Carbon nanotube-labeled antibody and immunochromatographic membrane

Abstract

To provide a carbon nanotube-labeled antibody excellent in visibility in immunochromatography.SOLUTION: A carbon nanotube-labeled antibody comprising an antigen-binding portion and a carbon nanotube portion, wherein the carbon nanotube-labeled antibody is used for detecting binding of the carbon nanotube-labeled antibody to an antigen via the antigen-binding portion by visually observing a color derived from the carbon nanotube portion.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a carbon nanotube-labeled antibody and an immunochromatographic membrane. [Background technology]

[0002] Immunochromatography is a conventionally known testing method that utilizes a specific reaction, such as an antigen-antibody reaction. Immunochromatography is applied to influenza test kits, pregnancy test kits, and the like, and uses an immunochromatographic membrane containing an antibody labeled with a coloring agent (labeled antibody) and a capture antibody. When a target antigen is present in a sample, the antigen binds to the labeled antibody to form a complex, which then moves along the immunochromatographic membrane by capillary action. When the complex reaches the test line on the immunochromatographic membrane, it is captured by the capture antibody immobilized on the test line, causing the test line to develop a color derived from the coloring agent. By visually confirming this color development, the presence of the antigen in the sample can be detected simply and quickly.

[0003] Antibodies labeled with gold nanoparticles as a coloring agent are widely used as labeled antibodies in immunochromatography (Patent Documents 1 and 2). When gold nanoparticles are used as a coloring agent in this way, the presence of an antigen in a sample is usually detected by the test line exhibiting a red color derived from the gold nanoparticles. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2013-195403 [Patent Document 2] Japanese Patent Application Laid-Open No. 2007-023384 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the above-mentioned conventional labeled antibodies had the problem that when a red sample such as blood was used, the immunochromatographic membrane was stained red, making it difficult to visually confirm that the test line was red (i.e., visibility was reduced).

[0006] Therefore, an object of the present invention is to provide a carbon nanotube-labeled antibody that is highly visible in immunochromatography, and an immunochromatographic membrane containing the carbon nanotube-labeled antibody. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above problems and have found that the use of a carbon nanotube-labeled antibody containing an antigen-binding moiety and a carbon nanotube moiety can improve visibility in immunochromatography, leading to the completion of the present invention.

[0008] That is, the present invention aims to advantageously solve the above-mentioned problems, and according to the present invention, there are provided carbon nanotube-labeled antibodies as described in [1] to [2] below, and an immunochromatographic membrane as described in [3] below.

[0009] [1] A carbon nanotube-labeled antibody comprising an antigen-binding portion and a carbon nanotube portion, which is used to detect that the carbon nanotube-labeled antibody binds to an antigen via the antigen-binding portion by visually observing the color derived from the carbon nanotube portion. By visually observing the color derived from the carbon nanotube (CNT) portion, it is possible to detect the binding of the CNT-labeled antibody to the antigen via the antigen-binding site, thereby improving the visibility in immunochromatography.

[0010] [2] The carbon nanotube-labeled antibody according to [1] above, which comprises a linker between the antigen-binding portion and the carbon nanotube portion, and the linker comprises a polyethylene glycol structure. The use of a CNT-labeled antibody containing a linker containing a polyethylene glycol structure between the antigen-binding portion and the CNT portion can further enhance visibility in immunochromatography.

[0011] [3] An immunochromatographic membrane comprising the carbon nanotube-labeled antibody according to [1] or [2] above and a capture antibody. An immunochromatographic membrane containing any of the above-mentioned CNT-labeled antibodies and a capture antibody has excellent visibility in immunochromatography. [Effects of the Invention]

[0012] According to the present invention, it is possible to provide a carbon nanotube-labeled antibody that is highly visible in immunochromatography, and an immunochromatographic membrane containing the carbon nanotube-labeled antibody. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a diagram showing a schematic configuration of an example of an immunochromatographic membrane according to the present invention. [Figure 2] 1 shows the results of evaluating visibility in Examples, in which "C" indicates a control membrane and "S" indicates a test membrane. [Figure 3] 1 shows the results of evaluating visibility in Examples. The horizontal axis shows the mass ratio of the total amount of SA-PEG and PL-PEG to the amount of CNT ((SA-PEG+PL-PEG) / CNT), and the vertical axis shows grayscale intensity. #1 to #4 in the figure represent the results of Examples 1 to 4, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0014] Hereinafter, embodiments of the present invention will be described in detail. The CNT-labeled antibody of the present invention is used to detect the specific binding between an antigen and an antigen-binding site in an immunochromatography method or the like by visually observing it. Further, the immunochromatography membrane of the present invention is characterized by containing the CNT-labeled antibody of the present invention.

[0015] (Carbon nanotube-labeled antibody) The CNT-labeled antibody of the present invention contains at least a CNT portion and an antigen-binding portion, and optionally further contains a linker. Note that the CNT-labeled antibody of the present invention may further contain portions other than the CNT portion, the antigen-binding portion, and the linker.

[0016] And since the CNT-labeled antibody of the present invention contains a CNT portion and an antigen-binding portion, the visibility in the immunochromatography method can be enhanced. Thus, although the reason for obtaining the above effects by using the CNT-labeled antibody of the present invention is not clear, it is presumed as follows.

[0017] In conventional immunochromatography tests, especially when using a red sample such as blood, the immunochromatography membrane may turn red, making it difficult to confirm the color development of the judgment line. In contrast, the present invention uses a CNT-labeled antibody containing a CNT portion and an antigen-binding portion, and utilizes the fact that the judgment line exhibits black color derived from the CNT portion. Therefore, even when the immunochromatography membrane turns red as described above, the fact that the judgment line exhibits black color can be clearly observed. For the above reasons, the CNT-labeled antibody of the present invention is considered to be excellent in visibility in the immunochromatography method.

[0018] <CNT portion> The CNT portion contained in the CNT-labeled antibody of the present invention may be composed of single-walled CNTs or multi-walled CNTs, but it is preferably composed of single-walled CNTs. Further, the CNT portion may be composed of one CNT, or may be composed of a CNT bundle formed by aggregating a plurality of CNTs in a bundle shape (in a bundle) along the long axis direction of the CNT.

[0019] The average diameter (Av) of the CNTs is preferably 0.5 nm or more, more preferably 1 nm or more, and is preferably 15 nm or less, more preferably 10 nm or less.

[0020] Furthermore, the CNTs may be those for which the t-plot obtained from the adsorption isotherm exhibits an upwardly convex shape, or those for which no aperture treatment has been performed and the t-plot exhibits an upwardly convex shape.

[0021] Generally, adsorption is the phenomenon in which gas molecules are removed from the gas phase onto a solid surface, and is classified into physical adsorption and chemical adsorption depending on the cause. The nitrogen gas adsorption method used to obtain t-plots utilizes physical adsorption. Generally, at a constant adsorption temperature, the higher the pressure, the greater the number of nitrogen gas molecules adsorbed onto CNTs. Furthermore, a plot of relative pressure (the ratio of the pressure P at adsorption equilibrium to the saturated vapor pressure P0) on the horizontal axis and the amount of nitrogen gas adsorption on the vertical axis is called an "isotherm." When the amount of nitrogen gas adsorption is measured while increasing the pressure, it is called an "adsorption isotherm," and when the amount of nitrogen gas adsorption is measured while decreasing the pressure, it is called a "desorption isotherm."

[0022] The t-plot can be obtained by converting the relative pressure into the average thickness t (nm) of the nitrogen gas adsorption layer in an adsorption isotherm measured by nitrogen gas adsorption. That is, the average thickness t of the nitrogen gas adsorption layer corresponding to the relative pressure is calculated from a known standard isotherm in which the average thickness t of the nitrogen gas adsorption layer is plotted against the relative pressure P / P0, and the above conversion is then performed to obtain a t-plot for CNTs (the t-plot method by de Boer et al.).

[0023] In a sample with pores on the surface, the growth of the nitrogen gas adsorption layer can be classified into the following processes (1) to (3): The slope of the t-plot changes depending on the processes (1) to (3) below. (1) The process of forming a monolayer of nitrogen molecules on the entire surface (2) Formation of multi-layer adsorption and the accompanying capillary condensation filling process in the pores (3) The process of multilayer adsorption on an apparently non-porous surface whose pores are filled with nitrogen

[0024] In the case of a t-plot showing an upward convex shape, the plot lies on a straight line passing through the origin in the region where the average thickness t of the nitrogen gas adsorption layer is small, but as t increases, the plot shifts downward from the straight line. CNTs with such a t-plot shape have a large ratio of their internal specific surface area to their total specific surface area, indicating that many openings are formed in the CNTs.

[0025] Here, the BET specific surface area of ​​the CNT is 600 m 2 / g or more, 800m 2 / g or more, and 2500m 2 / g or less, 1200m 2 / g or less.

[0026] CNTs having the above-described properties can be efficiently produced by, for example, supplying raw material compounds and a carrier gas onto a substrate having a catalyst layer for CNT production on its surface and synthesizing CNTs by chemical vapor deposition (CVD), by making a trace amount of oxidant (catalytic activator) present in the system to dramatically improve the catalytic activity of the catalyst layer (super-growth method; see WO 2006 / 011655), by forming a catalyst layer on the substrate surface using a wet process. Note that, hereinafter, carbon nanotubes obtained by the super-growth method may be referred to as "SGCNTs."

[0027] <Antigen-binding portion> The antigen-binding portion contained in the CNT-labeled antibody of the present invention is a portion that can specifically bind to the target antigen, and is usually composed of a protein that has binding specificity to the antigen.

[0028] The protein constituting the antigen-binding portion is not particularly limited as long as it can specifically bind to an antigen. Examples of proteins constituting the antigen-binding portion include monoclonal antibodies such as mouse antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies; antigen-binding fragments such as (Fab')2, Fab, Fab', and Fv; and biotin-binding proteins such as streptavidin and avidin. Note that the CNT-labeled antibody may contain one type of antigen-binding portion or may contain two or more types of antigen-binding portions, but typically contains one type of antigen-binding portion.

[0029] Furthermore, there is no particular limitation on the number of antigen-binding moieties contained in the CNT-labeled antibody, and the CNT-labeled antibody may contain only one antigen-binding moiety, or may contain two or more antigen-binding moieties.

[0030] <linker> The linker is a region interposed between the CNT portion and the antigen-binding portion. The structure of the linker is not particularly limited, but examples thereof include a structure containing polyethylene glycol and a structure containing DNA. Among these, the linker is preferably a compound represented by the following formula (1): [ka] (In formula (1), n ​​is an integer of 2 or more.) It is preferable that the polyethylene glycol structure is represented by the following formula:

[0031] When the CNT-labeled antibody contains a linker containing a polyethylene glycol structure, the polyethylene glycol structure in the linker functions as a dispersant, improving the dispersibility of the CNT-labeled antibody and further enhancing its visibility in immunochromatography. Note that the linker may contain a structure other than the polyethylene glycol structure (another structure).

[0032] The degree of polymerization of the polyethylene glycol structure in the linker (the value of n in the following formula (1)) can be adjusted appropriately within a range that achieves the desired effect. For example, the degree of polymerization can be set to 50 or more and 200 or less, or 100 or more and 150 or less.

[0033] Other structures that the linker may optionally contain include, for example, a phospholipid structure. An example of a phospholipid structure is a structure in which a fatty acid and a phosphoric acid are bonded to a central skeleton of glycerin, and an alcohol is further ester-bonded to the phosphoric acid. The linker may contain only one type of other structure, or may contain multiple types.

[0034] Preferred examples of the linker structure include the structure of the following formula (1): In formula (1), n ​​is an integer of 2 or more, preferably an integer of 50 to 200, and more preferably an integer of 100 to 150. [ka]

[0035] In the CNT-labeled antibody of the present invention, the binding mode of each of the above-mentioned portions (CNT portion, antigen-binding portion, and linker) may be either a covalent bond or a non-covalent bond, or a combination of a covalent bond and a non-covalent bond.

[0036] Examples of the covalent bond include a disulfide bond, an amide bond, a thioether bond, etc. Among these, the amide bond is preferred as the covalent bond.

[0037] Examples of non-covalent bonds include van der Waals forces, hydrogen bonds, electrostatic interactions, hydrophobic interactions, etc. Among these, van der Waals forces are preferred as non-covalent bonds.

[0038] <Suitable structure> Suitable structures of the CNT-labeled antibody of the present invention include, for example, the following structures (1) to (3): However, the structure of the CNT-labeled antibody of the present invention is not limited to these. (1) A structure in which the CNT portion and the antigen-binding portion are linked via a covalent bond; (2) A structure in which the antigen-binding moiety is linked to the linker via a covalent bond, and the linked antigen-binding moiety-linker is adsorbed to the CNT moiety via a non-covalent bond; and (3) A structure in which the antigen-binding portion is covalently linked to a linker, and the linker is further covalently linked to a CNT portion.

[0039] Among the above, from the viewpoints of further improving visibility in immunochromatography and improving the ease of CNT-labeled antibodies, structures (1) to (2) are preferred, structure (2) is more preferred, and a structure in which the antigen-binding moiety is linked to the linker via an amide bond and the linked antigen-binding moiety-linker bond is adsorbed to the CNT moiety by van der Waals forces is even more preferred.

[0040] <Preparation of carbon nanotube-labeled antibodies> The method for preparing the CNT-labeled antibody of the present invention is not particularly limited. For example, the CNT-labeled antibody having the structure (1) above can be prepared by reacting CNTs capable of forming a CNT portion with a protein molecule such as an antibody capable of forming an antigen-binding portion.

[0041] Functional groups used to form covalent bonds with antibodies and other materials can also be introduced onto the surface of the CNTs used in the reaction. Methods for introducing functional groups onto the CNT surface include known surface treatments, such as surface oxidation by strong acid treatment, chemical modification using functional groups introduced by surface oxidation, chemical modification using various addition reactions to the CNT surface, and physical modification using non-covalent bonds to the CNT surface. Functional groups used to form covalent bonds with CNTs can also be introduced into antibodies and other materials by site-specific mutation or chemical modification.

[0042] The CNT-labeled antibody having the structure (2) described above can be prepared, for example, through the steps of: subjecting a mixture containing CNTs and a dispersion medium to a dispersion treatment to obtain a CNT dispersion (dispersion step); linking an antibody or the like capable of forming an antigen-binding moiety with a reagent capable of forming a linker via a covalent bond to obtain an antigen-binding moiety-linker (linking step); and reacting the CNT dispersion with the antigen-binding moiety-linker to obtain a CNT-labeled antibody (labeling step).

[0043] <<Dispersion process>> In the dispersion step, a mixed liquid containing CNTs and a dispersion medium, and optionally further containing other components, is subjected to a dispersion treatment to obtain a CNT dispersion liquid.

[0044] The dispersion medium may contain only water, or may contain only an organic solvent (e.g., esters, ketones, alcohols), or may be a mixture of water and an organic solvent. From the viewpoint of improving the dispersibility of the CNT-labeled antibody and further enhancing the visibility when detecting an antigen-antibody reaction by immunochromatography, it is preferable that the dispersion medium contains water. The organic solvent may be used alone or in combination of two or more.

[0045] Examples of other components in the mixed solution include dispersants, antioxidants, and carbon materials other than CNTs (for example, particulate carbon materials such as carbon black, and fibrous carbon materials other than CNTs such as carbon nanohorns). Among these, it is preferable to use a dispersant as the other component, from the viewpoint of improving the dispersibility of the CNT-labeled antibody and further enhancing the visibility when detecting an antigen-antibody reaction by immunochromatography. The other components can be used singly or in combination of two or more. In the present invention, a carbon material is "particulate" when the aspect ratio, obtained by measuring the major and minor axes of 100 randomly selected carbon materials and dividing the average major axis by the average minor axis, is 1 or more and less than 5; and a carbon material is "fibrous" when the aspect ratio, obtained by measuring the major and minor axes of 100 randomly selected carbon materials and dividing the average major axis by the average minor axis, is 5 or more.

[0046] The dispersant is not particularly limited, and any known dispersant capable of assisting the dispersion of CNTs can be used. Among these, it is preferable to use a surfactant as the dispersant.

[0047] Here, examples of surfactants include ionic (cationic, anionic) surfactants and nonionic surfactants. Examples of cationic surfactants include quaternary ammonium salts such as dodecyltrimethylammonium bromide, cetyltrimethylammonium bromide, and distearyldimethylammonium chloride; and quaternary phosphonium salts such as tetrabutylphosphonium chloride, tetrapentylphosphonium chloride, trioctylmethylphosphonium chloride, and pentyltriphenylphosphonium chloride. Examples of anionic surfactants include sodium dodecyl sulfate, sodium deoxycholate, sodium cholate, sodium dodecylbenzenesulfonate, sodium dodecylbenzenesulfonate, and sodium dodecyldiphenyloxidedisulfonate. Examples of nonionic surfactants include ether-type nonionic surfactants such as polyoxyethylene alkyl ethers; ether ester-type nonionic surfactants such as polyoxyethylene ethers of glycerin esters; polyethylene glycol fatty acid esters; and glycerin esters. Among the surfactants mentioned above, anionic surfactants are preferred, and sodium cholate is more preferred, from the viewpoint of improving the dispersibility of the CNT-labeled antibody and further enhancing the visibility when detecting an antigen-antibody reaction by immunochromatography.

[0048] The proportion of the dispersant contained in the mixed solution is preferably 0.1% by mass or more, more preferably 0.2% by mass or more, and even more preferably 0.5% by mass or more, and preferably 10% by mass or less, more preferably 5% by mass or less, and even more preferably 2% by mass or less, assuming the total amount of the mixed solution to be 100% by mass. If the proportion of the dispersant in the mixed solution is within the above-mentioned range, the dispersibility of the CNT-labeled antibody can be improved, and the visibility when detecting an antigen-antibody reaction by immunochromatography can be further improved.

[0049] The dispersion treatment in the dispersion step is not particularly limited as long as it can disperse CNTs in the dispersion medium, and any known dispersion treatment can be used. Such dispersion treatment can be carried out using a mixer such as a wet jet mill, bead mill, ball mill, sand mill, pigment disperser, crusher, ultrasonic disperser, homogenizer, planetary mixer, or Filmix. Alternatively, ultracentrifugation can be carried out after the dispersion treatment, and the supernatant can be recovered and diluted to an appropriate concentration to prepare a CNT dispersion.

[0050] <<Connection process>> In the linking step, an antibody or the like capable of forming an antigen-binding moiety is covalently linked to a reagent capable of forming a linker to obtain an antigen-binding moiety-linker. The antibody or the like capable of forming an antigen-binding moiety is the antibody or antigen-binding fragment described above.

[0051] Furthermore, examples of reagents capable of forming linkers include reagents having a polyethylene glycol structure and a functional group capable of forming a covalent bond with an antibody or the like. Examples of functional groups capable of forming a covalent bond with an antibody or the like include functional groups capable of forming a covalent bond with a primary amine, such as an N-hydroxyester (NHS ester) group, an isothiocyanate group, an isocyanate group, an acyl azide group, a sulfonyl chloride group, an aldehyde group, a glyoxal group, an oxirane group, a carbonate group, an aryl halide group, an imide ester group, and a carbodiimide group; functional groups capable of forming a covalent bond with a thiol group, such as a thiol group and a maleimide group; and the like. Examples of such reagents include the SUNBRIGHT series manufactured by NOF Corporation.

[0052] The reaction conditions in the linking step are not particularly limited as long as the reaction proceeds sufficiently, and can be appropriately selected depending on the types of antibodies and reagents used.

[0053] <<Labeling process>> In the labeling step, the CNT dispersion obtained in the dispersing step is reacted with the antigen-binding moiety-linker obtained in the linking step to obtain a CNT-labeled antibody.

[0054] Here, when reacting the CNT dispersion with the antigen-binding portion-linker, it is preferable to add a second reagent that has a polyethylene structure but does not have a functional group that can form a covalent bond with an antibody or the like. Adding the second reagent can improve the labeling rate of the CNT-labeled antibody. This can further improve visibility in immunochromatography.

[0055] The reaction conditions (reaction temperature, reaction time, etc.) in the labeling step are not particularly limited as long as a sufficient labeling rate is obtained, but the reaction temperature is preferably 4 to 30°C, and the reaction time is preferably 1 to 24 hours.

[0056] Here, the ratio of the total amount of the antigen-binding moiety-linker and the second reagent to the amount of CNT (antigen-binding moiety-linker + second reagent / CNT) is preferably 1 or more, more preferably 2 or more, even more preferably 4 or more, and preferably 15 or less, and more preferably 10 or less, by mass. If the ratio of the total amount of the antigen-binding moiety-linker and the second reagent to the amount of CNT is within the above-mentioned range, the labeling rate of the CNT-labeled antibody can be improved. Therefore, visibility in immunochromatography can be further improved.

[0057] Furthermore, after the reaction, the reaction solution can be subjected to ultrafiltration, and then an appropriate buffer solution can be added and the ultrafiltration repeated to remove CNT-unlabeled antibody molecules (unreacted antibodies that are not labeled with CNTs) and other components such as surfactants used in the dispersion process.

[0058] (Immunochromatography membrane) The immunochromatographic membrane of the present invention contains at least the above-mentioned CNT-labeled antibody and capture antibody, and optionally further contains other components. Furthermore, since the immunochromatographic membrane of the present invention contains the CNT-labeled antibody of the present invention, it has excellent visibility in immunochromatography.

[0059] Hereinafter, an example of the structure of the immunochromatography membrane of the present invention will be further described with reference to FIG. 1. The immunochromatography membrane 1 shown in FIG. 1 includes a moving layer 2, a labeled antibody encapsulation layer 3 on the moving layer 2, and a lower part 4 of the sample drop on the labeled antibody encapsulation layer 3. On the moving layer 2, a judgment line 5 and a control line 6 are provided. A capture antibody 7 is immobilized on the judgment line 5, and a control antibody 8 is immobilized on the control line 6. The labeled antibody encapsulation layer 3 contains a CNT-labeled antibody 9. When a sample 11 containing an antigen 10 is dropped onto the lower part 4 of the sample drop, the dropped sample 11 binds to the CNT-labeled antibody 9 in the labeled antibody encapsulation layer 3 to form a complex. The complex moves through the moving layer 2 by capillary action and is captured by the capture antibody 7 immobilized on the judgment line 5 when it reaches the judgment line 5, presenting a black color derived from the CNT part in the CNT-labeled antibody 9. On the other hand, among the CNT-labeled antibodies 9 in the labeled antibody encapsulation layer 3 that did not bind to the antigen 10, they are not captured by the capture antibody 7 immobilized on the judgment line 5, but further move through the moving layer 2 and are captured by the control antibody 8 immobilized on the control line 6.

[0060] <CNT-labeled antibody> As the CNT-labeled antibody, the CNT-labeled antibody of the present invention described above is used.

[0061] <Capture antibody> The capture antibody is an antibody that specifically binds to an antigen. The capture antibody is immobilized on the judgment line of the moving layer and has a function of capturing the complex (antigen-CNT-labeled antibody) by binding to the antigen in the complex (antigen-CNT-labeled antibody) that has moved through the moving layer by capillary action.

[0062] <Control antibody> The control antibody is an antibody that specifically binds to the antigen-binding part in the CNT-labeled antibody. The control antibody is immobilized on the control line of the moving layer and has a function of binding to and capturing the antigen-binding part in the CNT-labeled antibody (free CNT-labeled antibody that has not bound to the antigen) that has moved through the moving layer by capillary action.

[0063] The materials for the mobile layer, the labeled antibody-containing layer, and the sample dropping section are not particularly limited, and known materials that are commonly used for immunochromatographic membranes can be used.

[0064] <Method for preparing immunochromatographic membrane> The immunochromatographic membrane of the present invention shown in Figure 1 can be produced, for example, by layering the above-mentioned mobile layer, labeled antibody-containing layer, and sample drop section in this order. A capture antibody and a control antibody are immobilized on the test line and control line of the mobile layer, respectively. This immobilization can be achieved, for example, by repeatedly dropping an antibody solution onto the test line or control line of the mobile layer and then drying. Furthermore, a method for incorporating a CNT-labeled antibody into the labeled antibody-containing layer includes, for example, impregnating the CNT-labeled antibody into the labeled antibody-containing layer and then drying it. [Example]

[0065] The present invention will be specifically described below based on examples, but the present invention is not limited to these examples. In the following description, "%" and "parts" representing amounts are based on mass unless otherwise specified. In the examples, the visibility was evaluated using the following method.

[0066] <Visibility> [Preparation of test membrane] The membrane for immunochromatography (manufactured by ADVANTECH, product name "IAB090") was cut into a size of 7 mm in width and 60 mm in height. At the center of the position 20 mm from the lower end (judgment line), 2 μL of the capture antibody (biotin-labeled anti-Iba1 rabbit antibody, manufactured by FUJIFILM Wako Pure Chemical Corporation) prepared at 40 μg / mL was dropped and air-dried. By repeating this dropping and air-drying three times, a membrane with the capture antibody immobilized on the judgment line was obtained. The membrane with the biotin-labeled antibody immobilized was gently shaken in a 1% BSA solution for 30 minutes, then washed three times with pure water and dried under reduced pressure to obtain a test membrane. [Preparation of Control Membrane] A control membrane was prepared in the same manner as the test membrane, except that a control antibody (anti-goat IgG rabbit antibody, manufactured by Rockland Immunochemicals) was used instead of the capture antibody. [Immunochromatography Test] 500 μL of the CNT-labeled antibody (CNT-PEG-SA) solution prepared in each example was placed in a 5 mL vial. Further, the test membrane was placed in this vial with the part where the capture antibody was immobilized facing down. Then, it was left standing until the CNT-labeled antibody solution flowing on the test membrane reached the upper end of the test membrane. When the CNT-labeled antibody solution reached the upper end of the test membrane, the test membrane was taken out of the vial and left standing to dry. The dried test membrane was photographed with a camera (manufactured by AS ONE, product name "PCS5000"). The obtained image was converted to grayscale, and the average signal intensity (ROI; Region of Interest) was calculated for each of the region where the capture antibody was immobilized and the background region. And the grayscale intensity was calculated as the difference between the ROI of the region where the capture antibody was immobilized and the ROI of the background region. The same test was performed using the control membrane instead of the test membrane. The higher the grayscale intensity, the better the visibility in the immunochromatography method.

[0067] (Example 1) [Preparation of CNT-Labeled Streptavidin (SA-PEG-CNT) Solution] [Dispersion process] A mixture was obtained by adding 3 mg of SGCNT (manufactured by Zeon Corporation, product name "ZEONANO (registered trademark) SG101"; CNT containing 90% or more single-walled CNT) to 10 mL of a 1% sodium cholate aqueous solution. The mixture was subjected to a dispersion treatment for 10 minutes using a tip-type ultrasonic homogenizer, and then ultracentrifugation (105,000 × g, 1 hour) was performed to recover the supernatant. The absorption spectrum of the recovered supernatant was measured, and the concentration was adjusted with a 1% sodium cholate aqueous solution so that the absorbance at a wavelength of 750 nm was 35, and this was used as a CNT dispersion (CNT concentration: 500 μg / mL). Note that, when measured with a Raman spectrophotometer, SGCNT exhibited a peak intensity of 100 to 300 cm, which is characteristic of single-walled CNT. -1 A radial breathing mode (RBM) spectrum was observed in the low wavenumber region. The BET specific surface area of ​​the SGCNT measured using a BET specific surface area meter (BEL Japan Co., Ltd., BELSORP (registered trademark)-max) was 1325 m 2 / g (unopened). [Connection process] 19.2 mg of streptavidin and 4.5 mg of PL-PEG-NHS (NOF Corporation, product name "SUNBRIGHT® DSPE-050GS") were added to 20 mL of 25 mM HEPES buffer (pH 7.0-7.6). The resulting solution was reacted overnight with stirring in a refrigerator at 5°C, then transferred to a centrifugal ultrafiltration unit (MWCO: 30 kDa) and centrifuged at 2000 × g for 60 minutes. An appropriate amount of purified water was added to the centrifugal ultrafiltration unit, and the unit was centrifuged at 2000 × g for 60 minutes. This addition of purified water and centrifugation was repeated five times to remove unreacted PL-PEG-NHS. The contents of the centrifugal ultrafiltration unit were then collected and lyophilized to obtain SA-PEG powder as the antigen-binding site-linker. SA-PEG has a structure in which the primary amine in the streptavidin molecule reacts with the N-hydroxyester (NHS ester) group of PL-PEG-NHS, linking them through an amide bond. [Labeling process] The CNT dispersion obtained in the dispersion process, PL-PEG (manufactured by Sunbright, product name "DSPE-050CN") as a second reagent dissolved in 25 mM HEPES buffer to a concentration of 4 mg / mL, and the SA-PEG powder obtained in the coupling process were mixed to obtain a reaction solution with a solid content of 0.2 g CNT, 0.8 g PL-PEG, and 0.18 g SA-PEG. The resulting reaction solution was left overnight at room temperature and then placed in a centrifugal ultrafiltration unit (MWCO: 3 kDa) and centrifuged at 12,300 × g for 15 minutes. An appropriate amount of 25 mM HEPES buffer was added to the centrifugal ultrafiltration unit, and the unit was centrifuged at 12,300 × g for 15 minutes. This addition of HEPES buffer and centrifugation was repeated three times. 25 mM HEPES buffer was then added, and the contents of the centrifugal ultrafiltration unit were collected to obtain a CNT-labeled streptavidin (SA-PEG-CNT) solution as a CNT-labeled antibody. SA-PEG-CNT has the structure of the following formula (2) as a linker between streptavidin, which serves as the antigen-binding moiety, and CNT (in formula (2), the value of n is 113 to 114). SA-PEG-CNT has a structure in which streptavidin is linked to the linker via an amide bond, and the linked streptavidin-linker (SA-PEG) is adsorbed to the CNT surface by van der Waals forces. [ka] This CNT-labeled streptavidin solution was used to evaluate visibility, and the results are shown in Figures 2 and 3.

[0068] Example 2 A CNT-labeled streptavidin (SA-PEG-CNT) solution was prepared as a CNT-labeled antibody in the same manner as in Example 1, except that in the labeling step of Example 1, the solid contents in the reaction solution were changed to 0.2 g of CNT, 1.6 g of PL-PEG, and 0.18 g of SA-PAGE. Evaluation was then carried out in the same manner as in Example 1. The results are shown in Figures 2 and 3.

[0069] Example 3 A CNT-labeled streptavidin (SA-PEG-CNT) solution was prepared as a CNT-labeled antibody in the same manner as in Example 1, except that in the labeling step of Example 1, the solid contents in the reaction solution were changed to 0.2 g of CNT, 1.72 g of PL-PEG, and 0.17 g of SA-PAGE. Evaluation was then carried out in the same manner as in Example 1. The results are shown in Figures 2 and 3.

[0070] Example 4 Instead of SGCNT, CNT (Merck, product name "CoMoCAT (registered trademark)"); A solution containing CNT-labeled streptavidin (SA-PEG-CNT) as a CNT-labeled antibody was prepared in the same manner as in Example 1, except that CNTs containing 90% or more single-walled CNTs were used. Evaluation was then carried out in the same manner as in Example 1. The results are shown in Figures 2 and 3.

[0071] 2 and 3, it can be seen that in Examples 1 to 4, black spots derived from the CNT portion were observed, which were generated by binding of CNT-labeled streptavidin (CNT-labeled antibody) to the immobilized biotin-labeled antibody (capture antibody). [Industrial Applicability]

[0072] According to the present invention, it is possible to provide a carbon nanotube-labeled antibody that is highly visible when detecting an antigen-antibody reaction by immunochromatography, and an immunochromatographic membrane containing the carbon nanotube-labeled antibody. [Explanation of symbols]

[0073] 1 Immunochromatographic membrane 2. Moving Layer 3. Labeled antibody encapsulation layer 4 Sample dropping section 5 Judgment Line 6. Control Line 7 Capture antibody 8 Control Antibodies 9 CNT labeled antibody 10 antigens 11 Sample

Claims

1. A carbon nanotube-labeled antibody comprising an antigen-binding portion and a carbon nanotube portion, A carbon nanotube-labeled antibody used to detect binding of the carbon nanotube-labeled antibody to an antigen via the antigen-binding portion by visually observing the color derived from the carbon nanotube portion.

2. a linker between the antigen-binding portion and the carbon nanotube portion; The carbon nanotube-labeled antibody of claim 1 , wherein the linker comprises a polyethylene glycol structure.

3. An immunochromatographic membrane comprising the carbon nanotube-labeled antibody according to claim 1 or 2 and a capture antibody.

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