or an active esterified carbonyl group. As the functional group of the nitrogen cation-containing group that reacts with these reactive groups, for example, a carboxyl group can be used. For example, N-hydroxy-succinimide ester and maleimide ester can be used for the carbonyl group which has been activated esterified. When N-hydroxy-succinimide is used and DCC is used as a condensing agent, the fluorescent dye and the biomolecule are bonded by an amide bond via the N-hydroxy-succinimide ester. As the carbodiimide group, a carbodiimide reagent such as N, N′-dicyclohexylcarbodiimide (DCC) or 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide can be used. A fluorescent dye and a biomolecule can be bound by an amide bond via a carbodiimide body.

  An anionic group or a cationic group can be used as the reactive group that forms an ionic bond. As the anionic group, for example, a sulfonyl group or a carboxyl group can be used. These anionic groups are ionically bonded to a cationic group of the biomolecule, such as an amino group. Further, as the cationic group, a nitrogen cation-containing group such as a quaternary ammonium group or a pyridinium group can be used. These cationic groups are ionically bonded to an anionic group of the biomolecule, such as a carboxyl group. In the present invention, the nitrogen cation-containing group bonded to the color forming portion can also serve as a cationic group as a reactive group.

In the azole derivative and imidazole derivative having a nitrogen cation-containing group, the combination of the linker and the nitrogen cation-containing group is not particularly limited, but — (CH ₂ ) n- (n is an integer of 1 to 4) is used for the linker. The nitrogen cation-containing group is preferably a pyridinium group, a piperidinium group, a piperazinium group or a quaternary amino group.

The measurement method using the diagnostic agent of the present invention is a method for detecting an antigen in a subject with an antibody labeled with a fluorescent dye, and not only a combination of an antigen and an antibody, but also a hapten-antihapten antibody, biotin-avidin, A Tag-anti-Tag antibody combination can also be used.

Specifically, a binding substance such as an antibody labeled with a fluorescent dye is allowed to act on an antigen or hapten present on a substrate, in solution, on a bead, or on an antibody, and the antigen or hapten-specific binding ability of the antibody. To detect a specific antigen or hapten. Antigens include proteins, polysaccharides, nucleic acids, peptides and the like, and haptens include low molecular weight molecules such as FITC and dinitrophenyl groups. Examples of the combination of an antigen or hapten and an antibody include GFP and an anti-GFP antibody, FITC and an anti-FITC antibody, and the like.
As a specific example, the following method can be used.
(1) A method of detecting by binding a fluorescently labeled antibody to an antigen (protein, polysaccharide, nucleic acid, peptide) present in a substrate or a solution.
(2) A method of detecting by binding a fluorescently labeled anti-hapten antibody to an antigen (protein, polysaccharide, nucleic acid, peptide) modified with a hapten in a substrate or a solution.
(3) A method in which fluorescence-labeled avidin is bound to an antigen (protein, polysaccharide, nucleic acid, peptide) modified with biotin in the substrate or solution for detection.
(4) A method in which an antibody is bound to an antigen (protein, polysaccharide, nucleic acid, peptide) present in the substrate or solution, and an antibody labeled with a fluorescent dye that specifically binds to the antibody is bound to perform detection.
(5) Detection is performed by binding an antibody modified with a hapten to an antigen (protein, polysaccharide, nucleic acid) present in the substrate or solution, and further binding an antibody labeled with a fluorescent dye that specifically binds to the hapten. Method.
(6) Detection is performed by binding an antibody modified with biotin to an antigen (protein, polysaccharide, nucleic acid) present in the substrate or solution, and further binding avidin labeled with a fluorescent dye that specifically binds to the biotin. Method.
(7) A method in which a tag (histidine or the like) is introduced into an antigen (protein, polysaccharide, nucleic acid) present in a substrate or a solution, and detection is performed with an anti-Tag antibody labeled with a fluorescent dye.
(8) A method in which an antibody is bound to a protein labeled with a fluorescent dye and bound to an antigen (protein, polysaccharide, nucleic acid) present in a substrate or a solution for detection.
These labeled products can be used in various measurement techniques such as immunostaining, ELISA, Western blotting, and flow cytometry.
Further, a specific example will be described. For example, as shown in FIG. 1, when an IgG antibody is treated with pepsin, a fragment called F (ab ′) ₂ is obtained. When this fragment is reduced with dithiothreitol or the like, a fragment called Fab ′ is obtained. Fab ′ fragments have one or two thiol groups (—SH). A specific reaction can be carried out by allowing a maleimide group to act on the thiol group. That is, as shown in FIG. 2, the antibody can be labeled with the fluorescent dye by reacting the fluorescent dye introduced with the maleimide group with the thiol group of the fragment. In this case, the physiological activity (antigen capturing ability) of the antibody is not lost. In addition, in the method of binding a fluorescent dye and an antibody to a protein such as albumin (BSA), two or more fluorescent dyes can be introduced into the protein, so that it can be detected with higher sensitivity when bound to an antigen. It becomes possible.

EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, the scope of the present invention is not limited by the following example.
Synthesis Example 1
As the organic EL dye, 1, 2, 5, -oxadiazolo- [3, 4-c] pyridine derivative was used.
The reaction examples of the active ester form of oxadiazolo- [3,4-c] pyridine with a spacer moiety introduced (Schemes 2 and 3) are shown below. In addition, an active ester having no spacer portion is abbreviated as EL-OSu, and an active ester having a spacer portion introduced is abbreviated as EL-OSu-Sp.

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Scheme 2.

  Next, the oxadiazolopyridine active ester (6) was reacted with alanine in DMF to synthesize a carboxylic acid (7) having a spacer moiety introduced. Thereafter, the carboxylic acid form (7) was reacted with N-hydroxysuccinimide in dioxane to synthesize an oxadiazolopyridine active ester form (8) into which a spacer portion was introduced. A reaction example is shown below.

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Scheme 3.

The reaction proceeded gently in each step, and the desired active ester (8) was obtained in high yield via the carboxylic acid (7).
(Synthesis procedure)
(1) Synthesis of diketone derivative (2)
In a 500 mL three-necked flask, 37.5 g (0.25 mol) of 4-methoxyacetophenone (1) and 0.15 g of sodium nitrite were dissolved in 100 mL of acetic acid. In a water bath, 100 mL of HNO ₃ dissolved in 100 mL of acetic acid was added dropwise over 2 hours. Thereafter, the mixture was stirred at room temperature for 2 days. The reaction mixture was slowly poured into 500 mL of water to produce a precipitate. The precipitate was filtered and dissolved in chloroform. The chloroform phase was washed with a saturated aqueous sodium bicarbonate solution and twice with a 10% NaCl aqueous solution. After dehydration with MgSO ₄ , chloroform was distilled off under reduced pressure to obtain 34.5 g (yield 78%) of oxadiazole-N-oxide (2).

(2) Synthesis of diketone derivative (3)
17.7 g (0.05 mol) of oxadiazole-N-oxide (2) was dissolved in 400 mL of acetonitrile in a 500 mL three-necked flask. Zn 12.0 g, AcOH 7 mL, and Ac ₂ O 20 mL were added thereto. The reaction was cooled in a water bath so that the reaction temperature did not exceed 30 ° C. The reaction was terminated by stirring for 12 hours. The reaction mixture was filtered to remove insolubles. Acetonitrile was distilled off under reduced pressure to obtain a residue. The residue was recrystallized from chloroform to obtain 10.2 g (yield 60%) of oxadiazole-N-oxide (3).

(3) Synthesis of oxadiazolopyridine ethyl ester (4)
In a 500 mL three-necked flask, 15.6 g (0.046 mol) of oxadiazole-N-oxide (3) was dissolved in 300 mL of butanol. Thereto was added 32.0 g (0.23 mol) of glycine ethyl ester hydrochloride. The mixture was heated under reflux for 24 hours. Butanol was distilled off under reduced pressure to obtain a residue. The residue was dissolved in 200 mL chloroform and washed with 10% HCl, saturated NaHCO ₃ , 10% NaCl. It was dried over MgSO ₄ and the solvent was distilled off. The obtained residue was recrystallized from chloroform to obtain 13.0 g (yield 70%) of oxadiazolopyridine ethyl ester (4).

(4) Hydrolysis of oxydiazolopyridine ethyl ester (4)
In a 500 mL three-neck flask, 3.0 g (0.007 mol) of oxadiazolopyridine ethyl ester (4) was dissolved in 200 mL of ethanol. KOH 0.62 g (0.01 mol) was added there. After heating at reflux for 5 hours, the reaction mixture was added to 200 mL of water. When concentrated hydrochloric acid was added dropwise to this aqueous solution to adjust to pH 1, precipitation occurred. The precipitate was filtered and dissolved in chloroform. The chloroform phase was washed with 10% aqueous NaHCO ₃ solution and water. Chloroform was distilled off to obtain a residue. The residue was recrystallized from water-ethanol (1: 1) to obtain 2.1 g (yield 81%) of oxadiazolopyridinecarboxylic acid (5).

(5) Synthesis of active ester (6)
In a 50 mL three-necked flask, 1.0 g (0.0026 mol) of oxadiazolopyridinecarboxylic acid (5) and 0.30 g (0.0026 mol) of N-hydroxysuccinimide were dissolved in 20 mL of DMF. N, N′-dicyclohexylcarbodiimide 0.54 g (0.0026 mol) was added dropwise thereto over 30 minutes. After dropping, the mixture was stirred at room temperature for 30 hours. DMF was distilled off under reduced pressure. The residue was isolated and purified by silica gel column chromatography (chloroform) to obtain 0.76 g (yield 62%) of an oxadiazolopyridine active ester (6).

(6) Synthesis of carboxylic acid form (7)
In a 50 mL three-necked flask, 100 mg (0.21 mmol) of oxadiazolopyridine active ester and 18.8 mg (0.21 mmol) of alanine were dissolved in 20 mL of DMF. Then, it stirred at room temperature for 12 hours. After completion of the reaction, DMF was distilled off under reduced pressure. The residue was isolated and purified by silica gel column chromatography (chloroform-methanol = 7: 3) to obtain 83 mg (yield 88%) of the carboxylic acid form (7).

(7) Synthesis of active ester (8) Next, 70 mg (0.16 mmol) of oxadiazolopyridinecarboxylic acid (7) and 18.0 mg (0.16 mmol) of N-hydroxysuccinimide were dissolved in 20 mL of DMF in a 50 mL three-necked flask. did. N, N'-dicyclohexylcarbodiimide 32.2 mg (0.16 mmol) melt | dissolved in DMF 5 mL was dripped at this over 30 minutes. After dropping, the mixture was stirred at room temperature for 30 hours. DMF was distilled off under reduced pressure. The residue was isolated and purified by silica gel column chromatography (chloroform) to obtain 75.8 mg (yield 89%) of active ester (8).

Synthesis Example 2
The oxadiazolopyridine derivative used in Synthesis Example 1 was used as the organic EL dye, and cysteic acid was used for the spacer portion. The oxadiazolopyridine active ester (6) was reacted with cysteic acid to synthesize a carboxylic acid (9) having a spacer moiety introduced. Thereafter, the carboxylic acid form (9) was reacted with N-hydroxysuccinimide in dioxane to synthesize an oxadiazolopyridine active ester form (10) having a spacer moiety introduced. A reaction example is shown below.

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Scheme 4.

Hereinafter, a synthesis procedure of only a portion different from Synthesis Example 1 is shown.
(1) Synthesis of carboxylic acid compound (9)
In a 50 mL three-necked flask, 100 mg (0.21 mmol) of oxadiazolopyridine active ester and 39 mg (0.23 mmol) of cysteic acid were dissolved in 20 mL of DMF. Then, it stirred at room temperature for 12 hours. After completion of the reaction, DMF was distilled off under reduced pressure. The residue was isolated and purified by silica gel column chromatography (chloroform-methanol = 7: 3) to obtain 98 mg (yield 88%) of the carboxylic acid form (9).

(2) Synthesis of active ester (10) Next, 80 mg (0.15 mmol) of oxadiazolopyridinecarboxylic acid (9) and 19 mg (0.17 mmol) of N-hydroxysuccinimide are dissolved in 20 mL of DMF in a 50 mL three-necked flask. did. To this, 35 mg (0.17 mmol) of N, N′-dicyclohexylcarbodiimide dissolved in 5 mL of DMF was added dropwise over 30 minutes. After dropping, the mixture was stirred at room temperature for 30 hours. DMF was distilled off under reduced pressure. The residue was isolated and purified by silica gel column chromatography (chloroform: methanol = 10: 1) to obtain 73 mg (yield 78%) of active ester (10).

Synthesis Example 3
The oxadiazolopyridine derivative used in Synthesis Example 1 was used as the organic EL dye, and serine was used for the spacer portion. The oxadiazolopyridine active ester (6) was reacted with serine to synthesize a carboxylic acid (11) having a spacer moiety introduced. Thereafter, the carboxylic acid form (11) was reacted with N-hydroxysuccinimide in dioxane to synthesize an oxadiazolopyridine active ester form (12) having a spacer moiety introduced. A reaction example is shown below.

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Scheme 5.

Hereinafter, a synthesis procedure of only a portion different from Synthesis Example 1 is shown.
(1) Synthesis of carboxylic acid compound (11)
In a 50 mL three-necked flask, 100 mg (0.21 mmol) of the oxadiazolopyridine active ester (6) and 26 mg (0.25 mmol) of serine were dissolved in 20 mL of DMF. Then, it stirred at room temperature for 12 hours. After completion of the reaction, DMF was distilled off under reduced pressure. The residue was isolated and purified by silica gel column chromatography (chloroform-methanol = 7: 3) to obtain 79 mg (yield 81%) of the carboxylic acid form (12).

(2) Synthesis of active ester (12) Next, 70 mg (0.15 mmol) of oxadiazolopyridinecarboxylic acid (11) and 19 mg (0.17 mmol) of N-hydroxysuccinimide were dissolved in 20 mL of DMF in a 50 mL three-necked flask. did. To this, 35 mg (0.17 mmol) of N, N′-dicyclohexylcarbodiimide dissolved in 5 mL of DMF was added dropwise over 30 minutes. After dropping, the mixture was stirred at room temperature for 30 hours. DMF was distilled off under reduced pressure. The residue was isolated and purified by silica gel column chromatography (chloroform: methanol = 10: 1) to obtain 61 mg (yield 72%) of the active ester (12).

Synthesis Example 4
The oxadiazolopyridine derivative used in Synthesis Example 1 was used as the organic EL dye, and alanylserine (Ala-Ser) was used as the peptide linker in the spacer portion. The oxadiazolopyridine active ester (6) was reacted with alanylserine to synthesize a carboxylic acid (13) having a spacer moiety introduced. Thereafter, the carboxylic acid form (13) was reacted with N-hydroxysuccinimide in dioxane to synthesize an oxadiazolopyridine active ester form (14) having a spacer moiety introduced. A reaction example is shown below.

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Scheme 6.

Hereinafter, a synthesis procedure of only a portion different from Synthesis Example 1 is shown.
(1) Synthesis of carboxylic acid form (13)
In a 50 mL three-necked flask, 100 mg (0.21 mmol) of the oxadiazolopyridine active ester (6) and 45 mg (0.25 mmol) of alanylserine were dissolved in 20 mL of DMF. Then, it stirred at room temperature for 10 hours. After completion of the reaction, DMF was distilled off under reduced pressure. The residue was isolated and purified by silica gel column chromatography (chloroform-methanol = 6: 4) to obtain 72 mg (yield 64%) of the carboxylic acid form (13).

(2) Synthesis of active ester (14) Next, oxadiazolopyridinecarboxylic acid (13) 60 mg (0.11 mmol) and N-hydroxysuccinimide 14 mg (0.12 mmol) were dissolved in DMF 15 mL in a 50 mL three-necked flask. did. To this, 25 mg (0.12 mmol) of N, N′-dicyclohexylcarbodiimide dissolved in 5 mL of DMF was added dropwise over 30 minutes. After dropping, the mixture was stirred at room temperature for 15 hours. DMF was distilled off under reduced pressure. The residue was isolated and purified by silica gel column chromatography (chloroform: methanol = 8: 2) to obtain 60 mg (yield 86%) of the active ester (14).

Synthesis Example 5
Hereinafter, a reaction example in the case of using a 3-ethyl ester pyridinium group bonded to an active ester as a nitrogen cation-containing group is shown.
The oxadiazolopyridine ethyl ester form (4) is subjected to a reduction reaction in the presence of NaBH ₄ to obtain a hydroxymethyl form (15), which is reacted with thionyl chloride to obtain a thiadiazolopyridine chloromethyl form (16). This was reacted with a 3-ethyl ester pyridine active ester (17) to synthesize a nitrogen cation (18).

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Scheme 7.

The synthesis procedure is shown below.
(1) Synthesis of chloromethyl compound (16)
In a 50 mL three-necked flask, 100 mg (0.35 mmol) of pyridine hydroxymethyl compound (15) was dissolved in 20 mL of SOCl ₂ . Then, it stirred at 60 degreeC for 10 hours. After completion of the reaction, SOCl ₂ was distilled off under reduced pressure. The residue was isolated and purified by silica gel column chromatography (chloroform-hexane = 8: 2) to obtain 72 mg (yield 62%) of chloromethyl compound (16).

(2) Synthesis of nitrogen cation (18) Thialazolopyridine chloromethyl (16) (100 mg, 0.3 mmol) and 3-ethyl ester pyridine active ester (17) (0.9 mmol, 206 mg) were combined with Toluene Dissolve in 5 ml and heat to reflux overnight. After completion of the reaction, suction filtration was performed, and the residue was vacuum-dried to obtain a nitrogen cation (18). The reaction is quantitative.
¹ H-NMR (CDCl ₃ ): 9.33 (s, 1H), 9.07 (d, 1H), 8.35-8.51 (m, 4H), 7.50-7.67 (m, 8H), 6.46 (s, 2H), 3.32 ( t, 2H), 3.10 (t, 2H), 2.80 (s, 4H).

Reference Example 1
(Labeling procedure)
300 μl of 6.5 mg / ml GFP antibody (SIGMA # G6539) solution was buffer-exchanged to 0.2 M sodium bicarbonate (pH 9.0) using Microcon YM-50. The oxadiazolopyridine active ester EL-Osu 6 was prepared as a 10 mg / ml DMSO solution, 25 μl of this was added, and the mixture was stirred at room temperature for 2 hours. After equilibrating the NAP-10 column with PBS (pH 7.4), the reaction solution was added, and the fraction containing the antibody labeled with the active ester EL-Osu 6 was collected in an Eppendorf tube (about 1 ml).

(Evaluation of labeled antibody activity)
Dispense 100 μl of His-Tag-attached EGFP (green fluorescent protein expressed from E. coli) solution (in PBS pH 7.4) to each well in a nickel titer plate (96 wells) and let stand at room temperature for 2 hours. The wells were washed 3 times with 100 μl of PBST buffer (buffer added with 0.05% Tween-20 in PBS) (using a plate washer) to immobilize EGFP on the plate. Then, 100 μl of GFP antibody solution (20 μg / ml, 2 μg / ml, 200 ng / ml, 20 ng / ml) modified with oxadiazolopyridine active ester EL-Osu 6 was dispensed into each well. After allowing to stand at 37 ° C. for 2 hours, each well was washed 3 times with 100 μl of PBST buffer (using a plate washer). Finally, the fluorescence of the plate was measured with a plate reader. The fluorescence was measured at an excitation wavelength of 400 nm and a fluorescence wavelength of 530 nm, and a filter of 515 nm was used.

(Measurement result)
The results of the activity test are shown in FIG. The GFP antibody labeled with EL-Osu was able to detect EGFP even when its concentration was 2 ng / 100 μL.

Comparative Example 1
The activity of the modified antibody was evaluated in the same manner as in Reference Example 1 except that Cy3 dye was used as the fluorescent dye. In order to detect EGPT, the concentration of the fluorescently labeled GFP antibody was required to be about 2 ng / 100 μL.

Reference Example 2
(Labeling procedure 1: labeling with fluorescent dye)
BSA (Bovine Serium Albumin) reacts with the oxadiazolopyridine active ester EL-OSu-Sp 8 containing the amino group of the lysine residue to form an amide bond and label the dye with BSA It was. Specifically, 400 μl of DMSO solution containing 35.82 μg of active ester (75.25 nmol) was added to 100 μl of carbonate buffer (pH 9.0) containing 1.0 mg (15.05 nmol) of BSA and shaken at room temperature for 24 hours. Thereafter, 0.1 M TEAA buffer (pH 7.0) was added so that the total amount was 1 ml, and 1.5 ml of components derived from BSA were collected using a NAP-10 column (GE healthcare Sephadex G-25). 100 μl of the resulting solution was analyzed by reverse phase HPLC.

  BSA labeled with EL-OSu was identified by MALDI TOF MS. The labeled BSA (FIG. 4A) had an increased molecular weight of about 2200 compared to the raw material (FIG. 4B), indicating that about 5 molecules of organic EL dye were bound.

(HPLC measurement conditions)
LC-2000plus series manufactured by JASCO Corporation was used as the HPLC apparatus.
Column: GL Science Inertsil ODS-3 Column 5μm, 4.6 mm x 250 mm
DNA gradient conditions
Eluting solvent A: 0.1M TFA aqueous solution (pH 7.0)
Eluting solvent B: 90% CH ₃ CN / 0.1M TFA aqueous solution (pH7.0)
Gradient (B%) 0 min (5%) → 20 min (50%) → 60 min (70%) → 70 min (100%) → 80 min (100%) → 90 min (5%)
Flow rate 0 min → 20 min, 60 min → 90 min: 1 mL / min, 20 min → 60 min: 0.5 mL / min
40 ℃

(Results of fluorescent dye labeling)
The HPLC results of BSA labeled with EL-OSu without a spacer part and EL-OSu-Sp with a spacer part are shown in FIGS. 5A and 5B, respectively. The labeling rate is shown in Table 1.

  By using EL-OSu-Sp having a spacer part, the labeling rate could be dramatically improved as compared with the case of using EL-OSu having no spacer part. It is considered that by introducing a spacer portion between the active ester which is a reactive group and the dye molecule, the steric hindrance between the labeling site of the BSA and the dye molecule is reduced, and thus the labeling rate is improved. Moreover, from the measurement result of TOF-MASS, it was confirmed that 5 molecules of organic EL dye were introduced into BSA labeled with EL-OSu. In the case of EL-OSu-Sp, it was possible to label almost quantitatively when 5 times mole was used to introduce 5 molecules.

(Labeling procedure 2: binding to antibody)
The labeled dye was prepared by binding the fluorescent dye-introduced BSA prepared in the above labeling procedure 1 to the GFP antibody in the same manner as in Reference Example 1.

(Evaluation of labeled antibody activity)
The same method as in Reference Example 1 was used.

(Results of labeled antibody activity)
As in Reference Example 1, the GFP antibody labeled with EL-Osu was able to detect EGFP even when its concentration was 2 ng / 100 μL.

Reference Example 3.
Reference Example 2 except that the active ester EL-Osu-Sp 10 of oxadiazolopyridine having a spacer portion synthesized in Synthesis Example 2 was used as the organic EL dye, and DMSO was changed to 10 μl to 10 vol% of the entire sample solution. A fluorescent dye was introduced into BSA by the same method. Binding to the antibody and activity evaluation of the labeled antibody were performed in the same manner as in Reference Example 1.

(result)
The labeling rate of the fluorescent dye was obtained at the same high labeling rate as in Reference Example 2. Furthermore, as for the activity of the labeled antibody, as in Reference Example 1, the GFP antibody labeled with EL-Osu-Sp 10 was able to detect EGFP even when its concentration was 2 ng / 100 μL. In Reference Example 2, DMSO was used at 80 vol% of the entire sample solution in order to dissolve the organic EL dye, but in this Reference Example, the organic EL dye was dissolved even at 10 vol% and showed excellent water solubility.

Reference Example 4
Reference Example 2 except that the active ester EL-Osu-Sp 12 of oxadiazolopyridine having a spacer moiety synthesized in Synthesis Example 3 was used as the organic EL dye and DMSO was made 10 μl to make 10 vol% of the entire sample solution. A fluorescent dye was introduced into BSA by the same method. Binding to the antibody and activity evaluation of the labeled antibody were performed in the same manner as in Reference Example 1.

(result)
The labeling rate of the fluorescent dye was obtained at the same high labeling rate as in Reference Example 2. Further, as for the activity of the labeled antibody, as in Reference Example 1, the GFP antibody labeled with EL-Osu-Sp 12 was able to detect EGFP even when its concentration was 2 ng / 100 μL. As in Reference Example 2, the fluorescent dye of this Reference Example also showed excellent water solubility.

Reference Example 5
Reference Example 2 except that the active ester EL-Osu-Sp 14 of oxadiazolopyridine having the spacer moiety synthesized in Synthesis Example 4 was used as the organic EL dye, and the DMSO was changed to 10 μl to 10 vol% of the entire sample solution. A fluorescent dye was introduced into BSA by the same method.

(result)
The labeling rate of the fluorescent dye was obtained at the same high labeling rate as in Reference Example 2. Further, as for the activity of the labeled antibody, as in Reference Example 1, the GFP antibody labeled with EL-Osu-Sp 14 was able to detect EGFP even when its concentration was 2 ng / 100 μL. As in Reference Example 2, the fluorescent dye of this Reference Example also showed excellent water solubility.

Example 1 .
1.0 mg of streptavidin was dissolved in 400 μl of 0.2 M sodium bicarbonate buffer (pH 8.3) to prepare a streptavidin solution. To the prepared streptavidin solution, 200 μl of a DMSO solution of a nitrogen cation (18) was added and stirred at room temperature for 1 hour. Thereafter, the reaction phase was filtered through Sephadex series G-25 to obtain labeled streptavidin.

  A rat spinal cord tissue piece was reflux fixed with 4% paraformaldehyde / 0.1 M Phosphate Buffer (pH 7.2 to 7.4), and then fixed by immersion for 3 hours. Washed with 0.1 M Phosphate Buffer containing 20% sucrose to prepare 10 μm frozen sections. After washing with 0.1 M Phosphate Buffer (pH 7.2 to 7.4), it was blocked with PBSTBF (0.1% PBS containing 0.8% fish gelatin, 1% bovine serum albumin, 0.2% Triton X-100). Next, an anti-glial fibrillary acidic protein (GFAP) mouse monoclonal antibody was placed, reacted at 4 ° C. for 14 hours, and washed with 0.1 M Phosphate Buffer. Biotin-labeled anti-mouse antibody / PBSTBF was placed and reacted at room temperature for 90 minutes. Then, it was washed with 0.1 M Phosphate Buffer. Next, nitrogen cation (18) -labeled streptavidin (1: 200 to 800) / PBSTBF was placed and reacted at room temperature for 90 minutes. Then, it was washed with 0.1 M Phosphate Buffer (pH 7.2 to 7.4). Then, it enclosed with glycerin: PBS (3: 1) and observed using the fluorescence microscope.

(result)
The results are shown in FIG. Blue fluorescence was detected in a piece of rat spinal cord that had been reacted with a biotin-labeled anti-mouse antibody and a nitrogen cation-labeled streptavidin.

  As described above, by using a fluorescent dye containing an organic EL dye, it is possible not only to have high fluorescence intensity even in a solid state, but also to reduce the amount of dye used for an antibody. Furthermore, by providing a spacer portion, in the case of conventional fluorescent dyes, a dye amount of about 200-fold mol was common sense, but this can be reduced to about 1/200. As a result, the amount of the fluorescent dye to be used can be greatly reduced, and the use of the diagnostic agent of the present invention can greatly reduce the cost of antigen detection. In addition, it is possible to eliminate a step of removing a large amount of unreacted dye after the labeling reaction, and detection can be performed in a shorter time.

Claims (13)

A diagnostic agent comprising at least an antibody and a fluorescent dye that labels the antibody,
Fluorescent dye, a coloring portion comprising an organic EL-dye, and a binding portion which binds to the antibody possess,
The organic EL dye is a condensed polycyclic compound composed of a 5-membered ring compound containing one or more heteroatoms, selenium atoms, or boron atoms and a 6-membered ring compound having a conjugated system,
The fused polycyclic compound is an azole derivative represented by any one of the following general formulas (9), (10) or (11), or the following general formulas (12), (13), (14), (15) or a diagnostic agent which is an imidazole derivative represented by any one of (16) .

(In the formula, R ₁ in (9) and (11), and _one of R ₁ and R _{4 in} (10) is represented by the general formula LM, and M is a pyridinium group which may have a substituent. Secondary amino group, tertiary amino group, quaternary amino group, piperidinium group, piperazinium group, imidazolium group, thiazolium group, oxazolium group, quinolium group, benzoimidazolium group, benzothiazolium group or benzoxazolium group A nitrogen cation-containing group which is a group, L is a direct bond or — (CH ₂ ) n— (n is an integer of 1 to 4), —NHCOO—, —CONH—, —COO—, —SO ₂ NH -, -HN-C (= NH) -NH-, -O-, -S-, -NR- (R is an alkyl group), -Ar- (Ar is an aromatic hydrocarbon group) and -CO-Ar- A linker consisting of one or more functional groups selected from the group consisting of NR-, linking M and the coloring moiety, the remainder of R ₁ and R _{4 in} (10) , and R ₂ and R ₃ are each independently Hydrogen atom, halogen source And may have an alkyl group, alkoxy group, alkyl ester group, phosphate ester group, sulfate ester group, nitrile group, hydroxyl group, cyano group, sulfonyl group, aromatic hydrocarbon group or heterocyclic group as a substituent. An aromatic hydrocarbon group, an aliphatic hydrocarbon group or a heterocyclic group; X represents an optionally substituted carbon atom, nitrogen atom, sulfur atom, oxygen atom, selenium atom or boron atom; and R 'is an aliphatic hydrocarbon group or aromatic hydrocarbon group consisting of an alkyl group which may contain an aromatic ring, an ^- is a halide ion, CF ₃ SO ₃ ^- shows a) ^-, BF ₄ ^- or PF _6.

(In the formula, _one of R ₁ and R ₄ in (12), (14) and (15) , and any one of R ₁ , R ₄ and R ₅ in (13) and (16) is represented by the general formula: M represents a pyridinium group, a secondary amino group, a tertiary amino group, a quaternary amino group, a piperidinium group, a piperazinium group, an imidazolium group, a thiazolium group, an oxazolium group, which may have a substituent. A nitrogen cation-containing group which is a quinolium group, a benzimidazolium group, a benzothiazolium group or a benzoxazolium group, and L is a direct bond or — (CH ₂ ) n— (n is an integer of 1 to 4) ), -NHCOO-, -CONH-, -COO-, -SO ₂ NH-, -HN -C (= NH) -NH-, -O-, -S-, -NR- (R is an alkyl group), A linker comprising at least one functional group selected from the group consisting of -Ar- (Ar is an aromatic hydrocarbon group) and -CO-Ar-NR-, and linking M and the coloring portion, (12), (14) and R ₁ and the balance R ₄ in (15), R (13) and (16) ₁ , the remainder of R ₄ and R ₅ , and R ₂ and R ₃ are each independently a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, an alkyl ester group, a phosphate ester group, a sulfate ester group as a substituent, A nitrile group, a hydroxyl group, a cyano group, a sulfonyl group, an aromatic hydrocarbon group or an aromatic hydrocarbon group which may have an aromatic group, an aliphatic hydrocarbon group or a heterocyclic group, R ′, R ″ Is an aliphatic hydrocarbon group or an aromatic hydrocarbon group composed of an alkyl group which may contain an aromatic ring, An ⁻ represents a halide ion, CF ₃ SO ₃ ⁻ , BF ₄ ⁻ or PF ₆ ⁻ )   Any one kind of reactivity in which the bonding part is selected from carboxylic acid group, isocyanate group, isothiocyanate group, epoxy group, halogenated alkyl group, triazine group, carbodiimide group, maleimide group and active esterified carbonyl group. The diagnostic agent according to claim 1, which has a group.   The diagnostic agent according to claim 1, wherein the fluorescent dye has a spacer portion that connects the coloring portion and the binding portion. The spacer part is -CH _2- , -NHCOO-, -CONH-, -COO-, -SO ₂ NH-, -HN-C (= NH) -NH-, -O-, -S-, -NR - (R is an alkyl _{group), - (CH 2 -CH 2} -O) n- (n is an integer of from 1 to 10), - CH = CH -, - C≡C -, - Ar- and -CO-Ar The diagnostic agent according to claim 3, comprising at least one functional group selected from the group consisting of -NR-. The diagnostic agent according to claim 4 , wherein the spacer portion is represented by the following general formula (I).
-(CHR ') pX- (CHR ") q- (I)
(Wherein, X is a direct bond or -NHCOO-, -CONH-, -COO-, -SO ₂ NH-, -HN-C (= NH) -NH-, -O-, -S-, -NR Represents at least one functional group selected from the group consisting of —, —CH═CH—, —C≡C—, —Ar—, and —CO—Ar—NR—, wherein R ′ and R ″ are each independently An aliphatic hydrocarbon group such as an alkyl group or an alkenyl group which may contain a hydrogen atom or an aromatic ring, or an aromatic hydrocarbon group, if necessary, from a sulfonyl group, a hydroxyl group, a quaternary amine group and a carboxyl group Represents an aryl group substituted with any one charged group selected from the group consisting of: Ar represents an aryl group, p and q each independently represents an integer of 0 to 20, and p + q ≧ 1 .) The diagnostic agent according to claim 3 , wherein the spacer portion is an amino acid or a peptide linker composed of 2 to 20 amino acids. The diagnostic agent according to claim 6 , wherein the spacer part is an amino acid, and the amino acid is a natural amino acid or a synthetic amino acid. The amino acid, cysteic acid, 2-amino-3-sulfonyl sulfur sulfonyl propanoic acid, 2-amino-3-sulphoxide propanoic acid, tyrosine, claim 7, wherein the one selected from the group consisting of threonine and serine Diagnostics. The diagnostic agent according to claim 6 , wherein the spacer portion is a peptide linker, and the peptide linker has at least one charged group selected from the group consisting of a sulfonyl group, a hydroxyl group, a quaternary amine group, and a carboxyl group. . The peptide linker includes at least one amino acid selected from the group consisting of cysteic acid, 2-amino-3-sulfosulfanylpropanoic acid, 2-amino-3-sulfoxypropanoic acid, tyrosine, threonine and serine The diagnostic agent according to claim 9 . Additional linker L _{is, - (CH 2) n- (} n is an integer from 1 to 4) and is claim 1, wherein the fluorescent dye. An assay method for detecting an antigen in a specimen, wherein the antibody labeled with the diagnostic agent according to claim 1 comprises a fluorescent dye having a coloring part composed of an organic EL dye and a binding part that binds to the antibody. It is reacted with, a method of measuring the fluorescence from the fluorescent dye. 2. The method for producing a diagnostic agent according to claim 1 , comprising at least an antibody and a fluorescent dye for labeling the antibody, wherein the fluorescent dye has a coloring part composed of an organic EL dye and a binding part that binds to the antibody. And at least one bond selected from the group consisting of an amide bond, an imide bond, a urethane bond, an ester bond and a guanidine bond between the organic EL dye and the antibody. Method.

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