JP7178623B2 - detector - Google Patents

Description

The present disclosure relates to detection devices for detecting influenza virus in samples.

Conventionally, a fluorescence method has been widely used as a technique for detecting a substance to be detected in a sample (see Patent Document 1, for example). In Patent Document 1, first, a substance to be detected to which an antibody labeled with a fluorescent substance (hereinafter referred to as a labeled antibody) is bound is immobilized on a sensor portion including a metal layer (hereinafter referred to as an immobilized antibody ) onto the sensor part. Then, by irradiating the sensor section with excitation light, plasmons are excited in the metal layer to generate an optical electric field enhanced by the plasmons. At this time, the amount of the substance to be detected can be detected by measuring the amount of fluorescence generated from the fluorescent substance of the labeled antibody in the enhanced optical field (hereinafter referred to as the enhanced electric field).

In addition, in Patent Document 1, a fragmented antibody (hereinafter referred to as a fragmented antibody) is used as the immobilized antibody. As a result, the distance between the fluorescent substance and the metal layer becomes shorter than when using a normal antibody, and the enhanced electric field can be efficiently used to detect optical signals.

In addition, Patent Documents 2 and 3 disclose antibodies that bind to influenza viruses.

JP 2010-043934 A U.S. Patent Application Publication No. 2014/0302063 JP 2018-58811 A

However, since the concentration of influenza viruses floating in the air is very small, it is difficult for the above-described conventional technology to detect, for example, influenza viruses collected from the air.

Accordingly, the present disclosure provides a detection device capable of detecting minute amounts of influenza virus.

In the detection device according to one aspect of the present disclosure, a first VHH antibody having a property of specifically binding to an intranuclear protein of influenza A virus is immobilized, and surface plasmons are generated by irradiation with excitation light. A metal microstructure, a second VHH antibody that has a property of specifically binding to the influenza A virus nuclear protein and is labeled with a fluorescent substance, and the influenza A virus nuclear protein. an introduction unit for introducing a sample to be obtained into the metal microstructure; a light irradiation unit for irradiating the metal microstructure into which the second VHH antibody and the sample have been introduced with the excitation light; a detection unit that detects the intranuclear protein of the influenza A virus based on the fluorescence generated from the fluorescent substance by light irradiation, wherein the first VHH antibody is composed of FR1, CDR1, Having structural domains arranged in the order FR2, CDR2, FR3, CDR3, FR4, wherein FR denotes framework regions, CDR denotes complementarity determining regions, and in said first VHH antibody, said CDR1 is , comprising the amino acid sequence shown in SEQ ID NO: 1, wherein said CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, said CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, and said second VHH antibody comprises having structural domains arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus, wherein said CDR1 has the amino acid sequence shown in SEQ ID NO: 4 in said second VHH antibody wherein said CDR2 comprises the amino acid sequence shown in SEQ ID NO:5 and said CDR3 comprises the amino acid sequence shown in SEQ ID NO:6.

In addition, these general or specific aspects may be realized by a system, method, integrated circuit, computer program, or a recording medium such as a computer-readable CD-ROM. and any combination of recording media.

According to the present disclosure, minute amounts of influenza virus can be detected.

FIG. 1 is a schematic configuration diagram of a detection system according to Embodiment 1. FIG. FIG. 2 is a configuration diagram of a detection device according to Embodiment 1. FIG. 3A is a perspective view of a detection region of a sensor cell according to Embodiment 1. FIG. 3B is an enlarged cross-sectional view of the metal microstructure according to Embodiment 1. FIG. FIG. 4 is a diagram showing a SAM on a metal microstructure according to Embodiment 1. FIG. 5 is a flowchart showing the operation of the detection device according to Embodiment 1. FIG. FIG. 6 is a diagram showing a SAM on a metal microstructure according to Embodiment 2. FIG. 7 is a graph showing the normalized NP binding amount for evaluating the competitiveness of VHH antibodies for NP in Example 2. FIG. 8 is a graph showing the amount of immobilized VHH antibody in Example 3. FIG. 9 is a graph showing the amount of immobilized VHH antibody and IgG antibody in Example 4. FIG. FIG. 10 shows the surface-enhanced fluorescence (SEF) detection results when a sequential sandwich assay was performed using Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Nucleoprotein in Example 5 as an antigen. It is a graph showing. 11 is a graph showing the results of detection of SEF when a sequential sandwich assay was performed using Influenza A H1N1 (A/California/07/2009) Nucleoprotein as an antigen in Example 5. FIG. FIG. 12 is a graph showing the detection results of SEF when a sequential sandwich assay was performed using Influenza A H3N2 (A/Switzerland/9715293/2013) Nucleoprotein in Example 5 as an antigen. 13 is a graph showing the results of detection of SEF when a sequential sandwich assay was performed using Influenza A H7N9 (A/Shanghai/2/2013) Nucleoprotein in Example 5 as an antigen. FIG. 14 is a graph showing the detection results of SEF when a pre-mixed reaction sandwich assay was performed using Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Nucleoprotein in Example 6 as an antigen. . 15 is a graph showing the results of SEF detection when sequential sandwich assays were performed using IgG antibodies as the immobilized antibody and the labeled antibody in Comparative Example 1. FIG.

(Findings on which this disclosure is based)
In the fluorescence method using the fragmented antibody described in Patent Document 1, for example, as an immobilized antibody and a labeled antibody, the labeling substance bound to the sensor part including the metal layer via the immobilized antibody and the sensor part The distance to the enhanced magnetic field generation surface is shorter than when binding via a normal antibody. As a result, in the prior art described in Patent Document 1, metal quenching can be effectively prevented, and the enhanced magnetic field can be efficiently used. However, with the conventional technique described in Patent Document 1, it is difficult to detect a low-concentration substance to be detected. The present inventors discovered that the reason for this is the reduction in antigen-capturing ability due to antibody fragmentation. A fragmented antibody is a portion of an antibody molecule. Specifically, fragmented antibodies are obtained by digesting normal antibodies with protease. A fragmented antibody also has at least one antigenic determinant that can specifically bind to a substance to be detected. In other words, since fragmented antibodies fragment antibodies, their antigen-capturing ability is lower than that of normal antibodies. Therefore, when a fragmented antibody is used as an immobilized antibody and a labeled antibody, the ability of the immobilized antibody to capture the substance to be detected is low, making it difficult to detect a substance to be detected at a low concentration. lead to a decline in

The antibodies described in Patent Documents 2 and 3 are antibodies containing VHH antibodies or composed of VHH antibodies, and bind to influenza virus. VHH antibodies are variable regions of heavy chain antibodies possessed by camelids (llamas, alpacas, etc.). Thus, VHH antibodies are naturally occurring single domain antibodies. VHH antibodies are characterized by having a lower molecular weight (that is, smaller size) than ordinary IgG antibodies, high structural stability, and a long CDR3 region, which is one of the complementarity determining regions. Therefore, VHH antibodies have a higher antigen-capturing ability than fragmented antibodies obtained by fragmenting IgG antibodies. Among such VHH antibodies, the VHH antibody described in Patent Document 3 is known to have high reactivity with the intranuclear protein of influenza A virus.

By the way, in the surface-enhanced fluorescence method in which the fluorescence based on the substance to be detected is enhanced by surface plasmon and detected, the decrease in the detection sensitivity in the fluorescence method can be reduced by using the VHH antibody as the immobilized antibody and the labeled antibody. can be done. At this time, if the same VHH antibody is used as the immobilized antibody and the labeled antibody, it is difficult to sandwich the antigen effectively.

Therefore, in the detection device according to one aspect of the present disclosure, the first VHH antibody having the property of specifically binding to the intranuclear protein of influenza A virus is immobilized, and surface plasmons are generated by irradiation with excitation light. a metal microstructure to be produced, a second VHH antibody that has the property of specifically binding to the influenza A virus nuclear protein and is labeled with a fluorescent substance, and the influenza A virus nuclear protein. and a sample that can contain the introduction unit for introducing into the metal microstructure, a light irradiation unit for irradiating the excitation light to the metal microstructure into which the second VHH antibody and the sample have been introduced; a detection unit that detects the intranuclear protein of the influenza A virus based on the fluorescence generated from the fluorescent substance by irradiation with the excitation light, wherein the first VHH antibody is composed of FR1, Having structural domains arranged in the order of CDR1, FR2, CDR2, FR3, CDR3, FR4, FR indicates a framework region, CDR indicates a complementarity determining region, and in the first VHH antibody, CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, said CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, said CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, said second VHH antibody has structural domains arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus, wherein in said second VHH antibody said CDR1 is shown in SEQ ID NO: 4 comprising an amino acid sequence, wherein said CDR2 comprises the amino acid sequence shown in SEQ ID NO:5 and said CDR3 comprises the amino acid sequence shown in SEQ ID NO:6.

According to this configuration, the first VHH antibody and the second VHH antibody each have a complementarity determining region containing different amino acid sequences, and the first VHH antibody and the second VHH antibody are of influenza A virus. It can bind to different sites of nuclear proteins (hereafter NP). Therefore, NP can be effectively sandwiched between the first VHH antibody and the second VHH antibody. As a result, the detection device according to an aspect of the present disclosure improves the NP trapping performance, thereby improving the NP detection sensitivity by the surface fluorescence enhancement method using the metal microstructure. Therefore, the detection device according to one aspect of the present disclosure can detect a small amount of influenza A virus with high sensitivity by having the above configuration.

For example, in the detection device according to an aspect of the present disclosure, the influenza A virus may be at least one of H1N1, H3N2, and H7N9, which are subtypes of influenza A virus . .

In this way, the detection device according to one aspect of the present disclosure can detect a plurality of influenza A subtypes, and thus can detect influenza A virus with high accuracy.

For example, in the detection device according to one aspect of the present disclosure, in the first VHH antibody, the FR1 contains the amino acid sequence shown in SEQ ID NO: 7, and the FR2 contains the amino acid sequence shown in SEQ ID NO: 8. , the FR3 may comprise the amino acid sequence shown in SEQ ID NO:9, and the FR4 may comprise the amino acid sequence shown in SEQ ID NO:10.

According to this configuration, for example, when the first VHH antibody is bound to the SAM linker molecule, that is, when immobilized on the SAM, the amino group of the lysine residue in any of the above FRs binds to the carboxyl group in the linker molecule. In addition, since the lysine residue does not exist in the CDR but exists in the FR and is sterically separated from the CDR, the first VHH antibody is fixed to the SAM. The first VHH antibody can retain its antigen-capturing ability even when modified.

For example, in the detection device according to one aspect of the present disclosure, in the second VHH antibody, the FR1 contains the amino acid sequence shown in SEQ ID NO: 11, and the FR2 contains the amino acid sequence shown in SEQ ID NO: 12. , the FR3 may comprise the amino acid sequence shown in SEQ ID NO: 13, and the FR4 may comprise the amino acid sequence shown in SEQ ID NO: 14.

According to this configuration, the second VHH antibody can stably maintain the CDR conformation (eg, loop structure). Therefore, the CDRs can exert antigen-trapping ability.

For example, in the detection device according to one aspect of the present disclosure, a self-assembled monolayer (SAM) containing linker molecules and non-linker molecules is formed on the surface of the metal microstructure. , the first VHH antibody may be immobilized on the metal microstructure via the linker molecule.

According to this configuration, a SAM containing linker molecules and non-linker molecules is formed on the surface of the metal microstructure. Therefore, the first VHH antibody can be immobilized by the linker molecule while the non-linker molecule prevents the second VHH antibody and the fluorescent substance from attaching to the SAM. In other words, it is possible to reduce non-specific adsorption while maintaining the immobilized amount of the first VHH antibody.

For example, in the detection device according to one aspect of the present disclosure, the linker molecule includes an alkyl chain having a thiol group at one end and a carboxyl group at the other end, and having 10 or more carbon atoms, and ethylene glycol. and said non-linker molecule may comprise an alkyl chain of 10 or more carbon atoms having a thiol group at one end and a hydroxyl group at the other end, and an ethylene glycol chain.

According to this configuration, the SAM can contain alkyl chains with 10 or more carbon atoms. As a result, a dense SAM can be achieved, and both improvement in the ability to immobilize the first VHH antibody and reduction in non-specific adsorption can be achieved. Additionally, the SAM can contain an ethylene glycol chain. As a result, it is possible to impart mobility to the carboxyl group located at the end of the linker molecule, and improve the binding property between the first VHH antibody bound to the carboxyl group and the substance to be detected, while the second VHH antibody is sterically hindered. non-specific adsorption of VHH antibodies and fluorescent substances can be reduced.

Embodiments of the present disclosure will be described below with reference to the drawings.

It should be noted that the embodiments described below are all comprehensive or specific examples. Numerical values, shapes, materials, components, arrangement positions and connection forms of components, steps, order of steps, and the like shown in the following embodiments are examples, and are not intended to limit the scope of the claims. In addition, among the constituent elements in the following embodiments, constituent elements that are not described in independent claims representing the highest concept will be described as arbitrary constituent elements.

Also, each figure is not necessarily strictly illustrated. In each figure, substantially the same configurations are denoted by the same reference numerals, and overlapping descriptions are omitted or simplified.

In addition, in the following embodiments, a case where influenza A viruses floating in the air are collected and detected will be described as an example, but the present invention is not limited to this. For example, influenza A virus contained in objects (for example, dust on the floor) on which coughs and vomits of an infected person are attached may be collected and detected.

(Embodiment 1)
A detection device according to this embodiment will be described below. The detection device according to the present embodiment is, for example, a device capable of detecting a minute amount of influenza A virus floating in the air. Here, detecting an influenza A virus means detecting a nuclear protein (NP: Nucleoprotein) of the influenza A virus. Detecting influenza A virus may also include detecting the presence or absence of the virus. The detection device may be installed in facilities where many people gather, such as hospitals, commercial facilities, airports, and schools, or may be installed in places where families gather, such as the living room of a house. In this case, the detection device is one configuration of a detection system that collects air around the detection device and detects influenza A virus contained in the collected air.

[Overview of detection system]
FIG. 1 is a schematic configuration diagram of a detection system 10 according to Embodiment 1. FIG. The detection system 10 is installed, for example, in a room where people enter and leave, such as a hospital waiting room. As shown in FIG. 1 , detection system 10 includes collection device 100 , detection device 200 , and controller 300 . Details of the collection device 100, the detection device 200, and the controller 300 will be described below.

[Configuration of collection device]
The collection device 100 collects fine particles that may contain influenza A virus in the air and mixes them with a collection liquid. As shown in FIG. 1, the collection device 100 includes an aspirator 102, a collection liquid tank 104, a pump 106, a cyclone 108, an air inlet 110, a cleaning liquid tank 112, a pump 114, and a waste liquid tank. 120 and a liquid channel 122 . Each component of the collection device 100 will be described below.

Aspirator 102 sucks ambient ambient air from air inlet 110 . Particles that may contain influenza A virus floating in the ambient air are sucked into the cyclone 108 from the air inlet 110 together with the air.

The collection liquid tank 104 is a container for holding a collection liquid for collecting influenza A viruses in the air.

Pump 106 supplies the collected liquid in collected liquid tank 104 to cyclone 108 .

The cyclone 108 is connected to the air inlet 110 and the collection liquid tank 104, and collects fine particles that may contain influenza A virus in the air sucked from the air inlet 110 by the aspirator 102 and the pump 106. It mixes with the collecting liquid supplied from the liquid tank 104 . Cyclone 108 is connected to detection device 200 via liquid flow path 122 . A collection liquid mixed with fine particles (hereinafter referred to as a sample) is discharged from the cyclone 108 through the liquid flow path 122 to the detection device 200 .

The cleaning liquid tank 112 is a container for holding cleaning liquid for cleaning the cyclone 108 and the liquid flow path 122 . The cleaning liquid tank 112 is connected to the cyclone 108 , and the cleaning liquid in the cleaning liquid tank 112 is supplied to the cyclone 108 by a pump 114 .

The waste liquid tank 120 is a container for storing unnecessary liquid.

Liquid flow path 122 is a path for guiding the sample output from cyclone 108 to detection device 200 .

[Configuration of detection device]
Next, the detection device 200 will be specifically described with reference to FIGS. 1 and 2. FIG. FIG. 2 is a configuration diagram of the detection device 200 according to Embodiment 1. As shown in FIG.

The detection device 200 detects influenza A virus from the sample obtained by the collection device 100 . As described above, the sample is a collected liquid mixed with fine particles that may contain influenza A virus floating in the air. As shown in FIGS. 1 and 2, the detection apparatus 200 includes a sensor device 202 , an introduction section 206 , a light source 208 , a beam splitter 210 , a lens 212 and a detection section 214 . Each component of the detection device 200 will be described below.

Sensor device 202 comprises a sensor cell 204 . Note that the sensor device 202 includes a single sensor cell 204 in FIG. 1, but may include a plurality of sensor cells.

In this embodiment, the sensor device 202 can detect NP of influenza A virus contained in the sample liquid 2061 in a concentration range of 0.1 pM to 100 nM. In addition, in this embodiment, a surface-enhanced fluorescence method is used to optically detect the amount of NP of the virus.

The sensor cell 204 enhances the fluorescence from the fluorescent substance bound to the influenza A virus NP by generating surface plasmons when irradiated with excitation light. As shown in FIG. 2, the sensor cell 204 comprises a channel 204a and a detection area 204b.

The channel 204a is a path for guiding the sample liquid 2061 dripped from the introducing portion 206 to the detection region 204b.

The detection region 204b is a region for optically detecting NP of influenza A virus using surface plasmons. A metal microstructure is arranged in the detection region 204b, and surface plasmons are generated by irradiating the excitation light from the light source 208 thereon. Also, the first VHH antibody is immobilized on the metal microstructure. The first VHH antibody is an immobilized antibody that specifically binds to NP of influenza A virus. Details of the detection area 204b will be described later with reference to FIGS. 3A and 3B.

The introduction unit 206 introduces a second VHH antibody labeled with a fluorescent substance (hereinafter referred to as a labeled antibody) and a sample into the sensor cell 204 . Specifically, the introduction unit 206 drips a sample liquid 2061 containing the labeled antibody and the sample onto the sensor cell 204 . The sample is a liquid that may contain NPs of influenza A virus, and in the present embodiment is a collected liquid discharged from the cyclone 108 . Details of the first VHH antibody and the second VHH antibody will be described later.

If the sample contains influenza A virus, the NP of the virus binds to the metal microstructure via the first VHH antibody. At this time, the NP of the virus also binds to the second VHH antibody labeled with a fluorescent substance. That is, a complex of a first VHH antibody, influenza A virus NP (hereinafter also simply referred to as NP), a second VHH antibody, and a fluorescent substance is bound to the metal microstructure. When the metal microstructure is irradiated with light in this state, fluorescence is emitted from the fluorescent substance indirectly bonded to the NP, and the fluorescence is enhanced by surface plasmons. Fluorescence enhanced by surface plasmons is hereinafter referred to as surface-enhanced fluorescence.

The light source 208 is an example of a light irradiation unit that irradiates the sensor cell 204 with excitation light. As the light source 208, any known technology can be used without particular limitation. For example, a laser such as a semiconductor laser or gas laser can be used as the light source 208 . The light source 208 preferably irradiates excitation light with a wavelength (for example, 400 nm to 2000 nm) that interacts less with NPs. Furthermore, the wavelength of the excitation light is preferably 600 nm to 850 nm, which can be used by the semiconductor laser.

Beam splitter 210 separates surface-enhanced fluorescence generated in detection region 204 b from excitation light emitted from light source 208 . Specifically, beam splitter 210 passes excitation light from light source 208 , separates surface-enhanced fluorescence generated in sensor cell 204 , and guides the separated surface-enhanced fluorescence to detection unit 214 .

The lens 212 converges the excitation light from the light source 208 that has passed through the beam splitter 210 onto the detection area 204b.

The detector 214 disperses the surface-enhanced fluorescence guided by the beam splitter 210 and detects light in a specific wavelength band, thereby outputting an electrical signal corresponding to the amount of NPs in the sample. The detection unit 214 can use any known technology without particular limitation as long as it can detect light in a specific wavelength band. For example, as the detection unit 214, an interference filter that transmits a specific wavelength band to disperse light, a Czerny spectroscope that disperses light using a diffraction grating, an Echelle spectroscope, or the like can be used. Furthermore, the detection unit 214 can be a notch filter for removing the excitation light from the light source 208, or can block the excitation light from the light source 208 and transmit the surface-enhanced fluorescence generated in the sensor cell 204. A long pass filter may be included.

[Controller configuration]
Controller 300 controls the overall operation of detection system 10 . Specifically, the controller 300 controls the collection device 100 and the detection device 200 .

More specifically, the controller 300 controls the initiation of the measurement, causes the aspirator 102 to begin aspirating ambient ambient air, and causes the pump 106 to pump the collected liquid from the collected liquid tank 104 to the cyclone 108 . to supply As a result, the collected liquid and the fine particles are mixed in the cyclone 108 and the sample is supplied from the cyclone 108 to the detection device 200 . Furthermore, the controller 300 causes the light source 208 to emit light and the detection unit 214 to detect surface-enhanced fluorescence.

For example, the controller 300 can control each pump to supply a predetermined volume of the sample liquid 2061 to the detection device 200 under preset conditions based on input parameters. Furthermore, the controller 300 has a timer function, and may generate and store information on the time required for each operation. The controller 300 may also receive the measured value from the detection device 200 and calculate the temporal change in the concentration of influenza A virus floating in the air based on the measured value and the time information.

Note that the controller 300 is realized by, for example, one or more dedicated electronic circuits. One or more dedicated electronic circuits may be integrated on a single chip or may be discretely formed on multiple chips. Controller 300 may also be implemented by a general-purpose processor (not shown) and memory (not shown) in which software programs or instructions are stored, instead of one or more dedicated electronic circuits. In this case, the processor functions as the controller 300 when the software program or instructions are executed.

[Structure of detection area of sensor cell]
Here, a detailed configuration of the detection area 204b of the sensor cell 204 will be specifically described with reference to FIGS. 3A and 3B.

3A is a perspective view of detection region 204b of sensor cell 204 according to Embodiment 1. FIG. FIG. 3B is an enlarged cross-sectional view of the metal microstructure 2041 according to Embodiment 1. FIG.

As shown in FIG. 3A, the detection region 204b is provided with a nanoscale metal microstructure 2041 for generating surface plasmons. In this embodiment, as shown in FIG. 3B, metal microstructure 2041 includes resin substrate 2042 and metal film 2043 .

The resin substrate 2042 has nanostructures formed by nanoimprinting or injection molding. Here, the nanostructure includes a plurality of pillars 2042a. In the plurality of pillars 2042a, the size ratio of the pillar height to the pitch between the pillars is preferably 1:1 to 1:3. In this embodiment, the wavelength of excitation light and the wavelength of fluorescence are 750 nm to 850 nm. Therefore, in this embodiment, it is desirable that the pillar height is approximately 200 nm, the pillar diameter is approximately 230 nm, and the pitch between the pillars is approximately 460 nm. Note that the nanostructure of the resin substrate 2042 is not limited to this, and may include fine projections such as a plurality of hemispheres and polygonal prisms instead of a plurality of pillars.

The metal film 2043 is produced by forming a metal film on the resin substrate 2042 . The metal film 2043 is formed with a plurality of protrusions 2043a corresponding to the plurality of pillars 2042a of the resin substrate 2042 . When the excitation light wavelength and fluorescence wavelength are 750 nm to 850 nm, the thickness of the metal film 2043 is preferably about 400 nm from the top of the pillar 2042a.

The material of the metal film 2043 is not particularly limited, and may be gold, silver, copper, aluminum, or an alloy containing any of these metals as a main component. Moreover, in this embodiment, sputtering is used as a method for forming the metal film 2043 . Note that the method for forming the metal film 2043 is not particularly limited, and may be, for example, electron beam (EB) vapor deposition or vacuum vapor deposition.

The distance between the first VHH antibody and the metal film 2043 is set on the metal film 2043 from the viewpoint of facilitating the immobilization of the first VHH antibody and the orientation control and reactivity of the second VHH antibody. Molecules (linkers) that can be properly secured may be attached. Molecules that can serve as linkers include, for example, thiol derivatives such as alkanethiols that form a self-assembled monolayer (hereinafter referred to as SAM), hydrophilic polymers containing polyethylene glycol chains (PEG chains), and phospholipids. MPC polymer, which is a polymer of MPC (2-methacryloyloxyethylphosphorylcholine) having a polar group, and the like. The SAM can be formed, for example, by immersing the metal microstructure 2041 in a thiol solution. The first VHH antibody is immobilized on the metal microstructure 2041 via this SAM. For example, the first VHH antibody is immobilized on the carboxyl group of the thiol compound modified on the metal microstructure 2041 via an amino group by an amine coupling method. FIG. 4 is a diagram showing the SAM 2044 on the metal microstructure 2041 in Embodiment 1. FIG.

As shown in FIG. 4, SAM 2044 is formed on metal microstructure 2041 . In the present embodiment, SAM2044 contains an alkyl chain having about 6 carbon atoms, for example. A first VHH antibody 2045 is immobilized on the metal microstructure 2041 via the SAM 2044 .

When the sample liquid 2061 contains influenza A virus NP2062 (hereinafter referred to as NP2062), the NP2062 binds to the first VHH antibody 2045 immobilized on the metal microstructure 2041 . Also bound to NP 2062 is a second VHH antibody 2063 labeled with a fluorescent substance 2064 .

When such a metal microstructure 2041 is irradiated with excitation light, fluorescence is emitted from the fluorescent material 2064 and the fluorescence is enhanced by surface plasmon generated on the metal microstructure 2041 . That is, surface-enhanced fluorescence corresponding to the amount of NP2062 is emitted.

[Operation of detector]
The operation of the detecting device 200 configured as above will be described with reference to FIGS. 2 and 5. FIG. FIG. 5 is a flow chart showing the operation of the detection device 200 according to the first embodiment.

First, the introduction unit 206 introduces the sample liquid 2061 that may contain the influenza A virus NP2062 into the metal microstructure 2041 (S102). Subsequently, the light source 208 irradiates excitation light to the metal microstructure 2041 (see FIG. 3A) into which the sample liquid 2061 has been introduced (S104). The detection unit 214 detects NP2062 of influenza A virus in the sample liquid 2061 by measuring the fluorescence generated from the fluorescent substance 2064 (see FIG. 4) by the irradiation of the excitation light and enhanced by the surface plasmon. (S106).

[First VHH antibody and second VHH antibody]
The first VHH antibody 2045 and the second VHH antibody 2063 are described in detail below with reference to FIG. 4 again.

As described above, the first VHH antibody 2045 is an immobilized antibody immobilized on the metal microstructure 2041 and the second VHH antibody 2063 is a labeled antibody labeled with a fluorescent substance 2064. The first VHH antibody 2045 and the second VHH antibody 2063 bind to influenza A virus, particularly to nuclear protein (hereinafter referred to as NP) 2062 of influenza A virus. First VHH antibody 2045 and second VHH antibody 2063 each bind to a different site in NP2062. The influenza A virus subtype to which the first VHH antibody 2045 and the second VHH antibody 2063 bind is at least one of H1N1, H3N2, and H7N9.

The first VHH antibody 2045 and the second VHH antibody 2063 each have structural domains arranged from N-terminus to C-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. FR indicates Framework Region, and CDR indicates Complementarity Determining Region. A framework region is a region that plays a role in structurally supporting a complementarity determining region, and includes highly conserved regions among antibody variable regions. A complementarity determining region is a region that directly contacts an antigen to form a complementary three-dimensional structure with the antigen, that is, a region that forms a binding site with the antigen, and is a region that determines the specificity for the antigen. Complementarity determining regions include sequence-variable antigen-recognition sites or random-sequence regions in antibody variable regions. Thus, the variable region of an antibody is composed of multiple CDRs and FRs. In addition, these CDRs and FRs are arranged in order from the N-terminal side, for example, the first complementarity determining region is CDR1, the second complementarity determining region is CDR2, and the first framework region is is expressed as FR1, and the second framework region as FR2.

As described later in Examples, the inventors obtained multiple clones of highly reactive influenza A virus antibodies by screening a VHH antibody library. Among them, as the first VHH antibody 2045 and the second VHH antibody 2063, influenza A virus antibodies having three complementarity determining regions of CDR1, CDR2 and CDR3 containing the following amino acid sequences are used in order from the N-terminal side. Therefore, it was found that a trace amount of NP2062 can be detected with high sensitivity.

The complementarity determining regions of first VHH antibody 2045 and second VHH antibody 2063 each comprise the following amino acid sequences.

In the first VHH antibody 2045, CDR1 comprises the amino acid sequence shown in SEQ ID NO:1, CDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:3.

In the second VHH antibody 2063, CDR1 comprises the amino acid sequence shown in SEQ ID NO:4, CDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and CDR3 comprises the amino acid sequence shown in SEQ ID NO:6.

Furthermore, the first VHH antibody 2045 and the second VHH antibody 2063 have four frame regions FR1, FR2, FR3 and FR4 containing the following amino acid sequences in order from the N-terminal side.

In the first VHH antibody 2045, FR1 comprises the amino acid sequence shown in SEQ ID NO:7, FR2 comprises the amino acid sequence shown in SEQ ID NO:8, FR3 comprises the amino acid sequence shown in SEQ ID NO:9, FR4 comprises the amino acid sequence shown in SEQ ID NO:10. Therefore, for example, when the first VHH antibody is bound to the linker molecule of the SAM, that is, when immobilized on the SAM, the amino group of the lysine residue in any of the above FRs is in the linker molecule. binds to the carboxyl group of In addition, since the lysine residue does not exist in the CDR but exists in the FR and is sterically separated from the CDR, the first VHH antibody is fixed to the SAM. The first VHH antibody can retain its antigen-capturing ability even when modified.

In the second VHH antibody 2063, FR1 comprises the amino acid sequence shown in SEQ ID NO: 11, FR2 comprises the amino acid sequence shown in SEQ ID NO: 12, FR3 comprises the amino acid sequence shown in SEQ ID NO: 13, FR4 comprises the amino acid sequence shown in SEQ ID NO:14. This allows the second VHH antibody to stably maintain the CDR conformation (eg, loop structure). Therefore, the CDRs can exert antigen-trapping ability.

As described above, the first VHH antibody 2045 is an antibody comprising the amino acid sequence shown in SEQ ID NO:15. Second VHH antibody 2063 is an antibody comprising the amino acid sequence shown in SEQ ID NO:16.

A method for binding the first VHH antibody 2045 to the end of the linker molecule of SAM2044 is, for example, the amine coupling method. In the amine coupling method, the carboxyl group at the end of the linker molecule is active-esterified, and the active-esterified carboxyl group at the end of SAM2044 and the amino group ( _NH2 ) are combined. As shown in the amino acid sequence of the first VHH antibody 2045 (SEQ ID NO: 15), the lysine residue (K) of the first VHH antibody 2045 is present in the FR but not in the CDR having antigen-binding ability, and It is arranged sterically apart from the CDR. Therefore, even when the first VHH antibody 2045 binds to SAM2044, it can be immobilized while maintaining its antigen-binding ability. As will be described later in Examples, the second VHH antibody 2063 is more difficult to immobilize on the metal microstructure 2044 than the first VHH antibody 2045 . Therefore, the detection device 200 uses the first VHH antibody 2045 as the immobilized antibody and the second VHH antibody 2063 as the labeled antibody, thereby detecting a minute amount of influenza A virus with high sensitivity. can be done.

As a method for labeling the second VHH antibody 2063 with the fluorescent substance 2064, a known method such as a physical adsorption method, a covalent bond method, an ionic bond method, a cross-linking method, or the like is used.

[Effects, etc.]
As described above, according to the detection device 200 according to the present embodiment, in the surface-enhanced fluorescence method in which fluorescence based on NP2062 of influenza A virus is enhanced by surface plasmon and detected, the immobilized antibody and the labeled antibody are VHH antibodies can be used. VHH antibodies are smaller than normal IgG antibodies and have a higher antigen-capturing ability than fragmented IgG antibodies. Therefore, the detection device 200 has a high antigen-capturing ability by using VHH antibodies as immobilized antibodies and labeled antibodies. In addition, since the VHH antibody is smaller than the IgG antibody, using the VHH antibody as the immobilized antibody increases the immobilized amount of the immobilized antibody immobilized on the metal microstructure 2041 . Moreover, when a VHH antibody is used as a labeled antibody, nonspecific adsorption like a labeled IgG antibody is less and noise is small. Therefore, the detection device 200 can detect a very small amount of antigen (here, influenza A virus NP2062) with high sensitivity.

In this way, the detection device according to one aspect of the present disclosure can detect a plurality of influenza A subtypes, and thus can detect influenza A virus with high accuracy.

(Embodiment 2)
Next, Embodiment 2 will be described. This embodiment differs from the first embodiment in the configuration of the SAM. The detection device according to the present embodiment will be described below, focusing on the differences from the first embodiment.

The configuration of the collection device and controller according to the present embodiment is substantially the same as that of the collection device and controller according to the first embodiment. Moreover, the configuration of the detection device according to the present embodiment is substantially the same as that of the detection device according to the first embodiment except for the SAM. Therefore, the detection system according to the present embodiment will be described mainly with respect to the SAM that differs from that of the first embodiment.

[Structure of SAM]
FIG. 6 is a diagram showing SAM 2044B on metal microstructure 2041A according to the second embodiment. As shown in FIG. 6, a SAM 2044B including linker molecules 2046 and non-linker molecules 2047 is formed on the surface of the metal microstructure 2041A. First VHH antibody 2045 is immobilized to metal microstructure 2041A via linker molecule 2046 .

Linker molecule 2046 has a thiol group 20461 at one end and a carboxyl group 20464 at the other end. The thiol group 20461 bonds with the surface of the metal microstructure 2041A. Carboxyl group 20464 binds to first VHH antibody 2045 .

Furthermore, the linker molecule 2046 includes an alkyl chain 20462 having 10 or more carbon atoms and an ethylene glycol chain 20463 between the thiol group 20461 and the carboxyl group 20464 . Specifically, alkyl chain 20462 is connected to thiol group 20461 and ethylene glycol chain 20463 , and ethylene glycol chain 20463 is connected to alkyl chain 20462 and carboxyl group 20464 .

Non-linker molecule 2047 has a thiol group 20471 at one end and a hydroxyl group 20474 at the other end. The thiol group 20471 bonds with the surface of the metal microstructure 2041A. Since the hydroxyl group 20474 is hydrophilic, it suppresses nonspecific adsorption of the second VHH antibody 2063 and the fluorescent substance 2064 .

Furthermore, the non-linker molecule 2047 includes an alkyl chain 20472 having 10 or more carbon atoms and an ethylene glycol chain 20473 between the thiol group 20471 and the hydroxyl group 20474 . Specifically, alkyl chain 20472 is connected to thiol group 20471 and ethylene glycol chain 20473 , and ethylene glycol chain 20473 is connected to alkyl chain 20472 and hydroxyl group 20474 .

In this embodiment, SAM 2044B contains fewer linker molecules 2046 than non-linker molecules 2047 .

[Effects, etc.]
As described above, according to detection device 200 according to the present embodiment, SAM 2044B including linker molecules 2046 and non-linker molecules 2047 is formed on the surface of metal microstructure 2041A. Therefore, the first VHH antibody 2045 can be immobilized by the linker molecule 2046 while the non-linker molecule 2047 prevents the second VHH antibody 2063 and the fluorescent substance 2064 from attaching to the SAM 2044B. That is, it is possible to reduce non-specific adsorption while maintaining the ability to immobilize the first VHH antibody.

Moreover, according to the detection device 200 according to the present embodiment, the SAM 2044B can contain alkyl chains 20462 and 20472 having 10 or more carbon atoms. As a result, a dense SAM 2044B can be realized, and both improvement in the ability to immobilize the first VHH antibody 2045 and reduction in non-specific adsorption can be achieved.

For example, in Embodiment 1, SAM2044 containing an alkyl chain having about 5 carbon atoms is used. By shortening the alkyl chain, the fluorescent substance 2064 is brought closer to the surface of the metal microstructure 2041, so that the effect of enhancing fluorescence by plasmon resonance can be improved. On the other hand, in this embodiment, in order to reduce non-specific adsorption, the number of carbon atoms is increased and the alkyl chain is lengthened as compared with the first embodiment. By lengthening the alkyl chain in this manner, the intermolecular force between adjacent alkyl chains increases, and a denser SAM can be formed. As a result, the hydrophobic surface of the SAM, which is the site of non-specific adsorption, is firmly included in the SAM, and non-specific adsorption can be reduced. Furthermore, since the dense SAM increases the binding sites of the immobilized antibody, the amount of NP that binds to the immobilized antibody also increases, resulting in an increase in signal intensity. As described above, since the signal is increased and the noise is reduced, it is possible to improve the detection sensitivity of NP.

In addition, in this embodiment, there is a disadvantage that the distance between the fluorescent substance and the surface of the metal microstructure increases due to the lengthening of the alkyl chain. However, the amount of increase in alkyl chain length when one carbon is increased is approximately 0.15 nm. In other words, even if the number of carbon atoms increases by five, the amount of increase in the length of the alkyl chain is about 0.75 nm. small.

Moreover, according to the detection device 200 according to the present embodiment, the SAM 2044B can contain the ethylene glycol chains 20463 and 20473 . As a result, the carboxyl group 20464 located at the terminal end of the linker molecule 2046 can be given mobility, and the binding property between the first VHH antibody 2045 bound to the carboxyl group 20464 and NP can be improved.

Specifically, the ethylene glycol chains 20463, 20473 provide flexibility to the carboxyl group 20464 located at the end of the linker molecule 2046 and the hydroxyl group 20474 located at the end of the non-linker molecule 2047. By binding the first VHH antibody 2045 to the carboxyl group 20464 connected to the ethylene glycol chain 20463 of the linker molecule 2046, the binding property between the first VHH antibody 2045 and NP2062 is improved.

Without the ethylene glycol chain, the mobility of the SAM-anchored first VHH antibody is reduced. This increases the possibility that the binding site for NP2062 in the first VHH antibody 2045 will not be exposed, and also increases the possibility that NP2062 and the first VHH antibody 2045 cannot bind.

According to detection device 200 according to the present embodiment, linker molecules 2046 are fewer than non-linker molecules 2047 in SAM 2044B. As a result, the first VHH antibody 2045 can be appropriately immobilized while reducing non-specific adsorption, so that the detection accuracy of NP2062 can be improved. Carboxyl group 20464 located at the end of linker molecule 2046 binds first VHH antibody 2045 . On the other hand, the hydroxyl group 20474 located at the end of the non-linker molecule 2047 is hydrophilic and suppresses non-specific adsorption of the second VHH antibody 2063 and the fluorescent substance 2064 . As a result of experiments on the mixing ratio of the linker molecule 2046 and the non-linker molecule 2047, it was found that by increasing the ratio of the non-linker molecule 2047 compared to the linker molecule 2046, the first VHH antibody can be obtained while reducing non-specific adsorption. It was found that 2045 could be appropriately immobilized and the detection accuracy of NP2062 was improved. The reason why the signal does not decrease significantly compared to the case where the linker molecule 2046 is 100% is that the first VHH antibody 2045 has a large size, so even if many carboxyl groups 20464 are densely present, all the carboxyl groups This is because the first VHH antibody 2045 is not fixed to 20464 .

Examples will be described below in more detail, but these examples do not limit the present disclosure in any way.

(Example 1)
A VHH antibody that binds to a nuclear protein of the H1N1 subtype of influenza A virus (ie, the variable region of the heavy chain of the heavy chain antibody) was prepared according to the following procedure.

[Immunization to Alpaca and Acquisition of Mononuclear Cells]
To prepare a VHH antibody gene library, alpacas were immunized using a recombinant nuclear protein (SEQ ID NO: 17) derived from the H1N1 subtype of influenza A virus (A/Puerto Rico/8/34/Mount Sinai) as an antigen. rice field. The recombinant nuclear protein was produced at Higeta Shoyu Co., Ltd. using the Brevibacillus expression system. Recombinant nuclear proteins were conditioned with adjuvants prior to administration to alpacas.

Specifically, 100 μg/mL of recombinant nuclear protein was administered to alpacas. Administration of the recombinant nuclear protein to alpacas was performed once a week for 5 weeks. Alpaca blood was collected one week after the last dose.

Mononuclear cells were then obtained from blood from immunized alpacas. Specifically, blood was collected from the immunized alpaca, treated with heparin, and mononuclear cells were obtained according to a conventional method. In this example, a mononuclear cell-containing fraction was prepared using a lymphocyte separation tube (Greiner Japan Co., Ltd., trade name: Leucosep) and a blood cell separation solution (Cosmo Bio Co., Ltd., trade name: Lymphoprep). A fraction (hereinafter referred to as mononuclear cell fraction) was obtained. Phosphate-buffered saline (hereinafter referred to as PBS) was used to dilute the heparinized blood. An RNA preservation solution (trade name: RNAlater) was used to preserve the obtained mononuclear cells.

[Preparation of VHH antibody cDNA gene library]
Total RNA was extracted from the obtained mononuclear cells, and cDNA (complementary DNA) was synthesized according to a conventional method. Specifically, cDNA was synthesized from total RNA by a reverse transcription reaction using a reverse transcriptase. The cDNA was then incorporated into a vector to create a VHH antibody cDNA gene library.

A TOTAL RNA extraction reagent (trade name: TRIzol Reagent) was used for the extraction of TOTAL RNA. 1 mL of TOTAL RNA extraction reagent was added to the mononuclear cell fraction, gently mixed, and allowed to stand at room temperature for 5 minutes. Chloroform (200 μL) was added to this mixture, vigorously shaken for 15 seconds, and allowed to stand at room temperature for 2-3 minutes. The chloroform mixture was centrifuged at 12,000 xg or less for 15 minutes at 4 degrees Celsius.

Supernatants were collected in new tubes. 200 μL each of RNase-free water and chloroform were then added to the tube, followed by 500 mL of isopropanol. After vigorously stirring the liquid contained in the tube with a vortex mixer, it was allowed to stand at room temperature for 10 minutes. The mixed liquid was then centrifuged at up to 12,000 xg for 15 minutes at a temperature of 4 degrees Celsius. After removing the supernatant, 1 mL of 75% ethanol was added to the pellet, and the pellet was resuspended and rinsed. The suspension was centrifuged at <7,500 xg for 5 minutes at a temperature of 4 degrees Celsius. After removing the supernatant, the remaining ethanol was dried to obtain TOTAL RNA. The obtained TOTAL RNA was dissolved in RNase-free water.

In this example, a kit containing a reverse transcriptase (Takara Bio Inc., trade name: PrimeScript II 1st strand cDNA Synthesis Kit) was used. Random 6mer and Oligo dT primer included in the kit were used as primers. cDNA was obtained according to the standard protocol of the kit.

Subsequently, PCR was performed using the obtained cDNA to obtain the VHH antibody gene derived from alpaca. In this example, Ex-taq (Takara Bio Inc.) was used as the Taq polymerase.

First, a PCR reaction solution was prepared. For the reaction solution, 10x buffer, dNTPs, Primer F, and Primer R were mixed at the following mixing ratio.
5 μL of 10x buffer
4 μL of dNTPs
Primer F 2 μL
Primer R 2 μL
Ex-taq 0.25 μL

cDNA (eg, 1 µL of cDNA template) was added to this reaction solution, mixed well, and then PCR was performed under the following conditions.

(1) Heat at 95 degrees Celsius for 2 minutes (2) Repeat 30 cycles of 96 degrees Celsius for 30 seconds, 52 degrees Celsius for 30 seconds, and 68 degrees Celsius for 40 seconds (3) 68 degrees Celsius for 4 minutes Keep warm at 4 degrees Celsius after heating

In this PCR method, primer 1 (SEQ ID NO: 18), primer 2 (SEQ ID NO: 19), primer 3 (SEQ ID NO: 20), primer 4 (SEQ ID NO: 21), primer 5 (SEQ ID NO: 22), and primer 6 (SEQ ID NO:23) was used.
（参考文献：ＷＡＮＧ Ｄａｎ ｅｔ． ａｌ．， “Ｉｓｏｌａｔｉｏｎ ａｎｄ Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ Ｒｅｃｏｍｂｉｎａｎｔ Ｖａｒｉａｂｌｅ Ｄｏｍａｉｎ ｏｆ Ｈｅａｖｙ Ｃｈａｉｎ Ａｎｔｉ－ｉｄｉｏｔｙｐｉｃ Ａｎｔｉｂｏｄｉｅｓ Ｓｐｅｃｉｆｉｃ ｔｏ Ａｆｌａｔｏｘｉｎ Ｂ _１ ”， Ｂｉｏｍｅｄｉｃａｌ ａｎｄ Ｅｎｖｉｒｏｎｍｅｎｔａｌ Ｓｃｉｅｎｃｅｓ， ＥＬＳＥＶＩＥＲ， ２０１４年， ｖｏｌ．２７， Ｎｏ．２ , pp. 118-121)

The following three rounds of PCR were performed.

Two types of samples, a primer set A consisting of cDNA, Primer 1 and Primer 3 and a primer set B consisting of cDNA, Primer 1 and Primer 4, were used for the first PCR.

For the second PCR, the gene amplified using the primer set A in the first PCR, the primer set C consisting of Primer 2 and Primer 3, and the primer set B amplified in the first PCR Two types of samples, a primer set D consisting of a gene, Primer 2, and Primer 4, were provided.

For the third PCR, the gene amplified using the primer set C in the second PCR, the primer set E consisting of Primer 2 and Primer 5, and the primer set D amplified in the second PCR Two types of samples, a primer set F consisting of a gene, Primer 2, and Primer 6, were provided.

Thus, a gene library of VHH antibodies was created. In other words, the VHH antibody gene library contained genes amplified using primer set E and primer set F.

[Preparation of phage library]
Then, the VHH antibody gene library was treated with the restriction enzyme SfiI to obtain VHH antibody gene fragments. A pUC119 plasmid vector (SEQ ID NO: 24) (4057 bp) was obtained by treating a plasmid pUC119 (manufactured by Takara Bio Inc.) with a restriction enzyme SfiI. The obtained VHH antibody gene fragment was ligated into the pUC119 plasmid vector (SEQ ID NO: 24). Specifically, a VHH antibody gene fragment and a pUC119 plasmid vector (SEQ ID NO: 24) were mixed at a ratio of 1:2, and an enzyme (Toyobo Co., Ltd., trade name: Ligation High ver.2) was added. . This mixed solution was allowed to stand at a temperature of 16 degrees Celsius for 2 hours. Then, the ligated pUC119 plasmid vector was introduced into competent cells for transformation. Escherichia coli (Takara Bio Inc., trade name: HST02) was used as competent cells.

Then, the transformed E. coli (transformant) was cultured on a 2YT plate medium containing 100 μg/mL ampicillin (hereinafter referred to as 2YTA medium) for 15 hours. Thus, a library of phages displaying proteins obtained from VHH antibody gene fragments contained in the VHH antibody gene library was obtained.

[Biopanning]
A VHH antibody that specifically binds to an influenza A virus nuclear protein was obtained from a phage library according to the following procedure.

Two rounds of biopanning were performed to screen phage that specifically bind to the antigen among the phage that expressed the VHH antibody.

Specifically, the transformant into which the VHH antibody gene fragment was introduced was placed in 100 mL of 2YTAG medium (containing 100 ug/mL ampicillin and 1% glucose) at 30 degrees Celsius until the absorbance (OD600 nm) reached 1.0. 2YT medium) to grow transformants. Helper phage (M13KO7) (Invitrogen) was added to this culture solution so that the multiplicity of infection (MOI) was about 20, and the culture was incubated at 37 degrees Celsius for about 30 minutes. The culture was then centrifuged at 4000 rpm for 10 minutes to collect transformants. Transformants were cultured overnight in 100 mL 2YTAK medium (2YT medium containing 100 ug/mL ampicillin and 50 ug/mL kanamycin) at 30 degrees Celsius and centrifugation at 213 rpm. This culture was then centrifuged at 4000 rpm for 10 minutes and the supernatant (20 mL) was recovered. The above series of operations were performed twice to obtain a supernatant (40 mL).

The resulting supernatant (40 mL) was added to 20% PEG solution (10 mL) containing NaCl (2.5 M) and mixed by inversion. The mixture was then chilled on ice for approximately 1 hour before being centrifuged at 4000 rpm for 10 minutes and the supernatant removed. PBS containing 10% glycerol was injected towards the precipitate. Finally, the precipitate was dissolved in PBS while loosening. A library of phage displaying VHH antibodies was thus obtained.

[Screening for VHH antibodies that specifically bind to NP]
(A) Antigen Immobilization NP was mixed with PBS to prepare a 2 μg/mL NP solution. The NP solution (2 mL) was injected into an immunotube (manufactured by Nunc) and allowed to stand overnight. This immobilized the NP inside the immunotube. Then, the inside of the immunotube was washed with PBS three times. This removed excess NP that was not immobilized inside the immunotube.

Then, the immunotube was filled with PBS containing 3% skim milk (Wako Pure Chemical Industries, Ltd.) and allowed to stand at room temperature for 1 hour. After that, the inside of the immunotube was washed with PBS three times. This blocked NP as an antigen inside the immunotube.

(B) Panning A mixture was prepared by mixing a library of phage displaying VHH antibodies (concentration: approximately 5E+11/mL) with 3 mL of PBS containing 3% skimmed milk. This mixture was injected into an immunotube on which NP antigen was immobilized.

The immunotube was capped with parafilm and placed on the rotator. Then, the mixed liquid in the immunotube was inverted and stirred for 10 minutes by a rotator. After that, the immunotube was allowed to stand at room temperature for 1 hour.

Then, the inside of the immunotube was washed ten times with PBS containing 0.05% tween20 (hereinafter referred to as PBST). After washing, the inside of the immunotube was filled with PBST and allowed to stand for 10 minutes. The inside of the immunotube was then washed 10 times with PBST.

A 100 mM trimethylamine solution (1 mL) was injected into the immunotube to elute the phage displaying VHH antibody bound to NP from the inside of the immunotube.

The immunotube was capped with parafilm and placed on the rotator. Then, the mixed liquid in the immunotube was turned upside down and stirred for 10 minutes by a rotator. The mixture was then transferred to a tube containing 1 mL of 0.5 M Tris/HCl (pH 6.8) to neutralize the mixture. The immunotube was again injected with 100 mM trimethylamine solution (1 mL) to elute the phage displaying VHH antibody bound to NP. Through the above operation, 3 mL of phage eluate was obtained.

After mixing the eluate (1 mL) and 9 mL of competent cells (here, Escherichia coli HST02), the mixture was allowed to stand at 30 degrees Celsius for 1 hour.

The mixture was then centrifuged and the precipitate collected. A plate (40 mL/plate) of 2YTA medium was inoculated with the precipitate and cultured overnight at 30 degrees Celsius. Thus, the first panning was performed.

A second round of panning was performed in exactly the same manner as the first round of panning. In other words, panning was repeated. Monoclonal phage displaying VHH antibodies were thus purified.

After the second round of panning, transformant colonies were picked with a toothpick. One colony picked was placed in one well of a 96-well flat bottom plate. This was repeated. 200 μL of 2YTAG medium was dispensed into one well. The 96-well flat-bottomed plate was then shaken at 30 degrees Celsius and 213 rpm to culture the transformants. From each well of this 96-well flat-bottom plate, 50 μL of the culture solution was collected per well and added to 2YTA medium dispensed into another 96-well flat-bottom plate. 50 μL of 2YTA medium was dispensed per well. Culture medium (50 μL) collected from one well was added to the corresponding well of a 96-well flat-bottom plate dispensed with 2YTA medium and mixed. This mixture was then allowed to stand at 37 degrees Celsius for 40 minutes. The 2YTA medium contained helper phages at a multiplicity of infection (MOI) of 20.

A 96-well flat-bottom plate containing the mixture was centrifuged to collect the precipitate. Escherichia coli was contained in the precipitate. This precipitate was mixed with 200 μL of 2YTAK medium and cultured overnight at 30 degrees Celsius. This culture was then centrifuged and the supernatant containing E. coli was recovered.

(C) Qualitative evaluation of phage-displayed VHH antibodies and antigens by ELISA To each well of a 96-well plate (Thermo Fisher Scientific, trade name: MaxiSorp (registered trademark)), a 2 μg/mL nuclear protein solution was added as an antigen. . The volume of HA protein solution in each well was 50 μL. The 96-well plate was left overnight at 4°C. Thus, NP antigen was immobilized to each well.

Each well was washed 3 times with PBS. PBS containing 3% skimmed milk was then injected into each well (200 μL/well). The 96-well plate was left at room temperature for 1 hour. Thus, nuclear proteins were blocked in each well. Each well was then washed 3 times with PBS.

Monoclonal phage displaying VHH antibodies were injected into each well (50 μL/well). The 96-well plate was then allowed to sit for 1 hour. Thus, VHH antibodies displayed on the surface of phage reacted with NP antigen.

Each well was washed 3 times with PBST. Next, an anti-M13 antibody (abcam, trade name: ab50370, 10000-fold dilution) was added to each well (50 μL/well). Each well was then washed 3 times with PBST.

A color developer (Thermo Fisher Scientific, trade name: 1-STEP ULTRA TMB-ELISA) was injected into each well (50 μL/well). The 96-well plate was allowed to stand for 2 minutes to allow the color developer to react with the antibodies. Then, 50 μL of an aqueous sulfuric acid solution (1N) was added to each well to stop the reaction between the coloring agent and the antibody. Then, the absorbance of this reaction solution was measured at a wavelength of 450 nm.

Multiple wells with good absorbance measurements were selected. The DNA sequences contained in the phage contained in these multiple wells were analyzed by Greiner. Among them, the following two types of VHH antibodies (first VHH antibody and second VHH antibody) having high antigen-binding ability were selected.

The gene sequence of the first VHH antibody is shown in SEQ ID NO:25. The protein synthesized from the gene sequence shown in SEQ ID NO:25 has the amino acid sequence of the first VHH antibody shown in SEQ ID NO:15. SEQ ID NO: 26 shows the gene sequence of the second VHH antibody. The protein synthesized from the gene sequence shown in SEQ ID NO:26 has the amino acid sequence of the second VHH antibody shown in SEQ ID NO:16.

(Example 2)
According to Example 1, two types of VHH antibodies (first VHH antibody and second VHH antibody) highly reactive with influenza A nuclear proteins were obtained. Subsequently, in this example, it was examined whether or not the antigen-binding sites of these two types of VHH antibodies do not overlap, that is, do not compete with each other.

[Evaluation of competitiveness]
In this example, whether the antigenic epitopes of the first VHH antibody and the second VHH antibody overlap and compete with each other was evaluated using a surface plasmon resonance (SPR) evaluation device.

Evaluation device: Biacore T200 (GE Healthcare Japan Co., Ltd.)
Sensor chip: CM5 (GE Healthcare Japan Co., Ltd.)
Immobilization reagents: N-hydroxysuccinimide (NHS) and ethyl(dimethylaminopropyl)carbodiimide (EDC)
Immobilization buffer: Phosphate buffered saline containing 0.05% (v/v) tween20 (hereinafter, PBST)
Running buffer: PBST
Antigen (NP): Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Nucleoprotein (Catalog Number: 11675-V08B, Sino Biological Inc.)
Evaluation sample:
(1) 2 nM NP-PBST solution (hereinafter, “NP only”)
(2) 2 nM NP + 0.5 μg/mL first VHH antibody-PBST solution (hereinafter, “NP + VHH1”)
(3) 2 nM NP + 0.5 μg/mL second VHH antibody-PBST solution (hereinafter, “NP + VHH2”)

Immobilization of the first VHH antibody to the sensor chip CM5 was carried out using N-hydroxysuccinimide (NHS) and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) using amine cup A ring method was used, and ethanolamine was used for blocking. The first VHH antibody was diluted to about 50 μg/mL with an immobilization buffer (10 mM CH ₃ COOH—CH ₃ COONa, pH 4.5) and immobilized on CM5 according to the protocol attached to Biacore T200. Next, evaluation sample (1) was added to CM5 on which the first VHH antibody was immobilized at a flow rate of 10 μL/min for 5 minutes to react with the first VHH antibody immobilized on CM5. Evaluation sample (2) and evaluation sample (3) were each reacted with the first VHH antibody immobilized on CM5 in the same procedure as for evaluation sample (1).

A series of steps of competitiveness evaluation using the Biacore system will be described in more detail. In the Biacore system, one of the substances for observing the interaction (here, the first VHH antibody) is immobilized on the gold thin film of the sensor chip, and the interface between the gold thin film and the glass from the back side of the sensor chip causes total reflection. is irradiated with light, a portion (SPR signal) where the reflection intensity is reduced is formed in a part of the reflected light. The other substance (here, the evaluation sample) of the substance whose interaction is to be observed is allowed to flow over the surface of the sensor chip, and when the first VHH antibody and the evaluation sample bind, the mass of the substance immobilized or bound to the sensor chip surface increases. and the refractive index of the solvent on the surface of the sensor chip changes. This change in refractive index shifts the position of the SPR signal (conversely, when the bond dissociates, the signal position returns). The Biacore system plots the shift amount, that is, the change in mass on the surface of the sensor chip on the vertical axis, and displays the change in mass over time as measurement data (sensorgram). From the sensorgram, the amount of binding of the NP in the evaluation sample to the ligand captured on the sensor chip surface (amount of change in response on the sensorgram before and after the evaluation sample interacts) is obtained.

For evaluation sample (2) and evaluation sample (3), the amount of NP binding was calculated from the reaction signal on the assumption that one antibody was bound to one NP. The results are shown in FIG. 7 is a graph showing the normalized NP binding amount for evaluating the competitiveness of VHH antibodies for NP in Example 2. FIG.

The vertical axis in FIG. 7 indicates the amount of NP binding when each of the evaluation samples (1) to (3) is flowed over the substrate, and the amount of NP binding when only the NP of the evaluation sample (1) is flowed over the substrate. (hereinafter referred to as normalized NP bond amount).

As shown in FIG. 7, the normalized NP binding amount of the evaluation sample (2), which is a mixed solution of NP and the first VHH antibody, was almost zero. That is, the NPs in the evaluation sample (2) did not bind to the immobilized antibody immobilized on the CM5 substrate. The reason is as follows. The antibody contained in the evaluation sample (2) is the first VHH antibody and is the same antibody as the immobilized antibody. Therefore, the antibody contained in the evaluation sample (2) binds to NP at the same binding site as the immobilized antibody. That is, the antibody contained in the evaluation sample (2) competes with the immobilized antibody. Therefore, the NPs contained in the evaluation sample (2) were bound to the first VHH antibody and could not bind to the immobilized antibody, resulting in no change in the SPR signal.

On the other hand, in the evaluation sample (3), which is a mixed solution of NP and the second VHH antibody, the normalized NP binding amount is about 1.1 times that of the evaluation sample (1) containing only NP. The results were equivalent to those of the evaluation sample (1). From the above results, it was confirmed that the first VHH antibody and the second VHH antibody do not compete with each other.

(Example 3)
Example 2 confirmed that the first VHH antibody and the second VHH antibody do not compete with NP. Subsequently, in this example, the amount of each of the first VHH antibody and the second VHH antibody immobilized on the substrate surface was evaluated.

[Evaluation of immobilized amount]
In this example, the amount of immobilization when the first VHH antibody and the second VHH antibody were each used as the immobilized antibody was evaluated using an SPR evaluation device.

Evaluation device: Biacore T200 (GE Healthcare Japan Co., Ltd.)
Sensor chip: C1 (GE Healthcare Japan Co., Ltd.)
Immobilization reagents: N-hydroxysuccinimide (NHS) and ethyl(dimethylaminopropyl)carbodiimide (EDC)
Immobilization buffer: PBST
Running buffer: PBST
Antibody concentration: 50 µg/mL in PBST

Each of the first VHH antibody and the second VHH antibody was immobilized on the sensor chip C1 by reaction at a concentration of 50 μg/mL for 10 minutes by the amine coupling method. The results are shown in FIG. 8 is a graph showing the amount of immobilized VHH antibody in Example 3. FIG.

As shown in FIG. 8, the first VHH antibody immobilized on the sensor chip C1 was 8.7 femtomol/mm ² and the second VHH antibody immobilized on the sensor chip C1 was 0.7 femtomol/mm 2 . It was 7 mol/ ^mm2 . In other words, the immobilized amount of the first VHH antibody was 10 times more than that of the second VHH antibody. From this, it was found that the first VHH antibody should be used as the immobilized antibody.

(Example 4)
From Example 3, it was found that, of the first VHH antibody and the second VHH antibody, it is preferable to use the first VHH antibody as the immobilized antibody. In this example, the immobilized amount of the first VHH antibody and the immobilized amount of IgG were compared.

[Comparison between the immobilized amount of the first VHH antibody and the immobilized amount of IgG]
In this example, Biacore T200 was used to compare and evaluate the immobilized amounts of the first VHH antibody and the IgG antibody.

Evaluation device: Biacore T200 (GE Healthcare Japan Co., Ltd.)
Sensor chip: SIA Kit Au (GE Healthcare Japan Co., Ltd.)
Immobilization reagents: N-hydroxysuccinimide (NHS) and ethyl(dimethylaminopropyl)carbodiimide (EDC)
Immobilization buffer: PBST
Running buffer: PBST
Concentration of first VHH antibody: 10 μg/mL in PBST
IgG antibody: Anti-Influenza A Virus Nucleoprotein antibody [B247M] (ab66191) (manufactured by abcam)
IgG antibody concentration: 100 μg/mL in acetate buf. (pH 5.5)

First, an unmodified gold substrate included in the sensor chip SIA Kit Au was immersed overnight in an ethanol solution of hydroxyEG3 undecanethiol and carboxyEG6 undecanethiol to form a SAM on the gold substrate. A gold substrate on which SAM is formed (hereinafter referred to as a SAM substrate) and a first VHH antibody PBST solution having a concentration of 10 μg/mL of the first VHH antibody were reacted for 30 minutes by an amine coupling method to form a SAM substrate. A first VHH antibody was immobilized. The IgG antibody was also immobilized on the SAM substrate by the same procedure as for the first VHH antibody. The results are shown in FIG. 9 is a graph showing the amount of immobilized VHH antibody and IgG antibody in Example 4. FIG.

As shown in FIG. 9, the first VHH antibody immobilized on the SAM substrate was 43.2 femtomol/mm ² and the IgG immobilized on the SAM substrate was 6.4 femtomol/mm ² . rice field. Thus, even if immobilization is performed at an equivalent molar concentration, the volume of the first VHH antibody is smaller than that of the IgG antibody. It is possible that there were more. Therefore, using a VHH antibody as an immobilized antibody can immobilize a larger amount than when using an IgG antibody, so detection sensitivity can be enhanced.

(Example 5)
In this example, the first VHH antibody was immobilized on the surface of the metal microstructure as the immobilized antibody, and the second VHH antibody was used as the labeled antibody to perform a sandwich assay.

[Fabrication of metal microstructure]
In this example, a resin substrate on which a nanostructure having a plurality of pillars was formed by nanoimprinting was used as the resin substrate for the metal microstructure. Each of the pillars had a height of 200 nm, a diameter of 230 nm, and a pitch of 460 nm. Then, a gold (Au) film was formed on the resin substrate by sputtering so that the thickness from the top of the pillar was 400 nm, thereby producing a metal microstructure.

[SAM formation and immobilization of immobilized antibody]
SAM was formed on the metal microstructure by immersing the substrate on which the metal microstructure was formed in the SAM solution in an incubator at 40° C. overnight. In addition, the SAM solution was prepared by the following procedures. Hydroxy EG3 undecanethiol and carboxy EG6 undecanethiol were each diluted with ethanol to 1 mM and mixed. A SAM solution was prepared by diluting the obtained mixed material 5-fold with ethanol.

Then, by the amine coupling method, the carboxyl group (COOH) terminal of the SAM and the amino group (NH2) of the first VHH antibody are peptide-bonded by EDC/NHS reaction, and the first VHH antibody is immobilized on the SAM. did. After immobilizing the first VHH antibody, blocking treatment was performed using a blocking solution containing bovine serum albumin as a main component (hereinafter referred to as AD solution).

[Detection of NP]
The first VHH antibody immobilized on the metal microstructure is bound to the nuclear protein (NP) of influenza A virus, which is an antigen, and further labeled with an organic fluorescent dye (emission wavelength: 800 nm), which is a fluorescent substance. A sandwich Assay was performed by binding the second VHH antibody prepared by the method to NP. In this example, NP and the second VHH antibody labeled with a fluorescent substance are sequentially added to cause an antigen-antibody reaction, and therefore, this is referred to as a sequential sandwich assay (hereinafter, sequential reaction).

A sample subjected to a sandwich assay of sequential reactions was irradiated with laser light having a wavelength of 785 nm to excite the organic fluorescent dye, and the intensity of fluorescence having a wavelength of 800 nm emitted by the organic fluorescent dye was measured.

As antigens, the following four types of influenza A virus nuclear proteins (NP) were used.

(1) Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Nucleoprotein (Catalog Number: 11675-V08B, Sino Biological Inc.)
(2) Influenza A H1N1 (A/California/07/2009) Nucleoprotein (Catalog Number: 40205-V08B, Sino Biological Inc.)
(3) Influenza A H3N2 (A/Switzerland/9715293/2013) Nucleoprotein (Catalog Number: 40499-V08B, Sino Biological Inc.)
(4) Influenza A H7N9 (A/Shanghai/2/2013) Nucleoprotein (Catalog Number: 40111-V08B, Sino Biological Inc.)

NP solutions were prepared at concentrations of 0 M (solvent only), 1 pM, 10 pM, 100 pM, and 1000 pM using the AD solution as a solvent.

The results of sequential sandwich assays performed using the antigens (1) to (4) above will be described below with reference to FIGS. 10 to 13. FIG.

FIG. 10 shows the surface enhanced fluorescence (SEF) when a sequential sandwich assay was performed using the Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Nucleoprotein in (1) above as an antigen. ) is a graph showing the detection results. As shown in FIG. 10, the detected SEF for the antigen (1) above is the detection signal in the case of no NP (“no antigen” in the figure) in the concentration range of NP concentration of 1 pM or more, A significant difference was confirmed from the detection signal at each of the above NP concentrations. It was also confirmed that the detected SEF signal increased depending on the NP concentration.

FIG. 11 is a graph showing the results of detection of SEF when a sequential sandwich assay was performed using the Influenza A H1N1 (A/California/07/2009) Nucleoprotein of (2) above as an antigen. As shown in FIG. 11, for the antigen (2) above, the detected SEF is the detection signal in the case of no NP (“no antigen” in the figure) in the concentration range of NP concentration of 10 pM or more, and A significant difference was confirmed from the detection signal at each of the above NP concentrations. It was also confirmed that the detected SEF signal increased depending on the NP concentration.

FIG. 12 is a graph showing the results of detection of SEF when a sequential sandwich assay was performed using Influenza A H3N2 (A/Switzerland/9715293/2013) Nucleoprotein in (3) above as an antigen. As shown in FIG. 12, for the antigen (3) above, the detected SEF is the detection signal in the case of no NP (“no antigen” in the figure) in the concentration range of NP concentration of 1 pM or more, A significant difference was confirmed from the detection signal at each of the above NP concentrations. It was also confirmed that the detected SEF signal increased depending on the NP concentration.

FIG. 13 is a graph showing the results of SEF detection when a sequential sandwich assay was performed using the Influenza A H7N9 (A/Shanghai/2/2013) Nucleoprotein in (4) above as an antigen. As shown in FIG. 13, for the antigen (4) above, the detected SEF is the detection signal in the case of no NP (“no antigen” in the figure) and 1 pM in the concentration range of NP concentration of 1 pM or more A significant difference was confirmed from the detection signal at each of the above NP concentrations. It was also confirmed that the detected SEF signal increased depending on the NP concentration.

(Example 6)
In this example, the metal microstructure was prepared, the SAM was formed, and the immobilized antibody was immobilized in the same manner as in Example 5.

In this example, unlike Example 5, NPs and a second VHH antibody labeled with a fluorescent substance (hereinafter, labeled antibody) were mixed in advance, and the NPs bound with the labeled antibody and the metal It binds with the first VHH antibody immobilized on the microstructure. In this example, the NP and the labeled antibody are mixed in advance and then added to allow the antigen-antibody reaction with the first VHH antibody, so this is called a premixed reaction sandwich assay.

In this example, the antigen used was Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Nucleoprotein (see Example 5), which is the nuclear protein of influenza A virus described in (1) above. .

The NP solution was adjusted to concentrations of 0 M (solvent only), 2 pM, 20 pM, 200 pM, and 2000 pM using the AD solution as the solvent, and the labeled antibody solution was adjusted to 1 ug/mL using the AD solution as the solvent. , the same amount of NP solution and labeled antibody solution were mixed.

FIG. 14 is a diagram showing the detection results of SEF when a premixed reaction sandwich assay was performed using Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Nucleoprotein in (1) above as an antigen. be. As shown in FIG. 14, for the antigen (1) above, the detected SEF is the detection signal in the case of no NP (“no antigen” in the figure) in the concentration range of NP concentration of 1 pM or more, A significant difference was confirmed from the detection signal at each of the above NP concentrations. It was also confirmed that the detected SEF signal increased depending on the NP concentration.

In comparison with the SEF detection results when the sequential sandwich assay shown in FIG. 10 was performed, the same detection results as in FIG. 10 were obtained in FIG. 14 as well. Therefore, in the sandwich assay, the reaction for forming the sandwich structure of the immobilized antibody, the NP, and the labeled antibody is either a sequential reaction or a premixed reaction, the detection device according to one aspect of the present disclosure. was able to detect NP with the same accuracy.

(Comparative example 1)
In Comparative Example 1, instead of the first VHH antibody and the second VHH antibody, the IgG antibodies shown below were used as the immobilized antibody and the labeled antibody, respectively. did The immobilized antibody was prepared to have a concentration ten times that of the immobilized antibody in Example 6 and used. As the antigen, A/Puerto Rico/8/34/Mount Sinai (H1N1) Nucleoprotein, which is the intranuclear protein of the influenza A virus described in (1) above, was used. NPs were prepared at concentrations of 0 M (solvent only), 1 pM, 10 pM, 100 pM and 1000 pM using acetate buffer (pH 5.5) as the solvent.

Immobilized antibody: Anti-Influenza A Virus Nucleoprotein antibody [B247M] (ab66191) (manufactured by abcam)
Labeled antibody: Anti-Influenza A Virus Nucleoprotein antibody [F8] (ab8261) (manufactured by abcam) labeled with a fluorescent substance

FIG. 15 is a graph showing the results of SEF detection (average of n=5) when a sequential sandwich assay was performed using IgG antibodies as the immobilized antibody and the labeled antibody. As shown in FIG. 15, the detection signal without NP (“no antigen” in the figure) was very high, and the noise due to non-specific adsorption of the labeled antibody was large. Therefore, it was found that the use of IgG antibodies as the immobilized antibody and the labeled antibody greatly reduced the detection sensitivity.

(summary)
As described above, the detection device according to one aspect of the present disclosure uses the first VHH antibody as the immobilized antibody and the second VHH antibody as the labeled antibody, thereby immobilizing the metal microstructure. It was confirmed that the immobilized amount of the antibody was improved and the ability of the labeled antibody to capture the antigen was enhanced. In addition, since the detection device according to one aspect of the present disclosure uses the VHH antibody as the immobilized antibody and the labeled antibody, it was confirmed that the noise due to non-specific adsorption of the labeled antibody is very small and the detection sensitivity is high. . In addition, regarding the sandwich assay, even if the NPs and the labeled antibody are added onto the metal microstructure in this order, or the NPs and the labeled antibody are mixed in advance and added onto the metal microstructure, the results are equivalent. It was confirmed that detection sensitivity was obtained.

Therefore, according to the above examples, it was confirmed that the detection device according to the present disclosure can detect a very small amount of influenza A virus with high sensitivity.

Although the detection device according to the present disclosure has been described above based on the embodiments and examples, the present disclosure is not limited to these embodiments and examples. As long as it does not deviate from the gist of the present disclosure, various modifications that a person skilled in the art can think of are applied to the embodiments and examples, and other forms constructed by combining some components in the embodiments and examples , are included in the scope of this disclosure.

The detection device according to the present disclosure can be used in a detection system or the like that detects the concentration of airborne viruses in the air of a room with high sensitivity in order to reduce the risk of virus infection to people staying in the room. can.

10 detection system 100 collection device 102 aspirator 104 collection liquid tank 106, 114 pump 108 cyclone 110 air inlet 112 cleaning liquid tank 120 waste liquid tank 122 liquid flow path 200 detection apparatus 202 sensor device 204 sensor cell 204a flow path 204b detection area 206 Introduction Part 208 Light Source 210 Beam Splitter 212 Lens 214 Detection Part 300 Controller 2041, 2041A Metal Microstructure 2042 Resin Substrate 2042a Pillar 2043 Metal Film 2043a Protrusion 2044, 2044B SAM
2045 first VHH antibody 2046 linker molecule 2047 non-linker molecule 2061 sample liquid 2062 influenza A virus NP (NP)
2063 second VHH antibody 2064 fluorescent substance 20461, 20471 thiol group 20462, 20472 alkyl chain 20463, 20473 ethylene glycol chain 20464 carboxyl group 20474 hydroxyl group

Claims (7)

a metal microstructure on which a first VHH antibody having a property of specifically binding to an intranuclear protein of influenza A virus is immobilized and which generates surface plasmons when irradiated with excitation light;
a second VHH antibody that has the property of specifically binding to the influenza A virus nuclear protein and is labeled with a fluorescent substance, and a sample that may contain the influenza A virus nuclear protein; an introduction part to be introduced into the metal microstructure;
a light irradiation unit that irradiates the excitation light onto the metal microstructure into which the second VHH antibody and the sample have been introduced;
a detection unit that detects the intranuclear protein of the influenza A virus based on the fluorescence generated from the fluorescent substance by irradiation with the excitation light;
with
said first VHH antibody has structural domains arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus;
FR indicates a framework region, CDR indicates a complementarity determining region,
In the first VHH antibody,
The CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1,
the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2,
said CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3,
said second VHH antibody has structural domains arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus;
In the second VHH antibody,
the CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4,
the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5,
said CDR3 comprises the amino acid sequence shown in SEQ ID NO: 6;
detection device. The influenza A virus is at least one of H1N1, H3N2, and H7N9, which are subtypes of influenza A virus ;
A detection device according to claim 1 . In the first VHH antibody,
The FR1 comprises the amino acid sequence shown in SEQ ID NO: 7,
The FR2 comprises the amino acid sequence shown in SEQ ID NO: 8,
The FR3 comprises the amino acid sequence shown in SEQ ID NO: 9,
said FR4 comprises the amino acid sequence shown in SEQ ID NO: 10;
3. A detection device according to claim 1 or 2. In the second VHH antibody,
The FR1 comprises the amino acid sequence shown in SEQ ID NO: 11,
The FR2 comprises the amino acid sequence shown in SEQ ID NO: 12,
The FR3 comprises the amino acid sequence shown in SEQ ID NO: 13,
said FR4 comprises the amino acid sequence shown in SEQ ID NO: 14;
The detection device according to any one of claims 1-3. A self-assembled monolayer containing linker molecules and non-linker molecules is formed on the surface of the metal microstructure,
wherein the first VHH antibody is immobilized on the metal microstructure via the linker molecule;
A detection device according to any one of claims 1 to 4. The linker molecule has a thiol group at one end and a carboxyl group at the other end, and includes an alkyl chain having 10 or more carbon atoms and an ethylene glycol chain,
The non-linker molecule has a thiol group at one end and a hydroxyl group at the other end, and includes an alkyl chain having 10 or more carbon atoms and an ethylene glycol chain,
6. A detection device according to claim 5. a first VHH antibody that has the property of specifically binding to an intranuclear protein of influenza A virus and is immobilized on a substrate surface;
A method for detecting influenza A virus using a second VHH antibody that has the property of specifically binding to the intranuclear protein of influenza A virus and is labeled with a fluorescent substance,
said first VHH antibody has structural domains arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus;
FR indicates a framework region, CDR indicates a complementarity determining region,
In the first VHH antibody,
The CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1,
the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2,
said CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3,
said second VHH antibody has structural domains arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminus to C-terminus;
In the second VHH antibody,
the CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4,
the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5,
said CDR3 comprises the amino acid sequence shown in SEQ ID NO: 6;
A method for detecting influenza A virus.

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