JP7602739B2 - Immobilized Fluorescence Counting of Viruses - Google Patents

Description

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

Currently, genetic testing using the PCR method is the mainstream for virus testing. This test involves taking mucus or phlegm from the back of the nose or throat and examining proteins such as antigen viruses contained therein. This PCR method is a highly accurate method in which a sample is taken, the genes contained in the sample are amplified, and testing 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 testing time is approximately six hours or more. 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.

Therefore, as a test to supplement the PCR method, a simple and rapid serological diagnostic method using immunochromatography and enzyme-linked immunosorbent assay (ELISA) to detect virus-specific antibodies in serum has 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 at the outpatient clinic or bedside, and it is desired to introduce it into clinical practice 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.

Under these circumstances, virus testing requires accuracy comparable to the PCR method for amplifying genes, and a simple and rapid test equivalent to the immunochromatography method. Therefore, the inventors have conducted intensive research to realize a method that can achieve these two goals: accuracy comparable to the PCR method, and a simple and rapid test 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. By using a primary antibody and a secondary antibody in sequence, the distribution of the primary antibody in tissues and cells, that is, the distribution of the antigen it recognizes, is observed 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, they learned that even if they were able to take the time to solidify the virus as a sample, pseudo-samples would likely exist among the solidified samples, which would trigger non-specific reactions (a phenomenon in which some biological component other than the target being measured reacts abnormally with components in the measurement reagent or the additives in the blood collection tube, resulting in measured values that are far removed from the pathological condition) and reduce the accuracy of the measurement.

Patent Application No. 2020-74439

KONIKAMINORUTA Report 2012

The present invention aims to realize a method and device that has 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 plasmons 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 the 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 the fluorescent image, and the number of viruses can be counted as the number of dot-like fluorescence (hereinafter referred to as the fluorescence counting method), while the fluorescence counting method can eliminate or reduce non-specific reactions, significantly improving accuracy.

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 in that the fluorescence counting system comprises the steps of agglomerating 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 fluorescent spots or particles above a predetermined threshold. In the present invention, an antibody (usually diluted with a buffer solution, the same below) is solidified (a state in which it is aggregated on a metal substrate by the electrode potential difference with the 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, usually diluted with a buffer solution; the same below) are mixed and dropped onto the solidified substrate, and the antigen is sandwiched between the antibody and the labeled antibody, a so-called antigen-antibody reaction is used. 1) In addition to the sandwich method, a viral antigen is solidified and then labeled with a labeled antibody, or an antibody is solidified and then labeled with a labeled antigen (labeled with a fluorescent substance, including antigens partially labeled; the same below), 2) a direct method, and 3) an indirect method are used in which a viral antigen is solidified and then an antibody and a secondary antibody are bound in order to label it, or an antibody is solidified and then a viral antigen is bound, and finally an antibody and a secondary antibody are bound in order to label it.

First, the present invention enhances the fluorescence of the labeled fluorescent molecules associated with the virus or its antibody captured by the antigen-antibody reaction by efficiently exciting surface plasmons using a plasmon metal complex immobilized at the same time, so that specimens such as viruses can be quantified as fluorescent dots. 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 field light induced on the surface of a gold film electrode. The fluorescent signal appears as dots or particles in a fluorescent image observed with a fluorescent microscope, and when this is binarized and the fluorescent dots or particles above a certain threshold are counted, it correlates with the number of viruses and antibodies, making it possible to accurately quantify the number of viruses and antibodies. 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 that tests 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 fluorescent counting method. Furthermore, unlike solid-phase substrates that are excited by localized light field induced on the gold film electrode surface, there is no need for microchannels, and spurious fluorescent signals due to non-specific reactions are also eliminated.

The difference between the conventional SPFS method and the fluorescence counting method of the present invention is that in the former, the specimen, such as a virus, is solidified as an organic molecule on a gold thin film, whereas in the latter, the specimen is formed and integrated by the aggregation of plasmon metal complex quantum crystals. The solidification technology of the specimen 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 technology. 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).

That is, the inventors have 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 measurement, the measured image shows that, as shown in FIG. 6, 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. This granular fluorescence is the fluorescence of the labeled antibody sandwiching the virus, and counting the number of fluorescent particles above a certain threshold correlates with the number of viruses (FIG. 6(a)). On the other hand, when there is no virus, the 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). 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 solidified 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 quantifying the number of viruses based on the presence or absence of viruses and the number of counts.

The present invention provides a novel solid-phase specimen fluorescence counting method that is excellent for image search observed with a fluorescence microscope in a surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) method, and can count the number of granular fluorescent particles in a fluorescent image to analyze diseases based on the presence or absence of viruses and the amount of counts.

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) is used, 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 for collecting specimens for PCR testing. Copan UTM is one example. The liquid culture medium of Copan UTM contains sucrose, gelatin, etc. in addition to HEPES buffer, and 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 inactivating solution, but it is preferable to inactivate the virus by mixing it with a buffer solution in order 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 sample taken from a human is immersed in the solution to inactivate it, and then the solution is 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 solution.

In another method of the present invention, the antigen is immobilized using the quantum crystal aggregation method of the plasmon metal complex, and the antigen is reacted with the antibody to form an antibody immobilized substrate having gaps or microchannels between the quantum crystals, and a labeled secondary antibody labeled with a fluorescent substance is then bound to the substrate.

In addition, in the present invention, metal powder may be used instead of the metal substrate. In this case, after cleaning, the remaining complex or labeled secondary antibody is irradiated with excitation light to excite the quantum crystal with surface plasmons, enhancing the fluorescence of the complex or the labeled secondary antibody, and the resulting fluorescent image is observed and the number of granular fluorescent particles in the image is counted and detected.

In the present invention, the antigen is usually sandwiched between fluorescently labeled antibodies (primary antibodies), 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.

According to the present invention, the number of fluorescent particles of the virus antigen, rather than the fluorescence intensity of the virus antigen, can be measured as the virus concentration. Moreover, since the quantum crystals forming the antibody or antigen solid-phase substrate have 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 crystals, which excites the surface plasmon and enhances the fluorescence of each complex or secondary labeled antibody, so that the granular fluorescence, rather than the overall fluorescence intensity, can be counted and detected with good reproducibility. 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 PCR tests, which require complicated pretreatment, have low sensitivity due to primers, have many protocols, and take a long time to test. In addition, it is revolutionary because it can not only determine the presence or absence of a disease, but also determine the severity of the disease because the count number corresponds to the number of viruses.

The present invention provides an antibody testing method that captures antibodies produced in the body that are effective against specific viruses. Here, an antigen is captured with a primary antibody and a fluorescently labeled secondary antibody, and the fluorescence becomes stronger when the primary antibody or the labeled secondary antibody is bound to the primary antibody or the labeled primary antibody, so by adding a labeled secondary antibody, it becomes possible to detect the fluorescence with higher sensitivity.

(Embodiment 1)
This method for quantifying the measurement target of virus or antibody in a specimen comprises: 1) a step of preparing a solid-phase substrate by a step of solidifying inactivated virus or its antibody, 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 point-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 virus in the specimen (Figs. 7 and 8).
(Embodiment 2)
In the solid-phase process, the inactivated virus or its antibody is taken up in a buffer solution, mixed with 1000 ppm to 5000 ppm, preferably 1000 to 3000 ppm, of an aqueous solution of a plasmon metal complex to neutralize it, and then 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 immobilized substrate increases with the increase in the 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 a fluorescently labeled antigen with a part of the antigen; 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 plasmon metal complex quantum crystal aggregates of about 100 nm that are aggregated with antigens or antibodies on a metal substrate (FIG. 9-1), the quantum crystals cause a surface plasmon excitation phenomenon, exciting the fluorescent labels of the viruses or their antibodies that are immobilized with the quantum crystals. This allows for measurements with fewer non-specific reactions, and the number of fluorescent spots with brightness values above a certain threshold is accurately obtained by surface plasmon excitation, which correlates with the virus or antibody concentration, making quantitative measurements possible (FIG. 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 of two or more visual field measurements, thereby enabling rapid quantitative measurement (Figure 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).

FIG. 1 is a schematic diagram showing steps (1) to (4) of the first method (sandwich method) of the present invention. FIG. 1 is a schematic diagram of steps (1) to (3) of the first method of the present invention when metal powder is used instead of a metal substrate. FIG. 1 is a schematic diagram of steps (4) to (5) of the first method of the present invention when metal powder is used instead of a metal substrate. FIG. 1 is a schematic diagram showing steps (1) to (4) of the second method (indirect method) of the present invention. 1A to 1C are process diagrams showing a method for producing a quantum crystal substrate according to the present invention. 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. (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. 1 is a graph showing influenza virus quantification performed using the fluorescence count number of the present invention. 1A to 1C are process explanatory diagrams illustrating a method for analyzing a fluorescent image according to the present invention. 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. 13 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 4000 ppm Ag reagent (silver thiosulfate aqueous solution) and phosphate buffer solution were used. 1 shows an SEM image and a component analysis graph of a solid-phase substrate when a phosphate buffer solution containing 4000 ppm Ag reagent and influenza antibody (50 μg/ml) was used. 1 shows an SEM image and a component analysis graph of a solid-phase substrate when a phosphate buffer solution containing 4000 ppm Ag reagent and influenza virus (50 μg/ml) was used. (a) is an image showing a method for producing an influenza antibody solid-phase substrate, and (b) is an image of a clear-view portion of the solid-phase substrate. FIG. 13 is a diagram showing a test process for the sensitivity of a solid-phase substrate. 13 is a graph showing the correlation between each concentration of FITC influenza antibody and the number of fluorescent spots counted in a fluorescent image. FIG. 1 is an image showing the relationship between immobilization of influenza antibodies and quantum crystal concentration. FIG. 1 is a process diagram of a method in which inactivated influenza virus antigens are immobilized and subjected to fluorescence counting by the method of the present invention (direct method). 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). FIG. 1 is a flow chart showing a method for fluorescent counting of inactivated Covid-19 virus using a patient sample by the method of the present invention (sandwich method). FIG. 1 is a schematic diagram showing an analysis method of the quantum crystal solid-phase fluorescence counting method of the present invention. 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. FIG. 1 is a schematic diagram of steps (5) to (7) of the third method of the present invention. FIG. 1 is a schematic diagram showing collection steps (1) to (3) using the specimen inactivation collection kit of the present invention. FIG. 1 is a schematic diagram showing collection steps (4) to (6) using the specimen inactivation collection kit of the present invention. 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. FIG. 1 is an outline diagram showing (3) the latter half of the fluorescent labeling process and (4) the surface plasmon excitation process when detecting two types of viruses using the sandwich method of the present invention.

In the present invention, the quantum crystal aggregation method is used to easily create a solid-phase substrate on a specified metal substrate and metal powder using the various materials listed below.

(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 a silver complex, a silver thiosulfate aqueous solution is aggregated on copper or a copper alloy with an electrode potential (high ionization tendency) lower than that of silver to form quantum crystals of the silver complex by adopting an electrodeposition method with an electrode potential difference. 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 a dispersant is used, its concentration should also be considered. It can usually be used in the range of 100 ppm to 1000 ppm, but 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 order to prepare nano-sized nanoclusters of 50 to 150 nm, which can be called nanoclusters 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 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 (log β) 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 plasmon 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 1)

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 (5 μg/ml) at quantum crystal concentrations of 1000, 2000, 3000, 4000, and 5000 ppm (solidification time 1 minute). Next, FITC-Avidin (5 μg/ml) was dropped onto the Biotin solid-phase substrate, and the image of Avidin labeled with FITC using Avidin-Biotin bonds was measured with a Keyence Corporation 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 there is a large amount of quantum crystals, 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: 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)

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 antigen-antibody reactions under a fluorescent microscope, and counted the fluorescent points 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
Equal amounts of influenza antibodies (100 μg/ml) were mixed with a buffer solution at quantum crystal concentrations of 2000, 4000, and 6000 ppm, 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 (10 μg/ml) and FITC-labeled influenza antibody (25 μg/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 viruses were detected when influenza was solid-phased with quantum crystals at 2000 ppm (total 1000 ppm). FIG. 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, 10x lens, 1-field measurement (here, 1-field measurement refers to a method of acquiring only one part of the chip, as shown in FIG. 16, unlike the case of Patent Application No. 2019-234330, which leads to a reduction in measurement time).
Equipment used: 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)

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 containing a virus and an antibody containing an antigen to be solidified, in addition to the quantum crystal metal complex aqueous solution, to solidify the quantum crystal aggregate. However, the antibody or antigen can be solidified using a quantum crystal aggregation method (JP 2016-197114 A) for producing a quantum crystal of a plasmon metal complex. Therefore, the method described in JP 2016-197114 A is cited and referenced in this specification. However, the virus and the antibody it produces are added to an inactivation liquid or buffer, mixed with a plasmon metal complex reagent (e.g., a silver thiosulfate aqueous solution) for solidification, and added to the solid-phased substrate to aggregate. Unlike the case of quantum crystals alone, the solidification of antigens and antibodies by quantum crystals is thought to be affected by interaction with the buffer and changes in pH.
FIG. 9 shows SEM images of a control (FIG. 9-1(a)) in which an equal amount of phosphate buffer 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 at 50 μg/ml each, and an equal amount of 2000 ppm of silver thiosulfate solution (Ag reagent) was mixed with this (FIG. 9-1(a)) and (FIG. 9-1(b)). When a buffer solution was added, it was found that the quantum crystals were 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 the quantum crystal silver complex is solidified. However, antibodies and antigens are not detected.
(Preparation of influenza antibody immobilized substrate and its sensitivity)

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, the antibody was solidified in about 1 minute, and the remaining liquid on the metal plate was blown off with air to obtain an antibody-immobilized substrate (Figure 10(a)). Figure 10(b) is a clear-view image of the solid-phase substrate.
Using the quantum crystal aggregation method, FITC-labeled influenza antibodies with different concentrations were immobilized to prepare antibody-immobilized substrates, and the number of fluorescent spots in each of the obtained 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 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)

FIG. 13 shows the steps of the direct method for testing influenza viruses (immobilization of inactivated influenza viruses).
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 number of fluorescent spots is 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 of equal amounts of the above-mentioned silver thiosulfate aqueous solution (Ag reagent) 2000 ppm and early influenza virus (50 μg/ml) in phosphate buffer (pH 7.4) was added to a phosphor bronze plate to create a virus-immobilized substrate (immobilization took about 1 minute). For comparison, a buffer solution without viruses was mixed with the quantum crystal Ag reagent to form an immobilized substrate. Next, FITC-labeled influenza antibody (25 μg/ml) was dropped onto the two immobilized substrates (reaction time was only 1 minute). Unbound complexes and FITC antibodies were washed away with water or buffer. 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 immobilized substrate.
(Immobilization of antibodies part 1)

FIG. 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 complex and FITC antibody 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 those for the immobilization of the virus.
(Immobilization of antibodies part 2)

The inspection process is shown in Figure 15. A complex of an inactivated COVID-19 patient sample and an 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 resulting fluorescent image.
COVID-19 antibody: GeneTex “SARS-COV-2 spike antibody” CatNo. GTX135356
FITC-labeled COVID-19 antibody: GeneTex 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 using a Keyence Corporation fluorescent microscope "BZ-X710," and the number of fluorescent points in the obtained fluorescent image that are equal to or greater than a predetermined threshold 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 as follows: Blank: count 8 (relative value 0), Sample 1: count 16 (relative value 8), 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.

(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 one minute, and then the solution was blown away. 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 were thin hexagonal columnar crystals of about 100 nm, and the surface had irregularities of the order of several nm. No facets specific to metal nanocrystals were observed. 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 three minutes, and the solution was blown away, only silver was detected.

(Quantum crystal agglomeration theory)
When a 2000 or 4000 ppm silver thiosulfate complex solution is dropped onto a phosphor bronze plate and left for one minute, quantum crystals are formed in the shape of hexagonal columns of about 100 nm, and each hexagonal column quantum crystal has irregularities on the order of several nm. The SEM image confirmed that the nanocrystals were made of silver, but the facets characteristic of metal nanocrystals were not observed. EDS elemental analysis detected elements derived from silver and the complex ligands, so the entire structure was determined to be a nanocrystal of silver complex. It is presumed that the unevenness that appears on the surface is due to the silver in the complex forming quantum dots as clusters and spreading out. The silver complex quantum crystal of the present invention is formed on a phosphor bronze plate, while on a copper substrate When we look at the phenomenon of the deposition of nanoparticles of only silver, the equilibrium potential of the silver thiosulfate complex is 0.33, which is equivalent to the electrode potential of copper (0.34), so only silver (0. 80) precipitated, and in the case of phosphor bronze, the electrode potential was 0.22, which is slightly less noble, so it is believed that silver complex crystals precipitated. Therefore, in order to create quantum crystals, 1) the complex aqueous solution must be in the dilute range of 500 to 2000 ppm, 2) the electrode potential of the supported metal must be slightly lower than the equilibrium potential of the metal complex aqueous solution, and 3) ) It is important that the metal complex is agglutinated by the electrode potential difference, but it has been found that it is desirable to use a higher concentration of Ag thiosulfate quantum crystal reagent of 2000 ppm or more for immobilizing the antigen and 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 through the aggregation of quantum crystals. This may affect the state of quantum crystal formation and affect the measured values. Therefore, in order to keep the quantum crystal formation area constant, as shown in Figure 4, 1) the substrate of the phosphor bronze plate is placed in the liquid drop area. 2) The area is left as is or polished with sandpaper, electrolytic polishing, or chemical polishing, and 3) Ag reagent solution (2000-4000 ppm silver thiosulfate solution) is dripped onto the area. The liquid is collected in the circular groove by surface tension, and 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 the crystals could be used as is, electrolytic polishing, chemical polishing, It was found that sandpaper polishing caused variation in the measurement results.

(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.

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 viruses, 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.

In the present invention, specimens collected from humans or animals and specimens containing inactivated viruses or fragmented parts are included as solid-phase targets, and body fluids collected from humans or animals, such as throat swabs, saliva, blood, and urine, are also included as specimens.

(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.)

(Example 2 of solid-phase substrate)
A solution of equal amounts of Ag reagent (2000ppm, 12.5μl) and hemagglutinin H1 influenza A antibody (25μg/ml, 12.5μl) for making 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 under 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.)

(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 immobilized substrate. The complex of the influenza virus and the FITC-labeled antibody binds to the immobilized antibody on the immobilized substrate, and when observed under a fluorescent microscope, the fluorescence of the FITC 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

(Method of analyzing fluorescent images)
The fluorescence images were analyzed as shown in Figure 8 (1) to (3).
Step (1) is an 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 grains). Here, as shown in Figure 16, only one 10x objective lens is photographed (one visual field measurement), and the photographing time is as short as 3.5 seconds if binarized and counted, but multiple images may be taken.
Step (2) is a binarization step, in which all fluorescent particles with a brightness value equal to or greater than 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 equal to or greater than 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 only fluorescent red particles (dashed line area) is calculated).

"Fluorescence image acquisition process"
Here, a technique can be used as a reference in which the fragmented DNA captured on the 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 Japanese Patent Application No. 2019-234330).

Example 4
Similarly, a liquid was prepared by mixing Ag reagent (2000ppm, 5μl) used to create quantum crystals with FITC-labeled H1N1 influenza virus antibodies (250μg/ml, 5μl), 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 500ppm to 10000ppm, and FITC-labeled H1N1 influenza virus antibodies have a range of 1pg/ml to 1g/ml.)

(Measurement Example 1)
The present invention applies the surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) method to detect viruses in patients, using throat swabs, saliva, urine, and feces in which viral antigens are present. Figure 1 shows the method consisting of steps (1) to (4). In step (1), an antibody solid-phase substrate is prepared using the quantum crystal aggregation method. In detail, a viral antibody is added to a buffer solution (phosphate buffer solution at pH 7.4) to prepare a solid-phase antibody solution. An equal amount of a plasmon metal complex solution with a concentration of 1000 to 10000, preferably 2000 to 4000 ppm is added to this solution 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 then 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 solid-phase substrate on which the viral antibody is solidified. 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 antibody includes influenza virus antibodies, monoclonal antibodies of 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 including five classes, 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.), proliferating the virus, 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 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 portions of antibodies 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. The specimen may be a throat swab, saliva, urine, or feces. Examples of fluorescent substances that label the viral antibody include those with an excitation light of 400-436 nm, such as Pacific Blue, 453-505 nm, such as FITC, 485-566 nm, such as TRITC, 488-706 nm, such as APC, and 732-784 nm, such as IRDye800.
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, 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), the labeled antibody-antigen complex remaining on the substrate is irradiated with excitation light, 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. Since 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, such a fluorescent measurement method is cited and referenced herein. In the present invention, the single visual field measurement conditions shown in FIG. 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 a chip, as shown in FIG. 16, unlike the case of Patent Application No. 2019-234330. It has been found that the single visual field measurement when using the solid-phase substrate according to the present invention gives almost the same average value of two or more single visual field measurements, and therefore the result of the single visual field measurement can be used without adopting the average value. This shows that the solid phase of the antigen or antibody is formed almost uniformly by the quantum crystal).

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 Fig. 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 the aqueous solution 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, where 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.

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 a virus or a fragmented part of a virus such as a viral nucleocapsid) and capturing an antibody produced in the body. As shown in Figure 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 plasmons, 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 an aqueous solution of a plasmon metal complex at a concentration of 500 to 10,000 ppm. Here, as the viral antigen or a part thereof, throat swabs, saliva, urine, feces, or a part of a non-infectious antigen is used.
Treatment of viral antigens or parts thereof can be performed using 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 utilizing 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) may 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 having a brightness value or greater 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.

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 specimens to be tested are pharyngeal swabs, saliva, urine, and feces. Fluorescent substances that label virus antibodies include those with excitation light of 400-436 nm, such as Pacific Blue, those with excitation light of 453-505 nm, such as FITC, those with excitation light of 485-566 nm, such as TRITC, those with excitation light of 488-706 nm, such as APC, and those with excitation light of 732-784 nm, such as IRDye800.
Combinations of primary and secondary labeled antibodies are made with a secondary labeled antibody that recognizes the animal species from which the primary labeled antibody originates. For example, if a primary labeled antibody derived from a mouse is used, a secondary labeled antibody that recognizes mouse antibodies should be used, 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 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.
(Actual measurement based on antigen-antibody reaction of influenza virus)

Equal amounts of Ag reagent (500-10,000 ppm), which is the basis 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 (5-1000 μg/ml) and FITC-labeled influenza antibody (5-1000 μg/ml) are mixed and dropped onto the quantum crystal substrate.
Reagent examples: Influenza antibody HyTest Monoclonal mouse anti-Influenza A haemagglutinin H1
Influenza A (H1N1) Virus (HyTest)
- FITC influenza antibody IBL Anti-Influenza A Virus (H1N1) FITC was used.

The excess FITC-labeled influenza antibody was washed with pure water, etc., and the antibody was irradiated with light from a light source (metal halide lamp 80W) and observed under a fluorescent microscope (Keyence fluorescent microscope BZ-X710).
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 FIG. 6(a). In contrast, the result is shown in FIG. 6(b) when no influenza antigen is included. 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 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 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)). On the other hand, when the virus is not present, no numerous granular fluorescent light appears (Figure 6(b)).
Using influenza antibody (25 μg/ml) and FITC influenza antibody (25 μg/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 x10, and the results shown in the table in Figure 7 were obtained. This was 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)

The present invention is suitable for a method of quickly performing virus testing on the spot at immigration inspections and hospital diagnosis, and may include inactivating or attenuating antigens in specimens (throat swabs, saliva, sputum, nasopharyngeal fluid, urine, etc.) collected from humans. (The same applies below) and then immobilizing the inactivated antigen on a substrate by a quantum crystal aggregation method, labeling the immobilized antigen by binding a labeled antibody to the antigen through an antigen-antibody reaction, washing the unbound labeled antibody with a buffer solution or pure water, irradiating the antibody label (fluorescent substance) from a light source with excitation light, and counting fluorescent particles on the substrate with a fluorescence microscope. Here, the quantum crystal aggregation method refers to an aggregation method for producing quantum crystals of plasmon metal complexes as shown in JP 2016-197114 A, and refers to a method in which plasmon metal complexes in a solution are aggregated as quantum crystals of metal complexes 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 using the quantum crystal aggregation method, and then the antigens are labeled by binding the labels to the substrate through an antigen-antibody reaction with a labeled antibody, after which the unbound labeled antibodies are washed with a buffer solution or pure water, and the fluorescent particles on the substrate are counted using a fluorescence microscope after irradiating the substrate with excitation light that matches the antibody label (fluorescent substance) from a light source.

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. Therefore, whether or not a virus is susceptible to inactivation by drugs depends on whether or not it has an envelope, but in general, enveloped viruses are sensitive to disinfectants, so the use of drugs 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 alteration 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.

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 narrow, 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.

According to the present invention, the 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 the 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 interacts with the photons incident by the excitation light and the free electrons of the plasmon metal particles forming the quantum crystal, exciting the surface plasmon to enhance 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 rapid testing in a short time of 2 to 5 minutes, providing highly accurate diagnostic results that can replace PCR testing, which requires complicated pretreatment, has low sensitivity due to primers, has many protocols, and takes a long time to test. Furthermore, this is revolutionary because it can not only determine whether or not a disease is present, but also determine the severity of the disease, since the count corresponds to the number of viruses.

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 samples)

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 quantum crystals was mixed and dropped onto a phosphor bronze plate, and the hemagglutinin H1 influenza A antibody was immobilized along with the quantum crystals aggregated on the plate. Next, a complex of an inactivated sample (25 μg/ml, 5 μl) and an FITC-labeled H1N1 influenza virus antibody (25 μg/ml, 5 μl) was dropped onto the hemagglutinin H1 influenza A antibody immobilized plate. If the H1N1 influenza virus was present in the inactivated sample, the complex of the influenza virus and the FITC-labeled antibody bound to the antibody immobilized on the immobilized plate, and FITC fluorescence was observed on the particles when observed under a fluorescent microscope. If the influenza virus was not present in the sample, no FITC fluorescence was observed on the particles. When the measurement images obtained by the fluorescence microscope were analyzed using Keyence Corporation's analysis software: BZ-X Analyzer with a threshold set at 57, a large difference was observed in the count value of the granular FITC fluorescence. As a result, when the fluorescent particles in the fluorescence images were counted, the number was 4 in the absence of the virus, whereas the number was 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

(Example 1 of measuring patient virus)
The fluorescence counting method of the present invention is applied to the detection of viruses in patients, and throat swabs, saliva, sputum, nasopharyngeal fluid, urine, and feces in which viral antigens are present are used. Figure 17 shows a method consisting of steps (1) to (7). In step (1), an inactivated sample is prepared using a sample inactivation and collection kit shown in Figure 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 the quantum crystal aggregation method. More specifically, the inactivated antigen is added to a plasmon metal complex aqueous solution with a concentration of 2000 to 6000 ppm to prepare a composite aqueous solution of the plasmon metal complex and the inactivated antigen, 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, 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 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 reference is made.
In step (5), the virus antigen in the solid-phase specimen is labeled with a virus antibody labeled with a fluorescent substance by utilizing an antigen-antibody reaction. The specimen may be a throat swab, saliva, sputum, nasopharyngeal fluid, urine, or 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, 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 (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 easily 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)

The following method is described as a measurement method for detecting two types of viruses using one measurement chip.
1) Mix equal amounts of a mixture of influenza antibody (containing multiple antibodies as influenza antibodies) buffer and coronavirus (Covid-19) antibody buffer with the Ag reagent (silver thiosulfate aqueous solution 3000 ppm) to prepare a solid-phase specimen containing 1000 ppm 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 prepare a solid-phase substrate (see FIG. 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 FIG. 19A(2)). Unbound complexes and labeled antibodies are washed away with water or a buffer solution (see FIG. 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 FIG. 19B(4)). In this way, two fluorescent images obtained from the two excitation lights are obtained with a fluorescent microscope, and the fluorescent points or particles on the images are counted to perform a quantitative count.
In addition, the filter in the green region: 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 (8)

A method for quantifying the virus or antibody to be measured in a specimen, comprising: 1) a solidification step in which inactivated viruses or antibodies thereof are coagulated together with plasmon metal complex quantum crystals on a metal substrate by the potential difference between the plasmon metal complex and the metal substrate, and then fixed together with the metal complex quantum crystals on the metal substrate to solidify the viruses or antibodies; 2) a labeling step in which the solidified viruses or antibodies are fluorescently labeled by an antigen-antibody reaction; 3) a fluorescence excitation step in which excitation light is applied to the fluorescently labeled viruses or antibodies to obtain a dot-like or granular fluorescent image of the fluorescently labeled viruses or antibodies by surface plasmon excitation; and 4) a fluorescence counting step in which the fluorescent points or particles in the fluorescent image are binarized, and fluorescent points or particles equal to or greater than a predetermined threshold are selected and counted. 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 1,000 to 3,000 ppm of a plasmon metal complex to neutralize it, and then dropped onto the metal substrate. The fluorescence counting method according to claim 1, wherein the object to be immobilized in the specimen is an antibody-producing virus (inactivated) or an antibody thereto, and the concentration of the virus in the specimen is 10 μg/ml or more. 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. 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. The fluorescence counting method according to claim 1, wherein the fluorescence counting step is a measurement image of a fluorescence microscope in one or more visual field measurements, and involves counting the number of fluorescent points having a predetermined brightness value or more, which are collected by binarizing the punctate fluorescence in any one visual field image. The fluorescence counting method according to claim 1, in which, in the solid-phase immobilization step, antibodies that bind to two or more different viruses through an antigen-antibody reaction are immobilized, in the labeling step, the viruses are labeled with labeled antibodies having different fluorescent wavelengths, and in the fluorescence counting step, one or more viruses are quantified. 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.