KR20150042553A - Test strip for diagnosing prostate cancer using peroxide of sarcosine metabolites, production method thereof and diagnosing method using it - Google Patents

Abstract

The present invention relates to a test strip for diagnosing prostate cancer with use of peroxide which is one of the sarcosine metabolites, a manufacturing method thereof and a method for diagnosing prostate cancer using the same, thereby accurately diagnosing prostate cancer quickly in a simple manner compared an existing method using a spectrophotometer where provided is to a test strip for diagnosing prostate cancer, including sarcosine oxidase which generates sarcosine metabolites; a peroxide chromogen which plays a chromogen role by reacting with peroxide of the sarcosine metabolites; and peroxidase which plays as a catalyst wherein the manufacturing method of the test strip for diagnosing prostate cancer is allowed to dissolve sarcosine oxidase and peroxidase in a buffer solution whose pH is between 7.5 and 10.0 and is allowed then to immerse and dry a cellulose test strip in the buffer solution.

Description

Technical Field [0001] The present invention relates to a method for diagnosing prostate cancer using peroxides in sarcoid metabolites, a method for preparing the same, and a method for diagnosing prostate cancer using the peroxide,

The present invention relates to a test strip for diagnosing prostate cancer and a method for diagnosing prostate cancer using the test strip. More particularly, the present invention relates to a test strip for diagnosing prostate cancer using peroxide generated by oxidation of sarcosine contained in urine of a prostate cancer patient, And a method for diagnosing prostate cancer using the same.

According to the World Health Organization (WHO), prostate cancer is the sixth most common cancer in men, followed by cancer, stomach cancer, liver cancer, colorectal cancer, and esophageal cancer. In the United States, about 186,000 patients develop each year, It is one of the most commonly diagnosed cancers among men who die of 9,000 people, but it is difficult to track the development of the disease. A typical diagnosis relies on a blood test to find a specific protein called Prostate Specific Antigen (PSA) in the blood, followed by a subsequent biopsy.

However, the value of existing prostate-specific antibodies is often elevated in prostate cancer, but this does not indicate the presence of a direct tumor, the high level of antibody does not mean that the degree of malignancy. In addition, the presence of trace amounts of prostate specific antibodies in the blood of healthy men has also been a reason to lower the reliability of prostate cancer diagnosis through prostate specific antibody testing.

Recently, Dr. Arul Chinayan of the University of Michigan Medical School says that the nature of the metabolism of prostate cancer cells, sarcoid, can be determined by how much urine is detected in prostate cancer and other organs.

Researchers have identified compounds in urine that can be used to detect prostate cancer in early stage, advanced, metastatic prostate cancer patients' urine, and differentiate positive and invasive forms of the disease. In addition, the concentration of sarcosine, the methylated form of the amino acid glycine, has been shown to result in a higher concentration of sarcosine in the urine of patients with prostate cancer, particularly when the cancer cells are more prone to spread to other organs, The authors reported that the value of sarcosine is more accurate than the prostate-specific antibody used for diagnosis at the time of diagnosis.

There have been various attempts to use it as a diagnostic index of prostate cancer by developing a method of detecting sarcosine in the urine.

Conventional methods include HPLC method and a method of using a spectrophotometer after treating sarcojenosis enzyme in a urine specimen. Both methods are possible to quantify, but there is a problem that special testers with professional skills must use expensive equipments and have a complicated process to process the sample in several stages, which limits the use.

Some of the existing techniques for diagnosing prostate cancer and related methods are as follows.

June 2, 2010 The invention described in Japanese Patent Application (2012-529021) filed by Charite Universitatsmedizin Berlin in Japan is an ex vivo method of diagnosing prostate cancer or its minor disease, Measuring at least one metabolite of only the test sample of the subject suspected of having prostate cancer and diagnosing prostate cancer or cancer thereof by the at least one metabolite. Such prior patents include collection of metabolites, data collection including the values of the metabolites, and storage media including the data collection. The prior art also provides a system for comparing the property values of a metabolite of a sample in operable form with a data storage medium. It also includes the use of one metabolite to produce diagnostic means, including at least one metabolite, and diagnostic means to diagnose prostate cancer.

This prior art relates to a method for classifying metabolites related to prostate cancer and describes the measurement of sarcojen content to diagnose prostate cancer.

This prior art has been used for the determination of sarcosine content in urine by liquid chromatography (LC) mass spectrometry and / or gas chromatography (GC) mass spectrometry in order to determine the direct content of sarcosine in the urine .

On the other hand, the present invention is different from the present invention in that it provides a test sheet for diagnosing prostate cancer using peroxides, which is a metabolite of sarcojen metabolite, and a method for diagnosing prostate cancer using the same.

The invention relating to "colorimetric methods and reagents for measuring enzymatic oxidation by enzymatic oxidation" (Registration No. 1992-0001449), filed and filed in Korea on Aug. 9, 1989 by Boehringer Mannheim GmbH, In the method of measuring the electron acceptor reduced by color formation as a colorimetric measurement of a specimen by the enzymatic oxidation of a specimen in the presence of a test substance and as a measurement value of the amount of the specimen, Wherein the specimen is oxidized by an appropriate oxidoreductase in the presence of a substance selected from the group consisting of nitrogen-containing compounds. The present invention also relates to a method for colorimetric determination of a specimen by enzymatic oxidation of a specimen.

Since the prior art uses a glucose oxidase, it is different from the present invention using sarcosine oxidase, and a colorimetric assay method for measuring the amount of a sample is described. However, the use of a sarcojen metabolite is not fundamentally used It differs from the present invention in that it does not.

In addition, the invention relating to "Methods for the diagnosis and treatment of preeclampsia or interspecies" (Publication No. 2007-0001991) filed by Beth Israel Deconis Medical Center on September 1, 2006 discloses a compound that increases VEGF or PlGF concentration or lt; RTI ID = 0.0 > sF1t-1 < / RTI > It also features a method of monitoring the treatment of preeclampsia or trauma by detecting sFlt-1, VEGF, or PlGF concentrations. It also features a method for diagnosing preeclampsia or trachoma by detecting the concentration of sFlt-1, VEGF, or PlGF in a subject.

In the above-mentioned prior art, a constitution for detecting VEGF or PlGF concentration in a urine sample for diagnosing preeclampsia or karyotypes has been described, while the present invention has been described in terms of using sarcosine oxidase for detecting sarcosine content for diagnosis of prostate cancer The present invention is different in that the colorimetric test method is used for diagnosis of disease, but the present invention is different in that the colorimetric test method for detecting peroxides generated by sarcosine oxidation is used.

In addition, the invention relating to "metabolic profiling method of prostate cancer" filed by Metaboton, Inc. on Aug. 15, 2008 (Japanese Patent Laid-Open No. 2010-537170) relates to a cancer test strip, Metabolites, and provides diagnostic, research, and therapeutic uses targeting cancer-specific metabolites.

The above-mentioned prior invention is somewhat similar in that it is described that sarcosine content is measured for diagnosing prostate cancer, but it is different from the present invention in that sarcosine oxidase is used for measuring sarcosine content, (Gas chromatography / mass spectrometry) and UHPLC-MS (ultrahigh performance liquid chromatography mass spectrometry) to determine the sarcosine content of sarcosine, the present invention relates to the use of peroxides produced by sarcosine oxidation In that it employs a colorimetric test method for detection.

SUMMARY OF THE INVENTION Accordingly, the present invention has been made to solve the above-mentioned problems of the related art, and it is an object of the present invention to provide a method and apparatus for qualitatively and quantitatively measuring sarcosine in the urine, So that the diagnosis method of prostate cancer can be provided without the need of skilled personnel and expensive equipment.

In order to achieve the above object, the present invention provides a diagnostic test sheet for diagnosing prostate cancer using peroxides, a method for producing the same, and a method for diagnosing prostate cancer using the same, wherein the sarcojenine metabolite Enzyme (Sarcosine Oxidase); A peroxide coloring material which reacts with the peroxide in the sarcosine metabolite to function as a coloring material; And peroxidase, which functions as a catalyst; The present invention provides a test strip for diagnosing prostate cancer.

The peroxide color material may be 3,3'-diaminobenzidine; 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetramethylbenzidine); 1,4-diaminobenzene; 1,2-dihydroxybenzene; 4-chloronapthol; 3-amino-9-ethylcarbazole; 2,7 '-diaminofluorene; N, N'-dimethylethylenediamine; N, N'-dimethylethylenediamine; N, N'-bis- (4-aminophenyl) -1,3-xylylenediamine); Is more preferable.

Also, a water-soluble polymer fixture that allows the addition reagents to be fixed; As shown in Fig.

Also, EDTA (Ethylenediaminetetraacetic Acid), which chelates and removes the metal ions to prevent the inhibition of the activity of sarcosine oxidase by metal ions; As shown in Fig.

Also, in the method for producing a test specimen for prostate cancer diagnosis, a method for producing a prostate cancer diagnosis test strip characterized in that a sarcojenosis oxidase and a peroxidase are dissolved in a buffer solution maintained in a pH range of 7.5 to 10.0, / RTI >

In addition, the concentration of the sarcosine oxidase is more preferably 200 to 500 unit / dL.

Further, it is preferable that the above-mentioned melt further contains a water-soluble polymer fixture for fixing the reagents.

Further, it is preferable that the above-mentioned melt further contains a dye in order to induce color development of the color material.

In addition, it is preferable that the lysate further contains EDTA which chelates the metal ion in order to prevent the inhibition of the activity of the sarcosine oxidase by the metal ion.

Further, a method for diagnosing prostate cancer using the test strip for diagnosing prostate cancer is provided.

The diagnostic test strips for diagnosing prostate cancer using peroxides, which are the metabolites of sarcosine metabolites according to the present invention, and the method for diagnosing prostate cancer using the test strips can be used for qualitative and quantitative determination of sarcosine in the urine by using a conventional HPLC method and a spectrophotometer It can be used as a diagnostic index of prostate cancer simply without need of professional manpower and expensive equipments such as analyzing it with HPCL or spectrophotometer after chemical reaction such as mixing sample and reagent in laboratory have.

1 is a graph showing changes in activity of sarcosine oxidase according to pH according to an embodiment of the present invention.
2 is a configuration view showing the appearance of a test paper according to an embodiment of the present invention;
3 is a photograph for explaining experimental results for implementation of an embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference will now be made in detail to the preferred embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. The present invention is capable of various modifications and various forms, and specific embodiments are illustrated in the drawings and described in detail in the text. It is to be understood, however, that the invention is not intended to be limited to the particular forms disclosed, but on the contrary, is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. Like reference numerals are used for like elements in describing each drawing. In the accompanying drawings, the dimensions of the structures are enlarged to illustrate the present invention, and are actually shown in a smaller scale than the actual dimensions in order to understand the schematic structure.

Also, the terms first and second, etc. may be used to describe various components, but the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, the first component may be referred to as a second component, and similarly, the second component may also be referred to as a first component. On the other hand, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

The diagnostic test site for prostate cancer using peroxidase, which is a metabolite of sarcojen metabolism and the method for diagnosing prostate cancer using the same, according to an embodiment of the present invention enables a diagnostic indicator of prostate cancer to be easily performed without expensive equipment and expert personnel.

Prostate Cancer The fact that the concentration of sarcosine in the urine of a patient increases is a fact that was published in Nature, issue 457,

In other words, sarcosine is metabolized by sarcosine oxidase to glycine, peroxide (H ₂ O ₂ ) and formaldehyde, and peroxides and formaldehyde produced by the metabolites of sarcosine are quantitatively , And thus it is possible to utilize it as a diagnostic index of prostate cancer. This metabolic process is represented by the following formula (1).

On the other hand, a conventional method for quantifying peroxides is widely known as the following formula (2).

When the reaction formula (3) below is configured to react on the test paper using the above formulas (1) and (2), the concentration of the sarcosine can be quantified through the color test paper reaction. The use of such a method is the basic technical content of the present invention.

Sarcosine produces peroxides and formaldehyde proportional to the concentration of sarcosine by sarcosine oxidase, as in [Formula 1] above. Factors affecting the activity of sarcosine oxidase at this time include the pH and concentration of the buffer solution and the reaction temperature.

At this time, the reaction temperature is sufficient at room temperature, the concentration of the buffer solution is about 1 to 2 molar, and the pH is preferably 8 to 9.5.

Tris, phosphate, citrate, borate, and the like can be used as the buffer solution.

Peroxidase, which catalyzes the peroxidation dehydrogenation reaction produced by sarcosine oxidase, is expressed specifically in the above formula (2), and the activity of about 50 to 80% of sarcosine oxidase Adding the amount is sufficient. Followed by 3,3'-diaminobenzidine; 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetramethylbenzidine); 1,4-diaminobenzene; 1,2-dihydroxybenzene; 4-chloronapthol; 3-amino-9-ethylcarbazole; 2,7 '-diaminofluorene; N, N'-dimethylethylenediamine; N, N'-dimethylethylenediamine; Peroxide color materials such as N, N'-bis- (4-aminophenyl) -1,3-xylylenediamine N, N'-bis- (4-aminophenyl) It becomes colored.

In this case, in the case of a color which is not easily recognized by eyes such as blue, an additional method of making the background color yellow by using yellow dye or the like, so that the tendency of color development is not blue but green, cyan or the like can be used.

Surfactants may also be added to allow the urine to be easily absorbed into the test strip, or a fixture (usually using a water soluble polymer) may be added to allow the addition reagents to be fixed to the cellulose test strip.

On the other hand, when using two types of enzymes, namely, sarcosine oxidase and peroxidase enzyme, the immersion solution should be prepared in two divided doses due to the difference in pH stability of each enzyme.

In the case of sarcosine oxidase, as shown in FIG. 1, the enzyme has the maximum activity at pH 8.0. In the case of the general spectrophotometric method, the concentration of the buffer solution is 0.05 to 0.1 mole, and the pH is in the range of 7.5 to 8.5. In the present invention, the test paper prepared by the colorimetry test method is dipped in a sample and exposed severely to the acidic pH and the like of the sample. To overcome this harsh exposure, the buffer solution of the primary solution is prepared by using a solution of borate-sodium hydroxide pH 8.0 to 9.5 at a concentration of 1.0 to 2.0 mol.

An excess amount of enzyme of 200 to 500 unit / dL of sarcosine oxidase should be added in consideration of the decrease in the enzyme activity when the test paper is dipped in the preparation solution and the test paper is thermally dried. If the amount of sarcosine oxidase is small at this time, the activity of the enzyme is deteriorated due to the activity of the enzyme in the drying process of the test strip, so that the enzyme does not have sufficient reactivity.

When the added amount of the reagent is not fixed to the test paper, the test paper is immersed in the sample. When the test paper is immersed in the sample, the reagent And the color contamination occurs. If the amount is large, even the water-soluble polymer interferes with the absorption of the sample.

When 3,3 ', 5,5'-tetramethylbenzidine is used as a coloring material, the color develops in the blue color system, but in the case of light blue color, it is not easy to visually confirm the difference. For this reason, when the color of the blue system is progressed from yellow to ground color, the color of the green system is displayed by the mixture of the yellow base and the blue color. Thus, 0.05-0.1 g / dL of tartrazine is used for easy visual confirmation. At this time, when the amount of tarragin is low, the color of the sample is buried in the color of the sample and it becomes green or blue. When the amount of tarragin is used, a light blue reaction by a small amount of sarcosine is buried in a deep yellow color.

As shown in [Table 1], the activity of sarcosine oxidase is greatly influenced by the metal ion. When excessive amount of metal ion is present in the sample, it is chelated and removed to lower the activity of sarcosine oxidase by the metal ion 0.0 > g / dL < / RTI > of EDTA is added. That is, EDTA acts as a metal adsorbent, in order to enable the enzymes to be activated well. In this embodiment, EDTA4NA is used, but other materials capable of chelating metal ions may be used. On the other hand, in the case of EDTA, if the amount of EDTA is small, it is not enough to remove the metal ion. Conversely, when the amount of EDTA is large, the coloring reaction is rather deteriorated.

After the reagent is sufficiently dissolved, the cellulose test paper is immersed, and the excess reagent is removed, followed by drying at 70 to 80 ° C for 30 minutes.

The secondary solution was prepared by adding 100 to 200 unit / dL of peroxidase, 3,3 ', 5,5'-tetramethylbenzidine 20 mM (mM) concentration. If the added amount of reagent is small, there is no difference in color between the medium concentration and high concentration sample. On the other hand, if the added amount is large, discoloration occurs in the natural state, resulting in deterioration of product or false positives.

When the reagent is sufficiently dissolved, the test paper which has been immersed in the first solution is dipped again in the second solution, and dried at 70 to 80 ° C for 30 minutes.

After that, a double-sided tape is attached to the test strip, and the test strip is cut and fixed on a support, and then molded into a shape as shown in FIG. 2. Since the resultant is sensitive to moisture, it is stored in a high- use.

[Figure 3] is an experimental result for diagnosing prostate cancer using the test strip prepared by the above method, and the results are shown in Table 1. [ This is a photo of sarcosine detection reaction using peroxidase when benzidine is used.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is clear that the present invention can be suitably modified and applied in the same manner. Therefore, the above description does not limit the scope of the present invention, which is defined by the limitations of the following claims.

Claims (10)

Sarcosine Oxidase, which produces the sarcosine metabolite;
A peroxide coloring material which reacts with the peroxide in the sarcosine metabolite to function as a coloring material; And
Peroxidase, which acts as a catalyst; Wherein the diagnostic test site is a prostate cancer diagnostic test site.
The method according to claim 1,
The peroxide color material,
3,3'-diaminobenzidine; 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetramethylbenzidine);1,4-diaminobenzene;1,2-dihydroxybenzene;4-chloronapthol;3-amino-9-ethylcarbazole; 2,7 '-diaminofluorene; N, N'-dimethylethylenediamine; N, N'-dimethylethylenediamine; N, N'-bis- (4-aminophenyl) -1,3-xylylenediamine); A test sample for diagnosing prostate cancer
The method according to claim 1,
A water soluble polymeric fixture that allows the addition reagents to be fixed; Further comprising a diagnostic test site for prostate cancer.
The method according to claim 1,
EDTA (Ethylenediaminetetraacetic Acid) which chelates and removes the metal ions to prevent the inhibition of the activity of sarcosine oxidase by the metal ion; Wherein the diagnostic test site is a prostate cancer diagnostic test site.
A method for producing a test strip for diagnosing prostate cancer,
Wherein the sarcojenosis oxidase and the peroxidase are dissolved in a buffer solution having a pH ranging from 7.5 to 10.0 to immerse the cellulosic test tissue and then to dry the test tissue.
6. The method of claim 5,
Wherein the concentration of the sarcosine oxidase is in the range of 200 to 500 unit / dL.
6. The method of claim 5,
Wherein the lysate further comprises a water-soluble polymer fixative to fix the reagents.
6. The method of claim 5,
Wherein the lysate further comprises a dye to induce color development of the color material.
6. The method of claim 5,
Wherein the lysate further comprises EDTA which chelates the metal ion in order to prevent the inhibition of the activity of the sarcosine oxidase by the metal ion.
A method for diagnosing prostate cancer using the test strip for prostate cancer diagnosis according to any one of claims 1 to 4.

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