JP2001302660A - Indicator for determination of protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a more sensitive indicator and a test piece for the determination of protein. SOLUTION: This indicator is 3', 3'',5',5''-tetraiodophenol-3,4,5,6- tetrabromosulfophthalein expressed by formula (1), and an indicator for the test piece for the determination of protein comprising the compound and the test piece for the determination of protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an indicator and a test strip for measuring a protein present in a biological sample such as serum, plasma, urine and the like.

[0002]

2. Description of the Related Art Measuring protein in a biological sample involves the steps of:
It is important in various pathological diagnoses. For example, albumin, which is abundantly contained in serum, is easily soluble in water, regulates the water balance between blood and tissues, and contains poorly water-soluble substances such as bilirubin, fatty acids, bile pigments, and drugs present in blood. It is responsible for transporting these together. Therefore, when the amount of serum albumin decreases due to a decrease in liver function, physiological disorders such as edema and accumulation of serum fluid are caused. Also, nephritis, nephrotic syndrome,
Urinary protein increases due to kidney and urinary tract diseases such as stones and tumors, diseases of the circulatory system and central nervous system, and the like. Therefore, measuring the protein can be a particularly important guide in the diagnosis of these diseases.

[0003] At present, in the field of protein measurement, measurement using a test piece impregnated with a protein error indicator is known as a simple method, and tetrabromophenol blue (TBPB) is used as a protein error indicator. The used urine test paper is widely used for primary screening. When a protein is present, TBPB dissociates a phenolic hydroxyl group at a low pH of about 3, which is not originally dissociated, and changes from yellow to blue. The test paper displays this change in color, so that the protein can be detected.

[0004] However, the test paper using TBPB as an indicator has a clinically required 10-20 mg / dl.
In some cases, the protein cannot be accurately detected due to insufficient detection sensitivity for a protein at a low concentration (trace amount). That is, in the visual judgment performed by comparing with a color chart, the color of the negative protein and the trace protein is close to each other, so that it is difficult to discriminate the judgment and the judgment is difficult. However, the determination is often erroneous due to low sensitivity. Therefore, there is a demand for the development of a compound other than TBPB that can serve as an indicator for such a purpose.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and an object of the present invention is to provide a highly sensitive indicator for protein measurement and a test strip.

[0006]

Means for Solving the Problems The present invention provides the following formula [1]

[0007]

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3 ', 3'',5', 5 ''-tetraiodophenol-3,4,5,6-tetrabromosulfonephthalein

Further, the present invention provides the following formula [1]

[0010]

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An indicator for a test strip for protein measurement comprising 3 ', 3 ", 5', 5" -tetraiodophenol-3,4,5,6-tetrabromosulfonephthalein It is an invention.

Further, the present invention provides the following formula [1]

[0013]

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A protein measuring test strip containing 3 ′, 3 ″, 5 ′, 5 ″ -tetraiodophenol-3,4,5,6-tetrabromosulfonephthalein as an indicator,
Invention.

That is, the present inventors have intensively studied to develop a compound which can be used as an indicator of a protein measurement test strip for measuring a protein in a biological sample, in particular, a low-concentration protein with high sensitivity. 3 ', 3'',5',
5 ''-tetraiodophenol-3,4,5,6-tetrabromosulfonephthalein (hereinafter abbreviated as TITBS) was found to be suitable as a target indicator,
The present invention has been completed.

The test piece for protein measurement in the present invention has the formula
This is a test strip comprising TITBS represented by [1] as an indicator.

The test strip for protein measurement of the present invention is obtained by impregnating an absorbent carrier with an impregnating liquid containing the indicator of the present invention, TITBS, a buffer, and, if necessary, a surfactant and a sensitizer. It can be prepared by drying it. The test piece may be used as it is, but may be cut into a desired shape and adhered to a non-absorbent if necessary.

The concentration of TITBS used is not particularly limited, but is usually 0.1 mM to 2 mM, preferably 0.3 mM to
0.6 mM.

Any buffer may be used as long as it has a good buffering capacity in the pH range of 2.5 to 4.5 and does not inhibit the reaction between the indicator TITBS and the protein. Acid buffer, succinate buffer,
Malate buffer, tartrate buffer and the like can be mentioned.

The concentration of the buffer used is not particularly limited, but is usually 0.1 M to 1.5 M, preferably 0.3 M to
1M. The pH of the solution is preferably set to a value slightly lower than the pKa value of TITBS, which is the indicator of the present invention, and is appropriately selected from the range of usually 2.5 to 4.5, preferably 3.0 to 4.0.

Any surfactant may be used as long as it does not have the property of inhibiting the measurement of protein.
Although not particularly limited, for example, polyoxyethylene alkylphenyl ethers such as polyoxyethylene isooctyl phenyl ether and polyoxyethylene nonyl phenyl ether, and polyoxyethylene alkyl phenyl ethers such as polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, and polyoxyethylene lauryl ether Nonionic surfactants such as oxyethylene alkyl ether, polyethylene glycol monolaurate, and tetraethylene glycol dodecyl ether, for example, amphoteric interface such as stearyl betaine and 2-alkyl-N-carboxylmethyl-N-hydroxyethylimidazolinium betaine Surfactants such as anionic surfactants such as cholic acid, deoxycholic acid and sodium polyoxyethylene alkylphenol ether sulfate; These surfactants may be used alone or in an appropriate combination of two or more.

The concentration of these surfactants is not particularly limited, but is appropriately selected and used in the impregnation solution so as to be usually 0.05 to 5 w / w%, preferably 0.1 to 1 w / w%.

Examples of the sensitizer include polypropylene glycol, polyethylene glycol, polycarbonate, polyvinyl ether and the like, and preferably polypropylene glycol.

The concentration of these sensitizers is not particularly limited, but is usually 0.05 to 5 w / w%, preferably 0.1 to 5 w / w% in the impregnating solution.
選 択 1 w / w% is appropriately selected and used.

The absorbent carrier includes, for example, porous sheets or films, foams (foamed bodies), woven fabrics, non-woven fabrics, and knitted fabrics that do not contain protein components. Examples of these materials include natural, semi-synthetic or synthetic fibrous materials, and these materials can be obtained by molding in a conventional manner into papermaking, film forming, foam molding, knitting, weaving, and the like. . Specific examples of these materials include, for example, cotton, hemp, cellulose, filter paper, rock wool, nitrocellulose, cellulose acetate, glass fiber, silica fiber, carbon fiber, boron fiber, polyamide, aramid, polyvinyl alcohol, polyvinyl acetate,
Rayon, polyester, nylon, polyacrylic acid,
Examples include polyacrylates and polyolefins.

The shape of these absorbent carriers is not particularly limited, but is generally rectangular or square or circular or elliptical.

Examples of the non-absorbent material include a sheet or film made of a material such as polyethylene terephthalate, polyester, polypropylene, polyethylene, polyvinyl chloride, polyvinylidene chloride, and polystyrene.

In order to produce 3 ′, 3 ″, 5 ′, 5 ″ -tetraiodophenol-3,4,5,6-tetrabromosulfonephthalein according to the present invention, for example, phenol and tetrabromo -O-sulfonebenzoic acid is reacted with a sulfuric acid or Lewis acid catalyst, if necessary using a suitable solvent, and then the obtained phenol-3,4,5,6-
Tetrabromosulfonephthalein and iodine may be reacted in a basic aqueous solution.

As the reaction solvent in the above reaction, specifically, for example, methyl chloride, dichloromethane, chloroform, tetrachloromethane, 1,2-dichloroethane, 1,1,1,2
Halogenated hydrocarbons such as 2,2-tetrachloroethane, phenol and the like, and these may be used alone,
Two or more kinds may be used in appropriate combination.

Examples of the Lewis acid catalyst include tin chloride,
Zinc chloride, aluminum chloride, titanium chloride and the like can be mentioned, and these may be used alone or in appropriate combination of two or more.

Examples of the basic aqueous solution include those in which a basic compound such as a hydroxide such as sodium hydroxide, potassium hydroxide or lithium hydroxide is dissolved in water.

The reaction time is generally 1 to 20 hours, preferably 1 to 10 hours. The reaction temperature varies depending on the reaction temperature and the amount of the starting material, but is usually 0 to 300 ° C, preferably 0 to 200 ° C.
° C. Reaction operations other than the above, post-treatments, and the like may be performed according to the same kind of reaction that is usually performed.

As described above, the TITBS-impregnated test piece of the present invention can measure protein with higher sensitivity than the test piece containing TBPB which has been conventionally used as an indicator.
It has the advantage that it can measure with high sensitivity even trace proteins of up to 20 mg / dl.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

[0035]

EXAMPLES Example 1 (1) Synthesis of phenol-3,4,5,6-tetrabromosulfonephthalein A mixture of 75.0 g (0.80 mol) of phenol and 50.0 g (0.1 mol) of tetrabromo-o-sulfonebenzoic acid was used. Add zinc chloride to the mixture.
4.9 g (0.92 mol) was added, and the mixture was heated and stirred at 150 to 160 ° C. for 3 hours. After completion of the reaction, the reaction solution was added to ice water, and then ethyl acetate was added.
500 ml was added, and the mixture was separated to extract a target substance. The obtained organic layer was acidified by adding 500 ml of 1N hydrochloric acid, and then 500 ml of 1N sodium hydroxide was added. Next, the obtained aqueous layer was acidified by adding 500 ml of 3N hydrochloric acid, and then 500 ml of ethyl acetate was added thereto. Ethyl acetate was distilled off, and phenol-3,
49.1 g (0.073 mol) of a crude product of 4,5,6-tetrabromosulfonephthalein was obtained. (Yield 73%) Elemental analysis (C ₁₉ H ₁₀ Br ₄ O ₅ S) Calculated (%): C34.06, H1.50 Actual (%): C34.03, H1.52

(2) Synthesis of 3 ′, 3 ″, 5 ′, 5 ″ -tetraiodophenol-3,4,5,6-tetrabromosulfonephthalein (TITBS) In 1.75 L of 0.48N aqueous sodium hydroxide solution 49.1 g (0.0729 mol) of the crude phenol-3,4,5,6-tetrabromosulfonephthalein obtained in the above (1) and 149.7 g of iodine (0.
599 mol), and the mixture was heated with stirring at 45 ° C. for 45 minutes. After completion of the reaction, the reaction solution was ice-cooled and made acidic by adding 500 ml of a 3N aqueous hydrochloric acid solution. Then, 500 ml of ethyl acetate was added thereto, and the mixture was separated to extract a desired product. To the obtained organic layer, 500 ml of an aqueous solution of sodium thiosulfate was added, and the mixture was separated and washed.
500 ml was added, and the mixture was separated again to extract the desired product. Then, ethyl acetate is distilled off, and the residue is crystallized with an acetic acid-methanol solvent, and 3 ′, 3 ″, 5 ′, 5 ″ -tetraiodophenol-3,4,5,6-tetrabromosulfonephthalein (TITBS) 26.1 g (0.022 mol) was obtained (yield 30.5%,
Melting point 300 ° C or higher). The target product thus crystallized had 1 mol of acetic acid per 1 mol of TITBS. Elemental analysis value (C ₁₉ H ₆ Br ₄ I ₄ O ₅ S · C ₂ H ₄ O ₂ ) Calculated value (%): C20.48, H0.82 Observed value (%): C20.18, H0.98 H ^1- NMR δppm (CD ₃ OD): 7.630 (4H, -C ₆ H ₂ (I ₂ ) (OH))

Example 2 Measurement of albumin using a test strip for protein measurement using TITBS as an indicator (1) Preparation of samples A urine sample containing no human albumin (Blank) and a urine sample containing human albumin (15, 100, respectively) 300 and 1000 mg / dl) were prepared respectively. (2) Preparation of test pieces ・ Solution A Citric acid monohydrate 5.68 g Trisodium citrate dihydrate 3.07 g Distilled water Total volume 50 ml (pH 3.3) ・ Solution B TITBS 46.9 mg (0.04 mmol) Polypropylene glycol 2000 (Diol type) 0.6g Methanol Total volume 50ml The above solution A and solution B are mixed to make an impregnating solution, which is immersed in cellulose filter paper for chromatography (0.4mm thick) and dried to prepare test pieces. did. (3) Measurements and results The test pieces prepared were immersed in each urine sample,
Measurement wavelength 635 by M-405 (manufactured by Wako Pure Chemical Industries, Ltd.)
The reflectance at nm (reference wavelength 760 nm) was measured. Table 1 shows the results.

Comparative Example 1 In Example 2 (2), 46.9 mg of TITBS of Solution B
A TBPB-impregnated test piece was prepared in the same manner as in Example 2 except that 39.4 mg (0.04 mmol) of TBPB was used instead of, and albumin measurement was performed in the same manner. The results are shown in Table 1.

[0039]

[Table 1]

As is clear from Table 1, the TITB of the present invention
Compared with the conventional TBPB-impregnated test specimen, the reflectance difference between the human albumin-containing urine sample and the blank is larger and the detection sensitivity is increased, and the detection sensitivity of the trace amount is particularly increased. I understood. Furthermore, in the visual judgment, the color of the blank and the trace amount of the sample could be clearly distinguished. Therefore, it was found that the use of the TITBS of the present invention as an indicator can measure protein (albumin) with high sensitivity over a wide range from trace amounts to proteins of 100 mg / dl or more.

[0041]

As described above, the present invention provides an indicator capable of measuring a protein in a biological sample, in particular, a test strip for measuring a protein. The present invention provides 3 ', 3'',5', 5 '
The use of a test strip containing 5 ''-tetraiodophenol-3,4,5,6-tetrabromosulfonephthalein (TITBS) as an indicator has a remarkable effect in that protein can be measured with high sensitivity. .

Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat II (Reference) G01N 33/68 G01N 33/68 (72) Inventor Ikuzo Mitama 6-1, Takada-cho, Amagasaki-shi, Hyogo Wako Pure Chemical Industries 2G042 AA01 BD12 BD18 CB03 DA08 FA11 FB07 FC01 2G045 AA13 AA16 BA11 BB29 BB48 BB52 CA25 CA26 CB03 DA36 DA38 FA29 FB11 FB17 GC11 HA10 2G054 AA07 AB07 BA05 CA01 CE01 EA

Claims (4)

[Claims] [Claim 1] The following formula [1] 3 ′, 3 ″, 5 ′, 5 ″ -tetraiodophenol-3,4,5,6-tetrabromosulfonephthalein represented by 2. The following formula [1] An indicator for a test strip for protein measurement comprising 3 ′, 3 ″, 5 ′, 5 ″ -tetraiodophenol-3,4,5,6-tetrabromosulfonephthalein 3. The following formula [1] A test piece for protein measurement containing 3 ′, 3 ″, 5 ′, 5 ″ -tetraiodophenol-3,4,5,6-tetrabromosulfonephthalein as an indicator, represented by the formula: 4. The test piece according to claim 3, comprising polypropylene glycol as a sensitizer.