WO2023090553A1 - Kit de diagnostic pour diagnostiquer un cancer colorectal à l'aide d'urine humaine comme échantillon - Google Patents

Kit de diagnostic pour diagnostiquer un cancer colorectal à l'aide d'urine humaine comme échantillon Download PDF

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Publication number
WO2023090553A1
WO2023090553A1 PCT/KR2022/007652 KR2022007652W WO2023090553A1 WO 2023090553 A1 WO2023090553 A1 WO 2023090553A1 KR 2022007652 W KR2022007652 W KR 2022007652W WO 2023090553 A1 WO2023090553 A1 WO 2023090553A1
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Prior art keywords
sample
line
colorectal cancer
urine
diagnostic
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PCT/KR2022/007652
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English (en)
Korean (ko)
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이상훈
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주식회사 스템바이오
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Publication of WO2023090553A1 publication Critical patent/WO2023090553A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon

Definitions

  • the present invention relates to a diagnostic kit capable of diagnosing colorectal cancer using human urine as a sample and a method for manufacturing the same, and more particularly, to a diagnostic kit for diagnosing colorectal cancer with the naked eye using an immunochromatography method. It is about diagnostic kits that can be used.
  • the large intestine is a place where digestion and absorption of food ingested orally takes place, and it is also a place where surplus food stays. In addition, this is where feces are made by absorbing moisture, and it is also a place where many kinds of bacteria live.
  • the length of the large intestine is about 2 m, and it is composed of the colon, rectum, and anus. Cancer can occur anywhere there is a mucous membrane of the large intestine, but the areas most prone to cancer are the S colon and the rectum.
  • colorectal cancer Currently, the incidence of colorectal cancer in Korea is increasing remarkably, and deaths due to colorectal cancer account for the fourth in males after stomach cancer, lung cancer, and liver cancer, and in females, it is similar. It is considered that environmental factors have a greater proportion than genetic factors influencing the occurrence of colorectal cancer, and it is recognized that the rapid westernization of dietary life, especially excessive intake of animal fat or protein, is the cause. However, it is known that around 5% of colorectal cancer is caused by a genetic predisposition.
  • colorectal cancer When detected early, colorectal cancer can be completely cured by endoscopic resection or surgical treatment, and even if it has progressed and metastasized (distant metastasis) to the liver or lungs, a complete cure by surgical treatment can be expected if surgery is possible. However, if the detection is delayed, surgical treatment cannot be used as in the above case because metastasis occurs to places where it is difficult to resect, such as the lung, liver, lymph nodes, or peritoneum.
  • colorectal cancer is almost 100% cured if it is early, but it is very difficult to detect it in an asymptomatic period because there are generally no subjective symptoms, and therefore, periodic examinations should be preceded.
  • the diagnosis of colorectal cancer is performed by rectal digital examination, fecal occult blood test, and colonography for patients with symptoms related to the large intestine. If necessary, a biopsy through colonoscopy is performed. It is done.
  • the present invention has been proposed to solve the above problems, and the object of the present invention is to provide a diagnostic kit that can easily check the occurrence of colorectal cancer by an immunological method using an indicator substance detected in human urine as a sample. is to have
  • Another object of the present invention is to provide a colorectal cancer diagnostic kit that can be easily checked and read for colorectal cancer with the naked eye using urine as a sample at a medical institution or home without special equipment, and a method for manufacturing the diagnostic kit.
  • the present invention provides a diagnostic strip for diagnosing colorectal cancer; Includes a urine sample inlet for protecting the diagnostic strip from various contaminants and injecting a sample into the diagnostic strip, and a result display unit formed on each diagnostic strip to indicate the diagnosis result of the urine injected through the urine sample inlet upper case to do;
  • a colorectal cancer diagnosis kit including a lower case in which a support is formed to support one surface of the diagnostic strip is provided.
  • the membrane that can be used for manufacturing the diagnostic strip is a material used as a material for a conventional diagnostic strip, for example, various synthetic polymers such as nitro cellulose, cellulose, cellulose acetate, and polyethylene. may be used, and reagents used for immunological analysis may include a suitable carrier, a detection label capable of generating a detectable signal, a solubilizing agent, and a detergent.
  • a suitable carrier may include a substrate capable of measuring enzyme activity and a reaction terminator, such as a soluble carrier, such as a physiologically acceptable buffer known in the art (eg, PBS), etc. may be used.
  • a reaction terminator such as a soluble carrier, such as a physiologically acceptable buffer known in the art (eg, PBS), etc.
  • the diagnostic antibody according to the present invention can use purified monoclonal anti-PSA, rabbit anti-PSA, and goat anti-mouse IgG, which are raw materials of commercially available antibodies that can be used for the manufacture of strips, preferably Preferably, goat-anti-mouse IgG polyclonal antibody and rabbit-anti-prion monoclonal antibody can be used.
  • kits for diagnosing colon cancer means a kit containing a composition for diagnosing colon cancer. Therefore, the expression “kit for diagnosing colon cancer” may be used interchangeably or interchangeably with “composition for diagnosing colon cancer”.
  • diagnosis refers to determining the susceptibility of a subject to a specific disease or disorder, determining whether a subject currently has a specific disease or disorder, or being susceptible to a specific disease or disorder Determining the prognosis of an affected subject (eg, determining the stage of colorectal cancer or predicting responsiveness to treatment), or using therametrics (eg, determining a subject's condition to provide information about treatment efficacy) including monitoring).
  • Colorectal cancer refers to various malignant tumors composed of cancer cells formed in the large intestine, and the present inventors confirmed that using the marker of the present invention can promptly and accurately diagnose the onset of colorectal cancer from a sample collected from an individual ( see Example 2).
  • colonal cancer diagnostic marker or colorectal cancer marker is a substance capable of diagnosing colorectal cancer, and a biomolecule that is increased in subjects with colorectal cancer compared to normal persons but decreased in urine.
  • the colorectal cancer diagnostic marker is a human prion protein.
  • the kit of the present invention forms an immunological complex through an immunoassay method, that is, an antigen-antibody reaction, an antigen-antibody reaction, and the formed immunological complex can be detected using an immunoassay method.
  • an immunoassay method that is, an antigen-antibody reaction, an antigen-antibody reaction, and the formed immunological complex can be detected using an immunoassay method.
  • numerous immunoassay methods used to detect or quantify antigens are known to those skilled in the art, see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York 1988, 556-612, Reiter RE., Gu Z., Watabe T., Thomas G., Prostate stem cell antigen: A cell surface marker overexpressed n prostate cancer. Proc Natl Acad Sci USA. 1998. Feb 17;95(4):1735-40].
  • the colorectal cancer diagnosis kit of the present invention is designed based on the immunochromatographic assay, and the principles of currently marketed colorectal cancer diagnosis kits can be applied as it is. It is a test method that combines the chromatographic assay and applies the specific immunoreactivity of the antibody to the antigen and the movement of molecules by the capillary phenomenon of the porous membrane.
  • the immunochromatography method integrates the color development process through the reaction of sample dilution, washing, and enzyme conjugate and substrate required in the existing multi-step immunoassay method into one, quickly and one-step. It is convenient to inspect, and also has advantages in the ease and economy of determining the inspection result without using specific equipment, and the speed of reading the inspection result.
  • a human urine sample is added to a microtiter plate coated with an antibody against human prion protein and reacted, the enzyme-secondary antibody conjugate is reacted, and the substrate of the enzyme is treated
  • Colorectal cancer can be diagnosed based on the result of measuring absorbance and comparing the absorbance with a standard curve to quantitatively compare prion proteins present in human urine.
  • the diagnostic kit provided herein can not only use urine as a sample, but also apply immunochromatography to blood as a sample to measure the specific antigen of human prion protein, thereby detecting colorectal cancer, colorectal cancer-related malignant and benign tumors, etc. It can also be applied to methods for diagnosing cancer diseases.
  • the present invention can be provided as a diagnostic kit capable of semi-quantitatively measuring the amount of prion protein in urine and blood by immunochromatography, and also immune response to urine components for accurate diagnosis of the present invention. It can be applied as a method of accurately diagnosing using a measurement reader, and its specific manufacturing method and application embodiments will be described in detail through the description of 'specific contents for carrying out the invention' below.
  • colorectal cancer is determined not by a doctor's opinion, but by a molecular diagnosis method.
  • the markers of the present invention are biomolecules that are present in high concentrations in colorectal cancer, but are characterized in that they are present in low concentrations in human urine, which is the sample of the present invention.
  • a treatment for colorectal cancer can be administered to a patient.
  • the present invention is a step of separating urine from the subject; 80 to 120 ⁇ l of urine was added to the sample pad of the colorectal cancer diagnostic kit of the present invention, and after 5 to 10 minutes, color development of the control line C (control line) and the result line T (test line) was judged, and only the control line was colored reddish purple. It provides a method for diagnosing colon cancer, including diagnosing colon cancer when indicated.
  • the present invention proposes prion protein in urine as a biomarker for colorectal cancer.
  • the marker of the present invention can be used to conveniently, quickly and accurately diagnose colon cancer as a urine marker.
  • 1 is a diagram showing a comparison of prion concentrations in the urine of a normal person and a colorectal cancer patient.
  • FIG. 2 is a graph showing a comparison of prion concentrations in the urine of a normal person and a colorectal cancer patient.
  • 3A is a diagram showing a colorectal cancer diagnostic kit strip of the present invention.
  • 3B is a diagram showing a schematic diagram of accessories constituting the colorectal cancer diagnosis kit of the present invention.
  • 3C is a diagram showing a colorectal cancer diagnostic kit strip and a lower case of the present invention.
  • FIG. 4 is a diagram showing the result of an enzyme immune reaction of prion protein in urine detected in a colorectal cancer patient by the colorectal cancer diagnosis kit of the present invention.
  • terminal used in this specification are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the contents throughout this specification. Throughout the specification, when a certain component is said to "include”, it means that it may further include other components without excluding other components unless otherwise stated.
  • the present invention collects urine from a total of 100 people (50 normal patients among patients visiting Ajou University and 50 colorectal cancer patients among patients visiting Chungnam National University) under the IRB review exemption number (P01-202108-31-006). recruited
  • Urine was collected from 50 normal control subjects and 50 colorectal cancer patients, and prion protein in the urine was quantitatively confirmed by an enzyme immunoassay method.
  • the average result of 50 normal groups showed a value of about 0.6255ng/ml.
  • the average result value of 50 patients with colorectal cancer was about 0.5366 ng/ml (see FIG. 1).
  • colorectal cancer can be diagnosed at an early stage by using prion protein present in human urine as a biomarker and detecting it using an enzyme immunization method using an antibody.
  • the antibody binding pad uses a polyester (100% Polyester) pad, and the antibody is evenly sprayed on the pad and dried to prepare and use the antibody binding pad.
  • polyester 100% Polyester
  • the sample pad glass microfiber or cotton fiber (Cotton Linter), but 0.1 to 3.0% Triton X-100 surfactant was evenly sprayed and used after drying.
  • a cotton fiber pad was used as an absorbent pad (100% Cotton Linter) to absorb unreacted substances in the sample and to move the sample solution including the analyte by capillary action.
  • a nitrocellulose membrane is used as the strip material, and two lines (line 1: Control, line 2: Test) are applied to the membrane, and the first control line (Control) is a goat-anti-mouse IgG polyclonal antibody with a polyclonal antibody of 1.5 to 10. It was spotted at a concentration of 1.7 mg/ml, and the second control (Test) line was spotted with a rabbit-anti-prion monoclonal antibody at a concentration of 0.5 to 1.0 mg/ml and used after drying.
  • Line 1 Control
  • line 2 Test
  • the diagnostic strip was provided with a nitrocellulose membrane, a sample pad to which 2 to 3 drops of urine were added, a bonding pad made of polyester, and an absorbent pad made of cotton fiber.
  • the nitrocellulose membrane has two lines.
  • the first line is a control line C-line in which goat-anti-mouse IgG polyclonal antibody is spotted at a concentration of 1.5 to 1.7 mg/ml
  • the second line is a rabbit-anti-prion single
  • the clonal antibody was applied dropwise at a concentration of 0.5 to 1.0 mg/ml, dried, and used, and provided in a configuration having a T-line, a result line provided to compare and observe the presence or absence of color development and the thickness of the concentration.
  • the bonding pad is provided by applying antibody to it, spraying it evenly on a polyester (100% Polyester) pad and then drying it. Diagnostic strips were used in a configuration provided that cotton fibers (100% Cotton Linter) were used to act as transfers.
  • the diagnostic kit is provided with a lower case and an upper case formed of a plastic housing, and the lower case is provided in a structure having an upper strip holder, a lower strip holder, a plurality of supports on the side, an upper locking jaw, and a lower locking jaw.
  • the absorbent pad of the diagnostic strip is located, and the sample pad of the diagnostic strip is located on the lower jaw, forming a structure that is stably placed / inserted into the upper strip holder and the lower strip holder, and the upper case has a handle portion at one end
  • the diagnostic kit has a urine sample inlet and a result display part displaying the control line C-line and the result line T-line of the diagnostic strip in the central part, and an entry direction display part interpolated to the reader. It is configured to diagnose colorectal cancer through color development.
  • test sample was dropped into the invented diagnostic kit using the principle of an enzyme immunization method for detecting prion protein, and the color development results of the test line and the control line were determined.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
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  • Urology & Nephrology (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention propose une protéine prion en tant que marqueur moléculaire pour le cancer colorectal. Le marqueur de la présente invention peut être utilisé en tant que marqueur d'urine pour diagnostiquer de manière commode, rapide et précise le cancer colorectal.
PCT/KR2022/007652 2021-11-18 2022-05-30 Kit de diagnostic pour diagnostiquer un cancer colorectal à l'aide d'urine humaine comme échantillon WO2023090553A1 (fr)

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KR1020210159457A KR102649112B1 (ko) 2021-11-18 2021-11-18 인간의 소변을 검체로 이용하여 대장암을 진단하는 진단키트
KR10-2021-0159457 2021-11-18

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090246798A1 (en) * 2008-03-25 2009-10-01 Man-Sun Sy Method of detecting cancer and evaluating cancer prognosis
KR20100044307A (ko) * 2008-10-22 2010-04-30 한국생명공학연구원 대장암 관련 마커를 이용한 대장암 진단 키트 및 이를 이용한 대장암 진단 방법
KR20100061192A (ko) * 2008-11-28 2010-06-07 한국생명공학연구원 대장암 진단 마커 및 이를 이용한 대장암 진단방법
JP2012018119A (ja) * 2010-07-09 2012-01-26 Toray Ind Inc 大腸癌検出用マーカーおよびそれを用いた大腸癌検出方法
KR20120124320A (ko) * 2011-05-03 2012-11-13 (주)에스에이치제약 인간의 소변을 검체로 이용하여 전립선암을 진단하는 진단키트 및 그 제조방법
KR20140067047A (ko) * 2011-08-29 2014-06-03 도레이 카부시키가이샤 대장암 또는 식도암의 검출용 마커 및 검사 방법
WO2017213246A1 (fr) * 2016-06-10 2017-12-14 株式会社日立製作所 Procédé de diagnostic de maladie basé sur un métabolite dans l'urine

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101212024B1 (ko) 2011-08-08 2012-12-13 한국생명공학연구원 대장암 및 전이에 대한 바이오마커, 이를 이용한 대장암 진단 및 치료제 스크리닝

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090246798A1 (en) * 2008-03-25 2009-10-01 Man-Sun Sy Method of detecting cancer and evaluating cancer prognosis
KR20100044307A (ko) * 2008-10-22 2010-04-30 한국생명공학연구원 대장암 관련 마커를 이용한 대장암 진단 키트 및 이를 이용한 대장암 진단 방법
KR20100061192A (ko) * 2008-11-28 2010-06-07 한국생명공학연구원 대장암 진단 마커 및 이를 이용한 대장암 진단방법
JP2012018119A (ja) * 2010-07-09 2012-01-26 Toray Ind Inc 大腸癌検出用マーカーおよびそれを用いた大腸癌検出方法
KR20120124320A (ko) * 2011-05-03 2012-11-13 (주)에스에이치제약 인간의 소변을 검체로 이용하여 전립선암을 진단하는 진단키트 및 그 제조방법
KR20140067047A (ko) * 2011-08-29 2014-06-03 도레이 카부시키가이샤 대장암 또는 식도암의 검출용 마커 및 검사 방법
WO2017213246A1 (fr) * 2016-06-10 2017-12-14 株式会社日立製作所 Procédé de diagnostic de maladie basé sur un métabolite dans l'urine

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Title
WIT, M. D. ET AL.: "Cell surface proteomics identifies glucose transporter type 1 and prion protein as candidate biomarkers for colorectal adenoma-to-carcinoma progression", GUT, vol. 61, 2012, pages 855 - 864, XP009545550 *

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