WO2017051938A1 - Procédé permettant de surveiller la progression et l'état de traitement d'une maladie cardiaque au moyen d'une table de sensibilité à une concentration standard d'un biomarqueur cardiaque et biopuce pour ce procédé - Google Patents

Procédé permettant de surveiller la progression et l'état de traitement d'une maladie cardiaque au moyen d'une table de sensibilité à une concentration standard d'un biomarqueur cardiaque et biopuce pour ce procédé Download PDF

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WO2017051938A1
WO2017051938A1 PCT/KR2015/010007 KR2015010007W WO2017051938A1 WO 2017051938 A1 WO2017051938 A1 WO 2017051938A1 KR 2015010007 W KR2015010007 W KR 2015010007W WO 2017051938 A1 WO2017051938 A1 WO 2017051938A1
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concentration
sensitivity
heart disease
standard
biochip
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PCT/KR2015/010007
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Korean (ko)
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김태선
송금수
김정훈
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(주)바이오메트릭스 테크놀로지
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Priority to CN201580002621.6A priority Critical patent/CN107076754A/zh
Priority to PCT/KR2015/010007 priority patent/WO2017051938A1/fr
Publication of WO2017051938A1 publication Critical patent/WO2017051938A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
    • C01G7/00—Compounds of gold
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
    • C07D219/02—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with only hydrogen, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the ring system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled 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
    • G01N33/543—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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a method for diagnosing heart disease by rapidly and quantitatively measuring the concentration of heart disease biomarkers using a concentration-sensitivity graph of a standard sample containing a heart disease biomarker, and a method for monitoring the progress and treatment status of heart disease. .
  • the present invention prepares a concentration-sensitivity graph of a standard sample containing a heart disease biomarker by using a biochip that can measure the sensitivity of the measuring means in proportion to the concentration of the heart disease biomarker.
  • Types of biochips are used to detect cardiac disease biomarkers in the blood samples of subjects and to quantitatively determine their concentrations in comparison to the standard sample concentration-sensitivity graph, thereby diagnosing heart disease and progressing and treating heart disease. It relates to how to monitor the status.
  • a heart that is about the size of a human fist has a great effect on the human body when its function is degraded, because not only the blood circulation is lowered, but also myocardial infarction, hypertension, heart failure, arrhythmia, cardiomyopathy, and other diseases are high.
  • WHO World Health Organization
  • Cardiovascular disease accounts for about one third of all deaths from vascular diseases in Korea, and is considered to be an important cause of acute death in the 40s and 50s.
  • acute myocardial infarction occurs in 80% of people who suddenly die from their normal health. It is also known that mortality increases rapidly if it is not detected early with age, and at the same time, regular check-ups are known as essential diseases. This disease is mainly caused by cardiovascular blockage, and most people die if they fail to judge the symptoms at the beginning and take proper treatment.
  • ACC / AHA American Heart Association
  • AMI acute myocardial infarction
  • AMI Acute myocardial infarction
  • basic examinations such as X-rays, electrocardiograms, and echocardiography require time and equipment, and about 20% of patients with AMI do not show electrocardiogram changes, so chest pain and electrocardiograms are the criteria for AMI diagnosis.
  • Cardiac Bio-Markers As an important diagnostic criterion for acute myocardial infarction has been further emphasized. Therefore, the development of a rapid blood diagnosis method has been required.
  • FRET fluorescence resonance transfer
  • Rapid and timely diagnosis and determination of rapid concentration changes in cardiac disease markers is critical for acute myocardial infarction (AMI). Especially in case of mild pain in the chest, it is not only necessary for self-diagnosis, hospital visit, rapid treatment, but also very useful as a method to confirm the completion of treatment by detecting the level of heart disease marker in the diagnosis and treatment process. .
  • Cardiac troponin I (cTnI), a biomarker that reflects cardiovascular diseases, is a biomarker useful for differential diagnosis of acute respiratory distress, diagnosis of heart failure, and prognostic determination. ) Is used a lot.
  • cTnI is 250pg / ml for distinguishing between normal and abnormal, 80pg / ml for NT proBNP, and 100-400pg / ml for BNP ( American Heart Association Circulation, 2002; 105: 2328-2331. Biosite Package Insert. , en.wikipedia.org/wiki/Troponin), so quantitative analysis of biomarkers from hundreds of pg / ml to tens of pg / ml is very important.
  • ELISA plates or NC membranes which have been widely used for the analysis of heart disease biomarker proteins, are mainly used for detecting protein concentrations by immobilizing antibody proteins on solid substrates by chemical bonding or physisorption.
  • the activity of immobilized protein decreases with time after immobilization, so it is difficult to secure high sensitivity.
  • immobilization by physisorption it is difficult to control the direction of immobilized protein and maintain protein activity after immobilization. And securing reproducibility is difficult.
  • proteins immobilized on solid substrates may generate denatured and nonspecific bonds of the immobilized protein over time, which can be used to distinguish a variety of cardiac disease biomarkers into abnormal and normal states, ranging from several hundred pg / ml to tens of pg /. Accurate analysis at the concentration level of ml has very difficult problems.
  • biochips such as protein chips as a method or means for detecting the heart disease biomarker protein and concentration measurement
  • the protein chip-based technology for labeling protein analysis mainly uses a variety of molecular recognition techniques to fix a large number of proteins in a narrow space on the chip surface, mostly used for research.
  • the analysis of the majority of labeled proteins was performed in the form of a sandwich of antibody-antigen-antibody between the binding antibody and the secondary antibody to which the substance required for analysis was attached.
  • the inventors of the present invention disclose a gene chip capable of detecting a desired gene quickly and sensitively using an aminocalixarene derivative and a continuous guanine-attached gene in Korean Patent Application No. 10-2005-0096322.
  • Application No. 10-2008-0093800 discloses a gene chip capable of hybridization at room temperature using an aminocalixamine derivative and a continuous guanine-attached gene.
  • the paper published by the inventors of the present invention discloses a technique for detecting biomarkers at the picogram level using the dielectric difference chip disclosed in the above-mentioned literature. have.
  • the technique is to rapidly bind a plurality of binding antibodies and secondary antibodies, etc. on each label protein and the solution, each protein is immobilized at a predetermined position in the chip using a gene-gene binding, in the existing protein chip based technology Most of the problems that appeared were solved.
  • Patent Document 1 Korean Patent Application No. 10-2008-0093800
  • Non-Patent Document 1 Chem. Commun., 2011, 47, 7716-7718
  • the inventors have developed a gene chip consisting of a substrate, an aminocalcoxamine derivative and a 9G-gene (hereinafter also referred to as 9G DNA Chip), using antibody-gene conjugates, antigens and marker-antibody conjugates to It has been reported that biomarker proteins such as CRP can be detected at picogram level by using amplification method, but from several hundred pg / ml, it is possible to distinguish between abnormal state and normal state of heart disease biomarker. Methods to quantitatively analyze or determine their concentration at the level of tens of pg / ml have not yet been developed.
  • the present inventors further studied the detection and concentration determination of the antigenic protein using the above-described gene chip, and as a result, the antibody-gene conjugate, the antigen and the label-antibody conjugate formed a complex to quantitatively bind to the gene chip.
  • concentration-sensitivity graphs of standard samples containing cardiac disease biomarkers could be constructed, and the same type of biochips were used to detect cardiac disease biomarkers in the blood samples of subjects to quickly diagnose heart disease.
  • concentrations of these were determined quantitatively at the picogram level, thereby discovering that the progress and treatment of heart disease could be monitored and completed the present invention.
  • the present invention it is possible to rapidly detect a variety of heart disease biomarker proteins at the same time and to quantitatively analyze their concentrations simultaneously at a picogram concentration level that makes it possible to distinguish between abnormal states and normal states of heart disease biomarker proteins.
  • the progress and treatment of heart disease can be monitored and cured judgments can be made.
  • 1 is a schematic of antibody-gene and antibody-phosphor synthesis.
  • FIG. 2 shows Nanodrop measurements for materials obtained in antibody-gene and antibody-phosphor synthesis.
  • Figure 3 is a schematic diagram of the shape and manufacturing method of the protein detection biochip.
  • Figure 5 shows the results measured using a standard sample and a concentration versus concentration graph.
  • Figure 6 is a graph showing the optimal internal standard spot sensitivity results and protein concentration versus internal standard spot sensitivity.
  • Figure 7 shows the location of the protein detection biochip and internal standard spot (IS: Internal Standard) and protein detection spot (PD: Protein Detection) applied to the internal standard spot.
  • IS Internal Standard
  • PD Protein Detection
  • FIG. 8 shows actual concentrations and measured concentrations of three heart disease biomarker proteins (cTnI, NT proBNP, and BNP) obtained by adjusting gain with an internal standard spot.
  • Figure 9 is the result measured at a concentration diluted from 1/2 to 1/32 of three heart disease biomarker proteins (cTnI, NT proBNP, BNP) in blood samples.
  • the aforementioned biochip may be selected from biochips capable of detecting heart disease biomarkers at the picogram level as well as quantitatively analyzing the concentration at the picogram level.
  • biochips of this type mention may be made, for example, of biochips comprising the following (a)-(d):
  • aminocalixarene derivatives having a means for attachment to a solid substrate and a recognition site for a continuous guanine base
  • a biochip comprising an antibody-gene conjugate in which a gene and an antibody are complementary to the immobilized gene.
  • the sample to be measured is measured before or after the measurement of the "label-antibody conjugate” (hereinafter referred to as component (f)) in addition to the "heart disease biomarker protein antigen” (hereinafter referred to as component (e)).
  • component (d) label-antibody conjugate
  • component (e) the "heart disease biomarker protein antigen”
  • component (d) described above may be included in the biochip from the beginning as mentioned above, or may be added separately before and after the addition of the sample to be measured.
  • the above-mentioned aminocalixarene derivative is attached to a solid substrate by chemical or physical bonding between the aforementioned attachment means and the surface of the solid substrate, and the immobilization gene described above is connected by a continuous guanine base. It is attached to the recognition site of the continuous guanine base of the immobilized gene described above.
  • step (1) for constructing the standard concentration-sensitivity graph described above may comprise:
  • the above-described standard concentration-sensitivity graph is a graph form combining the numerical value of the standard concentration and the numerical value of the standard sensitivity, and instead of the table form combining the numerical value of the standard concentration and the image of the standard sensitivity (standard Concentration-sensitivity spots) can be created and used.
  • component (d) may be added in advance in the manufacture of the biochip, but with a sample comprising components (e) and (f) in the biochip comprising component (d) or It can be added separately and made into a biochip comprising component (d).
  • the above-described cardiac disease biomarker protein antigen is at least one, preferably all three of cTnI, NT proBNP and BNP, and the above-mentioned antibody may be selected from the antibodies of said antigen. .
  • the continuous guanine base may be formed by consecutively combining seven or more, preferably nine, guanine (G) bases.
  • the target base is not particularly limited in length, and may be selected from 13mer or less, 14-17mer, 18-21mer, or 22mer or more, if necessary.
  • the solid substrate may be selected from the group consisting of metals such as gold, silver, platinum, glass, fiberglass sheets, silicon wafers, and crystal crystals, and the surface of the substrate may be modified with, for example, amine groups, siloxane groups, or the like. Can be.
  • amino callix arene derivative may be selected from the amino callix arene represented by the following formula (1) or (2):
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R ' 1 , R' 2 , R ' 3 , R' 4 , R ' 5 , R ' 6 , R' 7 and R ' 8 are independently of each other -H, -CH 3 , -C 2 H 5 , -C 3 H 7 , -OCH 3 , -Cl, -C 6 H 5 , -OH,- OCH 2 CH 3 , -Br, -CF 3 , -OCH 2 C 6 H 5 , -OC 6 H 5 , -OC 6 H 4 CH 3 , -OC 6 H 4 C (CH 3 ) 3 , -OC 6 H 4 CF 3 , —OC 6 H 4 Cl, —OCOCH 3 , —NHCOCH 3 , —CONHCH 3 , —CN, COOH, and —COOR, wherein
  • Substituents Y 1 , Y 2 , Y 3 and Y 4 in Chemical Formulas 1 and 2 may be represented as a means for attaching to a solid substrate through ionic bonds, covalent bonds, coordination bonds, etc. Serves to attach the compounds of 1 and 2.
  • Y 1 , Y 2 , Y 3 and Y 4 in Formula 1 or 2 may be selected from thiol groups.
  • the solid substrate is selected from the group consisting of glass, silicon wafers and quartz crystals to which amine functional groups are attached, one or more of Y 1 , Y 2 , Y 3 and Y 4 may be selected from aldehyde groups, thereby Linking of the substrate with the compound of formula 1 or 2 may be accomplished via an imine group or an amine group reduced thereto.
  • the above-described labeling substance may be selected from the group consisting of phosphors, enzymes, radioactive substances, microparticles and pigments.
  • Another object of the present invention is a method for quantitative analysis of antigenic proteins using the method described above, wherein one or more, preferably 3 to 10, antigenic proteins are selected from the group consisting of the marker proteins described in FIG. It is to provide a method for quantitative analysis of antigenic proteins for quantitative analysis.
  • Another object of the present invention is to simultaneously detect the concentration of cardiac disease marker proteins including cTnI, NT proBNP, BNP, etc. between 1,000 pg / ml and 25 pg / ml and use them for monitoring or early diagnosis, or It is to provide a biochip that can be used for the purpose.
  • a standard concentration-sensitivity graph or a standard concentration-sensitivity table is prepared as an internal standard, and the concentration is quantitatively determined from the measured sensitivity of the biomarker detected using the biochip, and the concentration of the biomarker thus determined is determined. It can be applied to the method of monitoring the progress and treatment state of the disease using the measured concentration.
  • the biochip of the present invention may have the following configuration:
  • a solid substrate which may be selected from the group consisting of metals such as gold, silver, platinum, glass, silicon wafers, and quartz crystals;
  • an aminocalixarene derivative attached to the solid substrate and having an attachment site to the solid substrate and a recognition site for the continuous guanine base, wherein the aforementioned continuous guanine base is specifically 5 or more, preferably Means at least 7, in particular at least 9 consecutive guanine bases;
  • linking gene attached to the aminocalixarene derivative, the linking gene having a continuous guanine base and a target base (sequence), wherein the linking gene is attached to the above-mentioned aminocalixarene derivative through the continuous guanine base has exist;
  • (d) antibody-gene conjugates, (e) antigens and (f) marker-attached secondary antibodies can detect conjugates formed by gene-gene binding.
  • a mixture of (e) an antigen and (f) a labeling substance-attached secondary antibody is added to the biochip comprising (a) to (d)
  • (d) the antibody-gene conjugate is (e) The antigen is attached, and (e) the antigen-fed secondary antibody (f) is attached to the antigen, thereby (d) the antibody-gene conjugate, (e) the antigen and (f) the label-attached 2 antibody.
  • a conjugate of primary antibodies can be formed.
  • amino Calixarene derivatives (Amino Calixarene derivatives) that recognizes the continuous guanine base in the present invention, the biochip attached to a solid substrate is called "9G gene chip," and at least 80% hybridization reaction at room temperature within 10 minutes Possible genetic chip technology (Chem. Commun., 2011, 47 / 7101-7103). Techniques related to such room temperature hybrid gene chips are disclosed in Korean Patent Application No. 10-2008-0093800 by the inventor, which is incorporated herein by reference.
  • the reaction between the biomarker protein and the antibody is carried out on a solution in which activity is maintained, and then the antibody-gene conjugate is hybridized on a 9G gene chip to confirm the result.
  • the cardiovascular biomarker protein is resolved at the picogram level, specifically 1 to 1000 pg / ml, more specifically 5 to 800 pg / ml, preferably 10 It was possible to detect up to a concentration of ⁇ 600 pg / ml, particularly preferably 25 to 400 pg / ml (see FIGS. 8-9).
  • the concentration can be determined using a dilution method or a standard concentration-sensitivity range can be determined even at concentrations higher than the picogram level, the upper limit of the concentration range is not important, and the heart disease biomarker protein is nano.
  • the present invention can be applied even when contained at the gram level or higher.
  • the concentration range of 25 pg / ml or less experimented in the present invention, the concentration of 5 pg / ml or less as described above can be measured by adjusting the gain (gain) of the scanner or by varying the type of labeling material, the concentration range
  • the lower limit of can also be sufficiently overcome by the method of the present invention.
  • probe genes immobilize them on the gene chip, analyze the fluorescence expressed on the chip using complementary genes with fluorescence, and select the probe gene with the highest specificity and sensitivity among them.
  • Biochips with immobilized genes were prepared to detect the concentrations of cTnI, NT proBNP, and BNP in the heart disease biomarker protein simultaneously or simultaneously on one chip (see FIG. 3).
  • the antibody-gene conjugate was prepared and purified as in Example 1 using a gene complementary to the gene immobilized on the gene chip, and the protein sugar for preparing the antibody-gene conjugate optimized for the present invention.
  • the optimal number of bindings for hybridization with the immobilized genes was selected by checking the number of genes bound per protein using the purified nano-protein conjugates using Nanodrop.
  • the quantitative analysis was performed by confirming the number of display materials bound to the secondary protein using Nanodrop. It was made possible.
  • the display material that can be used may be a phosphor, an enzyme, a radioactive material, a fine particle, or a dye, but the phosphor is used in the present invention.
  • a standard concentration-sensitivity graph for quantitative analysis is prepared by converting the sensitivity of the detected biomarker protein into the amount of protein by applying an internal standard using a standard spot using a certain ratio of the gene mixture fixing technology. A technique that could be provided (see FIGS. 5-6).
  • Internal standard is a technology for preparing a standard spot to detect the sensitivity expressed between 0% and 100% by mixing and immobilizing two or more genes, for example, by using a standard solution having a standard concentration. It can be implemented by the standard concentration-sensitivity graph or standard concentration-sensitivity table made by numerically graphing or imaging the standard sensitivity, and standardizing various detection equipments used to analyze biomarker proteins related to heart disease. At the same time, it can provide a system for the distribution and storage of biochips for diagnosing heart disease, and for the verification of potential problems. In addition, early detection of equipment problems that may occur when using detection equipment can provide accurate analysis results.
  • a standard solution having a concentration of 400 pg / ml, 200 pg / ml, 100 pg / ml, 50 pg / ml, and 25 pg / ml was prepared, and these standard solutions were prepared.
  • the measurement sensitivity was obtained by measuring with a specially prepared biochip for analyzing heart disease biomarker protein (see FIG. 7), and a standard concentration-sensitivity graph and / or table were prepared from the concentration and measurement sensitivity of the standard solution.
  • the sensitivity of the detected biomarker protein was converted to the amount of protein by adjusting the gain of the detector to compare the actual concentration with the detected concentration, and the correspondence was confirmed.
  • the accuracy of the concentration analysis technique was confirmed by the biochip for analyzing the heart disease biomarker protein used in the present invention and the internal standard (standard concentration-sensitivity graph) prepared according to the present invention (see FIG. 8).
  • the present invention provides a biochip technology capable of quantitatively analyzing one or three types of heart disease biomarker proteins to a level of pg / ml to monitor the treatment of heart disease and to cure them.
  • the conversion of the concentration can be estimated by extrapolation.
  • the blood sample can be measured by diluting it at a fixed rate, for example by 1/2. Such dilution may be performed until the measurement sensitivity is within the numerical range of the above-described graph, for example, diluting by diluting at a magnification of 1/2, 1/4, 1/8, 1/16, 1/32.
  • the biochip can be configured to measure simultaneously the samples taken.
  • the diluted concentration can be converted from the measured sensitivity obtained in the diluted sample, and the conversion concentration of the original blood sample can be obtained by multiplying the dilution ratio inversely.
  • the equivalent concentration of the cardiovascular biomarker protein obtained according to the method of the present invention can be used to diagnose acute myocardial infarction (AMI) or to monitor its progress and treatment.
  • AMI acute myocardial infarction
  • the occurrence of heart disease can be judged when the conversion level of one or more of the cardiovascular biomarker proteins exceeds a predetermined level, and the reduction of heart disease is determined by the stagnation or reduction of the conversion level. This can be lowered.
  • the above-mentioned fixed figures may be determined according to the official opinion of a professional institution or expert.
  • Lung cancer CEA, SCC, NSE, SLX
  • -Pancreatic cancer (gallbladder cancer, biliary tract cancer): CEA, CA 19-9, CA 50, DUPAN-2, Esterase 1, Span-1 Ag, SLX, NCC-ST-439
  • Prostate cancer PAP, PSA, ⁇ -Sm
  • Uterine cancer SCC Antigen, CA 125, CEA, TPA
  • Ovarian cancer CA 125, CEA, SCC, STN, CA 72-4, SLX
  • one or more, specifically 3 to 10 antigenic proteins are selected from the group consisting of the marker proteins of the primary organ or disease to perform the quantitative analysis method of the antigenic protein of the present invention.
  • the abnormality or abnormality of the primary organ may be determined or the disease may be diagnosed, and further, its progress and / or treatment may be monitored.
  • Substances used for antibody-gene binding are shown in Table 1 below, and the base sequences of the target genes (TD) used are shown in Table 2.
  • the target genes TD06, TD01, and TD10 were purchased from Bioneer, Inc., and an amine group (-NH 2 ) is attached to the 5'- side of each target gene.
  • Synthesis of cTnI Ab-TD06 consists of three steps as depicted in FIG. 1 and was carried out as follows.
  • the TD06-SMCC was purified using a Sephadex G-25 packed column (3ml size) of bioWORLD, and 1XPBS was used for the mobile phase.
  • TD06-SMCC Purified TD06-SMCC was verified by measuring purity and concentration using ND1000 UV / Vis spectrometer. The results are shown in FIG. 2 and 200ul of TD06-SMCC (concentration 202.4ng / ul) was obtained.
  • NT proBNP Ab-TD01 was synthesized in the same manner as in Example 1-1 using NT proBNP Ab (concentration 6 mg / ml) 42 ul to obtain 80 ul of NT proBNP Ab-TD01 (concentration 2.08 mg / ml). The results are shown in 2.
  • BNP Ab-TD10 was synthesized in the same manner as BNP Ab (concentration 6mg / ml) 41ul Example 1-1) to obtain 60ul of BNP Ab-TD06 (concentration 2.39mg / ml) and the results are shown in FIG. .
  • a labeling agent-secondary antibody conjugate As an amplification material of the antigen detection signal, a labeling agent-secondary antibody conjugate was prepared. Fluorescent material (Cy5) was used as the labeling material, and Table 3 lists the materials used for antibody-fluorescence binding.
  • cTnI AB-Cy5 was purified using a Sephadex G-25 packed column (3ml size) of bioWORLD, and mobile phase was 1XPBS.
  • NT proBNP AB-Cy5 was synthesized in the same manner as in Example 2-1 using NT proBNP AB (concentration 8mg / ml) 30ul to obtain 190ul NT proBNP AB-Cy5 (concentration 1.1mg / ml) The results are shown in 2.
  • BNP AB-Cy5 was synthesized in the same manner as in Example 2-1 using 33ul of BNP AB (concentration 7.6mg / ml) to obtain 150ul of BNP AB-Cy5 (concentration 1.4mg / ml). The results are shown.
  • 9G-oligo DNA having 9 consecutive guanine bases was immobilized on an aminocalixarene monolayer.
  • a 9G-DNA chip was prepared and used as a biochip for protein detection (see FIG. 3).
  • FIG. 4 Methods and experiments for detecting heart disease biomarkers as antigenic proteins are illustrated schematically in FIG. 4.
  • the heart disease biomarker protein three antigenic proteins cTnI, NP proBNP and BNP were prepared and used as standard samples.
  • Antigen standard samples were prepared by purchasing cTnI Ag (0.5 mg / ml), NT proBNP Ag (0.9 mg / ml), and BNP Ag (3.5 ng / ml) from Hytest.
  • the incubation mixture was prepared by mixing 100ul of the antibody mixture prepared in 2) and 300ul of the incubation buffer in a 1.5ml e-tube.
  • FIG. 6 shows a graph of standard concentration versus standard sensitivity (“concentration graph versus standard sample sensitivity”).
  • the concentration of the target protein contained in the blood sample of the subject can be analyzed.
  • an internal standard spot (standard concentration-sensitivity table) as shown in FIG. 5 was prepared to obtain these standard fluorescence values at the same value in all the scanning equipments, and the gain 90 (Gain 90) of FIG. Fluorescence sensitivity shown in) was determined as the standard sensitivity.
  • the internal standard spot (standard concentration-sensitivity graph or standard concentration-) can be determined by comparing the measured sensitivity with the sensitivity of the internal standard spot. Sensitivity table) and the materials used for internal standard spot immobilization and hybridization reactions are listed in Table 7 below.
  • the internal standard spot sensitivity was used as a mixed gene as shown in Table 8 below to show all sensitivity values of 65000 to 10000 at the concentration of 400 pg / ml to 25 pg / ml of the standard sample of Example 4, and standard spot 1 (IS 1).
  • standard spot 2 IS 2
  • standard spot 3 IS 3
  • standard spot 4 IS 4
  • Table 8 shows the mixed gene ratio (HC: NC) representing the standard concentration of the internal standard spots.
  • PCP06 Using the PCP06, PCP01 and PCP10 genes shown in Table 4 and the IS1, IS2, IS3 and IS4 mixed genes shown in Table 8, the same procedure as described in Example 11 of Korean Patent No. 10-0786577 was performed.
  • the biochip described in 7 was prepared.
  • HC TD-Cy5 which has a base sequence complementary to that of the HC gene, was used for concentration analysis of the sensitivity at gain 90 of FIG. 6 so that the fluorescence sensitivity after hybridization with four internal standard spots could represent all values of 65000 to 10,000 levels.
  • the internal standard spot sensitivity was determined as follows.
  • I. 1XPBS 30ul with a. Incubation buffer 30ul prepared in was mixed on the chip and incubated at room temperature for 2 hours.
  • the scanner gain was adjusted to compare the actual concentrations of cTnI, NT proBNP and BNP standard samples with those measured in the protein detection biochip according to the present invention. It was carried out in the same manner as in Example 4 using the standard samples 1 to 5 described in Table 5.
  • a plasma sample (sample 1) having a sensitivity of 65000 or more is prepared by measuring with a biochip, and the sample diluted from 1 XPBS to 1/2 to 1/32 as shown in Table 9 (samples 2 to 3). 6) was further prepared.
  • Concentration analysis showed that the cTnI and NT proBNP proteins, including the heart disease biomarker protein antigens, had a precisely reduced sensitivity by half in samples diluted in half concentrations for plasma samples of unknown concentration. It was confirmed that quantitative concentration analysis was possible in the concentration range of picogram level from high concentration of 400pgml to low concentration of 25pg / ml.
  • the sensitivity was 65000 or more and the concentration was more than 400 pg / ml, so it was difficult to accurately convert the concentration.
  • the concentration converted from the sensitivity measured in the half-diluted sample was 322 pg / ml.
  • the concentration of plasma sample was determined to be 644 pg / ml.
  • the present invention can be used industrially in the field of protein chip manufacturing and diagnostic and therapeutic using the same.

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Abstract

La présente invention concerne, à l'aide d'un graphe de sensibilité à une concentration standard et d'un procédé d'analyse de protéine selon lequel un composite d'un complexe anticorps-gène et d'un antigène et d'un complexe matériau de marqueur-anticorps secondaire est mesuré au moyen d'une biopuce d'un type de puce à ADN à neuf guanine (9G), un procédé permettant une détection rapide et simultanée, au niveau de picogrammes, de multiples types de protéines de biomarqueur cardiaque (cTnI, NT-proBNP et BNP) qui permet de différencier des états normaux et anormaux de la protéine de biomarqueur cardiaque, et une analyse quantitative de concentrations de ces derniers, ce qui permet de diagnostiquer des maladies cardiaques, et de surveiller la progression et son procédé de traitement et ce qui permet de déterminer si une récupération complète a été obtenue.
PCT/KR2015/010007 2015-09-23 2015-09-23 Procédé permettant de surveiller la progression et l'état de traitement d'une maladie cardiaque au moyen d'une table de sensibilité à une concentration standard d'un biomarqueur cardiaque et biopuce pour ce procédé WO2017051938A1 (fr)

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PCT/KR2015/010007 WO2017051938A1 (fr) 2015-09-23 2015-09-23 Procédé permettant de surveiller la progression et l'état de traitement d'une maladie cardiaque au moyen d'une table de sensibilité à une concentration standard d'un biomarqueur cardiaque et biopuce pour ce procédé

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Citations (4)

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KR20090051689A (ko) * 2007-11-19 2009-05-22 (주)바이오메트릭스 테크놀로지 상온 교잡반응이 가능한 유전자칩 및 칩 제조를 위한 탐침 유전자 디자인 신기술
JP2011513753A (ja) * 2008-03-05 2011-04-28 シンギュレックス・インコーポレイテッド 分子の高感度検出の方法および組成物
JP2014032062A (ja) * 2012-08-02 2014-02-20 Mitsubishi Chemical Medience Corp Ｂｎｐ測定用標準物質とその利用
KR20140059864A (ko) * 2011-10-17 2014-05-16 에프. 호프만-라 로슈 아게 트로포닌 및 ｂｎｐ 토대의 뇌졸중의 위험 환자 및 원인 진단

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KR20090051689A (ko) * 2007-11-19 2009-05-22 (주)바이오메트릭스 테크놀로지 상온 교잡반응이 가능한 유전자칩 및 칩 제조를 위한 탐침 유전자 디자인 신기술
JP2011513753A (ja) * 2008-03-05 2011-04-28 シンギュレックス・インコーポレイテッド 分子の高感度検出の方法および組成物
KR20140059864A (ko) * 2011-10-17 2014-05-16 에프. 호프만-라 로슈 아게 트로포닌 및 ｂｎｐ 토대의 뇌졸중의 위험 환자 및 원인 진단
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