WO2024128354A1 - Precise immunoassay automation system - Google Patents
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- WO2024128354A1 WO2024128354A1 PCT/KR2022/020471 KR2022020471W WO2024128354A1 WO 2024128354 A1 WO2024128354 A1 WO 2024128354A1 KR 2022020471 W KR2022020471 W KR 2022020471W WO 2024128354 A1 WO2024128354 A1 WO 2024128354A1
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- 238000001514 detection method Methods 0.000 claims abstract description 57
- 238000006243 chemical reaction Methods 0.000 claims abstract description 47
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- 230000008859 change Effects 0.000 abstract description 7
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- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 description 2
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- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 description 2
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Definitions
- Immune tests are based on antigen-antibody combinations to have biological specificity that occurs during biological recognition, and are widely used, like biomarkers, to detect disease-related substances in clinical diagnosis.
- a substance such as an enzyme that generates an optical signal
- the antigen which is a biomarker, is selectively detected due to the specific binding of the antibody to the antigen.
- Label-free immunoassay is a notable biomarker detection and analysis tool that not only has excellent convenience, speed, and sensitivity as it can directly measure antigen-antibody binding, but also has excellent economic efficiency due to reduced costs.
- a primary antibody capable of binding to an antigen is immobilized on the surface of the substrate, and a labeled antibody capable of binding to a specific antigen is used as a secondary antibody.
- a labeled antibody capable of binding to a specific antigen is used as a secondary antibody.
- antigen-antibody binding efficiency, selectivity, sensitivity, and signal amplification effects can be achieved by using primary and labeled secondary antibodies.
- This applied biosensor can be used in immunotests based on antigen-antibody combination. Immunological tests, like biomarkers, are widely used to detect disease-related substances in clinical diagnosis.
- Enzyme-based immunoassay ELISA, Enzyme Liked Imuuno-Sorbant Assay
- ELISA Enzyme Liked Imuuno-Sorbant Assay
- a primary antibody is fixed to the surface of a plate, and containers (wells) containing substances and antigens, secondary antibodies, enzymes, etc. that react with it are arranged, and the rod on which the plate is fixed is automatically moved vertically and horizontally to enable continuous reaction. It relates to an automated inspection system that measures optical or electrochemical signals generated by
- the object to be achieved by the present invention is to contain the necessary materials and washing reagents in a well, and to move the bound capture antibody as needed to react with or wash the solution contained in the well, depending on the detection marker.
- the goal is to provide a precise immune testing system using a detection rod that can control the position of the well and the solution.
- the present invention provides an automated immunoassay system in which an optical change occurs due to an enzyme bound to the micropattern after the final reaction through a reagent reaction, and the concentration of the specific marker is analyzed by counting the magnitude of the optical change.
- the present invention provides an automatic imaging inspection system in which optical changes occur in a plurality of micropatterns after the final reaction through a reagent reaction, and the micropatterns in which optical changes occur are counted to analyze the concentration of the specific marker.
- the present invention includes a detection rod; A capture antibody bound to a fine pattern on the lower surface of the detection rod; a container (well) each containing a substance that reacts with the capture antibody and a washing reagent; And the detection rod or the vessel is movable, and as the detection rod or the vessel moves, the relative position changes so that the detection rod is sequentially immersed in the vessel to react or wash, and in the final reaction vessel (well)
- a detection rod that measures reaction results.
- the present invention provides an automated system for precise immune testing using a detection rod, wherein the reaction is an enzyme-linked immune reaction (ELISA).
- ELISA enzyme-linked immune reaction
- the present invention provides a precise immune test system in which the reaction result is an optical signal.
- the present invention provides a precise immune test automation system in which the micropattern consists of a plurality of containers and measures optical signals by counting the number of containers in which an enzyme reaction has occurred.
- the necessary materials and washing reagents are contained in a container (well), and the bound capture antibody moves as needed to react with or wash the solution contained in the container, thereby forming the well according to the detection marker. It has the effect of controlling the location and solution.
- the present invention is to automatically perform immunoassay reactions such as antigen binding, secondary antibody binding, enzyme binding, and enzyme-substrate reaction by loading a rod with a fine pattern formed on a well equipped with a plurality of reagents in order, and It is characterized by quantitative analysis of concentration by measuring the size of the optical change that occurs due to the enzyme reaction.
- Figure 1 is a diagram corresponding to an example of a precision immune test system using a moving detection rod of the present invention.
- Figure 3 is a description of the immune response (ELISA) in the automatic imaging test system using the moving detection rod of the present invention.
- Figure 4 shows the color change that occurred due to the immunoassay reaction in the microchamber formed by the micropattern of the rod and the bottom of the well. It can be seen that the higher the concentration of the antigen, the greater the color reaction occurs.
- Figure 5 is a graph showing optical signals measured for various concentrations of antigen. It can be seen that as the concentration of antigen increases, the size of the optical signal also increases.
- Figure 6 shows an example consisting of a plurality of fine patterns, in which a plurality of vessels (wells) are finely patterned.
- Figure 8 is a graph showing the size of the optical signal measured according to the concentration of amyloid beta 40 (AB40), an Alzheimer's dementia marker, using the immune test system developed in the present invention. It can be seen that as the concentration of AB40 increases, the optical signal also increases.
- AB40 amyloid beta 40
- Figure 9 shows the results of measuring Tacrolimus, an immunosuppressant, in the blood using competitive ELISA as an example of the present invention.
- competition reaction ELISA it is a method of mixing specific substances that compete with the antigen, and the signal size decreases as the concentration of the antigen increases.
- the terms 'about', 'substantially', etc. are used to mean at or close to the numerical value when manufacturing and material tolerances inherent in the stated meaning are presented, and are used to enhance the understanding of the present invention. Precise or absolute figures are used to assist in preventing unscrupulous infringers from taking unfair advantage of stated disclosures.
- the present invention relates to an automatic imaging inspection system using a moving detection rod, and more specifically, a detection rod with a capture antibody fixed to a micro groove at the bottom, a transport unit for transporting the detection rod, and a reaction or washing reagent for detecting a specific marker. It is composed of a plurality of containers (wells), and an optical signal is generated in the well after the last reaction, and the size of the optical signal is analyzed to analyze the concentration of the specific marker.
- a precision immune test system using a moving detection rod. will be.
- Figure 1 is a diagram corresponding to an example of an automatic imaging inspection system using a moving detection rod of the present invention.
- the present invention includes a detection rod; A capture antibody bound to a micro groove on the lower surface of the detection rod; a container (well) each containing a substance reacting with the capture antibody and a washing reagent; and optical analysis devices.
- An optical device can be analyzed by counting the microgroove in which an optical reaction occurs among a plurality of microgrooves.
- the detection rod can be moved automatically by an external control device, and in particular, in order to react with the substance contained in the well and be cleaned by the cleaning reagent, it moves horizontally to a predetermined position and then moves vertically inside the container.
- the container can be set according to the reaction sequence and the marker of the detection target, and the container containing the reaction material and cleaning reagent can be appropriately placed. Through this, immunoassay reactions such as antigen binding, washing, secondary antibody binding, washing, enzyme binding, washing, and enzyme-substrate reaction are automatically performed, and the optical signal generated as a result of the enzyme-substrate reaction is measured to quantitatively determine the concentration of the antigen.
- the immune test reaction is automatically analyzed.
- a capture antibody is bound to the inside of a micro pattern on the lower surface of the detection rod.
- the degree of reaction with the antigen by the capture antibody in the final reaction well is determined by the presence or absence of reaction of the capture antibody located in each microgroove. It is desirable that the stable structure, which can minimize the fluid effect within the container without interfering with each other, be located inside the micro groove.
- the fine pattern is located at the bottom of the movable rod, is inserted into the container (well), and comes into contact with the bottom of the container to form a micro chamber. Because the microchamber has a large surface area per unit volume, it has the effect of accelerating the reaction rate and amplifying the signal when measuring target antigens.
- the above reaction is applicable to an enzyme-linked immune reaction (ELISA), but is not limited to this.
- ELISA enzyme-linked immune reaction
- Figure 1 is a diagram corresponding to an example of an automated immune test system using a moving detection rod according to the present invention.
- the optical analysis device quantitatively examines the concentration of the antigen to be measured by measuring the size of the optical signal according to the reaction in a container containing the enzyme substrate, which is the final reaction step, and quantifying the degree of change.
- Figure 2 is an example of the position of a container (well) in the immune test system using the moving detection rod of the present invention.
- a container in the immune test system using the moving detection rod of the present invention.
- the critical numerical value that determines the presence or absence of a reaction can be solved through a statistical approach using existing data values, and if necessary, accuracy can be increased using an artificial intelligence process.
- Figure 3 is a description of an example of an immune response (ELISA) that can be detected in the immune test system using the moving detection rod of the present invention.
- ELISA immune response
- the reaction carried out in the well is an enzyme-linked immunosorbent assay (ELISA) reaction.
- Enzyme immunoassay is the most widely used immunoassay method today.
- the disease factor antigen is simultaneously combined with the primary antibody and secondary antibody.
- the primary antibody is immobilized on the surface of the detection rod.
- the secondary antibody has an enzyme (Horsedox Peroxidase, hereinafter HRP) attached to it, and when it is moved to a well containing an enzyme substrate (3,3 ⁇ , 5,5 ⁇ -tetramethylbenzidine, hereinafter TMB) solution, the TMB develops color by the HRP enzyme.
- HRP Hydrophil Peroxidase
- TMB an enzyme substrate
- An optical signal is generated through a reaction. That is, in the present invention, the enzyme that generates the biochemical reaction is HRP, the reactant that reacts with it is the enzyme substrate TMB, and the concentration of the antigen is quantitatively measured by measuring the optical signal generated by the enzyme reaction.
- the present invention can also be applied to tests for Alzheimer's disease, which is a specific disease factor.
- Figure 4 shows the color change that occurred due to the immunoassay reaction in the microchamber formed by the micropattern of the rod and the bottom of the well. It can be seen that the higher the concentration of the antigen, the greater the color reaction occurs.
- Figure 5 is a graph showing optical signals measured for various concentrations of antigen. It can be seen that as the concentration of antigen increases, the size of the optical signal also increases.
- Figure 6 shows an example consisting of a plurality of fine patterns, in which a plurality of vessels (wells) are finely patterned.
- Figure 7 shows that due to the enzyme reaction combined inside a plurality of micropattern containers, the antigen concentration can be quantitatively measured by counting the number of containers in which optical signals are generated among all containers.
- Figure 8 is a graph showing the size of the optical signal measured according to the concentration of amyloid beta 40 (AB40), an Alzheimer's dementia marker, using the immune test system developed in the present invention. It can be seen that as the concentration of AB40 increases, the optical signal also increases.
- AB40 amyloid beta 40
- Figure 9 shows the results of measuring Tacrolimus, an immunosuppressant, in the blood using competitive ELISA as an example of the present invention.
- competition reaction ELISA it is a method of mixing specific substances that compete with the antigen, and the signal size decreases as the concentration of the antigen increases.
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The present invention relates to a precise immunoassay automation system using a detection rod, comprising: a detection rod; a capture antibody bound to a fine pattern on the lower surface of the detection rod; and vessels (wells) each containing a washing reagent and a substance that reacts with the capture antibody, wherein the detection rod or the vessels are movable, the relative positions change as the detection rod or the vessels move, so that the detection rod is sequentially immersed in the vessels to react or be washed, and the reaction result is measured in a final reaction vessel (well).
Description
본 발명은 미세 패턴이 형성된 이동 검출로드에 의한 면역 검사 시스템에 관한 것으로, 보다 자세하게는 하단부 미세 패턴에 포획 항체(capture antibody)가 고정된 검출로드와 이를 이송하는 이송부 및 특정 마커 검출을 위하여 반응 또는 세척 시약이 담겨진 복수개의 용기(well)로 구성되며, 마지막 반응후의 용기(well)에 광학 신호가 발생하고 광학 신호를 분석하여 상기 특정 마커의 농도를 분석하는 이동 검출로드에 의한 정밀 면역 검사 자동화 시스템에 관한 것이다. The present invention relates to an immune testing system using a moving detection rod with a fine pattern formed on it. More specifically, it relates to a detection rod with a capture antibody fixed to the bottom micropattern, a transport unit for transporting the detection rod, and a reaction or detection rod for detecting a specific marker. It consists of a plurality of wells containing washing reagents, and an optical signal is generated in the well after the last reaction, and the optical signal is analyzed to analyze the concentration of the specific marker. A precise immunoassay automation system using a moving detection rod. It's about.
면역검사는 생물학적 인식과정에서 발생하는 생물학적 특이성(Specificity)을 갖도록 항원-항체 결합을 기반으로하고, 바이오마커와 같이 임상진단에서 질병과 관련된 물질을 감지하는데 널리 사용되고 있다. 광학 신호를 발생시키는 효소 등의 물질을 항체에 연결하여 효소 반응으로 발생한 광학 신호를 측정하면, 항원에 대한 항체의 특이결합에 때문에 선택적으로 바이오마커인 항원을 검출한다.Immune tests are based on antigen-antibody combinations to have biological specificity that occurs during biological recognition, and are widely used, like biomarkers, to detect disease-related substances in clinical diagnosis. When a substance such as an enzyme that generates an optical signal is linked to an antibody and the optical signal generated by the enzyme reaction is measured, the antigen, which is a biomarker, is selectively detected due to the specific binding of the antibody to the antigen.
면역검사는 샌드위치형(sandwich-type) 면역검사와 비표지(label-free)면역 검사 두 가지 종류가 있다. 비표지면역 검사는 항원-항체 결합을 바로 측정할 수 있어 편이성, 신속성 및 민감도 등이 뛰어날뿐만 아니라 비용이 절감되어 경제성이 뛰어나 주목할만한 바이오마커 검출 분석 도구이다.There are two types of immune tests: sandwich-type immune tests and label-free immune tests. Label-free immunoassay is a notable biomarker detection and analysis tool that not only has excellent convenience, speed, and sensitivity as it can directly measure antigen-antibody binding, but also has excellent economic efficiency due to reduced costs.
이와 달리, 샌드위치형은 기재표면에 항원과 결합할 수 있는 일차(primary)항체가 고정되고, 특이항원과 결합할 수 있는 표지된 항체(labeled antibody)가 이차(secondary)항체로서 사용된다. 샌드위치형에서는 일차 및 표지된 이차항체를 사용함으로서 항원-항체 결합 효율, 선택성, 민감도 및 신호 증폭 효과를 얻을 수 있다. In contrast, in the sandwich type, a primary antibody capable of binding to an antigen is immobilized on the surface of the substrate, and a labeled antibody capable of binding to a specific antigen is used as a secondary antibody. In the sandwich type, antigen-antibody binding efficiency, selectivity, sensitivity, and signal amplification effects can be achieved by using primary and labeled secondary antibodies.
이를 응용한 바이오센서로 항원-항체 결합을 기반으로하는 면역검사에 활용될 수 있다. 면역검사는 바이오마커와 같이 임상진단에서 질병과 관련된 물질을 감지하는데 널리 사용되고 있다. 효소기반 면역분석법(ELISA, Enzyme Liked Imuuno-Sorbant Assay)는 효소가 연결된 항체가 항원에 특이적으로 결합하게 되고, 효소 반응으로 발생하는 물질을 측정하여 선택적으로 바이오마커인 항원을 검출하는 방법이다. This applied biosensor can be used in immunotests based on antigen-antibody combination. Immunological tests, like biomarkers, are widely used to detect disease-related substances in clinical diagnosis. Enzyme-based immunoassay (ELISA, Enzyme Liked Imuuno-Sorbant Assay) is a method in which an antibody linked to an enzyme binds specifically to an antigen and measures substances generated from the enzyme reaction to selectively detect antigens, which are biomarkers.
본 발명은 일차항체가 플레이트 표면에 고정되고 이와 반응하는 물질 및 항원, 이차항체와 효소등이 포함된 용기(well)을 나열함으로써 상기 플레이트가 고정된 로드를 자동으로 수직, 수평이동시켜 연속적인 반응으로 발생하는 광학 또는 전기화학 신호를 측정하는 자동화 검사 시스템에 관한 것이다. In the present invention, a primary antibody is fixed to the surface of a plate, and containers (wells) containing substances and antigens, secondary antibodies, enzymes, etc. that react with it are arranged, and the rod on which the plate is fixed is automatically moved vertically and horizontally to enable continuous reaction. It relates to an automated inspection system that measures optical or electrochemical signals generated by
본 발명이 이루고자 하는 과제는 용기(well)에 필요한 물질 및 세척시약을 담겨있고 결합된 포획 항체(capture antibody)가 필요에 따라 이동하여 상기 용기에 담겨져 있는 용액과 반응 또는 세척하는 것으로 검출마커에 따라 용기(well)의 위치 및 용액을 조절할 수 있는 검출로드에 의한 정밀 면역 검사 시스템을 제공하는 데 있다. The object to be achieved by the present invention is to contain the necessary materials and washing reagents in a well, and to move the bound capture antibody as needed to react with or wash the solution contained in the well, depending on the detection marker. The goal is to provide a precise immune testing system using a detection rod that can control the position of the well and the solution.
또한 본 발명은 시약 반응을 통해 마지막 반응후의 미세 패턴에 결합된 효소에 의해 광학적 변화가 발생하고 광학적 변화의 크기를 계수하여 상기 특정 마커의 농도를 분석하는 자동화된 면역 검사 시스템을 제공하는 데 있다. In addition, the present invention provides an automated immunoassay system in which an optical change occurs due to an enzyme bound to the micropattern after the final reaction through a reagent reaction, and the concentration of the specific marker is analyzed by counting the magnitude of the optical change.
또한 본 발명은 시약 반응을 통해 마지막 반응후의 복수의 미세 패턴에 광학적 변화가 발생하고 광학적 변화가 발생한 미세 패턴을 계수하여 상기 특정 마커의 농도를 분석하는 자동영상화된 검사 시스템을 제공하는 데 있다. In addition, the present invention provides an automatic imaging inspection system in which optical changes occur in a plurality of micropatterns after the final reaction through a reagent reaction, and the micropatterns in which optical changes occur are counted to analyze the concentration of the specific marker.
상기와 같은 문제를 해결하기 위하여 본 발명은 검출로드; 상기 검출로드 하부표면의 미세 패턴에 결합된 포획 항체(capture antibody);상기 포획 항체(capture antibody)와 반응하는 물질 및 세척시약이 각각 담겨진 용기(well); 및 상기 검출로드 또는 상기 용기는 이동이 가능하며,상기 검출로드 또는 상기 용기가 이동함에 따라 상대적인 위치가 달라져 순차적으로 상기 검출로드가 상기 용기에 담궈져 반응 또는 세척되며,최종 반응 용기(well)에서 반응결과를 측정하는 것에 특징이 있는 검출로드에 의한 정밀 면역 검사 시스템을 제공한다. In order to solve the above problems, the present invention includes a detection rod; A capture antibody bound to a fine pattern on the lower surface of the detection rod; a container (well) each containing a substance that reacts with the capture antibody and a washing reagent; And the detection rod or the vessel is movable, and as the detection rod or the vessel moves, the relative position changes so that the detection rod is sequentially immersed in the vessel to react or wash, and in the final reaction vessel (well) We provide a precise immune testing system using a detection rod that measures reaction results.
또한 본 발명은 상기 반응은 효소가 연결된 면역반응(ELISA)인 것에 특징이 있는 검출로드에 의한 정밀 면역 검사 자동화 시스템을 제공한다. Additionally, the present invention provides an automated system for precise immune testing using a detection rod, wherein the reaction is an enzyme-linked immune reaction (ELISA).
또한 본 발명인 상기 검출로드는 효소기질(Enzyme Substrate)이 포함된 용기에 담궈져 반응되는 것에 특징이 있는 검출로드에 의한 정밀 면역 검사 자동화 시스템을 제공한다. In addition, the present invention provides an automated system for precise immune testing using a detection rod, which is characterized in that the detection rod reacts by being immersed in a container containing an enzyme substrate.
또한 본 발명에서 상기 반응결과는 광학적 신호인 것에 특징이 있는 정밀 면역 검사 시스템을 제공한다. In addition, the present invention provides a precise immune test system in which the reaction result is an optical signal.
또한 본 발명인 상기 미세 패턴은 복수개의 용기로 구성되어서 효소 반응이 발생한 용기의 개수를 카운팅하는 방식으로 광학신호를 측정하는 것에 특징이 있는 정밀 면역 검사 자동화 시스템을 제공한다.In addition, the present invention provides a precise immune test automation system in which the micropattern consists of a plurality of containers and measures optical signals by counting the number of containers in which an enzyme reaction has occurred.
본 발명은 용기(well)에 필요한 물질 및 세척시약을 담겨있고 결합된 포획 항체(capture antibody)가 필요에 따라 이동하여 상기 용기에 담겨져 있는 용액과 반응 또는 세척하는 것으로 검출마커에 따라 용기(well)의 위치 및 용액을 조절할 수 있는 효과가 있다. In the present invention, the necessary materials and washing reagents are contained in a container (well), and the bound capture antibody moves as needed to react with or wash the solution contained in the container, thereby forming the well according to the detection marker. It has the effect of controlling the location and solution.
또한 본 발명은 미세 패턴이 형성된 로드가 복수개의 시약이 구비된 웰에 차례로 담지되어 항원 결합, 이차 항체 결합, 효소결합, 효소기질반응 등의 면역 측정 반응을 자동으로 수행하고, 미세 패턴에 결합된 효소 반응으로 발생한 광학적 변화의 크기를 측정하여 농도를 정량적으로 분석하는 것에 특징이 있다. In addition, the present invention is to automatically perform immunoassay reactions such as antigen binding, secondary antibody binding, enzyme binding, and enzyme-substrate reaction by loading a rod with a fine pattern formed on a well equipped with a plurality of reagents in order, and It is characterized by quantitative analysis of concentration by measuring the size of the optical change that occurs due to the enzyme reaction.
도 1은 본 발명인 이동 검출로드에 의한 정밀 면역 검사 시스템의 일예에 해당되는 도면이다. Figure 1 is a diagram corresponding to an example of a precision immune test system using a moving detection rod of the present invention.
도 2는 본 발명인 이동 검출로드에 의한 정밀 면역 검사 시스템에서 용기(well)의 위치에 대한 일예이다. Figure 2 is an example of the position of a container (well) in the precision immune test system using the moving detection rod of the present invention.
도 3은 본 발명인 이동 검출로드에 의한 자동영상화 검사 시스템에서 면역반응(ELISA)에 대한 설명이다. Figure 3 is a description of the immune response (ELISA) in the automatic imaging test system using the moving detection rod of the present invention.
도 4는 로드의 미세패턴과 웰의 바닥면이 형성한 미세챔버에서 면역 검사 반응으로 발생한 색변화를 보여준다. 항원의 농도가 높아질수록 발색 반응도 크게 발생하는 것을 알 수 있다. Figure 4 shows the color change that occurred due to the immunoassay reaction in the microchamber formed by the micropattern of the rod and the bottom of the well. It can be seen that the higher the concentration of the antigen, the greater the color reaction occurs.
도 5는 다양한 농도의 항원에 대하여 측정된 광학신호를 표시한 그래프이다. 항원의 농도가 높아질수록 광학신호의 크기도 커지는 것을 알 수 있다.Figure 5 is a graph showing optical signals measured for various concentrations of antigen. It can be seen that as the concentration of antigen increases, the size of the optical signal also increases.
도 6은 복수개의 미세 패턴으로 구성된 실시예로서 복수개의 용기(well)이 미세 패턴된 것이다. Figure 6 shows an example consisting of a plurality of fine patterns, in which a plurality of vessels (wells) are finely patterned.
도 7은 복수개의 미세패턴 용기 내부에 결합된 효소 반응으로 인하여, 전체 용기중에서 광학 신호가 발생되는 용기의 개수를 카운팅하는 방식으로 항원의 농도를 정량적으로 측정할 수 있다.Figure 7 shows that due to the enzyme reaction combined inside a plurality of micropattern containers, the antigen concentration can be quantitatively measured by counting the number of containers in which optical signals are generated among all containers.
도 8은 본 발명으로 개발된 면역 검사 시스템을 이용해 알츠하이머 치매 마커인 아밀로이드베타 40(Amyloid beta 40, 이하 AB40)의 농도에 따라 측정된 광학 신호의 크기를 표시한 그래프이다. AB40의 농도가 커질수록 광학신호도 커지는 것을 확인할 수 있다.Figure 8 is a graph showing the size of the optical signal measured according to the concentration of amyloid beta 40 (AB40), an Alzheimer's dementia marker, using the immune test system developed in the present invention. It can be seen that as the concentration of AB40 increases, the optical signal also increases.
도 9는 본 발명의 실시예로서 경쟁반응 ELISA (Competetive ELISA)를 이용해 혈중 면역억제제인 Tacrolimus를 측정한 결과이다. 경쟁반응 ELISA의 경우에는 항원과 경쟁하는 특정물질을 혼합하는 방식으로서 항원의 농도가 높아질수록 신호의 크기가 작아지는 결과를 보여준다.Figure 9 shows the results of measuring Tacrolimus, an immunosuppressant, in the blood using competitive ELISA as an example of the present invention. In the case of competition reaction ELISA, it is a method of mixing specific substances that compete with the antigen, and the signal size decreases as the concentration of the antigen increases.
이하 본 발명의 바람직한 실시예를 상세히 설명하기로 한다. 우선, 본 발명을 설명함에 있어, 관련된 공지기능 또는 구성에 대한 구체적인 설명은 본 발명의 요지를 모호하지 않게 하기 위하여 생략한다.Hereinafter, preferred embodiments of the present invention will be described in detail. First, in describing the present invention, detailed descriptions of related well-known functions or configurations are omitted in order to make the gist of the present invention unambiguous.
본 명세서에서 사용되는 정도의 용어 '약', '실질적으로' 등은 언급된 의미에 고유한 제조 및 물질 허용오차가 제시될 때 그 수치에서 또는 그 수치에 근접한 의미로 사용되고, 본 발명의 이해를 돕기 위해 정확하거나 절대적인 수치가 언급된 개시 내용을 비양심적인 침해자가 부당하게 이용하는 것을 방지하기 위해 사용된다.As used herein, the terms 'about', 'substantially', etc. are used to mean at or close to the numerical value when manufacturing and material tolerances inherent in the stated meaning are presented, and are used to enhance the understanding of the present invention. Precise or absolute figures are used to assist in preventing unscrupulous infringers from taking unfair advantage of stated disclosures.
본 발명은 이동 검출로드에 의한 자동영상화 검사 시스템에 관한 것으로, 보다 자세하게는 하단부 미세홈에 포획 항체(capture antibody)가 고정된 검출로드와 이를 이송하는 이송부 및 특정 마커 검출을 위하여 반응 또는 세척 시약이 담겨진 복수개의 용기(well)로 구성되며, 마지막 반응후의 용기(well)에 광학 신호가 발생하고 광학 신호의 크기를 분석하여 상기 특정 마커의 농도를 분석하는 이동 검출로드에 의한 정밀 면역 검사 시스템에 관한 것이다. The present invention relates to an automatic imaging inspection system using a moving detection rod, and more specifically, a detection rod with a capture antibody fixed to a micro groove at the bottom, a transport unit for transporting the detection rod, and a reaction or washing reagent for detecting a specific marker. It is composed of a plurality of containers (wells), and an optical signal is generated in the well after the last reaction, and the size of the optical signal is analyzed to analyze the concentration of the specific marker. A precision immune test system using a moving detection rod. will be.
도 1은 본 발명인 이동 검출로드에 의한 자동영상화 검사 시스템의 일예에 해당되는 도면이다. Figure 1 is a diagram corresponding to an example of an automatic imaging inspection system using a moving detection rod of the present invention.
본 발명은 검출로드; 상기 검출로드 하부표면의 미세홈에 결합된 포획 항체(capture antibody);상기 포획 항체(capture antibody)와 반응하는 물질 및 세척시약이 각각 담겨진 용기(well); 및 광학 분석 장치가 포함된다. 광학 장치는 복수개의 미세홈중에서 광학 반응이 발생한 미세홈을 카운팅하는 방식으로 분석할 수 있다.The present invention includes a detection rod; A capture antibody bound to a micro groove on the lower surface of the detection rod; a container (well) each containing a substance reacting with the capture antibody and a washing reagent; and optical analysis devices. An optical device can be analyzed by counting the microgroove in which an optical reaction occurs among a plurality of microgrooves.
상기 검출로드는 외부 제어장치에 의해 자동으로 이동가능하며, 특히 용기(well)에 담겨진 물질과 반응 및 세척시약에 의해 세척되기 위해서는 정해진 위치로 수평이동 후 수직이동으로 용기내부로 이동하게 된다. 상기 용기는 반응순서 및 검출대상의 마커에 따라 세팅이 가능하며, 반응물질 및 세척시약이 담겨진 용기를 적절하게 배치할 수 있다. 이를 통해 항원 결합, 세척, 이차 항체 결합, 세척, 효소결합, 세척, 효소기질반응 등의 면역 측정 반응을 자동으로 수행하고, 효소 기질 반응의 결과물로 발생하는 광학 신호를 측정하여 항원의 농도를 정량적으로 분석하는 면역 검사 반응을 자동으로 수행하게 된다.The detection rod can be moved automatically by an external control device, and in particular, in order to react with the substance contained in the well and be cleaned by the cleaning reagent, it moves horizontally to a predetermined position and then moves vertically inside the container. The container can be set according to the reaction sequence and the marker of the detection target, and the container containing the reaction material and cleaning reagent can be appropriately placed. Through this, immunoassay reactions such as antigen binding, washing, secondary antibody binding, washing, enzyme binding, washing, and enzyme-substrate reaction are automatically performed, and the optical signal generated as a result of the enzyme-substrate reaction is measured to quantitatively determine the concentration of the antigen. The immune test reaction is automatically analyzed.
본 발명은 포획 항체(capture antibody)가 검출로드 하부면 미세 패턴 내부에 결합된 상태에 있다. In the present invention, a capture antibody is bound to the inside of a micro pattern on the lower surface of the detection rod.
최종 반응 용기(well)에서 포획 항체(capture antibody)에 의해 항원과 반응정도는 각각의 미세홈에 위치한 포획 항체(capture antibody)의 반응유무에 의해 판단된다. 서로 간섭을 받지 않고 용기내의 유체영항을 가장 최소화시킬 수 있는 안정적인 구조는 미세홈 내부에 위치하는 것이 바람직하다. The degree of reaction with the antigen by the capture antibody in the final reaction well is determined by the presence or absence of reaction of the capture antibody located in each microgroove. It is desirable that the stable structure, which can minimize the fluid effect within the container without interfering with each other, be located inside the micro groove.
미세 패턴이 이동식 로드의 하부에 위치하여, 용기(Well)에 삽입되고 용기 바닥면과 맞닿게 되어 미세 챔버를 형성하게 된다. 미세 챔버는 단위 부피당 표면적이 넓기 때문에 타겟 항원을 측정할 때 반응속도를 촉진하고 신호를 증폭하는 효과가 있다.The fine pattern is located at the bottom of the movable rod, is inserted into the container (well), and comes into contact with the bottom of the container to form a micro chamber. Because the microchamber has a large surface area per unit volume, it has the effect of accelerating the reaction rate and amplifying the signal when measuring target antigens.
일반적으로 상기 반응은 효소가 연결된 면역반응(ELISA)것에 적용가능하나 이에 한정하는 것은 아니다. In general, the above reaction is applicable to an enzyme-linked immune reaction (ELISA), but is not limited to this.
도 1은 본 발명인 이동 검출로드에 의한 면역 검사 자동화 검사 시스템의 일예에 해당되는 도면이다. 상기 광학 분석 장치는 최종반응단계인 효소기질(Enzyme Substrate)이 포함된 용기에서 반응에 따른 광학 신호의 크기를 측정하여 변화의 정도를 수치화하여 측정하고자 하는 항원의 농도를 정량적으로 검사한다. Figure 1 is a diagram corresponding to an example of an automated immune test system using a moving detection rod according to the present invention. The optical analysis device quantitatively examines the concentration of the antigen to be measured by measuring the size of the optical signal according to the reaction in a container containing the enzyme substrate, which is the final reaction step, and quantifying the degree of change.
도 2는 본 발명인 이동 검출로드에 의한 면역 검사 시스템에서 용기(well)의 위치에 대한 일예이다. 정확도를 높이기 위해 반응유무를 결정하는 샘플을 용기 1개로 하는 것보다 바람직하게는 동일한 조건에서 반응하는 복수의 용기를 준비하여 평균적인 값으로 임계적 기준값과 비교하는 것이 바람직하다.Figure 2 is an example of the position of a container (well) in the immune test system using the moving detection rod of the present invention. In order to increase accuracy, rather than using a single container as the sample for determining the presence or absence of reaction, it is preferable to prepare multiple containers that react under the same conditions and compare the average value with the critical reference value.
또한 반응농도의 정확성을 높이기 반응유무를 결정하는 임계적 수치값을 기존의 데이터값을 이용하여 통계적인 접근으로 해결할 수 있으며, 필요하면 인공지능 프로세스를 이용하여 정확도를 높일 수 있다. In addition, to increase the accuracy of reaction concentration, the critical numerical value that determines the presence or absence of a reaction can be solved through a statistical approach using existing data values, and if necessary, accuracy can be increased using an artificial intelligence process.
도 3은 본 발명인 이동 검출로드에 의한 면역 검사 시스템에서 검출할 수 있는 일예로 면역반응(ELISA)에 대한 설명이다. Figure 3 is a description of an example of an immune response (ELISA) that can be detected in the immune test system using the moving detection rod of the present invention.
용기(well)에서 진행되는 반응은 효소면역정량법(ELISA)반응이다. 효소면역정량법은 오늘날 가장 널리 이용되는 면역정량법이다. 특정마커로 질병인자 항원이 1차항체 및 2차항체와 동시에 결합된다. 이때 1차 항체는 검출로드 표면에 고정되어 있다.The reaction carried out in the well is an enzyme-linked immunosorbent assay (ELISA) reaction. Enzyme immunoassay is the most widely used immunoassay method today. As a specific marker, the disease factor antigen is simultaneously combined with the primary antibody and secondary antibody. At this time, the primary antibody is immobilized on the surface of the detection rod.
상기 2차항체는 효소(Horseredox Peroxidase, 이하 HRP)가 붙여진 상태이며, 이후 효소기질 (3,3´, 5,5´-tetramethylbenzidine, 이하 TMB) 용액이 담겨진 웰로 이동시키면 TMB가 HRP효소에 의해 발색 반응을 통해 광학적 신호를 발생시킨다. 즉, 본 발명에서 생화학 반응을 발생시키는 효소는 HRP이고, 이와 반응하는 반응물질이 효소기질 TMB이며, 상기 효소 반응에 의해 발생되는 광학적 신호를 측정하여 정량적으로 항원의 농도를 측정한다. The secondary antibody has an enzyme (Horsedox Peroxidase, hereinafter HRP) attached to it, and when it is moved to a well containing an enzyme substrate (3,3´, 5,5´-tetramethylbenzidine, hereinafter TMB) solution, the TMB develops color by the HRP enzyme. An optical signal is generated through a reaction. That is, in the present invention, the enzyme that generates the biochemical reaction is HRP, the reactant that reacts with it is the enzyme substrate TMB, and the concentration of the antigen is quantitatively measured by measuring the optical signal generated by the enzyme reaction.
본 발명은 특정질병인자인 알츠하이머검사에도 응용될 수 있다.The present invention can also be applied to tests for Alzheimer's disease, which is a specific disease factor.
특정항원을 알츠하이머 질병인자 항원으로 가정하에 설명한다. 첫번째 용기(well)에 있는 분리된 혈장에 포함된 알츠하이머 질병인자 항원과 검출로드 표면에 결합된 포획 항체(capture antibody, 1차 항체)와 결합하는 단계; 상기 1차항체와 결합되지 않은 알츠하이머 질병인자 항원을 세척버퍼가 담겨진 용기(well)에서 세척되는 단계; 효소와 연결된 2차항체가 포함된 용기(well)에서 1차항체가 포함된 용기(well)에 검출로드 표면에 결합된 포획 항체(capture antibody, 1차 항체)를 담궈 반응하는 단계; 상기 검출로드를 이용시켜 세척버퍼가 담겨진 용기(well)에서 상기 1차항체와 결합되지 않은 효소와 연결된 2차항체를 세척하는 단계; 및 상기 검출로드를 이용시켜 효소기질이 포함된 용액의 용기(well)로 이동하여 상기 2차항체와 연결된 효소와 반응시켜 발생되는 광학 신호를 측정하고 광학 신호의 크기를 분석하여 검출할 수 있다. This explanation is based on the assumption that the specific antigen is an Alzheimer's disease factor antigen. A step of combining the Alzheimer's disease factor antigen contained in the separated plasma in the first well with a capture antibody (primary antibody) bound to the surface of the detection rod; Washing the Alzheimer's disease factor antigen that is not bound to the primary antibody in a well containing a washing buffer; reacting by dipping a capture antibody (primary antibody) bound to the surface of the detection rod into a well containing a primary antibody in a well containing a secondary antibody linked to an enzyme; Washing the secondary antibody linked to the enzyme that is not bound to the primary antibody in a well containing the washing buffer using the detection rod; And, using the detection rod, the solution can be moved to a well containing the enzyme substrate, reacted with the enzyme linked to the secondary antibody, measure the optical signal generated, and be detected by analyzing the size of the optical signal.
도 4는 로드의 미세패턴과 웰의 바닥면이 형성한 미세챔버에서 면역 검사 반응으로 발생한 색변화를 보여준다. 항원의 농도가 높아질수록 발색 반응도 크게 발생하는 것을 알 수 있다. Figure 4 shows the color change that occurred due to the immunoassay reaction in the microchamber formed by the micropattern of the rod and the bottom of the well. It can be seen that the higher the concentration of the antigen, the greater the color reaction occurs.
도 5는 다양한 농도의 항원에 대하여 측정된 광학신호를 표시한 그래프이다. 항원의 농도가 높아질수록 광학신호의 크기도 커지는 것을 알 수 있다.Figure 5 is a graph showing optical signals measured for various concentrations of antigen. It can be seen that as the concentration of antigen increases, the size of the optical signal also increases.
도 6은 복수개의 미세 패턴으로 구성된 실시예로서 복수개의 용기(well)이 미세 패턴된 것이다. Figure 6 shows an example consisting of a plurality of fine patterns, in which a plurality of vessels (wells) are finely patterned.
도 7은 복수개의 미세패턴 용기 내부에 결합된 효소 반응으로 인하여, 전체 용기중에서 광학 신호가 발생되는 용기의 개수를 카운팅하는 방식으로 항원의 농도를 정량적으로 측정할 수 있다.Figure 7 shows that due to the enzyme reaction combined inside a plurality of micropattern containers, the antigen concentration can be quantitatively measured by counting the number of containers in which optical signals are generated among all containers.
도 8은 본 발명으로 개발된 면역 검사 시스템을 이용해 알츠하이머 치매 마커인 아밀로이드베타 40(Amyloid beta 40, 이하 AB40)의 농도에 따라 측정된 광학 신호의 크기를 표시한 그래프이다. AB40의 농도가 커질수록 광학신호도 커지는 것을 확인할 수 있다.Figure 8 is a graph showing the size of the optical signal measured according to the concentration of amyloid beta 40 (AB40), an Alzheimer's dementia marker, using the immune test system developed in the present invention. It can be seen that as the concentration of AB40 increases, the optical signal also increases.
도 9는 본 발명의 실시예로서 경쟁반응 ELISA (Competetive ELISA)를 이용해 혈중 면역억제제인 Tacrolimus를 측정한 결과이다. 경쟁반응 ELISA의 경우에는 항원과 경쟁하는 특정물질을 혼합하는 방식으로서 항원의 농도가 높아질수록 신호의 크기가 작아지는 결과를 보여준다.Figure 9 shows the results of measuring Tacrolimus, an immunosuppressant, in the blood using competitive ELISA as an example of the present invention. In the case of competition reaction ELISA, it is a method of mixing specific substances that compete with the antigen, and the signal size decreases as the concentration of the antigen increases.
이상에서 설명한 본 발명은 전술한 실시예 및 첨부된 도면에 의해 한정되는 것이 아니고, 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 여러 가지 치환, 변형 및 변경이 가능함은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 있어서 명백할 것이다. The present invention described above is not limited to the above-described embodiments and the accompanying drawings, and various substitutions, modifications, and changes are possible without departing from the technical spirit of the present invention, as is commonly known in the technical field to which the present invention pertains. It will be clear to those who have the knowledge of.
[부호의 설명][Explanation of symbols]
1 : 항원 2 : 검출 항체(detection antibody)1: Antigen 2: Detection antibody
3 : 효소(enzyme) 4 : 세척버퍼3: Enzyme 4: Washing buffer
5 : 효소기질 6 : 광학 측정 장치5: Enzyme substrate 6: Optical measurement device
10 : 포획 항체(capture antibody)10: capture antibody
20 : 검출로드 30 : 미세홈 20: detection rod 30: fine groove
Claims (5)
- 검출로드; detection rod;상기 검출로드 하부표면의 미세 패턴에 결합된 포획 항체(capture antibody);A capture antibody bound to a fine pattern on the lower surface of the detection rod;상기 포획 항체(capture antibody)와 반응하는 물질 및 세척시약이 각각 담겨진 용기(well); 및 A container (well) containing a substance reacting with the capture antibody and a washing reagent, respectively; and상기 검출로드 또는 상기 용기는 이동이 가능하며,The detection rod or the container is movable,상기 검출로드 또는 상기 용기가 이동함에 따라 상대적인 위치가 달라져 순차적으로 상기 검출로드가 상기 용기에 담궈져 반응 또는 세척되며,As the detection rod or the container moves, the relative position changes and the detection rod is sequentially immersed in the container to react or wash,최종 반응 용기(well)에서 반응결과를 측정하는 것에 특징이 있는 검출로드에 의한 정밀 면역 검사 시스템.A precision immunoassay system using a detection rod that measures reaction results in the final reaction well.
- 제1항에 있어서,According to paragraph 1,상기 반응은 효소가 연결된 면역반응(ELISA)인 것에 특징이 있는 검출로드에 의한 정밀 면역 검사 자동화 시스템.An automated system for precise immune testing using a detection rod, wherein the reaction is an enzyme-linked immune reaction (ELISA).
- 제1항에 있어서,According to paragraph 1,상기 검출로드는 효소기질(Enzyme Substrate)이 포함된 용기에 담궈져 반응되는 것에 특징이 있는 검출로드에 의한 정밀 면역 검사 자동화 시스템.The detection rod is an automated system for precise immune testing using a detection rod, which is characterized in that the detection rod is immersed in a container containing an enzyme substrate and reacted.
- 제1항에 있어서,According to paragraph 1,상기 반응결과는 광학적 신호인 것에 특징이 있는정밀 면역 검사 시스템.A precision immune test system characterized in that the reaction result is an optical signal.
- 제1항에 있어서,According to paragraph 1,상기 미세 패턴은 복수개의 용기로 구성되어서 효소 반응이 발생한 용기의 개수를 카운팅하는 방식으로 광학신호를 측정하는 것에 특징이 있는 정밀 면역 검사 자동화 시스템.The micropattern is composed of a plurality of containers and is characterized by measuring optical signals by counting the number of containers in which an enzyme reaction occurred.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011501194A (en) * | 2007-10-25 | 2011-01-06 | アボット・ラボラトリーズ | How to perform an ultrasensitive immunoassay |
| KR20170050399A (en) * | 2015-10-30 | 2017-05-11 | 전자부품연구원 | Auto Immune Inspection System and method |
| JP2019048986A (en) * | 2009-03-03 | 2019-03-28 | アクセス メディカル システムズ，リミティド | Detection system and method for high-sensitivity fluorometric analysis |
| KR20190098504A (en) * | 2018-02-14 | 2019-08-22 | 중앙대학교 산학협력단 | Enzyme-Linked Immunosorbent Assay System and method of using thereof |
| KR102220357B1 (en) * | 2019-06-10 | 2021-02-25 | 한국전자기술연구원 | Immunodiagnostic kit and immunodiagnostic method using the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011501194A (en) * | 2007-10-25 | 2011-01-06 | アボット・ラボラトリーズ | How to perform an ultrasensitive immunoassay |
| JP2019048986A (en) * | 2009-03-03 | 2019-03-28 | アクセス メディカル システムズ，リミティド | Detection system and method for high-sensitivity fluorometric analysis |
| KR20170050399A (en) * | 2015-10-30 | 2017-05-11 | 전자부품연구원 | Auto Immune Inspection System and method |
| KR20190098504A (en) * | 2018-02-14 | 2019-08-22 | 중앙대학교 산학협력단 | Enzyme-Linked Immunosorbent Assay System and method of using thereof |
| KR102220357B1 (en) * | 2019-06-10 | 2021-02-25 | 한국전자기술연구원 | Immunodiagnostic kit and immunodiagnostic method using the same |
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