JP5938713B2 - High-sensitivity measurement method and reagent for hemoglobin - Google Patents

Description

The present invention relates to a highly sensitive measurement method and reagent that is used in the field of colorectal cancer screening or clinical diagnosis for diagnosing hematuria and quantitatively measures hemoglobin in feces or urine and is free from the influence of coexisting substances.

Conventionally, an inexpensive method for quantifying hemoglobin has been to measure cyanmethemoglobin using potassium cyanide and potassium ferricyanide as hemoglobin. However, since this method can only measure hemoglobin at a fairly high concentration of several g / dL level, it is mainly used for quantitative measurement of blood hemoglobin, and for quantitative measurement of low concentration hemoglobin in feces and urine. Could not be used. Specifically, fecal hemoglobin has been an important screening test for colorectal cancer, but a highly sensitive measurement method capable of detecting a small amount of hemoglobin of several hundred ng / dL is required. In the case of hemoglobin, it is necessary to detect hemoglobin of about 300 ng / mL, which is a level for initial diagnosis of kidney disease, that is, ± on the urine test paper. This concentration could not be detected by the conventional cyanmethemoglobin method.

At present, urine test paper is most used in detecting hemoglobin in urine. Although the urine test paper can be operated by a simple method, the determination of the result is mainly performed visually, and the qualitative diagnosis result is inevitably obtained.
In addition, false positive reactions and false negative reactions are generally cited as problems of urine test paper. Causes of false positive reactions include large amounts of strong bacterial urine and strong leukocyte urine semen Old urine and alkaline urine, etc. Causes of false negative reactions include strong reducing substances (such as ascorbic acid) High specific gravity urine, high There is proteinuria.
This is because the reaction of the urine test paper uses 100% moisture in the specimen, that is, the ratio of the specimen / reagent is infinite, and the specimen itself must be the medium of the reaction. This is because the influential substance directly enters the reaction system at a high concentration. In order to detect hemoglobin, the higher the analyte / reagent ratio, the higher the sensitivity, but on the other hand, influential substances will also enter in large quantities, and the cause of the above cannot be ignored. In order to eliminate this point, it is necessary to make the analyte / reagent ratio as small as possible, that is, a highly sensitive measurement method that obtains a large absorbance with a small amount of hemoglobin.
Urine test strips are not sensitive enough at low concentrations, and are not qualitative but qualitative, so if the urine test strip is judged to be ±, the patient will reexamine the number of red blood cells during urine examination as a microscope. It was necessary to have a test to count at. If the hemoglobin of about 300 ng / mL, which is taken as ±, can be detected quantitatively, the probability of retesting will be reduced, and the patient will not have to bother to visit for retesting. If quantification is possible with such a highly sensitive measurement method, the need for retesting will be greatly reduced. In the urine test paper test, the pseudoperoxidase action of hemoglobin is used to detect hemoglobin, and the coloration due to the oxidation reaction of hydroperoxide and a pigment source is measured. In this method, tetramethylbenzidine or o-tolidine is used as a dye source. However, these dye sources generate dyes by an oxidation reaction, but it is necessary to oxidize two hydrogen atoms to generate one molecule of dye, and the dye generation due to the pseudoperoxidase action of hemoglobin is slow, and the sensitivity Quantitative measurement is not possible due to lack.
In addition, urine contains a high concentration of ascorbic acid when drinks containing vitamin C are taken. Ascorbic acid inhibits the oxidative coloring reaction and reduces the colored pigment itself to make it colorless. There is a risk of false negatives. Normally, ascorbic acid oxidase is used to eliminate ascorbic acid, but the optimum pH of the oxidative coloring reaction using this dye source is in the range of 4-4.5, and ascorbic acid oxidase that eliminates ascorbic acid is used. In prescribing, it was difficult to ensure the stability of ascorbate oxidase under the conditions of this measurement reagent. For this reason, the influence of ascorbic acid cannot be eliminated sufficiently.

  As a solution to such a sensitive problem, a method of measuring turbidity by using an anti-hemoglobin antibody and measuring turbidity resulting from an antigen-antibody reaction in terms of absorbance or a method of measuring turbidity with a nephelometer have been proposed. However, since this method is an immunization method, there is no influence of ascorbic acid, but it is very expensive because a polyclonal antibody is used as a reagent, and a special instrument is required to measure turbidity. . Polyclonal antibodies are produced by immunizing large animals such as sheep and goats with long-term antigens and collecting and purifying blood. However, there are individual differences in animals, and there are problems with antibody reproducibility. It was.

  On the other hand, as a pigment source, a substance having a phenothiazine structure or a diphenylamine structure represented by Formula 1 or Formula 2 and a structure in which a pigment is formed by a decomposition reaction is used. A method for measuring the amount of hydroperoxide has been proposed, but conversely, a method for measuring hemoglobin using this pigment source and hydroperoxide has not been known.

None None

Junichi Kawai, Eiji Suzuki, et al. Journal of Japanese Society of Oral Surgery vol 31 No8 1854-1858 (1985) Alfresa Pharma Co., Ltd. Nescourt “Hemokit-N” Appendix David AA, Samuel S. Clin. Chem. Vol21 362-369 (1975 ) Crosby W.H.Furth FW Blood vol11 380-383 (1956) Watanabe Nobuko, Hashimoto Yoshiaki, etc Clinical Pathology vol 51 403-408 (2003) Fragrance Journal No.76 (1986) p31-34 Biochemistry International Vol.10 No. 2 February (1986) p205-211 Agric. Biol. Chem. 49 (9) (1985) 2799-2801 Isao Fujii, Noriaki Hirayama, Norihito Aoyama, Akira Miike Bull. Chem. Soc. Jpn. 69, 1423-1427 (1996)

  Low-cost measurement method that can measure hemoglobin in stool or urine with high sensitivity, is not affected by reducing substances coexisting in stool or urine, and can be applied to biochemical automatic analyzers for quantitative measurement And reagents were sought.

As a result of various studies, the inventor uses a compound represented by Formula 1 or Formula 2 having a phenothiazine structure or a diphenylamine structure and a hydroperoxide as a pigment source, and performs a reaction in which a pigment is generated by a decomposition reaction by the catalytic action of hemoglobin. The rate of the chromogenic reaction increases in proportion to the amount of hemoglobin under the condition where the measurement reagent PH is about 5.5-8, and the reaction is completed within about 5 minutes. A measurement system capable of quantitative measurement has been developed. In the reaction of the present invention, a reaction in which a dye is generated by decomposing one bond of a carbamoyl residue of a dye source molecule is applied instead of a conventional oxidation reaction of two hydrogen atoms in a dye source. That is, in order to generate one molecule of dye, it is only necessary to decompose one bond, and then the decomposed product is naturally oxidized to the dye by oxygen in the system, so that the dye is generated by the catalytic action of hemoglobin. Since the molecular absorption coefficient of the generated dye was large, it was found that highly sensitive measurement was possible, and the present invention was completed.
The present invention relates to the following 1 to 9.
1. Using a compound having a phenothiazine structure or diphenylamine structure represented by Formula 1 or Formula 2 and a hydroperoxide as a dye source in a buffer solution having a pH of 5.5-8, and utilizing the catalytic activity of hemoglobin. A method for measuring hemoglobin that produces dye and measures the absorbance of the generated dye.
2. A measuring method of 1, which is a reagent for measuring hemoglobin in stool or urine.
3. Measuring method of 1 or 2 whose hydroperoxide is cumene hydroperoxide or tertiary butyl hydroperoxide.
4. The measuring method of 2 or 3, wherein the reducing substance in the specimen is oxidized with iodate and / or eliminated with ascorbate oxidase.
5. Measurement method of 1 or 4, wherein the buffer is Good's buffer ADA and / or glycylglycine.
6. A reagent for measuring hemoglobin containing a compound having a phenothiazine structure or a diphenylamine structure represented by Formula 1 or Formula 2 as a pigment source, and hydroperoxide, and having a pH of 5.5-8.
7. Reagent of 6, wherein the buffer is Good's buffer ADA and / or glycylglycine.
8. The reagent according to 6, or 7, wherein the hydroperoxide is cumene hydroperoxide or tertiary butyl hydroperoxide.
9. 6, or 7, or 8 reagents containing iodate and / or ascorbate oxidase.
As a sample, in the case of urine, a sample diluted with a solution that stabilizes raw urine or hemoglobin as needed, and in the case of stool, several mg to several tens of mg of stool is stabilized with a fixed amount of pure water or hemoglobin. A solution obtained by dispersing and dissolving in a solution containing an agent is used as it is or a filtrate obtained by filtering insoluble components is used as a specimen.

According to the present invention, a highly sensitive measurement method that can be used for the initial diagnosis of kidney disease, that is, a low concentration of urinary hemoglobin that is ± on a urine test paper, and fecal hemoglobin performed in early colon cancer screening And provide reagents. The urine contains a high concentration of ascorbic acid when a drink containing vitamin C or the like is drunk, but the reagent of the present invention can have a pH in the range of 5.5 to 8, so that it is a representative reducing substance. Since ascorbate oxidase that can eliminate ascorbic acid is formulated in the reagent, there is no influence.

The urine contains a reducing substance in addition to ascorbic acid. Specific examples include cysteine having a thiol group, glutathione, uric acid, and bilirubin. In order to eliminate these effects, iodates can be added to the reagent of the present invention.

Diagram 1 of the structural formula of the dye source is a general expression of a phenothiazine type dye source, and Rn, Y, and Z have the following meanings. Formula 2 represents a diphenylamine type dye source in a general formula, and Rn and Y have the following meanings. R1, R2, R3 and R4 may be the same or different, and may be hydrogen, C1-C4 alkyl group, alkylene group, aryl group, hydroxyl group, carboxyl group, 1 or secondary amino group, halogen group, sulfonyl group, etc. A substituted C1-C4 alkyl group, alkylene group or aryl group; Z and Y may be the same or different and each represents an oxygen atom or a sulfur atom; R5 represents a hydrogen C1-C6 alkyl group, alkylene group or aryl group; Or a substituted alkyl group, a substituted alkylene group, or a substituted aryl group. The substituent is a hydroxyl group, a carboxyl group, a primary or secondary amino group, a halogen group, a sulfonyl group, or the like. R6 and R7 may be the same or different and each represents hydrogen, a halogen group, a nitro group, a carboxyl group, a hydroxyl group, an amino group, a substituted amino group, a sulfonyl group, a nitrile group, or a substituted or unsubstituted alkyl group of C1 to C3. The substituent here is the above-described substituent.

The measurement is performed by the following process.
(1) Add reagent source, pigment source, ascorbic acid oxidase, cyclodextrin or surfactant, reaction accelerator, iodate, etc. to reagent 5.5 as a reagent 1 The sample is added to this solution and heated at 37 ° C. for about 3 to 7 minutes. In this step, the influence substance which may be contained in the specimen is processed so as to be erased or not affected.
(2) Next, a reagent 2 containing hydroperoxide and a surfactant, and if necessary, a cyclodextrin or a surfactant and a reaction accelerator, etc. is added to (1) and mixed with a buffer solution of pH 5.5-8. And further warm at 37 ° C. for 3-7 minutes. In this process, a dye is produced by the catalytic action of hemoglobin in the presence of a dye source and hydroperoxide.
(3) The absorbance is measured, and the hemoglobin concentration is calculated by comparison with that of the standard solution.
In recent years, the operations of steps (1) to (3) are automatically performed by a biochemical automatic analyzer. Reagent 1 is set in the place corresponding to the first reagent of the automatic analyzer, and reagent 2 is set in the place corresponding to the second reagent.
Specifically, in step (1), ascorbic acid as a reducing effect substance is oxidized to dehydroascorbic acid by ascorbate oxidase to eliminate the influence, and cysteine and bilirubin have weak oxidizing power like potassium iodate. It is eliminated by oxidation with a compound. The turbidity of the sample is solubilized by the surfactant to eliminate the influence.
In step (2), pigment is produced by the catalytic action of hemoglobin in the presence of a pigment source and hydroperoxide, but the catalytic action and pigment production increase or decrease depending on the amount of hemoglobin present, with the result that the amount of hemoglobin is proportional to the amount of pigment. .
In step (3), the amount of dye purified in step (2) is detected by measuring the absorbance, and the absorbance is proportional to the amount of hemoglobin.

Physically, a compound represented by Formula 1 or Formula 2 is used as the color source.
Formula 1 is a derivative of a compound generally referred to as phenothiazine or phenoxazine, and is most preferably used.

Formula 2 is generally a diphenylamine derivative, but an electron donating group is desirable at the para position, and the sensitivity is slightly inferior to the above compounds, but the reagent blindness is small.

Specific examples of the dye source used in the present invention include, for example, Biochemistry
International Vol.10 No.2 February (1986) p205-211 and Agric. Biol. Chem. 49 (9) (1985) 2799-2801 or Bull. Chem. Soc. Jpn. 69,
The pigment sources shown in 1423-1427 (1996) and those commercially available as DA-67 (Wako Pure Chemical Industries) are representative. For example, 10N-methylcarbamoyl-3,7 dimethylaminophenothiazine (abbreviated as MCDP), 10N-carboxylmethylcarbamoyl-3,7 dimethylaminophenothiazine (Wako Pure Chemical Industries, abbreviated as DA-67), 10N-methylthiocarbamoyl 3,7dimethylaminophenothiazine (MTDP), 10N-carboxylmethylthiocarbamoyl-3,7dimethylaminophenothiazine, 10N-methylcarbamoyl-3,7dimethylamino-2-nitrophenothiazine, 10N-carboxylmethylcarbamoyl-3,7dimethyl Amino-2-nitrophenothiazine, 10N-methylcarbamoyl-3,7 dimethylamino-2-chlorophenothiazine, 10N-carboxylmethylcarbamoyl-3,7 dimethylamino-2 Chlorophenothiazine, 10N-phenylcarbamoyl-3,7dimethylaminophenothiazine, 10N-carboxylphenylcarbamoyl-3,7dimethylaminophenothiazine, 10N-phenylmethylcarbamoyl-3,7dimethylaminophenothiazine, 10N-hydroxymethylcarbamoyl-3,7 Dimethylaminophenothiazine, 10N-carboxylethylcarbamoyl-3,7dimethylaminophenothiazine, 4,4'-dimethylaminodiphenyl-N-methylcarbamoyl-amine, 4,4'-dimethylaminodiphenyl-N-methylthiocarbamoyl-amine, etc. Used. The concentration used is preferably in the range of 0.01 mg / mL to 0.5 mg / mL. Some of these have poor water solubility. The following surfactants and cyclodextrins having an entrapping action can be used for dissolving such substances in an aqueous solution.

As the buffer used in the present invention, glycylglycine buffer and Good buffer may be used in addition to organic acids such as phosphate, borate and citrate. Particularly preferred are glycylglycine buffer and Good buffer. The concentration used is usually 1-100 mM with a buffering effect.

In the present invention, a surfactant that solubilizes fat components in stool and turbidity in urine can also be used. For example, surfactants having a polyethylene oxide chain and medium to higher fatty acids, such as Tween 20, Tween 60, Tween 65, Tween 80, Triton X-100, Emulgens (Kao), Pluronics (Asahi Denka), Te Tronics (Asahi Denka) are also good, but there are some which decrease the absorbance obtained by addition, and they are added appropriately in view of the solubilization ability and the desired absorbance. In addition, sodium dodecyl sulfate (SDS) which is a sulfonate of higher fatty acid, potassium lauryl sulfate, sodium myristyl sulfate, potassium myristyl sulfate, sodium palmityl sulfate, sodium stearyl sulfate, sodium oleyl sulfate, triethanolamine lauryl sulfate and the like can be used. . In particular, SDS is effective in increasing the reaction rate and is preferably used.
Moreover, what has a benzimidazole structure is suitable as what further accelerates | stimulates the catalytic action of hemoglobin. Particularly, 2-methylbenzimidazole, thiabendazole and the like can be mentioned.

Preparation of measuring reagent β-cyclodextrin (Wako Pure Chemical Industries) 4 mg / mL, 2-methylbenzimidazole (Wako Pure Chemical Industries) 1 mg / mL in a pH 6.0 solution of ADA buffer 50 mM (Dojindo Laboratories) , Thiabendazole (Wako Pure Chemical Industries) 0.02 mg / mL, triethylenetetramine-hexaacetic acid (TTHA, Dojindo Laboratories) 1 mg / mL, potassium iodate (Wako Pure Chemical Industries) 1 mg / mL were added and dissolved again. The pH was adjusted to 6.0 and used as the basic buffer.
Next, 5 mg / mL of Tween 20 (Wako Pure Chemical Industries), 5 mg / mL of sodium dodecyl sulfate (SDS, Wako Pure Chemical Industries) and 5 mg of cumene hydroperoxide (Wako Pure Chemical Industries) are added to 100 mL of the basic buffer and stirred. -Reagent 2 was dissolved and homogenized. In addition, 10 mg of DA-67 (Wako Pure Chemical Industries) was added to 100 mL of the basic buffer, and the mixture was well stirred and dissolved to give reagent R1. Further, ascorbate oxidase (Wako Pure Chemical Industries) 500 was added to 50 mL of R1. The unit was added to make reagent B R1.
On the other hand, after adjusting the pH of the basic buffer to 4.5 for comparison, 5 mg of o-tolidine hydrochloride was dissolved in 50 mL of the basic buffer instead of the dye source DA-67, and ascorbate oxidase ( Wako Pure Chemical Industries, Ltd.) 500 units added were prepared as R1. In the measurement, specimens are pretreated with R1, B1 and C1 as the first reagent, respectively, and then R2 is added as the second reagent, and the catalytic reaction of hemoglobin proceeds.

感度としては一定量のヘモグロビンの触媒反応の結果生成する色素の吸光度とし、アスコルビン酸の影響としては、アスコルビン酸とヘモグロビン標準液を添加したものの吸光度とヘモグロビン標準液のみを添加したものの吸光度の比を百分率で表した％をアスコルビン酸の影響とした。また、尿酸の影響については尿酸とヘモグロビン標準液を添加したものの吸光度とヘモグロビン標準液のみを添加したものの吸光度の比を百分率で表した％を尿酸の影響とした。尿成分の影響は随時尿とヘモグロビン標準液を添加したものから尿単独のものの吸光度の差とヘモグロビン標準液のみを添加したものの吸光度の比を百分率で表した％を尿成分の影響とした。
色素を本発明のＤＡ―６７を使用した場合、ｏ−トリジンを使用した場合に比べて約１０〜１２倍になり、非常に高感度な分析試薬が構築できた。また、アスコルビン酸オキシダーゼの添加効果は、アスコルビン酸オキシダーゼを添加していない場合（イＲ１）、回収率が４５％大きく影響を受けたが、添加した場合（ロＲ１）の９８．８％では、大きく差が出た。尿酸の影響は色素が本発明の色素を使用すればほとんど影響はないが、色素が従来のテトラメチルベンジジンでは２６％と大きな影響もあった。尿の中には尿酸の他にアスコルビン酸、微量であるがシステインなどのＳＨ化合物も含まれているため、従来の色素では影響もかなりある。しかし、本発明の色素では、アスコルビン酸オキシダーゼを入れておけば回収率も高く、影響がほとんどなかった。 Measurement of hemoglobin The following experiment was performed using reagent 2 of Example 1 with i R1 and ro R1 prepared in Example 1 as reagent 1 respectively. Human blood-derived hemoglobin (Sigma-Aldrich) was dissolved and diluted with physiological saline to a concentration of 3.8 μg / mL, and this was used as a standard solution for hemoglobin. In addition, a 100 mg / dL solution of ascorbic acid and a 10 mg / dL solution of uric acid were also prepared as affected substances. A human urine was also prepared. Six test tubes containing 10 mL were prepared. (1) 200 μL of physiological saline, (2) 100 μL of hemoglobin standard solution and 100 μL of physiological saline, (3) 100 μL of ascorbic acid solution and 100 μL of hemoglobin standard solution, (4) 100 μL of uric acid solution and 100 μL hemoglobin standard solution, (5) 100 μL ad libitum urine and 100 μL physiological saline, (6) 100 μL ad libitum urine and 100 μL hemoglobin standard solution, 2 mL of R1 reagent 1 was added, and the mixture was stirred at 37 ° C. for 3 minutes. Next, 1 mL of R2 was added to each test tube and stirred at 37 ° C. for 7 minutes. When the absorbance of each solution was measured with a spectrophotometer (Shimadzu UV2600 type), the results shown in Table 1 were obtained.

The sensitivity is the absorbance of the dye produced as a result of the catalytic reaction of a certain amount of hemoglobin, and the effect of ascorbic acid is the ratio of the absorbance of the addition of ascorbic acid and hemoglobin standard solution to that of the addition of only hemoglobin standard solution. The percentage expressed as a percentage was defined as the effect of ascorbic acid. As for the effect of uric acid, the effect of uric acid was defined as the percentage of the ratio of the absorbance of the sample added with uric acid and hemoglobin standard solution to the absorbance of the sample added with only hemoglobin standard solution. The effect of the urine component was expressed as a percentage of the ratio of the absorbance difference between the urine and the hemoglobin standard solution added to the urine alone and the absorbance of the urine component alone with the addition of the hemoglobin standard solution as a percentage.
When DA-67 of the present invention was used as the dye, it was about 10 to 12 times as compared with the case of using o-tolidine, and a very sensitive analytical reagent could be constructed. Further, the effect of addition of ascorbate oxidase was greatly affected by the recovery rate of 45% when no ascorbate oxidase was added (I R1). There was a big difference. The effect of uric acid has little effect if the dye uses the dye of the present invention, but it has a significant effect of 26% when the dye is the conventional tetramethylbenzidine. In addition to uric acid, urine also contains ascorbic acid and trace amounts of SH compounds such as cysteine. Therefore, conventional dyes have a considerable influence. However, with the dye of the present invention, if ascorbate oxidase was added, the recovery rate was high and there was almost no effect.

The hemoglobin standard solution of Example 2 was diluted with physiological saline to prepare a hemoglobin solution having a concentration of 0.6 μ / mL determined as +1 on the urine test paper and a 0.3 μg / mL determined as ±. In order to obtain the sensitivity using the above R1 and R2, the operations of (1) and (2) were performed 5 times each, and the statistical processing of the sensitivity was performed, and the average was 0.066 ABS at 0.6 μg / mL, respectively. When the coefficient of variation was 3.1% and 0.3 μg / mL, the average was 0.032 ABS, and the coefficient of variation was 5.2%. With this level of sensitivity (absorbance) and reproducibility (variation coefficient), hemoglobin can be measured quantitatively.
On the other hand, “Uriase Kc” manufactured by Terumo Corporation is used as a urine test paper, and immersed in the liquids of 0.6 μg / mL, 0.7 μg / mL, 0.3 μg / mL, and 0.4 μg / mL described above. After 20 seconds, 0.6 μg / mL was almost +1, and 0.3 μg / mL was ±, but it was difficult to distinguish between 0.6 μg / mL and 0.7 μg / mL. ,. It was difficult to distinguish between 3 μg / mL and 0.4 μg / mL. In this case, the measurement using the test paper is not quantitative.

Synthesis of MTDP and MCDP 10 g of methylene blue (Wako Pure Chemical Industries, Ltd.) was dissolved in 1 L of deionized water at room temperature, 20 g of hydrosulfite sodium was added and stirred, and methylene blue was reduced to synthesize leucomethylene blue. To this solution, 300 mL of chloroform was further added and stirred slowly, and leucomethylene blue was extracted and dissolved in the chloroform layer. The chloroform layer was separated with a separatory funnel, and dried filter paper was thrown into the separated chloroform layer to dry the chloroform layer. The filter paper was taken out, and 15 g of methyl isothiocyanate (Sigma-Aldrich) was added to the chloroform layer and reacted at room temperature for 2 days to synthesize MTDP. 500 mL of silica gel 60 (Wako Pure Chemical Industries, Ltd.) was packed in a 5 cm inner diameter glass column, washed with chloroform, and the reaction solution was added to a column conditioned with a solution of chloroform / ethyl acetate / acetic acid = 70/30/2. Elution was performed with a solution of ethyl acetate / acetic acid = 70/30/2. A rich cut was obtained while confirming MTDP by silica gel thin layer chromatography, and the rich cut was concentrated under reduced pressure using a rotary evaporator. 200 mL of methanol was added to the concentrated residue and dissolved while heating. After dissolution, the solution was ice-cooled and recrystallized. Under reduced pressure, filter paper was applied to Nutsche, washed with methanol, and the residue containing methanol was filtered to obtain recrystallized MTDP. Drying under reduced pressure in a desiccator gave about 3.5 g of MTDP.
Further, 15 mL of methyl isocyanate was added to the chloroform layer instead of methyl isothiocyanate to obtain 5.2 g of MCDP recrystallized by the same operation method as described above.

本発明の色素源を使用してヘモグロビンを測定すれば、高感度なヘモグロビンの測定が出来、アスコルビン酸の影響や尿成分の影響も非常に少なく測定可能であった。また、尿試験紙で＋１と判定される濃度である０．６μ／ｍＬ、±と判定される０．３μｇ／ｍＬのヘモグロビン溶液ではそれぞれ吸光度が表２に示すように十分に感度がある結果であった。
The composition similar to Lo R1 of Example 2 to which ascorbate oxidase was added was used as the first reagent. Instead of DA-67 as the chromogenic source, a: MTDP synthesized in Example 4 was: b: 10N-methylcarbamoyl- The same experiment as in Example 2 was performed using 3,7 dimethylaminophenothiazine (MCDP), C: DA-64 (Wako Pure Chemical Industries). However, when MTDP and MCDP were dissolved in the basic buffer solution, 10 mg was dissolved in 2 mL of methanol in advance and then added to the buffer solution for dissolution. The same procedure was performed except that the wavelength of the spectrophotometer using MTDP and MCDP was 666 nm, and the wavelength of C using DA-64 was 720 nm.
The results are shown in Table 2.

If hemoglobin was measured using the pigment source of the present invention, highly sensitive hemoglobin could be measured, and the effects of ascorbic acid and urine components could be measured very little. In addition, in the hemoglobin solution of 0.6 μ / mL that is determined to be +1 on the urine test paper and 0.3 μg / mL that is determined to be ±, the absorbance is sufficiently sensitive as shown in Table 2. there were.

  The measurement method and the reagent of the present invention have a sensitivity capable of detecting and quantifying hemoglobin in a small amount of urine or stool, and there is a reagent and a measurement method that are hardly affected by the reducing property of ascorbic acid, which is said to be the most problematic. I was able to provide it. As a result, when it was determined as ± in the conventional urine test paper, it was necessary for the patient to undergo an examination to see the urine sediment under a microscope as a retest. However, about 300 ng / mL of hemoglobin, which is assumed to be ±, was generated. Since the detection can be performed quantitatively, the probability of re-examination decreases, and there is a possibility that the patient does not have to bother to visit for re-examination.

R1, R2, R3 and R4 may be the same or different, and may be hydrogen, C1-C4 alkyl group, alkylene group, aryl group, hydroxyl group, carboxyl group, 1 or secondary amino group, halogen group, sulfonyl group, etc. A substituted C1-C4 alkyl group, alkylene group or aryl group; Z and Y may be the same or different and each represents an oxygen atom or a sulfur atom; R5 is hydrogen, a C1-C6 alkyl group, alkylene group, aryl group; Or a substituted alkyl group, a substituted alkylene group or a substituted aryl group. The substituent is a hydroxyl group, a carboxyl group, a primary or secondary amino group, a halogen group, a sulfonyl group, or the like. R6 and R7 may be the same or different and each represents hydrogen, a halogen group, a nitro group, a carboxyl group, a hydroxyl group, an amino group, a substituted amino group, a sulfonyl group, or a nitrile group. Indicates.

Claims (9)

Formula 1 or Formula 2 as the dye source in a buffer with a pH of 5.5-8

(Wherein R1, R2, R3 and R4 may be the same or different and are hydrogen, C1-C4 alkyl group, alkylene group, aryl group, hydroxyl group, carboxyl group, 1 or secondary amino group, halogen group, sulfonyl group) Represents a C1-C4 alkyl group, alkylene group or aryl group substituted with a group, etc., Z and Y may be the same or different and each represents an oxygen atom or a sulfur atom; R5 represents a hydrogen C1-C6 alkyl group or alkylene group; An aryl group, a substituted alkyl group, a substituted alkylene group, or a substituted aryl group, which is a hydroxyl group, a carboxyl group, a primary or secondary amino group, a halogen group, a sulfonyl group, etc. R6 and R7 are the same. Or may be different, hydrogen, halogen group, nitro group, carboxyl group, hydroxyl group, amino group, substituted amino group, Ruhoniru group, a nitrile group, a substituted or unsubstituted alkyl group of C1 to C3. The substituent here represented by.) Showing the above-described substituents, the compound having a phenothiazine structure or diphenylamine structure, and an alkyl group Alternatively, a method for measuring hemoglobin in which an alkyl hydroperoxide having an aryl group is used, a dye is produced using the catalytic activity of hemoglobin, and the absorbance of the generated dye is measured.
The measuring method according to claim 1, which is a reagent for measuring hemoglobin in feces or urine. The method according to claim 1 or 2, wherein the hydroperoxide is cumene hydroperoxide or tertiary butyl hydroperoxide. 4. The measuring method according to claim 2, wherein the reducing substance in the specimen is oxidized with iodate and / or eliminated with ascorbate oxidase. 5. The method according to claim 1, wherein the buffer is Good's buffer ADA and / or glycylglycine. Formula 1 or 2 as the dye source

Wherein R 1, R 2, R 3 and R 4 may be the same or different and are hydrogen, C 1 -C 4 alkyl group, alkylene group, aryl group, hydroxyl group or carboxyl group, 1 or secondary amino group, halogen group, A C1-C4 alkyl group, alkylene group or aryl group substituted with a sulfonyl group, etc., Z and Y may be the same or different and each represents an oxygen atom or a sulfur atom; R5 represents a hydrogen C1-C6 alkyl group or alkylene; A substituent, an aryl group, a substituted alkyl group, a substituted alkylene group, or a substituted aryl group, which is a hydroxyl group, a carboxyl group, a primary or secondary amino group, a halogen group, a sulfonyl group, etc. R6 and R7 are The same or different, hydrogen, halogen group, nitro group, carboxyl group, hydroxyl group, amino group, substituted amino group Sulfonyl group, a nitrile group, a substituted or unsubstituted alkyl group of C1 to C3. The substituent here represented by indicating the aforementioned substituent.), A compound having a phenothiazine structure or diphenylamine structure, and an alkyl group Alternatively, a reagent for measuring hemoglobin containing an alkyl hydroperoxide having an aryl group and having a pH of 5.5-8.
The reagent of claim 6, wherein the buffer is Good's buffer ADA and / or glycylglycine. The reagent according to claim 6 or 7, wherein the hydroperoxide is cumene hydroperoxide or tertiary butyl hydroperoxide. The reagent according to claim 6, 7 or 8, which contains iodate and / or ascorbate oxidase.

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