WO2017171237A1 - Kit for detecting influenza viruses by using quantum dot-latex bead-influenza virus antibody complex, and detection method using same - Google Patents

Abstract

The present invention relates to: a composition for detecting influenza viruses, containing, as an active ingredient, a quantum dot-latex bead-influenza virus antibody complex having a hydrophilic compound-coated quantum dot coupled to a latex bead, and having an influenza virus antibody coupled to the surface of the latex bead where the quantum dot is not coupled; a kit for detecting influenza viruses, comprising the composition; and a method for detecting influenza viruses. The quantum dot is nanoparticles comprising a semiconductor material, the quantum dot nanoparticles emit light at a specific wavelength according to size, and various signals can be simultaneously acquired without the overlapping of signals by having a half-width narrower than that of a conventional fluorescent material. Particularly, according to the present invention, the quantum dot-latex bead-influenza virus antibody complex can be dried so as to be applicable to a kit, thereby being convenient, and very useful for long-term use.

Description

Kit for detecting influenza virus using quantum dot-latex bead-influenza virus antibody complex and detection method using same

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.

Currently, diagnostic tests for influenza infection are mainly based on virus detection and the patient's immune response to the virus. Accordingly, there are four types of diagnostic tests: influenza virus isolation (culture), detection of viral antigens (fast antigen test, immunofluorescence method), viral nucleic acid verification (RT-PCR), and 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.

Immunological methods using antibodies can diagnose diseases with high accuracy. Enzymes were used as markers at the time of the first development of immunodiagnostic methods, but organic fluorescent materials are now commonly used with the development of subsequent technologies. However, the organic fluorescent material is not only very sensitive to the pretreatment conditions of the sample, but also has a problem in that the sensitivity of the sensor is lowered because the brightness of the phosphor rapidly decreases due to photobleaching.

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.

As a technique using quantum dots, Korean Patent No. 1512484 discloses a method for rapidly detecting norovirus in food using magnetic beads and quantum dots, and 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.

In order to achieve the above object, 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.

In addition, the present invention comprises the steps of (a) coating the surface of the quantum dots (QDs) with a hydrophilic compound;

(b) binding the hydrophilic compound-coated quantum dots and latex beads through an amide bond reaction;

(c) replacing the surface of the latex bead with a carboxyl group by reacting an amino group and a succinic anhydride present on the surface of the latex beads to which the quantum dots are bound; And

(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.

In addition, 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 When a light band appears, 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.

2 is a fluorescence detection result according to the molar ratio mixing ratio of the quantum dots and latex beads coated with a hydrophilic 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.

4 shows (A) quantum dots (QD620nm), (B) latex beads, (C) quantum dots (QD520nm) -latex bead-influenza virus antibody complex, (D) quantum dots (QD580nm) -latex bead-influenza virus antibody complex, ( E) quantum dots (QD620nm) -latex bead-influenza virus antibody complex and (F) quantum dots (QD640nm) -latex bead-influenza virus antibody complex.

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. As 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. As 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.

9 is a result of confirming the limit of H5N3 influenza virus detection using an antibody complex against influenza virus nucleoprotein of quantum dot-latex bead-H1N1, H5N3, H7N7 or H9N2 having a light emission wavelength of 520 nm.

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. As 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. As a graph showing the change in the value calculated by dividing the value of the fluorescence intensity according to the H9N2 influenza virus content by the value of the fluorescence intensity measured at the control line.

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.

14 is a result of confirming the limit of the detection of H5N1 influenza virus HA antigen using the antibody complex against the quantum dot-latex bead-H5N1 influenza virus HA antigen having a light emission wavelength of 620nm, fluorescence according to the H5N1 influenza virus HA antigen content It is a graph showing the change in the value calculated by dividing the value of the intensity by the value of the fluorescence intensity measured at the control line.

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.

When 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.

The 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 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.

The term 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. In addition, 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.

In the present invention, 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. However, there is a problem that such binding may not be performed smoothly depending on the structure of the antibody. In order to solve this problem, 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.

In addition, the present invention comprises the steps of (a) coating the surface of the quantum dots (QDs) with a hydrophilic compound;

(b) binding the hydrophilic compound-coated quantum dots and latex beads through an amide bond reaction;

(c) replacing the surface of the latex bead with a carboxyl group by reacting an amino group and a succinic anhydride present on the surface of the latex beads to which the quantum dots are bound; And

(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.

Wherein the 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. It is an immunostrip or fluorescent immunochromatographic test kit (FICT) which is provided with the test line which fixed the other antibody which can be fixed, and the control line which fixed the secondary antibody which can detect the antibody contained in the composition.

In addition, in the present invention, if a sample suspected of containing influenza virus is added to the influenza virus detection kit and a fluorescent band appears on the test line and the control line of the test strip, the influenza virus infection is determined to be positive, and only the control line has a fluorescent band. When is indicated, the method relates to an influenza virus detection method characterized in that negatively determined influenza virus infection.

In the present invention, 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).

In addition, 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.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for explaining the present invention in more detail, it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

Example 1 Preparation of Influenza Virus Detection Composition Comprising a Hydrophilic Compound-Coated Quantum Dots ( QDs ) -Latex Bead - Influenza Virus Antibody Complex

(1) coated with a hydrophilic compound Quantum dots  And latex Bidga Combined  Preparation of the 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).

Specifically, washing buffer was added to 50 μl of 0.044 μM 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 μM quantum dots coated with Mn were mixed at a molar ratio of 1:90, and 200 μl of 0.01 M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 0.01 M N-hydroxysulfosuccinic acid were added. 300 μl of mid (Sulfo-NHS) was added and reacted at room temperature for 1 hour (FIG. 1 (A)). After the reaction was completed, the reaction was centrifuged (17000rpm, 5 minutes) to remove the unbound quantum dots, the precipitate was washed once with the addition of the wash buffer solution, and then centrifuged again to wash the buffer 200 By dispersing in a μl to prepare a composite in which a quantum dot coated with a hydrophilic compound and latex beads.

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).

(2) Preparation of influenza virus detection through the combination of influenza virus antibody and the quantum dot - latex ratio complex through a linker

In the quantum dot-latex bead complex, 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.

Hemaggluti of H5N1 influenza virus or antibodies against nucleoprotein (NP) of influenza virus of influenza virus of type H1N1, H5N3, H7N7 or H9N2 in the quantum dot-latex bead complex of latex beads not bound Antibody (1 mg / ml) against nin (HA) was mixed in a molar ratio of 1: 455, 100 μl of 0.01 M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 0.01 M 150 μl of N-hydroxysulfosuccinimide (Sulfo-NHS) was added and reacted at room temperature for 2 hours. In order to eliminate the nonspecific reaction, 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 μl 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).

Example  2. Influenza Virus Detection Of kit  making

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. In order to evaluate the effect on the kit, an influenza virus detection kit including the influenza virus detection composition was prepared.

Specifically, 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. At a predetermined interval from the sample pad, 2 μl 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 μg 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.

After setting the control line (C) at regular intervals from the test line, 2 μg of a secondary antibody (an antibody against mouse IgG) to the antibody specifically binding to influenza virus was added thereto and fixed. .

As a result, 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

(1) Detection limit of influenza virus detection kit containing composition for detecting H1N1 influenza virus

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.

75 μl of a H1N1 influenza virus sample was added to a sample injecting unit of the influenza virus detection kit prepared in Example 2, followed by adding 75 μl of the sample dilution solution, reacting in a dark room for 5 minutes, and measuring the emitted fluorescence intensity. It was.

At this time, the amount of H1N1 influenza virus contained in the sample is 2.5 × 10 ² PFU / ㎖, 5.0 × 10 ² PFU / ㎖, 1.0 × 10 ³ PFU / ㎖, 1.0 × 10 ⁴ PFU / ㎖ or 5.0 × 10 ⁴ PFU / Ml was set, the control was used that was not treated virus.

After the reaction, 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.

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. In detail, 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.

Through this, 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.

In addition, 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.

In addition, 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.

(2) Limits of detection of influenza virus detection kits comprising a composition for detecting H5N3 influenza virus

Using an influenza virus detection kit prepared in Example 2, 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.

After the reaction, 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 As a graph, 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.

This shows that 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 580 nm indicates about 3.1 × 10 ⁰ PFU / ml. Can be.

In addition, 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.

In addition, the 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.

9 shows the detection of 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.

(3) Detection limit of influenza virus detection kit containing composition for detecting H7N7 influenza virus

Using a kit for influenza virus detection prepared in Example 2, 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.

After the reaction, 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 As a graph, 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.

In addition, 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.

In addition, 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.

(4) Limits of detection of influenza virus detection kits comprising a composition for detecting H9N2 influenza virus

Using a kit for influenza virus detection prepared in Example 2, 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. Set to ⁰ PFU / 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.

After the reaction, 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 As a graph, 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.

In addition, 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.

In addition, 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.

(5) Limits of detection of influenza virus HA antigen detection kits comprising a composition for detecting H5N1 influenza virus

Using an influenza virus detection kit prepared in Example 2, 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 μg / ml, 2.5 × 10 ⁻² μg / ml, 5.0 × 10 ⁻² μg / ml, 5.0 × 10 ⁻¹ μg / ml or It was set to be 2.0 × 10 ² μg / 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). . Through this, it can be seen that the detection limit of the influenza virus detection kit including the H5N1 influenza virus HA antigen detection composition is about 5.0 × 10 ⁻² μg / 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.

Sensitivity testing for clinical positive samples:

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).

From the following formula (1) it can be seen that the sensitivity of the kit of the present invention is 100%.

Equation (1) Sensitivity (%) = (number of positive samples / number of positive samples used in testing) × 100

Therefore, the sensitivity (%) of the kit of the present invention is the number of positive 9 samples / 9 positive samples x 100 = 100%.

Specificity tests for clinical negative samples:

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).

From the following formula (2), it can be seen that the specificity of the kit of the present invention is 100%.

Equation (2) Specificity (%) = (Number of samples of negative results / number of negative samples used in testing) × 100

Therefore, the specificity (%) of the kit of the present invention = negative 16 samples / 16 negative samples x 100 = 100%.

Claims (17)

1. A composition for detecting 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 having an influenza virus antibody coupled to the latex bead surface as an active ingredient.
2. The composition of claim 1, wherein the latex beads and the hydrophilic compound-coated quantum dots are combined in a molar ratio of 1:50 to 1,000.
3. The composition of claim 1, wherein the latex beads and the influenza virus antibody are combined at a molar ratio of 1:50 to 500.
4. According to claim 1, wherein the diameter of the latex beads is influenza virus detection composition, characterized in that 10 ~ 2,000nm.
5. The composition of claim 1, wherein the hydrophilic compound-coated quantum dot has a diameter of 1 to 30 nm and a light emission wavelength of 450 to 700 nm.
6. The composition of claim 2, wherein the binding of 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.
7. The influenza virus detection composition according to claim 3, wherein the latex bead and the influenza virus antibody are bound to an amide bond between a carboxyl group on the surface of the latex bead and an amino group included in the influenza virus antibody.
8. The method of claim 1, wherein the quantum dots are cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium tellurium (CdTe), zinc tellurium (ZnTe), zinc selenide (ZnSe), zinc sulfide (ZnS) , Zinc oxide (ZnO), indium phosphide (InP), indium arsenide (InAs), mercury telium (HgTe) and mercury selenide (HgSe), characterized in that the composition for detecting influenza virus.
9. The influenza virus detection composition according to claim 1, wherein the influenza virus antibody is an antibody that specifically recognizes H1N1 type, H5N3 type, H7N7 type, H9N2 type or H5N1 type influenza virus.
10. The method of claim 1, wherein the hydrophilic compound is any one selected from cysteamine, mercaptosuccinic acid, mercapatopropionic acid, glutathione, cysteine and thiol-containing silane. Influenza virus detection composition, characterized in that one.
11. (a) coating the surface of quantum dots (QDs) with a hydrophilic compound;

    (b) binding the hydrophilic compound-coated quantum dots and latex beads through an amide bond reaction;

    (c) replacing the surface of the latex bead with a carboxyl group by reacting an amino group and a succinic anhydride present on the surface of the latex beads to which the quantum dots are bound; And

    (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. Method for producing a composition for detecting influenza virus comprising a.
12. 12. The method of claim 11, wherein the amide coupling reaction of steps (b) and (d) comprises 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide ( Method of producing a composition for detecting influenza virus, characterized in that carried out in the presence of Sulfo-NHS).
13. Influenza virus detection kit comprising a composition for detecting influenza virus according to any one of claims 1 to 12.
14. The kit of claim 13, wherein the kit for influenza virus detection comprises: a sample injecting unit for injecting a sample; A combination of influenza virus antigens comprising a composition for detecting influenza viruses that binds to influenza virus antigens 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 in which anti-mouse IgG is immobilized sequentially. A kit for detecting influenza virus in the form of a test strip.
15. The kit for detecting influenza virus according to claim 13, wherein the binding unit of the influenza virus antigen comprises the composition for detecting influenza virus in an amount of 0.2 × 10 ⁻⁶ to 1.0 × 10 ⁻⁵ nmole.
16. The kit for detecting influenza virus according to claim 13, wherein the sample is thickening or feces of human or animal.
17. When a sample suspected of containing influenza virus is added to the kit for detecting influenza virus of claim 13 and a fluorescent band appears on the test line and the control line of the test strip, it is determined that the influenza virus infection is positive, and only the control line has a fluorescent band. If is shown, influenza virus infection is negatively determined.

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