WO2017196139A1 - Kit de diagnostic - Google Patents

Kit de diagnostic Download PDF

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Publication number
WO2017196139A1
WO2017196139A1 PCT/KR2017/004970 KR2017004970W WO2017196139A1 WO 2017196139 A1 WO2017196139 A1 WO 2017196139A1 KR 2017004970 W KR2017004970 W KR 2017004970W WO 2017196139 A1 WO2017196139 A1 WO 2017196139A1
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WO
WIPO (PCT)
Prior art keywords
blood
antibody
peptide
target
kit
Prior art date
Application number
PCT/KR2017/004970
Other languages
English (en)
Korean (ko)
Inventor
박성규
이건호
최석원
심규영
Original Assignee
광주과학기술원
조선대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 광주과학기술원, 조선대학교 산학협력단 filed Critical 광주과학기술원
Publication of WO2017196139A1 publication Critical patent/WO2017196139A1/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
    • 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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G01N33/5304—Reaction vessels, e.g. agglutination plates
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the present invention relates to diagnostic kits, in particular kits for diagnosing autoimmune diseases.
  • kits using antigen-antibody reactions are mostly made of kits using antigen-antibody reactions, and are typically designed to diagnose substances as antigens in biological samples.
  • Pregnancy diagnostic kit one of the currently commercially available diagnostic kits, is designed to be diagnosed by human chorionic gonadotropin (Hcg) in blood, which corresponds to the antigen in the applied antigen-antibody reaction of the diagnostic kit.
  • Hcg human chorionic gonadotropin
  • Specific substances can be specifically combined with antibodies to enable diagnosis.
  • such a conventional diagnostic kit typically uses an ELISA method and an anti-human IgG antibody, in which case randomly binds to autoimmune antibodies in the blood, various autoimmune antibodies present in the blood (autoimmune antibodies) There is a problem that does not screen for disease-specific autoimmune antibodies to.
  • a conventional diagnostic kit since a very small amount of antigen is present in the blood when diagnosing an infectious disease such as HIV, such a conventional diagnostic kit has a limitation in that sensitivity is low.
  • the present invention is to provide a diagnostic kit using an antibody as a diagnostic factor to solve the problems of such a conventional diagnostic kit.
  • a diagnostic kit comprising a blood development membrane located at (D) and an absorbent pad region located at the rear end of the blood development membrane based on the direction of blood progression, wherein the blood development membrane has a (c1) test line and (c2) control line on its surface.
  • the binding pad region may include a biotinyl peptide capable of specific binding with a labeled globulin antibody and a target antibody, and a coloring peptide capable of specific binding with the target antibody. Tabidine is immobilized, and the globulin antibody is immobilized on the control line.
  • disease-specific autoimmune antibodies can be screened for various autoimmune antibodies present in blood, but also have an effect of significantly improved sensitivity than conventional diagnostic kits.
  • FIG. 1 is a schematic diagram of a conventional kit utilizing a conventional antigen as a diagnostic factor.
  • FIG. 2 is a schematic diagram of a diagnostic kit utilizing an antibody as a diagnostic factor according to an embodiment of the present invention.
  • FIG 3 is a plan view and a side view of the diagnostic kit according to an embodiment of the present invention, respectively, viewed from above and from the side.
  • Figure 4 is a schematic diagram for explaining the operating state of the diagnostic kit according to an embodiment of the present invention.
  • a dark band is formed in both the test line and the control line, it is diagnosed that the blood sample contains a target antibody above the detection limit. If a dark band is formed only in the control line, the concentration of the target antibody is below the detection limit As low or non-existent.
  • no dark bands are formed in either the test line or the control line, it means that the specific binding that underlies the diagnosis according to the present invention has failed for some reason. Can be interpreted.
  • the present invention relates to a kit capable of diagnosing a disease by confirming the presence and use of an autoimmune antibody present in the blood of such a patient and capable of determining the presence or absence of the disease.
  • the present invention provides a new diagnostic kit system capable of screening only specific autoimmune antibodies in the blood, and also provides a visualization system that can easily determine whether the diagnostic kit works well.
  • a diagnostic kit comprising a region, (D) an absorbent pad region located at the rear of the blood developing membrane relative to the direction of blood development, wherein the blood developing membrane comprises (c1) a test line and (c2) a control line on a surface thereof,
  • the binding pad region comprises a labeled globulin antibody and a biotinyl peptide capable of specific binding to a target antibody, and a chromogenic peptide capable of specific binding with the target antibody, and streptavidin is immobilized on the test line. And a globulin antibody is immobilized on the control line.
  • the diagnostic kit according to the present invention consists of four porous membranes or pads, each of which is a sample pad, a binding pad, a blood development membrane (eg, nitrocellulose membrane), and an absorption pad.
  • the four porous membranes may be fixed on a plastic or glass support membrane as shown in FIG. 3 to constitute a diagnostic kit, wherein the sample pads, binding pads, blood development membranes, and absorption pads may be maintained continuously without blood development.
  • the ends of the can also be fixed so that they overlap each other little by little, for example 3 mm.
  • the sample pad region is a region in which blood of a patient containing globulin and a target antibody is accommodated, and may be treated with a buffer reagent or the like in advance.
  • the blood contained in the sample pad region is developed and sequentially passes through the binding pad, the blood developing membrane, the test line and the control line (or the control line and the test line) formed thereon to reach the absorption pad.
  • the binding pad region includes a labeled globulin antibody, a biotinyl peptide capable of specific binding with the target antibody, and a chromogenic peptide capable of specific binding with the target antibody.
  • the globulin in the developing blood binds to the labeled globulin antibody, and because the target antibody is capable of binding two peptides, it may bind the first two biotinyl peptides, and the second two chromogenic peptides. Alternatively, or third, the biotinyl peptide and the chromogenic peptide may be combined.
  • streptavidin is bound so that the target antibody which binds to two biotinyl peptides and the target antibody which binds to both biotinyl peptide and the chromophore peptide among these three target antibodies passes through this line.
  • a line can be observed while the chromogenic peptide region of the target antibody bound to both the biotinyl peptide and the chromogenic peptide is developed.
  • the globulin antibody is immobilized on the control line, and the globulin bound with the chromogenic globulin antibody passing through the line also binds to the globulin antibody, and a line can be observed as the chromogenic globulin antibody region develops.
  • the absorbent pad region serves as a reservoir of blood samples flowing through the blood developing membrane.
  • the target antibody is an autoimmune antibody and the target disease is an autoimmune disease.
  • the present invention uses a target antibody generated due to a specific target disease and contained in blood as a diagnostic target.
  • the autoimmune antibody is contained in the blood of a patient suffering from an autoimmune disease, and in the present invention, when targeting an autoimmune disease, such an autoimmune antibody becomes a target antibody.
  • control line is located at the rear of the test line based on the blood flow direction.
  • the region excluding the biotinyl of the biotinyl peptide and the region except the chromophore of the chromogenic peptide may be the same or different from each other, but both regions may specifically bind to the target antibody.
  • Peptide epitopes As described above, one autoimmune antibody is used to bind two peptide epitopes. In the present invention, each of the autoimmune antibodies is labeled to be capable of coloring and binding at a specific position due to biotin to constitute a diagnostic kit.
  • the blood development direction rather than the test line is used to check whether the blood is developed from the test line to the control line. It is preferable to be located at the rear end, but such a position is not necessarily necessary and it is not excluded that it may be located at the front of the test line from time to time.
  • FIG. 1 is a schematic diagram of a kit for diagnosing an existing diagnostic kit using an antigen as an index
  • the diagnostic kit includes a nitrocellulose membrane and three pads (sample pad, binding pad, and absorption pad), a test line, and a control line. It is composed.
  • the nitrocellulose membrane acts as a development medium that spreads the blood to the absorbent pad, and the sample pad acts as the patient's blood drop.
  • the binding pad of the conventional diagnostic kit is composed of a coloring antibody and a coloring streptavidin for an antigen in the blood, and a test line is fixed with an antibody to an antigen in the blood, and a biotin that binds to streptavidin is fixed in the control line.
  • antigens When blood falls, antigens enter the sample pads and move to the binding pads. In the binding pad, the antigen binds to the chromogenic antibody for that antigen and moves to the test line with the chromogenic streptavidin in the binding pad.
  • Antigen complexes in blood bound to chromogenic antibodies bind to antibodies against antigens immobilized on the test line and are immobilized in the form of sandwiches, and the presence or absence of antigens is shown through the gland.
  • the chromogenic streptavidin appears as a line in combination with the biotin immobilized in the control line, which determines whether it works normally.
  • Figure 2 is a schematic diagram of a diagnostic kit according to an embodiment of the present invention, it is largely composed of a nitrocellulose membrane and three pads (sample pad, binding pad, absorbent pad), the test line and the control line Not very different from the kit.
  • the binding pad includes a chromophoric peptide, a biotinyl peptide, which binds to an autoimmune antibody, and a globulin antibody that binds to globulin Is present.
  • test line is immobilized with streptavidin capable of binding to biotin, and globulin antibodies are immobilized on the control line.
  • autoimmune antibodies bind specifically to the peptide and globulins bind to labeled globulin antibodies. Since one autoimmune antibody can bind two peptides, there are three types of autoimmune antibodies combined with two chromogenic peptides, autoimmune antibodies combined with two bionyl peptides, and autoimmune antibodies combined with chromogenic peptides and bioyl peptides. The case of bonding appears.
  • the blood passes through the test line, and biotin binds specifically to the fixed streptavidin and the rest passes.
  • the autoimmune antibodies which bind to the chromogenic peptide and the biotinyl peptide, respectively, are immobilized on the test line by biotin and lined by the chromogenic peptide.
  • the user can check whether the blood is normally deployed through the control line, and can check whether the product is defective or not.
  • the user can check the presence or absence of autoimmune antibodies in the blood through the presence of the line, through which it is possible to determine the disease.
  • one of the differences between the existing diagnostic kit and the diagnostic kit according to an embodiment of the present invention is an indicator to be used for a test and a coupling principle in a test line and a control line.
  • the diagnostic kit according to an embodiment of the present invention does not use the antigen in the blood as an indicator of the kit, but uses an autoimmune antibody as an indicator and uses the globulin in the blood to determine whether the kit works.
  • Antibodies are defenses made by B cells against specific antigens, and are much more sensitive than antigens. As a result, the diagnosis of infectious diseases such as HIV uses highly sensitive antibodies instead of antigens present in the blood, which is why the binding principle of the test line is very different from kits that use conventional antigens as indicators.
  • test line of the pregnancy diagnosis kit uses a sandwich ELISA method, that is, a method of determining antigen using two antibodies.
  • diagnostic kit according to an embodiment of the present invention used the structural features of the autoimmune antibodies (points having two binding sites) to measure autoimmune antibodies, one of which was responsible for color development, and the other was responsible for fixation. Peptides were conjugated to indicate whether an autoimmune antibody was indicated by a line.
  • control line coupling principle of the present invention is also different.
  • Pregnancy diagnostic kits use the combination of the color streptavidin present in the binding pad with biotin from the control line to determine normal operation by simple fluid flow.
  • the control line of the diagnostic kit according to an embodiment of the present invention was able to accurately determine whether the kit is operated by direct determination of the blood flow using the globulin antigen present in the blood.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un kit de diagnostic et, plus spécifiquement, un kit de diagnostic de maladie auto-immune. Selon différents modes de réalisation de la présente invention, des anticorps auto-immuns spécifiques de maladies peuvent être sélectionnés parmi différents anticorps auto-immuns présents dans le sang, et il existe un effet de sensibilité grandement améliorée par rapport à celle d'un kit de diagnostic conventionnel.
PCT/KR2017/004970 2016-05-13 2017-05-12 Kit de diagnostic WO2017196139A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2016-0058549 2016-05-13
KR1020160058549A KR102141591B1 (ko) 2016-05-13 2016-05-13 진단용 키트

Publications (1)

Publication Number Publication Date
WO2017196139A1 true WO2017196139A1 (fr) 2017-11-16

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PCT/KR2017/004970 WO2017196139A1 (fr) 2016-05-13 2017-05-12 Kit de diagnostic

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WO (1) WO2017196139A1 (fr)

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Publication number Priority date Publication date Assignee Title
KR102353452B1 (ko) * 2020-12-23 2022-01-21 한국과학기술원 에어로졸화된 비말 검출장치

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US20110008910A1 (en) * 2007-11-12 2011-01-13 Eurodiagnostica Ab Method for the immobilization of a capture molecule on a solid support
US20120141457A1 (en) * 2010-11-02 2012-06-07 Kypha, Inc. Lateral Flow Immunoassay for Complement Activation and Methods of Use for Point-of-Care Assessment of Complement-Associated Disorders
WO2015109255A1 (fr) * 2014-01-16 2015-07-23 Genisphere, Llc Dosages à flux latéral au moyen de dendrimères d'adn
US20150293086A1 (en) * 2014-04-14 2015-10-15 Abreos Biosciences, Inc. Lateral flow immunoassay

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US20040023210A1 (en) 2000-06-14 2004-02-05 Lalit Mahajan Diagnostic kit for invitro detection of hepatitis C
KR100796772B1 (ko) 2006-09-04 2008-01-22 주식회사 이뮨메드 쯔쯔가무시병 진단 키트
KR100868560B1 (ko) 2007-03-08 2008-11-13 주식회사 이뮨메드 렙토스피라증 진단 키트
KR101051435B1 (ko) 2008-10-22 2011-07-22 한국생명공학연구원 대장암 관련 마커를 이용한 대장암 진단 키트 및 이를 이용한 대장암 진단 방법
AU2011308615B2 (en) 2010-10-01 2015-02-05 Hologic, Inc. Immunoassay test strip for use in a diagnostic system

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Publication number Priority date Publication date Assignee Title
US20110008910A1 (en) * 2007-11-12 2011-01-13 Eurodiagnostica Ab Method for the immobilization of a capture molecule on a solid support
US20120141457A1 (en) * 2010-11-02 2012-06-07 Kypha, Inc. Lateral Flow Immunoassay for Complement Activation and Methods of Use for Point-of-Care Assessment of Complement-Associated Disorders
WO2015109255A1 (fr) * 2014-01-16 2015-07-23 Genisphere, Llc Dosages à flux latéral au moyen de dendrimères d'adn
US20150293086A1 (en) * 2014-04-14 2015-10-15 Abreos Biosciences, Inc. Lateral flow immunoassay

Non-Patent Citations (1)

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Title
KIKKAS, INGRID ET AL.: "A rapid lateral flow immunoassay for the detection of tyrosine phosphatase-like protein IA-2 autoantibodies in human serum", PLOS ONE, vol. 9, no. 7, 21 July 2014 (2014-07-21), pages 1 - 6, XP055599713, DOI: 10.1371/journal.pone.0103088 *

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Publication number Publication date
KR102141591B1 (ko) 2020-09-14
KR20170127833A (ko) 2017-11-22

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