Composition for detecting virus comprising gold nanoparticles bound with capture antibody or detection antibody as active ingredients

Abstract

The present invention relates to a composition for detecting viruses comprising gold nanoparticles conjugated with a capture antibody or a detection antibody as an active ingredient. By confirming that a mixture of gold nanoparticles conjugated with a capture antibody and gold nanoparticles conjugated with a detection antibody effectively detects viral antigens and that a diagnostic kit prepared from said mixture is more effective in diagnosing viral infectious diseases than a conventionally used diagnostic kit, the composition can be usefully utilized as a virus detection composition or a diagnostic kit for viral infectious diseases.

Description

Technology Field

[0001] The present invention relates to a composition for detecting viruses comprising gold nanoparticles combined with a capture antibody or a detection antibody as an active ingredient. Background Technology

[0002] Diseases caused by viral infections, including COVID-19, have been on the rise recently. Among viral diseases, influenza, commonly known as the "flu," is an acute respiratory illness caused by the influenza virus. Influenza causes outbreaks of varying severity worldwide, and it is a highly contagious disease, typically infecting 10 to 20 percent of the population within two to three weeks once an outbreak begins.

[0003] When infected with the influenza virus, healthy individuals recover after experiencing symptoms such as chills, fever, headache, and cough for several days; however, those with chronic lung disease, heart disease, or weakened immune systems may develop complications such as pneumonia, which can lead to death. Every year, 5–10% of the adult population and 20–30% of the pediatric population are infected with the influenza virus, reaching up to 1 billion people, of whom 150,000 to 500,000 die. Consequently, research is currently underway to develop technologies capable of detecting the virus. Prior art literature

[0004] 1. Republic of Korea Published Patent KR 10-2021-0020518 (Published Feb. 24, 2021) The problem to be solved

[0005] The objective of the present invention is to provide a composition for detecting viruses.

[0006] Another objective of the present invention is to provide a kit for diagnosing viral infectious diseases comprising the above-mentioned composition for virus detection.

[0007] Another objective of the present invention is to provide a method for providing information for diagnosing a viral infectious disease, comprising: a composition for detecting a virus; and a step of measuring the antigen-antibody reaction of an antigen. means of solving the problem

[0008] To achieve the above objective, the present invention provides a composition for detecting viruses comprising, as active ingredients, gold nanoparticles bound to a capture antibody; and gold nanoparticles bound to a detection antibody.

[0009] In addition, the present invention provides a kit for diagnosing viral infectious diseases comprising the above-mentioned composition for virus detection.

[0010] In addition, the present invention provides a method for providing information for diagnosing a viral infectious disease, comprising: a mixture of gold nanoparticles conjugated with a capture antibody and gold nanoparticles conjugated with a detection antibody; and a step of measuring the antigen-antibody reaction of an antigen. Effects of the invention

[0011] According to the present invention, a mixture comprising gold nanoparticles bound to a capture antibody and gold nanoparticles bound to a detection antibody effectively detects a viral antigen, and by confirming that a diagnostic kit prepared with said mixture is more effective in diagnosing viral infectious diseases than a conventionally used diagnostic kit, it can be usefully utilized as a composition for detecting viruses or as a kit for diagnosing viral infectious diseases. Brief explanation of the drawing

[0012] Figure 1 shows the results of analyzing the hydrodynamic diameter and absorbance of a gold nanoparticle (hereinafter referred to as GNP)-antibody conjugate (hereinafter referred to as GNP-Ab) mixture (prepared in Experimental Example 1-1). Figure 2 shows the results of analyzing the antibody detection effect of the GNP-Ab mixture (prepared in Experimental Example 1-1). Figures 3 and 4 show the results of analyzing hydrodynamic diameter and absorbance according to the weight ratio of the GNP-H1N1 influenza virus capture antibody conjugate (hereinafter referred to as GNP-A ab) and the GNP-H1N1 influenza virus detection antibody conjugate (hereinafter referred to as GNP-B ab) of the GNP-Ab mixture (prepared in Experimental Example 1-1). Specifically, Figure 3 shows the analysis results for the experimental group without antigen; and Figure 4 shows the analysis results for the experimental group with antigen. Figure 5 shows the results of analyzing the antigen detection effect according to the weight ratio of GNP-A ab and GNP-B ab in the GNP-Ab mixture (prepared in Experimental Example 1-1). Figure 6 shows the results of analyzing the stability of the GNP-Ab mixture (prepared in Experimental Example 1-1) against antibodies. Figure 7 shows the results of analyzing the H1N1 antigen-specific detection effect of the GNP-Ab mixture (prepared in Experimental Example 1-1). GNP-H1N1 Target Ab; GNP-antibody conjugate mixture (GNP-A Ab : GNP-B Ab = 1 : 1) Figure 8 shows the results of comparing and analyzing the antigen detection effects of a diagnostic kit (manufactured in Experimental Example 1-2) and a conventionally used diagnostic kit. Specific details for implementing the invention

[0013] The present invention will be described in more detail below.

[0015] The present invention provides a composition for detecting viruses comprising, as active ingredients, gold nanoparticles bound to a capture antibody; and gold nanoparticles bound to a detection antibody.

[0016] The diameter of the gold nanoparticles combined with the capture antibody or the gold nanoparticles combined with the detection antibody may be 50 to 70 nm.

[0017] The above-mentioned capture antibody and detection antibody may be antibodies to influenza A virus or coronavirus, but are not limited thereto.

[0018] The above influenza A virus antibody may be one or more selected from the group consisting of H1N1 virus antibody, H2N2 virus antibody, H3N2 virus antibody, H5N1 virus antibody, H7N9 virus antibody, H7N7 virus antibody, H1N2 virus antibody, H9N2 virus antibody, H7N2 virus antibody, H7N3 virus antibody, H5N2 virus antibody, and H10N7 virus antibody, but is not limited thereto.

[0019] The above coronavirus antibodies may be one or more selected from the group consisting of coronavirus 229E antibodies, coronavirus NL63 antibodies, coronavirus OC43 antibodies, coronavirus HKU1 antibodies, MERS-CoV (Middle East respiratory syndrome coronavirus) antibodies, SARS-CoV (Severe acute respiratory syndrome coronavirus) antibodies and SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) antibodies, but are not limited thereto.

[0021] In addition, the present invention provides a kit for diagnosing viral infectious diseases comprising the above-mentioned composition for virus detection.

[0022] The above virus may be an influenza A virus or a coronavirus.

[0023] The above viral infectious diseases may be one or more selected from the group consisting of influenza, the common cold, pharyngitis, bronchitis, pneumonia, high fever, cough, shortness of breath, diarrhea, renal failure, renal dysfunction, and septic shock, but are not limited thereto.

[0024] In this specification, “diagnosis” refers to identifying the existence or characteristics of a pathological condition, and for the purposes of the present invention, means identifying a virus, determining the susceptibility of a subject to a virus or at least one of its symptoms, and therametrics (e.g., monitoring the condition of a subject to provide information on therapeutic efficacy). It also includes the primary diagnosis of a clinical condition or the diagnosis of a relapsed disease.

[0025] The above kit can be used to diagnose the presence of a virus by measuring the expression level of the protein or the gene encoding it in a sample isolated from an individual suspected of having a virus.

[0026] In addition, the kit may include not only a preparation for measuring the expression level of the protein or the gene encoding it, but also one or more other component compositions, solutions, or devices suitable for the analysis method.

[0027] For example, the kit according to the present invention may be a kit comprising genomic DNA derived from a sample to be analyzed for performing PCR, a primer set specific to the marker gene of the present invention, an appropriate amount of DNA polymerase, a dNTP mixture, a PCR buffer solution, and water. The PCR buffer solution may include KCl, Tris-HCl, and MgCl2. In addition, components necessary for performing electrophoresis to verify whether the PCR product has been amplified may be additionally included in the kit of the present invention.

[0028] In addition, the kit according to the present invention may be a kit containing essential elements necessary for performing RT-PCR. In addition to each primer pair specific to a marker gene, the RT-PCR kit may include a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC-water, sterile water, etc. Additionally, it may include a primer pair specific to a gene used as a quantitative control.

[0030] In addition, the present invention provides a method for providing information for diagnosing a viral infectious disease, comprising: a mixture of gold nanoparticles conjugated with a capture antibody and gold nanoparticles conjugated with a detection antibody; and a step of measuring the antigen-antibody reaction of an antigen.

[0031] The above antigen and virus may be influenza A virus or coronavirus.

[0032] The above viral infectious diseases may be one or more selected from the group consisting of influenza, the common cold, pharyngitis, bronchitis, pneumonia, high fever, cough, shortness of breath, diarrhea, renal failure, renal dysfunction, and septic shock, but are not limited thereto.

[0034] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0036] [ Experimental Example 1] Diagnosis kit manufacturing

[0037] 1-1. GNP- Ab Preparation of mixtures

[0038] 60 μL of 1% potassium carbonate (K2CO3) and H1N1 influenza virus antibody [capture antibody (Influenza A H1N1 Polyclonal Antibody, purchased from Invitrogen™, product number: PA1-7221) or detection antibody] (Anti-Influenza A Virus Nucleoprotein antibody, purchased from Abcam, product number: ab20343) were added to 3 mL of GNP (gold nanoparticle) (absorbance=1) and stirred for 30 minutes at 25°C and 500 rpm. Then, 300 μL of 10% BSA (Bovine Serum Albumin) was added and stirred for an additional 15 minutes at 25°C and 500 rpm, followed by centrifugation for 20 minutes at 25°C and 8000 rpm to remove the supernatant. After removing the supernatant, 3 mL of 1% BSA (in 20 mM boric acid) was added, and the following two types of GNP-Ab prepared through the above process were mixed in a 1:1 weight ratio to prepare a mixture (total volume 6 mL).

[0039] 1) GNP-A ab (GNP-H1N1 influenza virus capture antibody conjugate)

[0040] 2) GNP-B ab (GNP-H1N1 influenza virus detection antibody conjugate)

[0041] Afterwards, 100 μL of the above GNP-Ab mixture, 50 μL of antigen (H1N1 influenza virus), and 50 μL of Lysis buffer were mixed and incubated at room temperature for 15 minutes to induce an antibody-antigen reaction. In the following examples, for the experimental group without antigen, the antigen and Lysis buffer were not used, and 100 μL of the above GNP-antibody conjugate mixture was incubated at room temperature for 15 minutes.

[0043] 1-2. Diagnosis kit manufacturing

[0044] A diagnostic kit was prepared using the gold nanoparticle-antibody conjugate mixture prepared in Experimental Example 1-1 above. A nitrocellulose membrane was aligned and attached to a backing pad, a conjugate pad was attached, a sample pad was attached so as to overlap the conjugate pad by 1 to 2 mm, and a wick pad was attached.

[0046] [ Examples 1] GNP- Ab Analysis of mixture characteristics

[0047] 1-1. Characterization Analysis

[0048] To confirm the characteristics of the above GNP-Ab mixture (GNP-A ab : GNP-B ab = 1 : 1), Dynamic Light Scattering (DLS) analysis (fluid-based diameter measurement) and absorbance analysis were performed. For comparison, the experimental groups were set as follows. In the experimental groups below, the antigen represents the H1N1 influenza virus.

[0049] 1) GNP-A ab(100μL)

[0050] 2) GNP-B ab(100μL)

[0051] 3) GNP-Ab mixture (GNP-A ab + GNP-B ab) (100 μL)

[0052] 4) GNP-Ab mixture (GNP-A ab + GNP-B ab) (100μL) + antigen (50μL) + lysis buffer (50μL)

[0053] As a result, as shown in Figure 1, the hydrodynamic diameter of GNP-Ab was found to be 55–70 nm. While there was no significant difference between the antigen-free experimental groups, the hydrodynamic diameter increased in the antigen-containing experimental groups compared to the antigen-free experimental groups. Additionally, the absorbance in all experimental groups was found to be around 525 nm, which is the intrinsic absorption wavelength of GNP. From the above results, it was confirmed that the GNP-Ab conjugate mixture is materially stable and that the antigen-antibody reaction (aggregation reaction) occurs well.

[0054] In addition, as a result of analyzing the antigen detection effect of the kit manufactured using the above GNP-Ab, as shown in Figure 2, no antigen-antibody reaction occurred when the antigen was not present in the kit, and when the antigen was reacted in the kit for 15 minutes, a positive reaction (antigen-antibody reaction) occurred in the kit. From the above results, it was confirmed that the above GNP-Ab mixture is effective for detecting H1N1 antigen.

[0056] 1-2. Analysis of Characteristics Based on GNP-A ab and GNP-B ab Weight Ratios

[0057] In Example 1-1 above, the characteristics of the GNP-Ab mixture (GNP-A ab : GNP-B ab = 1 : 1) were confirmed. To confirm the characteristics according to the weight ratio of GNP-A ab and GNP-B ab, the GNP-Ab mixture was prepared in the same manner as in Experimental Example 1-1 above, but with different weight ratios of GNP-A ab and GNP-B ab. The experimental groups were set as follows (the total volume of all experimental groups is 1 mL). In the experimental groups below, the volume of the GNP-Ab mixture is 100 μL for all, the antigen is H1N1 influenza virus for all, and the weight ratio is GNP-A ab : GNP-B ab.

[0058] 1) GNP-Ab mixture (10:0)

[0059] 2) GNP-Ab mixture (7:3)

[0060] 3) GNP-Ab mixture (5:5)

[0061] 4) GNP-Ab mixture (3:7)

[0062] 5) GNP-Ab mixture (0:10)

[0063] 6) GNP-Ab mixture (10:0) + antigen (50μL) + lysis buffer (50μL)

[0064] 7) GNP-Ab mixture (7:3) + antigen (50μL) + lysis buffer (50μL)

[0065] 8) GNP-Ab mixture (5:5) + antigen (50μL) + lysis buffer (50μL)

[0066] 9) GNP-Ab mixture (3:7) + antigen (50μL) + lysis buffer (50μL)

[0067] 10) GNP-Ab mixture (0:10) + antigen (50μL) + lysis buffer (50μL)

[0068] As a result, as shown in Fig. 3, the hydrodynamic diameter of the antigen-free experimental group was 55–70 nm, and the absorbance was 526–527 nm. Additionally, as shown in Fig. 4, the hydrodynamic diameter of the antigen-containing experimental group was 65–125 nm, and the absorbance was 526–529 nm. When comparing the characteristics based on the presence or absence of antigen, excluding the experimental group using the GNP-Ab mixture (0:10), the hydrodynamic diameter increased in the antigen-containing experimental group compared to the antigen-free experimental group, while no significant difference was observed in absorbance. Furthermore, the highest hydrodynamic diameter was observed in the GNP-Ab mixture (GNP-A ab : GNP-B ab = 5 : 5). From the above results, it was confirmed that the GNP-Ab mixture is materially stable, the antigen-antibody reaction (=aggregation reaction) occurs well, the antigen-antibody reaction does not occur well when only the detection antibody is present, and the antigen-antibody reaction occurs best when the weight ratio of GNP-A ab and GNP-B ab is 5:5 (1:1), making it most effective for detecting H1N1 antigen.

[0069] In addition, as a result of analyzing the antigen detection effect of the kit manufactured using the above GNP-Ab, as shown in Figure 5, no antigen-antibody reaction occurred when the antigen was not present in the kit, and a positive reaction was observed in the kit when the antigen was reacted with the kit for 15 minutes. From the above results, it was confirmed that the above GNP-Ab mixture is effective for detecting H1N1 antigen.

[0071] [ Examples 2] Stability analysis of antibodies

[0072] To confirm the stability of the GNP-Ab mixture (GNP-A ab : GNP-B ab = 1 : 1) against antibodies, the color change of the mixture was observed visually. The experimental group was set as follows.

[0073] 1) GNP(1mL)

[0074] 2) GNP-A ab(1mL)

[0075] 3) GNP-B ab(1mL)

[0076] 4) GNP-Ab mixture (GNP-A ab + GNP-B ab) (1 mL)

[0077] As a result, as shown in Figure 6, no significant color change was observed between the experimental groups. From the above results, it was confirmed that the GNP-Ab mixture exhibits stability (particle stability) regarding antibody attachment.

[0079] [ Examples 3] Analysis of Antigen-Specific Detection Effects

[0080] To confirm whether the above GNP-Ab mixture (GNP-A ab : GNP-B ab = 1 : 1) specifically detects only H1N1 antigen, H1N1 and its subtypes (H3N2 and H5N1) were added to the mixture, and the absorbance was analyzed. After manufacturing a kit using the mixture, the antigen detection effect was analyzed. The experimental group was set as follows.

[0081] 1) GNP-Ab mixture (100μL) + Lysis buffer (100μL)

[0082] 2) GNP-Ab mixture (100μL) + H1N1 antigen (50μL) + Lysis buffer (50μL)

[0083] 3) GNP-Ab mixture (100μL) + H3N2 antigen (50μL) + Lysis buffer (50μL)

[0084] 4) GNP-Ab mixture (100μL) + H5N1 antigen (50μL) + Lysis buffer (50μL)

[0085] As a result, as shown in Fig. 7, when the above mixture was treated with the H1N1 antigen, a positive reaction was observed in the kit, whereas all other experimental groups showed negative reactions. From the above results, it was confirmed that the GNP-Ab mixture exhibits a specific detection effect against the H1N1 antigen.

[0087] [ Examples 5] Existing Diagnosis kit Comparative analysis of differences in detection effects

[0088] In order to compare the antigen detection effect of a kit prepared using the above GNP-Ab mixture with that of a conventionally used diagnostic kit (Median diagnostic kit), H1N1 antigen was applied to the two types of kits at different concentrations, and the antigen detection effect was analyzed.

[0089] As a result, as shown in FIG. 8, the kit of the present invention is 2×10 -7 (2×10 -0.25 TCID 50 While a significant color change is observed up to ( / mL), existing diagnostic kits show 2×10 -4 (2×10 -0.25 TCID 50 Significant color change was observed up to / mL, showing a sensitivity difference of approximately 1000 times between the two kits. From the above results, it was confirmed that the kit prepared with the GNP-Ab mixture of the present invention has a superior H1N1 antigen detection effect compared to existing diagnostic kits.

[0091] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A composition for detecting viruses comprising, as active ingredients, gold nanoparticles bound to a capture antibody; and gold nanoparticles bound to a detection antibody. Claim 2 A composition according to claim 1, characterized in that the diameter of the gold nanoparticles bound to the capture antibody or the gold nanoparticles bound to the detection antibody is 50 to 70 nm. Claim 3 A composition according to claim 1, characterized in that the capture antibody and the detection antibody are antibodies to influenza A virus or coronavirus. Claim 4 A composition according to claim 3, wherein the influenza A virus antibody is one or more selected from the group consisting of H1N1 virus antibody, H2N2 virus antibody, H3N2 virus antibody, H5N1 virus antibody, H7N9 virus antibody, H7N7 virus antibody, H1N2 virus antibody, H9N2 virus antibody, H7N2 virus antibody, H7N3 virus antibody, H5N2 virus antibody and H10N7 virus antibody. Claim 5 A composition according to claim 3, wherein the coronavirus antibody is one or more selected from the group consisting of coronavirus 229E antibody, coronavirus NL63 antibody, coronavirus OC43 antibody, coronavirus HKU1 antibody, MERS-CoV (Middle East respiratory syndrome coronavirus) antibody, SARS-CoV (Severe acute respiratory syndrome coronavirus) antibody, and SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) antibody. Claim 6 A kit for diagnosing viral infectious diseases comprising the composition for detecting the virus of claim 1. Claim 7 A kit according to claim 6, characterized in that the virus is an influenza A virus or a coronavirus. Claim 8 A kit according to claim 6, characterized in that the viral infectious disease is one or more selected from the group consisting of influenza, the common cold, pharyngitis, bronchitis, pneumonia, high fever, cough, shortness of breath, diarrhea, renal failure, renal dysfunction, and septic shock. Claim 9 A method for providing information for the diagnosis of a viral infectious disease, comprising: a mixture of gold nanoparticles bound to a capture antibody and gold nanoparticles bound to a detection antibody; and a step of measuring the antigen-antibody reaction of an antigen. Claim 10 A method for providing information according to claim 9, characterized in that the antigen and virus are influenza A virus or coronavirus. Claim 11 A method of providing information according to claim 9, wherein the viral infectious disease is one or more selected from the group consisting of influenza, the common cold, pharyngitis, bronchitis, pneumonia, high fever, cough, shortness of breath, diarrhea, renal failure, renal dysfunction, and septic shock.

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