Immobilized Fluorescence Counting of Viruses

Abstract

To provide a quantitative method for fluorescence-counting quantities of inactivated antigens (virus) or antibodies in a sample using surface plasmon excitation enhanced fluorescence spectroscopy (SPFS).SOLUTION: A method for quantifying a measurement object of virus or an antibody in a sample comprises: (1) a solid-phasing step of coagulating an inactivated virus or an antibody with a potential difference between plasmon metal complexes and a metal substrate on the metal substrate together with plasmon metal complex quantum crystals, fixing the virus or antibody on the metal substrate together with the metal complex quantum crystals, and solid-phasing the virus or the antibody; (2) a fluorescence-labeling step of fluorescence-labeling the virus or the antibody solid-phased with the antigen antibody reaction; (3) a fluorescence-exciting step of obtaining a point-like fluorescent image of the virus or the antibody fluorescence-labeled with the surface plasmon excitation by irradiating the fluorescence-labeled virus or the antibody with excitation light; and (4) a fluorescence-counting step of binarizing and count-quantifying the fluorescent point or a dot in the fluorescent image.SELECTED DRAWING: Figure 19A

Description

[Technical field]

[0001] The present invention relates to a novel fluorescence counting method in which viruses or their antibodies are immobilized by a quantum crystal aggregation method and quantified by counting fluorescent spots that appear on a fluorescent screen using a surface plasmon excitation method. [Background technology]

[0002] Currently, genetic testing using the PCR method is the mainstream for virus testing. Mucus or phlegm from the back of the nose or throat is collected and examined for antigens, viruses, and other proteins contained therein. This PCR method is a highly accurate method in which a sample is collected, the genes contained in the sample are amplified, and a test is performed to see if they match a specific gene sequence. However, this method requires advanced pre-processing techniques and sophisticated testing equipment, and the test takes more than six hours. For this reason, a simple and rapid gene amplification method is desired, and the LAMP (Loop-Mediated Isothermal Amplification) method has been proposed. However, since the PCR method only uses gene amplification, it is not suitable for on-site testing, which requires rapidity. Moreover, the PCR method is a qualitative judgment of positive or negative, and has the disadvantage of lacking quantitativeness.

[0003] Therefore, as a test to supplement the PCR method, simple and rapid serological diagnostic methods using immunochromatography and enzyme-linked immunosorbent assay (ELISA) to detect virus-specific antibodies in serum have been proposed. In the case of general acute viral infections, antibodies in the blood are induced about one week after the onset of symptoms. Therefore, in this type of serological diagnosis, it is necessary to measure the blood antibody titers during the acute and recovery phases of the disease and compare the antibody transitions. Therefore, it is relatively difficult to incorporate a serum-specific antibody detection method into the diagnostic method for acute viral infections, which require testing and diagnosis promptly after onset. However, the blood samples required for serological diagnosis are relatively easy to collect, and the risk of secondary infection to medical personnel during sample collection is relatively low. Furthermore, the virus-specific antibody detection method using immunochromatography allows qualitative analysis by visual judgment, does not require special equipment, and can be tested quickly and easily in outpatient clinics and at the bedside, and it is desired to introduce it into clinical settings as soon as possible. However, in the case of COVID-19, it is currently difficult to detect virus-specific antibodies in the serum of COVID-19 patients up to 6 days after onset. It has also been revealed that the detection rate is only about 20% even in serum one week after onset. Furthermore, the antibody positivity rate increases over time, and after 13 days from onset, most patients' IgG antibodies in the serum are positive, while the detection rate of IgM antibodies is low, and there are many cases where only IgG antibodies are positive. For this reason, it is considered necessary to evaluate paired sera, one from up to 6 days after onset and the other from 13 days after onset, for the serological diagnosis of COVID-19 using this kit. Furthermore, antibody testing methods that do not use gene amplification may not be able to rule out nonspecific reactions, and interpretation of the results is unreliable and requires careful consideration based on a comprehensive assessment of multiple test results and clinical symptoms.

[0004] In the current situation, virus testing requires accuracy comparable to the PCR method for amplifying genes and simple and rapid testing equivalent to the immunochromatography method. Therefore, the present inventors have conducted intensive research to realize a method that can achieve these two goals: accuracy comparable to the PCR method and simple and rapid testing equivalent to the immunochromatography method. There is a method called the fluorescent antibody method for examining the distribution of antigens in tissues and cells using antibodies that specifically recognize the antigens. This method uses a primary antibody and a secondary antibody in sequence to observe the distribution of the primary antibody in tissues and cells, that is, the distribution of the antigen it recognizes, as the distribution of the fluorescently labeled secondary antibody. However, in order to use this method outside the tissue and cell system, the virus in the specimen collected from the patient must be solidified outside the tissue and cell system. In addition, even if it is possible to take the time to solidify the virus as a sample, it was found that pseudo-samples are likely to exist among the solidified samples, which causes non-specific reactions (a phenomenon in which some biological component other than the target of measurement reacts abnormally with components of the measurement reagent or additives in the blood collection tube, resulting in measured values ​​that are far removed from the pathological condition) and reduces the accuracy of the measurement. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent application No. 2020-74439 [Non-patent literature]

[0006] [Non-Patent Document 1] KONIKAMINORUTA Report 2012 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to realize a method and device that has an accuracy comparable to the PCR method and can perform a simple and rapid test equivalent to the immunochromatography method. In order to solve this problem, the inventors have conducted extensive research. As a result, when a method of agglomerating quantum crystals of a plasmon metal complex that excites and enhances surface plasmon is used, viruses are simultaneously agglomerated when the quantum crystals are agglomerated, and the viruses are agglomerated and dispersed together with the quantum crystals and solidified on a metal substrate. Then, due to the surface plasmon enhancement effect of the agglomerated plasmon metal complex, the fluorescence of the antibody of the labeled virus appears in a dot-like or granular form in a fluorescent image, and the number of viruses can be counted as the number of dot-like fluorescence (hereinafter referred to as a fluorescence counting method), while the fluorescence counting method can eliminate or reduce non-specific reactions, significantly improving accuracy. [Means for solving the problem]

[0008] The present invention has been made based on the above findings, and is a novel fluorescence counting system that quantifies a specimen (a virus or an antibody produced by it through the immune function in a human or animal body) in a fluorescence image by using fluorescent spots or particles, and is characterized by comprising the steps of agglutinating inactivated viruses or their antibodies together with a plasmon metal complex on a metal substrate by an electrode potential difference, fixing and solidifying the inactivated viruses or their antibodies together with a metal complex quantum crystal on the metal substrate, labeling the solidified viruses or the solidified antibodies using an antigen-antibody reaction, and binarizing the fluorescent spots or particles in a fluorescence image obtained by the surface plasmon excitation action of the quantum crystal, and counting and counting the fluorescent spots or particles above a predetermined threshold. In the present invention, not only the 1) sandwich method, which utilizes a so-called antigen-antibody reaction in which an antibody (usually diluted with a buffer solution, the same below) is immobilized (a state in which the antibody is aggregated on a metal substrate by an electrode potential difference with a metal substrate by the quantum crystal aggregation method) together with a quantum crystal (referring to a plasmon metal complex of 50 to 150 nm; the same below), and then a viral antigen (usually inactivated with ethanol or the like, and may be diluted with a buffer solution; the same below) and a labeled antibody (labeled with a fluorescent substance and usually diluted with a buffer solution; the same below) are mixed and dropped onto the above-mentioned immobilized substrate, and the antigen is sandwiched between the antibody and the labeled antibody, but also the 2) direct method, which immobilizes a viral antigen and then labels it with a labeled antibody, or immobilizes an antibody and then labels a labeled antigen (labeled with a fluorescent substance, including antigens partially labeled; the same below), and the 3) indirect method, which immobilizes a viral antigen, then binds an antibody and a secondary antibody in order to label it, or immobilizes an antibody, then binds a viral antigen, and finally binds an antibody and a secondary antibody in order to label it, are used. Effect of the Invention

[0009] First, the present invention enhances the fluorescence of a labeled fluorescent molecule associated with a virus or its antibody captured by an antigen-antibody reaction by efficiently exciting it with surface plasmon using a plasmon metal complex immobilized at the same time, so that a specimen such as a virus can be quantified as a number of fluorescent spots. This is different from the conventional surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) immunoassay method (Non-Patent Document 1), which detects the fluorescent signal excited by localized light induced on the surface of a gold film electrode, in that the fluorescent signal appears as spots or particles in a fluorescent image observed by a fluorescent microscope, and when this is binarized and the number of fluorescent spots or particles exceeding a certain threshold is counted, it correlates with the number of viruses and the number of antibodies, and accurate quantification of the viruses and antibodies is possible. This means that accurate quantification is possible, unlike the conventional method of measuring fluorescence intensity. In addition, unlike the qualitative PCR method of positive or negative, this is a valuable quantitative test that can tell the current state of onset, progression, and healing of the disease. In addition, unlike the immunochromatography method of testing immune antibodies, it is possible to quickly and accurately determine whether or not there is an infection by quantifying the amount of virus using the fluorescence counting method. Furthermore, unlike a solid-phase substrate that is excited by localized light field induced on the gold film electrode surface, there is no need for a microchannel, and spurious fluorescent signals due to non-specific reactions are also eliminated.

[0010] The difference between the conventional fluorescence spectroscopy (SPFS) method and the fluorescence counting method of the present invention is that in the former, the specimen, such as a virus, is solidified on a gold thin film with organic molecules, whereas in the latter, the specimen is formed and integrated by the aggregation of plasmon metal complex quantum crystals. The solidification technique for specimens is usually complicated, and the SPFS measurement, which is a substrate-type reaction field of this kind, is disadvantageous in terms of reaction efficiency, so a fine channel (microchannel) is applied as a highly efficient reaction promotion technique. However, the use of this microchannel makes the measurement by the surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) method complicated and difficult. In the present invention, the solidification required for virus measurement can be easily and quickly achieved by the aggregation of quantum crystals of plasmon metal complexes. In other words, a new method can be provided that makes it easy to solidify antibodies or antigens in the reaction field, and enables highly reproducible surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) without using a microchannel (Patent Document 1).

[0011] That is, the present inventors found that such a fluorescence counting method is excellent in quantitative detection of viruses. For example, when performing fluorescence spectroscopy on influenza viruses by antigen-antibody reaction (sandwich method), unlike conventional solid-phase, the measured image shows that, if a virus is present, it is sandwiched between the solidified antibody on the quantum crystal and the labeled antibody, and emits a large number of granular fluorescence, as shown in FIG. 6. This granular fluorescence is the fluorescence of the labeled antibody sandwiching the virus, and counting fluorescent particles above a certain threshold correlates with the number of viruses (FIG. 6(a)). On the other hand, when no virus is present, the inventors newly found that a large number of granular fluorescence does not appear (FIG. 6(b)). Therefore, when examining the details of the present invention, the following features were found. By selecting the electrodeposition substrate potential, the plasmon metal complex in the solution aggregates as a quantum crystal of the metal complex on a metal substrate having an electrode potential near the reduction potential (hereinafter referred to as the quantum crystal aggregation method), and at that time, if an antigen or antibody coexists in the solution, the antigen or antibody aggregates on the substrate or particles together with the metal complex, forming a solid-phase plasmon reaction field. Therefore, unlike conventional plasmon metal thin films, metal complex crystals of about 100 nm are regularly arranged, and antigens or antibodies are physically or chemically immobilized between the quantum crystals at regular intervals, resulting in a structure similar to that of a microchannel, making it possible to enhance surface plasmon excitation.As a result, in such a surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) method, it is effective to count the number of granular fluorescent particles observed with a fluorescence microscope and analyze diseases by determining the presence or absence of viruses and the number of counts to determine the quantity of viruses.

[0012] The present invention provides a novel sample solid-phase fluorescence counting method in a surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) method, which is excellent in image search for observations made with a fluorescence microscope, and which is capable of counting the number of granular fluorescent particles in a fluorescence image to determine the presence or absence of a virus and to analyze a disease based on the number of counts.

[0013] In the present invention, a plasmon metal complex is solidified together with an antibody using a quantum crystal aggregation method to provide a surface plasmon excitation effect, and the fluorescence of the complex labeled by surface plasmon excitation can be observed as granular fluorescence in the fluorescence image by irradiation with excitation light, and the number of granular fluorescence particles can be detected as the amount of virus. Here, the quantum crystal aggregation method is a method in which a plasmon metal complex in a solution is aggregated as a quantum crystal of a metal complex on a metal substrate having an electrode potential near the reduction potential by selecting an electrodeposition substrate potential (hereinafter referred to as a quantum crystal aggregation method), and at that time, if an antigen or antibody coexists in the solution, the antigen or antibody is aggregated on the substrate or particles together with the metal complex to form a solidified plasmon reaction field, and this refers to an aggregation method in which metal complex crystals of about 100 nm are regularly arranged, and the antigen or antibody is physically or chemically solidified between the quantum crystals at regular intervals (see JP 2016-197114 A). In particular, in the present invention, not only the Ag reagent (silver complex aqueous solution containing silver thiosulfate aqueous solution: usually 1000 to 5000 ppm silver thiosulfate aqueous solution, pH about 5), but also this is mixed with a buffer solution containing inactivated viruses or antibodies (usually phosphate buffer solution, pH 7 or higher). Then, the mixed solution usually transitions from the neutral range to the weakly alkaline range, and the aggregation of the quantum crystal and the specimen on the metal substrate tends to disperse due to the buffering action. In addition, a commercially available kit (wipe stick and container containing culture medium) is used as a throat swab used to collect specimens for PCR testing. Copan UTM is one example. In addition to HEPES buffer, sucrose and gelatin are added to the liquid culture medium of Copan UTM, which is suitable for collecting, storing, and transporting viruses, chlamydia, mycoplasma, and ureaplasma. It also functions as a cryoprotectant for clinical viruses including cytomegalovirus and varicella-zoster virus. In this test method, the specimen collected from a human is inactivated before measurement. In this case, ethanol is usually used as the inactivation solution, but it is preferable to inactivate the virus by mixing it with a buffer solution to make the virus more stable. Specifically, 70% ethanol is mixed with a buffer solution (phosphate buffer solution, pH 7.4) to create a 50% ethanol solution (diluted buffer solution), and a human sample is immersed in the solution to inactivate it, which is then used for detection in this test method.This allows the pH of the inactivated sample taken from a human to be kept constant by the action of the buffer.

[0014] In another method of the present invention, the antibody can be produced by immobilizing an antigen using a quantum crystal aggregation method of a plasmon metal complex, reacting the antigen with an antibody to form an antibody-immobilized substrate having gaps or microchannels between the quantum crystals, and further binding a labeled secondary antibody labeled with a fluorescent substance.

[0015] In the present invention, metal powder may be used instead of the metal substrate. In this case, after washing, the remaining complex or labeled secondary antibody is irradiated with excitation light to excite the quantum crystal by surface plasmon, enhancing the fluorescence of the complex or the labeled secondary antibody, and the fluorescence image is observed and the number of granular fluorescent particles in the image is counted for detection.

[0016] In the present invention, the antigen is usually sandwiched between fluorescently labeled antibodies (primary antibodies) for production, but if the antigen is captured with a fluorescently labeled primary antibody and a fluorescently labeled secondary antibody as labeled antibodies and the fluorescence is imaged and analyzed, a more appropriate and accurate fluorescent image can be obtained.

[0017] According to the present invention, the number of fluorescent particles of the virus antigen, not the fluorescence intensity of the virus antigen, can be measured as the virus concentration. Moreover, since the quantum crystal forming the antibody or antigen solid-phase substrate has nanometer-sized gaps or microchannels between the quantum crystals, an interaction occurs between the photons incident by the excitation light and the free electrons of the plasmon metal particles forming the quantum crystal, and the fluorescence of each complex or secondary labeled antibody is enhanced by surface plasmon excitation, so that the granular fluorescence can be counted and detected with good reproducibility, not the overall fluorescence intensity. Therefore, since the surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) method can be used to perform a rapid test in a short time of 2 to 5 minutes, it is possible to provide a highly accurate diagnostic result that can replace the PCR test, which requires complicated pretreatment, has low sensitivity due to primers, has many protocols, and takes a long time to test. In addition, since the count number corresponds to the number of viruses, it is possible to determine the severity of the disease, which is revolutionary, in addition to the presence or absence of the disease.

[0018] According to the present invention, it is possible to provide an antibody testing method that captures antibodies produced in the body that are effective against a specific virus. Here, an antigen is captured with a primary antibody and a fluorescently labeled secondary antibody, and when the primary antibody or the labeled secondary antibody is bound to the primary antibody or the labeled primary antibody, the fluorescence becomes stronger, so that the fluorescence can be detected with higher sensitivity by adding a labeled secondary antibody.

[0019] (Embodiment 1) This method quantifies the amount of virus or antibody in a specimen, and includes 1) a step of preparing a solid-phase substrate by a step of solidifying inactivated viruses or their antibodies, 2) a labeling step of fluorescently labeling the virus or antibody solidified by antigen-antibody reaction, 3) a fluorescence excitation step of irradiating the fluorescently labeled virus or antibody with excitation light to obtain a dot-like fluorescent image of the fluorescently labeled virus or antibody by surface plasmon excitation, and 4) a fluorescence counting step of binarizing the fluorescent points or particles in at least one visual field measurement in the fluorescent image, selecting fluorescent points or particles above a predetermined threshold, and counting and quantifying them. The number of fluorescent points correlates with the number of viruses in the specimen (Figures 7 and 8). (Embodiment 2) In the solid-phase process, the inactivated virus or its antibody is collected in a buffer solution, mixed with a plasmon metal complex aqueous solution of 1000 ppm to 5000 ppm, preferably 1000 to 3000 ppm, to make it neutral, and dropped onto a metal substrate. The virus in the specimen is uniformly dispersed and solidified in the fluorescent image, and accurate measurement can be achieved by measuring one visual field without the need to calculate the average value of two or more visual fields (Figure 16). (Embodiment 3) The target of immobilization in the specimen is an antibody-producing virus (inactivated) or its antibody, and the concentration of the antibody in the specimen is 10 μg / ml or more. The sensitivity of the immobilization substrate improves with increasing concentration of the antibody immobilized (Figure 7). (Embodiment 4) In the present invention, the sandwich method is generally used for fluorescent labeling, but 2) a direct method in which a viral antigen is immobilized and then labeled with a labeled antibody, or an antibody is immobilized and then labeled antigen (including those labeled with a fluorescent substance and partially labeled antigens; the same applies below), or 3) an indirect method in which a viral antigen is immobilized and then labeled by binding an antibody and a secondary antibody in order, or an antibody is immobilized and then bound to a viral antigen, and finally an antibody and a secondary antibody are bound in order for labeling can also be used. The viral antigen is usually inactivated with ethanol or the like, and preferably diluted with a buffer solution. The labeled antibody is preferably labeled with a fluorescent substance and usually diluted with a buffer solution. (Embodiment 5) When excitation light is applied to a plasmon metal complex quantum crystal aggregate of about 100 nm that is aggregated with an antigen or antibody on a metal substrate (Figure 9-1), the quantum crystal causes a surface plasmon excitation phenomenon, exciting the fluorescent label of the virus or its antibody that is immobilized with the quantum crystal. This allows for measurements with fewer non-specific reactions, and the number of fluorescent dots with a brightness value above a certain threshold is accurately obtained by surface plasmon excitation, which correlates with the virus or antibody concentration, making quantitative measurement possible (Figure 11(b)). (Embodiment 6) In the fluorescence counting process according to the present invention, the quantification in one visual field measurement is equivalent to the average value of two or more visual field measurements, which allows rapid quantitative measurement (FIG. 16). (Embodiment 7) In the solid-phasing process, antibodies that bind to two or more different viruses through an antigen-antibody reaction are solidified, and in the labeling process, the viruses are labeled with labeled antibodies of different fluorescent wavelengths, making it possible to quantify two or more viruses in a sample in a single measurement (Figures 19A and B). (Embodiment 8) When embodiment 7 is applied to influenza and Covid-19 viruses, it is possible to distinguish and detect the viruses with a single measurement (Figures 19A and B). [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a schematic diagram showing steps (1) to (4) of the first method (sandwich method) of the present invention. [Figure 2A] FIG. 1 is a schematic diagram of steps (1) to (3) when metal powder is used instead of a metal substrate in the first method of the present invention. [Figure 2B] FIG. 2 is a schematic diagram consisting of steps (4) and (5) when metal powder is used instead of the metal substrate in the first method of the present invention. [Diagram 3] FIG. 1 is a schematic diagram showing steps (1) to (4) of the second method (indirect method) of the present invention. [Figure 4] 1A to 1C are process diagrams showing a method for producing a quantum crystal substrate according to the present invention. [Diagram 5] FIG. 1 is a schematic diagram showing a virus detection method using a primary labeled antibody and a secondary labeled antibody capable of binding to each other as the labeled antibodies in the first and second methods of the present invention. [Figure 6] (a) is a fluorescent image taken when the influenza virus is present, and (b) is a fluorescent image taken when the influenza virus is not present. [Figure 7] 1 is a graph showing influenza virus quantification performed using the fluorescence count number of the present invention. [Figure 8] 1A to 1C are process explanatory diagrams illustrating a method for analyzing a fluorescent image according to the present invention. [Figure 9-1] These are 25,000x SEM images of quantum crystal solid-phase substrates. (a) is an SEM image of the solid-phase substrate when 2000 ppm Ag reagent (silver thiosulfate aqueous solution) and phosphate buffer were used, (b) is an SEM image of the solid-phase substrate when 2000 ppm Ag reagent and phosphate buffer containing influenza antibody (50 μg / ml) were used, and (c) is an SEM image of the solid-phase substrate when 2000 ppm Ag reagent and phosphate buffer containing influenza virus (50 μg / ml) were used. [Figure 9-2] 1 is a graph showing the quantum crystal state and component analysis results of each solid-phase substrate, and shows SEM images and component analysis graphs of solid-phase substrates when using 4000 ppm Ag reagent (silver thiosulfate aqueous solution) and phosphate buffer solution. [Figure 9-3] 1 shows an SEM image and a graph of component analysis of a solid-phase substrate when a phosphate buffer solution containing 4000 ppm Ag reagent and influenza antibody (50 μg / ml) was used. [Figure 9-4] 1 shows an SEM image and a component analysis graph of a solid-phase substrate when using a phosphate buffer solution containing 4000 ppm Ag reagent and influenza virus (50 μg / ml). [Figure 10] (a) is an image showing the method for creating an influenza antibody solid-phase substrate, and (b) is an image of the clear-view area of ​​the solid-phase substrate. [Figure 11A] FIG. 13 is a diagram showing a test process for the sensitivity of a solid-phase substrate. [Figure 11B] 1 is a graph showing the correlation between each concentration of FITC influenza antibody and the number of fluorescent spots counted in a fluorescent image. [Figure 12] FIG. 1 is an image showing the relationship between immobilization of influenza antibodies and quantum crystal concentration. [Figure 13] FIG. 1 is a process diagram showing a method in which inactivated influenza virus antigens are immobilized and subjected to fluorescence counting by the method of the present invention (direct method). [Figure 14] FIG. 1 is a process diagram showing a method for fluorescent counting of inactivated influenza virus antigens by the method of the present invention (sandwich method). [Figure 15] FIG. 1 is a process diagram showing a method for fluorescent counting of inactivated Covid-19 viruses using patient samples by the method of the present invention (sandwich method). [Figure 16] FIG. 1 is a schematic diagram showing an analysis method of the quantum crystal solid-phase fluorescence counting method of the present invention. [Figure 17A] FIG. 1 is a schematic diagram showing steps (1) to (4) of the third method (direct method) using an inactivated sample of the present invention. [Figure 17B] FIG. 1 is a schematic diagram of steps (5) to (7) of the third method of the present invention. [Figure 18A] FIG. 1 is a schematic diagram of collection steps (1) to (3) using a specimen inactivation collection kit of the present invention. [Figure 18B] FIG. 2 is a schematic diagram showing collection steps (4) to (6) using the specimen inactivation collection kit of the present invention. [Figure 19A] FIG. 1 is a schematic diagram showing (1) the solid-phase step and (2) the first half of the fluorescent labeling step when detecting two types of viruses using the sandwich method of the present invention. [Figure 19B] FIG. 1 is an outline diagram showing (3) the latter half of the fluorescent labeling step and (4) the surface plasmon excitation step when detecting two types of viruses using the sandwich method of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] In the present invention, the quantum crystal aggregation method is used, and a solid-phase substrate can be easily produced on a predetermined metal substrate and metal powder by using the various materials listed below.

[0022] (Quantum Crystal Agglomeration Reaction) When silver complex quantum crystals are aggregated as a solid-phase substrate, it is preferable to use a copper or copper alloy substrate, particularly a phosphor bronze substrate, as the aggregation substrate. The substrate having the plasmonic metal quantum crystal region used in the method of the present invention is called a biochip. The method for producing the biochip is as follows. 1) A metal complex aqueous solution is chemically reduced by an electrode potential difference on a metal substrate with an electrode potential (high ionization tendency) lower than that of the metal forming the complex to aggregate quantum crystals (metal complex crystals of nano size). In the case of silver complexes, quantum crystals of silver complexes are formed by employing an electrode potential difference electrodeposition method by aggregating a silver thiosulfate aqueous solution on copper or a copper alloy with an electrode potential (high ionization tendency) lower than that of silver. In detail, the concentration of the metal complex in the aqueous solution should be determined mainly in consideration of the size of the quantum crystals to be formed, and when using a dispersant, the concentration should also be considered. Usually, a range of 100 ppm to 1000 ppm can be used, but depending on the virus containing the antigen used in the antigen-antibody reaction or the antibody produced by the virus in the immune reaction, in order to prepare a nano-sized cluster of 50 to 150 nm, it is preferable to use a quantum crystal aqueous solution of 500 to 1000 ppm, preferably 1000 to 5000 ppm, preferably 1000 to 300 ppm. In addition, in the solidification of antigens and antibodies of the present invention, the solidification target is mixed with an inactivation liquid and a buffer solution and aggregated together with the quantum crystal aqueous solution, so unlike aggregation only from the quantum crystal aqueous solution, the quantum crystals tend to disperse on the solidification substrate (see Figures 9-1(a), (b) and (c)). 2) The metal complex that forms the quantum crystal is selected so as to have a complex stability constant (logβ) greater than or equal to the formula (I) that correlates with the electrode potential E of the supported metal. Formula (I): E° = (RT / |Z|F) ln (βi) (Here, E° is the standard electrode potential, R is the gas constant, T is the absolute temperature, Z is the ionic charge, and F is the Faraday constant.) Here, when the metal complex is a complex of a plasmonic metal selected from Au, Ag, Pt, or Pd, it has a localized surface plasmon resonance enhancing effect against the excitation light. In particular, when the metal complex is a silver complex, it is preferably formed by a reaction between a silver complexing agent having a stability constant (formation constant) (logβi) of 8 or more and a silver halide, and the silver halide is preferably silver chloride, and the complexing agent is preferably one selected from thiosulfate, thiocyanate, sulfite, thiourea, potassium iodide, thiosalicylate, and thiocyanurate. The silver complex has quantum dots consisting of nanoclusters with an average diameter of 5 to 20 nm, and the size of the quantum crystal is 50 to 150 nm. (Solid phase concentration study part 1)

[0023] In the solid-phase technique using quantum crystals, the concentration of quantum crystal reagent (Ag reagent) is very important. Therefore, biotin is solidified by changing the concentration of quantum crystal reagent to be solidified, and FITC-labeled Avidin is detected by fluorescence microscopy using Avidin-Biotin bond. FITC-Avidin VEC “FLUORESCEIN AVIDIN D” CatNo.A-2001 Biotin Wakosha "(+)-Biotin" Cat No. 023-08711 A solid-phase substrate was prepared by solidifying Biotin (5μg / ml) at quantum crystal concentrations of 1000, 2000, 3000, 4000, and 5000ppm (solidification time 1 minute). Next, FITC-Avidin (5μg / ml) was dropped onto the Biotin solid-phase substrate, and the FITC-labeled Avidin was measured using the Avidin-Biotin bond with a Keyence fluorescence microscope "BZ-X710", and the average brightness value of the obtained fluorescent image was calculated (reaction time 1 minute). As a result, it can be concluded that biotin was immobilized in large amounts at 1000 ppm (average image brightness value 54), 2000 ppm (69), 3000 ppm (62), 4000 ppm (59), and 5000 ppm (59), and that FITC-Avidin bound most at 2000 ppm (concentration after adding an equal amount of biotin was 1000 ppm), which had the highest average brightness. This is probably because when the amount of quantum crystals is small, the amount of immobilized biotin is also small, and when the amount of quantum crystals is large, the immobilized biotin is buried, resulting in less detection of FITC-Avidin. The instruments used to measure each quantum crystal are as follows: Equipment used Equipment: Keyence BZ-X710 fluorescence microscope Light source: Metal halide lamp 80W Fluorescence filter: BZ-X filter GFP (525±25) Analysis software: BZ-X Analyzer (Solid phase concentration study part 2)

[0024] Next, we investigated the optimal concentration of quantum crystal reagents for influenza virus detection using antigen-antibody reactions. We immobilized influenza antibodies by changing the concentration of quantum crystals to be immobilized, measured influenza viruses and FITC-labeled influenza antibodies using an antigen-antibody reaction under a fluorescent microscope, and counted the fluorescent spots from the obtained fluorescent images (antigen-antibody reaction - sandwich method). Influenza antibody: Hytest "Monoclonal Mouse anti-influenza A haemogglutinin H1" Cat No. 3AH1 Influenza virus: HyTest "Influenza A (H1N1) virus" Cat No. IN73-3 FITC influenza antibody (ARP "Anti-Influenza A virus (H1N1) FITC") Cat No. 12-6250-3 An equal amount of influenza antibody (100μg / ml) was mixed with a buffer solution at quantum crystal concentrations of 2000, 4000, and 6000ppm, and the mixture was dropped onto a metal substrate to create a solid-phase substrate (solid-phase time 1 minute). Next, a complex formed by mixing inactivated influenza virus (10μg / ml) with FITC-labeled influenza antibody (25μg / ml) was dropped onto the solid-phase substrate (reaction time 1 minute). Unbound complexes and FITC antibodies were washed away with water or buffer. This chip was measured with a Keyence Corporation fluorescence microscope "BZ-X710", and the number of fluorescent points above a certain threshold in the obtained fluorescent image was counted. As a result, as in the case of Avidin-Biotin binding, the most complexes containing influenza virus were detected when influenza was solid-phased with quantum crystals at 2000ppm (total 1000ppm). Figure 12 shows images of the immobilization of influenza antibodies at quantum crystal concentrations of 2000, 4000, and 6000 ppm. Quantum crystal concentration (ppm) and count number 2000 (228 counts), 4000 (159 counts), 6000 (47 counts) Measurement conditions: Threshold 62, no blurring filter, 1-field measurement with a 10x lens (here, 1-field measurement refers to a method of acquiring only one part of the chip, as shown in Figure 16, unlike the case of Patent Application No. 2019-234330, which leads to shorter measurement time). Equipment used Equipment: Keyence BZ-X710 fluorescence microscope Light source: Metal halide lamp 80W Fluorescence filter: BZ-X filter GFP (525±25) Analysis software: BZ-X Analyzer (Preparation of solid-phase substrate)

[0025] The solid-phased substrate of the present invention has a different aggregation action in that it is mixed with an inactivation liquid and / or buffer solution containing a virus containing an antigen to be immobilized and an antibody to be immobilized, in addition to the quantum crystal metal complex aqueous solution, to immobilize the quantum crystal aggregate. However, the antibody or antigen can be immobilized using a quantum crystal aggregation method (JP Patent Publication No. 2016-197114) for producing a quantum crystal of a plasmon metal complex. Therefore, the method described in JP Patent Publication No. 2016-197114 is cited and referenced in this specification. However, the virus and the antibody it produces are added to an inactivation liquid or buffer solution, mixed with a plasmon metal complex reagent (e.g., a silver thiosulfate aqueous solution) for immobilization, and added to the solid-phased substrate to aggregate. Unlike the case of quantum crystals alone, the immobilization of antigens and antibodies by quantum crystals is thought to be affected by interaction with the buffer solution and changes in pH. Figure 9 shows SEM images of a control (Figure 9-1(a)) in which an equal amount of phosphate buffer solution was mixed with 2000 ppm of silver thiosulfate solution (Ag reagent), and a control in which an antibody (influenza antibody: ARP "Anti-Influenza A virus (H1N1) FITC" Cat No. 12-6250-3) and an antigen (inactivated influenza antigen: influenza virus: HyTest "Influenza A (H1N1) virus" Cat No. IN73-3) were added to the phosphate buffer solution at 50 μg / ml each, and then mixed with an equal amount of 2000 ppm of silver thiosulfate solution (Ag reagent) (Figure 9-1(a)) and (Figure 9-1(b)). When a buffer solution was added, the quantum crystals diffused throughout, and the antibody and antigen were bound to the quantum crystals and solidified. It is believed that the dispersion and aggregation of such quantum crystals creates a situation where it is possible to count fluorescent spots in a fluorescence image; thus, when a substrate on which antigens and antibodies are immobilized is labeled, surface plasmons are excited by irradiating it with excitation light, and the fluorescence of the complex can be observed as granular fluorescence in the fluorescence image, and the number of granular fluorescence can be detected as the amount of virus. This is because, as shown in Figures 9-2, 9-3, and 9-4, in addition to the Cu and Sn components of the phosphor bronze substrate, Ag components are detected in each quantum crystal, and it can be confirmed that solidification is performed by the quantum crystal silver complex. However, antibodies and antigens are not detected. (Influenza antibody immobilized substrate preparation and sensitivity)

[0026] The above silver thiosulfate aqueous solution (Ag reagent) 2000 ppm (pH 5.2) and influenza antibody (50 μg / ml) 0.1 When an equal volume mixture (pH 7.2) of mol / L phosphate buffer (pH 7.4) was added to the phosphor bronze plate, it was solidified in about 1 minute, and the remaining liquid on the metal substrate was blown off with air to obtain an antibody-immobilized substrate (Figure 10(a)). Figure 10(b) is a clear image of the solid-phase substrate. Using the quantum crystal aggregation method, FITC-labeled influenza antibodies with different concentrations were immobilized to create antibody-immobilized substrates, and the number of fluorescent spots in each of the resulting fluorescent images was counted. FITC influenza antibody (ARP "Anti-Influenza A virus (H1N1) FITC") Cat No. 12-6250-3) was used. An equal amount of the above-mentioned antibody (250, 125, 62.5, 31.25 μg / ml) was mixed with a 2000 ppm silver thiosulfate solution (Ag reagent) and dropped onto a substrate to prepare various solid-phase substrates (FIG. 11(a)). The time required was about 1 minute. The solid-phase substrate was counted using a fluorescent microscope (Keyence BZ-X710) with a fluorescence sensitivity of a predetermined threshold or higher, as shown in FIG. 11(b). As a result, it was found that the solid-phase substrate of the present invention increases the count number in a concentration-dependent manner. That is, the solid-phase substrate of the present invention has the antibody quantitatively solidified, and is quantified as shown in FIG. 12. Therefore, it can be seen that the present invention can quantitatively solidify antigens and antibodies. (Virus immobilization part 1)

[0027] Figure 13 shows the process of direct influenza virus testing (immobilization of inactivated influenza virus). Influenza viruses inactivated using the quantum crystal aggregation method are immobilized on a substrate, and FITC-labeled influenza antibodies are dropped onto the substrate, and the fluorescent spots are counted from the resulting fluorescent image (this is called the direct method using the antigen-antibody reaction). Influenza virus: HyTest "Influenza A (H1N1) virus" Cat No. IN73-3 FITC influenza antibody (ARP "Anti-Influenza A virus (H1N1) FITC") Cat No. 12-6250-3 A mixture (pH 7.4) of equal volumes of the above-mentioned silver thiosulfate aqueous solution (Ag reagent) and early influenza virus (50 μg / ml) in phosphate buffer solution was added to a phosphor bronze plate to create a virus-immobilized substrate (immobilization took approximately 1 minute). For comparison, a buffer solution without virus was mixed with the quantum crystal Ag reagent to form a solid-phase substrate. Next, FITC-labeled influenza antibody (25 μg / ml) was dropped onto the two solid-phase substrates (reaction time was only 1 minute). Unbound complexes and FITC antibodies were washed away with water or buffer solution. The remaining liquid on the metal substrate was blown away with air to obtain an antibody-immobilized substrate (Figure 10(a)). Figure 10(b) shows a fluorescent image of the solid-phase substrate. (Immobilization of antibodies part 1)

[0028] Figure 14 shows the process of sandwich testing for influenza viruses (immobilization of influenza antibodies). Influenza antibodies are immobilized on a substrate using quantum crystal aggregation techniques, and then FITC-labeled influenza antibodies and inactivated influenza viruses are combined and dropped onto the substrate. The number of fluorescent spots is counted from the fluorescent image obtained by the antigen-antibody reaction. Influenza antibody: Hytest "Monoclonal Mouse anti-influenza A haemogglutinin H1" Cat No. 3AH1 Influenza virus: HyTest "Influenza A (H1N1) virus" Cat No. IN73-3 FITC influenza antibody (ARP "Anti-Influenza A virus (H1N1) FITC") Cat No. 12-6250-3 An equal mixture (pH 7.2) of the above-mentioned silver thiosulfate aqueous solution (Ag reagent) and influenza antibody (50 μg / ml) in phosphate buffer is added to a phosphor bronze plate to create an antibody immobilized substrate (immobilization takes about 1 minute). Next, FITC-labeled influenza antibody (25 μg / ml) is mixed with influenza virus to form a complex, which is then dropped onto the immobilized substrate (reaction time is only 1 minute). Unbound complexes and FITC antibodies are washed away with water or buffer. This measurement chip is measured with a Keyence Corporation fluorescence microscope "BZ-X710," and the number of fluorescent points above a certain threshold in the obtained fluorescent image is counted. The measurement conditions and equipment used are the same as for the immobilization of the virus. (Immobilization of antibodies part 2)

[0029] The inspection process is shown in Figure 15. A complex of an inactivated COVID-19 patient sample and a FITC-labeled COVID-19 antibody is dropped onto a substrate on which COVID-19 antibodies have been immobilized using quantum crystals, and the number of fluorescent spots is counted from the obtained fluorescent image. COVID-19 antibody: GeneTex “SARS-COV-2 antibody spike” CatNo.GTX135356 FITC-labeled COVID-19 antibody: GeneTex's "SARS-COV-2 spike antibody" Cat. No. GTX135356 labeled with FITC (labeling rate 8.64) An equal amount of COVID-19 antibody (50 μg / ml) is mixed with 2000 ppm quantum crystal reagent (Ag thiosulfate) and dropped onto a phosphor bronze substrate to create an antibody immobilized substrate. Next, throat swabs obtained from COVID-19 patients are inactivated with 70% ethanol, and the complex formed by mixing with FITC-labeled COVID-19 antibody (34.5 μg / ml) is dropped onto the immobilized substrate. Unbound complexes and FITC antibodies are washed away with water or buffer solution. This measurement chip is measured with a Keyence fluorescence microscope "BZ-X710," and the number of fluorescent points above a certain threshold in the obtained fluorescence image is counted. The measurement conditions and equipment used are the same as those for virus immobilization part 1. As a result, the virus was detected in both samples taken from throat swabs of two COVID-19 patients. The counts indicated the patient's symptoms. 70% ethanol was used as the blank. The results were blank: count 8 (relative value 0), sample 1: count 16 (relative value 8), and sample 2: count 51 (relative value 43). The relative values ​​are the counts when the blank count is set to 0. If the sample is saliva, the results are similar, although the counts are slightly lower.

[0030] (Quantum crystal manufacturing) A 2000 or 4000 ppm aqueous solution of silver thiosulfate was prepared, and one drop of the solution was dropped onto a phosphor bronze plate, left for about 1 minute, and then the solution was blown off. When viewed in an SEM image, quantum crystals were created. In the photographs showing various SEM images of the nanoparticle aggregates (quantum crystals) produced in Example 1, the crystals are thin hexagonal columnar crystals of about 100 nm, and the surface has irregularities of the order of several nm. No facets specific to metal nanocrystals were confirmed. The correlation between the time left on the phosphor bronze plate after dropping and the quantum crystal shape is shown. First, hexagonal quantum crystals were generated and were observed to grow while maintaining their shape. In the graph showing the results of EDS spectrum (elemental analysis) of the quantum crystals, elements derived from silver and complex ligands were detected in the crystals formed on the phosphor bronze plate, but when a 1000 ppm aqueous solution of silver thiosulfate was prepared on a copper plate, one drop of the solution was dropped, left for about 3 minutes, and the solution was blown off, only silver was detected.

[0031] (Quantum crystal agglomeration theory) When a 2000 or 4000 ppm silver thiosulfate complex aqueous solution is dropped onto a phosphor bronze plate and left for 1 minute, quantum crystals are formed in the shape of hexagonal columns of about 100 nm, and it has been confirmed from SEM images that each hexagonal columnar quantum crystal has an unevenness of the order of several nm, but facets specific to metal nanocrystals cannot be confirmed, and elements derived from silver and complex ligands were detected by EDS elemental analysis, so it is presumed that the whole is a nanocrystal of a silver complex, and the unevenness appearing on the surface is due to the silver in the complex forming quantum dots as clusters and spreading. Looking at the phenomenon in which the silver complex quantum crystal of the present invention is formed on a phosphor bronze plate, while nanoparticles of only silver are precipitated on a copper substrate, it is considered that only silver (0.80) is precipitated on the copper substrate because the equilibrium potential of the silver thiosulfate complex is 0.33, which is equivalent to the electrode potential of copper (0.34), and the electrode potential of phosphor bronze is 0.22, which is slightly less noble, so silver complex crystals are precipitated. Therefore, in order to create quantum crystals, it is important that 1) the complex aqueous solution is in the dilute range of 500 to 2000 ppm, 2) the electrode potential of the supported metal is slightly less noble than the equilibrium potential of the metal complex aqueous solution, and 3) the metal complex is aggregated by the electrode potential difference.However, it was found that it is desirable to use a higher concentration of Ag thiosulfate quantum crystal reagent of 2000 ppm or more to solidify the antigen-antibody. The substrate is polished with sandpaper to physically remove the surface oxide film, and a silver thiosulfate solution is dropped onto it to form a solid-phase substrate by the aggregation of quantum crystals. The physical state of the substrate surface may affect the formation state of the quantum crystals and the measured values. Therefore, in order to make the quantum crystal formation area constant, as shown in Figure 4, 1) a circular groove is processed (etched) in the substrate liquid drop area of ​​the phosphor bronze plate, 2) the area is used as is or subjected to sandpaper polishing, electrolytic polishing, or chemical polishing, 3) Ag reagent solution (2000 to 4000 ppm silver thiosulfate solution) is dropped and the solution is pooled in the circular groove by surface tension, and 4) it is then removed to ensure the aggregation state of the quantum crystals. The aggregation state was observed and the variation in the measurement results was examined, and it was found that there was variation in the measurement results when using it as is, electrolytic polishing, chemical polishing, and sandpaper polishing.

[0032] (Target for solidification) The objects of immobilization include not only viruses, bacteria, fungi, etc. that cause antibodies to be produced by the immune system of humans and animals, but also heavy metals and proteins, and also antibodies produced by such viruses. Examples of antibodies include monoclonal antibodies of the five classes, IgA, IgD, IgE, IgG, and IgM, of animal species such as rats, mice, chickens, rabbits, and humans; polyclonal antibodies and antibodies such as immunoglobulins that include the five classes, IgA, IgD, IgE, IgG, and IgM, of animal species such as rabbits, guinea pigs, goats, sheep, rats, mice, and chickens; fragmented antibody parts such as Fc regions, Fab regions, heavy chains, light chains, antigen-binding sites, and hinge regions; recombinant antibodies and fragmented parts; and human antibodies obtained by infecting human B lymphocytes with a virus (such as EBV), allowing them to grow and cloning the antibody gene.

[0033] Examples of viruses include animal viruses that infect animals, such as coronaviruses, influenza viruses, avian influenza viruses, and adenoviruses, plant viruses that infect plants, such as tobacco mosaic virus, and bacterial viruses that infect bacteria, such as bacteriophages, as well as fragmented parts of viruses, such as spikes on the surface of viruses and viral nucleocapsids, and recombinant viruses and fragmented parts thereof.

[0034] In the present invention, specimens to be immobilized include specimens collected from humans or animals and specimens containing inactivated viruses or fragmented parts thereof, and specimens include body fluids collected from humans or animals, such as throat swabs, saliva, blood, and urine.

[0035] (Example 1 of solid-phase substrate) An example will be given in which quantum crystals and biotin, which has a binding ability to avidin, are immobilized. A solution of Ag reagent (1000 ppm, 20 μl) and biotin (5 μg / ml, 20 μl) for creating quantum crystals is prepared and dropped onto a phosphor bronze plate, and biotin is immobilized onto the quantum crystals that are refined on the substrate. Next, avidin labeled with FITC, which has the ability to bind to biotin, is dropped onto the biotin-immobilized substrate. The biotin and avidin labeled with FITC bind, and the fluorescence of the FITC on the particles is observed when observed under a fluorescent microscope. It can be seen that by mixing quantum crystals and biotin and dropping them onto the substrate, a solid-phase substrate is created in which biotin is immobilized on the quantum crystals. This shows that molecular compounds can be immobilized on the quantum crystal substrate. (The Ag reagent has a range of 500ppm to 10,000ppm, and the immobilized biotin has a range of 1pg / ml to 1g / ml.)

[0036] (Example 2 of solidified substrate) A solution of equal amounts of Ag reagent (2000 ppm, 12.5 μl) and hemagglutinin H1 influenza A antibody (25 μg / ml, 12.5 μl) for creating quantum crystals was prepared and dropped onto a phosphor bronze plate, and the hemagglutinin H1 influenza A antibody was immobilized on the quantum crystals refined on the substrate. Next, a complex of H1N1 influenza virus, which binds to the hemagglutinin H1 influenza A antibody through an antigen-antibody reaction, and FITC-labeled H1N1 influenza virus antibody was dropped onto the hemagglutinin H1 influenza A antibody immobilized substrate. Then, the complex of influenza virus and FITC-labeled antibody bound to the immobilized antibody on the immobilized substrate, and FITC fluorescence was observed on the particles when observed with a fluorescent microscope. As a result, it was found that by mixing the quantum crystals and the hemagglutinin H1 influenza A antibody and dropping it, a solid-phase substrate was created in which the hemagglutinin H1 influenza A antibody was immobilized on the quantum crystals. This shows that antibodies and other substances can be immobilized on the quantum crystal substrate. (The Ag reagent has a range of 500ppm to 10,000ppm, and the immobilized hemagglutinin H1 influenza A antibody has a range of 1pg / ml to 1g / ml.)

[0037] (Solid-phase Substrate Example 3) Next, a solution was prepared by mixing equal amounts of Ag reagent (4000 ppm, 12.5 μl) to create the quantum crystals and hemagglutinin H1 influenza A antibody (100 μg / ml, 12.5 μl), and this was dropped onto a phosphor bronze plate to immobilize the hemagglutinin H1 influenza A antibody onto the quantum crystals that were refined on the substrate. Next, a complex of the H1N1 influenza virus (100μg / ml, 5μl) that binds to the hemagglutinin H1 influenza A antibody through an antigen-antibody reaction and the FITC-labeled H1N1 influenza virus antibody (50μg / ml, 5μl) is dropped onto the hemagglutinin H1 influenza A antibody solid-phase substrate. The complex of the influenza virus and the FITC-labeled antibody binds to the solid-phase antibody on the solid-phase substrate, and when observed under a fluorescent microscope, FITC fluorescence on the particles is observed as shown in the image below. The equipment used is as follows: Equipment: Keyence BZ-X710 fluorescence microscope Light source: Metal halide lamp 80W Fluorescence filter: BZ-X filter GFP (525±25) Analysis software: BZ-X Analyzer

[0038] (Method of analyzing fluorescent images) The fluorescence images were analyzed as shown in Figure 8 (1) to (3). Step (1) is the image acquisition step, where the fluorescence image obtained by measurement is imported into the image analysis software "BZ-X Analyzer" and analyzed (fluorescence is observed as round particles). Here, as shown in Figure 16, only one 10x objective lens is photographed (one field of view measurement), and the photographing time is as short as 3.5 seconds if binarized and counted, but multiple images may also be taken. Step (2) is a binarization step, in which all fluorescent particles with a brightness value above a set value are extracted from the entire range of the fluorescent image and binarized (red particles are extracted fluorescent particles with a brightness value above a set value). Step (3) is a counting step, in which the extracted fluorescent particles having a brightness value equal to or greater than a set value are counted (the number of fluorescent red particles only (dashed line area) is calculated).

[0039] "Fluorescence image acquisition process" Here, we can refer to a technology in which the fragmented DNA captured on the above-mentioned plasmonic metal nanocrystal substrate is irradiated with excitation light, the autofluorescence of the captured fragmented DNA is enhanced by the surface plasmon enhancement effect, and the fluorescent colonies are obtained as fluorescent images (see the fluorescence measurement method in Patent Application No. 2019-234330).

[0040] Example 4 Similarly, a liquid was prepared by mixing Ag reagent (2000 ppm, 5 μl) used to create quantum crystals with FITC-labeled H1N1 influenza virus antibodies (250 μg / ml, 5 μl), and dropping it onto a phosphor bronze plate, immobilizing the FITC-labeled H1N1 influenza virus antibodies onto the quantum crystals produced on the substrate. When this immobilized substrate was observed under a fluorescent microscope, the fluorescence of the FITC particles was observed. This means that fluorescent labels such as FITC can be immobilized onto quantum crystal substrates. (Ag reagents have a range of 500 ppm to 10,000 ppm, and FITC-labeled H1N1 influenza virus antibodies have a range of 1 pg / ml to 1 g / ml.)

[0041] (Measurement example 1) The present invention applies surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) to detect viruses in patients, using throat swabs, saliva, urine, and feces in which viral antigens are present. FIG. 1 shows a method consisting of steps (1) to (4). In step (1), a quantum crystal aggregation method is used to prepare an antibody-immobilized substrate. In detail, a viral antibody is added to a buffer solution (phosphate buffer solution at pH 7.4) to prepare an immobilized antibody solution. An equal amount of the immobilized antibody solution is added to an aqueous solution of a plasmon metal complex having a concentration of 1000 to 10000, preferably 2000 to 4000 ppm to prepare a composite aqueous solution of the plasmon metal complex and the viral antibody. The plasmon metal complex solution containing the viral antibody is dropped onto a metal substrate having an electrode potential close to the reduction potential of the plasmon metal complex to aggregate the plasmon metal complex quantum crystals to which the antibody is bound, thereby preparing a viral antibody-immobilized substrate. Here, one selected from palladium, platinum, gold, silver, and copper is selected as the plasmon metal, a metal substrate having an electrode potential close to the redox potential of the plasmon metal complex is selected, and when a quantum crystal of a silver thiosulfate complex is used, copper or a copper alloy, in particular phosphor bronze, is selected as the substrate. Here, the antibodies include influenza virus antibodies, monoclonal antibodies of the five classes of IgA, IgD, IgE, IgG, and IgM of animal species such as rats, mice, chickens, rabbits, and humans, which are produced from viral antigens or hybridomas, polyclonal antibodies including the five classes of IgA, IgD, IgE, IgG, and IgM of animal species such as rabbits, guinea pigs, goats, sheep, rats, mice, and chickens, and antibodies such as immunoglobulins, fragmented parts of antibodies such as Fc regions, Fab regions, heavy chains, light chains, antigen-binding sites, and hinge regions, recombinant antibodies and fragmented parts, and human antibodies obtained by infecting human B lymphocytes with a virus (EBV, etc.), allowing them to grow and cloning the antibody gene. Examples of coronavirus antibodies include monoclonal antibodies of the five classes (IgA, IgD, IgE, IgG, and IgM) produced from viral antigens or hybridomas of animal species such as rats, mice, chickens, rabbits, and humans; polyclonal antibodies and antibodies such as immunoglobulins including the five classes (IgA, IgD, IgE, IgG, and IgM) of animal species such as rabbits, guinea pigs, goats, sheep, rats, mice, and chickens; fragmented antibody portions such as Fc regions, Fab regions, heavy chains, light chains, antigen-binding sites, and hinge regions; recombinant antibodies and fragmented portions; and human antibodies obtained by infecting human B lymphocytes with a virus (such as EBV), allowing them to grow and cloning the antibody gene. In step (2), a complex is formed between the viral antibody labeled with a fluorescent substance and the viral antigen in the specimen by utilizing an antigen-antibody reaction. Here, the specimen may be a throat swab, saliva, urine, or feces. Examples of the fluorescent substance that labels the viral antibody include fluorescent substances with an excitation light of 400 nm to 436 nm such as Pacific Blue, an excitation light of 453 to 505 nm such as FITC, an excitation light of 485 to 566 nm such as TRITC, an excitation light of 488 to 706 nm such as APC, and an excitation light of 732 to 784 nm such as IRDye800. In step (3), the above complex is dropped onto the antibody-immobilized substrate by utilizing an antigen-antibody reaction, the complex is bound to the antibody on the substrate, and the unbound complex and antibody are washed away with pure water, a buffer solution, etc. Here, a neutral phosphate buffer solution was used as the buffer solution, but PBS, HEPES, TRIS, BIS-TRIS, CAPS, CAPSO, Glycylglycine, MES, MOPS, PIPES, etc. can also be used. In step (4), excitation light is applied to the complex of the labeled antibody and antigen remaining on the substrate, and the resulting fluorescent image is observed with a fluorescent microscope or a fluorescent reader due to surface plasmon excitation. Fluorescent particles having a brightness value of an arbitrary value or more within an arbitrary range of the obtained fluorescent image or from the entire image are binarized and the number of particles obtained is counted. The fluorescent measurement method of the same applicant's Japanese Patent Application No. 2019-234330 can be used to binarize and count fluorescent particles having a brightness value of a certain threshold or more in the fluorescent image, and such a fluorescent measurement method is herein cited and referenced. In the present invention, the single visual field measurement conditions shown in Figure 16 are: threshold 62, no blurring filter, single visual field measurement with ×10x lens (here, single visual field measurement refers to a method of acquiring only a portion of the chip, as shown in Figure 16, unlike the case of Patent Application No. 2019-234330. It has been found that when the solid-phase substrate of the present invention is used, the average value of two or more single visual field measurements is almost equivalent, and therefore the result of the single visual field measurement can be used without adopting the average value. This shows that the solid-phase formation of the antigen or antibody is almost uniform by the quantum crystal).

[0042] In the above embodiment, a substrate on which an antibody was immobilized was used, but by using the same type of metal powder, it is possible to detect a viral antigen by utilizing an antigen-antibody reaction in liquid. The method uses throat swabs, saliva, urine, and feces containing viral antigens, and consists of steps (1) to (5) in Figure 2. In step (1), an antibody-immobilized metal powder is prepared using the quantum crystal aggregation method. More specifically, a viral antibody is added to an aqueous solution of a plasmon metal complex with a concentration of 500 to 10,000 ppm, and a carrier metal powder is added to this and mixed. The plasmon metal complex aggregates with the viral antibody and the metal powder having an electrode potential close to the reduction potential of the plasmon metal complex, forming a viral antibody-immobilized metal powder in which the viral antibody, plasmon metal complex, and carrier metal powder are integrated. On the other hand, in step (2), a complex is formed between a viral antibody labeled with a fluorescent substance and a viral antigen in a specimen by utilizing a first antigen-antibody reaction. Here, the specimen and the fluorescent substance are the same as those in the first method. Next, in step (3), the complex is added to the antibody-immobilized powder liquid, and the antibody-immobilized powder and the complex are bound together by utilizing a second antigen-antibody reaction. In step (4), the combination of the antibody-immobilized powder and the complex is filtered, and unbound complex and antibody are washed away with pure water, a buffer solution, or the like. Finally, in step (5), excitation light is irradiated onto the complex of labeled antibody and antigen remaining on the substrate, and the resulting fluorescent image is observed using a fluorescent microscope or a fluorescent reader due to surface plasmon excitation. Fluorescent particles with a brightness value equal to or greater than a given value within a given range of the obtained fluorescent image or from the entire image are binarized, and the number of particles obtained is counted.

[0043] The second method of the present invention is a method of immobilizing a viral antigen or a part thereof (a non-infectious part of an antigen, for example, a spike on the surface of the virus or a fragmented part of the virus such as a viral nucleocapsid) and capturing an antibody produced in the body. As shown in FIG. 3, the antigen or a part thereof is immobilized using a quantum crystal aggregation method of a plasmon metal complex, and then the antigen or the part thereof is reacted with an antibody to form an antigen or part thereof immobilized substrate having gaps or microchannels between the quantum crystals, while an antibody labeled with a fluorescent substance is dropped onto the antigen or part thereof immobilized substrate to bind the two. After washing away the unbound labeled antibody, the labeled antibody remaining on the substrate is irradiated with excitation light to excite the quantum crystals by surface plasmon, thereby enhancing the fluorescence of the labeled antibody, and the fluorescence is detected. The quantum crystal aggregation method of the plasmon metal complex is used to solidify an antigen or a part thereof, and then the antigen or a part thereof is reacted with an antibody to form an antigen or a part thereof solid-phase substrate having gaps or microchannels between quantum crystals, while a labeled antibody labeled with a fluorescent substance is dropped onto the antigen or the part thereof solid-phase substrate to bind the two, and after washing off the unbound labeled antibody, the labeled antibody remaining on the substrate is irradiated with excitation light to excite the quantum crystals with surface plasmon, and the fluorescent image is observed with a fluorescent microscope or a fluorescent reader, and fluorescent particles with a brightness value of an arbitrary value or more within an arbitrary range of the obtained fluorescent image or from the entire image are binarized, and the number of obtained particles is counted and detected. That is, in step (1), the quantum crystal aggregation method is used to prepare an antigen or a part thereof solid-phase substrate. More specifically, a viral antigen or a part thereof is added to a plasmon metal complex aqueous solution having a concentration of 500 to 10,000 ppm. Here, as the viral antigen or a part thereof, throat swabs, saliva, urine, feces, or parts of non-infectious antigens are used. Examples of processing methods for viral antigens or parts of them include autoclaving (high-pressure steam sterilization at 121°C for 15 minutes or more), immersion in 0.01% or more sodium hypochlorite for 1 hour or more, immersion in 4% formaldehyde solution, or immersion in 70% ethanol. A composite aqueous solution of a plasmon metal complex and a viral antigen or a portion thereof is prepared, and the plasmon metal complex solution containing the viral antigen or a portion thereof is dropped onto a metal substrate having an electrode potential close to the reduction potential of the plasmon metal complex to aggregate the plasmon metal complex quantum crystals to which the antigen or a portion thereof is bound, thereby preparing a viral antigen or a portion thereof solid-phase substrate in which the viral antigen or a portion thereof is solid-phased. Next, in step (2), a complex is formed between the immobilized antigen or a part of it and the virus antibody in the blood by utilizing a first antigen-antibody reaction. Here, blood, serum, or plasma is used as the sample containing the virus antibody. In step (3), a labeled antibody is prepared and dropped onto an antigen-antibody solid-phase substrate using a second antigen-antibody reaction to bind the complex to the antibody on the substrate, and unbound labeled antibody is washed away with pure water, a buffer solution, or the like. However, the sample in step (2) and the labeled antibody in step (3) can also be mixed in advance. In step (4), excitation light is irradiated onto the complex of labeled antibody and antigen remaining on the substrate, and the resulting fluorescent image is observed using a fluorescent microscope or a fluorescent reader due to surface plasmon excitation. Fluorescent particles with a brightness value equal to or greater than a given value within a given range of the obtained fluorescent image or from the entire image are binarized, and the number of particles obtained is counted and detected.

[0044] In the sandwich method, a primary labeled antibody and a secondary labeled antibody capable of binding to each other are simultaneously used as labeled antibodies to enhance the fluorescence intensity. In the method of Fig. 4, a primary labeled antibody and a secondary labeled antibody capable of binding to each other are simultaneously used in steps (1) to (4) of the first method of the present invention. In step (1), an antibody-immobilized substrate is prepared using the quantum crystal aggregation method, which is similar to step (1) in FIG. In step (2), a primary labeled antibody and a secondary labeled antibody are simultaneously used as viral antibodies labeled with a fluorescent substance, utilizing an antigen-antibody reaction, to form a complex with the viral antigen in the sample. Alternatively, the first labeled antibody may be bound followed by the second labeled antibody. Here, the specimen may be a throat swab, saliva, urine, or feces. Fluorescent substances for labeling virus antibodies include those with excitation light of 400 to 436 nm, such as Pacific Blue, those with excitation light of 453 to 505 nm, such as FITC, those with excitation light of 485 to 566 nm, such as TRITC, those with excitation light of 488 to 706 nm, such as APC, and those with excitation light of 732 to 784 nm, such as IRDye800. As a combination of primary and secondary conjugated antibodies Combine with a secondary labeled antibody that recognizes the animal species from which the primary labeled antibody was derived. For example, if a primary labeled antibody derived from a mouse is used, use a secondary labeled antibody that recognizes mouse antibodies, and similar combinations can be made for other animal species. In step (3), the complex is dropped onto the antibody-immobilized substrate by utilizing an antigen-antibody reaction, the complex is bound to the antibody on the substrate, and the unbound complex and antibody are washed away with pure water or a buffer solution, etc. Here, PBS, HEPES, TRIS, BIS-TRIS, CAPS, CAPSO, Glycylglycine, MES, MOPS, PIPES, etc. are used as the buffer solution. In step (4), excitation light is irradiated onto the complex of labeled antibody and antigen remaining on the substrate, and the resulting fluorescent image is observed using a fluorescent microscope or a fluorescent reader due to surface plasmon excitation. Fluorescent particles with a brightness value equal to or greater than a given value within a given range of the obtained fluorescent image or from the entire image are binarized, and the number of particles obtained is counted. (Actual measurement using antigen-antibody reactions of influenza viruses)

[0045] Equal amounts of Ag reagent (500-10,000 ppm) which is the source of quantum crystals and influenza antibody (5-1,000 μg / ml) are mixed, and the mixture is dropped onto a phosphor bronze plate to immobilize the quantum crystals and antibody on the phosphor bronze plate. Next, equal amounts of influenza virus (5 to 1000 μg / ml) and FITC-labeled influenza antibody (5 to 1000 μg / ml) are mixed and dropped onto the quantum crystal substrate. As an example of a reagent Influenza antibody HyTest Monoclonal mouse anti-Influenza A haemagglutinin H1 Influenza A (H1N1) Virus (HyTest) -FITC influenza antibody IBL's Anti-Influenza A Virus (H1N1) FITC was used. The excess FITC-labeled influenza antibody was washed with pure water, etc., and the specimen was then exposed to light from a light source (metal halide lamp 80 W) and examined under a fluorescent microscope (Keyence BZ-X710 fluorescent microscope). The measurement was performed using a fluorescence microscope. The image was observed using a fluorescence microscope and analyzed using a BZ-X Analyer. The result is shown in Figure 6(a). In comparison, the result for the case where influenza antigens were not included is shown in Figure 6(b). Surface plasmon excitation is performed and the resulting fluorescent image is observed using a fluorescence microscope or a fluorescence reader. Fluorescent particles with a brightness value of a given value or higher within a given range of the obtained fluorescent image or from the entire image are binarized, and the number of particles obtained is counted and detected. When a virus is present, it is sandwiched between the immobilized antibody on the quantum crystal and the labeled antibody, emitting numerous granular fluorescent light. This granular fluorescent light is the fluorescence of the labeled antibody that has sandwiched the virus (Figure 6(a)). However, when the virus is not present, no numerous granular fluorescent light appears (Figure 6(b)). Using influenza antibody (25μg / ml) and FITC influenza antibody (25μg / ml), the virus concentration and the fluorescent particles in the fluorescent image with a brightness value of 57 or more were binarized and counted using analysis software (BZ-X) using a Keyence BZ-X710 objective lens at 10x, and the results shown in the table in Figure 7 were obtained. These results were linearized to produce the graph in Figure 7. It can be seen from the image obtained that there is a relative relationship between the count number and the virus concentration. (Testing of samples collected on-site)

[0046] The present invention is suitable for a method of quickly performing a virus test on the spot at the time of immigration inspection or hospital diagnosis, and may include inactivating or attenuating antigens in a specimen (throat swab, saliva, sputum, nasopharyngeal fluid, urine, etc.) collected from a human. (The same applies below), then the inactivated antigen is immobilized on a substrate by a quantum crystal aggregation method, and a labeled antibody is bound to the immobilized antigen by an antigen-antibody reaction to label it, and the unbound labeled antibody is washed with a buffer solution or pure water, and an excitation light that matches the label (fluorescent substance) of the antibody is irradiated from a light source, and the fluorescent particles on the substrate are counted with a fluorescence microscope. Here, the quantum crystal aggregation method refers to an aggregation method for producing quantum crystals of a plasmon metal complex shown in JP 2016-197114 A, and refers to a method in which a plasmon metal complex in a solution is aggregated as a quantum crystal of a metal complex on a metal substrate having an electrode potential near the reduction potential by selecting an electrodeposition substrate potential. In this case, the antigens in the collected specimen are inactivated and bound to the substrate by an antigen-antibody reaction on a substrate on which antibodies have been immobilized in advance using quantum crystal aggregation method, and then the antigens are labeled by binding to the label through an antigen-antibody reaction with a labeled antibody to label the antigens.The unbound labeled antibodies are then washed with a buffer solution or pure water, and excitation light that matches the antibody label (fluorescent substance) is irradiated from a light source, and the fluorescent particles on the substrate are counted using a fluorescence microscope.

[0047] In the present invention, the viruses to be inactivated in the specimen are basically composed of either DNA or RNA nucleic acid and a shell protein (capsid) that protects it, and are classified into small spherical viruses that are enveloped in a membrane containing lipids and those that do not have an envelope. Therefore, whether or not a virus is susceptible to inactivation by a drug depends on whether or not it has an envelope, but since enveloped viruses are generally sensitive to disinfectants, the use of a drug is preferable. Other inactivation methods that are effective against most viruses include boiling (98°C or higher) for 15 to 20 minutes, 2 w / v% glutaral, 0.05 to 0.5 w / v% (500 to 5,000 ppm) sodium hypochlorite, 76.9 to 81.4 v / v% disinfectant ethanol, 70 v / v% isopropanol, 2.5 w / v% povidone iodine, 55 w / v% phthalaral, and 0.3 w / v% peracetic acid. In addition, many viruses are inactivated by the denaturation of capsid proteins at 56°C for 30 minutes, and enveloped viruses are easily inactivated by lipid solvents such as ether, chloroform, and fluorocarbon. In addition, by using antibodies that recognize nucleosides, nucleotides, and nucleocapsids present inside the virus, it is possible to detect inactivated virus antigens that have been broken down into small pieces by destroying the membrane or shell. Therefore, as the inactivation method according to the present invention, from the viewpoint of not affecting or having little effect on the antigen-antibody reaction, a drug method using ethanol, formalin, or AVL buffer, and an inactivation method such as heat treatment, SD treatment (chemical treatment), acid treatment, alkali treatment, and radiation treatment can be used. In the present invention, the preparation can be performed by using metal powder instead of the substrate. In addition, in the above method, if a fluorescently labeled primary antibody and a fluorescently labeled secondary antibody are simultaneously used as the labeled antibodies and imaged and analyzed, a more appropriate and accurate fluorescent image can be obtained.

[0048] In the present invention, it is preferable to use a specimen inactivation collection kit so that it can be applied to on-site collection and on-site testing, and it is preferable to inactivate the specimen using a chemical. As shown in FIG. 18(1), a tube 10 containing a chemical liquid L such as ethanol and a rod-shaped specimen collection part 20 are set, and the specimen collection part is composed of an absorbent material such as nonwoven fabric or gauze. Next, (2) a specimen is collected using the rod-shaped specimen collection part. The specimen S is saliva, phlegm, throat swab, nasopharyngeal fluid, etc. (3) After collecting the specimen S, the collection part 20 is inserted into the tube 10. (4) The back of the tube 10 is a narrow part, and when the specimen collection part 20 is inserted, the specimen collection part 20 is compressed by the narrow wall surface, and the specimen S (saliva) is dispersed in the chemical liquid L such as ethanol. (5) When the parts other than the specimen collection part 20 are removed, the specimen collection part 20 remains in the tube 10. (6) The specimen S is inactivated by the chemical L and remains in the specimen collection part 20.

[0049] According to the present invention, a virus antigen is collected on-site, inactivated, immobilized on a substrate by the quantum crystal aggregation method, and a labeled antibody is bound to the virus antigen, or the virus antigen inactivated by an antigen-antibody reaction is bound to a labeled antibody on a substrate on which an antibody has been immobilized in advance, and the number of fluorescent particles of the virus antigen is counted instead of the fluorescence intensity to measure the virus concentration. Moreover, the quantum crystal forming the antibody or antigen immobilized substrate causes an interaction between the photons incident by the excitation light and the free electrons of the plasmon metal particles forming the quantum crystal, which excites the surface plasmon and enhances the fluorescence of the labeled antibody, so that the granular fluorescence can be counted and detected with good reproducibility instead of the overall fluorescence intensity. Therefore, the surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) method can be used to perform a rapid test in a short time of 2 to 5 minutes, providing a highly accurate diagnostic result that can replace PCR tests, which require complicated pretreatment, have low sensitivity due to primers, have many protocols, and take a long time to test. In addition, since the count number corresponds to the number of viruses, it is possible to determine the severity of the disease, which is revolutionary, in addition to determining whether the disease is present or not.

[0050] According to the specimen inactivation collection kit of the present invention, collected viruses can be inactivated and stored in a tube, so that they can be sent to a required testing location and taken out for testing at any time. In the present invention, inactivated antigens and the like are directly immobilized by the quantum crystal agglutination method, but antibodies may be immobilized in advance, and the inactivated antigens may be bound to labeled antibodies by antigen-antibody reaction for detection, and the direct method, sandwich method, and indirect method of the fluorescent antibody method may be adopted. (Virus detection from inactive specimens)

[0051] A solution was prepared by mixing equal amounts of Ag reagent (2000 ppm, 12.5 μl) and hemagglutinin H1 influenza A antibody (25 μg / ml, 12.5 μl) to create quantum crystals, and the solution was dropped onto a phosphor bronze plate. The hemagglutinin H1 influenza A antibody is immobilized along with the quantum crystals. A complex of the inactivated specimen (25μg / ml, 5μl) and FITC-labeled H1N1 influenza virus antibody (25μg / ml, 5μl) is dropped onto a hemagglutinin H1 influenza A antibody solid-phase substrate. If the H1N1 influenza virus is present in the inactivated specimen, the complex of the influenza virus and the FITC-labeled antibody binds to the solid-phased antibody on the solid-phase substrate, and the fluorescence of the FITC on the particles is observed when observed under a fluorescent microscope. If the influenza virus is not present in the specimen, the fluorescence of the FITC on the particles is not observed. When the measurement image obtained by the fluorescent microscope was analyzed with the Keyence Corporation analysis software: BZ-X Analyzer, with a threshold set to 57, a large difference was obtained in the count value of the fluorescence of the granular FITC. As a result, when the fluorescent particles from the fluorescent image were counted, the number was 4 in the absence of the virus and 145 in the presence of the virus. The equipment used is as follows: Equipment: Keyence BZ-Z710 fluorescence microscope Light source: Metal halide lamp 80W Fluorescence filter: BZ-X filter GFP (525±25) Analysis software: BZ-X Analyzer

[0052] (Example 1 of measuring patient virus) The fluorescence counting method of the present invention is applied to the detection of viruses in patients, and uses throat swabs, saliva, sputum, nasopharyngeal fluid, urine, and feces in which viral antigens are present. FIG. 17 shows a method consisting of steps (1) to (7). In step (1), an inactivated sample is prepared using a sample inactivation collection kit shown in FIG. 18. In step (2), the inactivated antigen in the sample is mixed with an Ag reagent (silver thiosulfate complex solution). In step (3), a solid-phase substrate of the inactivated antigen is prepared using a quantum crystal aggregation method. More specifically, the inactivated antigen is added to a plasmon metal complex aqueous solution having a concentration of 2000 to 6000 ppm, a composite aqueous solution of the plasmon metal complex and the inactivated antigen is prepared, and the plasmon metal complex solution containing the inactivated antigen is dropped onto a metal substrate having an electrode potential near the reduction potential of the plasmon metal complex to aggregate the plasmon metal complex quantum crystals, thereby preparing a solid-phase substrate on which the inactivated antigen is solidified (step (4)). Here, the plasmon metal is selected from palladium, platinum, gold, silver, and copper, a metal substrate having an electrode potential close to the redox potential of the plasmon metal complex is selected, and when using a quantum crystal of a silver thiosulfate complex, copper or a copper alloy, particularly phosphor bronze, is selected as the substrate. As a method for producing the quantum crystal, the method described in JP 2016-197114 A is cited and referenced. In step (5), the virus antigen in the solid-phased specimen is labeled with a virus antibody labeled with a fluorescent substance by utilizing an antigen-antibody reaction. The specimens are pharyngeal swabs, saliva, sputum, nasopharyngeal fluid, urine, and feces. Examples of the fluorescent substance that labels the virus antibody include fluorescent substances with an excitation light of 400 nm to 436 nm such as Pacific Blue, excitation light of 453 to 505 nm such as FITC, excitation light of 485 to 566 nm such as TRITC, excitation light of 488 to 706 nm such as APC, and excitation light of 732 to 784 nm such as IRDye800. In step (6), unbound complexes and antibodies are washed off from the substrate with pure water or a buffer solution, etc. In addition to neutral phosphate buffer, PBS, HEPES, TRIS, BIS-TRIS, CAPS, CAPSO, Glycylglycine, MES, MOPS, PIPES, etc. can be used as the buffer solution. In step (7), excitation light is irradiated onto the complex of labeled antibody and antigen remaining on the substrate, and the resulting fluorescent image is observed with a fluorescent microscope or a fluorescent reader due to surface plasmon excitation. Fluorescent particles having a brightness value of a given value or more within a given range of the obtained fluorescent image or from the entire image are binarized and the number of particles obtained is counted. Fluorescent particles having a brightness value of a given threshold or more in the fluorescent image are binarized and counted. According to the method of the present invention, it is possible to simply and quickly perform highly accurate detection comparable to the PCR method in detecting viruses. Therefore, it is possible to perform rapid testing on site at immigration inspections, hospitals, etc. (Detection of two types of viruses)

[0053] The following method is described as a measurement method for detecting two types of viruses using one measurement chip. 1) Mix the Ag reagent (silver thiosulfate aqueous solution 3000 ppm) with equal amounts of a mixture of influenza antibody (containing multiple antibodies as influenza antibodies) buffer and coronavirus (Covid-19) antibody buffer to prepare a solid-phase specimen containing 1000 ppm of Ag reagent. Or mix the three in equal amounts in sequence to prepare a solid-phase specimen. Drop the mixture onto a metal substrate to create a solid-phase substrate (see Figure 19A(1)). 2) Next, a sample (throat swab or saliva, etc.) taken from a human and inactivated with ethanol or other substances is mixed with a mixture of green-labeled coronavirus antibodies and red-labeled influenza antibodies. If either virus is present in the sample, the virus and the labeled antibody form a complex. Then, the complex is dropped onto a solid-phase substrate. 3) The complex binds to the antibody immobilized on the substrate through an antigen-antibody reaction (see Figure 19A(2)). Unbound complexes and labeled antibodies are washed away with water or a buffer solution (see Figure 19B(3)). Here, the coronavirus antibody is labeled with a green label such as FITC or Cy2, and the influenza antibody is labeled with a red label such as Cy5 or APC, and the fluorescent regions are selected so that they do not overlap. The light source irradiates the specimen with green and red wavelength excitation light. If influenza virus is present in the specimen, no fluorescence is observed with green excitation, but fluorescence from red-labeled influenza antibodies is observed with red excitation. On the other hand, if coronavirus is present in the specimen, no fluorescence is observed with red excitation, but fluorescence from green-labeled coronavirus antibodies is observed with green excitation (see Figure 19B(4)). In this way, two fluorescent images obtained from the two excitation lights are acquired with a fluorescent microscope, and the fluorescent points or particles on the images are counted to perform a quantitative analysis. In addition, the green filter: excitation wavelength 470±20 nm, fluorescence wavelength 525±25 nm Red region filter: excitation wavelength 620±20 nm, fluorescence wavelength 700±37.5 nm.

Claims

1. A method for quantifying the amount of a virus or antibody to be measured in a specimen, comprising: 1) a solidification step of agglutinating an inactivated virus or antibody together with a plasmon metal complex quantum crystal on a metal substrate by the potential difference between the plasmon metal complex and the metal substrate, and fixing the virus or antibody together with the metal complex quantum crystal on the metal substrate, thereby solidifying the virus or antibody; 2) a labeling step of fluorescently labeling the virus or antibody that has been solidified by an antigen-antibody reaction; 3) a fluorescence excitation step of irradiating the fluorescently labeled virus or antibody with excitation light to obtain a dot-like or granular fluorescent image of the fluorescently labeled virus or antibody by surface plasmon excitation; and 4) a fluorescence counting step of binarizing the fluorescent points or particles in the fluorescent image, selecting fluorescent points or particles that are equal to or greater than a predetermined threshold, and counting and quantifying the same.

2. 2. The fluorescence counting method according to claim 1, wherein in the immobilization step, the inactivated virus or its antibody is taken up in a buffer solution, mixed with an aqueous solution of 1000 to 3000 ppm of a plasmon metal complex to neutralize it, and then dropped onto the metal substrate.

3. 2. The fluorescence counting method according to claim 1, wherein the object to be immobilized in the specimen is an inactivated virus which produces an antibody or an antibody thereto, and the concentration of said virus in the specimen is 10 .mu.g / ml or more.

4. 2) a direct method in which the viral antigen is immobilized and then labeled with a labeled antibody, or an antibody is immobilized and then labeled with a labeled antigen (including antigens labeled with a fluorescent substance and antigens partially labeled; the same applies below); or 3) an indirect method in which the viral antigen is immobilized and then labeled with an antibody and a secondary antibody in sequence, or an antibody is immobilized and then labeled with the viral antigen, and then labeled with the antibody and a secondary antibody in sequence, or an antibody is immobilized and then labeled with the viral antigen, and finally labeled with the antibody and a secondary antibody in sequence, or a viral antigen is immobilized and then bound to the antibody and a secondary antibody in sequence, and finally labeled.

5. 2. The fluorescence counting method according to claim 1, wherein the fluorescence excitation step comprises irradiating excitation light onto quantum crystal aggregates of about 100 nm aggregated on a metal substrate, thereby exciting the fluorescent label of a virus or its antibody immobilized together with the quantum crystal.

6. 2. The fluorescence counting method according to claim 1, wherein the fluorescence counting step is a measurement image obtained by measuring one or more visual fields using a fluorescence microscope, and involves counting the number of fluorescent points having a predetermined brightness value or more, which are obtained by binarizing and collecting point-like fluorescence in any one of the visual field images.

7. 2. The fluorescence counting method according to claim 1, wherein in the solid-phase immobilization step, antibodies that bind to two or more distinct viruses through an antigen-antibody reaction are solidified, in the labeling step, the viruses are labeled with labeled antibodies having distinct fluorescent wavelengths, and in the fluorescence counting step, one or more viruses are quantified.

8. The fluorescence counting method according to claim 1, wherein the two or more distinct viruses are influenza and Covid-19, the fluorescent wavelengths of the labeled antibodies are different, and either of the viruses can be detected.

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