Method for producing a nitrocellulose membrane on which antibodies, etc., are immobilized via lactoferrin, concanavalin A, lysozyme, and / or hemoglobin, and method for enhancing antigen binding properties, etc.

Abstract

To provide a nitrocellulose membrane with enhanced activity based on an antibody, enzyme, receptor, and lectin, and a method of enhancing the activity of the nitrocellulose membrane.SOLUTION: A method of producing a first protein-immobilized nitrocellulose membrane is provided, comprising (1) bringing a solution containing a linked product of a first protein and a second protein into contact with a nitrocellulose membrane, and (2) drying the nitrocellulose membrane that has been contacted by the linked product in the step (1), where the first protein is at least one selected from a group consisting of an antibody, receptor, lectin (excluding concanavalin A), and enzyme, and the second protein is at least one selected form a group consisting of lactoferrin, concanavalin A, lysozyme, and hemoglobin.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing a nitrocellulose membrane on which an antibody or the like is immobilized via lactoferrin, concanavalin A, lysozyme and / or hemoglobin, and a method for enhancing antigen-binding ability and the like.

Background Art

[0002] Nitrocellulose (NC) membranes have been conventionally known as carriers for immobilizing antibodies, enzymes, etc. in various immunological techniques such as immunochromatography, ELISA (Enzyme-Linked Immunosorbent Assay), and Western blotting. For example, immunochromatography is also used in POCT tests (Point of Care Testing; on-site clinical immediate testing) such as influenza tests because the results of antigen-antibody reactions can be obtained relatively quickly and easily.

[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 using recombinant antibodies such as single-chain antibodies (scFv) and single-domain antibodies (VHH). Furthermore, it is known that the three-dimensional structure of antibodies is easily lost, and the activity of antibodies is easily lost 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. Also, this was a problem as it contributed to an increase in inspection costs (Non-Patent Document 1).

[0004] In the field of molecular immunology, not only antibodies but also enzymes, receptors, and lectins are used for specific detection of target substances and their activities. However, when using these in techniques using NC membranes, there was a problem that, similar to antibodies, their activities were easily lost on the NC membrane, and thus the detection sensitivity based on enzymes, receptors, and lectins was very low.

[0005] Thus, many aspects of the protein adsorption mechanism in NC membranes remain unclear, and the development of better adsorption methods is needed. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Naoto Shimetani, "Current Status and Future Challenges of POCT (Point of Care Testing)," Medical Engineering Vol. 80, No. 4, pp. 37-44 (2010) [Overview of the project] [Problems that the invention aims to solve]

[0007] The objective is to provide a nitrocellulose membrane with enhanced activity based on antibodies, enzymes, receptors, and lectins. Furthermore, the objective is to provide a method for enhancing such activity in a nitrocellulose membrane. [Means for solving the problem]

[0008] The present inventors have conducted extensive research on immunological methods using nitrocerillose (NC) membranes over a long period and have surprisingly discovered that by pre-binding at least one protein selected from the group consisting of lactoferrin, concanavalin A, lysozyme, and hemoglobin to an antibody, and then contacting the resulting binding product with the NC membrane, the detection sensitivity of antigens on the NC membrane can be increased. Similarly, they have found that by pre-binding the same protein to an enzyme, and then contacting the resulting binding product with the NC membrane, the detection sensitivity of enzyme activity on the NC membrane can be increased. The present invention was completed through further research based on these findings, and this disclosure includes, for example, the inventions described below. Section 1. A method for producing a first protein-immobilized nitrocellulose membrane, comprising the following steps: (1) A step of bringing a solution containing a linkage between the first protein and the second protein into contact with a nitrocellulose membrane, and (2) A step of drying the nitrocellulose film that has been in contact with the connecting material in step (1) above, Here, the first protein is at least one selected from the group consisting of antibodies, receptors, lectins (excluding concanavalin A), and enzymes. The second protein is at least one selected from the group consisting of lactoferrin, concanavalin A, lysozyme, and hemoglobin. Item 2. The method for producing the product according to Item 1, wherein the first protein is at least one selected from the group consisting of a whole antibody, scFv, VHH, Fab, F(ab)'2, and a bispecific antibody. Item 3. A method for producing a protein-immobilized nitrocellulose membrane in which the activity due to the first protein is enhanced, as described in item 1 or 2. Here, if the first protein is an antibody, the enhancement of its activity is an enhancement of its antigen-binding ability. If the first protein is a receptor, the enhancement of its activity is an enhancement of its binding to the agonist and / or antagonist. If the first protein is a lectin, the enhancement of its activity is an enhancement of its glycosylation ability. If the first protein is an enzyme, then the enhancement of its activity is an enhancement of the enzyme activity. Section 4. A method for enhancing the activity of a first protein-immobilized nitrocellulose membrane, comprising the following steps: (a) A step of bringing a solution containing a linkage between the first protein and the second protein into contact with a nitrocellulose membrane, and (i) A step of drying the nitrocellulose membrane that has been in contact with the connective material in step (a) above to obtain a first protein-immobilized nitrocellulose membrane. Here, the first protein is at least one selected from the group consisting of antibodies, receptors, lectins (excluding concanavalin A), and enzymes. The second protein is at least one selected from the group consisting of lactoferrin, concanavalin A, lysozyme, and hemoglobin. Here, if the first protein is an antibody, the enhancement of its activity is an enhancement of its antigen-binding ability. If the first protein is a receptor, the enhancement of its activity is an enhancement of its binding to the agonist and / or antagonist. If the first protein is a lectin, the enhancement of its activity is an enhancement of its glycosylation ability. If the first protein is an enzyme, then the enhancement of its activity is an enhancement of the enzyme activity. Item 5. A first protein-immobilized nitrocellulose membrane, in which the first protein linked to the second protein is immobilized on the nitrocellulose membrane via the second protein. Here, the first protein is at least one selected from the group consisting of antibodies, receptors, lectins (excluding concanavalin A), and enzymes. The second protein is at least one selected from the group consisting of lactoferrin, concanavalin A, lysozyme, and hemoglobin. [Effects of the Invention]

[0009] By pre-linking an antibody to at least one protein (second protein) selected from the group consisting of lactoferrin, concanavalin A, lysozyme, and hemoglobin, and then contacting the resulting linkage with a nitrocellulose membrane, the antibody can be immobilized on the nitrocellulose membrane via the second protein. This provides an antibody-immobilized nitrocellulose membrane with enhanced antigen-binding properties. Furthermore, by pre-linking an enzyme to a second protein and then contacting the resulting linkage with a nitrocellulose membrane, the enzyme can be immobilized on the nitrocellulose membrane via the second protein. This provides an enzyme-immobilized nitrocellulose membrane with enhanced enzyme activity. Additionally, by pre-linking a receptor to a second protein and then contacting the resulting linkage with a nitrocellulose membrane, the receptor can be immobilized on the nitrocellulose membrane via the second protein. This provides a receptor-immobilized nitrocellulose membrane with enhanced binding to agonists and / or antagonists, which is an activity attributed to the receptor. Furthermore, by pre-linking the second protein and the lectin, and then contacting the resulting linkage with a nitrocellulose membrane, the lectin can be immobilized on the nitrocellulose membrane via the second protein. This makes it possible to provide a lectin-immobilized nitrocellulose membrane with enhanced glycosylation activity, which is an activity derived from the lectin. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows the antigen-binding properties of an antibody immobilized on an NC membrane without being linked to the second protein. [Figure 2] Figure 2 shows the antigen-binding properties when the antibody is immobilized on the NC membrane by linking it to the second protein (concanavalin A). [Figure 3] Figure 3 shows the antigen-binding properties when an antibody is immobilized on an NC membrane by linking it to the second protein (lactoferrin). [Figure 4]FIG. 4 is a diagram showing that when a conjugate of a second protein and an antibody is immobilized on a NC membrane, the antigen-binding property is improved compared to the case where the antibody (unconjugated) is immobilized on the NC membrane without being conjugated to the second protein. [Figure 5] FIG. 5 is a diagram showing that when a conjugate of a second protein and an antibody is immobilized on a NC membrane, the antigen-binding property is improved compared to the case where the antibody (unconjugated) is immobilized on the NC membrane. [Figure 6] FIG. 6 is a diagram showing that when a conjugate of a second protein and an antibody is immobilized on a NC membrane, the antigen-binding property is improved compared to the case where the antibody (unconjugated) is immobilized on the NC membrane. [Figure 7] FIG. 7 is a diagram showing that when a conjugate of a second protein and an antibody is immobilized on a NC membrane, the antigen-binding property is improved compared to the case where the antibody (unconjugated) is immobilized on the NC membrane. [Figure 8] FIG. 8 is a diagram showing that when a conjugate of a second protein and an enzyme is immobilized on a NC membrane, the enzyme activity is improved compared to the case where the enzyme (unconjugated) is immobilized on the NC membrane.

Mode for Carrying Out the Invention

[0011] Hereinafter, 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".

[0012] Method for manufacturing a protein-immobilized nitrocellulose membrane (Type 1) The present disclosure provides a method for producing a first protein-immobilized nitrocellulose (NC) membrane, which includes the following steps: (1) A step of contacting a solution containing a conjugate of a first protein and a second protein with a NC membrane, and (2) A step of drying the NC membrane contacted with the conjugate in the step (1). Here, the first protein is at least one selected from the group consisting of an antibody, a receptor, a lectin (excluding concanavalin A), and an enzyme. The second protein is at least one selected from the group consisting of lactoferrin, concanavalin A, lysozyme, and hemoglobin.

[0013] Process (1) The method for producing a first protein-immobilized NC membrane according to this disclosure includes the step of (1) bringing a solution containing a linkage between a first protein and a second protein into contact with an NC membrane. In this application, the first protein-immobilized NC membrane means an NC membrane on which the first protein is immobilized.

[0014] The nitrocellulose membrane is not limited as long as it is one that has been conventionally used as a support for immobilizing antibodies, enzymes, etc., in immunological techniques such as immunochromatography. In this respect, the thickness and shape of the NC membrane are also not limited. Although not limiting to this disclosure, the thickness of the NC membrane can be, for example, about 80 to 500 μm, preferably about 80 to 400 μm. Also, although not limiting to this disclosure, the average pore size of the NC membrane can be, for example, about 0.1 to 5 μm, preferably about 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) and Amersham Protran (manufactured by Cytiva).

[0015] In this disclosure, the first protein is at least one selected from the group consisting of antibodies, receptors, lectins, and enzymes. The first protein is different from the second protein. Also, in this disclosure, concanavalin A is not included as a lectin.

[0016] In this disclosure, the antibody (first protein) immobilized on the NC membrane is not limited and may be any single-chain antibody (scFv), single-domain antibody (VHH), full-length antibody (whole antibody), Fab, F(ab')2, bispecific antibody, etc. While not limiting to this disclosure, single-chain antibodies and single-domain antibodies are preferred examples of antibodies. These may be used individually or in combination of two or more.

[0017] Antibodies may or may not be modified by fluorescent labeling, enzyme labeling, metal labeling (such as metal colloids), biotin, streptavidin, secondary antibodies, latex (such as colored latex), etc., to the extent that they do not interfere with the effects of this disclosure. In conventional rapid testing methods such as influenza testing, antibodies are often immobilized on NC membranes without modification, and from this viewpoint, and also from the standpoint of simplicity, the antibodies may be immobilized on NC membranes without modification. The same applies to other first proteins below.

[0018] The antibody (protein 1) may be any antibody against viruses or bacteria such as anti-influenza antibodies (type A, type B, etc.), anti-norovirus antibodies, anti-human chorionic gonadotropin antibodies, anti-Helicobacter pylori antibodies, anti-SARS antibodies (-CoV-2 antibodies, etc.), anti-E. coli O-157 antibodies, anti-rotavirus antibodies, anti-adenovirus antibodies, anti-cytomegalovirus antibodies, anti-Legionella antibodies, etc. 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 type B antibodies, etc.). These may be used individually or in combination of two or more.

[0019] In this disclosure, the receptor (first protein) immobilized on the NC membrane is not limited as long as it is a receptor usable in the field of molecular immunology. The receptor is also called a receptor, and the recognized substance (the substance recognized by the receptor) that the receptor recognizes and can specifically bind to directly to form a complex is conventionally known. The recognized substance may be an agonist or an antagonist to the receptor.

[0020] Examples of receptors include T cell receptors, hormone receptors, and cytokine receptors, although these do not limit the scope of this disclosure.

[0021] T cell receptors are antigen receptor molecules expressed on the cell membrane of T cells and are conventionally known as receptors that specifically recognize and bind to antigen molecules bound to MHC molecules. T cell receptors are usually heterodimeric molecules that recognize antigens through the association of α and β chains, but single-chain T cell receptors (single-chain TCRs), in which the extracellular domains of the α and β chains are fused with a flexible linker (Gly4Ser)3, are also known to have antigen specificity equivalent to that of the aforementioned heterodimeric molecules. T cell receptors, and single-chain T cell receptors, are proteins belonging to the immunoglobulin superfamily, just like the variable region (Fv) of antibodies and single-chain antibodies (scFv), and form a β-sheet-rich three-dimensional structure.

[0022] Therefore, in this disclosure, a heterodimer T cell receptor may be used as the T cell receptor, or a single-stranded T cell receptor may be used. Although not limited to this disclosure, a single-stranded T cell receptor is preferred as an example.

[0023] The target of a T cell receptor is not limited as long as the T cell receptor can recognize and specifically bind to it. Many target substances that T cell receptors specifically recognize and bind to are known, and examples include target antigens of various diseases, including cancer and infectious diseases. In this disclosure, the target substance can be appropriately selected according to the combination of conventionally known T cell receptors and their target substances. As an example, T cell receptors that can specifically bind to the HER2 antigen found in breast cancer, the MAFE-A4 antigen found in esophageal cancer, head and neck cancer, ovarian cancer, melanoma, etc., and the NY-ESO-1 antigen found in osteosarcoma, esophageal cancer, ovarian cancer, melanoma, multiple myeloma, head and neck cancer, etc. are exemplified.

[0024] Furthermore, although not limiting to this disclosure, examples of substances recognized by hormone receptors include thyroid hormones, pituitary hormones, adrenocortical hormones, pancreatic hormones, gastrointestinal hormones, and gonadal hormones. Although not limiting to this disclosure, examples of thyroid hormones include thyroxine, triiodothyronine, and thyroglobulin. Examples of pituitary hormones include gonadotropins (luteinizing hormone, follicle-stimulating hormone), growth hormone, adrenocorticotropic hormone, thyroid-stimulating hormone, and prolactin (lactogenic hormone). Examples of adrenocortical hormones include cortisol and aldosterone. Examples of pancreatic hormones and gastrointestinal hormones include insulin, C-peptide, glucagon, and gastrin. Examples of gonadal hormones include hCG (human chorionic gonadotropin), hPL (human placental lactogen), estrogen, and testosterone. Other examples of hormones include calcitonin, parathyroid hormone (PTH), renin, angiotensin I, angiotensin II, enkephalin, erythropoietin, and somatostatin. Therefore, in this disclosure, the receptors for each of these hormones are exemplified as hormone receptors.

[0025] Without limiting this disclosure, examples of substances recognized by cytokine receptors include interferons, interleukins, tumor necrosis factor, colony-stimulating factors (such as erythropoietin), and cell growth factors (such as epidermal growth factor, fibroblast growth factor, and tumor growth factor). Therefore, in this disclosure, cytokine receptors are exemplified by receptors for each of these cytokines.

[0026] While not limiting this disclosure, examples of receptors include, but are not limited to, T cell receptors, and more preferably, single T cell receptors. Receptors may be used individually or in combination of two or more types.

[0027] In this disclosure, the lectin (protein 1) immobilized on the NC membrane is not limited to any lectin available in the field of molecular immunology. Lectins, also known as agglutinins, are proteins that specifically bind to certain glycans. Recognized substances (sequences or structures of glycans to which lectins specifically bind; glycan-holding substances) that lectins can recognize and specifically bind to directly to form complexes are well known.

[0028] Although not limiting the present disclosure, lectins include DBA (Dolichos biflorus Agglutinin), PHA-E (Phaseolus vulgaris Erythroagglutinin), PHA-L (Phaseolus vulgaris Leucoagglutinin), PNA (Peanut Agglutinin Arachis hypogaea peanuts), LCA (Lens culinaris Agglutinin), PSA (Pisum sativum Agglutinin), AAL (Aleuria aurantia Lectin), Lotus (LTL, Lotus tetragolonobus Lectin), WGA (Wheat Germ Agglutinin Triticum vulgaris), SSA (Sambucus sieboldiana agglutinin), MAM (Maackia amurensis), ABA (Agaricus bisporus), ECA (ECL, Erythrina) Examples of lectins include cristagalli lectin, PHA-E4 (Phaseolus vulgaris), PHA-P (Phaseolus vulgaris lectin), SBA (Glycine max lectin), UEA-I (Ulex europaeus lectin), DSA (Datura stramonium lectin), and PWM (Phytolacca Americana lectin). While not limiting this disclosure, DBA, PHA-E, PHA-L, PNA, LCA, PSA, AAL, Lotus, WGA, SSA, MAM, etc., are preferred examples of lectins. Lectins may be used individually or in combination of two or more. Lectins are commercially available from companies such as J-Chemical Co., Ltd. Although concanavalin A is considered a type of lectin, it is not included in the definition of lectins in this disclosure.

[0029] In this disclosure, the enzyme (protein 1) immobilized on the NC membrane is not limited, and examples of enzymes usable in the field of molecular immunology are provided. While not limiting to this disclosure, preferred examples of enzymes include digestive enzymes such as horseradish peroxidase (HRP), alkaline phosphatase, amylase, cellulase, trypsin, and chymotrypsin. The enzyme may be used individually or in combination of two or more.

[0030] Thus, each of the first proteins may be used individually or in combination of two or more.

[0031] In this disclosure, the second protein is at least one selected from the group consisting of lactoferrin, concanavalin A, lysozyme, and hemoglobin.

[0032] Lactoferrin is a conventionally known iron-binding glycoprotein with a molecular weight of approximately 80 kDa, and is known to be present in the milk, tears, saliva, and blood of mammals. In this disclosure, lactoferrin may be of animal origin or may be produced using genetic engineering techniques. Examples of mammals include humans, cattle, sheep, goats, pigs, mice, buffalo, camels, yaks, horses, donkeys, and llamas. In this disclosure, lactoferrin may be of any of these origins. In this disclosure, examples of lactoferrin include natural lactoferrin, free-form (apo-type) lactoferrin obtained by removing iron from lactoferrin by conventional methods, metal-bound lactoferrin obtained by partially chelating apolactoferrin with metals such as iron, copper, zinc, and manganese, metal-saturated (holo-type) lactoferrin obtained by completely chelating such metals, and fragments thereof. Lactoferrin is not limited to these examples, but for example, natural-form lactoferrin derived from cattle is preferably exemplified. The PDB number for lactoferrin is 1BLF. Thus, as long as the effects of this disclosure are obtained, the molecular weight of lactoferrin can be limited to approximately 20-80 kDa, 30-80 kDa, 40-80 kDa, 50-80 kDa, 60-80 kDa, 70-80 kDa, etc. Lactoferrin is commercially available, and examples include bovine-derived natural lactoferrin, trade name 129-04121, Lactoferrin from Bovine Milk (CAS number: 146897-68-9, lactoferrin, derived from milk, Wako Pure Chemical Industries, Ltd.). Lactoferrin may be used alone or in combination of two or more types.

[0033] Concanavalin A is conventionally known as a protein derived from sword bean. Concanavalin A is a dimeric protein with a molecular weight (dimer) of approximately 52 kDa. Concanavalin A may be of the natural form or may be manufactured (including fragments) using genetic engineering methods as described above. The PDB number for concanavalin A is 1JBC. As long as the effects of this disclosure are obtained, examples of molecular weights for concanavalin A include approximately 15-52 kDa, 25-52 kDa, 30-52 kDa, 35-52 kDa, 40-52 kDa, and 45-52 kDa. Concanavalin A is commercially available, for example, under the trade name L7647-250G, Concanavalin A Canavalia ensiformis (derived from Canavalia cordata) Type VI, lyophilized powder (CAS number: 11028-71-0, EC number: 234-258-2, Sigma-Aldrich). Concanavalin A may be used alone or in combination of two or more types.

[0034] Lysozyme is a carbohydrate hydrolase (EC number 3.2.1.17) with a molecular weight of approximately 15 kDa, and is a known protein that hydrolyzes the β-(1→4) glycosidic bond formed between N-acetylmuramic acid and N-acetylglucosamine, which constitute peptidoglycan. The PDB number for lysozyme is 1DPX. Lysozyme may be in its natural form or manufactured (including fragments) using genetic engineering techniques as described above. As long as the effects of this disclosure are obtained, examples of lysozyme's molecular weight include approximately 8-15 kDa, 10-15 kDa, 12-15 kDa, 13-15 kDa, and 14-15 kDa. Lysozyme is commercially available, for example, trade name 122-02673, Lysozyme, from Egg White (CAS number: 12650-88-3, lysozyme, derived from egg white, Wako Pure Chemical Industries, Ltd.). Lysozyme may be used alone or in combination of two or more types.

[0035] Hemoglobin is a red pigment protein found in red blood cells, with a molecular weight of approximately 65 kDa. The PDB number for hemoglobin is 2QSP. Hemoglobin may be of the natural form or may be manufactured (including fragments) using genetic engineering methods as described above. As long as the effects of this disclosure are obtained, examples of hemoglobin molecular weights include approximately 20-65 kDa, 30-65 kDa, 40-65 kDa, 50-65 kDa, and 60-65 kDa. Hemoglobin is commercially available, for example, trade name 081-03492, Hemoglobin, from Bovine (CAS number: 9008-02-0, hemoglobin, bovine, Wako Pure Chemical Industries, Ltd.). Hemoglobin may be used alone or in combination of two or more types.

[0036] While not limiting this disclosure, examples of preferred secondary proteins include lactoferrin and concanavalin A. The secondary protein may be used alone or in combination of the two above.

[0037] The linkage between the first protein and the second protein is obtained by linking the first protein and the second protein, and is not limited to this extent.

[0038] When an antibody is used as the first protein, the second protein is linked to a region of the antibody that does not affect its antigen-binding ability. Similarly, when a receptor is used as the first protein, the second protein is linked to a region of the receptor that does not affect its binding ability to agonists and / or antagonists. Furthermore, when a lectin is used as the first protein, the second protein is linked to a region of the lectin that does not affect its glycosylation ability. Finally, when an enzyme is used as the first protein, the second protein is linked to a region that does not affect its enzyme activity. These sites are conventionally known.

[0039] Examples of conventionally known methods that do not affect the activity include amine coupling, thiol coupling, gene fusion, streptavidin-biotin interaction, charged polymer method, and DNA hybridization. The disclosure is not limited to these methods, but preferably examples include amine coupling, thiol coupling, and gene fusion. This links the first protein and the second protein. The first protein and the second protein may be directly linked or indirectly linked, as long as they are linked, and it is sufficient that they form a linked product together when in contact with the NC membrane.

[0040] Without limiting the scope of this disclosure, one example is that the groups may be linked via a primary amine group (-NH2), a thiol group, a disulfide bond, etc., by an amine coupling method or a thiol coupling method, and conventionally known crosslinking agents may be used as appropriate. For example, examples of amine coupling agents include EDC (ethyldimethylaminopropylcarbodiimide) / NHS (N-hydroxysuccinimide) mixture, glutaraldehyde, and DSP (Dithiobis(succinimidyl propionate)). For example, examples of thiol coupling agents include combinations such as SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate) / iminothiolane (hydrochloride), SPDP (N-Succinimidyl 3-(2-pyridyldithio)propionate) / iminothiolane (hydrochloride), and SMCC / iminothiolane (hydrochloride). Instead of SMCC, EMCS (N-(6-Maleimidocaproyloxy)succinimide), GMCS (N-(4-Maleimidobutyryloxy)succinimide), etc. may be used.

[0041] Furthermore, although this disclosure does not limit our scope, examples of gene fusion methods include a method of synthesizing the genes for the first and second proteins in a fused form, a method of linking them by ligation and transfecting them into cells to create a fusion protein in which the first and second proteins are linked as a single polypeptide.

[0042] The content of the first protein and the second protein in the linked product of the first protein and the second protein is not particularly limited. Without limiting this disclosure, for example, the content of the first protein (total amount) and the second protein (total amount) in the linked product may be exemplified by a mass ratio of 1 to 3, more preferably 1 to 2, and even more preferably 1 to 1.5, of the second protein (total amount) per 1 unit of the first protein (total amount) in the linked product.

[0043] In this disclosure, the second protein may be linked to only one selected from the group consisting of antibodies, receptors, lectins, and enzymes, or to two or more, three or more, or all four. Preferably, only one or two selected from the group consisting of antibodies, receptors, lectins, and enzymes are linked to the second protein. Furthermore, a second protein linked to an antibody, a second protein linked to a receptor, a second protein linked to a lectin, and a second protein linked to an enzyme may be used in any combination.

[0044] Contact between the connector and the NC membrane is achieved by bringing a solution containing the connector into contact with the nitrocellulose membrane.

[0045] The solution containing the connecting material (hereinafter sometimes referred to as the NC film contact solution) is a solution used when bringing the connecting material into contact with the NC film, and is not limited as long as the connecting material is fixed to the NC film after contact and drying, as long as it does not interfere with the effects of this disclosure.

[0046] The pH of the NC membrane contact solution is not limited as long as the effects of this disclosure are obtained, but preferably a pH of 1 to 14 is exemplified, and more preferably a pH of 5 to 13.5 is exemplified. Furthermore, the pH may be any of 1 to 7, 2 to 6, 7 to 13, 10 to 13, 11 to 13, etc. Thus, the pH can be appropriately determined within the range of pH 1 to 14. The pH is the value measured at 25°C using a pH meter (product name LAQUA, manufactured by Horiba, Ltd.), and is the pH of the solution after mixing with the binder, and represents the pH at the time of contact with the NC membrane.

[0047] Furthermore, although this disclosure does not limit the NC membrane contact solution, conventionally known buffers may be used, and as an example of a buffer, a buffer prepared by conventional methods using various conventionally known Good's buffers is provided. Without limiting this disclosure, the following buffers may be used: MES(2-(N-morpholino)ethanesulfonic acid), Bis-Tris(Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane), ADA(N-(2-Acetamido)iminodiacetic acid), PIPES(piperazine-1,4-bis(2-ethanesulfonic acid)), ACES(N-(2-Acetamido)-2-aminoethanesulfonic acid), MOPSO(2-Hydroxy-3-morpholinopropanesulfonic acid) collamine salt, BES(N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), TES(N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) acid), DlPSO(3-[N,N-Bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), TAPSO(2-Hydroxy-N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), POPSO(Piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) HEPPSO(2-Hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid,Examples include monohydrate, EPPS (3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid), acetamidoglycine, tricine, glycinamide, pidine, 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.

[0048] Without limiting this disclosure, to give general examples, a buffer using ADA, PIPES, or POPSO as the buffering agent is a buffer prepared by mixing and dissolving the buffer with water and sodium hydroxide, and then adjusting the pH with sodium hydroxide. A buffer using Bis-Tris as the buffering agent is a buffer prepared by dissolving the buffer in water and adjusting the pH with hydrochloric acid. Other buffers specifically listed above are buffers prepared by dissolving each buffer in water and adjusting the pH with sodium hydroxide.

[0049] In addition to these, other known solutions for NC membrane contact include glycine-HCl buffer (approximate pH 1-3), acetate buffer (approximate pH 4), MES-NaOH buffer (approximate pH 5-6), Tris-HCl buffer (approximate pH 7-8), glycine-NaOH buffer (approximate pH 9-12), potassium chloride-NaOH buffer (approximate pH 13), and other known solutions such as aqueous sodium hydroxide solution, aqueous potassium hydroxide solution, and conventionally known phosphate-buffered saline.

[0050] Without limiting this disclosure, to give some general examples, glycine-hydrochloride buffer is a buffer prepared by dissolving glycine in water and adjusting the pH with hydrochloric acid. Acetate buffer is a buffer prepared by dissolving sodium acetate and acetic acid. MES-sodium hydroxide buffer is a buffer prepared by dissolving MES in water and adjusting the pH with sodium hydroxide. Tris-hydrochloride buffer is a buffer prepared by dissolving Tris in water and adjusting the pH with hydrochloric acid. Glycine-sodium hydroxide buffer is a buffer prepared by dissolving glycine in water and adjusting the pH with sodium hydroxide. Potassium chloride-sodium hydroxide buffer is a buffer prepared by mixing hydrochloric acid and sodium hydroxide in water. Sodium hydroxide aqueous solution is a buffer prepared by mixing sodium hydroxide and water. Potassium hydroxide aqueous solution is a buffer prepared by mixing potassium hydroxide and water. Phosphate buffer solution (PBS) can be used by adjusting the pH as appropriate according to generally known compositions.

[0051] As exemplified above, the NC membrane contact solution in this disclosure is prepared by mixing water and a buffer, etc., and adjusting the pH to a desired value, according to a conventionally known procedure. Although not limiting to this disclosure, the concentration of the buffer component in the NC membrane contact solution is typically 10 to 200 mM, preferably 10 to 100 mM, and more preferably 10 to 50 mM.

[0052] The solution may be used alone or in combination of two or more types.

[0053] Furthermore, although not limiting to this disclosure, the NC membrane contact solution may or may not contain conventional additives used for protein immobilization, such as monosaccharides and disaccharides like trehalose, mannitol, and sorbitol; ionic surfactants (such as sodium deoxycholate and sodium dodecyl sulfate (SDS)); amphoteric surfactants (such as CHAPS (3-(3-cholamidepropyl)dimethylammonio-1-propanesulpHonate)); and nonionic surfactants (such as Triton X-100 and Tween 20), as long as they do not impair the effects of this disclosure. If added, these may be used individually or in combination of two or more. The amount added may also be determined appropriately according to conventional procedures, such as within ranges of 0-5 w / v%, 0.0001-3 w / v%, 0.01-1 w / v%, 0.01-0.5 w / v%, etc., depending on the type of additive. Without limiting this disclosure, the NC membrane contact solution may contain electrolytes such as sodium chloride and potassium chloride, to the extent that they do not impair the effects of this disclosure. The amount of electrolytes is not limited, but examples include ranges of 0 to 500 mM and 0 to 300 mM.

[0054] The amount of the binder in the NC membrane contact solution containing the binder is not limited, as long as the effects of this disclosure are obtained. For example, the content of the first protein (total amount) in the solution is exemplified as 5 μg / ml or more, more preferably 10 μg / ml to 10 mg / ml, even more preferably 100 μg / ml to 5 mg / ml, and particularly preferably 500 μg / ml to 5 mg / ml.

[0055] In this disclosure, since antigens can be efficiently bound by using an NC membrane immobilized with antibodies, the antibody concentration in the solution may be relatively low, such as 10 μg / ml or less. Similarly, in this disclosure, since the activity of receptors can be efficiently detected by using an NC membrane immobilized with receptors, the receptor concentration in the solution may be relatively low, such as 10 μg / ml or less. Similarly, in this disclosure, since the activity of lectins can be efficiently detected by using an NC membrane immobilized with lectins, the lectin concentration in the solution may be relatively low, such as 10 μg / ml or less. Similarly, in this disclosure, since enzyme activity can be efficiently detected by using an NC membrane immobilized with enzymes, the enzyme concentration in the solution may be relatively low, such as 10 μg / ml or less.

[0056] The contact between the NC membrane contact solution containing the linking material and the NC membrane is not limited to any means as long as contact is achieved. This contact may be, for example, by dropping the solution onto the NC membrane, immersing the NC membrane in the solution, inkjet coating, capillary coating (coating, etc.), capillary coating (coating, etc.), etc. Furthermore, the contact may be performed on the entire NC membrane or only on a part of it, and should be appropriately determined depending on the location on the NC membrane where activity attributable to the first protein is to be displayed.

[0057] The contact temperature is not restricted, and the procedure can be carried out at room temperature (25°C) due to its simplicity. Furthermore, the amount of the NC membrane contact solution containing the binder that comes into contact with the NC membrane can be set as appropriate. This disclosure is not limited, and depends on the amount of the first protein in the solution, but for example, when the NC membrane contact 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 by capillary coating, the amount of the NC membrane contact solution 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.

[0058] Process (2) The first method for producing a protein-immobilized nitrocellulose membrane according to the present disclosure includes (2) a step of drying the nitrocellulose membrane that has been in contact with the connective in step (1).

[0059] Drying may be done 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 the first protein immobilized (first protein immobilized NC membrane) is obtained. Furthermore, according to the manufacturing method of this disclosure, the first protein linked to the second protein is immobilized on the NC membrane via the second protein. The first protein immobilized NC membrane is obtained in this way. From this, it can be said that in this disclosure, the second protein is substantially directly immobilized on the NC membrane by adsorption (adsorption immobilization). Furthermore, from this, it can be said that this disclosure provides a first protein immobilized NC membrane in which the first protein linked to the second protein is immobilized on the NC membrane via the second protein.

[0060] As will be explained in the examples described later, the inventors diligently investigated means to improve the antigen-binding properties of antibodies while using an NC membrane, and as an example, they also investigated various proteins. As a result, they found that the activity was improved when the second protein was used. This is thought to be because the second protein adsorbs to the NC membrane preferentially over the first protein, such as antibodies and enzymes, and as a result, the adsorption of the first protein to the NC membrane is inhibited, and its activity is efficiently maintained.

[0061] In this disclosure, confirmation that the first protein linked to the second protein is immobilized on the NC membrane via the second protein can be performed by cleaving the linkage between the first and second proteins with a reducing agent or endoprotease, obtaining the protein extracted from within the membrane using an immunological method such as Western blotting, and confirming its type (the second protein).

[0062] In the first protein-immobilized NC membrane of this disclosure, as described above, the first protein linked to the second protein is immobilized on the NC membrane via the second protein. However, this does not mean that there are no first proteins immobilized on the NC membrane without the second protein; there may be first proteins immobilized on the NC membrane without the second protein. This disclosure can be said to enhance the activity of the first protein by positioning the first protein immobilized on the NC membrane via the second protein.

[0063] The first protein immobilized NC membrane of this disclosure can be used as appropriate depending on the characteristics of the first protein immobilized on the NC membrane.

[0064] Furthermore, the first protein-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, by using a neutral buffer containing approximately 0.1-5% BSA (Bovine Serum Albumin) or casein.

[0065] When the first protein is an antibody, the antibody-immobilized NC membrane of this 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 is in contact with the antigen. Preferably, the antigen is an antigen corresponding to the antibody.

[0066] When the first protein is a receptor, the antibody-immobilized NC membrane of this disclosure is used by contacting it with at least one selected from agonists and antagonists. Such contact is not limited to the extent that the receptor immobilized on the NC membrane, the antigen, and at least one selected from agonists and antagonists are in contact.

[0067] When the first protein is a lectin, the antibody-immobilized NC membrane of this disclosure is used by contacting it with a glycan-holding substance. This contact is not limited to the lectin immobilized on the NC membrane and the glycan-holding substance.

[0068] When the first protein is an enzyme, the antibody-immobilized NC membrane of this disclosure is used by contacting it with a substrate. This contact is not limited to the enzyme immobilized on the NC membrane and the substrate.

[0069] In this disclosure, the product is typically used in contact with at least one substance selected from the group consisting of antigens, agonists, antagonists, glycan-sequence-holding substances, and substrates (hereinafter sometimes referred to as the "recognized substance"). However, a sample in which the presence of the recognized substance is suspected may be applied to the NC membrane, and the sample may be brought into contact with the first protein immobilized on the NC membrane. In such a case, since the purpose is generally to confirm whether or not the recognized substance is present in the sample, the sample does not necessarily need to contain the recognized substance.

[0070] The contact may be carried out in any procedure as long as it allows contact between the first protein and the substance to be recognized. For example, the contact may be performed by dropping a solution containing the substance to be recognized (including a sample in which the presence of the substance to be recognized is suspected, the same applies hereinafter) onto the first protein immobilized NC membrane; immersing the first protein immobilized NC membrane in a solution containing the substance to be recognized; or bringing a solution containing the substance to be recognized into contact with a portion of the first protein immobilized NC membrane, allowing the substance to penetrate from the contact area and come into contact with the first protein immobilized on the NC membrane; or by inkjet supply (coating, etc.); capillary supply (coating, etc.), etc., and should be carried out in accordance with conventional immunological methods. Furthermore, the contact may be performed on the entire first protein immobilized NC membrane or only on a portion thereof, and should be appropriately determined depending on the location on the NC membrane where the first protein to be reacted is to be desired.

[0071] Because the contact is simple, it can be carried out at room temperature (25°C). Furthermore, as long as the effects of this disclosure are obtained, the amount of solution containing the substance to be recognized that comes into contact with the NC membrane is not limited. Although not limiting to this disclosure, for example, when using an immunochromatography strip as shown in the examples below, the amount of contact with the NC membrane 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 determined appropriately by referring to these values. By making contact in this way, the first protein immobilized on the NC membrane can be bound to the substance to be recognized. After contact, drying may or may not be performed.

[0072] Without limiting this disclosure, the amount of the substance to be recognized may be adjusted. For example, if the first protein is an antibody, the amount of antigen in the solution may be 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 this value if necessary. When using other first proteins, the amount of the substance to be recognized may be determined appropriately, taking into consideration this value and conventional procedures.

[0073] The detection of the recognized substance in the NC membrane, i.e., the detection of the activity, may be carried out according to conventionally known procedures. When the first protein is an antigen, although this disclosure is not limited, the detection of the antigen (antigen-binding ability) may be carried out according to conventionally known antigen-antibody reaction detection procedures using, for example, a labeling substance such as a fluorescent substance, an enzyme, a metal (such as gold colloid), biotin, avidin, streptavidin, PEG (polyethylene glycol) label, or a polymer (such as colored latex) label, or a secondary antibody. The substance may be used alone or in combination of two or more.

[0074] Similarly, when the first protein is a receptor, the detection of agonists and / or antagonists (binding) can be performed using the various substances mentioned above as needed, following conventionally known detection procedures.

[0075] Similarly, when the first protein is a lectin, the detection of glycan-holding substances (glycan-binding properties) can be performed using the various substances mentioned above as needed, following conventionally known detection procedures.

[0076] Similarly, if the first protein is an enzyme, the detection of enzyme activity can be carried out according to conventionally known methods. For example, although not limiting this disclosure, enzyme activity can be detected by reacting it with a substrate appropriate to the enzyme. Although not limiting this disclosure, when HRP is used as the enzyme, examples of substrates include TMB (3,3',5,5'-tetramethylbenzidine), luminol, and DAB (3,3'-diaminobenzidine tetrahydrochloride). Also, when amylase, cellulase, trypsin, chymotrypsin, etc. are used as enzymes, substrates that react specifically with each enzyme are known. Furthermore, enzyme activity may be detected using commercially available enzyme activity kits, etc. This disclosure does not limit the kits, but for example, for amylase, there are "α-Amylase Analysis Kit (Ceralpha method)" (Amylase HR reagent (blocked p-nitrophenyl-maltoheptaoside (BPNPG7))), "α-Amylase SD Analysis Kit (High Sensitivity Method)" (4,6-O-ethylidene-α-4-nitrophenyl-maltoheptaoside (EtPNPG7), etc.), "β-Amylase Analysis Kit (Betamyl-3 method)" (all from Nippon Biocon Co., Ltd.), and for cellulase, there are "Cellulase Analysis Kit (CellG5 method)" (CellG5 reagent (4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-cellopentaoside (BPNPG5)), etc.), "Cellulase Analysis Kit (CellG3 method)" Examples include the CellG3 reagent (benzylidene group-blocked 2-chloro-4-nitrophenyl-β-D-serotrioside (BCNPG3)), etc. (both from Nippon Biocon Co., Ltd.), the Trypsin Activity Assay Kit (BioVision), and the Chymotrypsin Activity Assay Kit (Sigma-Aldrich).

[0077] In this disclosure, the detection of activity includes the meaning of measurement, and if necessary, the activity may be measured according to conventional methods. Detection (measurement) can be performed using the various substances mentioned above as needed, and using detection (measurement) devices such as visual observation, fluorescence microscopy, spectrophotometer, immunochromatograph, fluorescence scanner, luminometer, etc.

[0078] Furthermore, the first protein-immobilized NC membrane obtained by the method disclosed herein can be used in techniques that utilize NC membranes, such as immunochromatography, ELISA (enzyme-mediated immunoassay (direct, indirect, sandwich, competitive)), fluorescence immunoassay, and radioimmunoassay.

[0079] In particular, when the first protein is an antibody, the increased sensitivity in immunochromatography is useful for the development of POCT (Point-of-Cognitive Testing) and other applications. Furthermore, when the first protein is a lectin or receptor, it becomes possible to concentrate a variety of biomolecules on the membrane that are inherently difficult to separate and recover with antibodies, and its use in protein chips and affinity separation membranes can be expected. In addition, when the first protein is an enzyme, the labeled enzyme-immobilized NC membrane of this disclosure is also useful, for example, as an immobilized enzyme separation membrane for biomass and food processing.

[0080] As can be seen from the examples described later, when an antibody-immobilized NC membrane obtained by contacting an antibody linked to the second protein with the NC membrane was used, antigen detection was possible with higher sensitivity than when an antibody-immobilized NC membrane obtained by contacting an antibody not linked to the second protein (hereinafter referred to as unlinked) with the NC membrane was used. Surprisingly, the inventors have found that the antigen-binding ability of the antibody-immobilized NC membrane is enhanced by the use of the second protein. Antigen-binding ability is an activity attributable to the antibody.

[0081] Similarly, when an enzyme was used as the first protein, enzyme activity could be detected with higher sensitivity when an enzyme-immobilized NC membrane obtained by contacting an enzyme linked to the second protein with the NC membrane was used compared to when an enzyme-immobilized NC membrane obtained by contacting an unlinked enzyme with the NC membrane was used. Enzyme activity is the activity attributable to the immobilized enzyme.

[0082] Furthermore, since the detection sensitivity in the NC membrane improved when antibodies or enzymes linked to the second protein were used in this manner, it can be understood that even when receptors or lectins are used as the first protein instead of antibodies or enzymes, the detection sensitivity of activity caused by receptors or lectins in the NC membrane will similarly improve compared to when unlinked receptors or lectins are used.

[0083] Based on these findings, the disclosure also provides a method for producing a first protein-immobilized NC membrane with enhanced activity attributable to the first protein, comprising steps (1) and (2) described above. The activities attributable to the first protein are as described above.

[0084] In this disclosure, enhanced activity means that a smaller amount of the substance to be recognized can be detected, which means increased sensitivity of activity detection.

[0085] Furthermore, in conventional immunological methods, including rapid (simple) tests such as POCT tests using immunochromatography, samples such as blood, saliva, urine, nasal secretions, sputum, stool, mucous membranes (nasal mucosa, oral mucosa, etc.), tap water, food (including beverages) (including cotton swab samples, etc.) have been used, and these have been used to detect the aforementioned first proteins, such as antigens, contained in these samples. 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, as in the conventional method, a variety of samples can be targeted.

[0086] As described above, this disclosure enables highly sensitive detection of activity derived from the first protein even when using an NC membrane. In the field of immunology, various detection methods such as ELISA, dot blot assays, and immunochromatography have been conventionally known, but protein activity tends to be easily lost on NC membranes, limiting the use of the first protein on NC membranes. The first protein immobilized NC membrane of this disclosure is also useful in various means such as antibody chips, protein chips, membrane reactors, and affinity separation membranes.

[0087] Method for enhancing the activity of a protein-immobilized nitrocellulose membrane. As described above, the first protein-immobilized NC membrane of this disclosure has enhanced activity due to the first protein. Therefore, this disclosure also includes a method for enhancing the activity of the first protein-immobilized NC membrane, comprising the following steps: (a) A step of bringing a solution containing a linkage between the first protein and the second protein into contact with a nitrocellulose membrane, and (i) A step of drying the nitrocellulose membrane that has been in contact with the connective material in step (a) above to obtain a first protein-immobilized nitrocellulose membrane. Here, the first protein is at least one selected from the group consisting of antibodies, receptors, lectins, and enzymes. The second protein is at least one selected from the group consisting of lactoferrin, concanavalin A, lysozyme, and hemoglobin. Here, if the first protein is an antibody, the enhancement of its activity is an enhancement of its antigen-binding ability. If the first protein is a receptor, the enhancement of its activity is an enhancement of its binding to the agonist and / or antagonist. If the first protein is a lectin, the enhancement of its activity is an enhancement of its glycosylation ability. If the first protein is an enzyme, then the enhancement of its activity is an enhancement of the enzyme activity.

[0088] The first protein, the second protein, the linker, contact, drying, the first protein immobilized NC membrane, activity, enhancement, etc., are all explained in the same way as described above.

[0089] Therefore, process (a) is explained in the same way as process (1) above.

[0090] Step (a) is a step to obtain a first protein-immobilized NC film by drying the NC film in contact with the connective material, in the same manner as in step (2). For this reason, the drying will be described in the same manner as in step (2), and the acquisition of the first protein-immobilized NC film by this will also be described in the same manner as in the "Method for Producing a First Protein-Immobilized Nitrocellulose Film". Blocking treatment may also be performed as described above if necessary.

[0091] The enhancement of activity is also explained in the same way as the "Method for Producing a First Protein-Immobilized Nitrocellulose Membrane" described above. That is, if the first protein is an antibody, the enhancement of activity is an enhancement of antigen-binding ability; if the first protein is a receptor, the enhancement of activity is an enhancement of binding ability to at least one selected from the group consisting of agonists and antagonists; if the first protein is a lectin, the enhancement of activity is an enhancement of glycosylation ability; and if the first protein is an enzyme, the enhancement of activity is an enhancement of enzyme activity.

[0092] This enhancement means that the activity obtained when using a first protein-immobilized NC membrane produced according to the method of this disclosure is higher than the activity obtained when using an antibody-immobilized NC membrane obtained by contacting the first protein (without linkage to the second protein) with the NC membrane. In other words, the former allows for more sensitive detection of the activity than the latter. Thus, this enhancement is based on this activity as an indicator.

[0093] The comparison may be performed based on the presence or absence and amount (strength) of the activity detected in the first protein-immobilized NC membrane. As long as the activity can be detected (measured), it may be performed using conventionally known immunological methods, such as contacting the first protein with the recognized substance as described above, and may be based on either an absolute amount (absolute value) or a relative amount (relative value). Preferably, the methods described in the examples below are exemplified. When a receptor or lectin is used as the first protein, it may be used in place of antibodies or enzymes in the examples below. Furthermore, as described above, the comparison is not limited as long as it can be compared, but usually, the type of first protein used, contact, drying, and other conditions are substantially the same, except for the presence or absence of linkage of the second protein.

[0094] According to this disclosure, since the activity originating from the first protein can be enhanced in the first protein-binding NC membrane in this way, the target object can be detected with higher sensitivity.

[0095] Conventionally, the antigens used in NC membranes have been limited to certain full-length antibodies, resulting in a lack of versatility. Furthermore, although the mechanism is unknown, there has been a problem with the relatively low detection sensitivity (antigen binding ability of the antibody) despite the antibody being immobilized on the NC membrane. To address this, measures have been taken, such as contacting a large amount of antibody with the NC membrane during the manufacturing process, in order to immobilize a larger amount of antibody. However, such measures have problems in terms of manufacturing costs due to the large amount of antibody used. In addition, the use of enzymes in combination with NC membranes has been limited in versatility because their activity is easily deactivated when applied to the NC membrane. The method disclosed herein is useful in mitigating these problems because it can enhance activity.

[0096] Furthermore, while antibody-immobilized NC membranes have conventionally been used for rapid (simple) tests for influenza and other diseases, as mentioned above, the antibodies used have been limited to certain types of full-length antibodies, making it impossible to use single-chain antibodies or single-domain antibodies to a practical degree. However, this disclosure enables highly efficient detection of antigens even when using single-chain antibodies or single-domain antibodies, which increases the range of usable antibodies in rapid (simple) tests utilizing NC membranes, and furthermore, in conventional immunological methods utilizing NC membranes. This also significantly reduces the cost of various immunological tests, such as POCT tests, and greatly contributes to the widespread adoption of POCT tests. This widespread adoption will also be useful in strengthening disease control systems in regions with insufficient medical facilities, such as emerging countries.

[0097] Furthermore, based on the fact that enzyme activity could be detected with high efficiency even when not only antibodies but also enzymes were immobilized on the NC membrane according to this disclosure, it was understood that even when receptors, which are proteins like antibodies and enzymes, are immobilized on the NC membrane via the second protein, the binding affinity of the receptor to agonists and antagonists can be further enhanced. It was also understood that even when lectins, which are proteins like antibodies and enzymes, are immobilized on the NC membrane via the second protein, their glycosylation affinity can be further enhanced. From this, it can be said that the first protein immobilized NC membrane of this disclosure is useful in various means such as antibody chips, protein chips, membrane reactors, and affinity separation membranes. [Examples]

[0098] 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.

[0099] The inventors had been investigating a technique for highly sensitive detection of antigens and enzyme activity in NC membranes for a long period of time, and surprisingly, they discovered that the detection sensitivity in NC membranes could be increased by using lactoferrin, concanavalin A, lysozyme, and hemoglobin.

[0100] Test Example 1 1-1) Test Procedure 1-1-1) Procedure for linking antibody scFv with lactoferrin or concanavalin A The antibody scFv (anti-influenza A virus antibody) was linked to lactoferrin or concanavalin A according to a conventionally known procedure. Specifically, the antibody and lactoferrin or concanavalin A were linked by a coupling reaction according to the following procedure.

[0101] (1) A solution containing 2 mg / ml lactoferrin and a solution containing 2 mg / ml concanavalin A were prepared (both containing 1 mM EDTA (ethylenediaminetetraacetic acid), 100 mM MES (2-Morpholinoethanesulfonic acid, monohydrate), and pH 7.0). The pH was measured at 25°C using a pH meter (product name LAQUA, manufactured by Horiba, Ltd.). (2) SMCC (succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate) was dissolved in DMF (N,N-dimethylformamide) to obtain an SMCC solution. (3) To 1 mL of each solution prepared in (1), 10 μl of the solution prepared in (2) was added so that the number of moles of SMCC was 100 times the number of moles of lactoferrin and concanavalin A in the solution, and the mixture was incubated in a rotator at room temperature (25°C) for 30 minutes. (4) Ultrafiltration (4°C, 9,000×g, 10 min) was repeated three times using an Amicon Ultra Centrifugal Filter (10 K) to remove unreacted SMCC. (5) To a solution containing Ant-NP-A scFv prepared to 2 mg / ml (2 mM EDTA, 100 mM MES, pH 7.0), 2-iminothiolane hydrochloride solution (Traut's Reagent (2-Iminothiolane·HCl) (2 mM EDTA, 100 mM MES, pH 7.0) was added in an amount 100 times the number of moles of scFv, and the solution was incubated at room temperature in a rotator for 1 hour. (6) The scFv-containing solution obtained in (5) was added to each of the unreacted SMCC removal solutions obtained in (4), and the mixture was incubated overnight at 4°C to allow it to react. (7) The reaction was confirmed by SDS-PAGE. In this way, linked products of scFv and concanavalin A, and linked products of scFv and lactoferrin were prepared.

[0102] The lactoferrin and concanavalin A used in this study were commercially obtained as follows: Lactoferrin: 129-04121, Lctoferrin from Bovine Milk, Wako Concanavalin A: L7647-250G, Concanavalin A Canavalia ensiformis (derived from Canavalia cordata) Type VI, lyophilized powder, Sigma

[0103] 1-1-2) Immunochromatography Test (1) The linked product obtained as described above was diluted in each pH buffer so that the scFv concentration was 0.5 mg / ml. Each pH buffer was prepared according to the following procedure. (1-1) Each pH buffer at 100 mM was prepared (Table 1). Specifically, Gly-HCl buffer (Gly-HCl in the table) was prepared by dissolving glycine in ultrapure water and adjusting the pH with HCl. 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. (1-2) An additive solution (2 w / v% CHAPS (3-(3-cholamidepropyl)dimethylammonio-1-propanesulpHonate) aqueous solution) was prepared. (1-3) The buffer obtained in (1-1) and the additive solution obtained in (1-2) were mixed as appropriate to prepare two solutions for each pH range from 1 to 13 (CHAPS 0 w / v%, CHAPS 0.2 w / v%) (CHAPS 0 w / v%) for lactoferrin, as shown in Figure 1 below. The concentration of the additive in the solution was determined by referring to the antibody immobilization conditions of Denka Co., Ltd. (2) Inactivated influenza B virus was diluted to 406.3 pfu / ml using isolation medium. (3) To 89 μl of the sample suspension, 7.0 μl of each solution prepared in (1) above was added and mixed by pipetting to obtain the linked substance-containing solution. (Final concentration 28.4 pfu / ml) (4) 4 μl of commercially available antibody-immobilized blue latex was added to the mixture obtained in (3) above. (Latex final concentration 0.0172 w / v%) (5) 2 μl of each binder-containing solution obtained in (3) was spotted onto a nitrocellulose membrane (NC membrane, High-Flow plus HF180 (Merck KGaA)) of an immunochromatography strip cut to a width of 5 mm, and air-dried at room temperature (25°C). This obtained an NC membrane with each binder immobilized. (6) After air drying, the strip was inserted into the solution obtained in (4) and an immunochromatographic test was performed for 10 minutes. (7) After air drying, the images were taken using a GT-X830 scanner (manufactured by Epson Corporation).

[0104] [Table 1]

[0105] NC membranes immobilized with scFv linked to concanavalin A (hereinafter sometimes referred to as "scFv-ConA") were designated as Example 1 (CHAPS 0 w / v%) and Example 2 (CHAPS 0.2 w / v%), and NC membranes immobilized with scFv linked to lactoferrin (hereinafter sometimes referred to as "scFv-LF") were designated as Example 3 (CHAPS 0 w / v%). In addition, as Comparative Example 1, scFv without linkage to either lactoferrin or concanavalin A was used and prepared in the same manner, and an immunochromatographic test was performed.

[0106] 1-2) Results The results are shown in Figures 1-3. Figure 1 shows the results for scFv-fixed NC membranes without concanavalin A or lactoferrin linkage (Comparative Example 1). Figure 2 shows the results for ConA-scFv (Examples 1 and 2). In each figure, the upper vertical axis shows the results using a CHAPS 0 w / v% solution, and the lower vertical axis shows the results using a CHAPS 0.2 w / v% solution. The horizontal axis shows pH. Figure 3 shows the results for LF-scFv (Example 3). As shown in Figures 1 and 2, the spots appeared more intensely in the scFv-fixed NC membrane with concanavalin A (ConA) linkage (Figure 2, scFv-ConA) compared to the scFv-fixed NC membrane without lactoferrin or concanavalin A linkage (Figure 1, scFv). Also, as shown in Figure 3, the spots appeared more intensely in the scFv-fixed NC membrane with lactoferrin (LF) linkage (LF-scFv). The intensity of the color of each spot correlates with the antigen-binding ability of the antibody; the darker the spot, the more antigen is present in that area, indicating a higher antigen-binding ability of the antibody. From this, it was understood that linking ConA or LF to scFv with the antibody enhances the antigen detection ability of the antibody immobilized on the NC membrane.

[0107] Test Example 2

[0108] 2-1) Test Procedure Antigen detection was performed in the same manner as in Test Example 1, except that the scFv was changed to an scFv for influenza A virus (anti-NP A scFV). The antigen dose was 56.9 pfu / mL, and the test was performed without the addition of CHAPS.

[0109] 2-2) Results The results are shown in Figure 4. As shown in Figure 4, even when using scFv for influenza A virus, the spots appeared more vividly in the scFv-fixed NC membrane with LF linked (Figure (B), Example 4) and the scFv-fixed NC membrane with ConA linked (Figure (C), Example 5) compared to the scFv-fixed NC membrane without LF or ConA linked (Figure (A), Comparative Example 2), similar to Test Example 1. From this, it was understood that even with antibodies different from those in Test Example 1, linking LF or ConA to the antibody via scFV enhances the antigen detection ability of the antibody immobilized on the NC membrane.

[0110] Test Example 3 3-1) Test Procedure Antigen detection was performed in the same manner as in Test Example 1, except that scFv was changed to VHH for influenza A virus (anti-NP A VHH). The antigen dose was 56.9 pfu / mL, and the test was performed without the addition of CHAPS.

[0111] 3-2) Results The results are shown in Figure 5. As shown in Figure 5, even when VHH was used, the spots appeared more vividly in the VHH-fixed NC membrane with LF linked (Figure (B) VHH-LF, Example 6) and the VHH-fixed NC membrane with ConA linked (Figure (C) VHH-ConA, Example 7) compared to the VHH-fixed NC membrane without LF or ConA linked (Figure (A), Comparative Example 3), similar to Test Examples 1 and 2. From this, it was understood that the antigen detection ability of the antibody immobilized on the NC membrane is enhanced not only when scFV is used as the antibody, but also when VHH is used, by linking LF or ConA to the antibody.

[0112] Test Example 4 4-1) Test Procedure In the aforementioned test examples 1-3, it was confirmed that linking the antibody to LF or ConA enhanced the antigen detection ability of the antibody. Therefore, further tests were conducted by reducing the antigen concentration. Specifically, tests were performed at four antigen concentrations: 28.4 pfu / mL, 2.84 pfu / mL, 0.28 pfu / mL, and 0 pfu / mL.

[0113] 4-2) Results The results are shown in Figure 6. In Figure 6, (B) shows the results for the scFv-fixed NC membrane with LF linked (Example 8), and (C) shows the results for the scFv-fixed NC membrane with ConA linked (Example 9). In both Examples 8 and 9, dark spots appeared at an antigen concentration of 28.4 pfu / mL. Furthermore, spots were observed even when the antigen concentration was further reduced to 2.84 pfu / mL and 0.28 pfu / mL. In contrast, in the scFv-fixed NC membrane without LF or ConA linked (Comparative Example 4), shown in Table (A), at an antigen concentration of 28.4 pfu / mL, only significantly lighter spots appeared compared to Example 8 (B) in the same table using the same antigen concentration. When the antigen concentration was further reduced to 2.84 pfu / mL and 0.28 pfu / mL, no spots were observed. Thus, compared to the case without LF or ConA linked, the activity was greatly improved by linking LF or ConA. This demonstrates that linking LF or ConA to an antibody can enhance the antigen-binding ability of antibodies immobilized on an NC membrane. Comparative Examples 4, 8, and 9 show the results of comparing detection sensitivity under optimal immobilization conditions, and although the pH during immobilization differs, as shown in Figure 1, when LF and ConA are linked, the antigen-binding signal of scFv is significantly improved over a wide pH range, suggesting that the usable pH range of the present invention is relatively wide.

[0114] Test Example 5 5-1) Test Procedure The test was conducted in the same manner as in Test Example 4, except that the antibody used was scFv for influenza A, and the antigen concentrations were set to four levels: 56.9 pfu / mL, 5.69 pfu / mL, 0.57 pfu / mL, and 0 pfu / mL.

[0115] 5-2) Results The results are shown in Figure 7. In Figure 7, (B) shows the results for the scFv-fixed NC membrane with LF linked (Example 10), and (C) shows the results for the scFv-fixed NC membrane with ConA linked (Example 11). Similar to Figure 6, spots were observed in both cases at antigen concentrations of 56.9 pfu / mL, 5.69 pfu / mL, and 0.57 pfu / mL. In contrast, in the scFv-fixed NC membrane without either LF or ConA linked (Comparative Example 5), shown in Table (A), spots were observed at antigen concentrations of 56.9 pfu / mL and 5.69 pfu / mL, but these spots were lighter in color than the spots observed in Examples 10 and 11. Furthermore, in Comparative Example 5, no spots were observed at an antigen concentration of 0.57 pfu / mL. From this, it can be understood that linking LF or ConA to the antibody enhances the antigen detection ability of the antibody immobilized on the NC membrane.

[0116] Furthermore, although not shown in the results, when the test was performed using HVV for influenza A instead of the scFv for influenza A, darker spots were obtained when LF or ConA was linked to the antibody compared to when LF or ConA was not linked. From this, it was understood that regardless of the type of antibody, such as scFv or HVV, linking LF or ConA enhances the antigen detection ability of antibodies immobilized on the NC membrane.

[0117] Test Example 6 The following tests confirmed that activity could be enhanced even when an enzyme was used instead of an antibody. Furthermore, the following tests confirmed that activity could be enhanced even when lysozyme and hemoglobin were linked to lactoferrin and concanavalin A. In these tests, HRP (horseradish peroxidase) was used as the enzyme.

[0118] 6-1) Test Procedure 6-1-1) Preparation of linkages between enzymes (HRP) and secondary proteins (lactoferrin, concanavalin A, lysozyme, hemoglobin)

[0119] 6-1-1-1) Preparation of biotinylated protein II (1) Lactoferrin, concanavalin A, lysozyme, and hemoglobin were each dissolved in 0.1 M carbonate buffer (pH 8.3) to a concentration of 4.0 mg / ml, and these were prepared as the second protein solution. (2) Biotinamide caproic acid N-hydroxysuccinimide (Biotin-NHS) was dissolved in N,N-dimethylformamide (DMF) to a concentration of 10 mg / ml. (3) To each of the two protein solutions obtained in (1) above, the solution from (2) above was added so that the molar ratio of each protein to the biotinylation reagent was 1:10, and the mixture was incubated at room temperature (25°C) in a rotator for 1 hour. (4) To 1 ml of the reaction solution obtained in this manner, 100 μl of 1.5 M Tris-HCl (pH 8.5) was added, and the reaction (biotinylation) was stopped by incubation at 25°C for 1 hour in a rotator. (5) I started AKTA Purifier (GE Healthcare). (6) A HiTrap Desaulting (5 ml) (Cytiva) column for gel filtration was supplied with buffer (1 × PBS (pH 8.0)) to equilibrate the column. (7) Each biotinylated protein solution obtained in (4) above was injected at a rate of 2 ml / min at a rate of 1 ml to a 2 ml sample loop and filtered by gel. (8) The fractions were collected in 0.5 ml increments. (9) Protein quantification was performed using the DC protein assay (standard assay).

[0120] In this way, biotinylated lactoferrin, biotinylated concanavalin A, biotinylated lysozyme, and biotinylated hemoglobin were prepared. Hereafter, these will be referred to as biotinylated lactoferrin, biotinylated concanavalin A, biotinylated lysozyme, and biotinylated hemoglobin.

[0121] The lactoferrin and concanavalin A used in this test example are the same as those used in Test Example 1, etc. Hemoglobin and lysozyme are as follows: Lysozyme: 122-02673, from Egg White, Wako Hemoglobin: 081-03492, from Bovine, Wako

[0122] 6-1-1-2) Linking of HRP with lactoferrin, concanavalin A, lysozyme, and hemoglobin (1) HRP-labeled streptavidin (SA) was interacted with biotin. Specifically, biotinylated lactoferrin obtained as described above, commercially available SA-HRP (SA50141, (Vector)), and buffer were mixed in an Eppendorf tube to prepare a mixed solution containing 100 μg / ml biotinylated lactoferrin and 0.62 μg / ml SA-HRP. By interacting streptavidin (SA) and biotin in the Eppendorf tube, lactoferrin and HRP were linked via SA and biotin. This resulted in obtaining a linkage between lactoferrin and the enzyme (HRP). (2) Similarly, for biotinylated concanavalin A, biotinylated lysozyme, and biotinylated hemoglobin, SA-HRP and the buffer were mixed in an Eppendorf tube to allow streptavidin (SA) and biotin to interact. This resulted in the acquisition of concanavalin A-HRP linkages, lysozyme-HRP linkages, and hemoglobin-HRP linkages, respectively, via SA and biotin.

[0123] 6-1-2) Evaluation of HRP enzyme activity on NC membrane (1) As described above, 2 μl of the solution containing the prepared linkage (1xPBS (137 mmol / l NaCl, 10 mmol / l Na2HPO4, 2.68 mmol / l KCl, 2 mmol / l KH2PO4), pH 8.0, CHAPS concentrations 0, 0.2, 2.0%) was applied to each NC membrane (Amersham Protran, (cytiva)) and air-dried at room temperature. (2) After air drying, the films were blocked by shaking in 2% BSA-PBS (pH 8.0) at room temperature for 1 hour. This created NC films (i-iv of Example 12) with each linkage immobilized. (3) After washing five times with 0.1% PBST, the samples were colored using TMB (1-Step® Ultra TMB-Blotting Solution), a substrate for HRP. (4) After drying, the images were captured using a GT-X830 scanner.

[0124] As Comparative Example 6, unbiotinylated lactoferrin, SA-HRP, and buffer were mixed in an Eppendorf tube to prepare a mixed solution containing 100 μg / ml lactoferrin and 0.62 μg / ml SA-HRP (Comparative Example 1). The same procedure was followed for concanavalin A, lysozyme, and hemoglobin (Comparative Example 6, i-iv).

[0125] 6-2) Results The results are shown in Figure 8. In this study, instead of antibodies, lactoferrin (Figure (i)), concanavalin A (Figure (ii)), lysozyme (Figure (iii)), and hemoglobin (Figure (iv)) were linked to HRP (horseradish peroxidase), a protein known as a labeling enzyme.

[0126] As shown in Figure 8, even when enzymes were used, when lactoferrin (labeled i in the figure as "linked" (Example 12-i)) and concanavalin A (labeled ii in the figure as "linked" (Example 12-ii)) were linked, spots originating from enzyme activity appeared, allowing for the detection of enzyme activity. On the other hand, when lactoferrin and concanavalin A were not linked (labeled i and ii in the figure as "unlinked" (Comparative Example 6-i and Comparative Example 6-ii, respectively)), no spots originating from enzyme activity appeared, and enzyme activity could not be detected. In Figure 8, when the CHAPS concentration was 0 w / v%, even when the enzyme was linked to lactoferrin, etc., only faint spots were observed, but no spots were observed at all for enzymes that were not linked to lactoferrin, etc. From this, it was confirmed that enzyme activity improved not only at CHAPS concentrations of 0.2 w / v% and 2 w / v%, but also at 0 w / v%.

[0127] This confirms that, not only with antibodies but also with enzymes, linking lactoferrin and concanavalin A enhances enzyme activity. Furthermore, although the above procedure is an example of linking enzymes and lactoferrin via biotin-streptavidin interaction, even when lactoferrin and enzymes are linked by an amine coupling method using N-hydroxysuccinimide without using biotin-streptavidin (not shown in the figure), a similar enhancement of enzyme activity was observed compared to when lactoferrin was not linked. Similarly, when concanavalin A was used, linking concanavalin A enhanced enzyme activity. From these findings, it could be inferred that the orientation of antibodies on the NC membrane was improved.

[0128] Furthermore, as shown in Figure 8, when lysozyme (iii in the figure labeled "linked" (Example 12-iii)) and hemoglobin (iv in the figure labeled "linked" (Example 12-iv)) were linked to the enzyme instead of lactoferrin and concanavalin A, spots derived from enzyme activity appeared, and enzyme activity could be detected. On the other hand, when lysozyme and hemoglobin were not linked (iii and iv in the figure labeled "unlinked" (Comparative Examples 6-iii and iv)), spots derived from enzyme activity did not appear, and enzyme activity could not be detected. From this, it was confirmed that enzyme activity is enhanced even when lysozyme and hemoglobin are linked to the enzyme or antibody instead of lactoferrin and concanavalin A.

[0129] Furthermore, the inventors conducted similar tests using more than a dozen known proteins, including ovalbumin, as substances to be linked to antibodies or labeled enzymes, but no significant enhancement of antigen binding or enzyme activity was observed for any substance other than lactoferrin, concanavalin A, lysozyme, and hemoglobin.

[0130] Furthermore, these results demonstrate that detection sensitivity can be improved even when receptors and lectins, which are also proteins, are used in place of enzymes and antibodies. More specifically, it was found that the binding affinity of receptors to agonists and antagonists specifically recognized by the receptor can be enhanced, and that the glycosylation affinity of lectins specifically recognized by the lectin can be enhanced.

Claims

1. A method for producing a first protein-immobilized nitrocellulose membrane, comprising the following steps: (1) A step of bringing a solution containing a linkage between the first protein and the second protein into contact with a nitrocellulose membrane, and (2) A step of drying the nitrocellulose film in which the connecting material was in contact in step (1), Here, The first protein is an antibody, and the second protein is at least one selected from the group consisting of lactoferrin and concanavalin A. The first protein is an enzyme, and the second protein is at least one selected from the group consisting of concanavalin A, lysozyme, and hemoglobin.

2. The first protein is a full-length antibody (Whole antibody), scFv, VHH, Fab, F(ab') 2 The method for producing an antibody according to claim 1, wherein the antibody is at least one selected from the group consisting of and a bispecific antibody.

3. A method for producing a first protein-immobilized nitrocellulose membrane in which the activity due to the first protein is enhanced, according to claim 1 or 2. Here, If the first protein is an antibody, the enhancement of its activity is an enhancement of its antigen-binding ability. If the first protein is an enzyme, then the enhancement of its activity is an enhancement of the enzyme activity.

4. A method for enhancing the activity of a first protein-immobilized nitrocellulose membrane, comprising the following steps: (a) A step of bringing a solution containing a linkage between the first protein and the second protein into contact with a nitrocellulose membrane, and (i) A step of drying the nitrocellulose membrane in which the connective material was brought into contact in step (a) above to obtain a first protein-immobilized nitrocellulose membrane. Here, The first protein is an antibody, and the second protein is at least one selected from the group consisting of lactoferrin and concanavalin A. The first protein is an enzyme, and the second protein is at least one selected from the group consisting of lactoferrin, concanavalin A, lysozyme, and hemoglobin. If the first protein is an antibody, the enhancement of its activity is an enhancement of its antigen-binding ability. If the first protein is an enzyme, then the enhancement of its activity is an enhancement of the enzyme activity.

5. A first protein is linked to a second protein, and the first protein is immobilized on a nitrocellulose membrane via the second protein, in a first protein immobilized nitrocellulose membrane. Here, The first protein is an antibody, and the second protein is at least one selected from the group consisting of lactoferrin and concanavalin A. The first protein is an enzyme, and the second protein is at least one selected from the group consisting of lysozyme and hemoglobin.

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