WO2017171237A1 - Trousse de détection de virus de la grippe au moyen d'un complexe point quantique-bille de latex-anticorps contre le virus de la grippe, et procédé de détection utilisant celle-ci - Google Patents

Trousse de détection de virus de la grippe au moyen d'un complexe point quantique-bille de latex-anticorps contre le virus de la grippe, et procédé de détection utilisant celle-ci Download PDF

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WO2017171237A1
WO2017171237A1 PCT/KR2017/001811 KR2017001811W WO2017171237A1 WO 2017171237 A1 WO2017171237 A1 WO 2017171237A1 KR 2017001811 W KR2017001811 W KR 2017001811W WO 2017171237 A1 WO2017171237 A1 WO 2017171237A1
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influenza virus
composition
quantum dot
kit
latex
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Korean (ko)
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박현
여선주
강호만
최학수
정점규
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원광대학교산학협력단
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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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • 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
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus

Definitions

  • the present invention relates to a kit for influenza virus detection using a quantum dot-latex bead-influenza virus antibody complex and a detection method using the same.
  • Influenza is a respiratory disease caused by the influenza virus.
  • Influenza viruses which are the causative agent, are classified into A, B and C types by the antigenicity of the nucleoprotein (NP) of the virus.
  • the main causes of disease in humans are types A and B (Sandrock C, Kelly T. Clinical review: update of avian influenza A infections in humans. Crit Care. 2007; 11 (2): 209).
  • Influenza viruses are spherical (80-120 nm in diameter) and are divided into several subtypes depending on the types of hemagglutinin (HA) and neuraminidase (NA) glycoproteins, the major antigens on the surface. Are classified. In the case of influenza A, a total of 16 HAs and 9 NAs are known, and a combination of these can theoretically generate 144 subtypes.
  • HA hemagglutinin
  • NA neuraminidase glycoproteins
  • influenza virus isolation culture
  • detection of viral antigens fast antigen test, immunofluorescence method
  • viral nucleic acid verification RT-PCR
  • serological tests Lee Chang-seop. Journal of the Korean Medical Association, 2010; 53 (1): pp.43-51).
  • Virus culturing methods require a time of 2-10 days and require skilled professionals and have a high possibility of false negatives.
  • PCR methods have the disadvantage of requiring primers for each subtype.
  • a semiconductor quantum dot is a spherical material with unique optical and electronic properties. Its particle size is 2 to 20 nm, and it emits fluorescence at different wavelengths depending on the particle size. It varies from the ultraviolet region to the near infrared region. Quantum dots that emit light of various wavelengths can be excited at the same time with only one excitation light source. In addition, it is relatively stable even in a strong light source such as a laser, and since the half width is narrow to about 20 to 30 nm, it is advantageous to detect multiple viruses because various signals can be used simultaneously without overlapping signals unlike when using an organic phosphor.
  • Korean Patent No. 1512484 discloses a method for rapidly detecting norovirus in food using magnetic beads and quantum dots
  • a domestic journal (Bulletin of Food Tecnology Vol. 22, No. 3, pp. 577-586) discloses the synthesis and application of quantum dot labeled fluorescent microbeads, but the kit for detecting influenza virus using the quantum dot nanoparticle-latex bead complex of the present invention and a detection method using the same have not been disclosed.
  • the present invention is derived from the above requirements, the present invention is a quantum dot-latex bead-influenza virus antibody complex in which a quantum dot is coated with a hydrophilic compound coated on the latex beads, the influenza virus antibody is further added to the latex beads.
  • Influenza virus detection composition comprising as an active ingredient, an influenza virus detection kit and influenza virus detection method comprising the same, the quantum dot-latex bead-influenza virus antibody complex according to the invention is influenza virus subtypes H1N1, H7N7, It was confirmed that H9N2 or H5N3 virus and H5N1 influenza virus HA antigens can be detected, and the sensitivity and specificity of the influenza virus detection kit are calculated by applying the H1N1 H1N1 virus infection patient sample. Completed the command.
  • the present invention is an influenza virus comprising a quantum dot coated with a hydrophilic compound to the latex beads, the quantum dot- latex bead- influenza virus antibody complex conjugated to the latex bead influenza virus antibody as an active ingredient It provides a composition for detection.
  • the present invention comprises the steps of (a) coating the surface of the quantum dots (QDs) with a hydrophilic compound;
  • step (d) combining the carboxyl group on the surface of the latex beads substituted in step (c) with the amino group included in the influenza virus antibody through an amide coupling reaction, and then centrifuging to obtain a quantum dot-latex bead-influenza virus antibody complex. It provides a method for producing a composition for detecting influenza virus comprising a.
  • the present invention also provides a kit for influenza virus detection comprising the influenza virus detection composition.
  • the present invention is to put a sample suspected of containing influenza virus in the influenza virus detection kit and when a fluorescent band appears in the test line and the control line of the test strip, it is determined that the influenza virus infection is positive, only the control line fluorescent
  • an influenza virus detection method is provided, characterized by negatively determining an influenza virus infection.
  • the present invention is a composition for detecting influenza virus comprising a quantum dot coated with a hydrophilic compound coated on a latex bead, and a quantum dot-latex bead-influenza virus antibody complex conjugated with an influenza virus antibody on the surface of the latex bead, including the same.
  • the present invention relates to a kit for detecting influenza virus and a method for detecting influenza virus, which can effectively detect influenza virus even when a small amount of sample is used, and it is convenient to apply a dry quantum dot-latex bead-influenza antibody complex to the kit. It has the advantage of long term storage.
  • Figure 1 (A) is a schematic diagram showing the principle of manufacturing a composition for detecting influenza virus combined with a quantum dot-latex-antibody coated with a hydrophilic compound provided in the present invention
  • Figure 1 (B) is a hydrophilic provided by the present invention It represents the light emission wavelength of the quantum dots coated with the compound.
  • Figure 3 is the result of confirming the size of latex beads, quantum dot-latex beads, quantum dot-latex bead-influenza virus antibody complex according to the present invention.
  • QD620nm quantum dots
  • B latex beads
  • C quantum dots
  • D quantum dots
  • QD580nm quantum dots
  • E quantum dots
  • QD620nm latex bead-influenza virus antibody complex
  • F quantum dots
  • Figure 5 is a graph showing the fluorescence signal value according to the concentration of the antibody complex for the influenza virus nucleoprotein of QD640nm-latex beads-H1N1 type, H5N3 type, H7N7 type or H9N2.
  • FIG. 6 shows the results of confirming the limitation of H1N1 influenza virus detection using antibody complexes against influenza virus nucleoproteins of quantum dots-latex-H1N1, H5N3, H7N7 or H9N2 with luminescence wavelengths of 580 nm, 620 nm and 640 nm.
  • a graph showing the change in the value calculated by dividing the value of the fluorescence intensity according to the H1N1 influenza virus content by the value of the fluorescence intensity measured at the control line.
  • FIG. 7 shows the results of confirming the limitation of H1N1 influenza virus detection using an antibody complex against influenza virus nucleoprotein of quantum dot-latex bead-H1N1, H5N3, H7N7 or H9N2 having a luminescence wavelength of 520 nm.
  • FIG. 8 shows the results of confirming the limitations of H5N3 influenza virus detection using antibody complexes against influenza virus nucleoproteins of quantum dot-latex bead-H1N1, H5N3, H7N7 or H9N2 with luminescence wavelengths of 580 nm, 620 nm and 640 nm.
  • a graph showing the change in the value calculated by dividing the value of the fluorescence intensity according to the H5N3 type influenza virus content by the value of the fluorescence intensity measured at the control line.
  • FIG. 10 shows the results of confirming the limitation of H7N7 influenza virus detection using antibody complexes against influenza virus nucleoproteins of quantum dots-latex-H1N1, H5N3, H7N7 or H9N2 with luminescence wavelengths of 580 nm, 620 nm and 640 nm.
  • a graph showing the change in the value calculated by dividing the value of the fluorescence intensity according to the H7N7 type influenza virus content by the value of the fluorescence intensity measured at the control line.
  • 11 is a result of confirming the limit of H7N7 influenza virus detection using antibody complex against influenza virus nucleoprotein of quantum dot-latex bead-H1N1 type, H5N3 type, H7N7 type or H9N2 type with emission wavelength of 520 nm. .
  • FIG. 12 shows the results of confirming the limitation of H9N2 influenza virus detection using antibody complexes against influenza virus nucleoproteins of quantum dots-latex-H1N1, H5N3, H7N7 or H9N2 with emission wavelengths of 580 nm, 620 nm and 640 nm.
  • FIG. 13 shows the results of confirming the limit of H9N2 influenza virus detection using antibody complex against influenza virus nucleoprotein of quantum dot-latex bead-H1N1 type, H5N3 type, H7N7 type or H9N2 having an emission wavelength of 520 nm.
  • FIG. 15 is a fluorescence detection kit using an antibody complex against influenza virus nucleoprotein of quantum dot-latex bead-H1N1, H5N3, H7N7 or H9N2 having a luminescence wavelength of 640 nm, and H1N1 H1N1 influenza via RT-PCR It is a graph showing fluorescence values for thickening samples of people confirmed to be infected with the virus and thickening samples of people confirmed not to be infected with the H1N1 influenza virus.
  • the present invention is an influenza virus comprising a quantum dot coated with a hydrophilic compound on a latex bead, and a quantum dot-latex bead-influenza virus antibody complex in which an influenza virus antibody is bound to a surface which does not bind a quantum dot in the latex beads as an active ingredient. It relates to a composition for detection.
  • the latex beads used in the present invention are not particularly limited as long as they can be used as mediators to which quantum dots and antibodies can bind, but are preferably latex beads having a plurality of reactive amino groups to which quantum dots and antibodies can bind.
  • the diameter of the latex beads is preferably 10 to 2,000 nm, more preferably 50 to 1,000 nm, even more preferably 100 nm, but is not limited thereto.
  • Quantum dots coated with the latex beads and the hydrophilic compound are preferably bonded in a molar ratio of 1:40 to 1,000, more preferably in a molar ratio of 1:70 to 800, and even more preferably in a molar ratio of 1:90. But is not limited thereto.
  • the molar ratio of the latex beads and the quantum dots is less than 1:40, since the quantum dots are not sufficient, there is a problem that the amount of fluorescence emitted is low, and the fluorescence noise is severe at a molar ratio exceeding 1: 1000, and the influenza virus with respect to the latex beads There is a problem that the binding ratio of the antibody is lowered.
  • the latex beads and influenza virus antibodies are preferably bound in a molar ratio of 1:50 to 500, more preferably in a molar ratio of 1: 100 to 470, and even more preferably in a molar ratio of 1: 455. But is not limited thereto.
  • the bond between the latex beads and the quantum dots coated with a hydrophilic compound is an amide bond between an amino group on the surface of the latex beads and a carboxyl group included in the quantum dots, and the bond between the latex beads and the influenza virus antibody is a carboxyl group on the surface of the latex beads. And an amide bond between the amino group contained in the influenza virus antibody.
  • the quantum dots include cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium tellurium (CdTe), zinc telium (ZnTe), zinc selenide (ZnSe), zinc sulfide (ZnS), zinc oxide (ZnO) , At least one selected from indium phosphide (InP), indium arsenide (InAs), mercury tellerium (HgTe), and mercury selenide (HgSe), but is not limited thereto.
  • the diameter of the quantum dot is preferably 1 to 30 nm, more preferably 2 to 20 nm, but is not limited thereto.
  • the emission wavelength of the quantum dot is preferably 450 nm to 700 nm, but is not limited thereto.
  • influenza virus antibody preferably specifically recognizes nucleoprotein (NP) of influenza virus of type H1N1, H5N3, H7N7 or H9N2, or hemagglutinin (HA) of H5N1 influenza virus, but in particular
  • NP nucleoprotein
  • HA hemagglutinin
  • the present invention is not limited thereto, and any target virus-specific antibody may be applied without any limitation.
  • the hydrophilic compound is preferably any one selected from cysteamine, mercaptosuccinic acid, mercaptopropionic acid, glutathione, cysteine, and thiol-containing silane. It is not limited.
  • antibody of the present invention is a term known in the art and is a specific immunoglobulin directed against an antigenic site.
  • the form of the antibody includes all polyclonal antibodies, monoclonal antibodies and recombinant antibodies, and may include all immunoglobulin antibodies as well as special antibodies such as humanized antibodies.
  • the antibodies include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains.
  • a functional fragment of an antibody molecule means a fragment having at least antigen binding function and may be Fab, F (ab '), F (ab') 2, Fv, and the like.
  • the antibody is not particularly limited as long as it can bind with latex directly or indirectly using a linker, and all kinds of antibodies can be used.
  • the antibody may be directly bound to the latex by reacting the carboxyl group of the antibody with the reactive amino group of the latex to form an amide bond.
  • a linker such as a peptide, glutaraldehyde, or succinic anhydride may be used to indirectly bind the antibody and the latex, and the linker used may be specifically designed to mediate the binding between the antibody and the latex. It is not limited.
  • the present invention comprises the steps of (a) coating the surface of the quantum dots (QDs) with a hydrophilic compound;
  • step (d) combining the carboxyl group on the surface of the latex beads substituted in step (c) with the amino group included in the influenza virus antibody through an amide coupling reaction, and then centrifuging to obtain a quantum dot-latex bead-influenza virus antibody complex. It relates to a method for producing a composition for detecting influenza virus comprising a.
  • the amide coupling reaction of steps (b) and (d) was carried out in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS). It is preferably performed under, but not limited to.
  • the present invention also relates to an influenza virus detection kit comprising the composition for detecting influenza virus.
  • the influenza virus detection kit includes a sample injecting unit for injecting a sample; An influenza virus binding unit comprising a composition for detecting influenza virus that binds to an influenza virus antigen in a sample located at a point spaced apart from the sample injection unit; A test line in which specific antibodies of the influenza virus are fixed at positions spaced a predetermined distance from the binding unit; And a control line to which the anti-mouse IgG is fixed is preferably provided sequentially, but is not limited thereto.
  • influenza virus composition for detection is 0.2 ⁇ 10 -6 ⁇ 1.0 ⁇ 10 -5 nmole which it is preferred, more preferably from 1.0 ⁇ 10 -6 ⁇ 6.0 ⁇ 10 -6 nmole and, still more preferably from 1.76 ⁇ 10 - 6 nmole, but is not limited to such.
  • the influenza virus detection composition may be dried or in a solution state, and preferably in a dry state, but is not limited thereto.
  • the drying is preferably natural drying at room temperature for 1 hour, but is not limited thereto.
  • the kit to which the dry composition for influenza virus detection is applied is stable and can be stored for a long time.
  • Kit for detecting influenza virus provided by the present invention by combining a glass fiber, cotton or cellulose pad to the nitrocellulose membrane in the form of a strip to prepare a thick sample or fecal sample of human or animal
  • a sample injection unit which can be injected is provided, and a binding portion of the influenza virus antigen including the quantum dot-latex bead-influenza virus antibody complex and the same antigen as the influenza virus antibody can be detected while maintaining a predetermined distance from the sample injection unit.
  • FICT fluorescent immunochromatographic test kit
  • the method relates to an influenza virus detection method characterized in that negatively determined influenza virus infection.
  • the sensitivity is an index indicating how well the test selects a positive sample as a probability that the test result is positive in a positive sample (influenza virus infected sample).
  • specificity is a probability that a test result is negative in a negative sample (a sample not infected with influenza virus) in the differential test, and is an index indicating how well the test selects a negative sample.
  • Example 1 Preparation of Influenza Virus Detection Composition Comprising a Hydrophilic Compound-Coated Quantum Dots ( QDs ) -Latex Bead - Influenza Virus Antibody Complex
  • a composite in which a quantum dot coated with a hydrophilic compound and latex beads were combined was prepared by the method described below (FIG. 1A).
  • washing buffer was added to 50 ⁇ l of 0.044 ⁇ M amino latex (amine latex, life technology) having a size of about 100 nm and washed once with 0.1 M Phosphate Buffered Saline (8.77 g / L NaCl), and washed once.
  • 0.1 ⁇ M quantum dots coated with Mn were mixed at a molar ratio of 1:90, and 200 ⁇ l of 0.01 M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 0.01 M N-hydroxysulfosuccinic acid were added.
  • 300 ⁇ l of mid (Sulfo-NHS) was added and reacted at room temperature for 1 hour (FIG.
  • the light emission wavelength of the composite in which the quantum dot coated with the hydrophilic compound and the latex beads is combined is 520 nm, 580 nm, 620 nm or 640 nm (FIG. 1B).
  • a succinic anhydride was bonded to an amino group of the latex bead surface to which the quantum dot was not bonded to replace the latex bead surface with a carboxyl group.
  • Antibody (1 mg / ml) against nin (HA) was mixed in a molar ratio of 1: 455, 100 ⁇ l of 0.01 M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 0.01 M 150 ⁇ l of N-hydroxysulfosuccinimide (Sulfo-NHS) was added and reacted at room temperature for 2 hours.
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide
  • Sulfo-NHS N-hydroxysulfosuccinimide
  • the reaction was carried out for 30 minutes in a blocking solution containing 0.1% BSA, 1% sucrose, 1% gelatin, and 0.01% casein. After the reaction was completed, the reaction was centrifuged (15,000 rpm, 5 minutes) to remove unbound influenza virus antibody, and the precipitate was washed once by adding the wash buffer solution (pH7.4), and then again. Centrifugation was carried out in 500 ⁇ l of the wash buffer solution (pH7.4) to prepare a composition for influenza virus detection comprising a quantum dot-latex bead-influenza virus antibody complex as an active ingredient (FIGS. 3 to 5).
  • the antibody complex for quantum dot-latex bead-influenza virus nucleoprotein (NP) prepared in Example 1 or the antibody complex for quantum dot-latex bead-H5N1 influenza virus hemagglutinin (HA) is used for influenza virus detection.
  • an influenza virus detection kit including the influenza virus detection composition was prepared.
  • a sample injecting unit capable of injecting a thickened steel sample may be provided by bonding a sample pad made of glass fiber, cotton or cellulose to one end of a strip-shaped nitrocellulose membrane.
  • 2 ⁇ l of the 0.88 nM influenza virus detection composition according to Example 1 was added thereto, and then, at a predetermined interval, a test line (T) was set therein, followed by an influenza on the test line.
  • Antibodies to the virus 2.5 ⁇ g was added to fix.
  • Antibodies against influenza viruses immobilized on the test line are antibodies against nucleoproteins (NPs) of H1N1, H5N3, H7N7 or H9N2 or hemagglutinin (HA) of H5N1 influenza virus.
  • NPs nucleoproteins
  • H1N1, H5N3, H7N7 or H9N2 hemagglutinin
  • a fluorescence detection kit of a form in which a sample pad, a composition for detecting influenza virus, an inspection line and a control line were sequentially arranged on a strip-shaped nitrocellulose membrane was prepared.
  • Example 3 Measurement of detection limit of an influenza virus detection kit comprising a composition for influenza virus detection
  • H1N1 influenza virus was prepared as a sample, and sample dilutions (25 mM HEPES, 100 mM NaCl, 2.5 mM MgCl 2 , 0.1% NP40, pH7.5) were separately prepared.
  • the amount of H1N1 influenza virus contained in the sample is 2.5 ⁇ 10 2 PFU / ml, 5.0 ⁇ 10 2 PFU / ml, 1.0 ⁇ 10 3 PFU / ml, 1.0 ⁇ 10 4 PFU / ml or 5.0 ⁇ 10 4 PFU / Ml was set, the control was used that was not treated virus.
  • the detection kit was excited at a wavelength of 375 nm, and the fluorescence intensities of the test and control lines were measured with a fluorescence reader (aGcare TRF, Medisensor).
  • the emission wavelength of the composition for detecting influenza virus was 520 nm, 580 nm, 620 nm or 640 nm.
  • FIG. 6 is a change in fluorescence intensity according to H1N1 type influenza virus content using an influenza virus detection kit comprising an antibody complex for latex bead-influenza virus nucleoproteins using quantum dots having a light emission wavelength of 580 nm, 620 nm or 640 nm, respectively.
  • the graph shows the change in the value calculated by dividing the fluorescence intensity values of the control line C and the inspection line T by dividing the fluorescence intensity values of the control line C.
  • the detection limit for H1N1 influenza virus of the influenza virus detection kit including the antibody complex against the quantum dot-latex bead-influenza virus nucleoprotein having a luminescence wavelength of 580 nm was about 9.3 ⁇ 10 2 PFU / ml. Able to know.
  • the detection limit for the H1N1 influenza virus of the influenza virus detection kit containing the antibody complex against the quantum dot-latex bead-influenza virus nucleoprotein having a luminescence wavelength of 620 nm indicates about 5.1 ⁇ 10 2 PFU / ml. Able to know.
  • the detection limit for the H1N1 influenza virus of the influenza virus detection kit containing the antibody complex against the quantum dot-latex bead-influenza virus nucleoprotein having a luminescence wavelength of 640 nm indicates about 5.2 ⁇ 10 2 PFU / ml. Able to know.
  • FIG. 7 shows that the emission wavelength of the influenza virus detection kit including the antibody complex against the 520 nm quantum dot-latex bead-influenza virus nucleoprotein is detected on the UV light, and the emission wavelength is 520 nm. It can be seen that the H1N1 influenza virus detection limit of the influenza virus detection kit including the antibody complex against quantum dot-latex bead-influenza virus nucleoprotein represents about 5.0 ⁇ 10 4 PFU / ml.
  • H5N3 type influenza virus was prepared as a sample.
  • the amount of H5N3 influenza virus contained in the sample was 1.0 ⁇ 10 0 PFU / mL, 2.5 ⁇ 10 0 PFU / mL, 5.0 ⁇ 10 0 PFU / mL, 1.0 ⁇ 10 1 PFU / mL, or 5.0 ⁇ 10 1 PFU / mL Set to be Except for this, the detection method of the influenza virus detection kit was measured in the same manner as in Example (1). At this time, the control was used that did not process the virus.
  • the detection kit was excited at a wavelength of 375 nm, and the fluorescence intensities of the test and control lines were measured with a fluorescence reader (aGcare TRF, Medisensor).
  • the emission wavelength of the composition for detecting influenza virus was 520 nm, 580 nm, 620 nm or 640 nm.
  • Figure 8 shows the change in fluorescence intensity according to the H5N3 type influenza virus content using an influenza virus detection kit comprising an antibody complex for a quantum dot-latex bead-influenza virus nucleoprotein having a light emission wavelength of 580 nm, 620 nm or 640 nm
  • an influenza virus detection kit comprising an antibody complex for a quantum dot-latex bead-influenza virus nucleoprotein having a light emission wavelength of 580 nm, 620 nm or 640 nm
  • the change in the value calculated by dividing the fluorescence intensity values of the control line C and the inspection line T by dividing the fluorescence intensity values of the control line C is shown in detail.
  • the detection limit of H5N3-type influenza virus using an influenza virus detection kit comprising an antibody complex against a quantum dot-latex bead-influenza virus nucleoprotein having a luminescence wavelength of 620 nm indicates that the detection limit of H5N3 influenza virus is about 2.4 ⁇ 10 0 PFU / ml. Able to know.
  • H5N3-type influenza virus detection limit using an influenza virus detection kit comprising an antibody complex against a quantum dot-latex bead-influenza virus nucleoprotein having a luminescence wavelength of 640 nm indicates that the limit of detection of H5N3 influenza virus is about 4.4 ⁇ 10 0 PFU / ml. have.
  • H5N3 influenza virus on UV using an influenza virus detection kit including an antibody complex against a quantum dot-latex bead-influenza virus nucleoprotein having a light emission wavelength of 520 nm. It can be seen that the detection limit for H5N3 type influenza virus of the influenza virus detection kit including the antibody complex against phosphorus quantum dot-latex bead-influenza virus nucleoprotein is about 5.0 ⁇ 10 2 PFU / ml.
  • H7N7 influenza virus was prepared as a sample.
  • the amount of H7N7 influenza virus contained in the sample was 2.5 ⁇ 10 ⁇ 1 PFU / mL, 5.0 ⁇ 10 ⁇ 1 PFU / mL, 1.0 ⁇ 10 0 PFU / mL, 5.0 ⁇ 10 1 PFU / mL, or 1.0 ⁇ 10 1 PFU. Set to / ml. Except for this, the detection method of the influenza virus detection kit was measured in the same manner as in Example (1). At this time, the control was used that did not process the virus.
  • the detection kit was excited at a wavelength of 375 nm, and the fluorescence intensities of the test and control lines were measured with a fluorescence reader (aGcare TRF, Medisensor).
  • the emission wavelength of the composition for detecting influenza virus was 520 nm, 580 nm, 620 nm or 640 nm.
  • Figure 10 shows the change in fluorescence intensity according to the H7N7 type influenza virus content using an influenza virus detection kit comprising an antibody complex for a quantum dot-latex bead-influenza virus nucleoprotein having a light emission wavelength of 580 nm, 620 nm or 640 nm
  • an influenza virus detection kit comprising an antibody complex for a quantum dot-latex bead-influenza virus nucleoprotein having a light emission wavelength of 580 nm, 620 nm or 640 nm
  • the change in the value calculated by dividing the fluorescence intensity values of the control line C and the inspection line T by dividing the fluorescence intensity values of the control line C is shown in detail.
  • the H7N7 influenza virus detection limit using an influenza virus detection kit comprising an antibody complex against a quantum dot-latex bead-influenza virus nucleoprotein having a light emission wavelength of 580 nm indicates that the detection limit is about 6.0 ⁇ 10 ⁇ 1 PFU / ml. Able to know.
  • the H7N7 influenza virus detection limit using an influenza virus detection kit comprising an antibody complex against a quantum dot-latex bead-influenza virus nucleoprotein having a luminescence wavelength of 620 nm indicates that the detection limit is about 5.0 ⁇ 10 ⁇ 1 PFU / ml. Able to know.
  • the H7N7 type influenza virus detection limit using an influenza virus detection kit comprising an antibody complex against a quantum dot-latex bead-influenza virus nucleoprotein having a luminescence wavelength of 640 nm indicates about 7.0 ⁇ 10 ⁇ 1 PFU / ml. Able to know.
  • Figure 11 shows the detection of H7N7 influenza virus on the UV using an influenza virus detection kit comprising a light-emitting wavelength of the antibody complex for the 520nm quantum dot-latex bead-influenza virus nucleoprotein, through the H7N7 influenza virus It can be seen that the detection limit is about 5.0 ⁇ 10 1 PFU / ml.
  • H9N2 influenza virus was prepared as a sample.
  • the amount of H9N2 influenza virus contained in the sample was 2.5 ⁇ 10 ⁇ 2 PFU / mL, 5.0 ⁇ 10 ⁇ 2 PFU / mL, 1.0 ⁇ 10 ⁇ 1 PFU / mL, 5.0 ⁇ 10 ⁇ 1 PFU / mL, or 1.0 ⁇ 10.
  • the detection method of the influenza virus detection kit was measured in the same manner as in Example (1). At this time, the control was used that did not process the virus.
  • the detection kit was excited at a wavelength of 375 nm, and the fluorescence intensities of the test and control lines were measured with a fluorescence reader (aGcare TRF, Medisensor).
  • the emission wavelength of the composition for detecting influenza virus was 520 nm, 580 nm, 620 nm or 640 nm.
  • Figure 12 shows the change in fluorescence intensity according to H9N2 type influenza virus content using an influenza virus detection kit comprising an antibody complex for the quantum dot-latex bead-influenza virus nucleoprotein with a luminescence wavelength of 580 nm, 620 nm or 640 nm
  • an influenza virus detection kit comprising an antibody complex for the quantum dot-latex bead-influenza virus nucleoprotein with a luminescence wavelength of 580 nm, 620 nm or 640 nm
  • the change in the value calculated by dividing the fluorescence intensity values of the control line C and the inspection line T by dividing the fluorescence intensity values of the control line C is shown in detail.
  • the detection limit of H9N2-type influenza virus using an influenza virus detection kit containing an antibody complex against a quantum dot-latex bead-influenza virus nucleoprotein having a luminescence wavelength of 580 nm was about 2.6 x 10 -1 PFU / ml. It can be seen that.
  • the H9N2-type influenza virus detection limit was about 1.0 ⁇ 10 ⁇ 1 PFU / mL using an influenza virus detection kit containing an antibody complex against a quantum dot-latex bead-influenza virus nucleoprotein having a luminescence wavelength of 620 nm. It can be seen.
  • the H9N2-type influenza virus detection limit was about 6.0 ⁇ 10 ⁇ 2 PFU / ml using an influenza virus detection kit containing an antibody complex against a quantum dot-latex bead-influenza virus nucleoprotein having a luminescence wavelength of 640 nm. It can be seen.
  • FIG. 13 shows the detection of H9N2 influenza virus on UV using an influenza virus detection kit comprising an antibody complex for 520 nm quantum dot-latex bead-influenza virus nucleoprotein, wherein the emission wavelength is 520 nm. It can be seen that the UV phase detection limit of an influenza virus detection kit comprising an antibody complex against phosphorus quantum dot-latex bead-influenza virus nucleoprotein is about 1.0 ⁇ 10 1 PFU / ml.
  • H5N1 influenza virus HA antigen was prepared as a sample.
  • the HA antigen content of the H5N1 influenza virus included in the sample was 1.25 ⁇ 10 ⁇ 2 ⁇ g / ml, 2.5 ⁇ 10 ⁇ 2 ⁇ g / ml, 5.0 ⁇ 10 ⁇ 2 ⁇ g / ml, 5.0 ⁇ 10 ⁇ 1 ⁇ g / ml or It was set to be 2.0 ⁇ 10 2 ⁇ g / ml. Except for this, the detection method of the influenza virus detection kit was measured in the same manner as in Example (1). In this case, the control group was used without any antigen.
  • FIG. 14 shows H5N1 influenza virus hemagglutinin using an influenza virus detection kit comprising an antibody complex for quantum dot-latex bead-H5N1 influenza virus hemagglutinin (HA) using a quantum dot having a luminescence wavelength of 620 nm.
  • a graph showing a change in fluorescence intensity according to the (HA) content, and specifically, a change in the value calculated by dividing the fluorescence intensity values of the control line (C) and the inspection line (T) by dividing the fluorescence intensity values of the control line (C). .
  • the detection limit of the influenza virus detection kit including the H5N1 influenza virus HA antigen detection composition is about 5.0 ⁇ 10 ⁇ 2 ⁇ g / ml.
  • Example 4 Checking the Sensitivity and Specificity of an Influenza Virus Detection Kit Comprising a Composition for Influenza Virus Detection
  • Thickening specimens of humans infected with H1N1 type in influenza virus were applied to the fluorescent H1N1 type influenza virus detection kit prepared in Example 2, and the detection reaction was performed, and the detection sensitivity was confirmed.
  • a kit for influenza virus detection comprising a composition for detecting H1N1 influenza virus using the method of Example 3, except that a thickened specimen of a patient infected with H1N1 influenza virus was used in place of the H1N1 influenza virus.
  • the fluorescence intensity emitted from the control line (C) and the inspection line (T) was measured. At this time, the control group was used as a thickening specimen of a normal person.
  • the kit for detecting H1N1 influenza virus HA antigen was positive for 9 samples for 9 samples that were determined to be positive for H1N1 virus infection by RT-PCR (FIG. 15).
  • Sensitivity (%) (number of positive samples / number of positive samples used in testing) ⁇ 100
  • the kit for detecting H1N1 influenza virus HA antigen was negative for 16 samples determined as negative for H1N1 virus infection via RT-PCR (FIG. 15).
  • Equation (2) Specificity (%) (Number of samples of negative results / number of negative samples used in testing) ⁇ 100

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Abstract

La présente invention concerne : une composition pour détecter des virus de la grippe, contenant, en tant que substance active, un complexe point quantique-bille de latex-anticorps contre le virus de la grippe comportant un point quantique revêtu de composé hydrophile couplé à une vie de latex, et comportant un anticorps contre le virus de la grippe couplé à la surface de la bille de latex où le point quantique n'est pas couplé; une trousse pour détecter des virus de la grippe, comprenant celle-ci; et un procédé de détection de virus de la grippe. Le point quantique est constitué de nanoparticules comprenant un matériau semi-conducteur, les nanoparticules de point quantique émettent une lumière à une longueur d'onde spécifique en fonction de la taille, et différents signaux peuvent être acquis simultanément sans le chevauchement de signaux en ayant une largeur à mi-hauteur plus étroite que celle d'un matériau fluorescent conventionnel. En particulier, selon la présente invention, le complexe point quantique-bille de latex-anticorps contre le virus de la grippe peut être séché de façon à être applicable à une trousse, de façon à être pratique, et très utile pour une utilisation à long terme.
PCT/KR2017/001811 2016-03-29 2017-02-17 Trousse de détection de virus de la grippe au moyen d'un complexe point quantique-bille de latex-anticorps contre le virus de la grippe, et procédé de détection utilisant celle-ci WO2017171237A1 (fr)

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