Antibody Immobilization Substrate and Method for Manufacturing Antibody Immobilization Substrate

Abstract

To provide a novel antibody immobilization substrate and a method for producing the antibody immobilization substrate. [Solution] The present invention provides, A substrate for antibody immobilization having a resin plate with a first surface, The resin plate has a first groove structure on its first surface in a first direction parallel to the first surface. The arithmetic mean roughness Ra of the surface of the first groove structure in a cross section perpendicular to the first direction is 0.5 μm or more and 10 μm or less, and / or the surface roughness Rz of the first groove structure in a cross section perpendicular to the first direction is 5 μm or more and 20 μm or less. We provide a substrate for antibody immobilization.

Description

Technical Field

[0001] The present invention broadly relates to a substrate for antibody immobilization and a method for manufacturing the substrate for antibody immobilization.

Background Art

[0002] Point of care testing (POCT) technology for performing disease diagnosis and health management quickly and on-site at any time, as represented by antigen tests for influenza and coronavirus, and blood glucose analysis at home, is widely used, and its demand is increasing.

[0003] One of the POCT technologies, "lateral flow immunoassay (LFIA)", is a method for simply and inexpensively detecting target substances (such as viruses and proteins) contained in blood, urine, saliva, nasal mucus, etc. LFIA is a method in which a sample dropped onto a measurement device is transported by capillary force and an antigen is captured and detected by an antibody immobilized in the device. In existing LFIA devices, a membrane made of nitrocellulose is mainly used as a substrate that enables simultaneous liquid transport by capillary force and antibody immobilization. However, nitrocellulose membranes have problems such as being flammable, being easily affected by humidity and having a short expiration date, being likely to be damaged, shrunk, or scratched due to low strength, and having large variations in membrane properties and being unsuitable for accurate quantitative evaluation (Non-Patent Documents 1 and 2). Therefore, there is a need for a substrate for LFIA that can solve these problems.

[0004] As a substrate for antibody immobilization that replaces a normal nitrocellulose membrane, for example, Non-Patent Document 3 discloses an immunoassay device using a micropillar structure made of cycloolefin polymer (COP) produced by injection molding as a substrate.

[0005] Non-Patent Document 4 discloses an immunoassay device material having a nanopillar structure with a high aspect ratio using a polystyrene substrate.

[0006] Patent Document 1 discloses a substrate for an immunoassay device, which consists of a polycarbonate film on which numerous microcone structures are arranged. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2020 / 230572 [Non-patent literature]

[0008] [Non-Patent Document 1] R. Wong, et al., "Lateral Flow Immunoassay" DOI 10.1007 / 978-1-59745-240-3 [Non-Patent Document 2] J. Kong et al., Anal. Chim. Acta, 2020, 1118, 10. [Non-Patent Document 3] J. Melin, et al., Lab Chip, 2008, 8, 1891. [Non-Patent Document 4] A. Miyauchi, et al., Appl. Phys. Lett., 2008, 93, 033904. [Overview of the project] [Problems that the invention aims to solve]

[0009] However, the manufacture of immunoassay devices as disclosed in patent and non-patent literature requires complex processes, resulting in high costs and labor. Furthermore, complex chemical processes are required to immobilize antibodies onto these immunoassay devices.

[0010] The present invention aims to provide a novel antibody immobilization substrate and a novel method for producing the antibody immobilization substrate. [Means for solving the problem]

[0011] As a result of diligent research by the inventors, it was discovered that a substrate for antibody immobilization can be easily manufactured by forming a groove structure parallel to the surface of a resin plate using a polishing device.

[0012] In other words, this application encompasses the following inventions. [1] A substrate for antibody immobilization having a resin plate with a first surface, The resin plate has a first groove structure on its first surface in a first direction parallel to the first surface. The arithmetic mean roughness Ra of the surface of the first groove structure in a cross section perpendicular to the first direction is 0.5 μm or more and 10 μm or less, and / or the surface roughness Rz of the first groove structure in a cross section perpendicular to the first direction is 5 μm or more and 20 μm or less. Substrate for antibody immobilization. [2] The resin plate has a second groove structure on its first surface in a second direction that is parallel to the first surface and intersects with respect to the first direction. The arithmetic mean roughness Ra of the surface of the second groove structure in a cross section perpendicular to the second direction is 0.5 μm or more and 10 μm or less, and / or the surface roughness Rz of the second groove structure in a cross section perpendicular to the second direction is 5 μm or more and 20 μm or less. [1] The antibody immobilization substrate described above. [3] The density of the first groove structure in a cross section perpendicular to the first direction is 10 or more grooves per mm in the planar direction of the first surface, and / or The average length of the first groove structure is 1 mm or more. The antibody immobilization substrate described in [1] or [2]. [4] The density of the second groove structure in a cross section perpendicular to the second direction is 10 or more grooves per mm in the planar direction of the first surface, and / or The average length of the second groove structure is 1 mm or more. The substrate for antibody immobilization described in [2]. [5] The substrate for antibody immobilization according to [1] or [2], wherein the resin flat plate is made of polycarbonate, polyethylene terephthalate, or polyethylene naphthalate. [6] A method for producing a substrate for antibody immobilization having a resin flat plate with a first surface, the method including a step of forming a first groove structure on the first surface of the resin flat plate using a polishing device or a polishing tool capable of performing processing in a certain direction in a first direction parallel to the first surface. The method according to [6], wherein a belt sander with a number of #60 to #400 is used to form the first groove structure. [7] The method according to [6], wherein the resin flat plate is made of polycarbonate, polyethylene terephthalate, or polyethylene naphthalate. [8] The method according to [6], further including a step of forming a second groove structure on the first surface of the resin flat plate in a second direction parallel to the first surface and intersecting the first direction. [9] The method according to any one of [6] to [9], further including a step of immersing the resin flat plate in an organic solvent.

[10] The method according to

[10] , wherein the organic solvent is acetone and / or ethanol.

[11] The method according to

[10] , wherein the organic solvent is a mixture of acetone and ethanol, and the volume ratio of acetone in the organic solvent is 0.2 to 0.8.

[12] In [1] to [5], the substrate for antibody immobilization may be a substrate for lateral flow immunoassay, and in [6] to

[12] , the method for producing the substrate for antibody immobilization may be a method for producing a substrate for lateral flow immunoassay.

Advantages of the Invention

[0013] According to the present invention, it is possible to provide a novel method for manufacturing an antibody immobilization substrate and a novel antibody immobilization substrate. [Brief explanation of the drawing]

[0014] [Figure 1] The images show surface views of polycarbonate sheets with (a) a linear pattern and (b) a grid pattern, as captured by a scanning electron microscope (SEM). [Figure 2] The antibody adsorption capacity of polycarbonate sheets was evaluated using fluorescence intensity as an indicator, and the results are shown below. [Figure 3] The results of evaluating the flow velocity of capillary flow in polycarbonate sheets having linear and grid-like groove patterns are shown. [Figure 4] The results of an immunoassay using C-reactive protein (CRP) as a model target substance are shown. (a) Fluorescence micrographs of polycarbonate sheets observed from above after performing an immunoassay using a fluorescently labeled antibody as a secondary antibody and buffers containing different concentrations of CRP are shown. (b) Results of measuring relative fluorescence intensity are shown. [Figure 5] The results of detecting CRP in serum using an immunoassay are shown. [Figure 6] (a) Results of evaluating the antibody adsorption capacity of sheets treated with organic solvents are shown. (b) A photograph of the sheets treated with organic solvents is shown. [Figure 7] The results of an immunoassay, performed on sheets treated with and without organic solvent treatment, to detect CRP in the buffer are shown. [Figure 8] The results of immunoassays performed on sheets treated with and without organic solvents to detect serum CRP are shown. [Figure 9] The results of comparing (a) autofluorescence and (b) antibody adsorption capacity for sheets made of different resins are shown. [Figure 10] The images show cross-sectional views of flat and linear patterned polycarbonate sheets, taken with an optical microscope. [Figure 11]The results of measuring (a) arithmetic mean roughness Ra and (b) surface roughness Rz from a cross-sectional view of a polycarbonate sheet are shown. [Modes for carrying out the invention]

[0015] The following describes embodiments of the present invention (hereinafter referred to as "these embodiments"), but the scope of the present invention is not limited to these embodiments.

[0016] (Substrate for antibody immobilization) The antibody immobilization substrate of this embodiment is A substrate for antibody immobilization having a resin plate with a first surface, The resin plate has a first groove structure on its first surface, in a first direction parallel to the first surface. The arithmetic mean roughness Ra of the surface of the first groove structure in a cross section perpendicular to the first direction is 0.5 μm or more and 10 μm or less, and / or the surface roughness Rz of the first groove structure in a cross section perpendicular to the first direction is 5 μm or more and 20 μm or less. This is a substrate for immobilizing antibodies.

[0017] In this embodiment, the groove structure is represented by the arithmetic mean roughness Ra of the surface of the groove structure in a cross-section of a resin plate perpendicular to the direction of the groove, and / or the surface roughness Rz of the groove structure in a cross-section of a resin plate perpendicular to the direction of the groove.

[0018] Arithmetic mean roughness Ra is the average value of the height distance (absolute value) from the average position in the height direction of the contour curve used for roughness evaluation (JIS B0601:2001). In this embodiment, Ra may be 0.5 μm or more and 10 μm or less, preferably 0.5 μm or more and 8 μm or less, and more preferably 0.5 μm or more and 5 μm or less.

[0019] Surface roughness Rz refers to the maximum height roughness, and is calculated by extracting a portion of the roughness curve measured by a roughness meter at a reference length, and it corresponds to the sum of the highest point (maximum peak height: Rp) and the deepest point (maximum valley depth: Rv) (JIS B0601:1994). In this embodiment, Rz may be 1 μm or more and 20 μm or less, preferably 5 μm or more and 18 μm or less, and more preferably 5 μm or more and 15 μm or less.

[0020] The method for producing the antibody immobilization substrate of this embodiment is: A method for manufacturing an antibody immobilization substrate having a resin plate having a first surface, the method comprising the step of forming a first groove structure on the first surface of the resin plate using a polishing device or polishing instrument that enables processing in a certain direction in a first direction parallel to the first surface. In this embodiment, a resin plate is used to manufacture a substrate for antibody immobilization.

[0021] (resin flat plate) The resin sheet used in this embodiment may be a sheet made of resin. Examples of resins include polycarbonate, polyethylene terephthalate, polyethylene naphthalate, polyimide, polyphenylene sulfide, polyether ketone, polyvinyl chloride, polystyrene, polyurethane, polyethylene, polypropylene, acrylic resin, and polyacetal, among which polycarbonate, polyethylene terephthalate, or polyethylene naphthalate are preferred. Commercially available resin sheets may be used, for example, polycarbonate sheets (3-8901-01), polyethylene terephthalate sheets (3-2160-05), polyethylene naphthalate sheets (3-2161-05), polyimide sheets (3-1966-08), polyphenylene sulfide sheets (3-8898-03), and polyether ether ketone sheets (67-2190-91) (AS ONE). The manufacturing method for the resin sheet is not particularly limited; generally, a sheet can be made by stretching resin onto a flat surface, molding it, and then cutting it into predetermined sizes. Each component of the resin may be the main component of the resin sheet, and the resin sheet may also contain components other than the main component. The components other than the main component may be general-purpose components.

[0022] The shape of the resin plate can be arbitrary as long as grooves can be formed in the plate, and can be determined appropriately by a person skilled in the art, but a rectangular shape is preferred. The size of the resin plate can be arbitrary and can be determined appropriately by a person skilled in the art; for example, it may be approximately 3 cm x approximately 12 cm. The thickness of the resin plate may be arbitrary and can be determined appropriately by a person skilled in the art, for example, it may be in the range of about 0.05 mm to 5 mm.

[0023] (Polishing device or polishing equipment) In this embodiment, the resin plate is processed to form a groove structure in a specific direction using any polishing device or tool. The polishing device or tool can be any known device that enables processing of the resin substrate in a specific direction, and can be appropriately selected by a person skilled in the art. Examples of polishing devices or tools include belt sanders, sandpaper, cloth sandpaper, metal files, ceramic files, and disc sanders. While not particularly limited, the following explanation will use a belt sander as an example of a polishing device or tool. A belt sander can polish an object by rotating a ring-shaped sanding belt with a grinding surface around multiple rollers and pressing the rotating grinding surface against the object. A commercially available belt sander may be used, for example, the BDS-1010 (Kyocera), YS1N (Yodogawa Electric Works), and SB10V2 100V (HiKOKI).

[0024] The grit size of the belt sander can be arbitrary and can be determined appropriately by a person skilled in the art. For example, the grit size of the belt sander can be selected from #60, #80, #120, #240, and #400, with #60 to #400 being preferred, and #120 or #240 being more preferred.

[0025] The rotational speed of the belt sander and the size of the sanding belt can be arbitrary and can be appropriately determined by a person skilled in the art.

[0026] The step of forming a first groove structure on the first surface of a resin plate in a first direction parallel to the first surface using a belt sander may be carried out by pressing the resin plate against the belt sander. A groove is formed by pressing the resin plate against the belt sander. While pressing the resin plate against the belt sander, the resin plate may be moved up and down to form a groove in a desired portion of the resin plate.

[0027] In this embodiment, forming a groove structure on the first surface of the resin plate in a first direction parallel to the first surface may mean forming a groove structure in a certain direction on the first surface of the resin plate. Alternatively, the groove structure may be formed substantially parallel to the longitudinal direction of the resin plate. The term "substantially parallel to the longitudinal direction" is determined according to the shape of the resin plate, but for example, when used as a substrate for antibody immobilization, it is preferable that the grooves are formed along the flow direction of the test solution.

[0028] The antibody immobilization substrate of this embodiment may be used in assays, such as immunoassays, to detect target substances (antigens) in a sample by immobilizing antibodies on the substrate. The sample can be appropriately determined by those skilled in the art, but may be, for example, blood, urine, or saliva.

[0029] In this embodiment, the process may further include a step of cleaning the resin plate after processing it to form a groove structure, and the cleaning step may be carried out by a known method. For example, the process may further include a step of ultrasonically cleaning the resin plate after processing it with a belt sander. Ultrasonic cleaning may be carried out by immersing the resin plate in water and irradiating it with ultrasound having a frequency of 20kHz to 200kHz, 20kHz to 100kHz, or 20kHz to 50kHz for 10 seconds to 5 minutes, 10 seconds to 1 minute, or 10 seconds to 30 seconds.

[0030] In this embodiment, an organic solvent may be used as a processing means for the resin plate, and the method for producing the antibody immobilization substrate in this embodiment may include a step of immersing the resin plate in an organic solvent. By immersing the resin plate in an organic solvent, fine cracks are formed on the surface of the resin plate. As used herein, "organic solvent" is not particularly limited as long as it causes cracks in the resin plate, but may be at least one selected from the group consisting of methanol, ethanol, 2-propanol, butanol, pentanol, ethylene glycol, propylene glycol, acetone, methyl ethyl ketone, diethyl ketone, and acetophenone. In this embodiment, the organic solvent preferably contains acetone and / or ethanol, and more preferably contains acetone and ethanol. When the organic solvent is a mixture of acetone and ethanol, the volume ratio of acetone in the organic solvent may be 0.2 to 0.8, preferably 0.4 to 0.8, and more preferably 0.5 to 0.8.

[0031] In this embodiment, the immersion time of the resin plate in the organic solvent can be appropriately determined by a person skilled in the art depending on the type and size of the resin plate, and is preferably 5 seconds to 12 hours, more preferably 10 seconds to 1 hour, and even more preferably 20 seconds to 1 minute. For example, the immersion time may be 5 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 6 hours, or 12 hours.

[0032] The first embodiment is a method for producing an antibody immobilization substrate having a resin plate having a first surface, the method comprising the step of forming a first groove structure on the first surface of the resin plate in a first direction parallel to the first surface. The aspects described above in the first embodiment also apply to the other embodiments described below.

[0033] In a second embodiment, a method for manufacturing an antibody immobilization substrate having a resin plate having a first surface is provided, further comprising the step of forming a second groove structure on the first surface of the resin plate in a second direction that is parallel to the first surface and intersects with respect to the first direction.

[0034] In this embodiment, the direction of the first groove structure and the direction of the second groove structure are formed to intersect. The acute angle of the angle between the two directions may be greater than 0 degrees and less than 90 degrees. Preferably, this angle is greater than 0 degrees and 60 degrees or less, more preferably greater than 0 degrees and 45 degrees or less, and even more preferably greater than 0 degrees and 30 degrees or less.

[0035] The method for producing an antibody immobilization substrate, provided as a second embodiment, is also applicable to other embodiments described below.

[0036] In a third embodiment, a method for producing an antibody immobilization substrate having a resin plate having a first surface is provided, further comprising the step of immersing the resin plate in an organic solvent.

[0037] In this embodiment, the process may further include a step of washing the resin plate after immersing it in an organic solvent, and the washing step may be carried out by a known method. For example, the process may further include a step of ultrasonically cleaning the resin plate after immersing it in an organic solvent.

[0038] In this embodiment, the steps of forming a groove structure on a resin plate, immersing the resin plate in an organic solvent, and cleaning the resin plate may be performed in any order. The step of cleaning the resin plate after forming the groove structure on the resin plate may be further included. For example, the steps may be performed in the order of forming the groove structure, cleaning the resin plate, immersing the resin plate in an organic solvent, and cleaning the resin plate, or in the order of immersing the resin plate in an organic solvent, cleaning the resin plate, forming the groove structure, and cleaning the resin plate. However, it is preferable to perform the step of immersing the resin plate in an organic solvent after performing the step of forming the groove structure.

[0039] In this embodiment, the average length of the groove structure may be 1 mm or more.

[0040] In this embodiment, the density of grooves in the cross-section of the resin plate perpendicular to the direction of the grooves may be 10 or more grooves per millimeter in the surface direction of the resin plate. Here, "groove" means a cross-sectional shape in which the groove width is 1 μm or more and 50 μm or less, and the depth is 1 μm or more and 50 μm or less, and the recessed portion extends intermittently or continuously over a long length.

[0041] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0042] <Test Example 1: Fabrication of a resin plate 1> A 3cm x 12cm polycarbonate (PC) sheet (AS ONE, 3-8901-01) was pressed against a belt sander (coarseness: #60, #80, #120, #240, #400, Kyocera Corporation) with the rotation direction and longitudinal direction aligned. The sander was moved up and down at a speed of approximately 5cm / s, applying enough force to push the sheet about 1mm into the sander's surface. This process was repeated 10 times over approximately 30 seconds, removing about 8cm of the sheet's height. The sheet was then ultrasonically cleaned to obtain a sheet with a linear groove pattern.

[0043] <Test Example 2: Fabrication of Resin Flat Sheets 2> A 3cm x 12cm PC sheet was pressed against a belt sander (grit: #60, #80, #120, #240, #400) and moved up and down at a speed of approximately 5cm / s, applying enough force to push the sheet about 1mm into the sander's surface. After one pass over a sheet height of approximately 8cm with the longitudinal direction tilted 15 degrees to the right relative to the sander's rotation direction, another pass over a sheet height of approximately 8cm with the longitudinal direction tilted 15 degrees to the left relative to the sander's rotation direction. A total of 5 sets were performed over approximately 30 seconds to sand the sheet. Afterwards, the sheet was ultrasonically cleaned to obtain a sheet with a grid-like pattern of grooves.

[0044] <Test Example 3: Fabrication of Resin Flat Sheets 3> A sheet with a linear groove pattern was obtained in the same manner as in Test Example 1, except that polyethylene terephthalate (PET) sheet (3-2160-05), polyethylene naphthalate (PEN) sheet (3-2161-05), polyimide (PI) sheet (3-1966-08), polyphenylene sulfide (PPS) sheet (3-8898-03), and polyetheretherketone (PEEK) sheet (67-2190-91) (AS ONE) were used instead of PC sheet, and a belt sander (roughness: #120, Kyocera Corporation) was used. Figure 1 shows photographs of PC sheets with (a) a linear pattern and (b) a grid pattern, taken with a scanning electron microscope (SEM).

[0045] <Test Example 4: Evaluation of Antibody Adsorption Capacity> Unprocessed PC sheets, sandblasted PC sheets (particle coarseness: #120), and PC sheets prepared in Test Example 1 were each subjected to 1 μL of a buffer (50 mM Tris-HCl buffer containing 2% trehalose) containing 0.1 mg / mL of fluorescently labeled antibody (Thermo Fisher Scientific, Goat anti-rat IgG cross-absorbed secondary antibody, Alexa Fluor 488, A11006), and then dried. After washing, fluorescence microscope images were captured using a fluorescence microscope and camera, and fluorescence intensity was calculated using image analysis software (Image J). The excitation wavelength was 470-495 nm, and the fluorescence wavelength was 510-550 nm. The results are shown in Figure 2. We evaluated sheets that had been processed with a belt sander and sheets that had been sandblasted using particles with a particle size of #120, in comparison with the evaluation results of an unprocessed flat sheet. Sheets that underwent sandblasting and belt sanding showed increased fluorescence intensity compared to untreated sheets. This result suggests that the microstructure formed on the sheet promoted antibody adsorption. Furthermore, sheets processed with belt sanders of roughness #120 and #240 showed a significant increase in fluorescence intensity.

[0046] <Test Example 5: Evaluation of Capillary Flow Velocity> 10 μL of running buffer for LFIA (2% Triton X-100 / PBS) was dropped onto the PC sheets (3 mm wide, 30 mm long) prepared in Test Examples 1 and 2. The time from dropping to reaching the antibody immobilization area (distance from the dropping point: 10 mm) was measured, and the average flow rate was calculated. The results are shown in Figure 3. These results showed that sheets processed with belt sanders of roughness #120 and #240 exhibited a significantly increased flow velocity compared to other sheets. Furthermore, sheets with a grid-like groove pattern had a higher flow velocity than sheets with a straight groove pattern.

[0047] <Test Example 6: Immunoassay> To verify whether the present invention can detect specific antigens, the following immunoassay was performed using C-reactive protein (CRP) as a model target substance. In Test Examples 1 and 2, a sheet (3 mm wide, 30 mm long) prepared using a #120 belt sander was fitted with a piece of filter paper (5 mm wide, 30 mm long, cut from Advantec's circular qualitative filter paper, No. 6) to absorb the solution, overlapping by 10 mm in the length direction, and secured with adhesive tape. An anti-CRP antibody (Bethyl Laboratories' Human C-reactive protein polyclonal antibody, A80-125A) was immobilized on the upper surface near the center of the sheet. Samples were added to buffer (2% Triton X-100 / PBS) to achieve CRP concentrations of 0 μg / mL, 0.1 μg / mL, 1 μg / mL, and 10 μg / mL. These samples were dropped onto the sheet at a point 5 mm from the end opposite to the end where the filter paper was fixed, and left to stand at room temperature for 10 minutes to capture CRP. After washing with 10 μL of buffer (2% Triton X-100 / PBS), 10 μL of buffer (2% Triton X-100 / PBS) containing a fluorescently labeled secondary antibody (Bethyl Laboratories, Goat x-human C-reactive protein, FITC labeled, A80-125F) at a concentration of 0.02 mg / mL was added three times. Subsequently, the sample was washed with 10 μL of buffer (2% Triton X-100 / PBS). Fluorescence intensity was calculated in the same manner as in Test Example 4. The results are shown in Figure 4. These results confirmed that fluorescence intensity increased with increasing antigen (CRP) concentration. The detection limit, indicated by the standard deviation of the blank multiplied by 3, was approximately 100 ng / mL, demonstrating that it is sufficiently applicable even in medical diagnostics (CRP concentration of several μg / mL).

[0048] <Test Example 7: Detection of CRP in Serum> In Test Example 1, a sheet (3 mm wide, 30 mm long) prepared using a #120 belt sander was fitted with a piece of filter paper (5 mm wide, 30 mm long, cut from Advantec circular qualitative filter paper, No. 6) to absorb the solution, overlapping by 10 mm in the length direction, and secured with adhesive tape. Anti-CRP antibody was immobilized on the upper surface near the center of the sheet. Human serum (CRP-depleted serum, Assaypro, Human C-Reactive Protein (CRP) defficient serum, D100110) was mixed with CRP to prepare initial CRP concentrations of 0 μg / mL, 0.1 μg / mL, 1 μg / mL, and 10 μg / mL. Equal volumes of the sample and buffer (4% Triton X-100 / PBS) were mixed and dropped onto the sheet at a point 5 mm from the end opposite to the end where the filter paper was fixed, and left to stand at room temperature for 10 minutes to capture CRP. Buffer (2% Triton After washing with 10 μL of X-100 / PBS, 10 μL each of buffer (2% Triton X-100 / PBS) containing a fluorescently labeled secondary antibody (Bethyl Laboratories, Goat x-human C-reactive protein, FITC labeled, A80-125F) at a concentration of 0.02 mg / mL was added for a total of three times. Subsequently, 10 μL of buffer (2% Triton X-100 / PBS) was added for washing. Fluorescence intensity was calculated in the same manner as in Test Example 4. The results are shown in Figure 5. In this figure, the horizontal axis represents the initial CRP concentration. These results demonstrate that CRP can also be detected in serum. The detection limit is approximately 60 ng / mL, indicating that it is sufficiently applicable in medical diagnosis.

[0049] <Test Example 8: Treatment of Sheets with Organic Solvents> Acetone and ethanol were mixed so that the volume mixing ratios of acetone were 0, 0.2, 0.4, 0.45, 0.5, 0.55, 0.6, 0.8, and 1.0. The sheets prepared in Test Example 1 using a #120 belt sander were immersed in the prepared organic solvent for 20 seconds. After washing, fluorescently labeled antibodies were immobilized on the sheets, and the antibody adsorption capacity was evaluated. The results are shown in Figure 6. These results indicate that antibody adsorption increases with increasing volume mixing ratio of acetone. Furthermore, sheets with an acetone volume mixing ratio of 0.6 showed reduced curling compared to sheets with a ratio of 0.8.

[0050] <Test Example 9: Immunoassay 2> Acetone and ethanol were mixed so that the volume mixing ratio of acetone was 0.6. The sheet prepared using a #120 belt sander in Test Example 1 was immersed in the prepared organic solvent for 20 seconds. The same immunoassay as in Test Example 6 was performed on the obtained sheet and the sheet prepared using a #120 belt sander in Test Example 1. The results are shown in Figure 7. These results indicate that the fluorescence intensity increased in sheets immersed in organic solvent (with cracks) compared to sheets not immersed in organic solvent (without cracks). The detection limit was approximately 110 ng / mL for sheets with cracks and approximately 80 ng / mL for sheets without cracks.

[0051] <Test Example 10: Detection of CRP in Serum 2> Acetone and ethanol were mixed so that the volume mixing ratio of acetone was 0.6. The sheet prepared using a #120 belt sander in Test Example 1 was immersed in the prepared organic solvent for 20 seconds. The detection ability of the obtained sheet for CRP added to human serum was evaluated in the same manner as in Test Example 7. The results are shown in Figure 8. These results demonstrate that CRP can also be detected in serum. The detection limit is approximately 60 ng / mL, indicating that it is sufficiently applicable in medical diagnosis.

[0052] <Test Example 11: Comparison of Antibody Adsorption Capacity of Resin Plates> For the sheets obtained using a belt sander (roughness #120) in Test Example 1 and the sheets obtained in Test Example 3, (a) the fluorescence intensity of the sheet's autofluorescence and (b) the fluorescence intensity after immobilizing a fluorescently labeled antibody, similar to Test Example 4, were measured to evaluate the antibody adsorption capacity. The results are shown in Figure 9. In (b), the value obtained by subtracting the autofluorescence intensity from the fluorescence intensity when the antibody is adsorbed is shown. These results showed that PC sheets, PET sheets, PEN sheets, and PI sheets exhibited low autofluorescence, with PC sheets and PI sheets showing particularly low autofluorescence. Furthermore, PC sheets, PET sheets, and PEN sheets demonstrated high antibody adsorption capacity.

[0053] <Test Example 12: Quantitative Evaluation of Surface Roughness> The PC sheets obtained in Test Example 1, as well as the belt-sanded and unprocessed sheets treated with an organic solvent under the conditions of immersion in an organic solvent with an acetone volume mixing ratio of 0.6 for 20 seconds in Test Example 8, were embedded in OCT compound (Sakura Finetech Japan Co., Ltd.) and frozen at -80°C. Subsequently, frozen sections (thickness: 10 μm) of the PC sheets were prepared using a cryostat. The frozen sections were photographed with an optical microscope, and the obtained images were subjected to image analysis. The obtained images are shown in Figure 10, and the image analysis results are shown in Figure 11. These results indicate that (a) Ra has a maximum value around roughness #80 and increases with organic solvent treatment, and (b) Rz increases with belt sanding and tends to decrease slightly with increasing belt sander grit numbers.

Claims

1. A substrate for antibody immobilization having a resin plate with a first surface, The resin plate has a first groove structure on its first surface in a first direction parallel to the first surface. The arithmetic mean roughness Ra of the surface of the first groove structure in a cross section perpendicular to the first direction is 0.5 μm or more and 10 μm or less, and / or the surface roughness Rz of the first groove structure in a cross section perpendicular to the first direction is 5 μm or more and 20 μm or less. Substrate for antibody immobilization.

2. The resin plate has a second groove structure on its first surface in a second direction that is parallel to the first surface and intersects with respect to the first direction. The arithmetic mean roughness Ra of the surface of the second groove structure in a cross section perpendicular to the second direction is 0.5 μm or more and 10 μm or less, and / or the surface roughness Rz of the second groove structure in a cross section perpendicular to the second direction is 5 μm or more and 20 μm or less. The antibody immobilization substrate according to claim 1.

3. The density of the first groove structure in a cross section perpendicular to the first direction is 10 or more grooves per mm in the planar direction of the first surface, and / or The average length of the first groove structure is 1 mm or more. The antibody immobilization substrate according to claim 1 or 2.

4. The density of the second groove structure in a cross section perpendicular to the second direction is 10 or more grooves per mm in the planar direction of the first surface, and / or The average length of the second groove structure is 1 mm or more. The antibody immobilization substrate according to claim 2.

5. The antibody immobilization substrate according to claim 1 or 2, wherein the resin plate is made of polycarbonate, polyethylene terephthalate, or polyethylene naphthalate.

6. A method for producing an antibody immobilization substrate having a resin plate with a first surface, A method comprising the step of forming a first groove structure on the first surface of the resin plate using a polishing device or polishing tool that enables processing in a certain direction in a first direction parallel to the first surface.

7. The method according to claim 6, wherein the first groove structure is formed using a belt sander with a grit of #60 to #400.

8. The method according to claim 6, wherein the resin plate is made of polycarbonate, polyethylene terephthalate, or polyethylene naphthalate.

9. The method according to claim 6, further comprising the step of forming a second groove structure on the first surface of the resin plate in a second direction that is parallel to the first surface and intersects with respect to the first direction.

10. The method according to any one of claims 6 to 9, further comprising the step of immersing the resin plate in an organic solvent.

11. The method according to claim 10, wherein the organic solvent is acetone and / or ethanol.

12. The method according to claim 10, wherein the organic solvent is a mixture of acetone and ethanol, and the volume ratio of acetone in the organic solvent is 0.2 to 0.8.

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