WO2017200308A1 - Method for detecting antigen on membrane by using fluorescence resonance energy transfer immunoassay - Google Patents

Method for detecting antigen on membrane by using fluorescence resonance energy transfer immunoassay Download PDF

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WO2017200308A1
WO2017200308A1 PCT/KR2017/005148 KR2017005148W WO2017200308A1 WO 2017200308 A1 WO2017200308 A1 WO 2017200308A1 KR 2017005148 W KR2017005148 W KR 2017005148W WO 2017200308 A1 WO2017200308 A1 WO 2017200308A1
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antigen
antibody
fluorescence
sample
spectrum
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PCT/KR2017/005148
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French (fr)
Korean (ko)
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김민곤
오현경
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광주과학기술원
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Publication of WO2017200308A1 publication Critical patent/WO2017200308A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G01N33/54389—Immunochromatographic test strips based on lateral flow with bidirectional or multidirectional lateral flow, e.g. wherein the sample flows from a single, common sample application point into multiple strips, lanes or zones
    • 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
    • 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
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi

Definitions

  • the present invention relates to an antigen detection method using a fluorescence resonance energy transfer immunoassay, and more particularly, by using the respective antibodies that specifically bind to specific antigens on the membrane by binding them with the antigen fluorescence resonance energy transfer phenomenon It relates to an antigen detection method using the same.
  • a method mainly used for antigen detection includes an enzyme-linked immunosorbent assay (ELISA), which is widely used as an immunoassay of antigen / antibody reaction, which is excellent in sensitivity, quick and simple, and economical. It is put to practical use.
  • ELISA enzyme-linked immunosorbent assay
  • Commonly known indirect enzyme immunoassay is another antibody-enzyme conjugate that coats the antigen-producing material on the bottom and then adds a cell culture supernatant to bind the antibody present in the supernatant to the antigen and subsequently recognize the antibody bound to the antigen. Color development using to confirm whether or not the cell line producing the desired antibody.
  • a direct competitive enzyme immunoassay or an indirect competitive enzyme immunoassay is used.
  • a direct competition enzyme immunoassay generates a competition reaction between a target substance and a target substance-enzyme conjugate.
  • Indirect competitive enzyme immunoassay requires screening for fusion cell lines producing monoclonal antibody-coated detectable-enzyme conjugates and antibodies that compete well with the detected target. Since there is no step to confirm the competence, there is no guarantee that the final antibody is capable of competing for the detected substance.
  • indirect enzyme immunoassay can be said to be suitable only when the substance to be detected is a substance of high molecular weight, such as a protein.
  • a substance having a large molecular weight is easy to coat when using enzyme immunoassay, and a low molecular weight material (e.g., a toxin such as aflatoxin, okratoxin, geralenone, biomarker such as neooptin, biopterin, polycyclic aromatic carbon, drugs, etc.).
  • Etc. is not suitable for using indirect enzyme immunoassay because of poor coating. Therefore, if the substance to be detected is a low molecular substance, the indirect enzyme-immunoassay method cannot directly coat the low molecular substances, and a protein having a high molecular weight, that is, a coatable substance, should be combined with the detectable substance as a carrier and coated. .
  • antigen detection methods using antibodies include lateral flow immunoassay (LFA), electrochemical immunoassay, piezoelectric immunosensors, and fluorescence polarization immunoassays. exist.
  • LFA lateral flow immunoassay
  • electrochemical immunoassay piezoelectric immunosensors
  • fluorescence polarization immunoassays exist.
  • fungal toxin (mycotoxin) is a secondary metabolite produced by mold, which causes diseases and abnormal physiological effects in humans and livestock, and is mainly used in foods that are easy to breed grains, nuts and molds. Occurs. Therefore, fungal toxin contamination in foodstuffs is an unavoidable phenomenon, and inspection of agricultural products and their products is essential.
  • Fungal toxins are largely divided into Aspergillus genus, Penicillium genus, and Fusarium genus toxin. Representative examples also include Aflatoxin, Ochratoxin (OTA) and Geralenone, which are toxins which are potent pathogenic agents, generally having very low solubility in water, Soluble in polar organic solvents such as chloroform, methanol, acetonitrile and the like.
  • OTA Ochratoxin
  • Geralenone which are toxins which are potent pathogenic agents, generally having very low solubility in water, Soluble in polar organic solvents such as chloroform, methanol, acetonitrile and the like.
  • the existing analytical methods have many limitations in terms of equipment and manpower in effectively detecting fungal toxins among many samples. Following this situation, therefore, the introduction of a method that can efficiently analyze it is urgently required.
  • the present invention has been devised to solve this problem, and an object of the present invention is to detect fluorescence characteristics of antibodies in various antigen / antibody complexes more effectively by using a membrane, and to simultaneously detect when two or more antigens are present. It is to provide an antigen detection method that can be.
  • the antigen detection method (a) a sample containing the antigen to be detected at the other end of the diagnostic device formed of a membrane containing the antibody to one end of the antigen to be detected Contacting and reacting with the antibody; (b) irradiating the antigen-antibody complex formed by step (a) with light to cause fluorescence resonance energy transfer, thereby obtaining a spectrum thereof; And (c) determining the presence of the antigen to be detected and measuring the concentration by analyzing the spectrum obtained in the step (b).
  • the diagnostic device is in contact with the reaction membrane downstream of the reaction membrane and the reaction membrane including a sample contact portion for contacting the sample and injecting the sample, a test line containing the antibody and a control line for checking whether the sample is inserted. It may be formed to include an absorbent pad for absorbing the contacted sample.
  • the reaction membrane may be separated from one sample contact and branched into two or more branches.
  • the antigen may be a fungal toxin, biomarker or polycyclic aromatic carbon having an absorption wavelength from 300 to 400 nm.
  • the fungal toxin may be aflatoxin, okratoxin A (Ochratoxin, OTA) or geralenone.
  • the biomarker may be neoopterin, biopterin, nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphoric acid (NADPH).
  • NADH nicotinamide adenine dinucleotide
  • NADPH nicotinamide adenine dinucleotide phosphoric acid
  • the polycyclic aromatic carbon may be benzopyrene, dibenzoanthracene, pyrene or benzofluorene.
  • the step (a) may further comprise the step of drying the diagnostic device after the antigen-antibody reaction.
  • the step (a) may further comprise the step of washing by contacting the buffer solution after the antigen-antibody reaction.
  • the fluorescence spectrum analysis may be performed by comparing the fluorescence intensity at the wavelength indicating the maximum fluorescence intensity of the fluorescence spectrum of the antigen with the maximum fluorescence intensity of the fluorescence spectrum of the antibody.
  • the fluorescence spectrum analysis is a ratio of the fluorescence intensity (I antigen ) at the wavelength showing the maximum fluorescence intensity of the fluorescence spectrum of the antigen to the fluorescence time (I antibody ) at the wavelength showing the maximum fluorescence intensity in the fluorescence spectrum of the antibody .
  • I antigen / I antibodies can be calculated and performed.
  • the fluorescence spectrum can be easily observed even in a dark color sample by contacting a sample having an antigen to be detected on the membrane, and thus, an antigen in a sample such as coffee or wine can be easily detected.
  • the detectable signal intensity decreases as the concentration of the antigen increases, while the fluorescence resonance energy transfer method is used. Do.
  • FIG. 1 shows a representative embodiment of an immunochromatographic assay strip.
  • FIG. 2 is a simplified view of the membrane diagnostic apparatus used in the antigen detection method according to the present invention.
  • FIG. 3 is a view briefly showing the OTA detection process in the antigen detection method according to the present invention.
  • Figure 4 is a view showing the form of various membrane diagnostics that can be used in the antigen detection method according to the present invention.
  • FIG. 5 is a view showing an experimental procedure according to the antigen detection method according to the present invention.
  • Figure 6 is a graph showing the results of spectral measurement according to the presence or absence of the antigen according to the antigen detection method according to the present invention.
  • FIG. 7 is a graph showing the results of spectral measurement according to antigen concentration according to the antigen detection method according to the present invention.
  • FIG. 8 is a graph showing the fluorescence intensity according to the antigen concentration according to the antigen detection method according to the present invention.
  • FIG. 9 is a graph showing the ratio of the fluorescence intensity according to the antigen concentration according to the antigen detection method according to the present invention.
  • FIG. 10 is a graph showing the results of spectral measurements of coffee samples added with OTA according to the antigen detection method according to the present invention.
  • Fluorescence resonance energy transfer refers to a phenomenon in which two fluorescence materials having different wavelength ranges are adjacent to each other, where a resonance phenomenon in which one fluorescence energy is transferred to another phosphor material is generated. It uses the phenomenon that other fluorescence (acceptor or quencher) occurs.
  • the FRET phenomenon is a physical phenomenon in which energy is transferred through a non-radiative process from one excited dye molecule to another by long-range dipole-dipole interactions, where the energy-giving molecule is called a donor. A molecule is called an acceptor or quencher.
  • the donor molecule When the donor molecule is excited by using light of a specific wavelength, when the receptor molecule is present nearby, the energy of the donor molecule is transferred to the receptor molecule, thereby reducing the fluorescence of the donor molecule, and at the same time, the fluorescence of the receptor molecule appears, or Only the fluorescence of the donor molecule is reduced.
  • This energy transfer phenomenon occurs when the donor and the receptor are spatially located at a distance of about 1 to 10 nm.
  • the basic requirement for the energy transfer to occur is that the fluorescence spectrum of the donor molecule and the absorption spectrum of the receptor molecule must overlap each other. to be.
  • Trp tryptophan
  • Immunochromatographic assay is a method that allows you to qualitatively and quantitatively test analytes using a property of biological or chemical attachment to each other. Mounted and assembled immunoassay kits are commonly used. 1 shows a representative embodiment of an immunochromatographic assay strip. As shown in FIG. 1, an assay strip used for immunochromatographic analysis is provided on an adhesive plastic support 60 with a sample pad 40, a conjugate pad 30, a signal detection pad 20 and an absorbent pad ( 50). The sample pad 40 absorbs a liquid sample (or analyte) and ensures a uniform flow of the liquid sample.
  • the conjugate pad 30 includes a fluid conjugate that specifically binds to an analyte contained in the liquid sample, and the liquid sample introduced through the sample pad 40 is connected to the conjugate pad 30. ) Specific binding between the analyte and the fluid conjugate.
  • the signal detection pad 20 typically includes a detection zone 21 and a control zone 22.
  • the detection area 21 is an area for checking whether an analyte is present in the liquid sample
  • the control area 22 is for checking whether the liquid sample has normally passed through the detection area 21. It is an area for.
  • An absorbent pad 50 is positioned on the signal detection pad 20. The absorbent pad 50 absorbs the liquid sample passing through the signal detection pad 20 and assists capillary flow of the liquid sample in the analysis strip.
  • the assay strip is attached to the adhesive plastic support 60 in order of the sample pad 40, the conjugate pad 30, the signal detection pad 20, and the absorption pad 50, and the liquid sample is attached to the sample pad.
  • the signal is moved from the 40 to the absorption pad 50 via the signal detection pad 20, and the immunoassay is performed by detecting the signal at the signal detection pad 20.
  • the conjugate and the signal detector may be integrated into a single porous pad.
  • the sample pad, the conjugate pad, the signal detection pad, and the absorption pad are overlapped with each other or arranged at a predetermined interval on the plastic support. In the latter case, the liquid sample is transferred to the sample pad, the conjugate pad, and the signal detection pad by capillary action using another medium.
  • the detection is performed through a chromatographic technique in a signal detection pad consisting of a membrane, there is an advantage in that a sample of dark coffee, black tea, wine, or the like can be used.
  • the antigen detection method (a) reacts with the antibody by contacting a sample containing the antigen to be detected to the other end of the diagnostic device formed of a membrane containing an antibody to the antigen to be detected at one end And fluorescence resonance energy transfer by irradiating light to the antigen-antibody complex formed by step (a), obtaining a spectrum according thereto, and (c) analyzing the spectrum obtained in step (c). Determining the presence or absence of the antigen to be detected and measuring the concentration.
  • any antigen capable of causing FRET can be detected without limitation, but it is not limited thereto, but is a fungal toxin, biomarker having an absorption wavelength at 300 to 400 nm. Or polycyclic aromatic carbons.
  • fungal toxin examples include Aflatoxin, Olatatoxin A (Ochratoxin, OTA) or Geralenone (zearalenone), and specific examples of the biomarker may include neopterin and biopterin ( biopterin), nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphoric acid (NADPH), and specific examples of the polycyclic aromatic carbon include benzopyrene, dibenzoanthracene, pyrene or benzofluorene.
  • the tryptophan residue of the antibody protein exhibits maximum absorption at 280 nm and maximum emission at 330 nm.
  • Analysis of the fluorescence spectrum is performed by excitation using light of 280 nm wavelength and emission spectrum observation at 330 nm. Can be.
  • quenching occurs due to the binding of the fungal toxin and the antibody.
  • the higher the fungal toxin in the sample the greater the degree of quenching.
  • the decrease in fluorescence intensity is also more pronounced.
  • quenching does not occur, thus resulting in greater fluorescence intensity at 330 nm.
  • the analysis of the fluorescence spectrum may be performed by analyzing the fluorescence intensity at the wavelength representing the maximum fluorescence intensity in the fluorescence spectrum of the antigen or the fluorescence intensity at the wavelength representing the maximum fluorescence intensity in the fluorescence spectrum of the antibody.
  • the analysis of the fluorescence spectrum is based on the fluorescence intensity at the wavelength indicating the maximum fluorescence intensity in the fluorescence spectrum of the antigen with respect to the fluorescence intensity at the wavelength indicating the maximum fluorescence intensity in the fluorescence spectrum of the antibody (I antibody ). It may also be carried out by measuring the ratio of (I antigen ) (I antigen / I antibody ).
  • FIG. 2 is a simplified view of the membrane diagnostic apparatus used in the antigen detection method according to the present invention.
  • 3 is a view briefly showing the OTA detection process in the antigen detection method according to the present invention.
  • OTA an anti-OTA antibody capable of binding to the antigen of interest
  • the sample is brought into contact with the anti-OTA antibody after a predetermined time.
  • Anti-OTA antibody shows the maximum absorption at 280nm and the maximum emission at 330nm.
  • FIG. 4 is a view showing the form of various membrane diagnostics that can be used in the antigen detection method according to the present invention. As shown in FIG. 4, when the membrane is divided into several branches and an antibody capable of responding to a separate antigen is positioned for each branch, the diagnosis can be performed through one diagnosis. Do.
  • FIG. 5 is a view showing an experimental procedure according to the antigen detection method according to the present invention.
  • Absorbent pad (AP222), PALL, Grade 222 is attached on the top of the nitrocellulose membrane (180 membrane card, Millipore, HF180MC100), and then the interval is fixed by using automatic belt loop cutter (CuTex, TBC-50). (3.8 mm) to prepare a strip.
  • Anti-OTA was dissolved in 2 mM Borate buffer (pH 8.5) to prepare 0.44 mg / ml.
  • Antigen (ochratoxin A, Sigma, O1877) was prepared by dissolving in 100% methanol (methanol, Millipore, 106007), 25 mM MES (pH 6) (MES, Sigma, M8250) + 0.3% Tween-20 (Tween20, Sigma, P1379) Buffer was prepared in the composition. 1 ⁇ l of the antibody is dropped into the prepared strip and dried for about 15 minutes at a temperature of about 37 ° C. using a thermo-hygrostat. Separately, 10 ⁇ l of antigen and 90 ⁇ l of buffer are mixed to prepare a reaction solution (sample to be detected) in the microplate well.
  • FIG. 6 is a graph showing the results of measuring the spectrum in accordance with the antigen detection method according to the present invention. As shown in FIG. 6, it can be seen that when OTA is not present, the peak at 330 nm is decreased and the peak at 440 nm, which is an emission spectrum, is increased.
  • FIG. 7 is a graph showing the results of spectral measurement according to antigen concentration according to the antigen detection method according to the present invention.
  • (a) is a graph analyzing the spectrum in the entire wavelength region.
  • (b) is the graph which analyzed the spectrum in the 330 nm vicinity area
  • (c) is a 440 nm vicinity area
  • FIG. 7 it can be seen that as the concentration of OTA increases, the emission spectrum near 330 nm decreases and light of 330 nm generated by the antibody is absorbed by the antigen again. It can also be seen that the emission spectrum near 440 nm increases as the concentration of OTA increases.
  • the fluorescence intensity at 330 nm and the fluorescence at 440 nm are shown in FIG. 8 according to the OTA concentration so that the amount of antigen can be quantitatively identified.
  • the fluorescence intensity at 330 nm is difficult to measure quantitatively as the amount decreases as the concentration of the antigen increases.
  • the fluorescence intensity at 440 nm increases as the antigen concentration increases, and as shown in FIG. 9, the fluorescence intensity at 440 nm versus fluorescence intensity at 330 nm is shown in FIG. 9. Expressing this as the ratio of fluorescence intensity improves the sensitivity of the measured value according to the amount of antigen, which is more effective for quantitative measurement.

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Abstract

A method for detecting an antigen according to an embodiment of the present invention comprises the steps of: (a) contacting an antibody against an antigen to be detected with a specimen containing an antigen to be detected on a diagnostic device to react the antibody and the sample, the diagnostic device being formed of a membrane containing the antibody at one end thereof and the specimen at another end thereof; (b) irradiating light on the antigen-antibody conjugate formed in step (a) to give rise to fluorescence resonance energy transfer to obtain a resulting spectrum; and (c) analyzing the spectrum obtained in step (b) to determine the presence or absence of the antigen to be detected and to measure a concentration of the antigen.

Description

멤브레인상에서 형광 공명 에너지 전이 면역 분석법을 이용한 항원 검출방법Antigen Detection Using Fluorescence Resonance Energy Transfer Immunoassay on Membrane
본 발명은 형광 공명 에너지 전이 면역 분석법을 이용한 항원 검출방법에 관한 것으로, 보다 상세하게는 멤브레인 상에서 특정 항원들과 특이적인 결합을 하는 각각의 항체들을 사용하여 이를 항원들과 결합시켜 형광 공명 에너지 전이 현상을 발생시키고 이를 이용한 항원 검출방법에 관한 것이다.The present invention relates to an antigen detection method using a fluorescence resonance energy transfer immunoassay, and more particularly, by using the respective antibodies that specifically bind to specific antigens on the membrane by binding them with the antigen fluorescence resonance energy transfer phenomenon It relates to an antigen detection method using the same.
종래 항원 검출을 위해서 주로 사용되고 있는 방법으로는 효소면역측정법(enzyme-linked immunosorbent assay, ELISA)이 있는 데, 이는 항원/항체 반응의 면역기법을 이용함으로써, 감도가 뛰어나고 신속 간편하면서도 경제적인 분석법으로서 널리 실용화되고 있다. 일반적으로 알려진 간접 효소면역측정법은 항원이 되는 물질을 바닥에 코팅한 다음, 세포배양 상등액을 첨가하여 상등액에 존재하는 항체를 항원과 결합시키고, 이어서 항원과 결합한 항체를 인식하는 또 다른 항체-효소접합체를 이용하여 발색시킴으로써, 원하는 항체를 생산하는 세포주인지 여부를 확인한다.Conventionally, a method mainly used for antigen detection includes an enzyme-linked immunosorbent assay (ELISA), which is widely used as an immunoassay of antigen / antibody reaction, which is excellent in sensitivity, quick and simple, and economical. It is put to practical use. Commonly known indirect enzyme immunoassay is another antibody-enzyme conjugate that coats the antigen-producing material on the bottom and then adds a cell culture supernatant to bind the antibody present in the supernatant to the antigen and subsequently recognize the antibody bound to the antigen. Color development using to confirm whether or not the cell line producing the desired antibody.
그러나, 이러한 방법은 실험의 단순성에 비해 많은 단점을 지니고 있다. 첫째로, 검출대상 물질을 정량적으로 분석하기 위해서는 직접경합 효소면역측정법 또는 간접경합 효소면역측정법이 사용되는데, 직접경합 효소면역측정법은 검출대상물질과 검출대상물질-효소접합체에 대한 경합반응이 발생하고, 간접경합 효소면역측정법에서는 생산된 단클론 항체가 코팅된 검출대상물질-효소접합체 및 검출대상물질과 경합반응이 잘 일어나는 항체를 생산하는 융합세포주를 선별해야 하지만, 간접경합 효소면역측정법의 경우 항체의 경합능력을 확인하는 단계가 없기 때문에 최종적으로 생산되는 항체가 검출대상물질에 대한 경합능력을 갖추고 있다고 보장할 수 없다. 둘째로, 간접 효소면역측정법은 검출대상물질이 단백질과 같은 분자량이 큰 물질인 경우에만 적합하다고 할 수 있다. 이는, 분자량이 큰 물질이 효소면역측정법 이용 시 코팅이 용이하기 때문이며, 저분자물질 (예, 아플라톡신, 오크라톡신, 제랄레논 등 곰팡이 독소, 네옵테린, 바이옵테린과 같은 바이오마커, 다환 방향족 탄소, 마약류 등)은 코팅이 잘 되지 않기 때문에 간접 효소면역측정법을 사용하기에 적합하지 않다. 따라서, 검출대상물질이 저분자 물질인 경우, 간접 효소면역측정법을 이용한다면 저분자 물질들을 직접 코팅할 수는 없고, 분자량이 큰 단백질, 즉 코팅이 가능한 물질을 담체로서 검출대상물질과 결합시켜 코팅하여야 한다. 이후 세포배양 상등액을 반응시키면 항원을 인식하는 항체도 결합할 수 있지만, 담체를 인식하는 부분의 항체, 또한 항원과 담체의 사이를 인식하는 항체도 존재할 수 있고, 이러한 항체들이 2차 항체에 의해 발색을 야기하게 된다. ELISA 방법 외에도 항체를 이용한 항원 검출 방법에는, 측류 면역분석법 (lateral flow immunoassay, LFA), 전기화학적 면역분석법 (electrochemical immunoassay), 압전 면역센서 (piezoelectric immunosensors) 및 형광 극성 면역분석법 (fluorescence polarization immunoassays) 등이 존재한다.However, this method has many disadvantages compared to the simplicity of the experiment. First, in order to quantitatively analyze a substance to be detected, a direct competitive enzyme immunoassay or an indirect competitive enzyme immunoassay is used. A direct competition enzyme immunoassay generates a competition reaction between a target substance and a target substance-enzyme conjugate. Indirect competitive enzyme immunoassay requires screening for fusion cell lines producing monoclonal antibody-coated detectable-enzyme conjugates and antibodies that compete well with the detected target. Since there is no step to confirm the competence, there is no guarantee that the final antibody is capable of competing for the detected substance. Second, indirect enzyme immunoassay can be said to be suitable only when the substance to be detected is a substance of high molecular weight, such as a protein. This is because a substance having a large molecular weight is easy to coat when using enzyme immunoassay, and a low molecular weight material (e.g., a toxin such as aflatoxin, okratoxin, geralenone, biomarker such as neooptin, biopterin, polycyclic aromatic carbon, drugs, etc.). Etc.) is not suitable for using indirect enzyme immunoassay because of poor coating. Therefore, if the substance to be detected is a low molecular substance, the indirect enzyme-immunoassay method cannot directly coat the low molecular substances, and a protein having a high molecular weight, that is, a coatable substance, should be combined with the detectable substance as a carrier and coated. . Subsequent reaction of the cell culture supernatant may bind the antibody that recognizes the antigen, but there may be an antibody that recognizes the carrier, and also an antibody that recognizes the antigen and the carrier, and these antibodies are developed by the secondary antibody. Will cause. In addition to ELISA methods, antigen detection methods using antibodies include lateral flow immunoassay (LFA), electrochemical immunoassay, piezoelectric immunosensors, and fluorescence polarization immunoassays. exist.
특히, 검출 대상이 되는 저분자 항원 중에서도 곰팡이 독소 (mycotoxin)는 곰팡이가 생산하는 2차 대사 산물로서, 사람과 가축에 질병이나 이상생리작용을 유발하며, 곡류, 견과류 및 곰팡이가 번식하기 쉬운 식품에서 주로 발생한다. 따라서, 식품류에서 곰팡이 독소 오염은 피할 수 없는 현상이고, 농산물과 그들 생산품에 대한 검사가 필수적이다.In particular, among the low-molecular antigens to be detected, fungal toxin (mycotoxin) is a secondary metabolite produced by mold, which causes diseases and abnormal physiological effects in humans and livestock, and is mainly used in foods that are easy to breed grains, nuts and molds. Occurs. Therefore, fungal toxin contamination in foodstuffs is an unavoidable phenomenon, and inspection of agricultural products and their products is essential.
곰팡이 독소는 크게 생산균에 따라 아스페르길루스 (Aspergillus) 속, 페니실륨 (Penicillium) 속, 및 푸사리움 (Fusarium) 속 곰팡이 독소 등으로 구분한다. 또한, 대표적인 예로서, 아플라톡신 (Aflatoxin), 오크라톡신 A (Ochratoxin, OTA) 및 제랄레논 (zearalenone)을 들 수 있으며, 이러한 곰팡이 독소들은 강력한 병원성 물질로서, 일반적으로 물에서의 용해도가 매우 낮으며, 클로로포름, 메탄올, 아세토니트릴 등과 같은 극성 유기용매에 잘 용해된다. 특히, 세계적 농산물 수출입량의 급격한 증가로 인해서 곰팡이 독소들의 검출에 대한 관심도가 높아지고 있음에도 불구하고, 기존의 분석방법으로는 많은 시료들 중에서 곰팡이 독소를 효과적으로 검출함에 있어서, 장비, 인력 등의 측면에서 많은 제약이 따르는 실정이고, 따라서 이를 효율적으로 분석할 수 있는 방법의 도입이 절실히 요구되고 있다.Fungal toxins are largely divided into Aspergillus genus, Penicillium genus, and Fusarium genus toxin. Representative examples also include Aflatoxin, Ochratoxin (OTA) and Geralenone, which are toxins which are potent pathogenic agents, generally having very low solubility in water, Soluble in polar organic solvents such as chloroform, methanol, acetonitrile and the like. In particular, despite the increasing interest in the detection of fungal toxins due to the rapid increase in global agricultural imports and exports, the existing analytical methods have many limitations in terms of equipment and manpower in effectively detecting fungal toxins among many samples. Following this situation, therefore, the introduction of a method that can efficiently analyze it is urgently required.
상기한 배경기술로서 설명된 사항들은 본 발명의 배경에 대한 이해 증진을 위한 것일 뿐, 이 기술분야에서 통상의 지식을 가진 자에게 이미 알려진 종래기술에 해당함을 인정하는 것으로 받아들여져서는 안 될 것이다.The matters described as the background art are only for the purpose of improving the understanding of the background of the present invention and should not be taken as acknowledging that they correspond to the related art already known to those skilled in the art.
본 발명은 이러한 문제점을 해결하기 위해 안출된 것으로, 본 발명의 목적은 다양한 항원/항체 복합체에서 항체의 형광특성을 멤브레인을 이용하여 보다 효과적으로 검출하고, 2개 이상의 항원이 존재하는 경우에 동시에 검출할 수 있는 항원 검출방법을 제공하는 데 있다.The present invention has been devised to solve this problem, and an object of the present invention is to detect fluorescence characteristics of antibodies in various antigen / antibody complexes more effectively by using a membrane, and to simultaneously detect when two or more antigens are present. It is to provide an antigen detection method that can be.
위 목적을 달성하기 위하여 본 발명의 일 실시예에 따른 항원 검출방법은 (a) 검출대상 항원에 대한 항체가 일단에 함유되어 있는 멤브레인으로 형성된 진단기의 타단에 검출대상이 되는 항원이 함유된 시료를 접촉시켜 상기 항체와 반응 시키는 과정; (b) 상기 (a)과정에 의해 형성된 항원-항체 복합체에 광을 조사함으로써 형광 공명 에너지 전이를 야기하고, 이에 따른 스펙트럼을 얻는 과정; 및 (c) 상기 (b) 과정에서 획득한 스펙트럼을 분석함으로써 검출 대상 항원의 존부를 판단하고 농도를 측정하는 과정을 포함한다.In order to achieve the above object, the antigen detection method according to an embodiment of the present invention (a) a sample containing the antigen to be detected at the other end of the diagnostic device formed of a membrane containing the antibody to one end of the antigen to be detected Contacting and reacting with the antibody; (b) irradiating the antigen-antibody complex formed by step (a) with light to cause fluorescence resonance energy transfer, thereby obtaining a spectrum thereof; And (c) determining the presence of the antigen to be detected and measuring the concentration by analyzing the spectrum obtained in the step (b).
상기 진단기는 상기 시료를 접촉하여 상기 시료를 주입할 수 있는 시료접촉부와 상기 항체가 함유되어 있는 테스트 라인과 시료의 투입여부를 확인하는 콘트롤 라인을 포함하는 반응 멤브레인과 상기 반응멤브레인의 다운 스트림에 접촉하여 형성되어 상기 접촉된 시료를 흡수하는 흡수패드를 포함할 수 있다.The diagnostic device is in contact with the reaction membrane downstream of the reaction membrane and the reaction membrane including a sample contact portion for contacting the sample and injecting the sample, a test line containing the antibody and a control line for checking whether the sample is inserted. It may be formed to include an absorbent pad for absorbing the contacted sample.
상기 반응멤브레인은 하나의 시료접촉부에서 분리되어 두 개 이상의 가지로 분기되어 형성될 수 있다.The reaction membrane may be separated from one sample contact and branched into two or more branches.
상기 항원은 300 ~ 400nm 에서 흡수 파장을 갖는 곰팡이 독소, 바이오마커 또는 다환 방향족 탄소일 수 있다.The antigen may be a fungal toxin, biomarker or polycyclic aromatic carbon having an absorption wavelength from 300 to 400 nm.
상기 곰팡이 독소는 아플라톡신(Aflatoxin), 오크라톡신 A(Ochratoxin, OTA) 또는 제랄레논일 수 있다.The fungal toxin may be aflatoxin, okratoxin A (Ochratoxin, OTA) or geralenone.
상기 바이오마커는 네옵테린 (neopterin), 바이옵테린 (biopterin), 니코틴아미드아데닌디뉴클레오티드 (NADH) 또는 니코틴아미드아데닌디뉴클레오티드 인산 (NADPH)일 수 있다.The biomarker may be neoopterin, biopterin, nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphoric acid (NADPH).
상기 다환 방향족 탄소는 벤조피렌, 디벤조안트라센, 피렌 또는 벤조플루오렌일 수 있다.The polycyclic aromatic carbon may be benzopyrene, dibenzoanthracene, pyrene or benzofluorene.
상기 (a) 과정은 항원-항체 반응 후에 진단기를 건조시키는 과정을 더 포함할 수 있다.The step (a) may further comprise the step of drying the diagnostic device after the antigen-antibody reaction.
상기 (a) 과정은 항원-항체 반응 후에 버퍼용액을 접촉시켜 워싱하는 과정을 더 포함할 수 있다.The step (a) may further comprise the step of washing by contacting the buffer solution after the antigen-antibody reaction.
상기 형광 스펙트럼 분석은 상기 항원의 형광 스펙트럼 중 최대 형광 세기를 나타내는 파장에서의 형광세기와 상기 항체의 형광 스펙트럼 중 최대 형광 세기를 비교분석함으로써 수행될 수 있다.The fluorescence spectrum analysis may be performed by comparing the fluorescence intensity at the wavelength indicating the maximum fluorescence intensity of the fluorescence spectrum of the antigen with the maximum fluorescence intensity of the fluorescence spectrum of the antibody.
상기 형광 스펙트럼 분석은 상기 항체의 형광 스펙트럼 중 최대 형광 세기를 나타내는 파장에서의 형광시기(I항체)에 대한 상기 항원의 형광 스펙트럼 중 최대 형광 세기를 나타내는 파장에서의 형광 세기(I항원)의 비율인 I항원/I항체를 계산하여 수행될 수 있다.The fluorescence spectrum analysis is a ratio of the fluorescence intensity (I antigen ) at the wavelength showing the maximum fluorescence intensity of the fluorescence spectrum of the antigen to the fluorescence time (I antibody ) at the wavelength showing the maximum fluorescence intensity in the fluorescence spectrum of the antibody . I antigen / I antibodies can be calculated and performed.
본 발명에 의한 항원 검출방법에 따르면 다음과 같은 효과가 있다.According to the antigen detection method according to the present invention has the following effects.
첫째, 멤브레인상에서 검출대상이 되는 항원이 존재하는 시료를 접촉시켜 색상이 짙은 시료의 경우에도 형광 스펙트럼 관찰이 용이하므로, 커피나 와인 등의 시료내의 항원을 용이하게 검출할 수 있다.First, the fluorescence spectrum can be easily observed even in a dark color sample by contacting a sample having an antigen to be detected on the membrane, and thus, an antigen in a sample such as coffee or wine can be easily detected.
둘째, 멤브레인 진단기를 다단으로 구성하여 시료 내에 다양한 항원이 존재하는 경우에 한번의 시료채취로도 각 항원의 존부를 판단할 수 있어 다중 진단이 가능하다.Secondly, when a variety of antigens are present in a sample by configuring a membrane diagnostic apparatus in multiple stages, the presence or absence of each antigen can be determined even with a single sampling, thereby enabling multiple diagnosis.
셋째, 통상적인 competitive ELISA 방식에서는 항원의 농도가 커짐에 따라 검출할 수 있는 신호의 세기가 낮아 지는 반면에 형광 공명 에너지 전이방법으로 사용함으로써 항원의 농도가 클수록 검출 신호가 커짐으로써 정량적인 분석이 가능하다.Third, in the conventional competitive ELISA method, the detectable signal intensity decreases as the concentration of the antigen increases, while the fluorescence resonance energy transfer method is used. Do.
도 1은 면역크로마토그래피 분석 스트립의 대표적 구현 예를 보여주는 도면이다.1 shows a representative embodiment of an immunochromatographic assay strip.
도 2는 본 발명에 따른 항원 검출방법에서 사용되는 멤브레인 진단기를 간략하게 나타낸 도면이다. Figure 2 is a simplified view of the membrane diagnostic apparatus used in the antigen detection method according to the present invention.
도 3은 본 발명에 따른 항원 검출방법에서 OTA검출과정을 간략히 나타낸 도면이다. 3 is a view briefly showing the OTA detection process in the antigen detection method according to the present invention.
도 4은 본 발명에 따른 항원 검출방법에서 사용할 수 있는 다양한 멤브레인 진단기의 형태를 나타낸 도면이다.Figure 4 is a view showing the form of various membrane diagnostics that can be used in the antigen detection method according to the present invention.
도 5는 본 발명에 따른 항원 검출방법에 따른 실험과정을 나타낸 도면이다.5 is a view showing an experimental procedure according to the antigen detection method according to the present invention.
도 6은 본 발명에 따른 항원 검출방법에 따라 항원유무에 따른 스펙트럼 측정 결과를 나타낸 그래프이다.Figure 6 is a graph showing the results of spectral measurement according to the presence or absence of the antigen according to the antigen detection method according to the present invention.
도 7은 본 발명에 따른 항원 검출방법에 따라 항원농도에 따른 스펙트럼 측정 결과를 나타낸 그래프이다.7 is a graph showing the results of spectral measurement according to antigen concentration according to the antigen detection method according to the present invention.
도 8은 본 발명에 따른 항원 검출방법에 따라 항원농도에 따른 형광강도를 나타낸 그래프이다.8 is a graph showing the fluorescence intensity according to the antigen concentration according to the antigen detection method according to the present invention.
도 9 은 본 발명에 따른 항원 검출방법에 따라 항원농도에 따른 형광강도의 비를 나타낸 그래프이다.9 is a graph showing the ratio of the fluorescence intensity according to the antigen concentration according to the antigen detection method according to the present invention.
도 10은 본 발명에 따른 항원검출 방법에 따라 OTA를 첨가한 커피시료의 스펙트럼 측정결과를 나타낸 그래프이다.10 is a graph showing the results of spectral measurements of coffee samples added with OTA according to the antigen detection method according to the present invention.
여기서 사용되는 전문용어는 단지 특정 실시예를 언급하기 위한 것이며, 본 발명을 한정하는 것을 의도하지 않는다. 여기서 사용되는 단수 형태들은 문구들이 이와 명백히 반대의 의미를 나타내지 않는 한 복수 형태들도 포함한다. 명세서에서 사용되는 "포함하는"의 의미는 특정 특성, 영역, 정수, 단계, 동작, 요소 및/또는 성분을 구체화하며, 다른 특정 특성, 영역, 정수, 단계, 동작, 요소, 성분 및/또는 군의 존재나 부가를 제외시키는 것은 아니다.The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a,” “an,” and “the” include plural forms as well, unless the phrases clearly indicate the opposite. As used herein, the term "comprising" embodies a particular characteristic, region, integer, step, operation, element, and / or component, and other specific characteristics, region, integer, step, operation, element, component, and / or group. It does not exclude the presence or addition of.
다르게 정의하지는 않았지만, 여기에 사용되는 기술용어 및 과학용어를 포함하는 모든 용어들은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 일반적으로 이해하는 의미와 동일한 의미를 가진다. 보통 사용되는 사전에 정의된 용어들은 관련기술문헌과 현재 개시된 내용에 부합하는 의미를 가지는 것으로 추가 해석되고, 정의되지 않는 한 이상적이거나 매우 공식적인 의미로 해석되지 않는다.Unless defined otherwise, all terms including technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Commonly defined terms used are additionally interpreted to have a meaning consistent with the related technical literature and the presently disclosed contents, and are not interpreted in an ideal or very formal sense unless defined.
이하, 첨부된 도면을 참조하여 본 발명의 바람직한 실시예에 의한 항원 검출방법에 대하여 설명하기로 한다.Hereinafter, an antigen detection method according to a preferred embodiment of the present invention with reference to the accompanying drawings will be described.
형광 공명 에너지 전이 (fluorescence resonance energy transfer, FRET)이란 두 개의 서로 다른 파장 영역을 갖는 형광물질이 서로 인접하였을 경우, 하나의 형광을 일으키는 에너지 (donor)가 다른 형광물질에 전달되는 공명 현상이 발생함으로써 다른 형광 (acceptor or quencher)이 발생되는 현상을 이용한 것이다. FRET 현상은 장거리 쌍극자-쌍극자 상호작용에 의하여 하나의 들뜬 염료분자에서 다른 염료분자로 비복사 과정을 통해 에너지가 전이되는 물리적인 현상이며, 이때 에너지를 주는 분자를 공여체 (donor)라 하고 에너지를 받는 분자를 수용체 (acceptor or quencher)라고 한다.Fluorescence resonance energy transfer (FRET) refers to a phenomenon in which two fluorescence materials having different wavelength ranges are adjacent to each other, where a resonance phenomenon in which one fluorescence energy is transferred to another phosphor material is generated. It uses the phenomenon that other fluorescence (acceptor or quencher) occurs. The FRET phenomenon is a physical phenomenon in which energy is transferred through a non-radiative process from one excited dye molecule to another by long-range dipole-dipole interactions, where the energy-giving molecule is called a donor. A molecule is called an acceptor or quencher.
특정 파장의 빛을 사용하여 공여체 분자를 들뜨게 했을 때, 주위에 수용체 분자가 가까이 존재할 경우, 공여체 분자의 에너지가 수용체 분자로 전이되어 공여체 분자의 형광이 감소되는 동시에 수용체 분자의 형광이 나타나거나, 또는 공여체 분자의 형광이 감소되는 현상만 나타난다. 이러한 에너지 전이현상은 공간적으로 공여체와 수용체가 1~10nm 정도의 거리에 존재할 경우 발생되는데, 에너지 전이가 발생되기 위한 기본적인 요건은 우선 공여체 분자의 형광 스펙트럼과 수용체 분자의 흡수스펙트럼이 서로 중첩되어야 한다는 점이다. 항체와 같은 단백질의 경우에는, 트립토판 (tryptophan, Trp)이 존재함으로 인해 260nm ~ 290nm, 대략 280nm에서 최고 흡수파장을 가지며, 또한 이러한 파장대에서 빛을 야기시켰을 때 300nm ~ 400nm, 대략 340nm 근처에서 가장 강한 형광시그널을 나타낸다. 따라서, 전술한 바와 같이, 형광특성을 갖는 항원, 예를 들어 아플라톡신, 오클라톡신, 제랄레논 등과 같은 곰팡이 독소들의 흡수파장과 단백질인 항체의 형광스펙트럼이 서로 중첩된다는 사실을 알 수 있으며, 이러한 현상을 응용할 경우, 형광특성을 갖는 항원들을 검출할 수 있는 방법을 제공할 수 있게 된다.When the donor molecule is excited by using light of a specific wavelength, when the receptor molecule is present nearby, the energy of the donor molecule is transferred to the receptor molecule, thereby reducing the fluorescence of the donor molecule, and at the same time, the fluorescence of the receptor molecule appears, or Only the fluorescence of the donor molecule is reduced. This energy transfer phenomenon occurs when the donor and the receptor are spatially located at a distance of about 1 to 10 nm. The basic requirement for the energy transfer to occur is that the fluorescence spectrum of the donor molecule and the absorption spectrum of the receptor molecule must overlap each other. to be. For proteins such as antibodies, the presence of tryptophan (Trp) has the highest absorption wavelength from 260 nm to 290 nm, approximately 280 nm, and also the strongest at around 300 nm to 400 nm and approximately 340 nm when light is generated in these wavelengths. Fluorescence signal is shown. Therefore, as described above, it can be seen that the absorption spectrum of fungal toxins such as aflatoxin, oclatoxin, geralenone and the like and the fluorescence spectrum of the antibody, which are proteins, overlap each other. When applied, it is possible to provide a method capable of detecting antigens having fluorescent properties.
면역크로마토그래피 분석(immunochromatographic assay, ICA)은 생물학적 물질 또는 화학적 물질이 서로 특이적으로 부착하는 성질을 이용하여 분석 물질을 단시간에 정성 및 정량적으로 검사할 수 있는 방법으로, 분석 스트립을 플라스틱 하우징 내부에 장착해 조립한 형태의 면역분석 키트가 일반적으로 사용된다. 도 1은 면역크로마토그래피 분석 스트립의 대표적 구현 예를 보여주는 도면이다. 도 1에 도시된 바와 같이, 면역크로마토그래피 분석에 사용되는 분석 스트립은 접착성 플라스틱 지지체(60) 상에 검체 패드(40), 컨쥬게이트 패드(30), 신호검출 패드(20) 및 흡수 패드(50)를 포함하여 이루어진다. 상기 검체 패드(40)는 액상 검체(또는 분석시료)를 흡수하고 액상 검체의 균일한 유동을 보장한다. 상기 컨쥬게이트 패드(30)는 상기 액상 검체에 함유되어 있는 분석물질과 특이적으로 결합하는 유동성 컨쥬게이트를 포함하고 있으며, 상기 검체 패드(40)를 통해 도입된 액상 검체가 상기 컨쥬게이트 패드(30)를 통과하면서 분석물질과 유동성 컨쥬게이트 사이의 특이적 결합이 이루어진다. 상기 신호검출 패드(20)는 통상 검출영역(detection zone)(21)과 대조영역(control zone)(22)을 포함하여 이루어진다. 상기 검출영역(21)은 상기 액상 검체에 분석물질이 존재하는 지의 여부를 확인하기 위한 영역이며, 상기 대조영역(22)은 액상 검체가 상기 검출영역(21)을 정상적으로 통과하였는지의 여부를 확인하기 위한 영역이다. 상기 신호검출 패드(20)의 상부에, 흡수 패드(50)가 위치한다. 상기 흡수 패드(50)는 상기 신호검출 패드(20)를 통과한 액상 검체를 흡수하며, 상기 분석 스트립에서의 상기 액상 검체의 모세관 유동을 도와준다. 정리하면, 상기 분석 스트립은 접착성 플라스틱 지지체(60)에 검체 패드(40), 컨쥬게이트 패드(30), 신호검출 패드(20) 및 흡수 패드(50) 순서로 부착하여, 액상 검체를 검체 패드(40)로부터 신호검출 패드(20)를 경유해, 흡수 패드(50)까지 이동시키고, 신호검출 패드(20)에서의 신호검출을 통해 면역분석을 수행한다. 변형된 다른 방법으로는 상기 컨쥬게이트와 신호검출물질을 하나의 다공성 패드에 통합하기도 한다. 그리고, 검체 패드, 컨쥬게이트 패드, 신호검출 패드 및 흡수 패드는 서로 중첩되어 배치되거나 또는 일정한 간격을 두고 플라스틱 지지체에 배열되기로 한다. 후자의 경우, 액상검체가 다른 매개체를 이용하여 모세관 현상으로 검체 패드와 컨쥬게이트 패드, 신호검출 패드로 이동한다. 이와 같이 멤브레인으로 구성되는 신호검출 패드에서 크로마토 기법을 통하여 검출하는 경우 색이 진한 커피나 홍차, 와인 등의 샘플을 사용할 수 있는 이점이 존재한다. Immunochromatographic assay (ICA) is a method that allows you to qualitatively and quantitatively test analytes using a property of biological or chemical attachment to each other. Mounted and assembled immunoassay kits are commonly used. 1 shows a representative embodiment of an immunochromatographic assay strip. As shown in FIG. 1, an assay strip used for immunochromatographic analysis is provided on an adhesive plastic support 60 with a sample pad 40, a conjugate pad 30, a signal detection pad 20 and an absorbent pad ( 50). The sample pad 40 absorbs a liquid sample (or analyte) and ensures a uniform flow of the liquid sample. The conjugate pad 30 includes a fluid conjugate that specifically binds to an analyte contained in the liquid sample, and the liquid sample introduced through the sample pad 40 is connected to the conjugate pad 30. ) Specific binding between the analyte and the fluid conjugate. The signal detection pad 20 typically includes a detection zone 21 and a control zone 22. The detection area 21 is an area for checking whether an analyte is present in the liquid sample, and the control area 22 is for checking whether the liquid sample has normally passed through the detection area 21. It is an area for. An absorbent pad 50 is positioned on the signal detection pad 20. The absorbent pad 50 absorbs the liquid sample passing through the signal detection pad 20 and assists capillary flow of the liquid sample in the analysis strip. In summary, the assay strip is attached to the adhesive plastic support 60 in order of the sample pad 40, the conjugate pad 30, the signal detection pad 20, and the absorption pad 50, and the liquid sample is attached to the sample pad. The signal is moved from the 40 to the absorption pad 50 via the signal detection pad 20, and the immunoassay is performed by detecting the signal at the signal detection pad 20. Alternately, the conjugate and the signal detector may be integrated into a single porous pad. In addition, the sample pad, the conjugate pad, the signal detection pad, and the absorption pad are overlapped with each other or arranged at a predetermined interval on the plastic support. In the latter case, the liquid sample is transferred to the sample pad, the conjugate pad, and the signal detection pad by capillary action using another medium. As such, when the detection is performed through a chromatographic technique in a signal detection pad consisting of a membrane, there is an advantage in that a sample of dark coffee, black tea, wine, or the like can be used.
즉, 본 발명자들은 항원들과 특이적인 결합을 하는 각각의 항체를 사용하여 이를 항원들과 멤브레인 상에서 반응시켜 형광 공명 에너지 전이 현상을 발생시키고, 이에 의해서 항원의 존재 여부를 판독할 수 있는 항원 검출 방법을 개발하였다. 본 발명의 일 실시예에 따른 항원 검출 방법은 (a) 검출대상 항원에 대한 항체가 일단에 함유되어 있는 멤브레인으로 형성된 진단기의 타단에 검출대상이 되는 항원이 함유된 시료를 접촉시켜 상기 항체와 반응 시키는 과정, 상기 (a)과정에 의해 형성된 항원-항체 복합체에 광을 조사함으로써 형광 공명 에너지 전이를 야기하고, 이에 따른 스펙트럼을 얻는 과정 및 (c) 상기 (c)과정에서 획득한 스펙트럼을 분석함으로써 검출 대상 항원의 존부를 판단하고 농도를 측정하는 과정을 포함한다.That is, the present inventors react with the antigens on the membrane using the respective antibodies that specifically bind to the antigens to generate a fluorescence resonance energy transfer phenomenon, thereby detecting the presence of the antigen. Developed. The antigen detection method according to an embodiment of the present invention (a) reacts with the antibody by contacting a sample containing the antigen to be detected to the other end of the diagnostic device formed of a membrane containing an antibody to the antigen to be detected at one end And fluorescence resonance energy transfer by irradiating light to the antigen-antibody complex formed by step (a), obtaining a spectrum according thereto, and (c) analyzing the spectrum obtained in step (c). Determining the presence or absence of the antigen to be detected and measuring the concentration.
본 발명에 따른 항원 검출 방법을 사용하여 검출 가능한 항원으로는, FRET 현상을 야기할 수 있는 항원이라면 제한 없이 검출가능하며, 이에 제한되는 것은 아니지만, 300~400nm 에서 흡수 파장을 갖는 곰팡이 독소, 바이오마커 또는 다환 방향족 탄소를 예로 들 수 있다. 특히, 상기 곰팡이 독소의 구체적인 예로는 아플라톡신 (Aflatoxin), 오크라톡신 A (Ochratoxin, OTA) 또는 제랄레논 (zearalenone)을 들 수 있고, 상기 바이오마커의 구체적인 예로는 네옵테린 (neopterin), 바이옵테린 (biopterin), 니코틴아미드아데닌디뉴클레오티드 (NADH) 또는 니코틴아미드아데닌디뉴클레오티드 인산 (NADPH)을 들 수 있으며, 상기 다환 방향족 탄소의 구체적인 예로는 벤조피렌, 디벤조안트라센, 피렌 또는 벤조플루오렌을 들 수 있다.As an antigen detectable using the antigen detection method according to the present invention, any antigen capable of causing FRET can be detected without limitation, but it is not limited thereto, but is a fungal toxin, biomarker having an absorption wavelength at 300 to 400 nm. Or polycyclic aromatic carbons. In particular, specific examples of the fungal toxin include Aflatoxin, Olatatoxin A (Ochratoxin, OTA) or Geralenone (zearalenone), and specific examples of the biomarker may include neopterin and biopterin ( biopterin), nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphoric acid (NADPH), and specific examples of the polycyclic aromatic carbon include benzopyrene, dibenzoanthracene, pyrene or benzofluorene.
특히, 항체 단백질의 트립토판 잔기는 280nm에서 최대 흡수특성을 나타내며, 330nm에서 최대 방출특성을 나타내는 바, 형광 스펙트럼의 분석은 280nm 파장의 광을 사용한 여기 (excitation) 및 330nm에서의 방출 스펙트럼 관찰에 의해서 수행될 수 있다. 즉, 시료 중에 곰팡이 독소가 존재하는 경우에는 곰팡이 독소와 항체와의 결합에 의해서 소광 (quenching) 현상이 발생되는 바, 시료 중에 곰팡이 독소가 높을수록 그 소광의 정도가 더욱 커지고, 이에 따른 330nm에서의 형광 강도 감소 현상도 더욱 두드러지게 된다. 이와는 대조적으로, 시료 중에 곰팡이 독소가 존재하지 않는 경우에는 이러한 소광 현상이 발생되지 않고, 따라서 330nm에서의 형광 강도가 더 커지게 된다.In particular, the tryptophan residue of the antibody protein exhibits maximum absorption at 280 nm and maximum emission at 330 nm. Analysis of the fluorescence spectrum is performed by excitation using light of 280 nm wavelength and emission spectrum observation at 330 nm. Can be. In other words, if a fungal toxin is present in the sample, quenching occurs due to the binding of the fungal toxin and the antibody. The higher the fungal toxin in the sample, the greater the degree of quenching. The decrease in fluorescence intensity is also more pronounced. In contrast, in the absence of a fungal toxin in the sample, such quenching does not occur, thus resulting in greater fluorescence intensity at 330 nm.
그러므로, 상기 형광 스펙트럼의 분석은 상기 항원의 형광 스펙트럼 중 최대 형광 세기를 나타내는 파장에서의 형광 세기 혹은 상기 항체의 형광 스펙트럼 중 최대 형광 세기를 나타내는 파장에서의 형광 세기를 분석함으로써 수행될 수 있다.Therefore, the analysis of the fluorescence spectrum may be performed by analyzing the fluorescence intensity at the wavelength representing the maximum fluorescence intensity in the fluorescence spectrum of the antigen or the fluorescence intensity at the wavelength representing the maximum fluorescence intensity in the fluorescence spectrum of the antibody.
또 다른 방법으로는, 상기 형광 스펙트럼의 분석은 상기 항체의 형광 스펙트럼 중 최대 형광 세기를 나타내는 파장에서의 형광 세기 (I항체)에 대한 상기 항원의 형광 스펙트럼 중 최대 형광 세기를 나타내는 파장에서의 형광 세기 (I항원)의 비율 (I항원/I항체)을 측정함으로써 수행될 수도 있다.In another method, the analysis of the fluorescence spectrum is based on the fluorescence intensity at the wavelength indicating the maximum fluorescence intensity in the fluorescence spectrum of the antigen with respect to the fluorescence intensity at the wavelength indicating the maximum fluorescence intensity in the fluorescence spectrum of the antibody (I antibody ). It may also be carried out by measuring the ratio of (I antigen ) (I antigen / I antibody ).
곰팡이 독소로 OTA를 예를 들어 항원 검출과정을 보다 자세하게 설명한다. 도 2는 본 발명에 따른 항원 검출방법에서 사용되는 멤브레인 진단기를 간략하게 나타낸 도면이다. 도 3은 본 발명에 따른 항원 검출방법에서 OTA검출과정을 간략히 나타낸 도면이다. OTA를 예를 들어 항원 검출과정을 설명한다. 본 발명에서는 멤브레인으로 형성된 진단기에 검출대상의 항원인 OTA에 결합할 수 있는 Anti-OTA 항체를 함유시킨 뒤에 일단에 시료를 접촉시키게 되면 일정 시간 후에 이동하여 Anti-OTA 항체에 접촉하게 된다. Anti-OTA 항체의 경우에는 280nm에서 최대 흡수 특성을 나타내며, 330nm에서 최대 방출특성을 나타낸다. 이 때 형광 스펙트럼의 분석은 280nm파장의 광을 사용한 여기(exicitation) 및 330nm에서의 방출 스펙트럼의 관찰에 의해 수행될 수 있다. 도 4은 본 발명에 따른 항원 검출방법에서 사용할 수 있는 다양한 멤브레인 진단기의 형태를 나타낸 도면이다. 도 4에 도시한 바와 같이 멤브레인을 여러 갈래로 나누어 각각의 가지마다 별도의 항원에 반응할 수 있는 항체를 위치시킨 후 진단을 수행하게 되면 다양한 항원의 존재 여부를 한번의 진단으로 통하여 검출하는 것이 가능하다.Fungal toxins are described in more detail, for example, in the antigen detection process. Figure 2 is a simplified view of the membrane diagnostic apparatus used in the antigen detection method according to the present invention. 3 is a view briefly showing the OTA detection process in the antigen detection method according to the present invention. We describe the antigen detection process using OTA as an example. In the present invention, when the diagnostic agent formed into a membrane contains an anti-OTA antibody capable of binding to the antigen of interest, OTA, the sample is brought into contact with the anti-OTA antibody after a predetermined time. Anti-OTA antibody shows the maximum absorption at 280nm and the maximum emission at 330nm. In this case, the analysis of the fluorescence spectrum may be performed by excitation using light of 280 nm wavelength and observation of the emission spectrum at 330 nm. Figure 4 is a view showing the form of various membrane diagnostics that can be used in the antigen detection method according to the present invention. As shown in FIG. 4, when the membrane is divided into several branches and an antibody capable of responding to a separate antigen is positioned for each branch, the diagnosis can be performed through one diagnosis. Do.
이하 실시예를 통하여 본 발명에 대하여 보다 자세하게 설명한다. 도 5는 본 발명에 따른 항원 검출방법에 따른 실험과정을 나타낸 도면이다. 나이트로셀룰로오스 멤브레인 (180 membrane card, Millipore, HF180MC100) 위쪽에 흡수패드 (Absorbent Pad (AP222), PALL, Grade 222) 를 부착 후 커터기 (automatic belt loop cutter, CuTex, TBC-50) 를 사용하여 일정 간격 (3.8mm) 으로 잘라 스트립을 준비하였다. Anti-OTA를 2mM의 Borate buffer(pH 8.5)에 녹여 0.44mg/ml로 준비하였다. 항원 (ochratoxin A, Sigma, O1877) 은 100% 메탄올 (methanol, Millipore, 106007) 에 녹여 준비하고, 25mM MES (pH 6) (MES, Sigma, M8250) + 0.3% Tween-20 (Tween20, Sigma, P1379) 조성으로 버퍼를 준비하였다. 제조된 스트립에 항체를 1㎕ 떨어뜨리고, 항온항습기를 사용하여 약 37℃의 온도에서 약 15분간 건조시킨다. 별도로 항원 10㎕와 버퍼 90㎕을 혼합하여 반응 용액(검출대상시료)을 microplate well에 준비한다. 스트립 상의 항체가 다 건조된 후에 해당 스트립을 버퍼가 준비된 microplate well에 위치시키고, 상온에서 약 15분간 반응시킨다. 별도의 microplate well에 버퍼 100㎕를 준비한 후에 항원과 스트립상의 항체와 반응이 완료되고 나면 스트립을 버퍼가 준비된 microplate well에 위치시키고 다시 상온에서 약 15분간 반응시킨다. 모든 반응이 완료되고 난 후 항온항습기에서 다시 약 37℃에서 약 15분간 다시 건조시킨다. 건조된 스트립을 마이크로 리더기(Infinite 200 PRO NanoQuant Microplate Readers from TECAN)를 통하여 형광을 측정하였다. 도 6는 본 발명에 따른 항원 검출방법에 따라 스펙트럼을 측정한 결과를 나타낸 그래프이다. 도 6에 도시한 바와 같이 OTA 가 존재하지 않는 경우에는 330nm 에서의 피크가 감소하고 방출 스펙트럼인 440nm 의 피크가 증가하는 것을 알 수 있다. Hereinafter, the present invention will be described in more detail with reference to the following examples. 5 is a view showing an experimental procedure according to the antigen detection method according to the present invention. Absorbent pad (AP222), PALL, Grade 222 is attached on the top of the nitrocellulose membrane (180 membrane card, Millipore, HF180MC100), and then the interval is fixed by using automatic belt loop cutter (CuTex, TBC-50). (3.8 mm) to prepare a strip. Anti-OTA was dissolved in 2 mM Borate buffer (pH 8.5) to prepare 0.44 mg / ml. Antigen (ochratoxin A, Sigma, O1877) was prepared by dissolving in 100% methanol (methanol, Millipore, 106007), 25 mM MES (pH 6) (MES, Sigma, M8250) + 0.3% Tween-20 (Tween20, Sigma, P1379) Buffer was prepared in the composition. 1 μl of the antibody is dropped into the prepared strip and dried for about 15 minutes at a temperature of about 37 ° C. using a thermo-hygrostat. Separately, 10 μl of antigen and 90 μl of buffer are mixed to prepare a reaction solution (sample to be detected) in the microplate well. After the antibody on the strip is dried, the strip is placed in a microplate well prepared with buffer, and reacted for about 15 minutes at room temperature. After preparing 100 μl of buffer in a separate microplate well, after completion of the reaction with the antigen and the antibody on the strip, the strip is placed in the prepared microplate well and reacted for about 15 minutes at room temperature. After all reactions are complete, dry again in a thermo-hygrostat at about 37 ° C for about 15 minutes. The dried strips were measured for fluorescence through a micro reader (Infinite 200 PRO NanoQuant Microplate Readers from TECAN). Figure 6 is a graph showing the results of measuring the spectrum in accordance with the antigen detection method according to the present invention. As shown in FIG. 6, it can be seen that when OTA is not present, the peak at 330 nm is decreased and the peak at 440 nm, which is an emission spectrum, is increased.
또한 항원 농도에 따라 스펙트럼 검출결과를 확인하기 위해 동일한 방법으로 항원의 농도를 1~1000 ng/ml로 달리하여 실험을 진행하였다. 도 7은 본 발명에 따른 항원 검출방법에 따라 항원농도에 따른 스펙트럼 측정 결과를 나타낸 그래프이다. (a)는 전체 파장영역에서의 스펙트럼을 분석한 그래프이다. (b)는 330nm 부근 영역, (c)는 440nm 부근 영역에서의 스펙트럼을 분석한 그래프이다. 도 7에 도시한 바와 같이 OTA의 농도가 증가할수록 330nm 부근의 방출 스펙트럼이 감소하고 항체에 의해 생성되는 330nm 의 빛이 다시 항원에 의해 흡수되는 것을 알 수 있다. 또한 OTA의 농도가 증가할 수록 440nm 부근의 방출 스펙트럼이 증가하는 것을 알 수 있다. In addition, the experiment was carried out by varying the concentration of the antigen to 1 ~ 1000 ng / ml by the same method to confirm the spectrum detection results according to the antigen concentration. 7 is a graph showing the results of spectral measurement according to antigen concentration according to the antigen detection method according to the present invention. (a) is a graph analyzing the spectrum in the entire wavelength region. (b) is the graph which analyzed the spectrum in the 330 nm vicinity area | region, and (c) is a 440 nm vicinity area | region. As shown in FIG. 7, it can be seen that as the concentration of OTA increases, the emission spectrum near 330 nm decreases and light of 330 nm generated by the antibody is absorbed by the antigen again. It can also be seen that the emission spectrum near 440 nm increases as the concentration of OTA increases.
또한, 항원의 양을 정량적으로 확인할 수 있도록 OTA 농도에 따른 330nm 에서의 형광강도와 440nm에서의 형광광도를 도8에 나타내었다. 도 8에 도시한 바와 같이, (a)에서는 330nm에서 형광강도는 항원의 농도가 증가함에 따라 그 양이 감소하여 정량적으로 측정하기 어려운 것을 알 수 있다. 그러나 (b)에 도시한 바와 같이 440nm에서의 형광광도는 항원의 농도가 증가함에 따라 그 강도가 증가하는 것을 알 수 있으며, 도 9에 도시된 바와 같이, 330nm에서의 형광강도에 대한 440nm에서의 형광강도의 비로 이를 나타내면 보다 항원의 양에 따른 측정값의 민감도를 향상시켜 정량측정에 더욱 효과적이다.In addition, the fluorescence intensity at 330 nm and the fluorescence at 440 nm are shown in FIG. 8 according to the OTA concentration so that the amount of antigen can be quantitatively identified. As shown in Figure 8, in (a) it can be seen that the fluorescence intensity at 330 nm is difficult to measure quantitatively as the amount decreases as the concentration of the antigen increases. However, as shown in (b), the fluorescence intensity at 440 nm increases as the antigen concentration increases, and as shown in FIG. 9, the fluorescence intensity at 440 nm versus fluorescence intensity at 330 nm is shown in FIG. 9. Expressing this as the ratio of fluorescence intensity improves the sensitivity of the measured value according to the amount of antigen, which is more effective for quantitative measurement.
또한, 본 발명에서는 멤브레인 진단기를 사용하여 크로마토그래피 방식을 채택하고 있으므로 용액 방식의 형광 공명 에너지 전이현상을 이용한 방법에서는 측정하기 어려운 커피를 시료로 사용하여 OTA의 존부를 검출하였다. OTA를 커피액에 첨가하여 시료를 제조하였고 그 외에는 상기 실험과 동일한 방법으로 수행하였다. 도 10은 본 발명에 따른 항원검출 방법에 따라 OTA를 첨가한 커피시료의 스펙트럼 측정결과를 나타낸 그래프이다. 도10에 도시한 바와 같이, 진한 색을 띠는 커피 시료내에서도 OTA에 대한 형광강도의 변화를 확인할 수 있었다.In addition, in the present invention, since the chromatographic method is adopted by using a membrane diagnostic machine, the presence of OTA was detected using coffee, which is difficult to measure in the method using a solution-type fluorescence resonance energy transfer phenomenon, as a sample. Samples were prepared by adding OTA to the coffee liquor and otherwise performed in the same manner as the above experiment. 10 is a graph showing the results of spectral measurements of coffee samples added with OTA according to the antigen detection method according to the present invention. As shown in FIG. 10, the change in fluorescence intensity for OTA was confirmed even in a dark coffee sample.
이상 첨부된 도면을 참조하여 본 발명의 실시예를 설명하였지만, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자는 본 발명이 그 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다.Although embodiments of the present invention have been described above with reference to the accompanying drawings, those skilled in the art to which the present invention pertains may implement the present invention in other specific forms without changing the technical spirit or essential features thereof. I can understand that.
그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허청구범위에 의하여 나타내어지며, 특허청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변경된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the present invention is shown by the following claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention. .

Claims (11)

  1. (a) 검출대상 항원에 대한 항체가 일단에 함유되어 있는 멤브레인으로 형성된 진단기의 타단에 검출대상이 되는 항원이 함유된 시료를 접촉시켜 상기 항체와 반응 시키는 과정;(a) contacting a sample containing an antigen to be detected with the other end of a diagnostic device formed of a membrane containing an antibody to the antigen to be detected at one end to react with the antibody;
    (b) 상기 (a)과정에 의해 형성된 항원-항체 복합체에 광을 조사함으로써 형광 공명 에너지 전이를 야기하고, 이에 따른 스펙트럼을 얻는 과정; 및(b) irradiating the antigen-antibody complex formed by step (a) with light to cause fluorescence resonance energy transfer, thereby obtaining a spectrum thereof; And
    (c) 상기 (b)과정에서 획득한 스펙트럼을 분석함으로써 검출 대상 항원의 존부를 판단하고 농도를 측정하는 과정을 포함하는 항원 검출방법.(c) determining the presence or absence of the antigen to be detected by analyzing the spectrum obtained in step (b) and measuring the concentration.
  2. 청구항1에 있어서,The method according to claim 1,
    상기 진단기는 상기 시료를 접촉하여 상기 시료를 주입할 수 있는 시료접촉부와 상기 항체가 함유되어 있는 테스트 라인과 시료의 투입여부를 확인하는 콘트롤 라인을 포함하는 반응 멤브레인과 상기 반응멤브레인의 다운 스트림에 접촉하여 형성되어 상기 접촉된 시료를 흡수하는 흡수패드를 포함하는 항원 검출방법.The diagnostic device is in contact with the reaction membrane downstream of the reaction membrane and the reaction membrane including a sample contact portion for contacting the sample and injecting the sample, a test line containing the antibody and a control line for checking whether the sample is inserted. And an absorption pad formed to absorb the contacted sample.
  3. 청구항1에 있어서,The method according to claim 1,
    상기 반응멤브레인은 하나의 시료접촉부에서 분리되어 두 개 이상의 가지로 분기되어 형성되는 것을 특징으로 하는 항원 검출방법.The reaction membrane is an antigen detection method, characterized in that formed in one branch contact is separated into two or more branches.
  4. 청구항 1에 있어서,The method according to claim 1,
    상기 항원은 300 ~ 400nm 에서 흡수 파장을 갖는 곰팡이 독소, 바이오마커 또는 다환 방향족 탄소인 것을 특징으로 하는 항원 검출방법.The antigen is an antigen detection method, characterized in that the fungal toxin, biomarker or polycyclic aromatic carbon having an absorption wavelength at 300 ~ 400nm.
  5. 청구항 4에 있어서,The method according to claim 4,
    상기 곰팡이 독소는 아플라톡신(Aflatoxin), 오크라톡신 A(Ochratoxin, OTA) 또는 제랄레논 인 것을 특징으로 하는 항원 검출방법.The fungal toxin is Aflatoxin (Aflatoxin), Okratoxin A (Ochratoxin, OTA) or an antigen detection method characterized in that the geralenone.
  6. 청구항 4에 있어서,The method according to claim 4,
    상기 바이오마커는 네옵테린 (neopterin), 바이옵테린 (biopterin), 니코틴아미드아데닌디뉴클레오티드 (NADH) 또는 니코틴아미드아데닌디뉴클레오티드 인산 (NADPH)인 것을 특징으로 하는 항원 검출 방법.The biomarker is neoopterin (neopterin), biopterin (biopterin), nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphoric acid (NADPH) characterized in that the antigen detection method.
  7. 청구항 4에 있어서,The method according to claim 4,
    상기 다환 방향족 탄소는 벤조피렌, 디벤조안트라센, 피렌 또는 벤조플루오렌 인 것을 특징으로 하는 항원검출방법.The polycyclic aromatic carbon is benzopyrene, dibenzoanthracene, pyrene or benzofluorene characterized in that the antigen detection method.
  8. 청구항 1에 있어서,The method according to claim 1,
    상기 (a) 과정은 항원-항체 반응 후에 진단기를 건조시키는 과정을 더 포함하는 항원 검출방법.Said (a) step further comprises the step of drying the diagnostic device after the antigen-antibody reaction.
  9. 청구항 1에 있어서,The method according to claim 1,
    상기 (a) 과정은 항원-항체 반응 후에 버퍼용액을 접촉시켜 워싱하는 과정을 더 포함하는 항원 검출방법.Said step (a) further comprises the step of washing by contacting the buffer solution after the antigen-antibody reaction.
  10. 청구항 1에 있어서,The method according to claim 1,
    상기 형광 스펙트럼 분석은 상기 항원의 형광 스펙트럼 중 최대 형광 세기를 나타내는 파장에서의 형광세기와 상기 항체의 형광 스펙트럼 중 최대 형광 세기를 비교분석함으로써 수행되는 것을 특징으로 하는 항원 검출 방법.The fluorescence spectrum analysis is performed by comparing the fluorescence intensity at the wavelength indicating the maximum fluorescence intensity of the fluorescence spectrum of the antigen with the maximum fluorescence intensity of the fluorescence spectrum of the antibody.
  11. 청구항 10에 있어서,The method according to claim 10,
    상기 형광 스펙트럼 분석은 상기 항체의 형광 스펙트럼 중 최대 형광 세기를 나타내는 파장에서의 형광시기(I항체)에 대한 상기 항원의 형광 스펙트럼 중 최대 형광 세기를 나타내는 파장에서의 형광 세기(I항원)의 비율인 I항원/I항체를 계산하여 수행되는 것을 특징으로 하는 항원 검출방법.The fluorescence spectrum analysis is a ratio of the fluorescence intensity (I antigen ) at the wavelength showing the maximum fluorescence intensity of the fluorescence spectrum of the antigen to the fluorescence time (I antibody ) at the wavelength showing the maximum fluorescence intensity in the fluorescence spectrum of the antibody . Antigen detection method characterized in that performed by calculating the I antigen / I antibody .
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007064766A (en) * 2005-08-30 2007-03-15 Japan Advanced Institute Of Science & Technology Hokuriku Detection method of substance to be detected, sensitizer and kit for immunochromatography
KR20130037648A (en) * 2011-10-06 2013-04-16 한국생명공학연구원 A method of producing membrane sensor for multiple diagnosis using screen printing
JP2013120120A (en) * 2011-12-07 2013-06-17 Konica Minolta Medical & Graphic Inc Test strip for lateral flow type chromatography, and method for detecting or quantifying analyte using the same
KR20140058305A (en) * 2012-11-05 2014-05-14 광주과학기술원 Method for detection of antigen using fluorescence resonance energy transfer immunoassay

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3479100B2 (en) 1993-06-02 2003-12-15 帝国臓器製薬株式会社 Simple semi-quantitative immunochemical method and apparatus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007064766A (en) * 2005-08-30 2007-03-15 Japan Advanced Institute Of Science & Technology Hokuriku Detection method of substance to be detected, sensitizer and kit for immunochromatography
KR20130037648A (en) * 2011-10-06 2013-04-16 한국생명공학연구원 A method of producing membrane sensor for multiple diagnosis using screen printing
JP2013120120A (en) * 2011-12-07 2013-06-17 Konica Minolta Medical & Graphic Inc Test strip for lateral flow type chromatography, and method for detecting or quantifying analyte using the same
KR20140058305A (en) * 2012-11-05 2014-05-14 광주과학기술원 Method for detection of antigen using fluorescence resonance energy transfer immunoassay

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI, TAIHUA ET AL.: "Label-free Homogeneous FRET Immunoassay for the Detection of Mycotoxins that Utilizes Quenching of the intrinsic Fluorescence of Antibodies", BIOSENSORS AND BIOELECTRONICS, vol. 42, 2 November 2012 (2012-11-02), pages 403 - 408, XP055439321 *

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