JPH05287209A - Fluorescent labelling dye, biogenic substance labelled with the same, and reagent containing them - Google Patents

Abstract

PURPOSE:To obtain a reagent useful for analyzing various antigens and drugs in blood or for analyzing DNA sequence with a small semiconductor laser (670-780nm) by using a dye for fluorescent labeling comprising a compd. having a specified molecular structure. CONSTITUTION:This dye is represented by the formula, wherein A<1> and A<2> are each independently an arom. ring comprising carbon and hydrogen atoms or an arom. ring contg. nitrogen and/or oxygen and/or sulfur atoms in addition to carbon and hydrogen atoms; R<1>, R<2>, and R<3> are each independently an alkyl, alkoxy, aryloxy, aryl, or aralkyl group, etc.; X<1> is a sulfur, oxygen, or selenium atom, etc.; X<2> is a sulfur, oxygen, or selenium atom, C=0. CH=CH. etc.; and L is a polymethylene group. A reagent contg. the color or contg. a biogenic substance labeled with the color is applied in various analyses.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a fluorescent labeling dye, a biological substance labeled with the fluorescent labeling dye, and a reagent containing them.

[0002]

It has been reported that phthalocyanines can be used in various immunoassays (US Pat. No. 4,1).
60,645, US Patent 4,193,983, US Patent 4,220,450, US Patent 4,233,402, US Patent 4,235,869.
U.S. Pat. No. 4,256,834, U.S. Pat. No. 4,277,437, U.S. Pat. No. 4,318,70.
No. 7, US Pat. No. 4,483,929, US Pat. No. 4,540,660, US Pat. No. 4,540,6.
70, US Pat. No. 4,560,534, US
Patent 4,650,770, US Pat. No. 4,656,
129, US Pat. No. 4,659,676).

Further, phthalocyanines have been used as catalysts in chemiluminescent immunoassay systems [Bull. Che
m. Soc. Jpn. 56, 2965-2968 (1983), 56, 2267-2271 (1)
983), ibid., 57, 587-588 (198).
4), ibid. 57, 3009-3010 (198).
4), Vol. 58, p. 1299-1303 (198).
5)]. Hara et al. Have used iron phthalocyanine as a catalyst for the chemiluminescent reaction between luminol and hydrogen peroxide,
The analyte in the test sample is quantified from the amount of chemiluminescence signal. They investigated phthalocyanines of iron and cobalt and porphyrin complexes of iron, palladium, platinum, manganese and tin, and reported that iron phthalocyanine showed the best catalytic action and high sensitivity.

Fluorescent substances are widely used in addition to coloring substances in immunoassays. Furthermore, in enzyme immunoassay, fluorescent substances can be used more favorably than coloring substances because they can increase the sensitivity. Is becoming. The well-known fluorophore-enzyme pair is the alkaline phosphatase (al
kaline phosphatase) and 4-methylumbelliferyl phosphate, β-
Galactosidase (β-galactosidase) and 4-methylumbelliferyl-D-galactopyranoside (4-methy
lumbelliferyl-D-galactopyranoside), horseradish peroxidase and p-
P-hydroxyphenyl acetic aci
d) etc., and the detection sensitivity of these systems is 10 ⁻¹⁵ M. However, even if the detection sensitivity is further increased, the analytical characteristics of the fluorescent substance produced are limited.

Recently, reagents using phthalocyanines having a high fluorescence quantum yield and high solubility in water have been proposed (WO Patent 88/04777, WO 90/02747, and WO 90/02747). JP-A-1-233222).

[0006]

However, phthalocyanines have an absorption region and a fluorescence emission region of the Q-band of 65 or less.
Since it is in the region of 5 to 700 nm and overlaps with the absorption region (<700 nm) of heme and the like in the blood existing as an in-vivo substance, there is a defect that interferes with it.

It is considered that inexpensive and small semiconductor lasers (670 to 780 nm) will become the mainstream of radiation sources in the future, but when excited by a semiconductor laser of 670 to 690 nm, phthalocyanines have a fluorescent emission region similar to this. It is difficult to distinguish scattered light from the irradiation laser light and fluorescence emission because it is in the wavelength range of 700-700.
When excited with a semiconductor laser of 780 nm, the phthalocyanines are not excited because they cannot absorb light, and therefore do not serve as detection agents. Further, since phthalocyanines have a large π-conjugated system, they have a property of easily associating with each other, and there is a drawback that the fluorescence quantum yield is remarkably lowered when the associated product is formed.

The present invention is for measuring by using an inexpensive and small semiconductor laser (670 to 780 nm), which is not affected by in-vivo substances such as heme existing in blood and is expected to become the mainstream in the future. Another object of the present invention is to provide a reagent or a clinical test reagent useful for analysis of various antigens and drugs in blood, analysis of DNA base sequence, and the like.

[0009]

The present invention provides the following (1) to
It relates to (6). That is, (1) general formula (I)

[Chemical 2] [In the general formula (I), A ¹ and A ² are each independently an aromatic ring composed of a carbon atom and a hydrogen atom, or a nitrogen atom and / or an oxygen atom and / or a sulfur atom in addition to the carbon atom and the hydrogen atom. An aromatic ring consisting of R ¹ , R ² and R
³ are each independently an alkyl group, a heterocyclic residue, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an aryl group, an aralkyl group, an alkylcarbonyloxy group, an alkylcarbonyl group, an arylcarbonyl group, an alkyloxycarbonyl group. , An alkylamide group, an alkylsulfonamide group, an alkoxycarbonyl group, an aryloxycarbonyl group, an arylcarbonyloxy group, an arylamide group, an alkylamino group, an alkylcarbamoyl group, an alkylsulfamoyl group, an arylcarbamoyl group, an arylamino group, Aryl sulfonyl group, aryl sulfonamide group, hydroxyl group, carboxy group, halogen atom, aryl sulfamoyl group, cyano group, nitro group, sulfonic acid, sulfonate, carboxylate, this An alkyl group having these substituents,
Or a hydrogen atom, X ¹ is a sulfur atom, an oxygen atom, a selenium atom, NR ⁴ (provided that R ⁴ has the same meaning as R ^{1 to} R ³ )
X ² is a sulfur atom, an oxygen atom, a selenium atom, or C
═O, CH═CH, NR ⁵ or CR ⁶ R ⁷ (R ⁵ , R ⁶ and R ⁷ have the same meanings as R ^{1 to} R ³ ) and L represents a polymethylene group. ] The fluorescent labeling dye represented by (2) A reagent containing the fluorescent labeling dye of (1) above. (3) A biological substance labeled with the fluorescent labeling dye of (1) above. (4) A reagent containing the biological substance of (3) above. (5) The biological substance of (3) above, wherein the biological substance is a vitamin, an alkaloid or a nucleotide. (6) The reagent according to (4) above, wherein the biological substance is a vitamin, an alkaloid or a nucleotide.

In the compound represented by the general formula (I) of the present invention, benzene, naphthalene, anthracene, phenanthrene and the like are specific examples of the aromatic ring composed of carbon atoms and hydrogen atoms of A ¹ and A ² . Specific examples of the aromatic ring composed of a nitrogen atom and / or an oxygen atom and / or a sulfur atom in addition to the carbon atom and the hydrogen atom include pyridine, 1,
2-diazine, 1,3-diazine, 1,4-diazine,
Quinoline, isoquinoline, quinoxaline, 1,3-benzodiazine, 2,3-benzodiazine, 1,8-diazanaphthalene, 1,5-diazanaphthalene, 1,7-diazanaphthalene, 1,6-diazanaphthalene, Pyrrole,
Examples include imidazole, thiophene and furan. These aromatic rings can be fused at any possible position.

In the compound represented by the general formula (I) of the present invention, R ^{1 to} R ³ and an alkyl group of R ^{4 to} R ⁷ in X ¹ and X ² or an alkyl group-containing group Specific examples include methyl group, ethyl group, propyl group, sec-
There are propyl group, n-butyl group, iso-butyl group, t-butyl group, pentyl group, hexyl group and the like, and specific examples of the aryl group or the aryl group containing the aryl group include a phenyl group, a naphthyl group, There are anthranil group, phenanthrenyl group and the like, and specific examples of the heterocyclic residue are pyridyl group, pyridazyl group, pyrimidyl group, pyrazyl group,
Examples include a quinolyl group, an isoquinolyl group, a quinoxalyl group, a benzopyrimidyl group, a benzopyridazyl group, a diazanaphthyl group, a pyrrolyl group, an imidazolyl group, a thienyl group and a furyl group.

Examples of the polymethylene group represented by L include the groups represented by the following chemical formula 3.

[Chemical 3]

In the chemical formula 3, m and n are integers of 0 to 5,
Y is a hydrogen atom, a lower alkyl group such as a methyl group, a lower alkoxy group such as a methoxy group, a dimethylamino group, a diphenylamino group, a methylphenylamino group, a morpholino group, an imidazolidine group and an ethoxycarbonylpiperazine group. Group, alkylcarbonyloxy group such as acetoxy group, alkylthio group such as methylthio group,
It is a cyano group, a nitro group, a halogen atom or the like, and Z is a group for conjugating a double bond, specifically, -CH-, cyclohexadiene, cyclopentadiene, indene, 1,4-dihydro. Naphthalene, 1,2-
Dihydronaphthalene, 5,8-dihydroquinoxaline,
In addition to 5,6-dihydroquinoxaline, the groups shown in Chemical formula 4 and the like can be mentioned.

[Chemical 4] However, in Chemical formula 4, X ³ is N—R ⁸ (wherein R ⁸ represents an alkyl group), a sulfur atom or an oxygen atom.

In the present invention, the compound represented by the general formula (I) is synthesized by the method described in a reference book such as “Large Organic Chemistry, Nitrogen-containing Heterocyclic Compound I”, page 432 (Asakura Shoten). it can. Table 1 shows examples of the compound represented by the general formula (I).

[Table 1] Table 1 Examples of compounds represented by the general formula (I) ───────────────────────────────── ──── No. A ¹ X ¹ R ¹ L A ² X ² R ² R ³ ────────────────────────────── ─────── 1 of ₅ NC ₂ H 5 - reduction 9 of _{5 C (CH 3) 2 -} (CH 2) 3 SO 3 - 2 of ₅ NC ₂ H 5 - reduction 9 of 5 NC ₂ H _{5 - (CH 2) 3 SO 3} - 3 of ₅ NC ₂ H 5 - reduction 9 of _{6 S - (CH 2) 3} SO 3 - 4 of ₅ NC ₂ H 5 - reduction 9 of 6 C = O - (CH ₂ ) ₃ SO ₃ ^- 5 of 5 O CH ₃ of 10 of _{5 C (CH 3) 2 -} (CH 2) 3 SO 3 - 6 of 5 S CH ₃ of 10 of _{12 C (CH 3) 2 SO} 3 Na ( CH _{2) 4} SO ₃ ^- 7 of 5 Se CH ₃ of 10 of _{12 CH = CH SO 3 Na (} CH 2) 4 SO 3 - 8 of 6 N-CN - reduction 9 of _{13 CH = CH Cl (CH 2} ) ₂ COO ^- 9 of 6 N-CH ₃ - of 9 of _{5 C = O - (CH 2} ) 2 PO 3 - 10 of 7 of 8 CH ₃ of 9 of _{7 C (CH 3) 2 -} CH 3 · I - 11 of 7 of 8 - of 11 of _{7 N-CH 3 - C 2} H 5 · Cl - ───────────────────────── ────────── However, in Table 1, of 5 of 13 exhibits the following groups.

[Chemical 5]

[Chemical 6]

[Chemical 7]

Embedded image N- (CH ₂ ) ₃ COOH

Embedded image (CH = CH) ₂ -CH

[Chemical 10]

[Chemical 11]

[Chemical 12]

[Chemical 13]

The biological substances used in the present invention include proteins / peptides, nucleotides obtained from organisms such as animals, plants, microorganisms (including viruses),
There are sugars, lipids, hormones, vitamins, alkaloids, antibiotics, and their composites. These are extracted from nature, artificially completely synthesized, or artificially semi-synthesized. May be.

Specific examples of proteins / peptides include:
Serum albumin, IgG / IgA / IgM / IgD / I
Examples thereof include immunoglobulins such as gE, monoclonal antibodies against various proteins and leukocyte membrane antigens, enzymes such as peroxidase, glucose oxidase, and alkaline phosphatase. Specific examples of nucleotides include DNA, RNA, synthetic oligonucleotides, and synthetic polynucleotides. Nucleotides, ATP, CTP, GTP, TTP, UT
P, dATP, dCTP, dGTP, dTTP, dUT
P, ddATP, ddCTP, ddGTP, ddTT
P, ddUTP, derivatives thereof, and the like. Specific examples of the saccharide include glycogen, starch,
In addition to polysaccharides such as mannan, oligosaccharides, glucose, monosaccharides such as mannose are listed, lipids include phosphatidylcholine, phosphatidylethanolamine, fats and fatty acids, and hormones include insulin, growth hormone, oxytocin, vasopressin. , Peptide hormones such as secretin, epidermal growth factor, gastrin, glucagon and calcitonin, steroid hormones such as androgen, estrogen and hydrocortisone, and catecholamines such as adrenaline and noradrenaline. ₁ , B ₂ , B ₆ , B ₁₂ , p-aminobenzoic acid (hereinafter,
PABA), biotin, folic acid, vitamin C, vitamin D, vitamin E, and various other vitamins. Alkaloids include opium alkaloids such as morphine,
Tropane alkaloids such as atropine and scopolamine,
Examples include indole alkaloids such as vinblastine and vincristine, isoquinoline alkaloids such as lauren, and the like, and antibiotics include penicillin, cephalosporin, kanamycin, erythromycin, chloramphenicol, and the like.

In order to bind the fluorescent labeling dye to the biological substance, functional groups such as amino groups and hydroxyl groups in the biological substance and functional groups such as carboxyl group and sulfone group in the fluorescent labeling dye are used. Directly or ionically or covalently directly, or after chemical modification such as adding a linking group (linker) to a part of the biological substance so that the fluorescent labeling dye can react. , Just react. The biological substance labeled with the fluorescent labeling dye can be purified by a conventional separation means such as chromatography or recrystallization.

The compound represented by the general formula (I) has various absorption rings or fluorescence maximums in a long wavelength region (700 nm or more) by changing the aromatic ring or the polymethylene group, so that the maximum in the living body can be obtained. Since it is possible to eliminate the influence of various spectral interference factors, it can be used as a reagent or a clinical test reagent useful for analysis of various antigens and drugs in blood, analysis of nucleotide sequences of DNA, and the like.

[0019]

EXAMPLES The present invention will be described in more detail below with reference to examples. However, compound No. Is the compound N shown in Table 1.
o. Corresponding to. Example 1 Compound No. Synthesis of PABA adduct of 1 Compound N was prepared with reference to the method described in the literature (large organic chemistry, nitrogen-containing heterocyclic compound I, page 432 (Asakura Shoten)).
o. 1 was synthesized. Obtained compound No. 1 to 250 m
g, dissolved in chloroform, 2.0 ml of oxalyl chloride was added dropwise at 25 ° C., and the mixture was stirred at room temperature for 6 hours, then, the solvent was removed and Compound No. The solid sulfonyl chloride derivative (Chemical Formula 14) of 1 was obtained.

[Chemical 14] Further, 86 ml of sodium carbonate and p- in 1.5 ml of water.
50 mg of aminobenzoic acid (PABA) was added, and 51 mg of the sulfonyl chloride derivative of the above Chemical formula 14 was added to the solution dissolved by heating to 80 ° C., and after stirring at 80 ° C. for 6 hours,
The solvent was removed and the resulting solid was added with 10 wt% NH ₄ OH.
Dissolved in methanol and concentrated again to give compound N
o. A PABA adduct of 1 was obtained.

Example 2 Compound No. Synthesis of PABA adduct of 3 Compound N was prepared with reference to the literature method described in Example 1.
o. 3 was synthesized. Obtained compound No. 3 to 601 m
g, dissolve in chloroform, add dropwise 2.3 ml of oxalyl chloride at 25 ° C., stir at room temperature for 6 hours, remove the solvent, and remove compound No. A solid sulfonyl chloride derivative (Chemical Formula 15) of 3 was obtained.

[Chemical 15] Furthermore, 78 ml of sodium carbonate and p- in 1.5 ml of water.
Aminobenzoic acid (PABA) (50 mg) was added, and the solution obtained by heating to 80 ° C. and dissolving was added with the above-mentioned sulfonyl chloride derivative (50 mg) of Chemical formula 15 and stirred at 80 ° C. for 6 hours.
The solvent was removed and the resulting solid was added with 10 wt% NH ₄ OH.
Dissolved in methanol and concentrated again to give compound N
o. A PABA adduct of 3 was obtained.

Example 3 Compound No. Synthesis of PABA adduct of 5 Compound N was prepared with reference to the literature method described in Example 1.
o. 5 was synthesized. Obtained compound No. 5 to 510 m
g, dissolved in chloroform, 1.5 ml of oxalyl chloride was added dropwise at 25 ° C., and the mixture was stirred at room temperature for 6 hours, then, the solvent was removed and Compound No. Thus, the sulfonyl chloride derivative (Chemical Formula 16) of 5 was obtained.

[Chemical 16] Furthermore, 78 ml of sodium carbonate and p- in 1.5 ml of water.
50 mg of aminobenzoic acid (PABA) was added, and 49 mg of the sulfonyl chloride derivative of the above chemical formula 16 was added to the solution which was heated to 80 ° C. and dissolved, and after stirring at 80 ° C. for 6 hours,
The solvent was removed and the resulting solid was added with 10 wt% NH ₄ OH.
Dissolved in methanol and concentrated again to give compound N
o. A PABA adduct of 5 was obtained.

Example 4 Compound No. Synthesis of PABA adduct of 6 Compound N referring to the literature method described in Example 1
o. 6 was synthesized. Obtained compound No. 6 to 501 m
g, dissolved in chloroform, 1.5 ml of oxalyl chloride was added dropwise at 25 ° C., and the mixture was stirred at room temperature for 6 hours, then, the solvent was removed and Compound No. A sulfonyl chloride derivative (Chemical formula 17) of 6 was obtained.

[Chemical 17] Furthermore, 78 ml of sodium carbonate and p- in 1.5 ml of water.
Aminobenzoic acid (PABA) (50 mg) was added, and the solution obtained by heating to 80 ° C. and dissolving was added with the above-mentioned sulfonyl chloride derivative (50 mg) of Chemical formula 17 and stirred at 80 ° C. for 6 hours.
The solvent was removed and the resulting solid was added with 10 wt% NH ₄ OH.
Dissolved in methanol and concentrated again to give compound N
o. A PABA adduct of 6 was obtained.

Example 5 Compound No. 1, No. 3, N
o. 5 or No. Synthesis of Morphine Labeled with 6 Compound No. 6 obtained in Example 1 1 PABA adduct (10
0 mg) in triethylamine (1.5 ml),
At 0 ° C., 10 ml of ethyl chloroformate were added with stirring.
After stirring for 5 minutes, 3- (4-aminobutyl) morphine 4
0 mg was added, and the mixture was stirred at room temperature for 8 hours to give compound No. 1
The morphine labeled with was obtained. Similarly, compound N
o. Compound No. 1 instead of the PABA adduct. 3,
No. 5 and No. The reaction was performed using the PABA adduct of Compound No. 6 and compound No. 3, No. 5 and No. 6
The morphine labeled with was obtained.

Example 6 Affinity test for anti-morphine monoclonal antibody Morphine, aminomorphine and compound No. 1, N
o. 3, No. 5 or No. With respect to the morphine labeled with 6, the affinity for the anti-morphine monoclonal antibody (the affinity for morphine was 1.0, which is represented by a relative value) was measured by a competitive reaction with morphine labeled with tritium. The measurement results are shown in Table 2.

[Table 2] Table 2 Relative affinity of labeled morphine for antibody ────────────────────────── Compound (antigen) Relative affinity ── ──────────────────────── Morphine 1.0 Aminomorphine 1.0 Compound No. 1-labeled morphine 0.9 Compound No. 3 Labeled morphine 1.0 Compound No. 5 labeled morphine 1.0 Compound No. 6 Labeled morphine 1.0 ───────────────────────────

From the results shown in Table 2, there is almost no difference in the affinity depending on the compound (antigen), and even if morphine is labeled with the compound represented by the formula (I), the affinity with the antibody hardly changes. I understood.

Example 7 Compound No. Synthesis of oligonucleotide primer labeled with 1 and application to analysis of DNA base sequence (1) Synthesis of oligonucleotide primer to which linker is bound Primer (5 by an automatic DNA synthesizer using solid phase CED-phosphoramide method '-GTTTCCCAGT
CACGAC-3 ′) was synthesized. Phosphorylation of the synthesized primer was performed using 50 mM Tris-HCl (pH 7.6),
The incubation was carried out at 37 ° C. for 1 hour in a 100 μl reaction solution containing 10 mM magnesium chloride, 10 mM dithiothreitol, 3 mM ATP, and T ₄ -nucleotide kinase. The phosphorylated primer was separated by high performance liquid chromatography (HPLC) using a gel filtration column, the peaks of the phosphorylated primer were collected, and the solvent was removed by freeze-drying.

Next, this was mixed with 250 mM 1,2-diaminoethane (pH 6.0) and 200 mM ethyl-3 (3).
-Diethylaminopropyl) carbodiimide and 100
In 100 μl of a reaction solution containing mM N-methylimidazole (pH 6.0), the mixture was kept at 25 ° C. overnight and the linker [NH ₂ — (CH ₂ ) ₂ —N was added to the phosphate moiety of guanosine at the 5 ′ end.
H-] was attached.

(2) Compound No. Synthesis of oligonucleotide primer labeled with compound No. 1 Morphine labeled with 1 and an oligonucleotide primer having a linker bound to the phosphate moiety of the 5'-terminal guanosine synthesized in (1) above are mixed in a 0.2 M sodium carbonate buffer (pH 9.3), 25 ° C
After overnight incubation in the dark in the dark, it was purified by HPLC to give compound No. 1 via the linker. A primer to which 1 was bound was obtained.

(3) Analysis of DNA base sequence A DNA having a known base sequence was used as a sample and compound No. Sanger reaction was carried out with each of the four bases using the primer to which 1 was bound, followed by electrophoretic separation in separate lanes and analysis with a DNA sequencer equipped with a semiconductor laser with an oscillation wavelength of 780 nm. As a result, 99% of DNA up to 300 bases
Could be determined with the accuracy of.

[0030]

Industrial Applicability According to the present invention, a semiconductor laser (670 to 780 nm) which is not affected by in-vivo substances such as heme existing in blood and which is expected to become the mainstream in the future is required.
It was possible to provide a reagent or a clinical test reagent useful for the analysis of various antigens and drugs, the analysis of DNA base sequences, etc.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location G01N 33/533 8310-2J

Claims (6)

[Claims] 1. A compound represented by the general formula (I): [In the general formula (I), A ¹ and A ² are each independently an aromatic ring composed of a carbon atom and a hydrogen atom, or a nitrogen atom and / or an oxygen atom and / or a sulfur atom in addition to the carbon atom and the hydrogen atom. R ¹ , R ² and R ³ are each independently an alkyl group, a heterocyclic residue, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an aryl group, an aralkyl group, an alkylcarbonyloxy group. Group, alkylcarbonyl group, arylcarbonyl group, alkyloxycarbonyl group, alkylamide group, alkylsulfonamide group, alkoxycarbonyl group, aryloxycarbonyl group, arylcarbonyloxy group, arylamide group, alkylamino group, alkylcarbamoyl group, Alkylsulfamoyl group, arylcarbamoyl group, Arylamino group,
Arylsulfonyl group, arylsulfonamide group, hydroxyl group, carboxy group, halogen atom, arylsulfamoyl group, cyano group, nitro group, sulfonic acid,
A sulfonate, a carboxylate, an alkyl group having these as a substituent, or a hydrogen atom, X ¹ is a sulfur atom, an oxygen atom, a selenium atom, or NR ⁴ (wherein R ⁴ is R ^{1 to} R ³ ; X ² is a sulfur atom, an oxygen atom, a selenium atom, C═O, C
H = CH, NR ⁵ or CR ⁶ R ⁷ (provided that R ⁵ , R ⁶ and R ⁷ have the same meanings as R ^{1 to} R ³ ) and L represents a polymethylene group. ] The fluorescent labeling dye represented by 2. A reagent containing the fluorescent labeling dye according to claim 1. 3. A biological substance labeled with the fluorescent labeling dye according to claim 1. 4. A reagent containing the biological substance of claim 3. 5. The biological substance according to claim 3, which is a vitamin, an alkaloid or a nucleotide. 6. The reagent according to claim 4, wherein the biological substance is a vitamin, an alkaloid or a nucleotide.