Method for measuring antigen and method for producing immobilized antibody

Abstract

To provide a method for measuring an antigen that improves the detection sensitivity of the antigen in methods for measuring antigens in test samples using an antigen-antibody reaction, and a method for producing an immobilized antibody.SOLUTION: A method for measuring an antigen in a test sample includes the steps of: immobilizing a first antibody that immunologically bonds to the antigen in the test sample onto a first carrier; bonding the antigen in the test sample to the first antibody via a first epitope in the antigen; and detecting the antigen bonded to the first antibody via the first epitope. The step of immobilizing the first antibody that immunologically bonds to the antigen in the test sample to the first carrier is performed in a solution containing the first antibody and one or more proteins other than the antigen and the first antibody.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for measuring an antigen and a method for producing a solid-phase antibody. [Background technology]

[0002] A method for measuring an antigen in a test sample using an antigen-antibody reaction is widely used. In this method, an antigen is usually detected using an antibody immobilized on an insoluble carrier. When measuring an antigen in a test sample using this method, substances other than the antigen in the test sample are nonspecifically adsorbed to the carrier, and the background when measuring the antigen increases, which increases the detection limit concentration of the antigen (in other words, the detection sensitivity decreases). As a result, when the antigen is present in a trace amount in the test sample, it becomes impossible to detect or measure it. Therefore, in general, after the antibody is immobilized on the carrier, the carrier is blocked with a protein such as bovine serum albumin.

[0003] However, even if such blocking is performed, the non-specific adsorption cannot be completely suppressed. Therefore, in order to more effectively suppress non-specific adsorption, a technique has been reported in which the carrier is blocked with a non-polar amino acid such as alanine or valine, instead of a protein (Patent Document 1). In addition, a technique has been developed in which, when immobilizing an antibody on a carrier, an azide ion is made to coexist, or tris(hydroxymethyl)aminomethane is made to coexist with a non-polar amino acid, thereby measuring an antigen with high sensitivity and accuracy (Patent Document 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 5-288750 [Patent Document 2] JP 2007-178142 A Summary of the Invention [Problem to be solved by the invention]

[0005] An objective of the present invention is to provide a technique for improving the detection sensitivity of an antigen in a method for measuring an antigen in a test sample by utilizing an antigen-antibody reaction. [Means for solving the problem]

[0006] As a result of extensive investigations, the present inventors have found that the above-mentioned problems can be solved by making a specific protein coexist when immobilizing an antibody on a carrier, and have thus completed the present invention. The present invention is as follows.

[0007] The present invention relates to (a) immobilizing a first antibody that immunologically binds to an antigen in a test sample on a first carrier; (b) binding an antigen in the test sample to the first antibody via a first epitope in the antigen; and (c) detecting the antigen bound to the first antibody via the first epitope; the step (a) is carried out in a solution containing the first antibody and a protein other than the antigen and the first antibody; Method for measuring antigen in test sample to provide.

[0008] The method further comprises the step of: In a preferred embodiment, the weight ratio (said first antibody:said antigen and said protein other than said first antibody) is 1:1 or more and 1:19 or less. In a preferred embodiment of the method, the protein other than the antigen and the first antibody is one or more selected from the group consisting of bovine serum albumin, casein, gelatin, hemoglobin, and ovalbumin. In a preferred embodiment of the method, the first antibody is an anti-podoplanin antibody and the antigen is podoplanin.

[0009] The method also includes: the step (c) is a step of binding an antigen in the test sample to a labeled second antibody that recognizes a second epitope in the antigen different from the first epitope, via the second epitope, and detecting the antigen bound to the first antibody via the first epitope using the label; In a preferred embodiment, the step (a) is carried out in a solution containing the first antibody, the antigen, and a protein other than the first and second antibodies.

[0010] The present invention also provides The method includes a step of immobilizing an antibody that immunologically binds to an antigen in a test sample on a carrier, The solid-phase step is carried out in a solution containing the antibody, the antigen, and a protein other than the antibody. Method for producing immobilized antibodies that immunologically bind to antigens in test samples can be provided.

[0011] The manufacturing method further comprises the step of: and the weight ratio (the antibody:the antigen and the protein other than the antibody) is 1:1 or more and 1:19 or less. In a preferred embodiment of the production method, the protein other than the antigen and the antibody is one or more selected from the group consisting of bovine serum albumin, casein, gelatin, hemoglobin, and ovalbumin. In a preferred embodiment of the production method, the antibody is an anti-podoplanin antibody, and the antigen is podoplanin. Effect of the Invention

[0012] According to the present invention, a technique for improving the detection sensitivity of an antigen in a method for measuring an antigen in a test sample using an antigen-antibody reaction can be provided. This technique lowers the detection limit concentration of the antigen when measuring the antigen in the test sample, i.e., increases the detection sensitivity, thereby making it possible to detect and measure even a trace amount of the antigen in the test sample. [Brief description of the drawings]

[0013] [Figure 1] 1 is a graph showing the results of Example 1 and Comparative Example 1 according to one embodiment of the present invention. [Diagram 2] 1 is a graph showing the results of Example 1 and Comparative Example 1 according to one embodiment of the present invention. [Diagram 3] 1 is a graph showing the results of Example 1 and Comparative Example 1 according to one embodiment of the present invention. [Figure 4] 1 is a graph showing the results of Example 1 and Comparative Example 1 according to one embodiment of the present invention. [Diagram 5] 1 is a graph showing the results of Example 1 and Comparative Example 1 according to one embodiment of the present invention. [Figure 6] 1 is a graph showing the results of Example 1 and Comparative Example 1 according to one embodiment of the present invention. [Figure 7] 1 is a graph showing the results of Example 1 and Comparative Example 1 according to one embodiment of the present invention. [Figure 8] Graph showing the results of Example 2 according to one embodiment of the present invention. [Figure 9] 1 is a graph showing the results of Comparative Example 3 according to one embodiment of the present invention. [Figure 10] Graph showing the results of Example 3 according to one embodiment of the present invention. [Figure 11] 1 is a graph showing the results of Comparative Example 4 according to one embodiment of the present invention. [Figure 12] Graph showing the results of Example 3 and Comparative Example 4 according to one embodiment of the present invention. [Figure 13] 1 is a graph showing the results of Comparative Example 5 and Comparative Example 6 according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] In the present invention, an "antibody" may be an "antibody fragment containing an antigen-binding region", that is, a protein containing a portion of an antibody and capable of binding to an antigen. Examples of antibody fragments include F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide-linked Fv, single-chain antibody (scFv), and polymers thereof.

[0015] <Method for measuring antigens in test samples> One aspect of the present invention is (a) immobilizing a first antibody that immunologically binds to an antigen in a test sample on a first carrier; (b) binding an antigen in the test sample to the first antibody via a first epitope in the antigen; and (c) detecting the antigen bound to the first antibody via the first epitope; the step (a) is carried out in a solution containing the first antibody and a protein other than the antigen and the first antibody; This is a method for measuring an antigen in a test sample.

[0016] The method according to this embodiment includes step (a) of immobilizing a first antibody that immunologically binds to an antigen in a test sample on a first carrier, which is a step commonly performed in the art for immobilizing a first antibody that immunologically binds to an antigen in a test sample on a first carrier, in which the antigen and proteins other than the first antibody coexist. That is, step (a) includes a step of immobilizing the first antibody on the first carrier in a solution containing the first antibody, the antigen, and proteins other than the first antibody.

[0017] The origin of the test sample used in this embodiment is not particularly limited as long as it is a mammal containing an antigen that is measured using an antigen-antibody reaction that is commonly used in the technical field, and examples of the origin include humans, cows, goats, sheep, pigs, monkeys, dogs, cats, rats, mice, hamsters, and guinea pigs, etc. Preferably, the origin is humans.

[0018] Furthermore, when the test sample used in this embodiment is derived from a human, the human may be one suffering from a disease, one not suffering from a disease, or one suspected of suffering from a disease. The disease is not particularly limited as long as an antigen that serves as a marker for the disease can be measured by the method according to the present embodiment.

[0019] The antigen serving as a marker for the disease is preferably podoplanin, SCC, CYFRA, PSA, CA125, CEA, STN, NSE, N-acetyl-β-D-glucosaminidase, β2 microglobulin, α1 Microglobulin, KL-6, pulmonary surfactant protein-A, pulmonary surfactant protein-D, CK / CK-MB, troponin T, heart-type fatty acid binding protein, cardiac myosin light chain I, etc. are more preferred. The most common is podoplanin.

[0020] The diseases for which the antigen in the test sample used in this embodiment serves as a marker include, for example, cancer, nephritis (e.g., hereditary nephritis (Alport syndrome), acquired nephritis, glomerulonephritis, pyelonephritis, renal abscess, perinephritis, renal tuberculosis, renal failure, atrophic kidney, etc.), pneumonia (e.g., lobar pneumonia, bronchopneumonia, interstitial pneumonia, etc.), cardiomyopathy (e.g., hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, etc.), Examples of leukoplakia include leukoplakia (e.g., white keratosis, senile keratosis, Bowen's disease-like leukoplakia, etc.).

[0021] Examples of the cancer include mesothelioma, brain tumor, testicular tumor, prostate cancer, Kaposi's sarcoma, lymphangioma, cavernous hemangioma, angiosarcoma, lung squamous cell carcinoma, ovarian cancer, dysgerminoma, uterine cancer, cervical cancer, nerve tumors (glioma, etc.), germ cell tumors, head and neck cancer, lung cancer, bladder cancer, bone and soft tissue tumors (primary bone tumors (osteosarcoma, chondrosarcoma), soft tissue sarcoma), esophageal cancer, oral cancer, tongue cancer, pharyngeal cancer, vocal cord cancer, breast cancer, pancreatic cancer, and stomach cancer.

[0022] When the antigen in the test sample used in this embodiment is podoplanin, the disease is cancer, preferably mesothelioma, brain tumor, testicular tumor, prostate cancer, Kaposi's sarcoma, lymphangioma, cavernous hemangioma, angiosarcoma, lung squamous cell carcinoma, ovarian cancer, dysgerminoma, glioma, germ cell tumor, head and neck cancer, lung cancer, bladder cancer, bone and soft tissue tumor (primary bone tumor (osteosarcoma, chondrosarcoma), soft tissue sarcoma)), or esophageal cancer, more preferably lung squamous cell carcinoma.

[0023] When the antigen in the test sample used in this embodiment is SCC, the disease is cancer. Preferably, it is esophageal cancer, lung cancer, etc. When the antigen in the test sample used in this embodiment is CYFRA, the disease is cancer. and preferably, lung squamous cell carcinoma. When the antigen in the test sample used in this embodiment is PSA, the disease is cancer. , preferably prostate cancer. When the antigen in the test sample used in this embodiment is CA125 or CEA, the disease is cancer, preferably breast cancer, pancreatic cancer, or the like. When the antigen in the test sample used in this embodiment is STN, the disease is cancer. Preferably, it is stomach cancer or the like. When the antigen in the test sample used in this embodiment is NSE, the disease is cancer. , neurotumors, etc.

[0024] The antigen in the test sample used in this embodiment is N-acetylglucosamine, β-D-glucosaminidase, In the case of α1-microglobulin, the disease is nephritis.

[0025] When the antigen in the test sample used in this embodiment is KL-6, pulmonary surfactant protein-A, or pulmonary surfactant protein-D, the disease is pneumonia.

[0026] The antigen in the test sample used in this embodiment is CK / CK-MB, troponin T, heart-type fatty acid binding protein white, cardiac myosin light chain I, the disease is cardiomyopathy.

[0027] The test sample used in this embodiment is not particularly limited as long as it contains an antigen that can be measured by the method according to this embodiment, and examples of the test sample include body fluids, etc. Examples of the body fluids include blood (e.g., whole blood, serum, plasma, etc.), serous fluid, ascites, pleural fluid, lymphatic fluid, urine, cerebrospinal fluid, pleural fluid, peritoneal fluid, pericardial fluid, and mucosal secretions.

[0028] The test sample used in this embodiment may be diluted with a buffer solution or the like that is commonly used in the art. Examples of such buffer solutions include those that inhibit antigen-antibody reactions, such as Tris-HCl buffer, carbonate-bicarbonate buffer, phosphate buffer (e.g., PBS, D-PBS), and HEPES buffer. No denaturants in physiological buffers, e.g., SDS, urea, guanidine hydrochloride; surfactants such as ethanol ethoxylate, Tween 20, Tween 80, CHAPS; sodium azide peroxidase inhibitors such as glycerol; protein stabilizers such as osmolytes and sugars; Examples of blocking agents that can be added depending on the purpose include 0.098% (w / v) BSA, 0.0147 mol / L arginine, 0.00431% sodium azide, 0.049% SDS, and 0.049% Tw een 80, and a mixed solution containing 0.56% Lipidure BL802 (NOF Corporation).

[0029] The antigen in the test sample used in this embodiment is not particularly limited as long as it is a substance that binds to the first antibody via the first epitope in the antigen and is measured using an antigen-antibody reaction commonly used in the art. Examples include proteins (including peptides), sugars, lipids, etc. Preferably, it is a protein, and more preferably, it is an antigen that serves as a marker for the disease. When the disease is cancer, it may be a tumor marker. For example, podoplanin (also known as Aggrus, M2A, GP36, GP40, GP38, OTS8, T1A2, TI1A, T1A-2, HT1A-1, PA2.26, etc.), SCC, CYFRA, PSA, CA125, CEA, STN, NSE, etc. Examples include: The antigen in the test sample may be an immunoglobulin. The immunoglobulin is not particularly limited as long as it can be measured by the method according to the present embodiment, and any immunoglobulin may be used. For example, human immunoglobulins include IgG, IgA, IgM, IgD, and IgE. There are classes of antibodies, and IgG and IgA have subclasses. For example, the subclass of IgG is There are three subclasses of IgA: IgA1 and IgA2. do.

[0030] The first antibody used in this embodiment is not particularly limited as long as it binds to the antigen in the test sample via the first epitope in the antigen. The first antibody used in this embodiment may be a monoclonal antibody or a polyclonal antibody, and can be appropriately selected depending on the embodiment. One or more types of the first antibody may be used.

[0031] As the first antibody, for example, when the antigen in the test sample is podoplanin and the first antibody is a monoclonal antibody (i.e., an anti-podoplanin monoclonal antibody), for example, NZ-1.2 (Medical and Biological Laboratories Co., Ltd., D320-3), Japanese Patent No. 6737482 Further examples of the antibody include AP201 described in the specification of No. 6,336,414, and the like. In addition, EPR22182 (Abcam, ab236529), PMab-1 (Abcam, ab256559), EPR7072 (Abcam, ab128994), 18H5 (Abcam, ab10288), EPR7073 (Abcam, ab131216), LpMab-7 (Abcam, ab256566), RTD4E10 (Abcam, ab11936), LF3 / B7 / D5B27 (Novus Biologicals, LLC (R&D Systems Company), NB110-96423), RTD4E10 (Abcam, ab11936), 8.1.1 (Abcam, ab92319), E-1 (SANTA CRUZ BIOTECHNOLOGY, sc-376695), F-3 (SANTA CRUZ BIOTECHNOLOGY, sc-376962), B-11 (SANTA CRUZ BIOTECHNOLOGY, sc-166906), etc.

[0032] Furthermore, as the first antibody, for example, if the antigen in the test sample is podoplanin and the first antibody is a polyclonal antibody (i.e., an anti-podoplanin polyclonal antibody), examples of the first antibody include Anti-Podoplanin Antibody, Rabbit Polyclonal (Sino Biological Inc., 11065-T52) and Anti-Podoplanin antibody (Novopro, 106225). can be.

[0033] Furthermore, when the antigen in the test sample is an immunoglobulin and the first antibody is a monoclonal antibody, the first antibody is an anti-immunoglobulin monoclonal antibody. Specifically, for example, the immunoglobulin may be human IgG, IgA, IgM, IgD, or IgE. For example, the first antibody is an anti-human IgG monoclonal antibody, an anti-human IgA monoclonal antibody, an anti-human IgM monoclonal antibody, an anti-human IgD monoclonal antibody, or an anti-human IgE monoclonal antibody, respectively. In addition, if the immunoglobulin is human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2, the first antibody may be an anti-human IgG1 monoclonal antibody, an anti-human IgG2 monoclonal antibody, an anti-human IgG3 monoclonal antibody, an anti-human IgG4 monoclonal antibody, an anti-human IgG5 monoclonal antibody, an anti-human IgG6 monoclonal antibody, an anti-human IgG7 monoclonal antibody, an anti-human IgG8 monoclonal antibody, an anti-human IgG9 monoclonal antibody, an anti-human IgG1 monoclonal antibody, an anti-human IgG1 monoclonal antibody, an anti-human IgG2 monoclonal antibody, an anti-human IgG3 monoclonal antibody, an anti-human IgG4 ... The antibodies are anti-human IgA1 monoclonal antibody and anti-human IgA2 monoclonal antibody. The same applies when the mammal is a mammal other than a human. For example, if the antigen in the test sample is mouse IgG1, the first antibody is an anti-mouse IgG1 monoclonal antibody.

[0034] Furthermore, when the antigen in the test sample is an immunoglobulin and the first antibody is a polyclonal antibody, the first antibody is an anti-immunoglobulin polyclonal antibody. Specifically, for example, the immunoglobulin may be human IgG, IgA, IgM, IgD, or IgE. For example, the first antibody is an anti-human IgG polyclonal antibody, an anti-human IgA polyclonal antibody, an anti-human IgM polyclonal antibody, an anti-human IgD polyclonal antibody, or an anti-human IgE polyclonal antibody, respectively. Furthermore, if the immunoglobulin is human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2, the first antibody is an anti-human IgG1 polyclonal antibody, an anti-human IgG2 polyclonal antibody, an anti-human IgG3 polyclonal antibody, an anti-human IgG4 polyclonal antibody, an anti-human IgA1 polyclonal antibody, or an anti-human IgA2 polyclonal antibody, respectively. The same applies when the mammal is other than a human. For example, if the antigen in the test sample is mouse IgG1, the first antibody is an anti-mouse IgG1 polyclonal antibody.

[0035] The first carrier used in step (a) includes a conventional first carrier used in a method for measuring an antigen. The shape of the first carrier used in step (a) is not particularly limited as long as it is a carrier shape commonly used in the art, such as particles (beads), plates, tubes, gels, membranes (including fibers and filters), etc. The material of the first carrier used in step (a) is not particularly limited as long as it is a carrier material commonly used in the technical field and is insoluble in a solution commonly used in antigen-antibody reactions. For example, synthetic resin materials (e.g., polystyrene, polyurethane, polyethylene, polyethylene glycol, polypropylene, polyethylene terephthalate, polyvinyl alcohol, polyvinyl chloride, polyvinyl acetate, acrylic resins (polyacrylamide, etc.), polyurethane, polycarbonate, ABS resin, polyvinylidene fluoride, etc.), inorganic materials (e.g., , silicon, silica, glass, titanium, titanium oxide, iron, iron oxide, aluminum, aluminum oxide, zinc, zinc oxide, zirconium, zirconia, indium tin oxide, gold, silver, platinum, etc.), polysaccharides (e.g., agarose, cellulose, sepharose, nitrocellulose, chitin, etc.), proteins (e.g., gelatin, collagen, etc.), synthetic rubbers (e.g., styrene-butadiene rubber, isoprene rubber, butadiene rubber, chloroprene rubber, etc.). In addition, if the shape of the first carrier is a particle (bead), it may be a magnetic bead.

[0036] A typical step in the art for immobilizing a first antibody that immunologically binds to an antigen in a test sample on a first carrier includes contacting a buffer solution containing the first antibody with the first carrier at a predetermined temperature (e.g., 4°C, etc.) for a predetermined time (e.g., one day and one night, etc.) Examples of the buffer solution include phosphate buffer solutions (PBS, Dulbecco's Phosphate-Buffered Saline (D-PBS), etc.), carbonate buffer solutions, etc.

[0037] The concentration of the first antibody in a solution containing the first antibody, the antigen, and a protein other than the first antibody (sometimes referred to as a "solid-phased solution" in this specification) is not particularly limited as long as the first antibody is solid-phased to the first carrier. For example, any of 100 ng / mL, 200 ng / mL, 500 ng / mL, and 1000 ng / mL may be adopted as the upper or lower limit. The concentration may be

[0038] The concentration of the protein other than the antigen and the first antibody in the solid-phase solution is There are no particular limitations as long as, when the first antibody is immobilized on the first carrier and the test sample is applied thereto, nonspecific adsorption of substances other than the antigen in the test sample to the first carrier can be suppressed, an increase in background during antigen measurement can be suppressed, and the detection limit concentration of the antigen can be lowered (in other words, the detection sensitivity can be improved). The concentration in the solid-phase solution depends on the concentration of the first antibody in the solid-phase solution and the concentration of the antigen in the test sample, and may be, for example, 0.00001% (w / v), 0.00002% (w / v), 0.00004% (w / v), 0.00005% (w / v), 0.00008% (w / v), 0.00009% (w / v), 0.0001% (w / v), 0.00018% (w / v), 0.00019% (w / v), 0.0002% (w / v), 0.00038% (w / v), 0. ...5% (w / v), 0.00005% (w / v), 0.00005% (w / v), 0.0 %(w / v), 0.00045%(w / v), 0.0009%(w / v), 0.00095%(w / v), and 0.0019%(w / v). Either one of them may be used as the upper or lower limit.

[0039] Similarly, the weight ratio of the first antibody to the antigen and the protein other than the first antibody in the solid-phase solution (i.e., weight ratio (the first antibody:the antigen and the protein other than the first antibody)) is not particularly limited as long as it can lower the detection limit concentration of the antigen (in other words, improve the detection sensitivity) as described above. The weight ratio in the solid-phase solution depends on the concentration of the first antibody in the solid-phase solution and the concentration of the antigen in the test sample, but may be, for example, any of 1:1, 1:4, 1:9, 1:19, and 1:49 in total, which may be used as the upper or lower weight ratio.

[0040] The protein other than the antigen and the first antibody is not particularly limited as long as it can suppress non-specific adsorption of substances other than the antigen in the test sample to the first carrier when the first antibody is immobilized on the first carrier and then the test sample is applied thereto, suppress an increase in background when measuring the antigen, and lower the detection limit concentration of the antigen (in other words, improve the detection sensitivity). For example, a known blocking agent commonly used in the technical field can be mentioned. Specific examples include bovine serum albumin, casein, gelatin, hemoglobin, ovalbumin, etc. As the protein other than the antigen and the first antibody, one or more types may be used, and the composition may include this or these. For example, casein can be Block Ace (DS Pharma Biomedical Co., Ltd.).

[0041] In addition, the step (a) may include a step of subjecting the solid-phased first carrier to a blocking treatment according to a conventional method after the solidification, for example, a shaking treatment at room temperature for 2 hours using a blocking solution containing 1% (w / v) BSA and 0.05% Tween 20.

[0042] The step (b) of binding the antigen in the test sample to the first antibody via the first epitope in the antigen, which is included in the method according to this embodiment, may be a step commonly performed in the art for binding the antigen in the test sample to the first antibody via the first epitope in the antigen, for example, a step of contacting the first carrier on which the first antibody is immobilized, prepared in the step (a), with the antigen in the test sample at a predetermined temperature for a predetermined time (for example, overnight at 4°C, or one hour at room temperature).

[0043] The concentration of the antigen in the test sample is not particularly limited as long as the antigen in the test sample can be measured by the method according to the present embodiment. For example, any of 0.002 ng / mL, 0.004 ng / mL, 0.008 ng / mL, 0.016 ng / mL, 0.032 ng / mL, 0.064 ng / mL, 0.128 ng / mL, 0.256 ng / mL, 4 ng / mL, 8 ng / mL, 16 ng / mL, 32 ng / mL, 64 ng / mL, 128 ng / mL, 256 ng / mL, and 512 ng / mL may be used. The upper limit may be the upper or lower limit of the concentration. The upper limit is not particularly limited, but in clinical settings where a tumor marker for cancer is detected, it is sufficient to distinguish between a patient suffering from a disease and a healthy person, so the concentration does not need to be extremely high, for example, 1000. It may be less than ng / mL.

[0044] The S / N ratio (signal to noise ratio) achieved by the method according to this embodiment is The S / N ratio is a value obtained when the background signal is taken as 1.00, and in the case of the Examples described later, for example, the results are shown in Table 1-1 and the like. The S / N ratio is preferably 1.2 or more, more preferably 1.4 or more, and even more preferably 1.5 or more. On the other hand, although there is no particular upper limit, for example, in clinical settings such as detecting a tumor marker for cancer, it is sufficient to be able to distinguish between diseased individuals and healthy individuals, and therefore an extremely large S / N ratio is not acceptable. It is not necessary that the number be less than 1000, less than 100, or less than 54.63.

[0045] A combination of two or three selected from the group consisting of the concentration of the antigen in the test sample, the concentration of the first antibody, and the weight ratio (the first antibody: the antigen and a protein other than the first antibody) can be appropriately combined so as to obtain a larger S / N value than that of the conventional method. Cut. From this viewpoint, examples of the combination of the concentration of the first antibody and the weight ratio (the first antibody:the antigen and the protein other than the first antibody) include the following. For example, the concentration of the first antibody is 100 ng / mL or more and 1000 ng / mL or less, and the weight ratio is 1:1 or more and 1:19 or less. In addition, the concentration of the first antibody is 100 ng / mL or more and 1000 ng / mL or less, and the weight ratio is 1:9 or more and 1:19 or less, In addition, the concentration of the first antibody is 200 ng / mL or more and 1000 ng / mL or less, and the weight ratio is 1:1 or more and 1:19 or less, Further, the concentration of the first antibody is 500 ng / mL or more and 1000 ng / mL or less, and the weight ratio is 1:1 or more and 1:19 or less.

[0046] Examples of the combination of the concentration of the antigen, the concentration of the first antibody, and the weight ratio (the first antibody:the antigen and the protein other than the first antibody) are as follows. For example, when the concentration of the antigen is 0.004 ng / mL or more, The antibody concentration is from 100 ng / mL to 1000 ng / mL, and the weight ratio is from 1:1 to 1:19. Furthermore, for example, when the concentration of the antigen is 0.128 ng / mL or more, For example, the concentration of the first antibody is 100 ng / mL or more and 1000 ng / mL or less, and the weight ratio is 1:9 or more and 1:19 or less. Furthermore, for example, when the concentration of the antigen is 0.032 ng / mL or more, For example, the concentration of the first antibody is 200 ng / mL or more and 1000 ng / mL or less, and the weight ratio is 1:9 or more and 1:19 or less. In addition, when the concentration of the antigen is 0.016 ng / mL or more, For example, the concentration of the first antibody is 200 ng / mL or more and 1000 ng / mL or less, and the weight ratio is 1:9 or more and 1:19 or less. In addition, the concentration of the first antibody is 500 ng / mL or more and 1000 ng / mL or less, and the weight ratio is 1:4 or more and 1:9 or less. In any of the above cases, the upper limit of the antigen concentration is not particularly limited, and the above-mentioned values ​​can be used.

[0047] In addition, the "improvement in sensitivity compared to conventional methods" achieved by using the method according to the present embodiment is, in order of increasing preference, 2 times or more, 4 times or more, 8 times or more, and 16 times or more. The upper limit is not particularly limited, but may be, for example, 1000 times or less, 100 times or less, or 26.49 times or less. The above-mentioned "improvement in sensitivity compared to the conventional method" refers to a ratio when the S / N ratio is set to 1 when the concentration of the first antibody is a predetermined concentration, the concentrations of the antigen and proteins other than the first antibody are 0, and the concentration of the antigen is a predetermined concentration. For example, referring to Table 1-1 below, for example, in Comparative Example 1, The concentration was 100 ng / mL, the concentration of BSA was 0, and the concentration of Recombinant Human Podoplanin Fc was 100 ng / mL. If the result of "0.98" when the concentration is 0.128 ng / mL is defined as 1, the result of "2.38" when the concentration of NZ-1.2 is 100 ng / mL, the concentration of BSA is 1900 ng / mL, and the concentration of Recombinant Human Podoplanin Fc is 0.128 ng / mL in Example 1 is converted to "2.43". In this case, the "sensitivity improvement factor compared to the conventional method" is "2.43." Similarly, for example, if the result "0.81" in Comparative Example 1 where the NZ-1.2 concentration is 100 ng / mL, the BSA concentration is 0, and the Recombinant Human Podoplanin Fc concentration is 0.064 ng / mL is taken as 1, the result "1.40" in Example 1 where the NZ-1.2 concentration is 100 ng / mL, the BSA concentration is 1900 ng / mL, and the Recombinant Human Podoplanin Fc concentration is 0.064 ng / mL is converted to "1.73", and in this case the "improvement in sensitivity compared to the conventional method" is "1.73".

[0048] Furthermore, examples of the combination of the concentration of the first antibody and the weight ratio (the first antibody:the antigen and the protein other than the first antibody) include the following. For example, the improvement in sensitivity compared to the conventional method is 2-fold or more, and and the weight ratio is 1:9 or more and 1:19 or less. In addition, for example, the sensitivity is improved by 4 times or more compared to the conventional method, and The antibody concentration is 200 ng / mL or more and 500 ng / mL or less, and the weight ratio is 1:4 or more and 1:19 or less. The following are some of the reasons: In addition, the sensitivity is improved by 4 times or more compared to the conventional method, and The concentration is from 500 ng / mL to 1000 ng / mL, and the weight ratio is from 1:1 to 1:19. In addition, for example, the sensitivity is improved by 8 times or more compared to the conventional method, and The antibody concentration is 200 ng / mL or more and 500 ng / mL or less, and the weight ratio is 1:9 or more and 1:19 or less. The following are some of the reasons: In addition, the sensitivity is improved by 8 times or more compared to the conventional method, and The concentration is from 500 ng / mL to 1000 ng / mL, and the weight ratio is from 1:4 to 1:19. Also, for example, the improvement in sensitivity compared to the conventional method is 16 times or more, the concentration of the first antibody is 500 ng / mL or more and 1000 ng / mL or less, and the weight ratio is 1:4 or more and 1:9 or less. The following points can be mentioned. In any of the above cases, the upper limit of the rate of improvement in sensitivity is not particularly limited, and the above-mentioned numerical values ​​can be used.

[0049] The step (c) of detecting the antigen bound to the first antibody via the first epitope, which is included in the method according to this embodiment, may be a detection step in a method commonly used in the art, which is capable of detecting the antigen bound to the first antibody via the first epitope in the step (b).

[0050] For example, a step of labeling an antigen in the test sample in advance and detecting the antigen bound to the first antibody via the first epitope using the label can be mentioned. The label may be a label commonly used in the art, and examples thereof include labels using enzymes and labels using radioisotopes.

[0051] Examples of labeling using an enzyme include labeling using the enzyme alkaline phosphatase (AP) and labeling using the enzyme horseradish peroxidase (HRP). In addition, examples of the labeling include a labeling method in which a biotin-binding protein (such as avidin or streptavidin) labeled with the enzyme is bound to an antigen previously labeled with biotin via the biotin. The antigen bound to the first antibody via the first epitope can be detected by a method commonly used in the art, based on the type and properties of the label used.

[0052] Labeling with a radioisotope includes, for example, the radioisotope I 125 When such a label is used, the antigen bound to the first antibody via the first epitope can be detected by a method commonly used in the art, based on the type and properties of the label used.

[0053] In addition to these, examples of detection steps in methods commonly used in the technical field include a step of detecting an antigen in a surface plasmon resonance analysis method, a step of detecting an antigen in a latex agglutination method using one type of polyclonal antibody, and a step of detecting an antigen in a sandwich ELISA method. The first carrier may be subjected to a treatment that is commonly used in the technical field and is used in each of the steps.

[0054] Specifically, the surface plasmon resonance analysis method is as follows. For example, Biacore ( A method using a fluoroscopy technique ...

[0055] That is, the method comprises: (a) immobilizing the first antibody that immunologically binds to the antigen in the test sample on a sensor chip; (b) binding the antigen in the test sample to the first antibody via the first epitope in the antigen; and (c) detecting the antigen bound to the first antibody via the first epitope by measuring the refractive index of the solvent on the sensor chip surface; the step (a) is carried out in a solution containing the first antibody and a protein other than the antigen and the first antibody; This is a method for measuring an antigen in a test sample.

[0056] Specifically, the latex agglutination method using one type of polyclonal antibody is as follows: For example, the first polyclonal antibody that immunologically binds to the antigen in the test sample via an epitope in the antigen (including the first epitope) is immobilized on the first particles, the first particles are brought into contact with the test sample containing the antigen, the antigen in the test sample is bound to the first particles via the epitope, and the antigen in the test sample can be detected by a method commonly used in latex agglutination methods (for example, a method of measuring turbidity, etc.). In addition, the first polyclonal antibody binds to the antigen via the epitope, but since there are usually a plurality of first particles, the first polyclonal antibody on a certain first particle binds to the antigen via a first epitope among the epitopes, while the first polyclonal antibody on another first particle binds to the antigen via a second epitope among the epitopes. This can be easily understood by those skilled in the art.

[0057] That is, the method comprises: (a) immobilizing the first polyclonal antibody that immunologically binds to the antigen in the test sample on the first particles; (b) binding the antigen in the test sample to the first polyclonal antibody in a suspension via an epitope in the antigen; and (c) detecting the antigen bound to the first polyclonal antibody via the epitope by measuring the turbidity of the suspension; The step (a) is carried out in a solution containing the first polyclonal antibody, and a protein other than the antigen and the first polyclonal antibody. This is a method for measuring an antigen in a test sample.

[0058] Specifically, the sandwich ELISA method is as follows. The first antibody that immunologically binds to the antigen is immobilized on the first carrier, the antigen in the test sample and the first antibody are bound to each other via the first epitope in the antigen, the antigen in the test sample and a labeled second antibody that recognizes a second epitope in the antigen different from the first epitope are bound to each other via the second epitope, and the label is used to perform a method commonly used in sandwich ELISA (e.g., The method is capable of detecting an antigen in the test sample by using the above-mentioned method, such as a method for measuring fluorescence brightness.

[0059] That is, the method comprises: (a) immobilizing the first antibody that immunologically binds to the antigen in the test sample on the first carrier; (b) binding the antigen in the test sample to the first antibody via the first epitope in the antigen; and (c) binding the antigen in the test sample to a labeled second antibody that recognizes a second epitope in the antigen different from the first epitope via the second epitope, and detecting the antigen bound to the first antibody via the first epitope using the label; the step (a) is carried out in a solution containing the first antibody, the antigen, and a protein other than the first and second antibodies; This is a method for measuring an antigen in a test sample.

[0060] To describe the method in more detail, the second antibody is not particularly limited as long as it immunologically binds to the antigen in the test sample via a second epitope different from the first epitope and is labeled. The second antibody may be a monoclonal antibody or a polyclonal antibody, and can be appropriately selected depending on the embodiment. One or more types of the second antibody may be used. In addition, when the first antibody is a monoclonal antibody, the second antibody may be a labeled monoclonal antibody that recognizes the second epitope (i.e., an epitope different from the first epitope recognized by the first antibody). Also, if it can recognize the second epitope, it may be a labeled polyclonal antibody that can recognize the first epitope as well. This is because, in the latter case, the first epitope to which the first antibody binds and the second epitope to which the second antibody binds are actually different when the second antibody is detected by this method, and the first epitope and the second epitope are distinguished from each other. In addition, when the first antibody is a polyclonal antibody, the second antibody may be a labeled monoclonal antibody that recognizes an epitope that the first antibody can recognize, provided that it can recognize the second epitope. In addition, the second antibody may be a labeled polyclonal antibody that can recognize even an epitope that the first antibody can recognize, provided that it can recognize the second epitope. In the former case, the first antibody may be a labeled monoclonal antibody that recognizes an epitope that the first antibody can recognize, provided that it can recognize the second epitope. This is because the first epitope bound by the first antibody and the second epitope bound by the second antibody are actually different when the second antibody is detected by the method, and the first epitope and the second epitope are distinct.

[0061] As the second antibody, for example, when the antigen in the test sample is podoplanin and the second antibody is a monoclonal antibody (i.e., an anti-podoplanin monoclonal antibody), for example, NZ-1.2 (Medical and Biological Laboratories Co., Ltd., D320-3), Japanese Patent No. 6737482 Further examples of the antibody include AP201 described in the specification of No. 6,336,414, and the like. In addition, EPR22182 (Abcam, ab236529), PMab-1 (Abcam, ab256559), EPR7072 (Abcam, ab128994), 18H5 (Abcam, ab10288), EPR7073 (Abcam, ab131216), LpMab-7 (Abcam, ab256566), RTD4E10 (Abcam, ab11936), LF3 / B7 / D5B27 (Novus Biologicals, LLC (R&D Systems Company), NB110-96423), RTD4E10 (Abcam, ab11936), 8.1.1 (Abcam, ab92319), E-1 (SANTA CRUZ BIOTECHNOLOGY, sc-376695), F-3 (SANTA CRUZ BIOTECHNOLOGY, sc-376962), B-11 (SANTA CRUZ BIOTECHNOLOGY, sc-166906), etc. Furthermore, as the second antibody, for example, if the antigen in the test sample is podoplanin and the second antibody is a polyclonal antibody (i.e., an anti-podoplanin polyclonal antibody), examples of the second antibody include Anti-Podoplanin Antibody, Rabbit Polyclonal (Sino Biological Inc., 11065-T52) and Anti-Podoplanin antibody (Novopro, 106225). can be.

[0062] The above also applies when, for example, the antigen in the test sample is an immunoglobulin (including specific embodiments thereof) and the second antibody is a monoclonal antibody (i.e., an anti-immunoglobulin monoclonal antibody), and the second antibody can be selected by referring to the explanation regarding the first antibody already given. The same is true when, for example, the antigen in the test sample is an immunoglobulin (including specific embodiments thereof) and the second antibody is a polyclonal antibody (i.e., an anti-immunoglobulin polyclonal antibody), and the second antibody can be selected by referring to the explanation regarding the first antibody already given.

[0063] The label may be a label commonly used in sandwich ELISA, for example a fluorescent Examples of such labeling include labeling using dyes and labeling using enzymes. For example, enzyme-based labeling includes biotin-binding proteins labeled with alkaline phosphatase (AP) or horseradish peroxidase (HRP). Examples of such labels include a label in which a biotin-binding protein (e.g., avidin, streptavidin, etc.) is bound to a biotin-labeled second antibody via the biotin. In this case, the antigen bound to the first antibody via the first epitope can be detected by a method commonly used in the art based on the luminescence intensity generated by an enzymatic reaction between an enzyme bound to the second antibody and a substrate.

[0064] The step of detecting the antigen bound to the first antibody via the first epitope using the label in the step (c) may be a detection step that is commonly used in sandwich ELISA, depending on the type and properties of the label.

[0065] With regard to specific embodiments of the "protein other than the antigen and the first and second antibodies", the above-mentioned explanation of the "protein other than the antigen and the first antibody" is used to refer to the protein not including the second antibody.

[0066] Furthermore, the method according to this embodiment may include other steps before, after, or during the steps (a) to (c), such as an immunochromatography method.

[0067] Specifically, the immunochromatography method is as follows. For example, the first antibody (as a capture antibody) that immunologically binds to the antigen in the test sample via the first epitope in the antigen is immobilized on a detection line on a membrane (e.g., a cellulose membrane, etc.), and a second antibody that immunologically binds to the antigen via a second epitope in the antigen, which is different from the first epitope, and is labeled is prepared in the application section of the test sample containing the antigen. Then, the test sample containing the antigen is applied to the application section of the test sample, and the antigen and the second antibody are bound to each other via the second epitope to form a bound body, and the bound body is moved to the detection line by capillary action, and the bound body is bound to the first antibody via the first epitope. Thereafter, the antigen in the test sample can be detected by a method commonly used in immunochromatography (e.g., a method of measuring the color development of gold colloids) using the label. A third antibody that immunologically binds to the second antibody may be immobilized on a control line on the membrane, and the first antibody and the third antibody can be immobilized on the membrane by the step (a).

[0068] That is, the method comprises: (a) immobilizing the first antibody that immunologically binds to the antigen in the test sample on a detection line on a membrane; (d) providing a second antibody that immunologically binds to the antigen via a second epitope in the antigen that is different from the first epitope and that is labeled, at a portion of the membrane to which the test sample containing the antigen is applied; (e) applying the test sample containing the antigen to the application section, binding the antigen to the second antibody via the second epitope to form a conjugate, and moving the conjugate to the detection line; (b) binding the antigen in the test sample to the first antibody via the first epitope; and (c) detecting the antigen bound to the first antibody via the first epitope using the label; the step (a) is carried out using a solution containing the first antibody, the antigen, and a protein other than the first and second antibodies; This is a method for measuring an antigen in a test sample.

[0069] In this case, the method may include, prior to step (d), step (f) of immobilizing a third antibody that immunologically binds to the second antibody on a control line on the membrane in the same manner as step (a). In this case, "the first antibody, the antigen, and a protein other than the first and second antibodies" in step (a) is replaced with "a protein other than the antigen and the first, second, and third antibodies." Step (f) may be performed prior to step (a), or may be performed after step (a) as long as it is performed prior to step (d). Also, it may be performed simultaneously with step (a). Furthermore, when the method includes the step (f), the method may include, after the step (b) and before or after the step (c), or simultaneously with the step (c), a step (g) of detecting the second antibody immunologically bound to the third antibody using the label.

[0070] To describe the method in more detail, the second antibody is not particularly limited as long as it binds to the antigen in the test sample via a second epitope different from the first epitope and is labeled. The second antibody may be a monoclonal antibody or a polyclonal antibody, and can be appropriately selected depending on the embodiment. One or more types of the second antibody may be used. In addition, when the first antibody is a monoclonal antibody, the second antibody is the second A labeled monoclonal antibody that recognizes an epitope (i.e., an epitope different from the first epitope recognized by the first antibody) may be used. Also, a labeled polyclonal antibody that can recognize up to the first epitope may be used if it can recognize the second epitope. In the latter case, the first epitope to which the first antibody binds and the second epitope to which the second antibody binds are actually different when the second antibody is detected by the method, and the first epitope and the second epitope are distinguished. In addition, when the first antibody is a polyclonal antibody, the second antibody may be a labeled monoclonal antibody that recognizes an epitope that the first antibody can recognize, if it can recognize the second epitope. Also, the second antibody may be a labeled polyclonal antibody that can recognize even an epitope that the first antibody can recognize, if it can recognize the second epitope. This is because, in the former case, the first epitope to which the first antibody binds and the second epitope to which the second antibody binds are actually different when the second antibody is detected by this method, and the first epitope and the second epitope are distinguished. The same is true in the latter case.

[0071] A specific embodiment of the second antibody is the second antibody in the sandwich ELISA method. The explanation is cited.

[0072] The label may be a label that is commonly used in immunochromatography, and examples thereof include colloidal gold.

[0073] The step (c) of detecting the antigen bound to the first antibody via the first epitope using the label may be a detection step commonly used in immunochromatography, depending on the type and properties of the label.

[0074] With regard to a specific embodiment of the "protein other than the antigen and the first and second antibodies", the above explanation of the "protein other than the antigen and the first antibody" is also applicable as a protein not including the second antibody.

[0075] The third antibody is not particularly limited as long as the second antibody immunologically bound to the third antibody can be detected using the label. The third antibody may be a monoclonal antibody or a polyclonal antibody, and can be appropriately selected depending on the embodiment. One or more types of the third antibody may be used. The third antibody is, for example, an antibody against an anti-podoplanin antibody if the antigen in the test sample is podoplanin and the second antibody is an anti-podoplanin antibody, or an antibody against an anti-immunoglobulin antibody if the antigen in the test sample is immunoglobulin and the second antibody is an anti-immunoglobulin antibody.

[0076] The step (g) of detecting the second antibody immunologically bound to the third antibody using the label may be a detection step commonly used in immunochromatography, depending on the type and properties of the label.

[0077] The method according to this embodiment may also be a method using multiple types of carriers and multiple types of antibodies, for example, a method using multiple types of carriers and multiple types of monoclonal antibodies in a latex agglutination method, a method using multiple types of carriers and multiple types of polyclonal antibodies in a latex agglutination method, or a method using two types of carriers, one type of monoclonal antibody, and one type of polyclonal antibody in a latex agglutination method.

[0078] In the latex agglutination method, for example, a method using two types of carriers and two types of monoclonal antibodies is specifically as follows. For example, a first monoclonal antibody that immunologically binds to the antigen in the test sample through the first epitope in the antigen is immobilized on a first particle, and a second monoclonal antibody different from the first monoclonal antibody that immunologically binds to the antigen in the test sample through a second epitope different from the first epitope is immobilized on a second particle. Then, a suspension containing both is contacted with the test sample containing the antigen, the antigen in the test sample and the first particle are bound via the first epitope, and the antigen in the test sample and the second particle are bound via the second epitope, and the antigen in the test sample can be detected by a method usually used in the latex agglutination method (for example, a method of measuring turbidity, etc.).

[0079] That is, the method comprises: (a) immobilizing a first monoclonal antibody that immunologically binds to the antigen in the test sample on first particles; (a') immobilizing on second particles a second monoclonal antibody that immunologically binds to the antigen in the test sample and recognizes a second epitope different from the first epitope recognized by the first monoclonal antibody; (b) allowing the antigen in the test sample to bind to the first monoclonal antibody via the first epitope in a suspension; (b') binding the antigen in the test sample to the second monoclonal antibody via the second epitope in the suspension; and (c) detecting an antigen that binds to the first monoclonal antibody via the first epitope and to the second monoclonal antibody via the second epitope by measuring the turbidity of the suspension; The step (a) is carried out in a solution containing the first monoclonal antibody, the antigen, and a protein other than the first and second monoclonal antibodies; and the step (a') is carried out in a solution containing the second monoclonal antibody, the antigen, and a protein other than the first and second monoclonal antibodies; The order of steps (a) and (a') does not matter. Step (b) and step (b') may be performed in any order or simultaneously. This is a method for measuring an antigen in a test sample.

[0080] To describe the method in more detail, the second monoclonal antibody is not particularly limited as long as it immunologically binds to the antigen in the test sample via a second epitope that is different from the first epitope. One or more types of the second antibody may be used.

[0081] A specific embodiment of the second monoclonal antibody is The explanation for the second antibody being a monoclonal antibody will be repeated.

[0082] For specific embodiments of the second particles, the above description of the case where the "first carrier" is a particle is used. The first particles and the second particles may be the same or different, but are preferably the same.

[0083] As for a specific embodiment of "a protein other than the antigen and the first and second monoclonal antibodies," the above explanation of "a protein other than the antigen and the first antibody" is used to refer to "a protein other than the antigen and the first monoclonal antibody" which does not further include the second monoclonal antibody.

[0084] In the latex agglutination method, for example, a method using two types of carriers and two types of polyclonal antibodies is specifically as follows. For example, a first polyclonal antibody that immunologically binds to the antigen in the test sample through the first epitope in the antigen is immobilized on a first particle, and a second polyclonal antibody different from the first polyclonal antibody that immunologically binds to the antigen in the test sample through a second epitope different from the first epitope is immobilized on a second particle. Then, a suspension containing both is contacted with the test sample containing the antigen, the antigen in the test sample and the first particle are bound via the first epitope, and the antigen in the test sample and the second particle are bound via the second epitope, and the antigen in the test sample can be detected by a method usually used in the latex agglutination method (for example, a method of measuring turbidity, etc.).

[0085] That is, the method comprises: (a) immobilizing a first polyclonal antibody that immunologically binds to the antigen in the test sample on first particles; (a') immobilizing on second particles a second polyclonal antibody that immunologically binds to the antigen in the test sample and recognizes a second epitope different from the first epitope recognized by the first polyclonal antibody; (b) allowing the antigen in the test sample to bind to the first polyclonal antibody via the first epitope in a suspension; (b') binding the antigen in the test sample to the second polyclonal antibody via the second epitope in the suspension; and (c) detecting an antigen that binds to the first polyclonal antibody via the first epitope and to the second polyclonal antibody via the second epitope by measuring the turbidity of the suspension; The step (a) is carried out in a solution containing the first polyclonal antibody, the antigen, and a protein other than the first and second polyclonal antibodies; and the step (a') is carried out in a solution containing the second polyclonal antibody, the antigen, and a protein other than the first and second polyclonal antibodies; The order of steps (a) and (a') does not matter. Step (b) and step (b') may be performed in any order or simultaneously. This is a method for measuring an antigen in a test sample.

[0086] To describe the method in more detail, the second polyclonal antibody is not particularly limited as long as it immunologically binds to the antigen in the test sample via a second epitope that is different from the first epitope. One or more types of the second antibody may be used.

[0087] A specific embodiment of the second polyclonal antibody is The explanation for the case where the second antibody is a polyclonal antibody will be repeated.

[0088] For specific embodiments of the second particles, the above description of the case where the "first carrier" is a particle is used. The first particles and the second particles may be the same or different, but are preferably the same.

[0089] As for specific embodiments of "proteins other than the antigen and the first and second polyclonal antibodies," the above explanation of "proteins other than the antigen and the first antibody" is used to refer to "proteins other than the antigen and the first polyclonal antibody" that do not further include the second polyclonal antibody.

[0090] In the latex agglutination method, for example, a method using two types of carriers, one type of monoclonal antibody, and one type of polyclonal antibody is specifically as follows. For example, a monoclonal antibody that immunologically binds to an antigen in the test sample through the first epitope in the antigen is immobilized on a first particle, and a polyclonal antibody that immunologically binds to an antigen in the test sample through a second epitope different from the first epitope is immobilized on a second particle. Then, a suspension containing both is contacted with the test sample containing the antigen, the antigen in the test sample is bound to the first particle through the first epitope, and the antigen in the test sample is bound to the second particle through the second epitope, and the antigen in the test sample can be detected by a method usually used in the latex agglutination method (for example, a method of measuring turbidity, etc.).

[0091] That is, the method comprises: (a) immobilizing a monoclonal antibody that immunologically binds to the antigen in the test sample onto first particles; (a') immobilizing on second particles a polyclonal antibody that immunologically binds to the antigen in the test sample and recognizes a second epitope different from the first epitope recognized by the monoclonal antibody; (b) binding the antigen in the test sample to the monoclonal antibody via the first epitope in a suspension; (b') binding the antigen in the test sample to the polyclonal antibody via the second epitope in the suspension; and (c) detecting an antigen that binds to the monoclonal antibody via the first epitope and to the polyclonal antibody via the second epitope by measuring the turbidity of the suspension; The step (a) is carried out in a solution containing the monoclonal antibody and "a protein other than the antigen, the monoclonal antibody, and the polyclonal antibody", and the step (a') is carried out in a solution containing the polyclonal antibody and "a protein other than the antigen, the monoclonal antibody, and the polyclonal antibody"; The order of steps (a) and (a') does not matter. Step (b) and step (b') may be performed in any order or simultaneously. This is a method for measuring an antigen in a test sample.

[0092] To describe the method in more detail, the polyclonal antibody is not particularly limited as long as it immunologically binds to the antigen in the test sample via a second epitope different from the first epitope. One or more types of the second antibody may be used.

[0093] In addition, the polyclonal antibody may be a polyclonal antibody capable of recognizing the first epitope as long as it can recognize the second epitope, because the first epitope to which the monoclonal antibody binds and the second epitope to which the polyclonal antibody binds are actually different when the antigen is detected by the method of the present invention, and the first epitope and the second epitope are distinguished from each other. Furthermore, the monoclonal antibody may be a monoclonal antibody that recognizes an epitope that can be recognized by the polyclonal antibody, so long as it can recognize the first epitope, because the first epitope to which the monoclonal antibody binds and the second epitope to which the polyclonal antibody binds are actually different when the antigen is detected by the method, and the first epitope and the second epitope are distinguished from each other.

[0094] A specific embodiment of the polyclonal antibody is the second antibody in the sandwich ELISA method. The same explanation as for the case where the antibody is a polyclonal antibody is used.

[0095] For specific embodiments of the second particles, the above description of the case where the "first carrier" is a particle is used. The first particles and the second particles may be the same or different, but are preferably the same.

[0096] As for a specific embodiment of "a protein other than the antigen, the monoclonal antibody, and the polyclonal antibody," the above explanation of "a protein other than the antigen and the first antibody" is used to refer to a "protein other than the antigen and the monoclonal antibody" that does not further include the polyclonal antibody.

[0097] In addition, in the multi-assay method, for example, there is a method using multiple types of carriers and multiple types of antibodies. For example, in the Luminex method, for example, a method using two types of carriers and two types of antibodies is specifically as follows. For example, a first antibody that immunologically binds to a first antigen in the test sample is immobilized on a first particle. Separately, a second antibody that immunologically binds to a second antigen in the test sample is immobilized on a second particle. The first antigen in the test sample and the first antibody are bound via a first epitope in the first antigen, and the second antigen in the test sample and the second antibody are bound via a first epitope in the second antigen. Next, the first antigen in the test sample and a labeled third antibody that recognizes a second epitope different from the first epitope in the first antigen are bound via the second epitope in the first antigen. In addition, the second antigen in the test sample is bound to the labeled fourth antibody, which recognizes a second epitope in the second antigen that is different from the first epitope, via the second epitope in the second antigen, and the antigen in the test sample can be detected by a method commonly used in the Luminex method (e.g., a method of measuring fluorescence brightness) using the label. The first particle and the second particle contain a fluorescent dye that is excited by excitation light of the same wavelength, and contain the fluorescent dye so that they can be distinguished by the brightness of the fluorescence emitted when excited by the excitation light. An example of the latter is that the first particle and the second particle have different fluorescent dye contents. Furthermore, when measuring a third antigen in the test sample, the above explanation can be used to refer to the above, and the third antibody that immunologically binds to the third antigen in the test sample via the first epitope, the third particle, and a labeled fifth antibody that recognizes a second epitope different from the first epitope in the third antigen can be used to measure the third antigen. This also applies to the measurement of a fourth antigen, a fifth antigen, and subsequent antigens in the test sample.

[0098] That is, the method includes, for example, a method for measuring the first and second antigens in the test sample, (a) immobilizing a first antibody that immunologically binds to a first antigen in the test sample on first particles; (a') immobilizing a second antibody that immunologically binds to a second antigen in the test sample on second particles; (b) binding the first antigen in the test sample to the first antibody via a first epitope in the first antigen; (b') binding the second antigen in the test sample to the second antibody via the first epitope in the second antigen; and (c) the first antigen in the test sample and the first epitope in the first antigen. a labeled third antibody that recognizes a second epitope different from the first antigen is bound to the first antigen via the second epitope; and allowing the second antigen in the test sample to bind to the labeled fourth antibody, which recognizes a second epitope in the second antigen that is different from the first epitope, via the second epitope in the second antigen; detecting, using the label, the first antigen bound to the first antibody via the first epitope in the first antigen, and the second antigen bound to the third antibody via the first epitope in the second antigen; the step (a) is carried out in a solution containing the first antibody, the first antigen, and a protein other than the first, second, third, and fourth antibodies; and the step (a') is carried out in a solution containing the third antibody and a protein other than the antigen and the first, second, third and fourth antibodies; The order of steps (a) and (a') does not matter. Step (b) and step (b') may be performed in any order or simultaneously; the first particle and the second particle are excited with excitation light of the same wavelength and contain different amounts of fluorescent dye; This is a method for measuring an antigen in a test sample.

[0099] To describe this method in more detail, when focusing on a series of steps up to detection of the first antigen in the test sample, the above-mentioned sandwich ELISA method is performed using the first antigen as a carrier. This corresponds to the case where the above particles are used. The same applies to a series of steps up to the detection of the second antigen in the test sample. Therefore, for details, the above explanation of the sandwich ELISA method is used.

[0100] Regarding the specific aspects of the first particle and the second particle, the above description of the case where the "first carrier" is a particle is used, except that the first particle and the second particle contain a fluorescent dye that is excited by excitation light of the same wavelength, and contain the fluorescent dye so that they can be distinguished by the brightness of the fluorescence emitted when excited by the excitation light. For example, it is preferable that the first particle and the second particle are the same type, except that they contain a fluorescent dye that is excited by excitation light of the same wavelength, and the fluorescent dye content is different.

[0101] With regard to a specific embodiment of "proteins other than the antigen and the first, second, third, and fourth antibodies", the above explanation of "proteins other than the antigen and the first antibody" is also applied to proteins excluding the second, third, and fourth antibodies.

[0102] <Method of manufacturing solid-phase antibodies> Another aspect of the present invention is The method includes a step of immobilizing an antibody that immunologically binds to an antigen in a test sample on a carrier, The solid-phase step is carried out in a solution containing the antibody, the antigen, and a protein other than the antibody. This is a method for producing a solid-phase antibody that immunologically binds to an antigen in a test sample.

[0103] The "step of immobilizing an antibody that immunologically binds to an antigen in a test sample on a carrier" included in this embodiment is the same as the step (a) in the "method of measuring an antigen in a test sample" according to the previous embodiment. In this case, the "antibody" and the "carrier" in this step correspond to the "first antibody" and the "first carrier" in the step (a), respectively.

[0104] In addition, with regard to "the step of immobilizing the antibody in a solution containing the antibody, the antigen, and a protein other than the antibody," it is understood that "the step (a) is performed in a solution containing the first antibody, the antigen, and a protein other than the antibody" in the "method of measuring an antigen in a test sample" according to the above embodiment. The explanation for "the method for measuring an antigen in a test sample" is incorporated herein by reference. In this case, the "antibody" in this step corresponds to the "first antibody" in the "method for measuring an antigen in a test sample" according to the above embodiment. EXAMPLES

[0105] The present invention will be specifically described below using examples, but the present invention is not limited to these examples.

[0106] [Example 1] Sandwich ELISA method Anti-podoplanin antibody NZ-1.2 (MBL, N320-3) as the first antibody was diluted with D-PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 100 ng / mL, 200 ng / mL, 500 ng / mL, or 1000 ng / mL. At this time, BSA Reagent grade (Proliant, PRL68700-1) was added so that the weight ratio of NZ-1.2:BSA was 1:1, 1:4, 1:9, or 1:19 to prepare a solid-phase solution. The solid-phase solution was added to a white microtiter plate (Thermo Fisher Scientific, 436110) at 100 μL / well, and the plate was left to stand at 4° C. overnight to solidify NZ-1.2 onto the plate. The plate was washed four times with PBS-T, and a blocking solution containing 1% (w / v) BSA and 0.05% Tween 20 was added, followed by shaking at room temperature for 2 hours for blocking. Thereafter, the plate was washed four times with PBS-T to obtain a plate on which NZ-1.2 was immobilized.

[0107] Recombinant Human Podoplanin Fc (R&D Systems, 3670-PL-050) was added to commercially available healthy volunteer blood samples. The test samples were prepared by diluting the samples 50-fold with a mixture of dilution buffer (0.098% (w / v) BSA, 0.0147 mol / L arginine, 0.00431% sodium azide, 0.049% SDS, 0.049% Tween 80, and 0.56% Lipidure BL802 (NOF Corporation)). Thereafter, the test sample was added to the plate prepared above, and the plate was left to stand at 4°C for a whole day and night. Afterwards, the plate was washed four times with PBS-T. Recombinant Human Podoplanin Fc was diluted as described above. Therefore, the termination of Recombinant Human Podoplanin Fc in the test sample when it was added to the plate was The concentrations were 0.004 ng / mL, 0.008 ng / mL, 0.016 ng / mL, 0.032 ng / mL, 0.064 ng / mL, and 0.128 ng / mL, respectively. To this was added biotin-labeled anti-podoplanin antibody AP201-F(ab')2 as a second antibody, which had been diluted with 0.5% Lipidure BL802 / PBS to a final concentration of 800 ng / mL, and the mixture was shaken at room temperature for 1 hour. After that, the plate was washed six times with PBS-T. Then, HRP solution (final concentration 200 ng / mL Poly-HRP labeling) was added. Streptavidin (Fitzgerald, 85R-202), 5% (w / v) BSA, 200 mM arginine, 0.05% Tween 20) was added and shaken at room temperature for 20 minutes. Then, the plate was washed nine times with PBS-T. Super Signal ELISA Pico (Thermo Fisher Scientific, 37069) was added as a luminescence reagent, and the reaction was carried out at room temperature for 10 minutes, and the luminescence intensity was measured using a microplate reader (Molecular Devices, Spectra MAX iD3).

[0108] [Comparative Example 1] The procedure was the same as in Example 1, except that the solid-phase solution was prepared without adding BSA Reagent grade. .

[0109] [Comparative Example 2] The same procedure as in Example 1 was used except that the solid-phase solution did not contain NZ-1.2 or BSA Reagent grade. It was like this.

[0110] [result] The results are shown in Tables 1-1 to 1-4. The results in the tables are calculated assuming the background to be 1.00 under each condition, and represent the S / N ratio.

[0111] [Table 1-1]

[0112] [Table 1-2]

[0113] [Table 1-3]

[0114] [Table 1-4]

[0115] In Example 1, NZ-1.2 was diluted with D-PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 500 ng / mL, and BSA Reagent grade was added so that the BSA concentrations in the solid-phase solution were 0.00005% (w / v), 0.0002% (w / v), 0.00045% (w / v), and 0.00095% (w / v) (the weight ratios of NZ-1.2:BSA were 1:1, 1:4, 1:9, and 1:19, respectively). Figures 1 and 2 show the results when Recombinant Human Podoplanin Fc was dissolved in commercially available serum from a healthy subject to a final concentration of 6.4 ng / mL (the final concentration of Recombinant Human Podoplanin Fc in the test sample when added to the plate was 0.128 ng / mL). The results in each figure are shown as relative values ​​when the result of Comparative Example 1 is taken as 100%. The difference between Figures 1 and 2 is whether the values ​​on the horizontal axis are "BSA concentration in the solid-phase solution (% (w / v))" or "weight ratio (antibody NZ-1.2:BSA)." In Example 1, NZ-1.2 was diluted with D-PBS to a final concentration of 100 ng / mL, BSA Reagent grade was added to the solid-phase solution to give BSA concentrations of 0.00001% (w / v), 0.00004% (w / v), 0.00009% (w / v), and 0.00019% (w / v) (weight ratios of NZ-1.2:BSA of 1:1, 1:4, 1:9, and 1:19, respectively), and Recombinant Human Podoplanin Fc was dissolved in commercially available serum from a healthy subject to a final concentration of 6.4 ng / mL (the final concentration of Recombinant Human Podoplanin Fc in the test sample when added to the plate was 0.128 ng / mL). The results are shown in Figures 3 and 4. Each result is shown as a relative value when Comparative Example 1 is taken as 100%. The difference between Figures 3 and 4 is whether the value on the horizontal axis is "BSA concentration in the solid-phase solution (% (w / v)" or "weight ratio (antibody NZ-1.2:BSA)."

[0116] In Example 1, NZ-1.2 was diluted with D-PBS to a final concentration of 100 ng / mL, 200 ng / mL, 500 ng / mL, or 1000 ng / mL, and BSA Reagent grade was added so that the weight ratio of NZ-1.2:BSA was 1:9 (the BSA concentration in the solid-phase solution was 0.00009% (w / v), 0.00018% (w / v), respectively). , 0.00045% (w / v), 0.0009% (w / v) (black bars in FIG. 5 ), or without addition ( FIG. 5 The results are shown in Figure 5 when recombinant human podoplanin Fc was dissolved in commercially available serum from healthy subjects to a final concentration of 6.4 ng / mL (the final concentration of recombinant human podoplanin Fc in the test sample when added to the plate was 0.128 ng / mL). As shown in Figure 5, when BSA Reagent grade was added, the same detection sensitivity was achieved with approximately one-fifth the amount of antibody compared to when BSA Reagent grade was not added. Specifically, it was found that the same effect was obtained for the white bar at a final concentration of 1000 ng / mL (BSA concentration in the solid-phase solution is 0.0009% (w / v)) and the black bar at a final concentration of 200 ng / mL (BSA concentration in the solid-phase solution is 0.00018% (w / v)).

[0117] In Example 1, NZ-1.2 was diluted with D-PBS to a final concentration of 500 ng / mL, and BSA Reagent grade was added so that the weight ratios of NZ-1.2:BSA were 1:1, 1:4, 1:9, and 1:19 (the BSA concentrations in the solid-phase solution were 0.00005% (w / v), 0.0002% (w / v), 0.00045% (w / v), and 0.00095% (w / v), respectively). Recombinant Human Podoplanin Fc was added to commercially available healthy subject serum to a final concentration of 6.4 ng / mL (final concentration of Recombinant Human Podoplanin Fc in the test sample when added to the plate) The background results when the sample was dissolved at a concentration of 0.128 ng / mL are shown in Figures 6 and 7. The difference between Figures 6 and 7 is whether the horizontal axis value is "BSA concentration in the solid-phase solution (% (w / v)" or "weight ratio (antibody NZ-1.2:BSA)." As shown in Figures 6 and 7, when the BSA concentration in the immobilization solution is, for example, 0.0002% (w / v) to 0.00095% (w / v) (when the weight ratio (antibody NZ-1.2:BSA) is, for example, 1:4 to 1:19), the background is large. It was found that the concentration of

[0118] [Example 2] The same procedure was followed as in Example 1, except for the following: The first antibody, anti-podoplanin antibody NZ-1.2 (Medical and Biological Laboratories, Inc., N320-3), was diluted with D-PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 500 ng / mL, and BSA Reagent grade (Proliant Biologicals, Inc., PRL68700-1) was diluted with a final concentration of 4500 ng / mL (BSA concentration in the solid-phase solution is 0.00045% (w / v)) to prepare a solid-phase solution. Recombinant Human Podoplanin Fc (R&D, 3670-PL-050) was used at a final concentration of 0.2 ng / mL, 0.4 ng / mL, 0.8 ng / mL, 1.6 ng / mL, 3.2 ng / mL, 6.4 ng / mL, 12.8 ng / mL, or 25.6 ng / mL. In order to obtain a standard curve sample, commercially available healthy subject serum (Cosmo Bio Co., Ltd.) was dissolved in the serum in order to obtain a standard curve sample. In the test, the standard curve sample was diluted 100-fold with the dilution buffer used in Example 1. Because the standard curve samples were diluted in this manner, the final concentrations of Recombinant Human Podoplanin Fc in the standard curve samples were 0.002 ng / mL, 0.004 ng / mL, 0.008 ng / mL, 0.016 ng / mL, 0.032 ng / mL, 0.064 ng / mL, 0.128 ng / mL, and 0.256 ng / mL, respectively. In addition, plasma from a patient with stage I, III, or IV lung cancer (KAC Corporation) was diluted 100-fold with the dilution buffer used in Example 1 to prepare a test sample (test sample from a lung cancer patient). A detection test for planin was performed.

[0119] Serum or plasma from a healthy subject (KAC Corporation and Cosmo Bio Co., Ltd., respectively) was diluted 100-fold with the dilution buffer used in Example 1 to prepare test samples (test samples from healthy subjects). A podoplanin detection test was then performed.

[0120] [Comparative Example 3] The test samples used were from lung cancer patients and healthy subjects, as prepared in Example 2. A commercially available podoplanin quantification ELISA kit (Ray Bio's Podoplanin ELISA Kit) was used. In accordance with the package insert, a podoplanin detection test was performed by diluting the clinical specimen 15 times (recommended conditions based on the package insert).

[0121] [result] The results of Example 2 are shown in Figure 8, and the results of Comparative Example 3 are shown in Figure 9. In Comparative Example 3, false positives were detected and the amount of podoplanin in the blood could not be measured, whereas in Example 2, no false positives were detected in samples from healthy subjects, and a clearly higher concentration of podoplanin could be detected in the plasma of lung cancer patients than in healthy subjects.

[0122] [Example 3] The first antibody, anti-Goat Anti-Mouse IgG Fc antibody (Abcam, ab97261), was diluted with D-PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 200 ng / mL. At this time, the final concentration was 1800 ng / mL (weight ratio (anti-Goat Anti-Mouse IgG Fc antibody:casein) was 1:9, and the solid phase Block Ace (DS Pharma Biomedical Co., Ltd.) was added as casein to prepare a solid-phase solution so that the casein concentration in the solution was 0.00018% (x / v). The solid-phase solution was added to a white microtiter plate (Thermo Fisher Scientific, 436110) at 100 μL / well and allowed to stand overnight at 4° C., and the plate was coated with anti-Goat Anti-Mouse IgG Fc antibody. The plate was then washed four times with PBS-T, and a blocking solution containing 1% (w / v) Block Ace and 0.05% to Tween 20 was added, and the plate was shaken at room temperature for 2 hours for blocking. The plate was then washed four times with PBS-T to obtain a plate on which the anti-Goat Anti-Mouse IgG Fc antibody was immobilized.

[0123] Mouse IgG1 (mPG4D1) was diluted to a final concentration of 4 ng / mL, 8 ng / mL, 16 ng / mL, 32 ng / mL, 64 ng / mL, 128 ng / mL, 256 ng / mL, or 512 ng / mL in CD Forti CHO medium containing 0.05% Tween 20. The test samples were then diluted in PBS (Thermo Fisher Scientific) to prepare test samples. The test samples were then added to the plate prepared above and shaken at room temperature for 1 hour. The plate was then washed 4 times with PBS-T. . To this was added HRP-labeled anti-mouse IgG (Fab')2 antibody (Abcam, Inc.) as the second antibody, which was diluted with a solution containing 0.5% (w / v) Block Ace and 0.05% Tween 20 to a final concentration of 10 ng / mL. The plate was then shaken at room temperature for 20 minutes, and washed six times with PBS-T. Lumiflash Prime (Visual Protein, LF01-500) was added as a photoreagent, and the mixture was incubated at room temperature for 3 minutes. a Luminescence intensity was measured using MAX iD3).

[0124] [Comparative Example 4] The procedure was the same as in Example 3, except that the solid phase solution was prepared without adding Block Ace.

[0125] [Comparative Example 5] The same procedure as in Example 3 was carried out except that no test sample was used (i.e., the concentration of mouse IgG1 (mPG4D1) was zero).

[0126] [Comparative Example 6] The same procedure as in Comparative Example 4 was carried out except that no test sample was used (i.e., the concentration of mouse IgG1 (mPG4D1) was zero).

[0127] [result] The results of Example 3 are shown in FIG. 10, and the results of Comparative Example 4 are shown in FIG. The use of casein also confirmed an improvement in detection sensitivity.

[0128] In addition, in Example 3, mouse IgG1 (mPG4D1) was 512 ng / mL, and in Comparative Example 4, The results for the case where mouse IgG1 (mPG4D1) was 512 ng / mL are shown in FIG. Moreover, the results of Comparative Examples 5 and 6 are shown in Fig. 13. From the results in Fig. 13, it was confirmed that the detection sensitivity was improved and the background was reduced.

[0129] [Example 4] Mouse IgG1 antibody (mPG4D1) prepared from hybridoma culture supernatant as the first antibody was diluted with D-PBS (Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, or 1000 ng / mL. At this time, BSA Reagent grade (Proliant, PRL68700-1) was added so that the weight ratio of mouse IgG1 antibody:BSA was 1:1, 1:4, 1:9, 1:19, or 1:49 to prepare a solid-phase solution. The solid-phase solution was added to a black microtiter plate (Thermo Fisher Scientific, 437111) at 100 μL / well and left to stand at 4° C. overnight. The mouse IgG1 antibody was then immobilized on the plate. The plate was then washed four times with PBS-T. In this manner, a plate on which the mouse IgG1 antibody was immobilized was obtained.

[0130] To semi-quantitate the immobilized mouse IgG1 antibody, HRP-labeled anti-mouse IgG (Fab')2 antibody (Abcam, ab98659) was diluted with 1% (w / v) BSA and 0.05% Tween 20 to a final concentration of 1 ng / mL. ) was added and shaken at room temperature for 1 hour. Then, the plate was washed six times with PBS-T. Then, the plate was washed with luminescence reagent and Lumiflash Prime (Visual Protein, LF01-500) was then added, and the mixture was incubated at room temperature for 3 minutes. Luminescence was measured using a microplate reader (Molecular Devices, Spectra MAX iD3). The strength was measured.

[0131] [Comparative Example 7] The procedure was the same as in Example 4, except that the solid-phase solution was prepared without adding BSA Reagent grade. .

[0132] [result] The results are shown in Table 2 when the emission intensity of Comparative Example 7 is set to 1.0. The final results of the German ELISA method were obtained. In other words, the performance evaluation results of the analysis system including the overall effects of the first antibody (NZ-1.2), the antigen to be detected (Recombinant Human Podoplanin Fc), and a protein other than the first antibody (BSA) were obtained. On the other hand, in Example 4, since no antigen to be detected was used, the amount of the first antibody immobilized on the plate was evaluated.

[0133] [Table 2]

Claims

1. (a) immobilizing a first antibody that immunologically binds to an antigen in a test sample on a first carrier; (b) binding an antigen in the test sample to the first antibody via a first epitope in the antigen; and (c) detecting the antigen bound to the first antibody via the first epitope; the step (a) is carried out in a solution containing the first antibody and a protein other than the antigen and the first antibody; the concentration of the first antibody in the solution is 200 ng / mL or more and 500 ng / mL or less, the weight ratio (the first antibody:the antigen and the protein other than the first antibody) is 1:4 or more and 1:19 or less, and the concentration of the antigen in the test sample in the step (b) is 0.016 ng / mL or more. or The concentration of the first antibody in the solution is 500 ng / mL or more and 1000 ng / mL or less, a quantitative ratio (the first antibody:the antigen and the protein other than the first antibody) of 1:4 to 1:19, and a concentration of the antigen in the test sample in the step (b) is 0.008 ng / mL or less. Above, The protein other than the antigen and the first antibody is one or more selected from the group consisting of bovine serum albumin, casein, gelatin, hemoglobin, and ovalbumin. A method for measuring an antigen in a test sample.

2. The method of claim 1 , wherein the first antibody is an anti-podoplanin antibody and the antigen is podoplanin.

3. the step (c) is a step of binding an antigen in the test sample to a labeled second antibody that recognizes a second epitope in the antigen different from the first epitope, via the second epitope, and detecting the antigen bound to the first antibody via the first epitope using the label; The method of claim 1.

4. The method includes a step of immobilizing an antibody that immunologically binds to an antigen in a test sample on a carrier, the solid-phase step is carried out in a solution containing the antibody, the antigen, and a protein other than the antibody; The antibody concentration in the solution is 50 ng / mL or more and 500 ng / mL or less, and the weight ratio ( the ratio of the antibody to the antigen and the protein other than the antibody is 1:1 or more and 1:19 or less; or 、 The antibody concentration in the solution is 500 ng / mL or more and 1000 ng / mL or less, and the weight ratio ( the ratio of the antibody:the antigen and the protein other than the antibody is 1:1 or more and 1:9 or less, The protein other than the antigen and the antibody is one or more selected from the group consisting of bovine serum albumin, casein, gelatin, hemoglobin, and ovalbumin. A method for producing a solid-phase antibody that immunologically binds to an antigen in a test sample.

5. The method according to claim 4 , wherein the antibody is an anti-podoplanin antibody and the antigen is podoplanin.

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