JP7832652B2 - Method for producing antibody-immobilized nitrocellulose membrane and method for enhancing antigen binding - Google Patents

Description

This invention relates to a method for producing antibody-immobilized nitrocellulose membranes, a method for enhancing antigen binding, and a method for screening antibodies using antigen binding as an indicator.

Nitrocellulose (NC) membranes are conventionally known as antibody immobilization carriers in immunological techniques such as immunochromatography, ELISA (Enzyme-Linked Immunosorbent Assay), and Western blotting. Among these, immunochromatography is particularly useful because it provides antibody-antigen reaction results relatively quickly and easily, and is used, for example, in Point of Care Testing (POCT) tests such as influenza testing.

However, in antigen-antibody reaction tests using immunochromatography, such as POCT (Point-of-Care Testing), while antibodies are immobilized on the NC membrane, these antibodies are limited to certain types of full-length antibodies (whole antibodies), and there are very few examples of using recombinant antibodies such as single-chain antibodies (scFv) or single-domain antibodies (VHH). Furthermore, antibodies are prone to losing their three-dimensional structure, and not all antibodies can maintain their activity on the NC membrane even after immobilization. Therefore, when immobilizing antibodies on the NC membrane, it was necessary to apply a high-concentration antibody-containing solution to the NC membrane. POCT tests and similar tests are said to be expensive, and this is one of the reasons for the increased testing costs (Non-Patent Literature 1). Also, many aspects of the protein adsorption mechanism in NC membranes remain unclear, and the development of better methods is needed.

Naoto Shimetani, "Current Status and Future Challenges of POCT (Point of Care Testing)," Medical Engineering Vol. 80, No. 4, pp. 37-44 (2010)

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.

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.
Item 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 membrane 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 contacting an alkaline solution containing an antibody with a nitrocellulose membrane, and (b) a step of drying the nitrocellulose membrane that has been in contact with the alkaline solution in step (a) to obtain an antibody-immobilized nitrocellulose membrane.
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 membrane that was in contact with the alkaline solution in step (A) to obtain a candidate antibody-immobilized nitrocellulose membrane, 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).

By contacting an alkaline solution containing antibodies with an NC membrane and drying it, the antibodies can be immobilized on the NC membrane. This method of immobilizing antibodies on the 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.

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

The embodiments included in this disclosure will be described in further detail below. In this disclosure, "contains" also means "substantially consists of" or "consists of."

Method for manufacturing an antibody-immobilized nitrocellulose membrane This disclosure comprises a method for manufacturing 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 membrane that has been in contact with the alkaline solution in step (1).

Process (1)
The method for producing an antibody-immobilized nitrocellulose membrane according to this disclosure includes the step of (1) contacting an alkaline solution containing an antibody with a nitrocellulose membrane. In this disclosure, the antibody-immobilized nitrocellulose membrane means a nitrocellulose membrane on which an antibody has been immobilized.

The nitrocellulose (NC) membrane is not limited as long as it is one that has been conventionally used as an antibody immobilization carrier in immunological techniques such as immunochromatography. In this respect, the thickness and shape of the NC membrane are also not limited. While not limiting this disclosure, examples of NC membrane thickness include approximately 80 to 500 μm, preferably approximately 80 to 400 μm. Also, while not limiting this disclosure, examples of NC membrane average pore size include approximately 0.2 to 5 μm, preferably approximately 0.45 μm to 5 μm. In this disclosure, the average pore size is the value according to the product catalog. NC membranes are commercially available, and examples include High-Flow plus HF180 (manufactured by Merck KGaA).

The antibody immobilized on the NC membrane in this disclosure is not limited and may be any single-chain antibody (scFv), single-domain antibody (VHH), full-length antibody (whole antibody), sc(Fv) ₂ , Diabody, Fab, F(ab') ₂ , etc. While not limiting to this disclosure, examples of preferred antibodies include single-chain antibodies and single-domain antibodies. These may be used individually or in combination of two or more.

The antibody may be labeled by fluorescent labeling, enzyme labeling, metal labeling (such as gold colloid), biotin labeling, PEG (polyethylene glycol) labeling, polymer labeling (such as colored latex), etc., to the extent that it does not interfere with the effects of this disclosure, or it may be left unlabeled. In conventional clinical immediate testing methods such as influenza testing, the antibody is immobilized on the NC membrane without labeling (unmodified). Therefore, from the viewpoint of effort and cost, it is preferable in this disclosure that the antibody is immobilized on the NC membrane without labeling.

The pH of the alkaline solution containing the antibody used in step (1) 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, a pH of 10 to 13, 11 to 13, 12 to 13, etc., are exemplified for the pH of the alkaline solution containing the antibody. The pH can be appropriately determined within the range of pH 9 to 14 depending on the antibody immobilized on the NC membrane. The pH is the value measured at 25°C using a pH meter (product name LAQUA, manufactured by Horiba, Ltd.) and represents the pH at the time of contact with the NC membrane.

Examples of alkaline solutions include, but are not limited to this disclosure, 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 with buffering capacity in the alkaline range are also provided. Examples of such buffers, but are not limited to this disclosure, include Tricin, Bicine, TAPS (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), CHES (N-Cyclohexyl-2-aminoethanesulfonic acid), CAPSO (3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), and CAPS (N-Cyclohexyl-3-aminopropanesulfonic acid).

Without limiting this disclosure, to give general examples, 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. While the concentration of the buffer in these solutions is not limited, typically it is 10 to 200 mM, preferably 10 to 100 mM, and more preferably 10 to 50 mM.

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

The alkaline solution may contain, to the extent that it does not impair the effects of this disclosure, conventional additives 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.). If additives are included, they may be used individually or in combination of two or more. The amount of additive used can also be appropriately determined according to conventional procedures, for example, within ranges such as 0-5 w/v%, 0.0001-2 w/v%, 0.01-1 w/v%, or 0.02-0.5 w/v%, depending on the type of additive used.

By mixing the antibody with an alkaline solution, an alkaline solution containing the antibody is obtained. The amount of antibody in the alkaline solution is not limited as long as the effects of this disclosure are obtained. 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 binding can be efficiently achieved by using an NC membrane immobilized with the antibody, the antibody concentration in the alkaline solution may be relatively low, such as 10 μg/ml or less.

The contact between the alkaline solution containing the antibody and the NC membrane can be carried out by any means necessary, as long as contact is achieved. This contact may be achieved by, for example, dropping the alkaline solution containing the antibody onto the NC membrane, immersing the NC membrane in the alkaline solution, inkjet feeding (coating, etc.), capillary feeding (coating, etc.), or any other method. Furthermore, the contact may be applied to the entire NC membrane or only to a portion of it, depending on the location on the NC membrane where the antigen is to be reacted. The temperature of the contact is also not limited and can be carried out at room temperature (25°C) for convenience. The amount of alkaline solution containing the antibody that comes into contact with 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 in a chromatography strip, as shown in the examples below, by drawing a 5 mm long straight line using capillary coating, the amount of alkaline solution containing the antibody that contacts the NC membrane is preferably 0.2 to 2 μL per 5 mm straight line, and more preferably 0.3 to 1 μL.

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).

Drying may be performed by natural drying, or by 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. Therefore, in this disclosure, the immobilization of antibodies on the NC membrane occurs by adsorption (adsorption immobilization).

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

The antibody immobilized on the NC membrane is not limited as long as it is an antibody capable of binding 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. Furthermore, any antibody against troponin T, troponin I, CK-MB (creatine kinase-MB), myoglobin, LH (luteinizing hormone), E. coli verotoxin, etc. may be used. While not limiting 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.

The antibody-immobilized NC membrane manufactured in this manner according to the present disclosure 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 comes into contact with the antigen, 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 portion 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.); capillary supply (coating, etc.); etc. Furthermore, such contact may be performed on the entire antibody-immobilized NC membrane or only on a portion thereof, depending on the location on the NC membrane where the antibody is to be reacted. Preferably, the antigen corresponds to the aforementioned antibody.

The temperature of the contact is not restricted and, being simple, can be 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 antigen-containing solution is not restricted. Also, as long as the effects of this disclosure are obtained, the amount of antigen-containing solution that contacts the NC membrane is not restricted and can be appropriately determined considering the amount of antigen in the solution, etc. For 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 appropriate amount can be 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 that antibody. After contact, drying may or may not be performed.

While not 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.

Furthermore, while the antibody-immobilized NC membrane of this disclosure 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 this is generally done to confirm whether or not the antigen is present in the solution, the solution does not necessarily need to contain the antigen.

The detection of antigens bound to antibodies immobilized on NC membranes can be performed 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 (e.g., gold colloid), biotin, avidin, streptavidin, PEG (polyethylene glycol) labels, polymers (e.g., colored latex) labels, 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-mediated immunoassay). 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.

The antibody-immobilized NC membrane itself has been conventionally used in rapid (simple) tests for influenza and other diseases. In such tests, typically, an antigen (sample) is first placed on the antibody-immobilized NC membrane in a location 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 suitably applied to such methods. Therefore, from the perspective of simplifying detection according to conventionally known rapid (simple) tests, it is preferable to label the antigen with a labeling substance or other substance that facilitates detection before binding to the antibody. Thus, the antibody and antigen immobilized on the NC membrane may be directly bound or indirectly bound, and preferably directly bound.

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 allows for the detection of antigens (measurement of antigen quantity) using an antibody-immobilized NC membrane.

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 the NC membrane in a more stable state, minimizing changes in the three-dimensional structure of the antibodies. From this viewpoint, it has been considered good practice to use a neutral solution for antibody immobilization on an NC membrane. Nevertheless, surprisingly, the inventors have found that antigen binding is improved in antibody-immobilized NC membranes when using an alkaline solution rather than a neutral solution. Therefore, this disclosure also provides a method for producing an antibody-immobilized NC membrane with enhanced antigen binding, comprising steps (1) and (2) described above.

In this disclosure, enhanced antigen binding means that even a smaller amount of antigen can be detected, i.e., it means increased sensitivity in antigen detection.

Furthermore, in conventional rapid (simple) tests such as POCT (Point-of-Care Testing) 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). If the sample is not liquid, it is usually suspended in a solvent as appropriate before being used. In this disclosure, the same method as before can be used, and a variety of samples can be targeted.

Therefore, the antibody-immobilized NC membrane of this disclosure can be widely used in diagnostics and analyses utilizing antigen-antibody reactions, and consequently, the antibody-immobilized NC membrane can be preferably used as part of a diagnostic kit or analytical kit. Furthermore, although not limiting to 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.

Method for enhancing antigen binding of antibody-immobilized nitrocellulose membranes As described above, the antibody-immobilized NC membrane of this disclosure has enhanced antigen binding. Therefore, this disclosure also includes a method for enhancing antigen binding of antibody-immobilized nitrocellulose membranes, comprising the following steps:
(a) a step of contacting an alkaline solution containing an antibody with a nitrocellulose membrane, and (b) a step of drying the nitrocellulose membrane that has been in contact with the alkaline solution in step (a) to obtain an antibody-immobilized nitrocellulose membrane.

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

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 in contact with the alkaline solution will be explained in the same manner as in step (2), and the obtaining of an antibody-immobilized nitrocellulose film by this process will also be explained in the same manner as in the "Method for Producing an Antibody-Immobilized Nitrocellulose Film." Furthermore, blocking treatment may be performed as described above if necessary.

The antigen-binding enhancement described herein is 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 described herein 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 described herein is greater than that 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 described herein can detect smaller amounts of antigen (has a lower detection limit) than the antibody-immobilized NC membrane produced using the neutral solution.

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. While the comparison is not limited as long as it can be compared as described above, typically, the antibody, antigen, contact, drying, and other conditions are substantially the same, except that antibody-immobilized nitrocellulose membranes manufactured using an alkaline solution and those manufactured using a neutral solution are used. Although this disclosure is not limiting, the comparison is preferably performed according to the procedures described in Test Example 1 and Test Example 2 below.

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.

Antibody Screening Method 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 membrane that was in contact with the alkaline solution in step (A) to obtain a candidate antibody-immobilized nitrocellulose membrane, 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.

As mentioned above, conventionally, NC membranes have not been considered highly versatile as antibody immobilization carriers, and their use has been very limited. However, the antibody-immobilized NC membrane obtained by the method disclosed herein allows for more sensitive antigen detection even when 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.

Thus, this 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. Therefore, this 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, this antibody-immobilized NC membrane can be said to be useful for screening antibodies that are useful when immobilized on an NC membrane.

The antibody screening method of this disclosure includes the step of (A) contacting an alkaline solution containing a candidate antibody with a nitrocellulose membrane.

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 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).

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.

Furthermore, the antibody screening method of this disclosure includes (C) a step of contacting a candidate antibody on the antibody-immobilized nitrocellulose membrane obtained in step (B) with an antigen. This contact is not limited as long as the antigen-binding properties 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 for the use of a candidate antibody.

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

Therefore, the screening method of this 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).

The reference level may be determined as appropriate and does not limit this disclosure. However, 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 immobilized on the 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. 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 immobilized on an NC membrane. 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 immobilized on an NC membrane.

Furthermore, as one embodiment of the screening method of this disclosure, the antigen-binding level of an antibody that is already known to be useless may be used as the reference level (antigen-binding level of the reference antibody) when immobilized on an NC membrane and brought into contact with an antigen. In this case, if the antigen-binding level (test level) of a candidate antibody 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 not useful as an antibody (particularly not useful 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 not useful as an antibody immobilized on an NC membrane).

The antigen-binding level refers to the level of binding (degree of binding) between the antibody (candidate antibody) immobilized on the NC membrane and the antigen, i.e., it represents antigen-binding ability. Therefore, the screening method of this disclosure may further include such a determination step.

Furthermore, since the aforementioned level represents antigen-binding activity, it can be detected (measured) according to conventionally known methods, as described above, and is explained in the same way as the detection of antigen bound to the antibody 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.

Furthermore, while the comparison is not limited to the extent that they are comparable, it is preferably exemplified that it is carried out under substantially identical conditions, except that the candidate antibody used to obtain the test level differs from the antibody used to obtain the reference level. Also, without limiting this disclosure, the comparison may be based on the presence or absence of detection (detection amount) or on the detection limit.

Thus, the antibody screening method described herein allows for the convenient screening of useful antibodies using antigen-binding activity as an indicator.

Conventionally, the antibodies used in NC membranes have been limited to certain full-length antibodies. Furthermore, although the mechanism is unknown, there has been a problem with relatively low detection sensitivity (antigen binding ability of the antibody) despite the antibodies being immobilized on the NC membrane. The cause of this is still unclear. Therefore, 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 the antigen.

Furthermore, while NC membranes with immobilized antibodies have traditionally 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 antigen detection sensitivity. Therefore, this disclosure has the advantage of enabling antigen detection even with smaller amounts of antibody. Moreover, 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.

In particular, the method disclosed herein allows for the active immobilization of antibody fragments, such as single-chain antibodies and single-domain antibodies, which were previously practically unusable in rapid tests, onto NC membranes without requiring additional modifications to the antibodies. This makes them usable in immunochromatography. This, for example, leads to a significant reduction in the cost of rapid tests such as POCT (Point-of-Care Testing) and greatly contributes to the widespread adoption of POCT. Furthermore, the widespread adoption of POCT is useful for strengthening disease control systems in emerging countries and other nations with insufficient medical facilities.

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.

Test Example 1
1-1) Manufacturing procedure for antibody-immobilized NC membrane (1) Each 100 mM pH buffer was prepared according to a conventional method (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, and 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.

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.

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.

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. Next, these NC membranes were washed five times with PBST (PBS containing 0.1% Tween 20). Each of the NC membranes obtained in this way was scanned with a scanner GT-X830 (manufactured by Epson Corporation). The images obtained by scanning are the right-hand photographs (adsorption) in Figures 1 and 2.
(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 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 GT-X830 scanner (manufactured by Epson Corporation).

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).

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, a greater number of darker spots were observed when using the alkaline solution (Example A (pH 9 or higher)) than when using the 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 compared to 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.

Test Example 2
2-1) Manufacturing procedure for antibody-immobilized NC membranes

[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 test example, a total of 12 anti-influenza A antibody Ant-NP-A VHH clones were used, along with VHH1 and VHH2 used in Test Example 1. Antibodies were mixed into pH 7 and pH 13 buffers, prepared in the same manner as in Table 1 of Test 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 to 12 (VHH1 to VHH12). Each VHH-fixed NC membrane prepared using pH 7 buffer was designated as Comparative Examples 1 to 12 (VHH1 to VHH12).

2-2) Procedure for contact with antigen and detection (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.

2-3) 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 shows that antibody-immobilized NC membranes obtained using an alkaline solution can improve antigen detection sensitivity.

Test Example 3
3-1) Procedure for manufacturing 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 test was performed 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.

3-2) Results are shown in Figure 5. Even when whole antibodies and scFv were used, 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.

Test Example 4
4-1) Procedure for manufacturing antibody-immobilized NC membrane (1) Each 100 mM pH buffer was prepared in the same manner as in Test Example 1, 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 film of an immunochromatography strip prepared in the same manner as in 2-1) of Test Example 2, and air-dried at room temperature. Samples with a pH of 9 or higher were designated as Example B, and samples with a pH of 8 or lower were designated as Comparative Example B.

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 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 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.

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 tendency was observed when VHH1 and scFv were used. From this, it was understood that detection sensitivity can be improved by using an alkaline solution when immobilizing antibodies on the NC membrane.

Test Example 5
5-1) Manufacturing Procedure for Antibody-Immobilized NC Membrane 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 100 μg/ml using the pH 13 buffer 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 two μl was added to the NC film.
(3) The operation in (2) above was repeated twice to deposit each VHH onto the NC film (VHH immobilization amount: 1.2 μg/each spot). The VHH immobilized NC films obtained in this way were designated as Examples 16 to 23.

5-2) Procedure for contact with antigen and detection (1) Inactivated influenza A virus was added to the sample suspension so that the amount was 54-0 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 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.

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 (6)

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 membrane 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 . The manufacturing method according to claim 1, wherein the pH of the alkaline solution is 9 to 14. A method for producing an antibody-immobilized nitrocellulose membrane with enhanced antigen-binding properties, as described in claim 1 or 2 . A method for enhancing the antigen-binding properties of an antibody-immobilized nitrocellulose membrane, comprising the following steps:
(a) a step of contacting an alkaline solution containing an antibody with a nitrocellulose membrane, and (b) a step of drying the nitrocellulose membrane that has been in contact with the alkaline solution in step (a) to obtain an antibody-immobilized nitrocellulose membrane .
Here, the antibody is at least one selected from the group consisting of VHH, scFv, sc(Fv) ₂ , Diabody, Fab, and F(ab') ₂ . 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 membrane that was in contact with the alkaline solution in step (A) to obtain a candidate antibody-immobilized nitrocellulose membrane, 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 . 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).