Reagent for detecting or measuring serine protease

Abstract

To provide a novel reagent and method for detecting or measuring free and bound serine protease in a sample with high sensitivity and accuracy in a wide measurement range from low to high concentrations.SOLUTION: The reagent for measurement includes: (A) a surface active agent; and (B) latex particles to which an antibody against the serine protease or an antigen-binding fragment thereof is immobilized. The average particle diameter of the latex particles is in the range of 250 nm to 410nm.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a reagent for detecting or measuring a serine protease. [Background technology]

[0002] In the fields of medicine and clinical testing, attempts have been actively made to detect or measure trace amounts of specific substances related to diseases in biological samples such as blood with high accuracy and to judge the onset and severity of diseases. Immunological measurement methods that utilize antigen-antibody reactions are widely known as methods for detecting or measuring specific substances in biological samples.

[0003] In biological samples, substances are known to exist in a free state (free form) and in a bound form bound to a corresponding binding molecule. For example, trypsin is a type of serine protease, but it exists in the blood both as a free form and as a bound form with α1-antitrypsin, etc., and since the blood concentration of trypsin is higher in patients suffering from diseases such as pancreatic disease than in healthy people, it is useful as a marker for pancreatic disease. As another example, prostate-specific antigen (PSA), a type of serine protease, is known as a tumor marker for prostate cancer, but it is known to exist in the blood not only as a free form of PSA, but also as a bound form of PSA and α1-antichymotrypsin, etc.

[0004] For such substances, the ratio of the amount of free form to the amount of conjugate changes depending on the amount of binding molecule, the environment in the body, etc., so when measuring, it is necessary to measure both the free form and the conjugate. However, when measuring such substances by immunological measurement methods, the antibody binding rate differs between the free form and the conjugate, which makes it difficult to measure accurately.

[0005] As a conventional technique for solving such problems, for example, Patent Document 1 proposes a measurement method including a step (1) of reacting a specimen containing free and bound forms of the substance to be measured with latex 1 onto which monoclonal antibody 1 against the substance to be measured is immobilized to obtain reactant 1, and a step (2) of reacting reactant 1 with latex 2 onto which monoclonal antibody 2, which has a recognition site different from that of antibody 1 for the substance to be measured, is immobilized to obtain reactant 2.

[0006] Patent Document 2 proposes a PSA measurement reagent and a measurement method using the same, which comprises latex 1 to which monoclonal antibody 1 reactive with both free and conjugated PSA is immobilized, and latex 2 to which monoclonal antibody 2, which has a recognition site for PSA different from that of antibody 1 and is reactive with both free and conjugated PSA, is immobilized and has an average particle size different from that of latex 1.

[0007] In addition, as a reagent for measuring a trace amount of a specific substance related to a disease, it is important that the normal range of the substance can be accurately measured, and that the measurement range also includes high and low concentrations outside the normal range that are observed in samples derived from patients with the disease, etc. However, some measurement reagents have a narrow measurement range, making it difficult to accurately measure low and high concentrations of the target substance. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP 2007-163319 A [Patent Document 2] JP 2005-106609 A Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a novel reagent and method for detecting or measuring both free and bound serine proteases in a sample with high sensitivity and accuracy over a wide measurement range from low to high concentrations. [Means for solving the problem]

[0010] As a result of intensive research to solve the above-mentioned problems, the inventors have discovered that by detecting and measuring serine proteases in a sample using an antibody immobilized on latex particles having a specified particle size in the presence of a specified surfactant, the serine proteases can be released from their conjugates, and that all or a part of the immunologically measurable serine proteases contained in a sample can be detected or measured with high sensitivity and accuracy over a wide measurement range from low to high concentrations, regardless of the form they are in (free form, conjugate) and the ratio of their abundance, thereby completing the present invention. That is, the present invention includes the following aspects. [1] A reagent for detecting or measuring a serine protease that may exist in a living body in the form of a conjugate with a corresponding binding molecule, comprising: A and B below: (A) a surfactant, (B) Latex particles to which an antibody against the serine protease or an antigen-binding fragment thereof has been immobilized. Including, The average particle diameter of the latex particles is 250 to 410 nm. reagent. [2] The reagent according to item 1, wherein the content of the surfactant is 0.02% by weight or more. [3] The reagent according to item 1, Reagent A1 and Reagent B1 below: (A1) Reagent A1 containing a surfactant; (B1) Reagent B1, comprising latex particles on which an antibody against the serine protease or an antigen-binding fragment thereof is immobilized. Including, The average particle diameter of the latex particles is 250 to 410 nm, When measuring a sample, the sample, the reagent A1 and the reagent B1 are mixed. reagent. [4] The reagent according to item 3, wherein the content of the surfactant in the mixture of the sample, A1, and B1 is 0.02% by weight or more when the sample is measured. [5] The reagent according to any one of items 1 to 4, wherein the average particle diameter of the latex particles is 260 to 400 nm. [6] The reagent according to any one of items 1 to 5, wherein the surfactant comprises at least one selected from the group consisting of nonionic surfactants and amphoteric surfactants. [7] The reagent according to any one of items 1 to 5, wherein the surfactant comprises at least one selected from the group consisting of polyoxyethylene(20)sorbitan monolaurate, polyoxyethylenesorbitan monooleate, polyethylene glycol mono-4-octylphenyl ether, octylphenoxypoly(ethyleneoxy)ethanol, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. [8] The reagent according to any one of items 1 to 5, wherein the surfactant comprises polyoxyethylene (20) sorbitan monolaurate. [9] The reagent according to any one of items 1 to 8, wherein the serine protease is selected from the group consisting of trypsin, elastase, and prostate-specific antigen.

[10] The reagent according to any one of items 1 to 8, wherein the serine protease is trypsin.

[11] The reagent according to any one of items 1 to 10, for use in diagnosing a disease associated with serine protease.

[12] The reagent described in Item 11, wherein the serine protease is trypsin and the disease associated with the protease is a pancreatic disease.

[13] A method for detecting or measuring a serine protease that may exist in a living body in the form of a complex with a corresponding binding molecule, comprising the steps of: (1) Contacting a sample with the reagent according to any one of items 1 to 12; (2) Detecting or measuring serine proteases A method comprising:

[14] The method according to claim 13, wherein the sample is serum or plasma.

[15] A method for diagnosing a disease associated with a serine protease that may exist in a living body in the form of a conjugate with a corresponding binding molecule, comprising: (1) Contacting a sample with the reagent according to any one of items 1 to 12; (2) detecting or measuring serine proteases; (3) determining the state of a disease associated with the serine protease based on the obtained detection results or measured values; A method comprising:

[16] A method for assisting in the diagnosis of a disease associated with a serine protease that may exist in vivo in the form of a conjugate with a corresponding binding molecule, comprising: (1) Contacting a sample with the reagent according to any one of items 1 to 12; (2) detecting or measuring serine proteases; (3) Assisting in determining the state of a disease associated with the serine protease based on the obtained detection results or measured values. A method comprising:

[17] A method for collecting data for diagnosing a disease associated with a serine protease that may exist in a living body in the form of a conjugate with a corresponding binding molecule, comprising: (1) Contacting a sample with the reagent according to any one of items 1 to 12; (2) detecting or measuring serine proteases; (3) collecting data for determining the state of a disease associated with the serine protease based on the obtained detection results or measured values; A method comprising:

[18] The method according to any one of items 13 to 17, wherein the serine protease is trypsin, and the disease associated with the serine protease is a pancreatic disease.

[19] A method for producing a reagent for detecting or measuring a serine protease that may exist in a living body in the form of a conjugate with a corresponding binding molecule, comprising the steps of: (A) a surfactant, (B) Latex particles having an antibody against the serine protease or an antigen-binding fragment thereof immobilized thereon, the average particle diameter of the latex particles being 250 to 410 nm. Use of.

[20] A method for producing a reagent for use in diagnosing a disease associated with a serine protease, which can exist in a living body in the form of a conjugate with a corresponding binding molecule, comprising the steps of: (A) a surfactant, (B) Latex particles having an antibody against the serine protease or an antigen-binding fragment thereof immobilized thereon, the average particle diameter of the latex particles being 250 to 410 nm. Use of. Effect of the Invention

[0011] According to the present invention, all or some of the immunologically measurable serine proteases contained in a sample can be detected or measured with high sensitivity and accuracy in a wide measurement range from low to high concentrations, regardless of the form (free form, bound form) and the ratio of the forms. Furthermore, according to the present invention, serine protease in a sample can be detected or measured with high sensitivity and high accuracy over a wide measurement range, including high and low concentration sides outside the normal range. Therefore, by using the amount or concentration of serine protease as a cutoff value, the present invention can be suitably used for diagnosing diseases associated with serine proteases and determining the severity of such diseases. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of measuring standard solutions containing trypsin at various concentrations using measurement reagents (measurement reagents of Comparative Example 1, Comparative Example 2, Example 1, Example 2, and Comparative Example 3) prepared using each latex particle (average particle diameter: 189 nm, 239 nm, 302 nm, 351 nm, and 418 nm). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In the present specification, the term "serine protease that can exist in vivo in the form of a conjugate with a corresponding binding molecule" is not particularly limited as long as it is a serine protease that can exist in vivo alone or in a state bound to a corresponding binding molecule. Here, the "corresponding binding molecule" refers to a substance that has affinity for a serine protease and can bind to the serine protease in vivo to form a conjugate. Examples of such serine proteases include trypsin, elastase, prostate specific antigen (PSA), etc., and preferably trypsin. The corresponding binding molecules include, but are not limited to, for example, when the serine protease is trypsin, alpha 1 antitrypsin or alpha 2 macroglobulin, when the serine protease is elastase, alpha 1 antitrypsin, and when the serine protease is PSA, alpha 1-antichymotrypsin or protein C inhibitor.

[0014] As used herein, "diseases associated with serine protease" include, for example, when the serine protease is trypsin, pancreatitis such as acute pancreatitis, chronic pancreatitis, pancreatic cancer, and papillitis, or pancreatic diseases such as diseases that are prone to be complicated by pancreatic juice stasis, cholelithiasis, biliary tract cancer, and renal failure, and in particular pancreatic diseases. In the case of elastase, examples include pancreatic diseases such as acute pancreatitis, chronic pancreatitis, and pancreatic cancer, gastric cancer, liver cirrhosis, liver cancer, bile duct cancer, and lung cancer, and in particular pancreatic diseases. In the case of PSA, these include prostate cancer, prostatic hyperplasia, and prostatitis, and in particular prostate cancer.

[0015] As used herein, "an antibody against a serine protease, or an antigen-binding fragment thereof, which can exist in vivo in the form of a complex with a corresponding binding molecule" is not particularly limited as long as it has the ability to specifically bind to the serine protease. The above-mentioned "antibody" may be a monoclonal antibody or a polyclonal antibody. In addition, the antibody may be a commercially available product or may be produced by using a serine protease or a part thereof as an antigen according to a known method such as cell fusion technology, gene recombination technology, or phage display technology. The above-mentioned "antigen-binding fragment" refers to a fragment of the above-mentioned antibody that has the ability to bind to serine protease, and examples thereof include a Fab fragment obtained by partial digestion of the above-mentioned antibody with papain or the like, an F(ab')2 fragment obtained by partial digestion with pepsin or the like, and an Fab' fragment obtained by reducing the F(ab')2 fragment. Preferably, the antibody is a monoclonal antibody or an antigen-binding fragment thereof.

[0016] The term "latex particles" as used herein includes latexes known in the art, such as styrene-based latexes such as polystyrene latexes, acrylic acid-based latexes, various modified latexes (e.g., carboxylic acid-modified latexes in which carboxyl groups have been introduced into the above-mentioned polystyrene), colored latexes, fluorescent latexes, and the like. The latex particles can be produced by a known method, but may also be commercially available.

[0017] As used herein, the term "surfactant" includes, for example, nonionic surfactants, amphoteric surfactants, and the like. Specific examples of nonionic surfactants include polyoxyethylene (20) sorbitan monolaurate (trade name: Tween 20), polyoxyethylene sorbitan monooleate (trade name: Tween 80), polyethylene glycol mono-4-octylphenyl ether (trade name: Triton X-100), octylphenoxypoly(ethyleneoxy)ethanol (trade name: Nonidet (R) P40) etc. Specific examples of amphoteric surfactants include 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Preferred are nonionic surfactants, and in particular polyoxyethylene (20) sorbitan monolaurate.

[0018] As used herein, "diagnosis" refers to determining the current or future condition of a disease. As used herein, "severity" includes mild, moderate, severe, etc., and "aggravation" includes progression from mild to moderate or severe, and from moderate to severe. As used herein, the term "onset" refers to the appearance of symptoms of a disease.

[0019] The "sample" in this specification includes, for example, a biological sample collected from a human subject, particularly a blood sample prepared from blood. The "blood sample" in this specification means a sample containing at least a portion of blood components, and may be any of whole blood, serum, and plasma, or may be a diluted version of these. The blood sample is preferably serum or plasma, and more preferably serum. The blood sample can be prepared by a known method.

[0020] In one embodiment, the present invention provides a reagent for detecting or measuring a serine protease that may exist in a living body in the form of a conjugate with a corresponding binding molecule, comprising: A and B below: (A) a surfactant, (B) Latex particles to which an antibody against the serine protease or an antigen-binding fragment thereof has been immobilized. Including, The average particle diameter of the latex particles is 250 to 410 nm. Contains reagents.

[0021] The composition, shape, state, etc. of the reagent are not particularly limited. In addition, in the above-mentioned reagent, the above-mentioned antibody or antigen-binding fragment may be one type or two or more types. Both the first antibody and the second antibody may be antibodies, one may be an antibody and the other an antigen-binding fragment, or both may be antigen-binding fragments. Hereinafter, "antibodies or antigen-binding fragments thereof" will be collectively referred to as "antibodies" unless otherwise specified.

[0022] In the latex particles (B) on which the antibody against the serine protease is immobilized, the method for binding the antibody to the latex particles is not particularly limited, and known methods can be used. For example, the antibody can be bound to the latex by mixing the antibody and the latex in a buffer solution of pH 5.0 to 10.0, reacting for 2 to 3 hours at 20 to 30° C., and then carrying out known post-treatments such as centrifugation, blocking treatment, and heating (aging) treatment. The buffer solution used in this case may be the buffer solution contained in the above-mentioned reagent.

[0023] In the above reagent, two or more types of antibody-immobilized latex particles (B) can be used in combination.

[0024] The average particle diameter of the latex particles is 250 to 410 nm, preferably 260 to 400 nm, and particularly 270 to 390 nm. If the average particle diameter of the latex particles is smaller than the lower limit, it tends to be difficult to measure a low concentration of serine protease with high sensitivity and high accuracy, whereas if the average particle diameter is larger than the upper limit, it tends to be difficult to measure a high concentration of serine protease with high sensitivity and high accuracy. In this specification, the "average particle size" refers to a value measured by dynamic light scattering. In the above reagent, two or more types of latex particles having different average particle sizes may be used in combination.

[0025] In the above-mentioned reagent, the content of the surfactant is not particularly limited, but is, for example, 0.02% by weight or more of the total reagent, preferably 0.03 to 3% by weight, and particularly preferably 0.05 to 1% by weight.

[0026] The above reagent may contain a buffer solution. The buffer solution may be any solution that does not prevent the antibody from binding to the serine protease, and examples of the buffer solution include MES buffer solution, HEPES buffer solution, phosphate buffer solution, Tris buffer solution, Good's buffer solution, glycine buffer solution, borate buffer solution, etc., which have a buffering effect near neutral pH of 5.0 to 10.0, preferably pH 5.5 to 8.5. The concentration of the buffering agent in the buffer solution is appropriately selected from the range of usually 10 to 500 mM, preferably 10 to 300 mM.

[0027] The above reagents may contain additives known in the art in amounts that do not interfere with the binding of the antibody to the serine protease, such as buffers, preservatives (e.g., sodium azide), proteins (albumin), water-soluble polymers (e.g., sugars, polyethylene glycol, dextran), salts (e.g., sodium chloride, amino acids), etc.

[0028] The reagent may be in the form of a kit containing the reagent and other elements, such as a detection reagent for a labeled substance, a standard sample for serine protease, a reagent for preparing a blood sample, a diluent, a support, and instructions for use.

[0029] In the above-mentioned reagent, each component such as the antibody-immobilized latex particles, surfactant, buffer solution, additives, etc. may be contained separately in two or more separate reagents. As an example, for example, the following Reagent A1 and Reagent B1: (A1) Reagent A1 containing a surfactant; (B1) Reagent B1, comprising latex particles on which an antibody against the serine protease or an antigen-binding fragment thereof is immobilized. Including, The average particle diameter of the latex particles is 250 to 410 nm, When measuring a sample, the sample, the reagent A1 and the reagent B1 are mixed. Reagents, etc.

[0030] In the above-mentioned reagents, the sample, reagent A1, and reagent B1 are mixed when measuring the sample. The mixing conditions at that time are not particularly limited as long as they do not interfere with the binding reaction (antigen-antibody reaction) between the serine protease to be measured and the antibody.

[0031] The content of the surfactant in the mixture of the sample, reagent A1, and reagent B1 during the measurement of the sample is not particularly limited, but is, for example, 0.02% by weight or more, preferably 0.03 to 3% by weight, and particularly 0.05 to 1% by weight.

[0032] The above-mentioned reagent A1 may be a surfactant itself or a composition containing a surfactant, as long as the content of the surfactant in the mixture obtained by mixing the sample with reagent B1 during sample measurement falls within the above-mentioned specified range.

[0033] The above-mentioned reagent A1 may contain the above-mentioned buffer solution, additives, etc. in addition to the surfactant.

[0034] The antibody-immobilized latex particles in the above-mentioned reagent B1 may be the same as the above-mentioned antibody-immobilized latex particles (B). Furthermore, the above-mentioned reagent B1 may contain, in addition to the antibody-immobilized latex particles, the above-mentioned buffer solution, additives, etc. The components contained in these reagents A1 and B1 may be appropriately determined in consideration of the stability, handling, etc. of each reagent.

[0035] In the above-mentioned reagent, even if a serine protease is present in a sample in a bound state between the serine protease and the binding molecule, the action of the surfactant can be used to separate the serine protease from the binding molecule and liberate the serine protease. This makes it possible to detect or measure not only the serine protease present in a free form in the sample, but also the serine protease present in a bound state. Furthermore, by using antibody-immobilized latex particles having a predetermined average particle diameter, it becomes possible to measure serine proteases at both low and high concentrations, i.e., in a wide measurement range including the high and low concentration sides outside the normal range, with high sensitivity and high accuracy. Therefore, the above-mentioned reagent can accurately detect serine proteases, which exist in the body in both free and bound forms and whose abundance ratios may vary depending on the in vivo environment, and can also accurately measure their amounts (concentrations). Furthermore, since the above-mentioned reagent can accurately detect or measure serine protease in a sample, by using the amount or concentration of serine protease as a cutoff value, it can be used for diagnosing diseases associated with serine proteases and determining the severity of such diseases.

[0036] In one embodiment, the present invention provides a method for detecting or measuring a serine protease that may exist in a living body in the form of a complex with a corresponding binding molecule, comprising the steps of: (1) contacting the sample with the reagent; (2) Detecting or measuring serine proteases The present invention includes a method comprising the steps of:

[0037] The sample in the above method includes a sample to be measured using the above reagent. In the reagents used in the above-mentioned methods, the antibodies used may be one type or two or more types, and the surfactants used may be one type or two or more types. The above method can also be carried out using one or more of the above reagents.

[0038] The above step (1) is a step of contacting the above reagent with a sample to bind the antibody contained in the reagent to the serine protease contained in the sample. Therefore, the step is not particularly limited as long as it is performed under conditions that allow the serine protease to bind to the antibody. For example, the step can be performed according to a known immunological measurement method.

[0039] The above step (2) is a step of detecting or measuring the conjugate between the serine protease and the antibody obtained in the above step (1). The detection or measurement method for the conjugate can be performed by the latex agglutination method. For example, the degree of agglutination caused by an antigen-antibody reaction between serine protease and an antibody bound to latex particles can be measured using, for example, absorbance, and the concentration of serine protease in a sample can be determined from a calibration curve of a previously obtained standard. The wavelength for measuring absorbance is usually 340 to 1000 nm, preferably 500 to 900 nm. The degree of agglutination is not limited to absorbance, and known methods can be used, such as nephelometry and counting immunoassay. This makes it possible to detect the serine protease in the sample and to quantitate the amount (concentration) of the serine protease in the sample.

[0040] Based on the detection result or measurement value (concentration, amount) of the serine protease obtained in the above step (2), the state of a disease associated with the serine protease can also be judged. Thus, in one embodiment, the present invention provides a method for diagnosing a disease associated with a serine protease that may exist in vivo in the form of a conjugate with a corresponding binding molecule, comprising the steps of: (1) contacting the sample with the reagent; (2) detecting or measuring serine proteases; (3) determining the state of a disease associated with the serine protease based on the obtained detection results or measured values; The present invention includes a method comprising the steps of:

[0041] Steps (1) and (2) in the above diagnostic method can be carried out in the same manner as in the above detection or measurement method.

[0042] Step (3) in the above diagnostic method includes, for example, diagnosing using the detection result or measurement value (concentration, amount) of serine protease as an index, for example, determining a cutoff value and evaluating the severity based on the cutoff value, predicting the onset or aggravation, or evaluating the risk of onset or aggravation.

[0043] In one embodiment, the present invention provides a method for assisting in the diagnosis of a disease associated with a serine protease that may exist in a living body in the form of a conjugate with a corresponding binding molecule, comprising the steps of: (1) contacting the sample with the reagent; (2) detecting or measuring serine proteases; (3) Assisting in determining the state of a disease associated with the serine protease based on the obtained detection results or measured values. The present invention includes a method comprising the steps of:

[0044] Steps (1) and (2) in the above-mentioned diagnostic aiding method can be carried out in the same manner as in the above-mentioned detection or measurement method.

[0045] Step (3) in the above-mentioned method for assisting diagnosis includes, for example, assisting in the judgment of a physician in steps that require the physician's judgment, such as making a diagnosis using the detection result or measurement value (concentration, amount) of serine protease as an indicator, for example, determining a cutoff value and evaluating the severity based on the cutoff value, predicting the onset or aggravation, or evaluating the risk of onset or aggravation (e.g., collecting, organizing, and providing the detection result or measurement value to a physician).

[0046] Furthermore, in one embodiment, the present invention provides a method for collecting data for diagnosing a disease associated with a serine protease that may exist in a living body in the form of a conjugate with a corresponding binding molecule, the method comprising the steps of: (1) contacting the sample with the reagent; (2) detecting or measuring serine proteases; (3) collecting data for determining the state of a disease associated with the serine protease based on the obtained detection results or measured values; The present invention includes a method comprising the steps of:

[0047] Steps (1) and (2) in the above data collection method can be carried out in the same manner as in the above detection or measurement method.

[0048] Step (3) in the data collection method includes, for example, collecting data (e.g., detection results or measurement values) necessary for a doctor's judgment, such as making a diagnosis using the detection results or measurement values ​​(concentration, amount) of serine protease as an index, for example, determining a cutoff value and evaluating the severity based on the cutoff value, predicting onset or aggravation, or evaluating the risk of onset or aggravation. Furthermore, in addition to collecting data, this step also includes organizing the data and providing the data to a doctor.

[0049] In one embodiment, the present invention provides a method for producing a reagent for detecting or measuring a serine protease that may exist in a living body in the form of a conjugate with a corresponding binding molecule, comprising the steps of: (A) a surfactant, (B) Latex particles having an antibody against the serine protease or an antigen-binding fragment thereof immobilized thereon, the average particle diameter of the latex particles being 250 to 410 nm. Includes the use of.

[0050] Further, as one embodiment, the present invention relates to a method for producing a reagent for use in diagnosing a disease associated with a serine protease, which can exist in a living body in the form of a conjugate with a corresponding binding molecule, comprising the steps of: (A) a surfactant, (B) Latex particles having an antibody against the serine protease or an antigen-binding fragment thereof immobilized thereon, the average particle diameter of the latex particles being 250 to 410 nm. Includes the use of.

[0051] The reagent for detecting or measuring serine protease, the reagent for use in diagnosing a disease associated with serine protease, and (A) the surfactant and (B) the antibody or antigen-binding fragment thereof in the above uses are the same as those described for the reagents above. EXAMPLES

[0052] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these.

[0053] [Examples 1-2, Comparative Examples 1-3] <Production of monoclonal antibodies> Antibody A and Antibody B were obtained according to the following procedure. (immunogen) One mg of purified human trypsin (SCRIPPS, Cat. No. T0614, purity ≧95%) was dissolved in 5 mL of saline solution containing 1 mM TLCK (Tosyl-L-lysyl-chloromethane hydrochloride, Nakarai, Code 34219-94), heated at 37°C for 30 minutes, and then dialyzed against saline solution before use. (immunization method) The above immunogen was suspended in an adjuvant (Complete Friends' adjuvant: CFA, GIBCO), and BALB / c mice (5 weeks old, female) were immunized at 2-week intervals with 25 μg trypsin / mouse. Partial blood was collected during the immunization, and the degree of titer increase at the antiserum level was confirmed using the reactivity with the immunogen trypsin as an index. After confirming that the antibody titer had increased sufficiently through multiple immunizations, a saline solution of the above immunogen was administered intraperitoneally or intravenously. Antibody-producing cells were collected from the spleen and fused with myeloma (P3X63Ag8.653, ECACC). Cell fusion was performed using the PEG method, and the fused cells were seeded on a culture plate. The cells were then cultured in a carbon dioxide incubator at 37°C. (screening) The culture supernatant of each well into which the fused cells were dispensed was collected, and the antibody titer was confirmed based on the reactivity with trypsin. The antibody-producing clone cells were then subcultured and cloned by limiting dilution. The cells derived from the single colony obtained were used as anti-human trypsin monoclonal antibody-producing hybridomas. (Monoclonal antibody preparation) Next, the obtained monoclonal antibody-producing hybridoma was cultured in large quantities. Pristane (Sigma-Aldrich) was administered intraperitoneally beforehand, and the hybridoma was administered to BALB / c mice. After breeding for 10 to 25 days and waiting for ascites to accumulate, the ascites was collected, and the obtained ascites antibody was subjected to ammonium sulfate precipitation and affinity purification to obtain mouse anti-human trypsin monoclonal antibody. (Preliminary study of monoclonal antibody combinations) Each of the multiple monoclonal antibodies obtained above was sensitized to latex, and using the signal intensity of latex agglutination as an indicator, two combinations of antibodies A and B that gave large signals and were thought to have different epitopes were obtained.

[0054] <Antibody sensitization> Using the materials and methods described below, antibody A-sensitized latex α solution and antibody B-sensitized latex β solution were prepared for each latex having a different particle size. (material) Latex 188 solution: JSR, product name: IMMUNTEX, average particle size 188 nm, solid content: 10% Latex 239 solution: JSR, product name: IMMUNTEX, average particle size 239 nm, solid content: 10% Latex 302 liquid: JSR, product name: IMMUNTEX, average particle size 302 nm, solid content: 10% Latex 351 solution: JSR, product name: IMMUNTEX, average particle size 351 nm, solid content: 10% Latex 418 solution: JSR, product name: IMMUNTEX, average particle size 418 nm, solid content: 10% ·Antibody A solution: 5.0mg / mL Antibody A, 40mM NaCl, 20mM HEPES (pH7.4) ·Antibody B solution: 4.5mg / mL Antibody B, 40mM NaCl, 20mM HEPES (pH7.4) ·MES buffer: 1%BSA, 20mM MES (pH6.0), 0.05%NaN3 ·HEPES buffer: 2%BSA, 20mM HEPES (pH7.0), 150mM NaCl 0.05%NaN3 (method) 500 μL of latex 351 solution (10% w / vol) and 500 μL of antibody A solution (5.0 mg / mL) were mixed and shaken for 90 minutes at 20° C. Separately, 550 μL of antibody B solution (4.5 mg / mL) was added to a similarly prepared mixture and shaken for 90 minutes at 20° C. The resulting mixture was centrifuged at 10,000 rpm for 30 minutes at 10°C using a centrifuge (rotor: R15A (HI antibody ACH)). The supernatant was decanted, and 25 mL of MES buffer was added to the resulting sediment, which was then dispersed by sonication. The mixture was then shaken and stirred at 37°C for 60 minutes. The obtained dispersion was centrifuged at 10,000 rpm for 30 minutes at 10° C. using the same centrifuge. The supernatant was decanted, and 50 mL of HEPES buffer was added to the obtained precipitate, which was then dispersed by sonication to obtain antibody-sensitized latex α and β solutions. For latex liquids other than the above Latex 351 liquid, the same procedure was followed except for the amounts of antibody A solution and antibody B solution added during shaking and stirring to prepare antibody-sensitized latex α and β liquids for each latex liquid. The amounts of antibody A solution and antibody B solution for each latex liquid are as follows: Latex 188 solution: Antibody A solution 930 μL, Antibody B solution 1030 μL Latex 240 solution: 740 μL of antibody A solution, 810 μL of antibody B solution Latex 302 solution: 580 μL of antibody A solution, 640 μL of antibody B solution Latex 418 solution: Antibody A solution 420 μL, Antibody B solution 460 μL

[0055] <Preparation of measurement reagents> Reagent A and Reagent B having the following compositions were prepared. Reagent A was prepared by mixing each component to a specified amount or concentration. Reagent B was prepared by mixing equal amounts of each antibody-sensitized latex α and β liquid prepared using the above latex liquids (Latex 188 Liquid, Latex 239 Liquid, Latex 302 Liquid, Latex 351 Liquid, or Latex 418 Liquid) with the same average particle size, and further mixing other components to a specified amount or concentration. (1) Reagent A (composition) 0.3% Tween 20, 0.5% polyethylene glycol (PEG) (5000-50000), 0.5% bovine serum albumin (BSA), 150 mM NaCl, ·0.09%NaN3, ·100mM HEPES buffer (pH7.6) (2) Reagent B (composition) 0.5% BSA, 150 mM NaCl, ·0.09%NaN3, ·100mM HEPES buffer (pH7.0) Antibody A sensitized latex α liquid Antibody B sensitized latex β liquid

[0056] <Measurement of trypsin standard solution> (Measuring Reagents) [Table 1] (method) Using each of the measurement reagents of Examples 1-2 and Comparative Examples 1-3, trypsin standard solutions were measured. For each measurement reagent, 4 μL of trypsin standard solution was taken, to which 96 μL of reagent A was added, mixed, and heated at 37° C. for approximately 5 minutes. 24 μL of reagent B was added to this mixture, mixed, and after reacting at 37° C. for approximately 5 minutes, the absorbance at a wavelength of 700 nm was measured. The results are shown in FIG. 1. In addition, "188", "239", "302", "351", and "418" in FIG. 1 respectively represent the measurement reagents of "Comparative Example 1", "Comparative Example 2", "Example 1", "Example 2", and "Comparative Example 3". As shown in Figure 1, the absorbance varied greatly depending on the average particle size of the latex particles in the latex solution used for antibody binding. The reference normal value for trypsin is 210 to 570 ng / mL. 1) or 150-460ng / mL 2) There are reports such as: A measurement reagent must be able to accurately measure normal values, and must also include high-value samples from pancreatitis patients, etc., in the measurement range as much as possible. Taking these factors into consideration, when the measurement range was set to 50 ng / mL to 1600 ng / mL, the latex particle size of the antibody-sensitized latex solution showed appropriate absorbance of 302 nm (Example 1) and 351 nm (Example 2). 1) Shinnosuke Ueda et al.: Evaluation of basic performance and reference range of blood pancreatic enzyme measurement reagent "Libria Trypsin", Medicine and Pharmacology 2020;77(12):1659-1668. 2) Mikihiko Kono et al.: Study of serum trypsin measurement reagents using ELISA method, Medicine and Pharmacology 2005;53(5):635-641.

Claims

1. A reagent for detecting or measuring serine proteases that may exist in living organisms in the form of a conjugate with a corresponding binding molecule, A and B below: (A) Surfactants, (B) Latex particles immobilized with an antibody against the serine protease or its antigen-binding fragment. Includes, The average particle size of the latex particles is 250 to 410 nm. reagent.

2. The reagent according to claim 1, wherein the content of the surfactant is 0.02% by weight or more.

3. The reagent according to claim 1, The following reagents A1 and B1: (A1) Reagent A1 containing a surfactant, (B1) Reagent B1 comprising latex particles immobilized with an antibody against the serine protease or its antigen-binding fragment. Includes, The average particle size of the latex particles is 250 to 410 nm. During the measurement of the sample, the sample, reagent A1, and reagent B1 are mixed together. reagent.

4. The reagent according to claim 3, wherein, at the time of measurement of the sample, the amount of the surfactant in the mixture of the sample, A1, and B1 is 0.02% by weight or more.

5. The reagent according to claim 1 or 3, wherein the average particle size of the latex particles is 260 to 400 nm.

6. The reagent according to claim 1 or 3, wherein the surfactant comprises at least one selected from the group consisting of nonionic surfactants and amphoteric surfactants.

7. The reagent according to claim 1 or 3, wherein the surfactant comprises at least one selected from the group consisting of polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyethylene glycol mono-4-octylphenyl ether, octylphenoxy poly(ethyleneoxy)ethanol, and 3-[(3-coramidopropyl)dimethylammonio]-1-propanesulfonate.

8. The reagent according to claim 1 or 3, wherein the surfactant comprises polyoxyethylene (20) sorbitan monolaurate.

9. The reagent according to claim 1 or 3, wherein the serine protease is selected from the group consisting of trypsin, elastase, and prostate-specific antigen.

10. The reagent according to claim 1 or 3, wherein the serine protease is trypsin.

11. The reagent according to claim 1 or 3, for use in the diagnosis of serine protease-related diseases.

12. The reagent according to claim 11, wherein the serine protease is trypsin, and the disease associated with the protease is a pancreatic disease.

13. A method for detecting or measuring serine proteases that may exist in living organisms in the form of conjugates with corresponding binding molecules, (1) Contacting the reagent according to claim 1 or 3 with the sample, (2) Detect or measure serine proteases. Methods that include...

14. The method according to claim 13, wherein the sample is serum or plasma.

15. A diagnostic method for diseases related to serine proteases that may exist in the body in the form of a conjugate with a corresponding binding molecule, (1) Contacting the reagent according to claim 1 or 3 with the sample, (2) To detect or measure serine proteases, (3) To determine the state of the disease related to the serine protease based on the detection results or measurements obtained. Methods that include...

16. A method to assist in the diagnosis of diseases related to serine proteases that may exist in the body in the form of a conjugate with a corresponding binding molecule, (1) Contacting the reagent according to claim 1 or 3 with the sample, (2) To detect or measure serine proteases, (3) To assist in determining the state of disease related to the serine protease based on the obtained detection results or measurements. Methods that include...

17. A method for collecting data for diagnosing diseases related to serine proteases that may exist in the body in the form of conjugates with corresponding binding molecules, (1) Contacting the reagent according to claim 1 or 3 with the sample, (2) To detect or measure serine proteases, (3) To collect data for determining the state of disease related to the serine protease based on the detection results or measurements obtained. Methods that include...

18. The method according to claim 13, wherein the serine protease is trypsin, and the disease associated with the serine protease is a pancreatic disease.

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