Method for quickly measuring pancreatitis marker in acute pancreatitis

Abstract

The present invention pertains to a method which is capable of predicting the onset of acute pancreatitis in an early stage and enables appropriate therapeutic intervention in an early stage, and more specifically, to a method for providing data necessary for treating acute pancreatitis, the method comprising: (1) measuring a trypsin concentration in a specimen of a subject that is suspected to suffer from acute pancreatitis; and (2) providing data necessary for diagnosing acute pancreatitis based on the measured value, and recommending treatment for acute pancreatitis when the trypsin concentration is equal to or higher than a certain value.

Description

Rapid assay for pancreatitis markers in acute pancreatitis

[0001] This patent application claims priority to Japanese Patent Application No. 2023-107374, the entire contents of which are incorporated herein by reference. The present invention relates to a method for rapidly determining acute pancreatitis.

[0002] Acute pancreatitis is an acute inflammation of the pancreas that can sometimes affect other adjacent organs. The most common symptom of acute pancreatitis is upper abdominal pain, but the pain can also spread to the back and be accompanied by symptoms such as vomiting and fever. In rare cases, the condition can become severe, leading to multiple organ failure, including respiratory, circulatory, and renal failure, as well as infection. The two main causes of acute pancreatitis are alcohol and gallstones, but it is also known to occur in patients who have undergone endoscopic retrograde cholangiopancreatography (ERCP).

[0003] In general, acute pancreatitis is diagnosed when two or more of the following conditions are met: 1. There is acute abdominal pain and tenderness in the upper abdomen; 2. There is an increase in pancreatic enzymes in the blood or urine; and 3. There is an abnormal finding in the pancreas on ultrasound, CT, or MRI that is associated with acute pancreatitis, and other pancreatic diseases and acute abdominal conditions have been excluded. Furthermore, it is desirable to measure amylase and lipase as pancreatic enzymes (Non-Patent Document 1).

[0004] Acute Pancreatitis Treatment Guidelines 2021 (5th Edition), Kanehara Publishing Co., Ltd.

[0005] An object of the present invention is to provide a method that can predict the onset of acute pancreatitis at an early stage and enable early and appropriate therapeutic intervention.

[0006] As a result of extensive research to solve the above-mentioned problems, the present inventors have discovered that trypsin concentration can be used as an early predictor of acute pancreatitis (pancreatitis marker), and have completed the present invention. Specifically, the present invention includes the following aspects: [1] A method for providing data necessary for the treatment of acute pancreatitis, comprising: (1) measuring the trypsin concentration in a sample from a subject suspected of having acute pancreatitis; and (2) providing data necessary for diagnosing acute pancreatitis based on the measured trypsin concentration, and recommending treatment for acute pancreatitis if the trypsin concentration is equal to or greater than a certain value. [2] The method described in item 1, wherein treatment for acute pancreatitis is recommended if the trypsin concentration is 570 ng / mL or greater. [3] The method described in item 2, wherein, when the amylase concentration of the sample is measured simultaneously with the measurement of the trypsin concentration, the amylase concentration is 125 U / L or less. [4] The method described in item 2 or 3, wherein, when the lipase concentration of the sample is measured simultaneously with the measurement of the trypsin concentration, the lipase concentration is 55 U / L or greater. [5] The method of any one of items 1 to 4, wherein the treatment for acute pancreatitis is selected from fluid administration, drug administration, or surgical removal. [6] The method of any one of items 1 to 5, further comprising, after step (2), (3) determining the severity of the acute pancreatitis, and (4) recommending initiation of treatment within 3 hours if the result of step (3) indicates severe acute pancreatitis. [7] The method of any one of items 1 to 6, wherein the subject has undergone endoscopic examination, endoscopic surgery, or open surgery. [8] The method of any one of items 1 to 7, wherein the trypsin concentration is measured using an immunoassay reagent or immunoassay system. [9] The method of item 8, wherein the immunoassay reagent or immunoassay system is a reagent or assay system for using latex agglutination, ELISA, RIA, or chemiluminescence immunoassay.

[0007] According to the present invention, the onset or onset of acute pancreatitis can be predicted at an early stage, thereby enabling early and appropriate therapeutic intervention.

[0008] Figure 1 shows the results of comparing the trypsin, lipase, and amylase measurements of a group of patients diagnosed with acute pancreatitis the day after undergoing endoscopic retrograde cholangiopancreatography (ERCP) with a group of patients diagnosed without acute pancreatitis (no pancreatitis group) in terms of the ratio to the upper limit of normal (trypsin 570 ng / mL, lipase 55 U / L, amylase 125 U / L). The non-group was negative, and the pancreatitis group was positive. ROC curves were created for each marker (trypsin, lipase, amylase) immediately after the test, and the AUCs were calculated. The non-group was negative, and the pancreatitis group was positive. The AUCs were calculated for each marker (trypsin, lipase, amylase) two hours after the test, and the AUCs were calculated.

[0009] In this specification, "acute pancreatitis" is defined as a condition that meets two or more of the following criteria: (1) acute abdominal pain and tenderness in the upper abdomen, (2) elevated blood or urinary pancreatic enzymes, and (3) abnormal findings associated with acute pancreatitis in the pancreas on diagnostic imaging (ultrasound, CT, MRI). The causes of acute pancreatitis include, but are not limited to, alcohol, gallstones, and endoscopic retrograde cholangiopancreatography.

[0010] As used herein, "treating" or "treatment" means treating a disease state in a subject (patient), and includes suppressing (e.g., suppressing or delaying progression), alleviating, ameliorating, improving, curing, etc., the disease state. Furthermore, "treating acute pancreatitis" or "treatment of acute pancreatitis" as used herein includes fluid administration, drug administration, surgical removal, etc. Examples of the "fluid administration" include saline, Ringer's solution, etc. Examples of the "surgical removal" include surgical nexectomy, etc.

[0011] As used herein, "diagnosing" or "diagnosis" means determining (judging) the current or future state of a disease in a subject (patient), and includes predicting or determining whether or not a subject will contract a disease, predicting or determining whether or not a disease state will progress to a more severe state, determining whether or not a subject has a disease, determining the degree or progression of a disease state, determining the effectiveness of treatment for a disease, and determining whether or not there is a risk of the disease recurring after treatment.

[0012] As used herein, "severity" includes mild, moderate, severe, etc., and "severity" includes progression from mild to moderate or severe, and progression from moderate to severe. As used herein, "onset" refers to the appearance of symptoms of a disease.

[0013] As used herein, the term "subject" refers to a living organism to be examined, particularly an animal (e.g., a mammal such as a human, mouse, rat, hamster, guinea pig, monkey, cow, pig, horse, rabbit, sheep, goat, cat, or dog), and particularly includes a human (also referred to as a "test subject" in this case). Furthermore, the term "subject" (or "test subject" in the case of a human) as used herein includes a subject (patient) suffering from a disease, a subject (subject) suspected of having a disease, a subject (subject) at risk of having a disease, and a subject (healthy individual) who is not suffering from a disease, and particularly includes a subject who has undergone an endoscopic examination (e.g., an ERCP examination), endoscopic surgery, or open surgery.

[0014] The "sample" used herein is not particularly limited as long as it can contain the above-mentioned trypsin, and examples thereof include blood samples prepared from blood collected from a human subject. The term "blood sample" used herein refers to a sample containing at least a portion of blood components, and may be any of whole blood, serum, and plasma, or any of these diluted forms. The blood sample is preferably serum or plasma. Preparation of the blood sample can be carried out by known methods.

[0015] As used herein, "trypsin" refers to trypsin or its precursor (trypsinogen) that can exist in vivo either alone or in a state bound to a corresponding binding molecule. Examples of the corresponding binding molecule include, but are not limited to, α １ Antitrypsin or alpha ２ Hereinafter, unless otherwise specified, "trypsin or its precursor" and "a complex bound to a corresponding binding molecule" will be collectively referred to as "trypsin."

[0016] As used herein, the term "antibody or antigen-binding fragment thereof directed against a complex bound to trypsin or its corresponding binding molecule" is not particularly limited as long as it specifically binds to the above-mentioned complex bound to trypsin or its corresponding binding molecule. Examples of antibodies or antigen-binding fragments capable of specifically binding to "trypsin" include antibodies or fragments thereof capable of recognizing and binding to a portion of trypsin as an epitope. Examples of antibodies or antigen-binding fragments capable of specifically binding to a "complex" of trypsin and a binding molecule include antibodies or antigen-binding fragments thereof capable of recognizing and binding to a portion of trypsin present on the surface of the complex as an epitope, and antibodies or antigen-binding fragments thereof capable of recognizing and binding to a portion of the binding molecule as a partial or complete epitope. The "antibody" may be a monoclonal or polyclonal antibody. The antibody may be commercially available or may be produced by known methods such as cell fusion technology, genetic recombination technology, or phage display using trypsin or a portion thereof as an antigen. Furthermore, the above-mentioned "antigen-binding fragment of an antibody" refers to a fragment of the above-mentioned antibody that has the ability to bind to trypsin, and examples thereof include Fab fragments obtained by partial digestion of the above-mentioned antibody with papain or the like, F(ab')2 fragments obtained by partial digestion with pepsin or the like, and Fab' fragments obtained by reducing F(ab')2 fragments. Preferably, the antibody is a monoclonal antibody or an antigen-binding fragment thereof. Hereinafter, unless otherwise specified, "antibodies or antigen-binding fragments thereof" will be collectively referred to as "antibodies."

[0017] In one aspect, the present invention provides a method for providing data necessary for the treatment of acute pancreatitis, comprising: (1) measuring the trypsin concentration in a sample from a subject suspected of having acute pancreatitis; and (2) providing data necessary for diagnosing acute pancreatitis based on the measured value, and recommending treatment for acute pancreatitis if the trypsin concentration is equal to or higher than a certain value.

[0018] The above step (1) is a step of measuring the trypsin concentration in a sample from a subject suspected of having acute pancreatitis. The measurement of the trypsin concentration in step (1) may be performed by any method, but is preferably performed using an immunoassay reagent or an immunoassay system.

[0019] The immunoassay reagent or immunoassay system is not particularly limited, but examples include those that use an antibody or an antigen-binding fragment thereof against a complex bound to trypsin or its corresponding binding molecule. Specific examples include reagents or assay systems for enzyme-linked immunosorbent assay (ELISA), fluorescent enzyme immunoassay, chemiluminescent enzyme immunoassay, chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescent antibody assay, radioimmunoassay (RIA), Western blotting, immunoblotting, latex agglutination, immunochromatography, nephelometry, etc. Preferred are reagents or assay systems for latex agglutination, ELISA, RIA, or chemiluminescent immunoassay. These reagents or assay systems are not particularly limited in terms of their composition, shape, state, etc., and may be prepared according to known methods or commercially available.

[0020] In the above-mentioned reagent or measurement system, the antibody or antigen-binding fragment thereof may be one type, or two or more types. When two or more types of antibodies or antigen-binding fragments thereof are used, they may be, for example, two or more types of antibodies or antigen-binding fragments thereof that bind to different epitopes, or two or more types of antibodies or antigen-binding fragments thereof that bind to the same epitope. Furthermore, when trypsin may be present in a sample both in a free form and in a complex, at least one of the two or more types of antibodies may be an antibody or antigen-binding fragment thereof that binds to the complex. Examples include a combination of two or more antibodies or antigen-binding fragments thereof that can recognize and bind to different portions of trypsin as epitopes; a combination of at least one antibody that can recognize and bind to a portion of trypsin as an epitope and an antigen-binding fragment of at least one antibody that can recognize and bind to an epitope different from the epitope of the first antibody; a combination of at least one antibody or antigen-binding fragment thereof that can recognize and bind to a portion of trypsin as an epitope and at least one antibody or antigen-binding fragment thereof that can recognize and bind to a portion of the complex as part or all of the epitope; and a combination of at least one antibody or antigen-binding fragment thereof that can recognize and bind to a portion of trypsin as an epitope and an antibody or antigen-binding fragment thereof that can recognize and bind to a portion of the binding molecule as part or all of the epitope.

[0021] In the reagent or measurement system, the antibody may be immobilized on a support. The support can be selected appropriately depending on the method in which the reagent is used. Examples of supports include well plates (e.g., 96-well microplates), membranes (e.g., nitrocellulose membranes, polyvinylidene fluoride membranes), slide glasses, magnetic beads, and latex particles. The antibody can be immobilized on a support by a known method selected depending on the material of the support. Furthermore, when the antibody is immobilized on a support, a second antibody that binds to a different epitope of the trypsin may be included. In this case, both the first and second antibodies may be antibodies, one may be an antibody and the other an antigen-binding fragment, or both may be antigen-binding fragments.

[0022] The antibody may also be labeled. The labeling substance used for labeling is not particularly limited, and any known substance may be used. Examples of the labeling substance include enzyme labels such as peroxidase and alkaline phosphatase; fluorescent labels such as fluorescein isothiocyanate (FITC); radioisotope labels such as iodine-125; electrochemiluminescent labels such as ruthenium complexes; biotin; and metal nanoparticles. Labeling of the antibody can be performed using a known method selected according to the type of labeling substance.

[0023] The reagent or measurement system may be in the form of a kit. The kit may contain other elements in addition to the antibody. Examples of other elements include a detection reagent for the labeled substance, a standard sample for trypsin, a reagent for preparing a blood sample, a diluent, a buffer, a support, and instructions for use.

[0024] The reagent or measurement system can contain a surfactant. In a reagent or measurement system containing a surfactant, even if trypsin is present in a sample in the form of a conjugate between trypsin and a binding molecule, the surfactant can separate the trypsin from the binding molecule and liberate the trypsin, thereby making it possible to detect or measure not only trypsin present in the sample as a free form, but also trypsin present in the conjugate form. Therefore, in a reagent or measurement system containing the surfactant, trypsin, which exists in a living body in both a free form and a conjugate form and whose abundance ratio may vary depending on the living body environment, can be accurately detected and its amount (concentration) can also be accurately measured. An example of such a reagent or measurement system is a reagent or measurement system containing the following A and B: (A) a surfactant, (B) an antibody against the trypsin.

[0025] As used herein, the term "surfactant" refers to, for example, a nonionic surfactant, an amphoteric surfactant, etc. 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). Specific examples of amphoteric surfactants include 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Preferred are nonionic surfactants, particularly polyoxyethylene(20) sorbitan monolaurate.

[0026] The content of the surfactant in the reagent or measurement system is not particularly limited, but is, for example, 0.02% by weight or more of the total reagent or measurement system, preferably 0.03 to 3% by weight, and particularly 0.05 to 1% by weight.

[0027] The reagent or measurement system may contain a buffer solution. The buffer solution may be any solution that does not prevent the antibody from binding to trypsin, and examples thereof include buffer solutions that have a buffering effect near neutral pH 5.0 to 10.0, preferably pH 5.5 to 8.5, such as MES buffer, HEPES buffer, phosphate buffer, Tris buffer, Good's buffer, glycine buffer, and borate buffer. The concentration of the buffering agent in the buffer solution is generally selected from the range of 10 to 500 mM, preferably 10 to 300 mM.

[0028] The reagent or assay system may contain additives known in the art in amounts that do not interfere with antibody binding to trypsin, 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.

[0029] In the above-mentioned reagent or measurement system, each component such as the antibody, surfactant, buffer solution, additive, etc. may be contained separately in two or more separate reagents. An example thereof includes the following reagent A1 and reagent B1: (A1) reagent A1 containing a surfactant, (B1) reagent B1 containing the antibody against trypsin, and the sample, reagent A1, and reagent B1 are mixed when measuring the sample.

[0030] In the above-described reagent or measurement system, 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 trypsin and antibody being measured.

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

[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 at the time of measuring the sample falls within the above-mentioned predetermined range.

[0033] The reagent A1 may contain, in addition to the surfactant, the buffer solution, additives, etc. The reagent B1 may contain, in addition to the antibody, the buffer solution, additives, etc. The components contained in the reagents A1 and B1 may be appropriately determined taking into consideration the stability, handleability, etc. of each reagent.

[0034] Among the above-mentioned reagents or measurement systems, a reagent or measurement system for using the latex agglutination method includes (i) an antibody-sensitized latex solution containing latex particles to which at least one antibody or its antigen-binding fragment capable of recognizing and binding to a part of trypsin as an epitope is bound, and (ii) a binding molecule (e.g., α １ Examples of such reagents include a reagent containing an antibody-sensitized latex solution containing latex particles to which an antibody or an antigen-binding fragment thereof is bound, the antibody being capable of recognizing and binding to a part of the antigen-binding fragment (antitrypsin) as part or all of an epitope, or an assay system using such a reagent.

[0035] When the reagent is a reagent for use in latex agglutination (hereinafter referred to as "latex reagent"), latexes known in the art can be used. Examples include styrene-based latexes such as polystyrene latex, acrylic acid-based latexes, various modified latexes (e.g., carboxylic acid-modified latexes in which carboxyl groups have been introduced into the polystyrene), colored latexes, fluorescent latexes, etc. The latexes can be produced by known methods, but commercially available latexes may also be used. Two or more types of the latexes may also be used in combination.

[0036] The average particle size of the latex particles in the latex reagent is not particularly limited, but examples thereof include a size that allows aggregates formed by an antigen-antibody reaction between an antigenic substance to be measured and an antibody to be detected with the naked eye or optically. Specifically, the average particle size is, for example, 100 to 500 nm. The average particle size of the latex particles may be of two or more types. In this specification, the "average particle size" refers to a value measured by dynamic light scattering.

[0037] The latex reagent contains latex particles to which an antibody against trypsin is bound. The method for binding the antibody to latex 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 latex in a buffer solution of pH 5.0 to 10.0, reacting the mixture at 20 to 30°C for 2 to 3 hours, and then performing known post-treatments such as centrifugation, blocking, and heating (aging). The buffer solution used in this process may be the same as that contained in the reagent described above.

[0038] Measurement of trypsin concentration using the immunoassay reagent or immunoassay system can be carried out by a method comprising the following steps: (i) binding trypsin contained in the sample to an antibody contained in the reagent or assay system in the reagent or assay system, and (ii) measuring the amount or concentration of the trypsin-antibody complex obtained by step (i). Steps (i) and (ii) are not particularly limited and can be carried out as appropriate according to known protocols for the reagent or assay system used, or the package inserts of commercially available products.

[0039] For example, when the latex agglutination method is used as the reagent or measurement system, the degree of agglutination caused by the antigen-antibody reaction between trypsin and an antibody bound to latex particles can be measured, for example, using absorbance, and the concentration of trypsin in the sample can be determined from a previously determined calibration curve of a standard. The wavelength for measuring absorbance is typically 340 to 1000 nm, preferably 500 to 900 nm. The degree of agglutination is not limited to absorbance, and known methods can also be used, such as nephelometry and counting immunoassay.

[0040] Step (2) in one embodiment of the method of the present invention includes providing data necessary for diagnosing acute pancreatitis based on the measured trypsin concentration obtained in step (1), and recommending treatment for acute pancreatitis when the trypsin concentration is equal to or higher than a certain value.

[0041] Because the blood concentration of trypsin significantly increases in the early stages of acute pancreatitis, it can be used as an early predictor of acute pancreatitis (pancreatitis marker). That is, trypsin concentration can be used as an indicator for determining (judging) the onset of acute pancreatitis, whether acute pancreatitis will develop, and whether acute pancreatitis will progress to a more severe state. For example, a cutoff value can be determined by statistically processing data obtained from multiple patients, and based on this cutoff value, it is possible to predict whether acute pancreatitis will develop or whether acute pancreatitis will progress to a more severe state. For example, if the concentration is equal to or greater than a predetermined cutoff value, it can be predicted that acute pancreatitis will develop, and if it is below the cutoff value, it can be predicted that acute pancreatitis will not develop. Similarly, if the concentration is equal to or greater than a predetermined cutoff value, it can be predicted that acute pancreatitis will progress to a more severe state, and if it is below the cutoff value, it can be predicted that acute pancreatitis will not progress to a more severe state. Therefore, trypsin concentration can be used as data for predicting whether acute pancreatitis will develop or whether acute pancreatitis will progress to a more severe state.

[0042] Furthermore, the severity of acute pancreatitis can be evaluated based on the trypsin concentration. For example, data obtained from multiple patients can be statistically processed to determine a cutoff value (pathological condition identification value), and the severity of acute pancreatitis can be evaluated based on this cutoff value. For example, a value below a first cutoff value can be evaluated as mild, a value between the first cutoff value and a second cutoff value can be evaluated as moderate, and a value above the second cutoff value can be evaluated as severe. Therefore, the trypsin concentration can be used to evaluate the severity of acute pancreatitis, to assist in the evaluation, or as data for performing the evaluation.

[0043] Furthermore, the presence or absence, or the level, of the risk of developing or worsening acute pancreatitis can be determined based on the blood concentration of trypsin. For example, data obtained from multiple patients can be statistically processed to determine a cutoff value, and the presence or absence, or the level, of the risk of developing or worsening acute pancreatitis can be determined based on this cutoff value. For example, if the value is equal to or greater than a predetermined cutoff value, it can be determined that the risk of developing acute pancreatitis is present or high, and conversely, if the value is below the cutoff value, it can be determined that such risk is absent or low. Similarly, if the value is equal to or greater than a predetermined cutoff value, it can be determined that the risk of worsening acute pancreatitis is present or high, and conversely, if the value is below the cutoff value, it can be determined that such risk is absent or low. Therefore, the above-mentioned reagent can be used to evaluate the risk of developing or worsening acute pancreatitis, assist in such evaluation, collect data for such evaluation, etc.

[0044] Therefore, the data necessary for diagnosing acute pancreatitis include cutoff values ​​obtained by statistically processing data obtained from multiple patients to determine whether acute pancreatitis will develop, whether it will worsen, the severity, and the presence or absence or level of the risk of developing or worsening the disease.

[0045] In the above step (2), a judgment is made based on the data necessary for diagnosing acute pancreatitis as described above, and if the trypsin concentration is equal to or higher than a certain value, treatment of acute pancreatitis is recommended.

[0046] The recommended trypsin concentration for the treatment of acute pancreatitis is, for example, 570 ng / mL or higher.

[0047] Treatment for acute pancreatitis is not particularly limited, and examples thereof include treatments selected from transfusion, drug administration, and surgical removal. The treatment can be appropriately selected depending on the trypsin concentration, data on diagnostic indicators other than trypsin concentration, the severity and symptoms of acute pancreatitis, and the age, weight, sex, and medical history of the subject (patient).

[0048] Examples of data or diagnostic indicators necessary for diagnosing acute pancreatitis other than trypsin concentration include (1) the presence or absence of acute abdominal pain attacks and tenderness in the upper abdomen, (2) the presence or absence of elevated blood or urinary pancreatic enzymes (amylase, lipase, etc.), (3) the presence or absence of abnormal pancreatic findings associated with acute pancreatitis in imaging diagnostics (ultrasound, CT, MRI), and (4) possible causes (alcohol, gallstones, endoscopic examination (e.g., ERCP examination), endoscopic surgery, open surgery, etc.). Of these, the concentrations of pancreatic enzymes other than trypsin (amylase, lipase, etc.) are particularly important. Measurement of amylase and lipase concentrations is not particularly limited and may be performed using known reagents or measurement systems. Furthermore, the data or diagnostic indicators include various cutoff values ​​for determining whether acute pancreatitis will develop, whether the condition will worsen, and the severity, obtained by statistically processing data obtained from multiple patients. By taking into consideration such data or indicators in conjunction with trypsin concentration, it becomes possible to more accurately diagnose and determine the severity of acute pancreatitis, and to select a more appropriate treatment.

[0049] For example, when the amylase concentration of the sample is measured at the same time as the trypsin concentration of the sample, if the trypsin concentration is 570 ng / mL or higher and the amylase concentration is 125 U / L or lower, a diagnosis of acute pancreatitis can be made, and treatment can be initiated early.

[0050] Furthermore, for example, when the lipase concentration of the sample is measured at the same time as the trypsin concentration of the sample, if the trypsin concentration is 570 ng / mL or higher and the lipase concentration is 55 U / L or higher, a diagnosis of acute pancreatitis can be made, and treatment can be initiated early.

[0051] Furthermore, for example, when the amylase and lipase concentrations of the sample are measured simultaneously with the trypsin concentration of the sample, if the trypsin concentration is 570 ng / mL or higher, the amylase concentration is 125 U / L or lower, and the lipase concentration is 55 U / L or higher, a diagnosis of acute pancreatitis can be made, and treatment can be initiated early.

[0052] One aspect of the method of the present invention may include, after step (2), (3) determining the severity of the acute pancreatitis, and (4) recommending that treatment be initiated within 3 hours if the patient is determined to have severe acute pancreatitis as a result of the determination in step (3).

[0053] The assessment of the severity of acute pancreatitis in step (3) can be performed based on the data required for diagnosing acute pancreatitis, as described above. For example, trypsin concentration data obtained from multiple patients can be statistically processed to determine a cutoff value (pathological condition identification value), and the severity of acute pancreatitis can be assessed based on this cutoff value. Specifically, for example, a value below a first cutoff value can be assessed as mild, a value between the first cutoff value and a second cutoff value can be assessed as moderate, and a value above the second cutoff value can be assessed as severe.

[0054] Step (4) is a step of recommending treatment within 3 hours if the patient is diagnosed with severe acute pancreatitis as a result of the determination in step (3). Acute pancreatitis can become severe early, and if the patient is diagnosed with severe pancreatitis, it is preferable to start treatment as soon as possible. It is recommended to start treatment within 3 hours at the latest, preferably within 2 hours, and particularly preferably within 1 hour after the diagnosis.

[0055] The present invention will be explained in more detail below with reference to Reference Examples and Examples, but the present invention is not limited to these.

[0056] Reference Example 1 Production of Monoclonal Antibodies Antibody A and antibody B were obtained according to the following procedure. (Immunogen) 1 mg of human purified trypsin (SCRIPPPS, Cat. No. T0614, purity ≥ 95%) was dissolved in 5 mL of physiological 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 physiological saline solution before use. (Immunization Method) The immunogen was suspended in adjuvant (Complete Freund's adjuvant: CFA, GIBCO), and BALB / c mice (5-week-old, female) were immunized at 25 μg trypsin / mouse at 2-week intervals. Partial blood samples were taken during the immunization, and the degree of titer increase at the antiserum level was confirmed using the reactivity with the immunogen trypsin as an indicator. After confirming sufficient antibody titer increase through multiple immunizations, the immunogen was administered intraperitoneally or intravenously in saline. Antibody-producing cells were collected from the spleen and fused with myeloma cells (P3X63Ag8.653, ECACC). Cell fusion was performed using the PEG method, and the fused cells were seeded on culture plates. They were then cultured in a 37°C carbon dioxide incubator. (Screening) Culture supernatants were collected from each well containing fused cells, and antibody titers were confirmed using the reactivity with trypsin as an indicator. Antibody-producing clone cells were then subcultured and cloned by limiting dilution. Cells derived from the resulting single colonies were used as anti-human trypsin monoclonal antibody-producing hybridomas. (Monoclonal Antibody Preparation) The resulting monoclonal antibody-producing hybridomas were then mass-cultured. Pristane (Sigma-Aldrich) was administered intraperitoneally beforehand, and the hybridoma was then administered to BALB / c mice. The mice were kept for 10 to 25 days, and after 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 antibodies.(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.

[0057] <Antibody Sensitization> Antibody A-sensitized latex α solution and antibody B-sensitized latex β solution were prepared according to the following materials and methods. (Materials) Latex solution: Latex (manufactured by JSR, trade name: IMMUNTEX), average particle size 351 nm, solid content: 10% Antibody A solution: 4.9 mg / mL antibody A, 40 mM NaCl, 20 mM HEPES (pH 7.4) Antibody B solution: 4.5 mg / mL antibody B, 40 mM NaCl, 20 mM HEPES (pH 7.4) MES buffer: 1% BSA, 20 mM MES (pH 6.0), 0.05% NaN ３ ・HEPES buffer: 2% BSA, 20mM HEPES (pH 7.0), 150mM NaCl 0.05% NaN ３ (Method) 500 μL of latex solution (10% w / vol) and 500 μL of antibody A solution (4.9 mg / mL) were mixed and shaken at 20°C for 90 minutes. Separately, 550 μL of antibody B solution (4.5 mg / mL) was added to a similarly prepared mixture and shaken at 20°C for 90 minutes. The resulting mixture was centrifuged at 10,000 rpm and 10°C for 30 minutes using a centrifuge (rotor: R15A (HI antibody ACH)). The supernatant was decanted, and 25 mL of MES buffer was added to the resulting sediment and dispersed by sonication. The mixture was then shaken at 37°C for 60 minutes. The resulting dispersion was then centrifuged at 10,000 rpm and 10°C for 30 minutes using the same centrifuge. The supernatant was decanted, and 50 mL of HEPES buffer was added to the resulting sediment, which was then dispersed by sonication to obtain antibody-sensitized latex α and β solutions.

[0058] <Measurement Reagents> Reagent A and reagent B were prepared with the following compositions. Reagent A was prepared by mixing each component to a predetermined amount or concentration. Reagent B was prepared by mixing equal amounts of each antibody-sensitized latex α and β solution, and then mixing other components to a predetermined amount or concentration. (1) Reagent A (Composition) 0%, 0.3%, 0.5%, 0.75%, or 1.0% Tween 20, 0.5% polyethylene glycol (PEG) (5000-50000), 0.5% bovine serum albumin (BSA), 150 mM NaCl, 0.09% NaN ３ , ・100mM HEPES buffer (pH 7.6) (2) Reagent B (composition) ・0.5% BSA, ・150mM NaCl, ・0.09% NaN ３ 100 mM HEPES buffer solution (pH 7.0) Antibody A-sensitized latex α solution Antibody B-sensitized latex β solution

[0059] Example 1: Serum samples were collected from 54 patients (26 men and 28 women) undergoing endoscopic retrograde cholangiopancreatography (ERCP) before, immediately after, 2 hours after, and 24 hours after the test. The serum trypsin concentration was measured using the serum trypsin assay reagent constructed in Reference Example 1. Similarly, blood lipase and amylase concentrations were measured for the same sample group. Of the 41 samples for which measurements were possible, the group diagnosed with acute pancreatitis the day after the test according to the post-ERCP pancreatitis diagnostic guidelines (8 pancreatitis patients: 4 men and 4 women) and the group diagnosed without acute pancreatitis (33 unaffected patients: 16 men and 17 women) were compared. While there was no significant change in trypsin measurements in the control group, in the pancreatitis patient group, trypsin levels increased by more than 8 times immediately after the test and 2 hours after the test, and by more than 16 times 24 hours after the test compared to pre-test levels. Lipase and amylase levels also increased (Table 1).

[0060]

[0061] When trypsin values ​​were compared relative to the upper limit of normal (570 ng / mL), in the pancreatitis patient group, the increase was approximately 11.7 times immediately after testing, approximately 11.9 times two hours after testing, and approximately 23 times 24 hours after testing. Similarly, when lipase and amylase were compared relative to the upper limit of normal (lipase 55 U / L, amylase 125 U / L), the lipase and amylase levels immediately after testing were approximately 8.7 times and 1.6 times, respectively, 2 hours after testing were approximately 13.6 times and 2.5 times, respectively, and 24 hours after testing were approximately 29.4 times and 6.6 times, respectively (Figure 1). The ROC curves for each marker were created immediately after testing (negative for the non-patient group) and 2 hours after testing (positive for the pancreatitis patient group). AUCs were calculated. The values ​​for trypsin, lipase, and amylase were 0.84, 0.82, and 0.65, respectively, immediately after testing (Figure 2). Two hours after testing, the values ​​were 0.93, 0.89, and 0.79, respectively (Figure 3). The cutoff values ​​for trypsin, lipase, and amylase were 1487.1 ng / mL, 115.5 U / L, and 136.6 U / L, respectively, immediately after testing. Two hours after testing, the values ​​were 1701.3 ng / mL, 165.6 U / L, and 159.6 U / L, respectively. Trypsin was the marker that rose most rapidly in the blood of patients with pancreatitis, demonstrating high accuracy as a diagnostic aid for early pancreatitis.

Claims

1. 1. A method for providing data necessary for the treatment of acute pancreatitis, comprising: (1) Measuring the trypsin concentration in a blood sample from a subject suspected of having acute pancreatitis using an immunoassay reagent or an immunoassay system; (2) Providing data necessary for diagnosing acute pancreatitis based on the measured values ​​and recommending treatment for acute pancreatitis when the trypsin concentration is above a certain value; Including, The immunoassay reagent or immunoassay system is a reagent or assay system for use in a latex agglutination method, an immunochromatography method, or a chemiluminescence immunoassay method. method.

2. 10. The method of claim 1, wherein treatment for acute pancreatitis is recommended if the trypsin concentration is 570 ng / mL or greater.

3. 3. The method according to claim 2, wherein when the amylase concentration of the blood sample is measured simultaneously with the measurement of the trypsin concentration of the blood sample, the amylase concentration is 125 U / L or less.

4. 3. The method according to claim 2, wherein when the lipase concentration of the blood sample is measured simultaneously with the measurement of the trypsin concentration of the blood sample, the lipase concentration is 55 U / L or more.

5. 10. The method of claim 1, wherein the treatment for acute pancreatitis is selected from fluid administration, medication, or surgical removal.

6. The method described in claim 1, wherein the subject has undergone an endoscopic examination, endoscopic surgery or open surgery.

7. After the step (2), (3) assessing the severity of the acute pancreatitis; (4) If the result of the determination in step (3) is that the patient has severe acute pancreatitis, it is recommended that treatment be initiated within 3 hours. The method according to any one of claims 1 to 6, comprising: