Method for producing antibody-immobilized nitrocellulose membrane and method for enhancing antigen binding

Abstract

To provide an antibody-immobilized nitrocellulose membrane with enhanced antigen binging, an antigen binding enhancement method, a method of screening antibodies using antigen-binding affinity as an indicator, etc.SOLUTION: A method of producing an antibody-immobilized nitrocellulose membrane is provided, comprising (1) bringing an alkaline solution containing an antibody into contact with a nitrocellulose membrane, and (2) drying the nitrocellulose membrane that has been brought into contact with the alkaline solution in the step (1).SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing an antibody-immobilized nitrocellulose membrane, a method for enhancing antigen-binding properties, a method for screening an antibody using antigen-binding properties as an index, and the like.

Background Art

[0002] Nitrocellulose (NC) membranes have been conventionally known as carriers for antibody immobilization in immunological techniques such as immunochromatography, ELISA (Enzyme-Linked Immunosorbent Assay), and Western blotting. Among them, immunochromatography is useful in that the results of antibody-antigen reactions can be obtained relatively quickly and easily, and is also used in POCT tests (Point of Care Testing), such as influenza tests.

[0003] However, in antigen-antibody reaction tests using immunochromatography such as POCT tests, although antibodies are immobilized on the NC membrane, the antibodies are limited to some types of full-length antibodies (whole antibodies), and there are few examples of using recombinant antibodies such as single-chain antibodies (scFv) and single-domain antibodies (VHH). Furthermore, the three-dimensional structure of the antibody is easily lost, and not all antibodies can maintain their activity on the NC membrane just because they are immobilized on the NC membrane. Therefore, when immobilizing an antibody on the NC membrane, it was necessary to apply a high-concentration antibody-containing solution to the NC membrane. POCT tests and the like are said to have a high test cost, and this has also contributed to an increase in the test cost (Non-Patent Document 1). In addition, there are still many unclear points in the protein adsorption mechanism on the NC membrane, and the development of better means is required.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

[0005] The objective is to provide antibody-immobilized nitrocellulose membranes with enhanced antigen-binding properties, methods for enhancing antigen-binding properties, and methods for screening antibodies using antigen-binding properties as an indicator. [Means for solving the problem]

[0006] As a result of diligent research, the inventors discovered that when an antibody containing an alkaline solution is brought into contact with an NC membrane, the antibody is immobilized on the NC membrane, and the antigen can be detected with high sensitivity on the NC membrane. The present invention was completed based on this finding and further investigations, and this disclosure includes, for example, the inventions described below. Section 1. A method for producing an antibody-immobilized nitrocellulose membrane, comprising the following steps: (1) A step of bringing an alkaline solution containing an antibody into contact with a nitrocellulose membrane, and (2) A step of drying the nitrocellulose film that has been in contact with the alkaline solution in step (1). Item 2. The manufacturing method according to Item 1, wherein the pH of the alkaline solution is 9 to 14. Item 3. The method for producing an antibody according to item 1 or 2, wherein the antibody is at least one selected from the group consisting of VHH, scFv, sc(Fv)2, Diabody, Fab, and F(ab')2. Item 4. A method for producing an antibody-immobilized nitrocellulose membrane with enhanced antigen-binding properties, as described in any one of items 1 to 3. Section 5. A method for enhancing the antigen-binding properties of an antibody-immobilized nitrocellulose membrane, comprising the following steps: (a) A step of bringing an alkaline solution containing an antibody into contact with a nitrocellulose membrane, and (i) A step of drying the nitrocellulose film that has been in contact with the alkaline solution in step (a) above to obtain an antibody-immobilized nitrocellulose film. Section 6. A method for screening antibodies using antigen-binding activity as an indicator, comprising the following steps: (A) A step of contacting an alkaline solution containing a candidate antibody with a nitrocellulose membrane. (B) A step of drying the nitrocellulose film that was in contact with the alkaline solution in step (A) to obtain a candidate antibody-immobilized nitrocellulose film, and (C) A step of bringing the candidate antibody on the candidate antibody-immobilized nitrocellulose membrane obtained in step (B) into contact with the antigen. Item 7. The method for screening antibodies according to Item 6, further comprising (D) comparing the antigen-binding level of the candidate antibody immobilized on the nitrocellulose membrane with the antigen-binding level of a reference antibody after contact in step (C). [Effects of the Invention]

[0007] Antibodies can be immobilized on an NC membrane by contacting it with an alkaline solution containing antibodies and then drying it. This method of immobilizing antibodies on an NC membrane allows for the easy provision of antibody-immobilized NC membranes with enhanced antigen-binding properties. Furthermore, the enhanced antigen-binding properties enable screening of various candidate antibodies. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 shows the antigen-binding properties of antibody-immobilized NC membranes. [Figure 2] Figure 2 shows the antigen-binding properties of antibody-immobilized NC membranes. [Figure 3] Figure 3 is a schematic diagram of an experiment using immunochromatography. [Figure 4] Figure 4 shows the antigen-binding properties as determined by immunochromatography. [Figure 5] Figure 5 shows the antigen-binding properties as determined by immunochromatography. [Figure 6] Figure 6 is a diagram showing antigen-binding properties by immuno-chromatography. [Figure 7] Figure 7 is a diagram showing the test results of the detection limit on the antibody-immobilized NC membrane.

Embodiments for Carrying out the Invention

[0009] Hereinafter, the embodiments included in the present disclosure will be described in more detail. In the present disclosure, "containing" also includes the meanings of "substantially consisting of" and "consisting of".

[0010] Method for manufacturing antibody-immobilized nitrocellulose membranes The present disclosure includes a method for manufacturing an antibody-immobilized nitrocellulose membrane, which includes the following steps: (1) A step of bringing an alkaline solution containing an antibody into contact with a nitrocellulose membrane, and (2) A step of drying the nitrocellulose membrane that has been brought into contact with the alkaline solution in the step (1).

[0011] Process (1) The method for manufacturing an antibody-immobilized nitrocellulose membrane of the present disclosure includes a step (1) of bringing an alkaline solution containing an antibody into contact with a nitrocellulose membrane. In the present disclosure, an antibody-immobilized nitrocellulose membrane means a nitrocellulose membrane on which an antibody is immobilized.

[0012] The nitrocellulose (NC) membrane is not limited as long as it is used as a carrier for immobilizing antibodies in conventionally known immunological techniques such as immunochromatography. In this regard, the thickness, shape, etc. of the NC membrane are also not limited. Although not limiting the present disclosure, examples of the thickness of the NC membrane include about 80 to 500 μm, preferably about 80 to 400 μm. Also, although not limiting the present disclosure, examples of the average pore size of the NC membrane include about 0.2 to 5 μm, preferably about 0.45 μm to 5 μm. In the present disclosure, the average pore size is a value according to the product catalog. The NC membrane is commercially available, and examples include High-Flow plus HF180 (manufactured by Merck KGaA), etc.

[0013] The antibody immobilized on the NC membrane in the present disclosure is not limited and may be any of single-chain antibody (scFv), single-domain antibody (VHH), full-length antibody (whole antibody), sc(Fv)2, diabody, Fab, F(ab’)2, etc. Although not limiting the present disclosure, examples of the antibody preferably include single-chain antibody, single-domain antibody, etc. These may be used alone or in combination of two or more.

[0014] The antibody may be labeled or not labeled with a fluorescent label, enzyme label, metal label (such as gold colloid), biotin label, PEG (polyethylene glycol) label, polymer (such as colored latex) label, etc. as long as it does not interfere with the effects of the present disclosure. In conventional on-site rapid clinical testing methods such as influenza testing, the antibody is immobilized on the NC membrane without being labeled (unmodified). From the viewpoints of labor, cost, etc., in the present disclosure, preferably the antibody is immobilized on the NC membrane without being labeled.

[0015] The pH of the alkaline solution containing the antibody used in step (1) above is not limited as long as the effects of this disclosure are obtained, but a pH of 9 to 14 is preferably exemplified, and a pH of 9 to 13.5 is more preferably exemplified. Furthermore, the pH of the alkaline solution containing the antibody is more preferably exemplified by 10 to 13, 11 to 13, 12 to 13, etc. The pH can be appropriately determined within the range of pH 9 to 14 depending on the antibody to be immobilized on the NC membrane. The pH is the value measured using a pH meter (product name LAQUA, manufactured by Horiba, Ltd.) at 25°C, and represents the pH at the time of contact with the NC membrane.

[0016] Examples of alkaline solutions include, but are not limited to, known solutions such as aqueous sodium hydroxide solution, aqueous potassium hydroxide solution, potassium chloride-sodium hydroxide buffer (KCl-NaOH buffer), and glycine-sodium hydroxide buffer (Glycine-NaOH buffer). In addition to these, examples of buffers prepared by conventional methods using known Good's buffers that have buffering capacity in the alkaline range include, but are not limited to, Tricin, Bicine, TAPS (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), CHES (N-Cyclohexyl-2-aminoethanesulfonic acid), CAPSO (3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), CAPS (N-Cyclohexyl-3-aminopropanesulfonic acid), etc.

[0017] Without limiting this disclosure, to give a general example, an aqueous sodium hydroxide solution is a buffer prepared by mixing sodium hydroxide and water. An aqueous potassium hydroxide solution is a buffer prepared by mixing potassium hydroxide and water. A potassium chloride-sodium hydroxide buffer is a buffer prepared by mixing hydrochloric acid and sodium hydroxide in water. A glycine-sodium hydroxide buffer is a buffer prepared by dissolving glycine in water and adjusting the pH with sodium hydroxide. A tricine buffer is a buffer prepared by dissolving tricine in water and adjusting the pH with sodium hydroxide. Bicine buffer, TAPS buffer, CHES buffer, CAPSO buffer, and CAPS buffer are buffers prepared in the same manner as tricine buffer, except that tricine is replaced with these buffers, respectively. As exemplified by these, in this disclosure, alkaline solutions are prepared by mixing water and buffers, etc., according to conventionally known procedures and adjusting the pH to a desired value. The concentration of the buffer in these solutions is not limited, but typically it is 10 to 200 mM, preferably 10 to 100 mM, and more preferably 10 to 50 mM.

[0018] The solution may be used alone or in combination of two or more types.

[0019] The alkaline solution may contain additives conventionally used for protein immobilization, such as monosaccharides and disaccharides like trehalose, mannitol, and sorbitol; ionic surfactants (sodium deoxycholate, sodium dodecyl sulfate (SDS), etc.); amphoteric surfactants (CHAPS (3-(3-cholamidepropyl)dimethylammonio-1-propanesulpHonate), etc.); and nonionic surfactants (Triton X-100, Tween 20, etc.), to the extent that they do not impair the effects of the present disclosure. If additives are included, they may be used individually or in combination of two or more. The amount of additive used may also be determined appropriately according to conventional procedures, for example, within ranges such as 0-5 w / v%, 0.0001-2 w / v%, 0.01-1 w / v%, 0.02-0.5 w / v%, etc., depending on the type of additive used.

[0020] An alkaline solution containing the antibody is obtained by mixing the antibody with an alkaline solution. The amount of antibody in the alkaline solution is not limited as long as the effects of this disclosure are obtained, but for example, the antibody content in the alkaline solution is preferably 5 μg / ml or more, more preferably 10 μg / ml to 10 mg / ml, even more preferably 30 μg / ml to 2 mg / ml, and particularly preferably 100 μg / ml to 1 mg / ml. In this disclosure, since antigen can be efficiently bound by using an NC membrane on which the antibody is immobilized, the antibody content in the alkaline solution may be relatively low, such as 10 μg / ml or less.

[0021] Contact between the alkaline solution containing the antibody and the NC membrane can be performed by any means necessary, as long as contact is achieved. This contact may be, for example, by dropping the alkaline solution containing the antibody onto the NC membrane, immersing the NC membrane in the alkaline solution containing the antibody, inkjet feeding (coating, etc.), capillary feeding (coating, etc.), etc. Furthermore, contact may be performed on the entire NC membrane or only on a portion of it, depending on the location on the NC membrane where the antigen is to be reacted. The temperature of contact is also not limited and can be performed at room temperature (25°C) or similar for convenience. The amount of alkaline solution containing the antibody that contacts the NC membrane can also be set appropriately. Without limiting this disclosure, and depending on the amount of antibody in the alkaline solution, for example, when the alkaline solution is applied to an NC membrane used for chromatography strips as shown in the examples below by drawing a 5 mm long straight line by capillary coating, the amount of alkaline solution containing the antibody that comes into contact with the NC membrane is preferably 0.2 to 2 μL per 5 mm straight line, and more preferably 0.3 to 1 μL.

[0022] Process (2) The method for producing an antibody-immobilized nitrocellulose membrane according to the present disclosure includes (2) a step of drying the nitrocellulose membrane that has been in contact with an alkaline solution in step (1).

[0023] Drying may be done by natural drying, or it may be done using a device that allows adjustment of temperature, airflow speed, etc., such as a constant temperature bath. The drying temperature is not limited and can be 25 to 80°C, preferably 25 to 70°C, and more preferably 30 to 50°C. In this way, an NC membrane with immobilized antibodies (antibody-immobilized NC membrane) is obtained. From this, it can be said that in this disclosure, the immobilization of antibodies on the NC membrane occurs by adsorption (adsorption immobilization).

[0024] Furthermore, blocking treatment may or may not be performed on the antibody-immobilized NC membrane obtained in this manner. Blocking can be carried out according to conventionally known procedures, for example, by using a neutral buffer containing approximately 0.1-5% BSA (Bovine Serum Albumin) or casein.

[0025] The antibody immobilized on the NC membrane is not limited as long as it is an antibody that can bind to the antigen by an antigen-antibody reaction. Examples include antibodies against viruses and bacteria such as anti-influenza antibodies (type A, type B, etc.), anti-norovirus antibodies, anti-human chorionic gonadotropin antibodies, anti-E. coli O-157 antibodies, anti-SARS antibodies (SARS-CoV-2 antibodies, etc.), anti-rotavirus antibodies, anti-adenovirus antibodies, anti-cytomegalovirus antibodies, anti-Legionella antibodies, and anti-Helicobacter pylori antibodies. It may also be any antibody against troponin T, troponin I, CK-MB (creatine kinase-MB), myoglobin, LH (luteinizing hormone), E. coli verotoxin, etc. Although not limiting to this disclosure, preferred antibodies include anti-influenza antibodies (anti-influenza type A antibodies, anti-influenza B antibodies, etc.). These may be used individually or in combination of two or more.

[0026] The antibody-immobilized NC membrane of this disclosure, manufactured in this manner, is used by contacting it with an antigen. Contact with the antigen is not limited as long as the antibody immobilized on the NC membrane and the antigen come into contact as described above. For example, such contact may be achieved by dropping a solution containing the antigen onto the antibody-immobilized NC membrane; immersing the antibody-immobilized NC membrane in a solution containing the antigen; contacting a part of the antibody-immobilized NC membrane with a solution containing the antigen, allowing the antigen to penetrate from the contact area and come into contact with the antibody immobilized on the NC membrane; or by inkjet supply (coating, etc.) or capillary supply (coating, etc.). Furthermore, such contact may be performed on the entire antibody-immobilized NC membrane or only on a part of it, and can be appropriately determined depending on the location on the NC membrane where the antibody is to be reacted. Preferably, the antigen is an antigen corresponding to the antibody.

[0027] The temperature of the contact is not limited and can be easily carried out at room temperature (25°C), etc. Furthermore, as long as the effects of this disclosure are obtained, the amount of antigen in the solution containing the antigen is not limited. Also, as long as the effects of this disclosure are obtained, the amount of the solution containing the antigen that comes into contact with the NC membrane is not limited and can be appropriately determined considering the amount of antigen in the solution, etc. As an example, when using an immunochromatography strip as shown in the examples below, the contact amount of the solution can be 0.1 to 10 μL, 0.2 to 5 μL, 0.3 to 2 μL, 0.5 to 1 μL, etc. When using something other than a strip, the amount can be appropriately determined by referring to these values. By making contact in this way, the antibody immobilized on the NC membrane can be bound to the antigen corresponding to the antibody. After contact, drying may or may not be performed.

[0028] Without limiting this disclosure, the amount of antigen may be adjusted. For example, if the antibody is an anti-influenza virus antibody, examples of antigen amounts in the solution include 1 pfu / mL or more, 10 pfu / mL or more, 20-500 pfu / mL, 80-300 pfu / mL, 80-200 pfu / mL, etc. The amount of antigen in the solution may be set appropriately based on these values ​​if necessary.

[0029] While the antibody-immobilized NC membrane described herein is typically used in contact with an antigen, a solution (sample) in which the presence of an antigen is suspected may be applied to the NC membrane, bringing the solution into contact with the antibody immobilized on the NC membrane. In such cases, since the purpose is generally to confirm whether or not the antigen is present in the solution, the solution does not necessarily need to contain the antigen.

[0030] The detection of antigens bound to antibodies immobilized on NC membranes can be carried out according to conventionally known methods. While not limiting to this disclosure, examples include detection using conventionally known antigen-antibody reaction detection procedures utilizing labeling substances such as fluorescent substances, enzymes, metals (gold colloid, etc.), biotin, avidin, streptavidin, PEG (polyethylene glycol) labels, polymers (colored latex, etc.), and secondary antibodies. These substances may be used individually or in combination of two or more, following known procedures. Preferred examples of such detection methods include immunochromatography and ELISA (enzyme-linked immunosorbent assay). Furthermore, methods using colored latex, gold colloid, etc., are exemplified from the viewpoint of simple detection in immunochromatography, etc. Examples of such methods, while not limiting to this disclosure, include the method using colored latex shown in the examples below.

[0031] NC membranes with immobilized antibodies have been conventionally used in rapid (simple) tests for influenza and other diseases. In such tests, the antigen (sample) is usually placed in a location on the antibody-immobilized NC membrane that does not come into contact with the antibody. Before contact with the antibody, the antigen is directly or indirectly linked to a labeling substance (such as gold colloid) or a secondary antibody. Then, the antigen constituting the resulting linkage is brought into contact with and bound to the antibody on the antibody-immobilized NC membrane to detect the antigen. This disclosure can be preferably used in such methods. Therefore, in order to simplify detection according to conventionally known rapid (simple) tests, it is preferable to label the antigen with a labeling substance or the like before binding to the antibody. Thus, the antibody and antigen immobilized on the NC membrane may be directly bound or indirectly bound, and are preferably directly bound.

[0032] In this way, antigens bound to antibodies can be detected. This detection may be performed by visually confirming the result of the antigen-antibody reaction, as in conventional procedures, or by using any device capable of confirming the result. In this disclosure, detection encompasses measurement, and if necessary, the amount of binding between the antigen and antibody may be measured according to conventional methods. This makes it possible to detect antigens (measure the amount of antigen) using an antibody-immobilized NC membrane.

[0033] As can be seen from the examples described below, when an antibody-immobilized NC membrane obtained by contacting an NC membrane with an alkaline solution containing antibodies is used, antigen detection can be performed with higher sensitivity than when an antibody-immobilized NC membrane obtained by contacting an NC membrane with a neutral solution (pH 7) containing antibodies is used. The method using a neutral solution is a conventional and common method for immobilizing antibodies on an NC membrane. In this field, it has been conventionally considered desirable to immobilize antibodies on an NC membrane in a more stable state while minimizing changes in the three-dimensional structure of the antibodies, and from this viewpoint, it has been considered good to use a neutral solution for immobilizing antibodies on an NC membrane. Nevertheless, surprisingly, the present inventors have found that antigen binding is improved in antibody-immobilized NC membranes when an alkaline solution is used rather than when a neutral solution is used. From this, it can be said that this disclosure also provides a method for producing an antibody-immobilized NC membrane with enhanced antigen binding, comprising steps (1) and (2) above.

[0034] In this disclosure, enhanced antigen binding means that even a smaller amount of antigen can be detected, which in turn means increased sensitivity in antigen detection.

[0035] Furthermore, in conventional rapid (simple) tests such as POCT tests using immunochromatography, samples have been collected from blood, saliva, urine, nasal secretions, sputum, stool, mucous membranes (nasal mucosa, oral mucosa, etc.), tap water, food (including beverages), etc. (including cotton swab samples, etc.). If the sample is not in liquid form, it is usually suspended in a solvent as appropriate before being used as a sample. In this disclosure, the same method as in the past can be used, and a variety of samples can be targeted in this way.

[0036] Therefore, the antibody-immobilized NC membrane of this disclosure can be widely used in diagnostics and analyses that utilize antigen-antibody reactions, and thus, the antibody-immobilized NC membrane can preferably be used as part of a diagnostic kit or analytical kit. Furthermore, although not limiting this disclosure, the antibody-immobilized NC membrane of this disclosure can be widely used in various immunological methods, including ELISA, and is particularly preferably used as an antibody-immobilized NC membrane constituting an immunochromatography strip (test strip), or as a protein chip, antibody chip, etc.

[0037] Method for enhancing antigen binding of antibody-immobilized nitrocellulose membranes As described above, the antibody-immobilized nitrocellulose membrane prepared according to this disclosure has enhanced antigen-binding properties. Therefore, this disclosure also encompasses a method for enhancing the antigen-binding properties of an antibody-immobilized nitrocellulose membrane, comprising the following steps: (a) A step of bringing an alkaline solution containing an antibody into contact with a nitrocellulose membrane, and (i) A step of drying the nitrocellulose film that has been in contact with the alkaline solution in step (a) above to obtain an antibody-immobilized nitrocellulose film.

[0038] Step (a) is described in the same manner as step (1) above.

[0039] Step (a) is a step to obtain an antibody-immobilized nitrocellulose film by drying a nitrocellulose film that has been in contact with an alkaline solution in the same manner as in step (2). Therefore, the drying of the nitrocellulose film that has been in contact with an alkaline solution will be explained in the same manner as in step (2), and the obtaining of an antibody-immobilized nitrocellulose film by this will also be explained in the same manner as in the "Method for Producing an Antibody-Immobilized Nitrocellulose Film". Blocking treatment may also be performed as described above if necessary.

[0040] The enhanced antigen binding of this disclosure is also explained in the same manner as the "Method for Producing Antibody-Immobilized Nitrocellulose Membrane" described above. This enhancement means that when an antigen is brought into contact with an antibody-immobilized NC membrane produced according to the method of this disclosure and an antibody-immobilized NC membrane produced in the same manner except that a neutral solution (pH 7) containing the antibody is used instead of an alkaline solution containing the antibody, the amount of antigen-antibody binding on the antibody-immobilized NC membrane produced using the alkaline solution of this disclosure is greater than the amount of antigen-antibody binding on the antibody-immobilized NC membrane produced using the neutral solution. Alternatively, this enhancement means that the antibody-immobilized NC membrane produced using the alkaline solution of this disclosure can detect a smaller amount of antigen (has a lower detection limit) than the antibody-immobilized NC membrane produced using the neutral solution.

[0041] The comparison described above is based on the presence or absence and amount of antigen detected in the antibody-immobilized nitrocellulose membrane, and the means (contact between antibody and antigen, detection (measurement) of antigen, etc.) are described in the same manner as in the "Method for Producing Antibody-Immobilized Nitrocellulose Membrane". Furthermore, the comparison is not limited as long as these can be compared, and may be based on absolute amounts (absolute values) or relative amounts (relative values) as long as the binding affinity between antibody and antigen can be understood. Furthermore, as stated above, the comparison is not limited as long as these can be compared, but usually, the antibody, antigen, contact, drying, and other conditions used are substantially the same, except that antibody-immobilized NC membranes produced using an alkaline solution and antibody-immobilized NC membranes produced using a neutral solution are used. Although this does not limit the disclosure, the comparison is preferably performed according to the procedure described in Test Example 1 or Test Example 2 below.

[0042] According to this disclosure, since the antigen-binding properties of the antibody-conjugated NC membrane can be enhanced in this way, antigens can be detected with higher sensitivity.

[0043] Antibody screening methods This disclosure also includes a method for screening antibodies as an indicator of antigen binding, comprising the following steps: (A) A step of contacting an alkaline solution containing a candidate antibody with a nitrocellulose membrane. (B) A step of drying the nitrocellulose film that was in contact with the alkaline solution in step (A) to obtain a candidate antibody-immobilized nitrocellulose film, and (C) A step of bringing the candidate antibody on the candidate antibody-immobilized nitrocellulose membrane obtained in step (B) into contact with the antigen.

[0044] As mentioned above, conventionally, NC membranes have not been considered highly versatile as antibody immobilization carriers, and their use has been very limited. However, with the antibody-immobilized NC membrane obtained by the method disclosed herein, it is possible to detect antigens with higher sensitivity even while using an NC membrane. In particular, it has been found that this antibody-immobilized NC membrane can detect antigens even with smaller amounts of antigen.

[0045] Thus, the antibody-immobilized NC membrane is excellent for immobilizing antibodies on the NC membrane and performing antigen-antibody reactions, and it also allows for easy contact between antigens and antibodies. For this reason, the antibody-immobilized NC membrane can be said to be useful for screening antibodies using antigen-binding properties on the membrane as an indicator. In particular, the antibody-immobilized NC membrane can be said to be useful for screening antibodies that are useful when immobilized on an NC membrane.

[0046] A method for screening antibodies according to the present disclosure includes the step of (A) contacting an alkaline solution containing a candidate antibody with a nitrocellulose membrane.

[0047] The candidate antibody may be a natural antibody or an artificially produced antibody, and may be a known antibody or a newly produced antibody. Furthermore, the candidate antibody may be any of the following: a single-chain antibody (scFv), a single-domain antibody (VHH), a full-length antibody (whole antibody), sc(Fv)2, a diabody, Fab, F(ab')2, etc. The candidate antibody can be described in this way, and otherwise, step (A) is described in the same manner as step (1).

[0048] Furthermore, the screening method of this disclosure includes (B) a step of drying the nitrocellulose membrane that has been in contact with the alkaline solution in step (A) to obtain a candidate antibody-immobilized nitrocellulose membrane. Step (B) is a step of obtaining a candidate antibody-immobilized nitrocellulose membrane by drying the nitrocellulose membrane that has been in contact with the alkaline solution in the same manner as in step (2), except that a candidate antibody is used, and will be described in the same manner as above. In addition, blocking treatment may or may not be performed as described above, and blocking will be described in the same manner as above.

[0049] Furthermore, the antibody screening method of this disclosure includes (C) a step of contacting a candidate antibody on an antibody-immobilized nitrocellulose membrane obtained in step (B) with an antigen. This contact is not limited as long as the antigen-binding ability of the candidate antibody can be determined, and is described in the same manner as the antibody-antigen contact procedure described in the "Method for Producing an Antibody-Immobilized Nitrocellulose Membrane" section, except that a candidate antibody is used.

[0050] After contact, it is possible to determine whether the candidate antibody is useful as an antibody based on its antigen-binding level immobilized on the nitrocellulose membrane, and in particular, whether it is useful as an antibody to be conjugated to an NC membrane for use. Furthermore, when determining whether the candidate antibody is useful as an antibody based on this level, its usefulness may be determined by comparing it with a reference antigen-binding level.

[0051] Therefore, the screening method of the present disclosure may further include (D) a step of comparing the antigen-binding level of the candidate antibody immobilized on the nitrocellulose membrane with the antigen-binding level of the reference antibody after contact in step (C).

[0052] The reference level may be determined as appropriate and does not limit this disclosure, but as one embodiment of the screening method of this disclosure, for example, the antigen binding level of an antibody already known to be a desirable antibody when immobilized on an NC membrane and brought into contact with an antigen may be used as the reference level (antigen binding level of the reference antibody). In this case, if the antigen binding level (test level) of the candidate antibody when immobilized on the candidate antibody NC membrane is the same as or higher than the reference level, the candidate antibody may be determined to be highly likely to be useful as an antibody, and if the test level is lower than the reference level, the candidate antibody may be determined to be less likely to be useful as an antibody. In particular, if the test level is the same as or higher than the reference level, the candidate antibody may be determined to be highly likely to be useful as an antibody to be immobilized on an NC membrane, and if the test level is lower than the reference level, the candidate antibody may be determined to be less likely to be useful as an antibody to be immobilized on an NC membrane.

[0053] Furthermore, as one embodiment of the screening method of this disclosure, the antigen-binding level of an antibody that is already known to be an unusable antibody when immobilized on an NC membrane and brought into contact with an antigen may be used as the reference level (antigen-binding level of the reference antibody). In this case, if the antigen-binding level of a candidate antibody (test level) when the candidate antibody is immobilized on an NC membrane is the same as or lower than the reference level, it may be determined that the candidate antibody is likely to be unusable as an antibody (particularly unusable as an antibody immobilized on an NC membrane). If the test level is higher than the reference level, it may be determined that the candidate antibody is likely to be useful as an antibody (particularly unusable as an antibody immobilized on an NC membrane).

[0054] The antigen-binding level is the level (degree of binding) of the antibody (candidate antibody) immobilized on the NC membrane to the antigen, and thus represents antigen-binding ability. Therefore, the screening method of this disclosure may further include such a determination step.

[0055] Furthermore, since the aforementioned level represents antigen-binding activity, the level can be detected (measured) according to conventionally known methods, as described above, and is explained in the same way as the detection of antigens bound to antibodies as described in the "Method for Manufacturing Antibody-Immobilized Nitrocellulose Membrane" section. Moreover, the comparison may be based on the absolute value of the obtained detection value (measured amount) or on the relative value, and is not limited as long as the levels can be compared.

[0056] Furthermore, while such comparisons are not limited to those that can be compared, they are preferably and exemplified to be carried out under substantially the same conditions, except that the candidate antibody used to obtain the test level is different from the antibody used to obtain the reference level. Also, without limiting this disclosure, such comparisons may be based on the presence or absence of detection (detection amount) or on the detection limit.

[0057] Thus, the antibody screening method of this disclosure allows for the simple screening of useful antibodies using antigen-binding properties as an indicator.

[0058] Conventionally, the antibodies used in NC membranes have been limited to certain full-length antibodies, and despite the antibody being immobilized on the NC membrane (although the mechanism is unknown), the detection sensitivity (antigen-binding ability of the antibody) has been relatively low. The cause of this is still unclear. For this reason, even today, measures such as contacting the NC membrane with a high-concentration antibody-containing neutral solution during the manufacturing of antibody-immobilized NC membranes are taken to immobilize a larger number of antibodies. The method disclosed herein is useful in mitigating these problems because it allows for more sensitive detection of antigens.

[0059] Furthermore, while NC membranes with immobilized antibodies have conventionally been used in rapid (simple) tests such as influenza, pregnancy, and fecal occult blood tests, the enhanced antigen binding properties introduced in this disclosure improve the sensitivity of antigen detection. Therefore, this disclosure has the advantage of enabling antigen detection even with smaller amounts of antibody. In addition, as shown in the examples described later, the method of this disclosure is useful not only for full-length antibodies but also for single-chain antibodies, single-domain antibodies, etc., thus increasing the range of antibodies that can be used in immunological methods utilizing NC membranes.

[0060] In particular, the method disclosed herein allows antibody fragments such as single-chain antibodies and single-domain antibodies, which have conventionally been virtually unusable in rapid tests, to be immobilized onto NC membranes proactively and without requiring additional modifications to the antibodies, making them usable in immunochromatography. This will lead to a significant reduction in the cost of rapid tests such as POCT tests, and will greatly contribute to the widespread adoption of POCT tests. Furthermore, the widespread adoption of POCT tests will be useful in strengthening disease control systems in emerging countries and other countries with insufficient medical facilities. [Examples]

[0061] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below.

[0062] Test Example 1 1-1) Procedure for manufacturing antibody-immobilized NC membranes (1) Each 100 mM pH buffer was prepared according to conventional methods (Table 1). Specifically, Gly-HCl buffer (Gly-HCl in the table) was prepared by dissolving glycine (Gly) in ultrapure water and adjusting the pH with hydrochloric acid. Acetate buffer (Acetate in the table) was prepared by mixing sodium acetate and acetic acid in ultrapure water. Mes-NaOH buffer (Mes-NaOH in the table) was prepared by dissolving Mes(2-Morpholinoethanesulfonic acid, monohydrate) in ultrapure water and adjusting the pH with sodium hydroxide. Tris-HCl buffer (Tris-HCl in the table) was prepared by dissolving Tris(Tris(hydroxymethyl)aminomethan) in ultrapure water and adjusting the pH with hydrochloric acid. Gly-NaOH buffer (Gly-NaOH in the table) was prepared by dissolving glycine in ultrapure water and adjusting the pH with sodium hydroxide. KCl-NaOH buffer (KCl-NaOH in the table) was prepared by mixing hydrochloric acid and sodium hydroxide in ultrapure water. The pH was measured at 25°C using a pH meter (product name LAQUA, manufactured by Horiba, Ltd.). The pH measurement was performed in the same manner for subsequent measurements. (2) Sugars or surfactants were mixed with ultrapure water to prepare a total of four types of sugar solutions and surfactant solutions (20 w / v% trehalose aqueous solution, 20 w / v% sodium deoxycholate aqueous solution, 2 w / v% SDS (sodium dodecyl sulfate) aqueous solution, 2 w / v% CHAPS (3-(3-cholamidepropyl)dimethylammonio-1-propanesulpHonate) aqueous solution). These will be referred to as additive solutions below. (3) The buffer obtained in (1) above and the additive solution obtained in (2) above were mixed as appropriate to prepare five solutions for each pH range from 1 to 13, as shown in Figure 1 below. The concentration of sugars or surfactants in the solutions was determined by referring to the antibody immobilization conditions of Denka Co., Ltd. In the figure, "no additive" refers to a solution prepared without the additive solution shown in (2) above.

[0063] The solution obtained in this manner was mixed with the anti-influenza A antibody Ant-NP-A VHH clone (hereinafter referred to as VHH1) to an antibody concentration of 100 μg / ml. The resulting antibody-containing solution was then dropped onto NC membranes (product name High-Flow plus HF180 (manufactured by Merck KGaA)) in 2 μl increments and air-dried at room temperature (25°C). In this way, antibody-immobilized NC membranes (VHH1-immobilized NC membranes) were obtained.

[0064] Furthermore, VHH2-immobilized NC membranes were obtained in the same manner as described above, except that a different anti-influenza A antibody Ant-NP-A VHH clone (hereinafter referred to as VHH2) was used. Note that the antibody-containing solution used with VHH2 consisted of either no additives or only sodium deoxycholate as an additive.

[0065] [Table 1]

[0066] 1-2) Contact with antigen and detection procedure (1) The VHH1-fixed NC membrane and the VHH2-fixed NC membrane were each immersed in 2% BSA-PBS (PBS containing 2% bovine serum albumin (137 mmol / l NaCl, 10 mmol / l Na2HPO4, 2.68 mmol / l KCl, 2 mmol / l KH2PO4, pH 7.4)) and blocked by shaking at room temperature (25°C) for 1 hour. These NC membranes were then washed five times with PBST (PBS containing 0.1% Tween 20). Each of the NC membranes obtained in this way was scanned using a scanner GT-X830 (manufactured by Epson Corporation). The images obtained by scanning are shown in Figures 1 and 2, right-hand photographs (adsorption). (2) Next, 15 ml of inactivated influenza A virus, diluted to 203 pfu / ml in 0.2% BSA-PBST (PBS containing 0.2% bovine serum albumin and 0.1% Tween 20), was added dropwise to each NC membrane obtained as described above, and the mixture was shaken at room temperature for 1 hour. (3) Next, the NC membranes were washed five times with PBST, and then 15 ml each of biotinylated full-length antibody (Whole) antibody, diluted to 1 μg / ml with 0.2% BSA-PBST, was added dropwise to each NC membrane and shaken at room temperature for 1 hour. (4) Next, the NC membranes were washed five times with PBST, and then 15 ml each of HRP (horseradish peroxidase)-labeled streptavidin, diluted to 200 μg / ml with 0.2% BSA-PBST, was added dropwise to each NC membrane and shaken at room temperature for 1 hour. (5) Next, each NC film was washed five times with PBST and then colored with TMB (3,3',5,5'-tetramethylbenzidine). (6) After drying, the NC film was scanned using a scanner GT-X830 (manufactured by Epson Corporation).

[0067] 1-3) Results The results using VHH1 are shown in Figure 1, and the results using VHH2 are shown in Figure 2. In both Figures 1 and 2, the photograph on the right shows the antibody-fixed NC membrane obtained in 1-2) (1) above, before contact with the antigen (adsorption), and the photograph on the left shows the antibody-fixed NC membrane after contact with the antigen (inactivated influenza A virus) (antigen binding).

[0068] As shown in the right-hand photograph of Figure 1, before contact with the antigen, spots indicating the application area of ​​the antibody-containing solution were observed not only when using an alkaline solution, but also when using a neutral solution and an acidic solution. On the other hand, as shown in the left-hand photograph of Figure 1, after contact with the antigen, more dark spots were observed when using an alkaline solution (Example A (pH 9 or higher)) than when using a neutral solution or an acidic solution (Comparative Example A (pH 8 or lower)). The intensity of the color of each spot correlates with the antigen-binding activity of the antibody; the darker the spot, the more antigen is present in that area, indicating high antigen-binding activity of the antibody. Conventionally, neutral solutions containing antibodies are generally used for antibody immobilization on NC membranes. Therefore, it was expected that the spots using a neutral solution would have the highest antigen-binding activity. Surprisingly, however, a tendency for higher antigen-binding activity was observed when using an alkaline solution than when using a neutral solution, meaning that detection could be performed with high sensitivity. Thus, it was understood that using an alkaline solution containing antibodies for antibody immobilization on NC membranes enhances the antigen detection activity.

[0069] Test Example 2 2-1) Procedure for manufacturing antibody-immobilized NC membranes

[0070] [Preparation of strips for immunochromatography] As shown in Figure 3, the absorbent band, NC film, conjugate pad, and sample pad were attached to the packing sheet in that order. [Detection of antigens by immunochromatography] In this study, a total of 12 anti-influenza A antibody Ant-NP-A VHH clones were used, along with VHH1 and VHH2 used in Study Example 1. Antibodies were mixed into pH 7 and pH 13 buffers, prepared in the same manner as in Table 1 of Study Example 1, to achieve antibody concentrations of 100, 50, and 25 μg / ml, respectively, to prepare antibody-containing solutions. Next, 2 μl of the antibody-containing solution was dropped onto the NC membrane of the immunochromatography strip prepared as described above, and allowed to air dry at room temperature. This produced VHH-fixed NC membranes. Each VHH-fixed NC membrane prepared using pH 13 buffer was designated as Examples 1-12 (VHH1-VHH12). Each VHH-fixed NC membrane prepared using pH 7 buffer was designated as Comparative Examples 1-12 (VHH1-VHH12).

[0071] 2-2) Contact with antigen and detection procedure (1) Inactivated influenza A virus was added to the sample suspension to a concentration of 56.7 pfu / ml, and a commercially available red latex-labeled antibody was mixed in to a final concentration of 0.004% to prepare an antigen-containing solution. (2) The sample pad portion of the VHH-fixed NC film (strip) obtained as shown above was immersed in the solution obtained in (1), and the reaction solution was allowed to develop for about 20 minutes. (3) After drying, it was scanned using a GT-X830 scanner.

[0072] 2-3) Results The results are shown in Figure 4. In all types of VHH, the antigen binding was higher when the antibody was immobilized on the NC membrane using an alkaline solution (Examples 1-12) compared with the case where the antibody was immobilized on the NC membrane using a neutral solution (pH 7) (Comparative Examples 1-12). This confirms that antibody-immobilized NC membranes obtained using an alkaline solution can enhance antigen binding compared to antibody-immobilized NC membranes obtained using a neutral solution. This also indicates that antibody-immobilized NC membranes obtained using an alkaline solution can improve antigen detection sensitivity.

[0073] Test Example 3 3-1) Manufacturing procedure for antibody-immobilized NC membrane, contact with antigen, and detection procedure Antibody-immobilized NC membranes (strips) were manufactured in the same manner as in Test Example 2. The tests were conducted in the same manner as in Test Example 2, except that the type of antigen was changed to one type of full-length antibody (whole antibody) and two types of Ant-NP-A scFv (scFv1, scFv2), and the antigen concentration was set to 0 pfu / ml or 56.7 pfu / ml.

[0074] 3-2) Results The results are shown in Figure 5. Even when using whole antibodies and scFv, similar to the case with VHH, higher antigen binding was observed when the antibody was immobilized on the NC membrane using an alkaline solution (Examples 13-15) compared to when a neutral solution was used (Comparative Examples 13-15). From the results of Test Examples 2 and 3, it was confirmed that enhanced antigen binding, and consequently high-sensitivity detection, in antibody-immobilized NC membranes obtained using an alkaline solution was observed regardless of the type of antibody, such as VHH, whole antibodies, or scFv.

[0075] Test Example 4 4-1) Procedure for manufacturing antibody-immobilized NC membranes (1) Each 100 mM pH buffer was prepared in the same manner as in Test Example 1 above, according to Table 1. (2) Solutions of anti-influenza A antibody clones VHH1, VHH3, and scFv2, prepared to 100 μg / ml using each pH buffer, were dropped into the NC membrane of an immunochromatography strip prepared in the same manner as in 2-1) of Test Example 2, and allowed to air dry at room temperature. Examples with a pH of 9 or higher were designated as Example B, and comparative examples with a pH of 8 or lower were designated as Comparative Example B.

[0076] 4-2) Contact with antigen and detection procedure (1) Inactivated influenza A virus was added to the sample suspension to a concentration of 56 pfu / ml, and then commercially available red latex-labeled antibody was mixed in to a final concentration of 0.004% to prepare an antigen-containing solution. (2) The sample pad of the antibody-immobilized NC membrane (strip) obtained as described above was immersed in the solution obtained in (1) and the reaction solution was allowed to develop for about 20 minutes. (3) After drying, it was scanned using a GT-X830 scanner.

[0077] 4-3) Results The scan results for VHH3 are shown in Figure 6. From Figure 6, it was observed that the antigen binding tendency was higher when the antibody was immobilized on the NC membrane using an alkaline solution (Example B) compared with when the antibody was immobilized on the NC membrane using an acidic or neutral solution (Comparative Example B). Although not shown in the results, a similar trend was observed when VHH1 and scFv were used. From this, it was understood that the detection sensitivity can be improved by using an alkaline solution when immobilizing antibodies on the NC membrane.

[0078] Test Example 5 5-1) Procedure for manufacturing antibody-immobilized NC membranes From the above test examples, it became clear that the antigen detection sensitivity was increased when antibodies were immobilized on the NC membrane using an alkaline solution compared to when antibodies were immobilized on the NC membrane using a neutral solution. Therefore, using 8 clones of VHH, the antigen concentration was further reduced and the test was conducted as follows. (1) Eight clones of Anti-NP-A VHH were prepared to a concentration of 100 μg / ml using the pH 13 buffer mentioned above to create an antibody-containing solution. (2) Two μl of the antibody-containing solution obtained in (1) was added to the NC film of the immunochromatography strip prepared in the same manner as described above, and after air drying, another 2 μl was added to the NC film. (3) The operation in (2) above was repeated twice to deposit each VHH onto the NC film (amount of VHH immobilized: 1.2 μg / each spot). The VHH immobilized NC films obtained in this way were designated as Examples 16 to 23.

[0079] 5-2) Contact with antigen and detection procedure (1) An antigen-containing solution was prepared by adding inactivated influenza A virus to the sample suspension so that the amount was between 54 and 0 pfu / ml, and then mixing in a commercially available red latex-labeled antibody to a final concentration of 0.004%. (2) The antigen-containing solution from (1) was immersed in the VHH-fixed strip prepared as described above, and the reaction solution was allowed to develop for about 20 minutes. (3) After drying, it was scanned using a GT-X830 scanner.

[0080] 5-3) Results The results are shown in Figure 7. As shown in Figure 7, antibodies could be detected even when using antigen-containing solutions with very low concentrations of antigen, such as 54 pfu / ml and even 28 pfu / ml, in each example. Furthermore, depending on the clone, antibodies could be detected even with an extremely low antigen-containing solution of 3.5 pfu / ml. Although not shown in the results, detection was also possible at 1.75 pfu / ml. As can be seen from this, enhancing antigen binding is useful for detecting antigens with higher efficiency.

Claims

1. A method for producing an antibody-immobilized nitrocellulose membrane, comprising the following steps: (1) A step of bringing an alkaline solution containing an antibody into contact with a nitrocellulose membrane, and (2) A step of drying the nitrocellulose film that has been in contact with the alkaline solution in step (1), Here, the antibody is at least one selected from the group consisting of VHH, scFv, sc(Fv)2, Diabody, Fab, and F(ab')2.

2. The manufacturing method according to claim 1, wherein the pH of the alkaline solution is 9 to 14.

3. A method for producing an antibody-immobilized nitrocellulose membrane with enhanced antigen-binding properties, as described in claim 1 or 2.

4. A method for enhancing the antigen-binding properties of an antibody-immobilized nitrocellulose membrane, comprising the following steps: (a) A step of bringing an alkaline solution containing an antibody into contact with a nitrocellulose membrane, and (i) A step of drying the nitrocellulose film that has been in contact with the alkaline solution in step (a) above to obtain an antibody-immobilized nitrocellulose film. Here, the antibody is at least one selected from the group consisting of VHH, scFv, sc(Fv)2, Diabody, Fab, and F(ab')2.

5. A method for screening antibodies using antigen-binding activity as an indicator, comprising the following steps: (A) A step of bringing an alkaline solution containing a candidate antibody into contact with a nitrocellulose membrane. (B) A step of drying the nitrocellulose film that has been in contact with the alkaline solution in step (A) to obtain a candidate antibody-immobilized nitrocellulose film, and (C) A step of bringing the candidate antibody on the candidate antibody-immobilized nitrocellulose membrane obtained in step (B) into contact with the antigen. Here, the antibody is at least one selected from the group consisting of VHH, scFv, sc(Fv)2, Diabody, Fab, and F(ab')2.

6. Furthermore, the method for screening antibodies according to claim 5, comprising (D) a step of comparing the antigen-binding level of the candidate antibody immobilized on the nitrocellulose membrane with the antigen-binding level of a reference antibody after contact in step (C).

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