JPS6223825B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides photochemical photochemical analysis of antibodies or antigens, and
This invention relates to an immunoassay method for qualitative or quantitative detection. In order to qualitatively or quantitatively detect antigens or antibodies, methods of labeling antigens or antibodies with radioisotopes and enzymes have already been used. Furthermore, in JP-A No. 55-116259, the present applicant labeled an antigen or antibody with a spectral sensitizing dye instead of a radioactive element or an enzyme and caused an immunoreaction, and the labeled antigen or antibody was labeled with a spectral sensitizing dye. The labeled antigen-antibody reaction product is separated, and either the labeled antigen or antibody or the labeled antigen-antibody reaction product is brought into contact with silver halide and exposed to light at a wavelength absorbed by the spectral sensitizing dye. A photochemical inspection method for trace components has been proposed, which is characterized by developing the exposed silver halide and measuring the resulting density. Sensitizing dyes are used. The present invention similarly uses a spectral sensitizer as a labeling substance, but in this case, it is preferable to use a merocyanine dye having a carboxyl group in a heterocyclic ring, that is, an acidic nucleus or a basic nucleus, as a spectral sensitizer. This is a characteristic feature. The present invention further includes labeling an antigen or antibody using a merocyanine dye having a carboxyl-containing substituent on the heterocycle as a labeling substance, and subjecting the thus obtained labeled antigen or antibody to an immunoreaction to form a reaction product. After separating the unreacted substances, or simultaneously with the separation, either one of them is brought into contact with silver halide, exposed to light of a wavelength that is absorbed by the corresponding merocyanine dye, and then the exposed silver halide is developed. , relates to a method for photochemically detecting an antigen or antibody by labeling the antibody or antigen with a labeling substance, the method comprising measuring the concentration obtained. The merocyanine used in the present invention can be represented by the following general formula (). In the formula, p represents a numerical value of 2 or 3, n represents a numerical value of 1 or 2, and Z is a group of nonmetallic atoms necessary to complete a 5- or 6-membered nitrogen-containing heterocycle (basic nucleus);
Q also represents a nonmetallic atomic group necessary to form a 5- or 6-membered nitrogen-containing heterocycle (acidic nucleus), and the heteroatoms include nitrogen, sulfur, selenium, and oxygen. R
represents an alkyl group having 1 to 18 carbon atoms, preferably 1 to 7 carbon atoms, and may be substituted with an aromatic group, an OH group, a sulfo group, etc. In this general formula (), a group containing a carboxy group is bonded to a nitrogen-containing heterocycle. Regarding acidic nuclei and basic nuclei, “The Theory of the Photographic Process”
(4th ed.)” Edited by THJames (1977,
See Chapter 8 (published by Macmillan). As the basic nucleus containing Z, thiazole,
4-Methylthiazole, 4-phenylthiazole, 4,5-dimethylthiazole, 4,5-diphenylthiazole, benzothiazole 4-chlorobenzothiazole, 5-chlorobenzothiazole, 6-chlorobenzothiazole, 7-chlorobenzo Thiazole, 5-nitrobenzothiazole,
6-nitrobenzothiazole, 4-methylbenzothiazole, 5-methylbenzothiazole, 6-
Methylbenzothiazole, 5-bromobenzothiazole, 6-bromobenzothiazole, 5-iodobenzothiazole, 5-phenylbenzothiazole, 5-methoxybenzothiazole, 6-methoxybenzothiazole, 5-ethoxybenzothiazole, 5-ethoxy Carbonylbenzothiazole, 5-phenethylbenzothiazole, 5-fluorobenzothiazole, 5-chloro-6-nitrobenzothiazole, 5-trifluoromethylbenzothiazole, 5,6-dimethylbenzothiazole, 5-hydroxy-6- Methylbenzothiazole, tetrahydrobenzothiazole, 4-phenylbenzothiazole, 5-phenylbenzothiazole, naphtho[2,1-d]thiazole, naphtho[1,2-d]thiazole, naphtho[2,3-
d] Thiazole, 5-methoxynaphtho[1,2-
d] Thiazole, 7-ethoxynaphtho[2.1-
d] Thiazole, 8-methoxynaphtho[2.1-
d] Thiazole, 5-methoxynaphtho[2,3-
d] Thiazole, oxazole, 4-methyloxazole, 4-nitrooxazole, 5-methyloxazole, 4-phenyloxazole, 4.
5-diphenyloxazole, 4-ethyloxazole, benzoxazole, 5-chlorobenzoxazole, 5-methylbenzoxazole,
5-bromobenzoxazole, 5-fluorobenzoxazole, 5-phenylbenzoxazole, 5-methoxybenzoxazole, 5-nitrobenzoxazole, 5-trifluorobenzoxazole, 5-hydroxybenzoxazole, 6-methylbenzoxazole, 6-chlorobenzoxazole, 6-nitrobenzoxazole, 6-methoxybenzoxazole, 6-
Hydroxybenzoxazole, 5,6-dimethylbenzoxazole, 4,6-dimethylbenzoxazole, 5-ethoxybenzoxazole, naphtho[2,1-d]oxazole, naphtho[1,2-d]oxazole, naphtho[2, 3-
d] Oxazole, 5-nitronaphtho[2.1-
d] Oxazole, 4-methylselenazole, 4
-Nitroselenazole, 4-phenylselenazole, benzoselenazole, 5-chlorobenzoselenazole, 5-nitrobenzoselenazole, 5-
Methoxybenzoselenazole, 5-hydroxybenzoselenazole, 6-nitrobenzoselenazole, 5-chloro-6-nitrobenzoselenazole, naphtho[2,1-d]selenazole, naphtho[1,2-d]selenazole, 1-alkylimidazole, 1-alkyl-4-phenylimidazole, 1-alkylbenzimidazole, 1-alkyl-5-chlorobenzimidazole, 1-alkyl-5,6-dichlorobenzimidazole, 1-alkyl-5- Methoxybenzimidazole, 1-alkyl-5-cyanobenzimidazole, 1-alkyl-5-fluorobenzimidazole, 1-alkyl-5-trifluoromethylbenzimidazole, 1-alkylnaphtho[1.
2-d] imidazole, 1-aryl-5,6-
Dichlorobenzimidazole, 1-aryl-5
-Chlorbenzimidazole, 1-arylimidazole, 1-arylbenzimidazole, 1
-aryl-5-chlorobenzimidazole, 1
-Aryl-5,6-dichlorobenzimidazole, 1-aryl-5-methoxybenzimidazole, 1-aryl-5-cyanobenzimidazole, 1-arylnaphtho[1,2-d]imidazole, etc. are preferred; , alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, 2-hydroxyalkyl, 3-hydroxypropyl, etc., and aryl represents phenyl, halogen, substituted phenyl, methyl substituted phenyl, methoxy substituted phenyl group. . Basic nuclei include 2-pyridine, 4-pyridine, 5-methyl-2-pyridine, 3-methyl-4-pyridine, and quinoline nuclei (e.g., 2-quinoline, 3-pyridine).
Methyl-2-quinoline, 5-ethyl-2-quinoline, 6-methyl-2-quinoline, 6-nitro-2
-quinoline, 8-fluoro-2-quinoline, 6-
Methoxy-2-quinoline, 6-hydroxy-2-
Quinoline, 8-chloro-2-quinoline, 4-quinoline, 6-ethoxy-4-quinoline, 6-nitro-4-quinoline, 8-chloro-4-quinoline, 8
-fluoro-4-quinoline, 8-methyl-4-quinoline, 8-methoxy-4-quinoline). The residues represented by R include methyl, ethyl,
Propyl, butyl, benzyl, β-phenylethyl, 2-hydroxyethyl, 2-methoxyethyl, 2-(2-methoxyethoxy)ethyl, carboxymethyl, 2-carboxyethyl, 3-carboxypropyl, 4-carboxybutyl, 2- Sulfoethyl, 3-sulfopropyl, 3-sulfobutyl, 4-sulfobutyl, 2-(pyrrolidine-2
-on-1-yl)ethyl, tetrahydrofurfuryl, 2-acetoxyethyl, carbomethoxymethyl, 2-methanesulfonylaminoethyl, and the like. Representative examples of groups containing a carboxy group to be included in general formula () include -COOH, -
CH ₂ COOH, −(CH ₂ ) ₂ COOH, −NH−
_CH2COOH , -NH- _{CH2CH2COOH ,}

【formula】

[Formula] −NHCOCH ₂ CH ₂ COOH, −NHCOCH ₂ CH ₂ CH ₂ COOH,

【formula】

【formula】

[Formula] −CONHCH ₂ COOH,

[Formula] −C ₆ H ₄ −COOH

【formula】

[Formula] −CONHCH ₂ CH ₂ COOH, −CO
(NHCH ₂ CO) ₂ OH, −CO (NHCH ₂ CO) ₃ OH −C ₆ H ₄ −CH ₂ COOH, −C ₆ H ₄ CH ₂ CH ₂ COOH,

[In the formula, D ₁ and D ₂ represent a fused polycyclic aromatic heterocyclic residue or an aromatic heterocyclic substituted amino group, and these may include a -SO ₃ M group. M represents hydrogen, alkali metal or ammonium. -A- represents a divalent aromatic residue, which may contain a _-SO3M group. However, when the above D ₁ and D ₂ do not contain a -SO ₃ M group, -A- needs to contain a -SO ₃ M group. ]

When a compound represented by the following formula is mixed as a stabilizer, the antigen or antibody labeled with a spectral sensitizing dye has high stability even in an aqueous solution, which is advantageous in carrying out the present invention. In addition, in order to carry out the photochemical inspection method of the present invention, when developing silver halide, the general formula (H) In the formula, R ¹ is an aryl group or a substituted aryl group R ²
When a hydrazine compound represented by hydrogen, an alkyl group, a substituted alkyl group, an aryl group, or a substituted aryl group is allowed to coexist, the optical density of the phenomenon silver or coloring dye increases, which is convenient for measurement. In this case, the density enhancer may be added at any step before the development operation. Hereinafter, reference examples will be used to demonstrate the reactivity of the spectral sensitizing dye of the present invention in the synthesis of N-hydroxysuccinimide ester using N·N'-dicyclohexylcarbodiimide. Reference example Modification of phenylalanine with dye (-1) Add 3.3 g (10 mmol) of dye (-1) and 1.15 g (10 mmol) of N-hydroxysuccinimide to 60 ml of
Dissolved in DMF and cooled to 5°C. N・N'-dichlorohexylcarbodiimide 2.5g
(12.1 mmol) was added and stirred for 5 hours. Furthermore, it was left in the refrigerator overnight. The precipitated N·N'-dicyclohexyl urea was collected and washed with 100 ml of DMF. The liquid and washing liquid were combined, 30 ml of water was added to the mixture, and the precipitated crystals were collected. The amount of N-hydroxysuccinic acid amide ester obtained here was 2.5 g (yield 58.5%). 15ml of this active ester 213.5mg (500μmol)
200mg of phenylalanine dissolved in DMF
(1.2 mmol) and 0.02 M Tris-HCl buffer (PH7.5)
It was added dropwise into a solution consisting of 10 ml at room temperature for about 1 hour. Then, after stirring for 2 hours and leaving it overnight,
The reaction mixture was poured into 100 ml of water, and the precipitated crystals were collected. After washing with water, methanol/methylene chloride =
Recrystallization was performed at a ratio of 1/1 to obtain 108 mg (yield 42.8%) of the target compound. Mass spectrum (FD) m/z = 477 (M ⁺ ) In the same manner, compound (-2) was used to modify phenylalanine to obtain the corresponding modified compound in good yield. . Example 1 Purified porcine insulin (manufactured by Sigma) 20mg
(3.5 μmole) in 1 ml of 4M urea, and
Add 8 ml of DMF (N·N-dimethylformamide) and stir under ice cooling (0 to 4°C) (liquid A).
Prepare three sets of 1 mg (2.7 μmole) of dye (-2) dissolved in 1 ml of DMF, add 1 μ each of isobutyl chloroformate and 0.5 μ each of triethylamine while cooling to −15 to −20°C, and then cool the same solution. Add 0.5 mg each of N-hydroxysuccinimide to the bottom (solution B). Next, the above-mentioned solution B was added to solution A at 5-minute intervals while stirring on ice, and the mixture was kept at room temperature for 30 minutes under ice-cooling.
After reacting for a minute, the mixture was desalted using a Sephadex G-10 column equilibrated with 0.2N aqueous ammonia and freeze-dried to obtain dye-labeled insulin. yield
21.5 mg (94% yield), λ ² % ^SDS _nax = 712 nm ε
_712o m
≈8.9×10 (approximately 2 mol dye/mol insulin) In the amino terminal analysis of this labeled product, glycine and phenylalanine, which are the amino terminals of porcine insulin, were not detected. Furthermore, in chromatography on a Sephadex G-50 column (equilibrated with 1% SDS), this labeled substance showed a single peak. This dye, insulin, was spectrally sensitized to a silver chlorobromide emulsion (bromide content: 70%) with a maximum wavelength of about 760 nm. Example 2 Human lysozyme (purified from leukemia patient urine) 50
Dissolve mg in 2 ml of 2M urea and add 6 ml of DMF.
Add and stir under ice cooling (0 to 4°C). Pigment (
-2) Collect 2 mg of each into 3 small test tubes, and 2 ml of each
Add DMF and dissolve. -15 to -20℃
2μ each of isobutyl chloroformate and 1μ each of triethylamine are added to the mixture under cooling for activation.
Next, the activated dye (-2) is added and reacted with the human lysozyme solution at 5 minute intervals while being ice-cooled and stirred. After reacting for 30 minutes under ice cooling,
After desalting using a Sephadex G-10 column equilibrated with 0.2N aqueous ammonia, the product was freeze-dried to obtain dye-labeled human lysozyme. Yield: approximately 45 mg (yield 88%). λ ³ % ^SDS _nax = 712 nm ε ₇₁₂ o m≈4.6×10 ⁴ In the amino terminal analysis of this labeled product, lysine, which is the amino terminal of human lysozyme, was not detected. In addition, Sephadex G-50 (1% SDS
When chromatographed on a column (equilibrated with ), this preparation gave a single peak. Also, Micrococcus - Rhizodeikteikas
In terms of bacteriolytic activity using the bacterial cells of Lysodeikticus as a substrate, the enzyme exhibited almost the same activity as the unmodified enzyme. In addition, this dye-labeled hydrisozyme
AgBrCl emulsion (Br80 mol% average grain size 0.8μ
It exhibited spectral sensitization with a maximum of approximately 760 nm relative to m). Example 3 A two-antibody method using porcine insulin labeled with the dye (-2) described in Example 1, anti-porcine insulin guinea pig serum, and anti-guinea pig IgG rabbit serum as the first antibody and second antibody, respectively, Calibration curves for standard insulin at various concentrations were created using the following method. Dispense 0.1 ml of standard insulin solutions of various concentrations (0.2 ng to 12.8 ng/ml) into small test tubes,
Contains 0.1M NaCl1% bovine serum albumin (BSA)
Add 0.4 ml each of 0.1M Tris-HCl buffer PH8.5 (Solution C). Furthermore, 0.1 ml of each diluted solution of anti-pig insulin guinea pig serum with predetermined titer was added, and then dye (-2) labeled insulin was added to C.
Add 0.1 ml of each solution dissolved and diluted with liquid, stir well, and leave at 4°C for 16 hours. Next, add 0.1 ml of diluted anti-guinea pig IgG rabbit serum, stir thoroughly, and react at 4°C for 24 hours. The resulting precipitate was centrifuged (3000 rpm for 10 minutes), and 20 μ of each supernatant was dropped onto a 5 mm diameter area on a film coated with unexposed AgBrCl (Br 80 mol%, average particle size 0.8 μm) on a TAC support. After leaving it for 10 minutes,
Using a commercially available strobe (Guide No. 56), exposure from a distance of 30 cm through a Fujifilm SC-66 filter (equivalent to 10 ⁵ lux x 10 ^-3 seconds), and developing solution A with the following formulation at 20°C 10 After developing for 1 minute, the film was fixed using a conventional method, washed with water, and dried. The blackening density on the obtained film was measured using a Fujifilm photographic densitometer, and the following results were obtained (for each antiserum-labeled insulin). Dilution is standard insulin concentration 12.8n
The blackening density of the developed silver was determined to be between 2.0 and 2.5 (g/ml). Developer A Metol 3.1g Sodium sulfite 45g Hydroquinone 12g Anhydrous soda 67.5g KBr 1.9g Add water and Table 1 Insulin concentration (ng/ml) Blackening concentration 0 0.21 0.2 0.45 0.4 0.72 0.8 1.01 1.6 1.35 3.2 1.68 6.4 1.93 12.8 2.23 Example 4 In place of the dye (-2) in Example 1, the dye (
-5), dye-labeled porcine insulin was obtained in the same manner. At this time, the yield was 19.7 mg (yield: 86
%), λ ² % ^SDS _nax = 624 nm, ε ₆₂₄ = 2.00×10 ⁵
(about 2
Mol dye/mol insulin). Next, using the obtained labeled porcine insulin, satisfactory calibration curves for standard insulin at various concentrations were obtained by the two-antibody method in the same manner as in Example 3.

Claims (1)

[Scope of Claims] 1. An antigen characterized in that a merocyanine dye containing a carboxyl group or a substituent having a carboxyl group-containing substituent bonded to a heterocyclic ring is used as a spectral sensitizing dye as a labeling substance. Alternatively, a spectral sensitizing dye is bound to the antibody via a peptide bond, and the labeled antigen or antibody is subjected to immunoreaction, and free labeled antigen or antibody (F) is generated depending on the amount of the antigen or antibody to be measured. The labeled antigen-antibody reaction (B) is separated, and either the labeled antigen or antibody, or the labeled antigen-antibody reaction product is brought into contact with silver halide to absorb the spectral sensitizing dye. the amount of developed silver or color dye obtained, or the optical density of the developed silver or color dye. Photo chemical immunoassay method.