Method for measuring elastase 1 in feces

Abstract

Provided are a reagent for immunoanalysis of elastase 1 and an immunoanalysis method, by which elastase 1 (E1) present in human fecal samples can be analyzed in a simple manner that enables handling of large volumes of samples without the need for special facilities or equipment, within a short period of time fitting for emergency testing, and which enable quantitative analysis over a wide range of concentrations, i.e., from low to high concentration areas. This analysis method comprises: (a) a step for adding α1-antitrypsin (α1AT) to a fecal sample obtained from a subject; (b) a step for incubating the fecal sample to cause reaction between E1 and α1AT, thereby forming a complex; (c) a step for incubating the fecal sample with a solution containing a carrier on which an immunological partner capable of recognizing the E1-α1AT complex is immobilized; and (d) a step for analyzing a change caused by the reaction between the E1-α1AT complex and the immunological partner.

Description

Method for measuring fecal elastase 1

[0001] The present invention relates to a reagent and method for immunoassay of fecal elastase 1, and a method for detecting pancreatic diseases.

[0002] Pancreatic diseases are said to be difficult to diagnose, even with the development of various diagnostic methods. The reasons for this include the fact that the pancreas is located deep within the abdominal cavity, making it difficult to determine the presence or nature of disease using traditional diagnostic methods such as palpation, visual inspection, and auscultation, as well as X-ray examinations. Furthermore, the clinical symptoms of pancreatic diseases are similar to those of other digestive system diseases, and there is no simple and reliable diagnostic method (Patent Document 1). In particular, the five-year survival rate for pancreatic cancer is extremely short, and early detection is said to be essential to improving prognosis.

[0003] The pancreatic enzyme elastase 1 (CELA1; chymotrypsin-like elastase family, member 1, hereinafter sometimes referred to as E1) has been measured as a biomarker used in the diagnosis of pancreatic disease. E1 belongs to the serine protease family and is immunologically distinct from elastases also present in leukocytes, platelets, and the spleen. E1 is secreted from the pancreas into the duodenum in parallel with other digestive enzymes, but is leaked into the blood due to pancreatic duct stenosis or pancreatitis. In the blood, most of it is bound to α1-antitrypsin (hereinafter sometimes referred to as α1AT), and measurement of its blood concentration is considered clinically useful. In particular, E1 frequently shows abnormally high values ​​from a relatively early stage, reflecting pancreatitis associated with pancreatic cancer (particularly the head of the pancreas), making it useful as an indicator for diagnosing or monitoring the progress of pancreatic disease (Patent Document 2). However, because it is an invasive test, it is not widely practiced, and there are not many reports on blood E1 levels.

[0004] There is a linear correlation between E1 secretion and pancreatic lipase, amylase, and trypsin secretion, and furthermore, E1 secretion into the duodenum correlates with fecal E1 concentration (Patent Document 3). Pancreatic exocrine dysfunction is thought to be related to decreased E1 secretion, which results in decreased enzyme concentrations in the stool. Therefore, E1 has been reported to be used as a marker for diagnosing pancreatic exocrine insufficiency and monitoring pancreatic exocrine function in diabetes mellitus, cystic fibrosis, and chronic pancreatitis.

[0005] It has been reported that when E1 is used for diagnostic purposes in human fecal samples, its concentration is 5 to 6 times higher than in pancreatic juice. However, the details of this situation and its relationship to pancreatic diseases, such as whether the blood or fecal concentration is optimal, are not fully understood at present.

[0006] Reasons for the lack of accurate measurement and understanding of E1 in fecal samples include the influence of contaminants and the handling of fecal samples. Fecal samples typically contain moisture, food residues, intestinal mucosal cells, intestinal bacteria, etc., but also contain many components and solid contaminants that are not absorbed into the body and are excreted. Furthermore, fecal samples have unique properties, such as a wide variety of components, shapes, and pH depending on the excretion state. Therefore, the influence and causes on measurement results are diverse, and tests using fecal samples require different efforts than ordinary blood samples, such as suppressing nonspecific reactions. Regarding fecal sample handling, for example, Kampanis et al.'s dry extraction method for measuring elastase 1 in human feces is disclosed, but the actual handling is cumbersome and difficult to say that it is practically suitable for conducting large-scale sample tests (Non-Patent Document 1). In addition, in the case of wet extraction, wet or loose fecal samples must be dried, weighed, and finally diluted with an extraction solution, which is very labor-intensive and may cause hygiene problems. Furthermore, it is difficult to standardize the application of standard concentrations between extraction methods, which is one of the reasons why accurate measurement is difficult.

[0007] When using feces as a sample for clinical testing, the influence of fecal occult blood must be considered. Fecal occult blood refers to the presence of blood in the feces, but accurate measurements may be hindered by factors such as intestinal bleeding, anal fissures, and menstrual bleeding. The positive rate for fecal occult blood tests is generally estimated to be around 5-10%, but in addition to the influence of coexisting substances in the stool, the influence of substances in the blood due to fecal occult blood must also be considered depending on the subject.

[0008] In view of the above circumstances, there has been a need for the development of a method and reagent that can accurately measure E1 in human fecal samples.

[0009] Japanese Patent Application Laid-Open No. 63-73152 WO2002 / 079782 Japanese Patent Application Laid-Open No. 2017-516088

[0010] Ann. Clin. Biochem 46; 33-7, 2009

[0011] The object of the present invention is to provide an immunoanalysis reagent and an immunoanalysis method for E1 present in human fecal samples, which are simple and can handle large quantities of specimens without requiring special facilities or equipment, can analyze E1 in a short time so as to be suitable for emergency testing, and can quantitatively analyze a wide range of concentrations from low to high.

[0012] The present inventors have conducted extensive research to solve the above problems, and as a result have found that E1 in human fecal samples can be accurately measured using α1AT in a state where it has formed a complex with E1, thereby completing the present invention.

[0013] The present invention relates to an immunoassay method for analyzing E1 in a human fecal sample by forming a stabilized complex with its inhibitor, α1AT, and then analyzing the complex through an antigen-antibody reaction using a monoclonal antibody specific to the E1-α1AT complex.Furthermore, the present invention relates to a method for detecting pancreatic diseases by analyzing E1 using the immunoassay method.

[0014] That is, the present invention provides the following: [1] A method for measuring pancreatic elastase 1 present in a fecal sample, comprising the steps of: (a) adding α1-antitrypsin to a fecal sample obtained from a subject, (b) incubating the fecal sample to react pancreatic elastase 1 with α1-antitrypsin to form a complex, (c) incubating the fecal sample with a solution containing a carrier on which an immunological partner that recognizes the pancreatic elastase 1-α1-antitrypsin complex has been immobilized, and (d) analyzing changes resulting from the reaction between the pancreatic elastase 1-α1-antitrypsin complex and the immunological partner. [2] The method of [1], wherein the step (a) is carried out in a pretreatment step of extracting a fecal sample. [3] The method of [1] or [2], comprising a step of diluting the fecal sample before adding α1-antitrypsin to the fecal sample in the step (a). [4] The method of any of [1] to [3], wherein the amount of α1-antitrypsin added to the fecal sample in the step (a) is 20 μg or more. [5] The method of any of [1] to [4], wherein the amount of α1-antitrypsin contained in the reaction solution subjected to the reaction step in the step (b) is 100 ng or more. [6] A method for assisting in the detection of pancreatic diseases, which involves analyzing pancreatic elastase 1 by the measurement method of any of [1] to [5]. [7] The method of any of [1] to [6], wherein the analysis performed in the step (d) is any one of chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescent immunoassay, radioimmunoassay, immunochromatography, Western blotting, latex agglutination, and immunoturbidimetry. [8] A reagent for immunoassay, comprising a solid-phase carrier carrying an immunological partner that recognizes the pancreatic elastase 1-α1-antitrypsin complex, and α1-antitrypsin. [9] The immunoassay reagent of [8], wherein the reaction solution to be subjected to the reaction step contains 100 ng of α1-antitrypsin.

[10] A pretreatment extract containing α1-antitrypsin, used in a pretreatment step of a fecal sample to be subjected to a reagent for measuring pancreatic elastase 1 or pancreatic elastase 1-α1-antitrypsin.

[11] The pretreatment extract of

[10] , wherein the α1-antitrypsin is contained in an amount of 41 to 975 ng.

[0015] The method of the present invention, which measures E1 present in human fecal samples, can easily assist in the detection of pancreatic diseases. Accurate measurement of E1 in human fecal samples can be used as a marker for monitoring pancreatic exocrine function, which is expected to be useful in determining treatment strategies.

[0016] 1 is a diagram showing the results of E1 concentration measurement relative to the concentration of α1AT solution added. 2 is a diagram showing the recovery rate for each α1AT solution concentration relative to the theoretical value for E1 sample preparation. 3 is a diagram comparing the measured values ​​when α1AT was added using multiple measurement reagents.

[0017] The present invention relates to an immunoassay method for measuring E1 present in a human fecal sample in a state where it is complexed with α1AT, and also to an immunoassay reagent for measuring the E1-α1AT complex present in a human fecal sample.

[0018] Although an embodiment of a method for measuring E1 present in a human fecal sample will be described in detail below, the embodiments of the method of use are not limited thereto. For example, the present invention includes: a method for detecting pancreatic disease by measuring pancreatic elastase 1 present in a fecal sample; a method for assisting in the detection of pancreatic disease by measuring pancreatic elastase 1 present in a fecal sample; a method for measuring pancreatic elastase 1 present in a fecal sample to detect pancreatic disease; an in vitro method for detecting pancreatic disease by measuring pancreatic elastase 1 present in a fecal sample; use of an immunological partner that recognizes pancreatic elastase 1-α1-antitrypsin complex in the manufacture of a kit for detecting pancreatic disease; and a method for measuring pancreatic elastase 1 present in a fecal sample to provide information necessary for the detection of pancreatic disease. Note that, in this specification, "measurement" (in the broad sense) includes not only "measurement" (in the narrow sense) in which the amount of an analyte is quantitatively or semi-quantitatively determined, but also "detection" in which the presence or absence of an analyte is determined.

[0019] Samples used in the measurements of the present invention include human-derived fecal samples, intestinal lavage fluids, and the like. The specific form of the fecal sample is not particularly limited as long as it is derived from feces. For example, fecal samples can be used regardless of their shape, such as hardness (hard stool, normal stool, loose stool, diarrheal stool, watery stool, etc.), water content, etc. The term "intestinal lavage fluid" refers to a sample recovered through the intestinal lumen, and includes oral intestinal lavage fluid recovered from an orally ingested intestinal lavage agent through the intestinal lumen. The intestinal lavage fluid may be a sample excreted by a subject, or a sample retained in the subject's rectum just before excretion. In this specification, when the term "fecal sample" is used to mean a "sample" used in measurements (e.g., in claim 1), it includes not only fecal samples but also intestinal lavage fluids and the like.

[0020] Fecal samples can be pretreated using common techniques for extracting E1 and removing insoluble fractions. The extraction solution used in the extraction procedure can be physiological saline, but solutions containing buffers such as Good's buffer or phosphate, proteins such as BSA (bovine serum albumin), and surfactants may also be used. Those skilled in the art can appropriately determine the various conditions for the extraction solution, such as the concentration and pH of the buffer, and the concentrations of the surfactant and BSA.

[0021] The extraction procedure involves adding the extraction solution to the fecal sample and thoroughly dispersing it to elute E1 into the extraction solution. A homogenizer or vortex mixer may be used, if necessary. To further elute E1, the fecal sample may be allowed to stand for 30 minutes to 1 hour after dispersion in the extraction solution.

[0022] Centrifugation or filter filtration can be used to remove the insoluble fraction. The centrifugation conditions and the filter membrane used for filtration are not particularly limited as long as they are usable for pretreatment. For example, the carrier constituting the filter can include polypropylene (PP), polyvinylidene fluoride (PVDF), glass fiber (GF), polyethersulfone (PES), nylon (NY), polytetrafluoroethylene (PTFE), regenerated cellulose (RC), cellulose acetate (CA), and methacrylate butadiene styrene (MBS). Furthermore, a hybrid type composed of a combination of these components may also be used.

[0023] The above operations can also be performed using a general stool collection kit. An example of a stool collection kit that can be used is OC-HemoCatch (registered trademark) S (manufactured by Eiken Chemical Co., Ltd.), a stool collection kit for fecal occult blood. Furthermore, the pretreated stool sample is preferably stored in a cool, dark place or frozen. More preferably, it is stored in an ultra-low temperature freezer (-85 to -40°C), and the frozen stool sample can be thawed and used for measurement.

[0024] The extract used in these extraction procedures may contain α1AT, which will be described later.

[0025] The method for measuring E1 of the present invention is not particularly limited, and it is possible to use an immunological partner capable of measuring E1. Immunological protein detection methods include any commonly known method, such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescent immunoassay, radioimmunoassay, immunochromatography, and other immunoassays using labeled antibodies, as well as Western blotting, latex agglutination, and immunoturbidimetry.

[0026] The term "immunological partner" used in the present invention means a partner that specifically binds immunologically to the substance to be measured, for example, an immunological substance (i.e., an antigen or antibody) that can specifically bind to various proteins, polysaccharides, lipids, nucleic acids, haptens, and complexes or fragments thereof. When the immunological partner is an antibody, the antibody used may be a monoclonal antibody or a polyclonal antibody, or a fragment thereof treated with an enzyme or the like. The antibody fragment is preferably a functional fragment containing the antigen-binding region of the antibody or its variable region, for example, F(ab') ２ , Fab', Fab, etc. F(ab') ２ Fab' is an antibody fragment produced by treating an immunoglobulin with a proteolytic enzyme (e.g., pepsin or papain) across the disulfide bond present between the two H chains in the hinge region. Furthermore, multiple types of immunological partners may be used in combination.

[0027] The following describes, as an example, the use of an antibody as the immunological partner. In the method of the present invention, since the E1-α1AT complex is used as the analyte, the antibody used may be any antibody capable of recognizing the E1-α1AT complex, and may be an anti-E1 antibody that specifically recognizes E1, or an anti-E1-α1AT complex antibody that specifically recognizes the complex with E1-α1AT. The anti-E1 antibody can be appropriately selected and used as long as it does not recognize the E1-α1AT complex. The number of antibodies used may be a single antibody that specifically recognizes E1, or a combination of a first antibody that specifically recognizes E1 and a second antibody that recognizes E1 different from the first antibody. Furthermore, the number of antibodies used may be a single antibody that specifically recognizes the E1-α1AT complex, or a combination of a first antibody that specifically recognizes the E1-α1AT complex and a second antibody that recognizes an E1-α1AT complex different from the first antibody. These may also be used in combination (hereinafter, these may be collectively referred to as "anti-E1 antibodies"). There are no particular limitations on the anti-E1 antibodies as long as they specifically bind to different sites.

[0028] Anti-E1 antibodies can be produced, for example, using as an immunogen a polypeptide containing part or all of the amino acid sequence of E1 or the E1-α1AT complex. The antigen polypeptide may be a synthetic polypeptide chemically synthesized according to known methods or one produced by genetic recombination or the like.

[0029] The antibody used in the present invention can be used as an immobilized antibody supported on an insoluble carrier such as a solid phase carrier, or as a labeled antibody labeled with a labeling substance.

[0030] An immobilized antibody refers to an antibody that is supported on an insoluble carrier by physical adsorption, chemical bonding, or the like. These immobilized antibodies can be used to detect or quantify a substance to be measured contained in a sample. Examples of insoluble carriers that can be used to support antibodies include polymeric materials such as latex, rubber, polyethylene, polypropylene, polystyrene, styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, styrene-methacrylate copolymer, polyglycidyl methacrylate, acrolein-ethylene glycol dimethacrylate copolymer, polyvinylidene difluoride (PVDF), and silicone; agarose; gelatin; red blood cells; and inorganic materials such as silica gel, glass, inert alumina, and magnetic materials. These may be used alone or in combination.

[0031] The method of the present invention can be carried out, for example, by loading anti-E1 antibodies onto latex particles. The latex particles that can be used in this case are not particularly limited, as long as they are latex particles that can be used in conventional immunoassay reagents, and examples include polystyrene and styrene-styrene sulfonate copolymers. The average particle size of the latex particles can be appropriately selected depending on the detection concentration of the analyte or the measurement instrument. For example, particles having a particle size of 0.05 to 0.5 μm can be used. Using latex particles of different particle sizes is preferable, as it allows accurate analysis, particularly from low to high values. In particular, at high values, it is preferable to prevent misidentification due to reduced agglutination ability even at high concentrations of elastase 1 (the so-called prozone phenomenon, in which excessively high concentrations reduce agglutination ability, resulting in apparent reduced agglutination and resulting in a measurement result that is lower than the actual value). The average particle size of latex particles in the present invention refers to the value measured using an electron microscope.

[0032] When anti-E1 antibodies are immobilized on latex particles, the latex particles may contain, for example, two types of latex particles of different particle sizes carrying two types of anti-E1 antibodies with different specificities for E1 or the E1-α1AT complex, preferably at least (1) first latex particles carrying a first anti-E1 antibody against E1 or the E1-α1AT complex, and (2) second latex particles of a particle size different from that of the first latex particles carrying a second anti-E1 antibody against E1 or the E1-α1AT complex with a different specificity from that of the first anti-E1 antibody.

[0033] In the case of immunoassays using labeled antibodies, such as enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescent immunoassay, radioimmunoassay, and immunochromatography, it is preferable to use a labeling substance. The labeling substance is not particularly limited as long as it is a labeling substance that can be used in conventional immunoassays, and examples include enzymes, fluorescent substances, radioisotopes, insoluble granular substances, and the like. Examples of the labeling enzymes include alkaline phosphatase, peroxidase, glucose oxidase, tyrosinase, and acid phosphatase. Examples of fluorescent substances include fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), and luciferin. Examples of radioisotopes include: １２５ I, １４ C. ３２ Examples include P.

[0034] Furthermore, when the labeling substance is an enzyme, the labeling substance can be measured by carrying out a luminescence, fluorescence, or color reaction using a substrate for the enzyme. For example, when the enzyme is alkaline phosphatase, the substrate can be CDP-star (registered trademark) (2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)-tricyclo[3.3.1.13,7]decane}-4-yl)-1-phenylphosphate disodium), CSPD (registered trademark) (3-(4-methoxyspiro{1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane}-4-yl)phenylphosphate disodium), Chemiluminescent substrates such as 4-methylumbelliferyl phosphate (4-methylumbelliferyl phosphate) and the like; fluorescent substrates such as 4-methylumbelliferyl phosphate (4-methylumbelliferyl phosphate) and the like; chromogenic substrates such as p-nitrophenyl phosphate, BCIP (5-bromo-4-chloro-3-indolyl phosphate), NBT (4-nitroblue tetrazolium chloride) and INT (iodonitrotetrazolium) can be used.

[0035] The immunoassay method of the present invention can be carried out using an immunoassay reagent composed of one liquid or two or more liquids. When the reagent for carrying out the present invention is composed of one liquid, it can consist of, for example, a reaction reagent containing at least an insoluble carrier carrying an immunological partner for forming an immunological complex. A sample is reacted with the reagent according to a known method, and a signal is detected.

[0036] Since it is difficult to directly and accurately measure E1 present in a fecal sample, in the measurement of the present invention, E1 and α1AT are measured in a state in which they form a complex. The α1AT used in the present invention may be in any form that is ultimately mixed with the sample, and may be contained in a pretreatment reagent used in the pretreatment step, a stabilizing reagent, or a reagent containing the above-mentioned insoluble carrier. In the present invention, it has been found that in order to accurately measure E1 present in a fecal sample, it is important to add a certain concentration range of α1AT to the sample or reagent.

[0037] The α1AT used in the present invention may be added to a sample before the sample reacts with a reagent, or may be supplied in a state where it has been added to a reagent. It need only be contained in a reaction solution in which an immune reaction with the E1-α1AT complex, which is the substance to be measured, is carried out. For example, the α1AT may be added to any one of a diluent for pre-diluting the measurement sample, a reagent in which the measurement sample is mixed with an immobilized anti-E1 antibody bound to an insoluble carrier and the measurement sample is reacted with the immobilized anti-E1 antibody, and a reagent in which a labeled antibody is mixed with the measurement sample and reacted with the labeled antibody and the E1-α1AT complex, which is the substance to be measured, or to each of these reagents.

[0038] The α1AT used may be any α1AT that forms a complex with human pancreatic elastase 1, and preferably human-derived α1AT is used. Human-derived α1AT may be purified α1AT present in blood, or may be recombinantly expressed using cultured cells or the like, and those skilled in the art can appropriately select and use it.

[0039] The amount of α1AT added to form a complex with E1 is preferably a sufficient amount to form a complex with E1. The lower limit of the amount added may be 20 μg or more, and the upper limit may be set appropriately depending on the amount of E1 contained in the sample. For example, the lower limit is preferably 41 μg or more, and more preferably 49 μg or more. The upper limit is preferably 975 μg or less. The lower and upper limits can be combined as appropriate.

[0040] The lower limit of the amount of α1AT contained in the reaction solution may be set appropriately at a concentration sufficient to form a complex with E1 and confirm reactivity compared to when no α1AT is added, and may be 100 ng or more. For example, 514 ng or more is preferred, and 609 ng or more is more preferred. The upper limit of the amount of α1AT contained in the reaction solution is preferably 12,188 ng or less, more preferably 6,094 ng or less, and even more preferably 5,143 ng or less. Those skilled in the art can determine the optimal amount to be added for each measurement reagent, taking into account the E1 concentration expected to be contained in a fecal sample. The lower and upper limits can be combined as appropriate.

[0041] Alternatively, the amount of E1 to be added may be determined based on the weight ratio of E1 to α1AT, depending on the measurable range of the reagent used in the measurement. In this case, for example, by adding α1AT in an amount 6 to 2400 times the weight of E1, it becomes possible to sufficiently form a complex between E1 and α1AT, thereby enabling accurate measurement of E1 in a fecal sample.

[0042] When the reagent of the present invention is composed of two or more liquids, it can be composed of, for example, a stabilizing reagent and a reaction reagent containing an insoluble carrier carrying at least an antibody or antigen for forming an immunological complex. The stabilizing reagent is a reagent used to dilute the sample to an appropriate concentration or perform pretreatment, and can be prepared according to known methods. According to known methods, the sample is reacted with the stabilizing reagent, and then reacted with the reaction reagent, and a signal is detected. The α1AT used in the present invention is preferably added to the stabilizing reagent and / or reaction reagent before the sample reacts with the reaction reagent, but preferably can be supplied in a state where it has been added to the stabilizing reagent and / or reaction reagent.

[0043] It is known that nearly 90% of E1 present in blood forms a complex with α1AT, and measuring the E1-α1AT complex is used in clinical testing as a measure of E1. Serine protease inhibitors of the serpin superfamily, to which α1AT belongs, are covalently bonded to the serine residue in the active center of the protease molecule. Upon loop insertion, they are strongly attracted to the serpin molecule, disrupting the structure near the active center and preventing dissociation of the protease by hydrolysis. Thus, E1 and α1AT are thought to be covalently bonded and not easily dissociated. It was previously thought that E1 and α1AT formed a complex in fecal samples, so the addition of α1AT to fecal samples made it possible to measure E1, which was an unexpected effect.

[0044] The E1 measurement method of the present invention, which aids in the detection of pancreatic diseases, may be implemented by appropriately using original data or statistically processed data for calculating a determination threshold (cutoff value), such as a determination threshold (cutoff value) calculated from statistical data showing the correlation between E1 concentrations in fecal samples and various diseases. Those skilled in the art can appropriately set and use cutoff values ​​based on the correlation with pancreatic diseases. For example, a method for calculating these cutoff values ​​may involve creating and analyzing an ROC curve (Receiver Operating Characteristic Curve) from the E1 concentration, and setting the cutoff value within the range in which diagnostic sensitivity and specificity are effective.

[0045] The measured E1 concentration can also be used as a value for risk assessment. It can be used as an indicator to determine whether pancreatic inflammation or pancreatic cancer is improving through treatment such as medication. For example, if the E1 concentration remains high or increases, it suggests the need to reconsider the treatment plan.

[0046] In addition to the anti-E1 antibody-carrying latex particles, the reagent of the present invention may further contain various additives that can be added to a latex reagent, such as a buffer solution, an agglutination promoter (e.g., a water-soluble polymer such as polyethylene glycol), a non-specific reaction inhibitor (e.g., an alkali metal salt or a sugar), or a protein [e.g., bovine serum albumin (BSA)].

[0047] The buffer solution is preferably a buffer solution having a buffering capacity at pH 6 to 8.5. The buffer solution at pH 6 to 8.5 is a conventionally known buffer solution, such as Tris buffer solution, phosphate buffer solution, or Good's buffer solution.

[0048] When a Tris buffer solution is used in the reagent of the present invention, the Tris concentration in the Tris buffer solution is not particularly limited as long as it is a concentration that can achieve a predetermined Tris concentration described below in a system in which a latex agglutination reaction is carried out when the Tris buffer solution is used, but is preferably 0.1 to 0.5 mol / L.

[0049] The Tris concentration in the system in which the latex agglutination reaction is carried out is not particularly limited as long as it is a concentration that can suppress the self-agglutination reaction of latex particles, and can be appropriately selected depending on the concentrations of coexisting additives such as salts, proteins, and / or sugars. The Tris concentration in the system in which the latex agglutination reaction is carried out is preferably 0.1 to 0.5 mol / L, and more preferably 0.2 to 0.3 mol / L. If the Tris concentration is less than 0.1 mol / L, the latex particles may undergo self-agglutination, while if it exceeds 0.5 mol / L, the antigen-antibody reaction may be suppressed, resulting in poor detection sensitivity. The lower and upper limits of the Tris concentration can be appropriately combined, for example, between 0.1 and 0.3 mol / L.

[0050] The pH of the buffer solution is preferably 6 to 8.5. If the pH is outside this range, the latex particles may self-aggregate, and problems may arise in terms of measurement accuracy.

[0051] When the reagent of the present invention contains a buffer solution of pH 6 to 8.5, the state of each anti-E1 antibody-carrying latex particle and the pH 6 to 8.5 buffer solution in the reagent is not particularly limited, as long as the antibody-carrying latex particles, the pH 6 to 8.5 buffer solution, and the test sample can be in contact with each other during the latex agglutination reaction during use. That is, in this case, the form of the reagent of the present invention is not particularly limited, and it can be, for example, a one-liquid reagent containing both each anti-E1 antibody-carrying latex particle and a pH 6 to 8.5 buffer solution, or a two-liquid reagent consisting of a first reagent containing each anti-E1 antibody-carrying latex particle and a second reagent which is a pH 6 to 8.5 buffer solution. In the measurement method of the present invention, the antibody-carrying latex particles are brought into contact with the test sample, preferably under conditions of pH 6 to 8.5, to cause an antigen-antibody reaction and the resulting latex agglutination reaction, and the degree of agglutination is analyzed to analyze E1 in the test sample.

[0052] In the measurement method of the present invention, when a latex agglutination reaction is carried out under conditions of pH 6 to 8.5 using a buffer solution of pH 6 to 8.5, the order of contacting each anti-E1 antibody-carrying latex particle with the buffer solution of pH 6 to 8.5 and the test sample is not particularly limited, as long as the antigen-antibody reaction does not proceed in the absence of the buffer solution of pH 6 to 8.5 (i.e., the test sample is not contacted first with each anti-E1 antibody-carrying latex particle). For example, each anti-E1 antibody-carrying latex particle can be contacted in advance with a buffer solution of pH 6 to 8.5, and the test sample can be contacted with the mixture. Alternatively, the test sample can be contacted in advance with a buffer solution of pH 6 to 8.5, and the mixture can be contacted with each anti-E1 antibody-carrying latex particle.

[0053] The conditions for the antigen-antibody reaction in the measurement method of the present invention can be the same as those for carrying out a conventional immunological latex turbidimetric analysis method. For example, the reaction is preferably carried out at a pH of 6 to 8.5. The reaction temperature is preferably 0 to 50°C, more preferably 20 to 40°C. The reaction time can be determined appropriately; for example, measurement can be completed in 10 to 15 minutes using a general-purpose automatic analyzer. The lower and upper limits of the reaction temperature can be combined appropriately, for example, between 0 and 40°C.

[0054] The degree of agglutination caused by the antigen-antibody reaction can be analyzed by a known analytical method, for example, an optical analytical method. Examples of the optical analytical method include a method in which the reaction solution is irradiated with light and the scattered light or transmitted light is analyzed. More specifically, the analysis can be performed using an optical instrument that measures scattered light intensity, absorbance, or transmitted light intensity. The preferred measurement wavelength is 300 to 800 nm. Analysis using the optical instrument can be performed by measuring the increase or decrease in scattered light intensity, absorbance, or transmitted light intensity by selecting the size and / or concentration of the latex particles used and setting the reaction time according to a known method. It is also possible to use these methods in combination.

[0055] In the method for detecting pancreatic diseases according to the present invention, serum or plasma is used as a test sample, and E1 in a fecal sample is analyzed by the measurement method of the present invention, thereby enabling the detection (diagnosis) of pancreatic diseases (particularly acute pancreatitis).

[0056] From these findings, rapid and accurate measurement of E1 present in human fecal samples using the present invention will not only aid in the diagnosis of pancreatic diseases, but will also provide more reliable measurement values ​​in monitoring the progress of treatment, making it possible to provide extremely useful information.

[0057] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0058] [Example 1: Experiment on adding α1AT to fecal extract solution] Iatro IRE1II (hereinafter referred to as IRE1, manufactured by LSI Medience Corporation) was used as a reagent for measuring E1 in fecal samples, and it was confirmed whether measurement values ​​of Iatro IRE1II could be obtained by adding α1 antitrypsin to the fecal extract solution.

[0059] Purified human plasma-derived α1AT (Sigma-Aldrich) was dissolved in Tris buffer to prepare α1AT solutions with concentrations of 0, 16, 130, 325, 813, 3250, and 6500 μg / mL, calculated based on the extinction coefficient of α1AT. 150 μL of the prepared α1AT solution at each concentration was added to 150 μL of human fecal extract solution (hereinafter referred to as fecal extract solution) to prepare α1AT-spiked fecal extract solutions. The α1AT-spiked fecal extract solution was further diluted 80-fold to fit within the measurement range of the IRE1 reagent. Commercially available D-D dimer diluent (LSI Medience) was used for dilution. The 80-fold diluted α1AT-spiked fecal extract solution was measured at a multiplicity of 2 using the IRE1 reagent loaded on a Hitachi 7180-type automated analyzer (hereinafter referred to as H7180, Hitachi High-Tech Corporation).

[0060] Figure 1 shows the results of IRE1 reagent measurements relative to the concentration of α1AT solution added to the fecal extract solution. It was confirmed that adding α1AT to the fecal extract solution increased the measured value of the IRE1 reagent, with the maximum observed when the concentration of the α1AT solution was 325-3250 μg / mL. At 6500 μg / mL, the highest concentration among the experimental conditions, the measured value decreased by 7.5% compared to 3250 μg / mL, but the recovery rate was still sufficient for use in E1 measurement. The amounts of α1AT added to the samples subjected to the measurement process were 49, 122, 488, and 975 μg, respectively. However, because the samples were diluted 80-fold for measurement, the actual amounts were 609, 1523, 6094, and 12188 ng. This confirmed that adding α1AT enabled IRE1 reagent measurements to be obtained from fecal extract samples.

[0061] Example 2: Study of α1AT Solution Concentration The optimal concentration of α1AT to be added within the measurement range of 80 to 4000 ng / dL for the IRE1 reagent was investigated, and the α1AT solution concentration to be used in future experiments was determined. Elastase 1 purified from human pancreatic juice was used in phosphate buffer to prepare E1 samples with concentrations of 14, 57, 142, 285, and 571 ng / mL, calculated based on the extinction coefficient of E1. Similarly, α1AT solutions were prepared using phosphate buffer to prepare α1AT solutions with concentrations of 171, 875, 3429, 8571, 17143, and 34268 ng / mL, calculated based on the extinction coefficient of α1AT. 150 μL of α1AT solution was added to 150 μL of E1 sample to prepare an E1-α1AT mixed solution. The IRE1 reagent was loaded into an H7180, and the E1-α1AT mixed solution was measured at a multiplicity of 3. The measured value of a mixed solution of 571 ng / mL E1 sample 1 and 34,268 ng / mL α1AT solution 1 was taken as the theoretical preparation value (3,778 ng / dL) of E1 sample 1. The theoretical preparation value of each E1 sample was set based on the dilution ratio from E1 sample 1.

[0062] Figure 2 shows the recovery rates for each α1AT solution concentration relative to the theoretically prepared value for each E1 sample. For E1 samples 1-4, whose theoretically prepared values ​​were 378-3778 ng / dL, recoveries of 90% or more were obtained for α1AT solutions 1-4, whose α1AT concentrations were 3429-34286 ng / mL. For E1 sample 5, whose theoretically prepared value was 94 ng / dL, recoveries of 87.6% were observed for α1AT solutions 2-4, whose α1AT concentrations were 3429-17143 ng / mL, and a recovery rate of 69.7% was observed for the 34286 ng / mL α1AT solution, confirming sufficient recovery rates for E1 measurement. When targeting E1 concentrations ranging from 80-4000 ng / dL, which is the measurement range of the IRE1 reagent, recoveries of α1AT concentrations of 3429-17143 ng / mL were satisfactory relative to the theoretically prepared value. Specifically, the amounts of α1AT added when the recovery rate of E1 was 70% or higher were 514, 1286, 2571, and 5143 ng, respectively. Therefore, in the subsequent experiments, the concentration of the α1AT solution was provisionally set to 8600 ng / mL, and 1290 ng was contained in the sample used for measurement.

[0063] Example 3: Comparative experiment between IRE1 reagent and fPELA reagent The reactivity of an evaluation system in which α1AT is added using the IRE1 reagent was compared with that of an existing fecal elastase 1 LTX reagent. fPELA turbo (hereinafter referred to as fPELA, manufactured by BUHLMANN) was used as the existing fecal E1 measurement reagent.

[0064] E1 samples were prepared using purified E1 from human pancreatic juice and phosphate buffer, with concentrations of 14, 57, 142, 285, and 571 ng / mL, calculated based on the extinction coefficient of E1. Similarly, α1AT solutions were prepared using phosphate buffer, with a concentration of 8600 ng / mL, calculated based on the extinction coefficient of α1AT. E1-α1AT mixed solutions were prepared by mixing 200 μL of each prepared E1 solution with 200 μL of α1AT solution. Each E1 sample, E1-α1AT mixed solution, IRE1 reagent standard, and IRE1 reagent control were measured at a multiplicity of 2 using the fPELA reagent loaded on an automated analyzer, Cobas C501 (hereinafter, C501, manufactured by Roche). The E1-α1AT mixed solution was measured at a multiplicity of 2 using the IRE1 reagent loaded on the H7180.

[0065] Figure 3 plots the IRE1 values ​​versus the measured values ​​of each E1 sample, E1-α1AT mixed solution, IRE1 reagent standard, and IRE1 reagent control using the fPELA reagent. The measured values ​​of the E1-α1AT mixed solution in the IRE1 reagent and the values ​​measured using the fPELA reagent for the E1 sample showed a good linear concentration-dependent relationship. When the E1-α1AT mixed solution was measured using the fPELA reagent, the measured values ​​were lower than when the E1 solution was measured directly. The measured values ​​of the E1-α1AT mixed solution using the fPELA reagent were close to the values ​​measured using the IRE1 reagent standard and control. In the IRE1 reagent standard and control, E1 was contained as a complex with α1AT, indicating that the fPELA reagent is affected by α1AT. In the case of fecal samples, fecal occult blood may be present in the specimen, and α1AT in the blood may be present and affect the measurement value. However, it was confirmed that when a reagent that recognizes the E1-α1AT complex is used, the measurement value is not affected even in specimens where fecal occult blood is present. Since there is a correlation between the measurement value of the IRE1 reagent that recognizes the E1-α1AT complex and the measurement value of the fPELA reagent that recognizes and measures E1, it was confirmed that the results obtained by adding α1AT to fecal samples and measuring them are reliable data.

[0066] The use of the measuring reagent and measuring method of the present invention enables the measurement of the E1 concentration in fecal samples without being affected by measurement-interfering substances, and can be used to assist in the diagnosis of pancreatic diseases. Furthermore, since the E1 concentration in fecal samples from healthy individuals can be accurately determined, it can be used not only for the diagnosis of pancreatic diseases but also for the selection of appropriate treatments, monitoring of treatment effects, and prognosis prediction, thereby providing highly reliable measured values ​​and providing extremely useful information for medical treatment.

Claims

1. 1. A method for measuring pancreatic elastase 1 present in a fecal sample, comprising: (a) adding α1-antitrypsin to a fecal sample obtained from a subject; (b) incubating the stool sample to allow pancreatic elastase 1 and α1-antitrypsin to react and form a complex; (c) incubating the stool sample with a solution containing a carrier on which an immunological partner that recognizes the pancreatic elastase 1-α1-antitrypsin complex is immobilized; (d) analyzing the changes caused by the reaction of the pancreatic elastase 1-α1-antitrypsin complex with the immunological partner; A method comprising:

2. 2. The method of claim 1, wherein step (a) is carried out in a pretreatment step of extracting a fecal sample.

3. 2. The method of claim 1, further comprising the step of diluting the fecal sample before adding alpha 1-antitrypsin to the fecal sample in step (a).

4. The method according to claim 1, wherein the amount of α1-antitrypsin added to the fecal sample in step (a) is 20 μg or more.

5. 2. The method according to claim 1, wherein the amount of α1-antitrypsin contained in the reaction solution subjected to the reaction step in step (b) is 100 ng or more.

6. 2. The method of claim 1, wherein the analysis performed in step (d) is any one of a chemiluminescent immunoassay, an electrochemiluminescent immunoassay, a fluorescent immunoassay, a radioimmunoassay, immunochromatography, Western blotting, a latex agglutination assay, and an immunoturbidimetric assay.

7. A method for assisting in the detection of pancreatic diseases, which comprises analyzing pancreatic elastase 1 using the measurement method described in any one of claims 1 to 6.

8. A reagent for use in measuring pancreatic elastase 1 present in a fecal sample, comprising: A reagent for immunoassay, comprising a solid phase carrier carrying an immunological partner that recognizes a pancreatic elastase 1-α1-antitrypsin complex, and α1-antitrypsin to be added to a fecal sample.

9. 9. The reagent for immunoassay according to claim 8, wherein the amount of α1-antitrypsin contained in the reaction solution subjected to the reaction step is 100 ng or more.

10. A pretreatment extract containing α1-antitrypsin, which is used in the pretreatment step of a fecal sample to be used for a reagent for measuring pancreatic elastase 1 or pancreatic elastase 1-α1-antitrypsin.

11. The pretreatment extract solution according to claim 10, containing 41 to 975 μg of the α1-antitrypsin.