JPH03206896A - Determination of very small amount component in body fluid - Google Patents

Abstract

PURPOSE:To measure a very small amount of organism component in high sensitivity by using a specific compound as a coupler. CONSTITUTION:A composition (A) for determining a very small amount of body fluid, containing a compound shown by the formula [H is hydrogen; R1-4 are the same or different hydrogen, substituted phenyl or 1-5 (substituted) alkyl; A to C are the same or different sulfone, carboxyl, OH, nitro, halogen, 1-5C alkyl or alkoxy; n is 0 or 1-4]. The component A optionally containing FAD, acyl CoA oxidase, etc., and a buffer solution (B) comprising MgCl2, peroxidase, etc., are reacted with serum, etc., at about 37 deg.C for about 5 minutes to give a reaction coupler solution (C). Then absorbance of the component C is measured at 600nm wavelength to determine a very small amount component of free fatty acid in serum.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for quantifying trace components in body fluids and a quantitative composition used therefor.

[Conventional technology]

In recent years, enzymatic analysis methods have become increasingly popular and are attracting attention as a method for measuring trace components in body fluids in clinical tests. This involves performing an enzymatic reaction using an enzyme that specifically acts on the target components to be quantified in the blood, such as uric acid, glucose, cholesterol, cholinesterase, transaminase, triglyceride, and free fatty acids, and then measuring the resulting products. This method is used to quantify the target component. In particular,
A popular method is to use an oxidase as a hydrogen peroxide-producing enzyme to generate hydrogen peroxide, react it with a coloring agent in the presence of baroxidase, and quantify the coloration to determine the amount of the target component. are doing.

Conventionally, such color-forming reaction systems have mainly used phenol or its derivatives, aniline or its derivatives, etc. as objects to be condensed with 4-aminoantibiline. However, the color system based on this reaction has low sensitivity when quantifying trace components, and the maximum absorption wavelength is 50
It has a wavelength of 0 to 600 nm, and has the disadvantage of being affected by pigments such as bilirubin and hemoglobin.

In recent years, many methods have been reported that use triphenylmethane-based or diphenylnaphthylmethane-based leuco dyes as coloring agents to overcome these drawbacks [JP-A-56-26199, JP-A-56-31641, JP 60-194363, JP 60-218069, JP 60-256056, JP 62-296, JP 6293261, etc.].

However, when these leuco dyes are used in coloring systems, they have the following problems.

■ The measurement wavelength used in currently popular automatic analyzers is fixed, and most models use 600nm or 660n.
m, 700 nm, whereas coloring systems using these leuco dyes have a maximum absorption wavelength around 630 nm or around 750 nm, which is disadvantageous in terms of sensitivity, so measurements should be made at the shoulder of the absorption spectrum.

■Measurement sensitivity varies greatly depending on pH.

■ Insufficient water solubility.

■ Being affected by coexisting substances in the blood such as hemoglobin, protein, and uric acid.

Leuco crystal violet has also been reported as a triphenylmethane-based triamino derivative for the same purpose, but this dye is poorly soluble in water near neutrality and difficult to dissolve at the desired concentration. Not suitable for measuring trace components (Analytical Chem,
Vol, 42. No, 3 p410~411゜(1
970)).

[Problem to be solved by the invention]

In order to solve the above-mentioned problems of conventional leuco dyes, the present inventor conducted various studies and found that these problems were solved by introducing a hydrophilic group into a triphenylmethane-based triamino derivative. They have discovered that this is the case, and have completed the present invention.

[Means to solve the problem]

The present invention relates to the following formula (I): [In the formula, H represents hydrogen, and R1 to R6 may be the same or different and represent hydrogen, a substituted phenyl group, an alkyl group having an integer of 1 to 5, or a substituted alkyl group. , A to C may be the same or different and represent a sulfone group, a carboxyl group, a hydroxyl group, a nitro group, a halogen, an alkyl group or an alkoxy group having an integer of 1 to 5, and n is 0, 1.2.3 or 4. show. ] This is a method for quantifying trace components in body fluids, which is characterized by using a compound represented by the following as a coloring agent. Furthermore, the present invention
A composition for determining trace components in body fluids containing a compound of formula (I).

The principle of the present invention is that the compound represented by formula (I) [reduced form] and hydrogen peroxide react stoichiometrically in the presence of peroxidase to produce a dye [oxidized form], and the amount of this dye produced is This is based on the fact that it is proportional to the hydrogen peroxide content.

According to the present invention, when the target component to be quantified is a substrate, the enzyme necessary to decompose it to produce hydrogen peroxide is used. Quantifying the target substance by reacting the substrate and the enzyme necessary to generate hydrogen peroxide with a coloring agent of the compound represented by formula (I) in the presence of peroxidase, and measuring the color development. I can do it. Furthermore, when the antigen or antibody is labeled with peroxidase in enzyme-linked immunosorbent assay (ETA method), it is reacted with a coloring agent of the compound represented by formula (I) in the presence of excess hydrogen peroxide. The target component can be quantified by measuring its color development.

The compounds represented by formula (I) of the present invention are triphenylmethanetriamino derivatives, and these compounds have Ac1
d Violet 6B or Bri 1liant
A commercially available dye such as Blue G is reduced using water, sodium borohydride, etc., and purified by silica gel column chromatography, or a condensation reaction between a p-aminobenzaldehyde derivative and an aniline derivative described in the color index, or 4 It can be obtained by a synthetic method such as a condensation reaction between a 4'-diaminobenzhydrol derivative and an aniline derivative.

Examples of compounds obtained in this way are shown in Table 1.
The invention is not limited to these examples.

In addition, the symbols in Table 1 have the following definitions, and the numbers within ○ indicate the positions of substituents.

M knee CH3CM: E knee C2H3CE: P: n-C3H7cp: S knee 5O3HSP: C knee C0OHH3P: CH2C00H C2H4COOH n-C,H6C00H n-C3HsSO3H -CH2CHCH2SO,H H -CH2CHCH20H H HE: ~C2H4OHD HP: HP: n CsHsOH Table 2 shows the maximum absorption wavelength (λmax), sensitivity (molecular extinction coefficient), and influence of pl+ (absorbance percentage) when these compounds are used as color formers.

Sensitivity was measured using the following method.

(1) Preparation of reagent 50,000/0.4 mmol each of peroxidase A and compounds Nos. 1 to 42 as color formers
/i each, and as a comparative example, known coloring agent No. 1 to
4 (however, only N004 contains 0.1% Triton X-100) each at 0.025 mmol/A 50 mm
ol#'P[PE5-KOH buffer (pH 7,0) was dissolved to prepare a reagent solution. The influence of pH was examined by replacing the buffer in the above reagent solution with 50 mmol #7 citric acid-phosphate-borate buffer (pH 5.0, pH 7.0, and pH 9.0).

(2) Measurement method Separately take 3 ml each of the reagent solutions of Compound Nos. 1 to 42 prepared above and 3 ml each of the reagent solutions of Comparative Examples Nos. 1 to 4 into test tubes, and add 0. .6
2mm01/I! Add 50 μl of hydrogen peroxide aqueous solution, mix and heat at 37°C for 5 minutes to develop color. At the same time, perform the same operation using distilled water (for reagent blind testing). After color development, absorbance is measured using a reagent blind test as a control.

(Margins below this page) Table 2 (1) Table (2) E Comparative examples 1 and 2 are color index 42045 reduced type and 42090 reduced type described in Hazue Bureki No. 6-31641, and 3 and 4 are color index 42045 reduced type and 42090 reduced type. The compounds described in JP-A No. 62-296 are o, 27 and rJ No. 3.

Furthermore, Table 3 shows the results of a comparison of the solubility of typical compounds represented by formula (I) of the present invention with conventional coloring agents. This experiment was conducted in the following manner.

A 60mM DMF solution of each compound (0.057711) was added to 50mM citrate-phosphate-borate buffer (pH 4-9).
), and after mixing, the wavelength 800 was added to the buffer solution as a control.
Absorbance was measured at nm. Since the buffer solution of each compound and its oxidized product has no absorption at a wavelength of 800 nm, only the turbidity is reflected in the absorbance.

Table (margins below this page) Components in biological samples that can be measured by the method of the present invention include biological components that can be quantified by measuring hydrogen peroxide produced by an enzymatic reaction or labels used in enzyme immunoassay. All biological components that can be quantified by measuring the enzymatic activity of enzymes can be quantified, but this method is particularly advantageous for quantifying trace amounts of biological components.

Such trace amounts of biological components include, for example, cholesterol, bile acids, glucose, triglycerides, free fatty acids, uric acid, phospholipids, sialic acid, pyruvic acid, inorganic phosphorus, creatinine, creatine, polyamines, urea nitrogen, ammonia, GOT, GPT, monoamine oxidase, guanase, cholinesterase, HB virus,
α-FP.

Examples include CEA and CRP.

In the quantitative determination of biological components according to the present invention, the types and amounts of oxidase used as an enzyme for producing hydrogen peroxide, enzymes used for other purposes, and substrates and other substances involved in enzyme reactions are as described above. It may be appropriately selected according to a known quantitative method using an oxidizable coloring reagent depending on the object to be measured.

The amount of the compound represented by formula (I) according to the present invention, Tsuku-oxidase, and the amount of peroxide, such as hydrogen peroxide, used in the measurement of peroxidase activity are determined by the type and amount of the substance to be measured, and the amount of peroxidase used. They differ depending on the measurement method, ie, a rate assay method that measures the change in absorbance per unit time, an endpoint method that measures the absorbance after a certain period of time, etc., and are appropriately selected and determined accordingly. -Shotically, the compound represented by formula (I) of the present invention is 0.001 to
10mM, preferably 0.01 to 1mM, and the peroxidase is in the range of Q, Ql to 100U/ml, preferably 1 to 200/mj'. There are no particular limitations on the origin of peroxidase, and any peroxidase or peroxidase-like substance derived from plants, animals, or microorganisms can be used, but horseradish-derived peroxidase is usually used. Examples of peroxidase-like substances include hemoglobin and other heme compounds.

The pH according to the present invention can be carried out in a wide range of 4 to 10, and can be measured in accordance with the optimum pH of various substances to be measured in the subject or the optimum pH of the measurement method. Buffers that may be used include, but are not limited to, phosphate, citrate, acetate, borate, carbonate, Tris, and Gud buffers. Further, a surfactant such as Triton X-100 is used as necessary.

[Effect]

According to the present invention, the compound represented by formula (I) has the following advantages when used as a coloring agent for quantifying trace biological components.

(2) It has a maximum absorption wavelength near 600 nm and is extremely sensitive, making it particularly advantageous when using a general-purpose automatic analyzer compared to conventional color formers.

■ It is less affected by pH and can be measured with high sensitivity over a wide pH range.

■ Compared to conventional color formers, there is no decrease in color development sensitivity even when the concentration of the color former is increased, linearity is good, and the usable concentration range is very wide, so it is very advantageous in manufacturing. It is.

■ Almost unaffected by coexisting substances in the blood such as hemoglobin, protein, and uric acid.

■ Excellent water solubility.

Hereinafter, the present invention will be explained by reference examples and examples.

Reference example 1. Synthesis of Compound No. 1 10 g of sodium borohydride was dissolved in 100 ml of pure water, and an aqueous solution of Acid Violet 6B (color index No. 42640) was prepared.
olet 6B (15 g dissolved in 800 ml of pure water) was added dropwise under a nitrogen stream with stirring at room temperature. After dropping, stirring was performed at room temperature for 3 hours under a nitrogen stream to obtain Ac1d V
iole, t6B was reduced. After the reaction, under a nitrogen stream,
5N hydrochloric acid was gradually added to adjust the pH and decompose excess sodium borohydride. The pH was adjusted to 7 by adding 5N NaOH, and the mixture was concentrated to dryness under reduced pressure. Silica gel column chromatography, developing solvent ethanol:acetonitrile-1:
1.5, fractions containing compound No. 1 were collected and concentrated to dryness under reduced pressure to obtain the desired product.

Yield 3.0g, Yield 20% Melting point 315°C (decomposition) Reference Example 2. Synthesis of Compound No. 4 Using 15 g of Rilliant Blue R (color index No. 42660), compound No.
It was synthesized in the same manner as in 1.

Yield 3.8g, Yield 25% Melting point 225°C (decomposition) Reference Example 3. Synthesis of Compound No. 5 Using 15 g of Rilliant Blue G (color index No. 42655), compound No.
, was synthesized in the same manner as the synthesis of I.

Yield 3.0g, Yield 20% Melting point 215°C (decomposition) Reference Example 4. Synthesis of compound No. 20 [References: Acta Chem, 5cand,
Vol, 19. No, 6° p1381-1390.
(1965), ) 36% (w/w) hydrochloric acid 1.3 ml,
Add 0.30 g of urea (5 m
mol), P-diethylaminobenzaldehyde 0.
89 g (5 mmol) and 4.0 g (15 mmol) of N-ethyl-N-sulfopropylaniline sodium salt were sequentially added and dissolved. The mixture was refluxed with stirring at 90 to 100° C. for 24 hours under a nitrogen stream. After the reaction, 50ml of pure water
The pH was adjusted to 7 using a dilute NaOH aqueous solution, and under reduced pressure,
It was concentrated to dryness. Silica gel column chromatography The fraction containing compound No. 20 was collected using the developing solvent ethanol:chloroform = 5:1, and the fraction containing compound No. 20 was collected under reduced pressure.
The desired product was obtained by concentration to dryness.

Yield 2.4g, Yield 70% Melting point 217-223°C Reference Example 5. Synthesis of Compound No. 40 P-dimethylaminobenzaldehyde 0.75g (5m
m.

9 synthesis of compound No. 20 using (Reference Example 4)
Synthesis was performed in the same manner.

Yield 2.6g, Yield 78% Melting point 220-225°C Reference Example 6. Synthesis of Compound No. 41 4-dipropylamino-2-fluorobenzaldehyde 1. Compound No. 2 using 1 g (5 mmol)
Synthesis was carried out in the same manner as in the synthesis of 0 (Reference Example 4).

Yield 1.6g, Yield 45% Melting point 228-233°C Reference Example 7 - Compound No. Synthesis of 42 4-dipropylamino-2-methylbenzaldehyde l
, Compound No. 20 using 09 g (5 mmol)
It was synthesized in the same manner as in the synthesis (Reference Example 4).

Yield 1.3g, Yield 37% Melting point 208-216°C Reference Example 81 Absorption spectrum after color development (1) Preparation of reagent Peroxidase of 50000/A' and compound No. 1.20.41.42 as color former each 0
．． 5 mmol/12 each, and as a comparative example, known coloring agent No. 1.4. (However, No. 4 only 0.1%
(contains Triton
ol/lzutsu 50mmol/II B15-Tri
It was dissolved in S buffer (pH 7,0) to prepare a reagent solution.

(2) Measurement method Compound No. 1.20.41.42 prepared above
3 ml of each reagent solution, and Comparative Example No. 1.4
Separately take 3 ml of each reagent solution into test tubes and add 0.62 mmol/I! Hydrogen peroxide aqueous solution 5o1
Add ti, mix and heat at 37°C for 5 minutes to develop color. At the same time, perform the same operation using distilled water (for reagent blind testing).

After color development, the absorption spectrum is measured using a reagent blind test as a control. The measurement results are shown in FIG.

Example 1. Determination of free fatty acids (1) Preparation of reagent ■ MgCL 1 mmol#! , ATP 1 mmol/
47, CoA O, 1 mmol/I! , Asil Co
A synthetase 400U/j', peroxidase 10
000U/11 Triton
It was dissolved in H buffer (pH 7,0) to prepare reagent 1.

■ FAD 4 μmol#7, acyl-CoA oxidase 5000U#', Triton X-100℃, 03%,
Compounds No. 1, 20, 39, and 42 were used as coloring agents at 0.8 mmol/II and 50 mmol/each, respectively.
II PIPES-KOH buffer (pH 7,0) was dissolved to prepare Reagent 2.

(2) Measurement procedure Take 20 μl of serum in a test tube and add the above reagent 1 to it.
Add 3 ml, mix and warm at 37°C for 5 minutes, then add 1.3 ml of each of the above compound No. 1, 20, 39, 42 reagent 2 separately, and heat at 37°C after mixing.
Heat for 5 minutes to develop color. At the same time, perform the same operation using distilled water (for reagent blind testing) and standard solution. After color development, the absorbance was measured at a wavelength of 600 nm using a reagent blind test as a control.
The free fatty acid concentration in serum is determined from a previously prepared calibration curve (shown in Figure 2). The measurement results are shown in Figure 3.4.5.6 together with the values of the conventional method.

Example 2. Quantification of uric acid (1) Preparation of reagent ■KCI 50 mmol/C) Rydon X-1000,
03%, and 0.5 mmol/I! of compounds No. and 2.40.41 as color formers, respectively, in an amount of 0.5 mmol# and 50 mmol/I!
It was dissolved in PIPES-KOH buffer (1) H7, O) to prepare reagent 1.

■KCI 50mmol/l, peroxidase 1
2500 Ll/l, uricase 2500 U/l, Triton
mol/47 P[PE5-KOH buffer (pH 7,
0) and used as reagent 2.

(2) Measurement procedure Take 20 μl of serum in a test tube and add the above compound No.
2.40. Add 2.0 ml each of Reagent 1 of 41 separately, mix and warm at 37°C for 5 minutes, then add 0.5 ml of Reagent 2 prepared above, mix and heat at 37°C. Heat for 5 minutes to develop color. At the same time, perform the same operation using distilled water (for reagent blind testing) and standard solution. After color development, the absorbance is measured at a wavelength of 600 nm using a reagent blind test as a control, and the uric acid concentration in the serum is determined from a previously prepared calibration curve (shown in FIG. 7). This measurement result is combined with the value of the conventional method in Section 8.
Shown in Figure 9.10.

Example 3. Measurement of HBs antigen (1) Preparation of reagent ■ Preparation of 96-well flat bottom plate sensitized with anti-HBs antibody PBS
The purified anti-HBs mouse monoclonal antibody prepared at 5 μg/ml was dispensed in 50 μβ portions into each type of 96-well flat-bottomed microplate (manufactured by Nunc). After leaving this at 37°C for 3 hours, wash it with PBS, add 300 μl of 50 mmol/A' Tris-HCl buffer (pH 8,0) containing 0.5% (W/v) casein, and incubate at 4°C. The plate was left overnight to prepare a 96-well flat bottom plate sensitized with anti-HBs antibody.

■ Preparation of peroxidase-labeled anti-HBs antibody Add 5 mg of peroxidase to 1 ml of 0.3 mol/I! of sodium bicarbonate, and add 0.2 liters of 1% 2.4
- After adding dinitrofluorobenzene and stirring for 1 hour, 1 ml of 0.06 mol/I! Add sodium periodate, stir for 30 minutes, and add 1 ml of 0.16 mol.
Add /R ethylene glycol and stir for 1 hour. This is dialyzed day and night against carbonate buffer (pH 9,5). This was mixed with 1 rnl of anti-HBs monoclonal antibody (5 mg/mj) dissolved in carbonate buffer (pH 9,5), and after reacting at room temperature for 3 hours, (PBS dialysis) Sephacryl S-20
Peroxidase-labeled anti-HBs antibody is obtained by gel filtration at 0°C.

■ Preparation of substrate-coloring solution Hydrogen peroxide 1.45 mmol/l, compound No. 10
．． 0.1 mol/l to give a concentration of 5 mmol/l
1 phosphate buffer (1) H7.0) to serve as a substrate-coloring solution.

(2) Measurement procedure Anti-HBs antibody sensitized 96-well flat bottom plate at 200 ng/m
1400ng/m1.600ng/mj, 800ng
/m1.11000n/ml, 1200ng/ml
Take 50 μl of a sample containing HBs antigen at each concentration and incubate at 37°
The mixture was heated at C for 3 hours and washed with PBS. Next, add 50 μl of peroxidase-labeled anti-HBs antibody and incubate at 37°C.
The mixture was heated for 2 hours and washed with PBS. Then, add 100 μl of substrate-coloring solution and heat at 37°C for 30 minutes to develop color. At the same time, the same operation is performed using a 96-well flat bottom plate that has not been sensitized with anti-HBs antibody (for blind testing). After color development, the absorbance was measured using a microplate reader (MTP-32 manufactured by Corona Electric Co., Ltd.) at a wavelength of 600 nm using a blind test as a control. The results are shown in FIG.

〔Effect of the invention〕

As mentioned above, the compounds represented by formula (I) of the present invention all have maximum absorption wavelengths around 600 nm when coloring, so they are particularly suitable for general-purpose automatic analyzers, and are not affected by pH. It has remarkable effects when used as a coloring agent for measuring trace amounts of biological components, such as enabling highly sensitive measurements over a wide range of pl and having extremely good water solubility. .

[Brief explanation of drawings]

FIG. 1 shows the absorption spectrum in Reference Example 8, and FIG. 2 and 3. 4. 5. 6. The figure shows a calibration curve and a correlation diagram in Example 1, and FIGS. 7 and 8. 9.1
0 represents a calibration curve and a correlation diagram in Example 2, and FIG. 11 represents a calibration curve in Example 3.

Claims (1)

[Claims] 1. The following formula (I): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) [In the formula, H represents hydrogen, R_1 to R_6 may be the same or different, and hydrogen, Substituted phenyl group, integer 1 to 5
represents an alkyl group or substituted alkyl group, A to C may be the same or different, and represent a sulfone group, a carboxyl group,
It represents a hydroxyl group, a nitro group, a halogen, an alkyl group or an alkoxy group having an integer of 1 to 5, and n represents 0, 1, 2, 3 or 4. ] A method for quantifying trace components in body fluids, which is characterized by using a compound represented by the following as a coloring agent. 2. A composition for quantifying trace components in body fluids, which contains a compound represented by formula (I).