JP2023146555A - Test method, test drug, and test kit for aim-related diseases - Google Patents

Abstract

To find a form of existence of AIM having a high correlation with diseases such as acute kidney disorders and use the same as an indicator to enable high precision test of patients.SOLUTION: It has been discovered that immunologically detecting a full-length free AIM enables high-precision testing of AIM-related diseases.SELECTED DRAWING: None

Description

The present invention relates to a testing method, a testing agent, and a testing kit for AIM-related diseases using full-length free AIM as an indicator.

AIM (apoptosis inhibitor of macrophage; also referred to as CD5L, api6, Spα) is known as a secreted protein of approximately 50 kDa that is specifically produced by tissue macrophages (Non-Patent Document 1), and is associated with acute kidney injury, fatty liver, and hepatocytes. It has been shown that it has suppressive effects on various diseases such as cancer, obesity, fungal peritonitis, and multiple sclerosis, and has the potential to become a new therapeutic agent for a wide range of diseases.

The structure of AIM consists of three SRCR (scavenger receptor cysteine-rich) domains, which are specific sequences containing many cysteine residues, connected in tandem. It is thought that it has a compact spherical three-dimensional structure due to disulfide bonds.

AIM is known to interact with various molecules, and various molecules have been reported as its binding partners. For example, bind to LTA (LIPOTEICHOIC ACID) and LPS (LipopolysAcCharide) of fungi (PATHOGEN -ASSOCIATED MOLECULAR PATTERNS: PAMPS). It is known to have the ability to aggregate bacteria (non -patent documents) 2). In addition, there are many cells in the body that bind AIM to the cell surface or take it into the cell, and in addition to taking it into the producing cells, macrophages themselves, in adipocytes it is endowed with AIM through the scavenger receptor CD36. It has been reported that it is taken up by cytosis and induces lipolysis (Non-Patent Document 3).

Furthermore, AIM is known to bind to IgM in blood. In recent years, it has been reported that the binding of AIM and IgM is important for AIM to stably exist in the blood without being excreted in the urine. Furthermore, it has been reported that in serum samples, most of AIM exists as IgM-binding AIM, and almost never exists as a monomer (Non-patent Documents 4, 5). On the other hand, it has been revealed that AIM is dissociated from IgM and activated at the onset of a disease to promote healing of the disease (Non-Patent Document 4). Free AIM dissociated from IgM in the blood passes through the filtration membrane of the glomerulus and migrates to the proximal tubule. Free AIM adheres to dead cell clusters in renal tubules and contributes to the healing of acute kidney injury by promoting phagocytosis of surrounding cells, and at this time, free AIM is excreted in the urine. In fact, it has been reported that in patients with acute kidney injury, a significant increase in free AIM in the blood and AIM concentration in the urine is observed (Non-Patent Document 6).

However, the form of AIM present in human samples has not been sufficiently verified to date, and in order to test for AIM-related diseases with high accuracy, it is necessary to investigate the relationship between the form of AIM and the disease. , further elucidation was required.

Miyazaki T. et al. , J Exp Med 189:413-422, 1999 Sarrias MR et al. , J Biol Chem 280:35391-35398, 2005 Kurokawa J et al. , Cell Metab 11:479-492, 2010 Miyazaki et al., Nippon Rinsai Vol. 71, No. 9 (2013-9), pp. 1681-1689 Arai S. et al. , Science Direct 3(4):1187-1198, 2013 Kento Kitada et al., Japanese Society of Nephrology 58(8):1234-1237, 2016

The present invention was made in view of this situation, and its purpose is to elucidate the existence form of AIM, which has a high correlation with AIM-related diseases such as acute kidney injury, and to use this as an indicator to determine the existence form of AIM with high precision. Its purpose is to perform tests for AIM-related diseases.

In order to solve the above problems, the present inventors produced monoclonal antibodies capable of identifying each form of AIM, and analyzed AIM in the urine of patients who had developed acute kidney injury. As a result, they discovered that two forms of free AIM exist in patient urine: full-length free AIM and free AIM whose molecular weight has been reduced by truncating the C-terminus (hereinafter referred to as "Small AIM"). It was found that the concentrations of full-length free AIM and free AIM increased with onset of the disease. On the other hand, in the urine of subjects who did not develop acute kidney injury (non-acute kidney injury subjects), full-length free AIM was stable at a low concentration, but the concentration of free AIM significantly increased after surgery. It was found that this occurs. Based on these facts, the present inventors believe that it is possible to improve the accuracy of testing for AIM-related diseases by eliminating the influence of Small AIM and using only full-length free AIM as an indicator. I found out. Furthermore, the present inventors discovered that among the produced monoclonal antibodies, a full-length free AIM-specific monoclonal antibody exhibited excellent performance in this test, and the present invention was completed.

More specifically, the present invention provides the following.

[1] Testing method for AIM-related diseases comprising immunologically detecting full-length free AIM in human urine using the antibody described in (a) or (b) below (a) Specific to full-length free AIM (b) A combination of a monoclonal antibody specific for full-length AIM and a monoclonal antibody specific for free AIM.

[2] The method according to [1], wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 263 to 347 of human AIM.

[3] The method according to [1], wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 295 to 347 of human AIM.

[4] The method according to any one of [1] to [3], wherein the AIM-related disease is a renal disease.

[5] A test agent for AIM-related diseases comprising the antibody described in (a) or (b) below for immunologically detecting full-length free AIM in human urine (a) specific for full-length free AIM Monoclonal antibody (b) Combination of a monoclonal antibody specific for full-length AIM and a monoclonal antibody specific for free AIM.

[6] The test agent according to [5], wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 263 to 347 of human AIM.

[7] The test agent according to [5], wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 295 to 347 of human AIM.

[8] A test kit for AIM-related diseases, comprising the test drug according to any one of [5] to [7].

[9] The test agent according to any one of [5] to [7], or the test kit according to [8], wherein the AIM-related disease is a renal disease.

[10] A method for testing AIM-related diseases, including the step of detecting full-length free AIM.

[11] The method for testing an AIM-related disease according to [10], which comprises immunologically detecting full-length free AIM using a monoclonal antibody specific for full-length free AIM.

[12] The testing method according to [11], wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 263 to 347 of human AIM.

[13] The testing method according to [11], wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 295 to 347 of human AIM.

[14] A test agent for an AIM-related disease comprising (a) a monoclonal antibody specific for full-length free AIM or (b) a combination of a monoclonal antibody specific for full-length AIM and a monoclonal antibody specific for free AIM.

[15] The test agent according to [14], wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 263 to 347 of human AIM.

[16] The test agent according to [14], wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 295 to 347 of human AIM.

[17] Monoclonal antibody that binds to positions 263 to 347 of human AIM.

[18] A monoclonal antibody that binds to positions 295 to 347 of human AIM.

[19] A method for immunologically analyzing full-length free AIM in a sample, the method comprising contacting the monoclonal antibody according to [17] or [18] with the sample.

[20] A composition for immunologically analyzing full-length free AIM in a sample, comprising the monoclonal antibody according to [17] or [18].

[21] A solid phase for immunologically analyzing full-length free AIM in a sample, to which the monoclonal antibody according to [17] or [18] is bound.

[22] A kit for immunologically analyzing full-length free AIM in a sample, comprising the composition according to [20] or the solid phase according to [21].

[23] A method for producing a monoclonal antibody that specifically binds to full-length free AIM, comprising the steps of preparing a monoclonal antibody by immunizing with human AIM or a fragment containing positions 263 to 347 thereof, and A method comprising the step of selecting a monoclonal antibody that binds to position 347 from.

[24] A method for producing a monoclonal antibody that specifically binds to full-length free AIM, comprising the steps of preparing a monoclonal antibody by immunizing with human AIM or a fragment containing positions 295 to 347 thereof, and A method comprising the step of selecting a monoclonal antibody that binds to position 347 from.

According to the present invention, it is possible to conduct a highly accurate test for an AIM-related disease (eg, acute kidney injury, etc.) using full-length free AIM in a patient specimen as an indicator. The test of the present invention can be applied to patient urine specimens collected within a short period of time after the event precipitating the onset of the disease (eg, in acute kidney injury, cardiovascular surgery, drug administration, trauma, etc.). Therefore, the present invention is advantageous not only in its high accuracy but also in that it enables earlier diagnosis than conventional serum creatinine as an indicator. The test according to the present invention is an excellent means for assisting doctors in diagnosing AIM-related diseases.

FIG. 2 is a diagram showing the results of detecting the concentration of full-length AIM (rAIM) fractionated by gel filtration chromatography by a sandwich ELISA method using a commercially available kit. This is a diagram showing the results of detecting AIM in serum fractionated by gel filtration chromatography by bioluminescent enzyme immunoassay (BLEIA (registered trademark) method) using various antibody combinations (reagents 1 to 3). be. As a comparative example, the results of detection by sandwich ELISA using a commercially available kit are shown. FIG. 2 is a diagram showing the results of detecting full-length AIM (rAIM) and rSmall AIM by the BLEIA method using various antibody combinations (reagents 1 to 3). FIG. 3 is a diagram showing the results of Western blotting of AIM in serum. FIG. 2 is a diagram showing the results of epitope mapping of antibody clones. FIG. 2 is a diagram showing changes in urinary AIM concentration (median value corrected by creatinine concentration) in subjects who developed acute kidney injury and those who did not develop acute kidney injury. Free AIM was measured by the BLEIA method using Reagent 1, and full-length free AIM was measured by the BLEIA method using Reagent 2. FIG. 3 is a diagram comparing the urinary AIM concentration (value corrected by creatinine concentration) before and 3 hours after surgery in subjects who developed acute kidney injury and those who did not develop acute kidney injury, using U test. Free AIM was measured by the BLEIA method using Reagent 1, and full-length free AIM was measured by the BLEIA method using Reagent 2. FIG. 3 is a diagram showing the changes in urinary AIM concentration (value corrected by creatinine concentration) before and after surgery in each of the subjects (5 cases) who did not develop acute kidney injury. Free AIM was measured by the BLEIA method using Reagent 1, and full-length free AIM was measured by the BLEIA method using Reagent 2. FIG. 3 is a diagram showing the changes in urinary AIM concentration (value corrected by creatinine concentration) before and after surgery in each patient (four cases) who developed acute kidney injury. Free AIM was measured by the BLEIA method using Reagent 1, and full-length free AIM was measured by the BLEIA method using Reagent 2.

The present invention provides a method for testing AIM-related diseases, which includes the step of detecting full-length free AIM. The present invention also provides a method for testing AIM-related diseases, which includes immunologically detecting full-length free AIM in a human specimen.

"AIM" in the present invention is a secreted blood protein of about 40 kDa produced by tissue macrophages. A typical amino acid sequence of human-derived AIM is shown in SEQ ID NO: 1. Note that the DNA sequence of a gene encoding a protein may mutate in nature (ie, non-artificially) due to its mutation. Therefore, the AIM according to the present invention is not limited to the above-mentioned typical amino acid sequences, but also includes natural variants of these amino acid sequences. Human-derived AIM contains three cysteine-rich SRCR domains, the SRCR1 domain corresponds to positions 24 to 124 of SEQ ID NO: 1, and the SRCR2 domain corresponds to positions 138 to 238 of SEQ ID NO: 1. , the SRCR3 domain corresponds to positions 244-346 of SEQ ID NO:1.

In the present invention, "free AIM" refers to AIM that exists in a state that is not bound to IgM, and complex AIM that exists in a complex state with IgM (hereinafter referred to as "IgM-bound AIM"). Used in comparison with. Furthermore, "full-length free AIM" refers to AIM that has a full-length amino acid sequence among free AIMs, and is used in comparison with Small AIM, which is free AIM whose molecular weight has been reduced by truncating the C-terminal side. . Small AIM has, for example, positions 262 and below and positions 1 to 262 in the amino acid sequence of human AIM shown in SEQ ID NO: 1. Small AIM may be included in free AIM.

The "AIM-related disease" tested by the method of the present invention is not particularly limited as long as it is a disease for which AIM can be used as a marker, such as renal disease (particularly acute kidney injury), infectious disease, inflammation, arteriosclerosis, Examples include obesity-related inflammatory diseases, COPD, cancer, liver diseases (liver cancer, fatty liver, etc.), asthma, pulmonary tuberculosis, osteoarthritis, rheumatoid arthritis, sepsis, ischemic stroke, and obesity.

In the method of the present invention, the test sample (specimen) may be any human-derived sample, and human urine is particularly preferred. There are no particular restrictions on human urine, but for example, human urine may be collected from a patient within a relatively short period of time after the event that causes the onset of the disease (for example, in the case of acute kidney injury, cardiovascular surgery, drug administration, trauma, etc.). In the present invention, it is preferable to use urine from the viewpoint that more superior effects can be obtained. A relatively short period of time is, for example, within one week, preferably within two days, more preferably within six hours, and most preferably within three hours. This allows inspection to be performed with higher accuracy.

In the method of the present invention, an antibody specific for full-length free AIM is used as the first embodiment of the antibody for immunologically detecting full-length free AIM. Hereinafter, a monoclonal antibody specific to full-length free AIM will be disclosed as an antibody specific to full-length free AIM, but the present invention is not limited thereto, and a polyclonal antibody specific to full-length free AIM may also be used. Here, "specific for full-length free AIM" means that it does not substantially cross-react with IgM-bound AIM and Small AIM, that is, the reactivity for IgM-bound AIM is sufficiently lower than that for free AIM, and , which means that the reactivity to Small AIM is sufficiently lower than the reactivity to full-length AIM. "Sufficiently low" means 20% or less, preferably 15% or less, more preferably 10% or less with respect to reactivity with free AIM, and 20% or less with respect to reactivity with full-length AIM. , preferably 15% or less, more preferably 10% or less. The reactivity to these AIMs can be evaluated, for example, by a sandwich ELISA method using a monoclonal antibody to be evaluated. The antibody to be combined with the monoclonal antibody to be evaluated in the sandwich ELISA method is not particularly limited as long as it can recognize AIM, and may be either a polyclonal antibody or a monoclonal antibody. It is preferable that the monoclonal antibodies to be combined do not compete with the monoclonal antibody to be evaluated in binding to AIM (ie, recognize different epitopes).

The sandwich ELISA method can be performed, for example, according to the methods described in Examples 1 and 2. Specifically, first, a monoclonal antibody-immobilized plate is prepared in which the monoclonal antibody to be evaluated is bound (immobilized) to the plate, and a sample (a sample containing free AIM, a full-length sample) is placed on the monoclonal antibody-immobilized plate. A sample containing free AIM, a sample containing Small AIM, or a sample containing IgM-bound AIM) is added and reacted. Next, after washing, a polyclonal anti-AIM antibody is added and reacted, and after washing, a labeled secondary antibody is further added and reacted. After washing, the signal intensity of the label is finally measured. When horseradish peroxidase (HRP) is used as a label, the signal can be measured using a microplate reader after adding a chromogenic substrate.

One preferred embodiment of the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to the amino acid sequence consisting of positions 263 to 347, preferably consisting of positions 295 to 347, set forth in SEQ ID NO: 1. The monoclonal antibody typically recognizes a portion of the SRCR3 domain of AIM. Specific examples include antibody clone 3 and antibody clone 4 described in the Examples of the present application.

In the method of the present invention, a combination of an antibody specific for full-length AIM and an antibody specific for free AIM is used as the second embodiment of the antibody for immunologically detecting full-length free AIM. Hereinafter, monoclonal antibodies specific to full-length AIM and monoclonal antibodies specific to free AIM will be disclosed as antibodies specific to full-length AIM and antibodies specific to free AIM, but are not limited to these. A polyclonal antibody specific for full-length AIM or a polyclonal antibody specific for free AIM may be used. Here, "specific for full-length AIM" means that there is substantially no cross-reactivity to Small AIM, that is, the reactivity to Small AIM is sufficiently lower than the reactivity to full-length AIM. "Sufficiently low" refers to a reactivity with full-length AIM of 20% or less, preferably 15% or less, more preferably 10% or less. The full-length AIM-specific monoclonal antibody only needs to react with at least full-length free AIM, and may further react with full-length AIM bound to IgM. The monoclonal antibody specific for free AIM only needs to be specific for AIM that is not bound to IgM, and does not substantially cross-react with IgM-bound AIM. means an antibody that has sufficiently low reactivity to. A monoclonal antibody specific for free AIM only needs to react with at least full-length free AIM, and may further react with Small AIM. Therefore, by combining these monoclonal antibodies, full-length free AIM can be specifically detected.

The monoclonal antibody of the present invention may be produced by a generally known method. For example, as described in the Examples of the present application, first, a hybridoma is produced using recombinant AIM (rAIM) or a part thereof as an immunogen, and from the hybridoma, a monoclonal antibody showing high reactivity to the AIM is produced. The monoclonal antibodies produced by the selected hybridomas are analyzed for specificity to each form of AIM and epitope analysis to identify clones that produce monoclonal antibodies having the above-mentioned characteristics. .

According to the present invention, a monoclonal antibody is prepared by immunization with AIM or a fragment containing positions 263 to 347, preferably 295 to 347, of SEQ ID NO: 1, and By selecting a monoclonal antibody that binds to position 347, a monoclonal antibody that specifically binds to full-length free AIM can be efficiently produced.

Methods for producing monoclonal antibodies include hybridoma methods, typically the method of Kohler and Milstein (Kohler & Milstein, Nature, 256:495 (1975)). The antibody-producing cells used in the cell fusion step in this method include animals (e.g., mice, rats, hamsters, rabbits, monkeys, These include spleen cells, lymph node cells, and peripheral blood white blood cells from goats, sheep, donkeys, camels, alpacas, and chickens. It is also possible to use antibody-producing cells obtained by allowing an antigen to act in a medium on the above-mentioned cells or lymphocytes previously isolated from a non-immunized animal. Various known cell lines can be used as myeloma cells. Antibody-producing cells and myeloma cells may originate from different animal species as long as they can fuse, but are preferably from the same animal species. Hybridomas are produced, for example, by cell fusion between spleen cells obtained from mice immunized with an antigen and mouse myeloma cells, and subsequent screening yields hybridomas that produce monoclonal antibodies specific to the antigen. be able to. Monoclonal antibodies against the antigen can be obtained by culturing hybridomas or from the ascites of mammals to which the hybridomas have been administered.

Furthermore, if DNA encoding the desired monoclonal antibody can be obtained, it can also be produced by recombinant DNA methods. This method involves cloning the DNA encoding the above antibody from hybridomas, B cells, etc., inserting it into an appropriate vector, and injecting it into host cells (e.g., mammalian cell lines, E. coli, yeast cells, insect cells, plant cells, etc.). This is a method of introducing a recombinant antibody and producing it as a recombinant antibody (for example, P.J. Delves, Antibody Production: Essential Techniques, 1997, WILEY, P. Shepherd and C. Dean, Monoclonal Antibodies). dies, 2000, OXFORD UNIVERSITY PRESS, Vandamme A M. et al., Eur. J. Biochem. 192:767-775, 1990). For expression of DNA encoding an antibody, the DNA encoding the heavy chain or light chain may be separately incorporated into an expression vector and transformed into a host cell, or the DNA encoding the heavy chain and light chain may be combined into a single expression vector. It may be incorporated into an expression vector to transform host cells (see WO 94/11523). The recombinant antibody can be obtained in a substantially pure and homogeneous form by culturing the above-mentioned host cells, separating and purifying the host cells or from the culture solution. For isolation and purification of antibodies, methods commonly used for purification of polypeptides can be used. If a transgenic animal (cow, goat, sheep, pig, etc.) into which an antibody gene has been inserted is created using transgenic animal production technology, a large amount of monoclonal antibodies derived from the antibody gene can be produced from the milk of the transgenic animal. It is also possible to obtain

The monoclonal antibody of the present invention may be not only a complete antibody but also an antibody fragment as long as it can recognize the antigen. Examples of antibody fragments include, but are not limited to, F(ab') ₂ , Fab', Fab, Fv, single chain antibodies, and the like.

In the present invention, "detection" includes not only the detection of the presence or absence of full-length free AIM, but also the detection (for example, quantification) of the extent of the presence.

Examples of methods for immunologically detecting full-length free AIM include immunoassays using antibodies labeled with labeling substances (labeled immunoassays), and immunoassays using labeled detection antibodies and labeled antibodies against detection antibodies. It is a method of measurement. For example, immunoagglutination methods (latex agglutination method, colloidal gold agglutination method, etc.) using immunophelometry, immunoturbidimetry, etc., enzyme immunoassay (EIA method) using an enzyme as a label, radiation using a radioactive isotope as a label. immunoassay (RIA), chemiluminescent immunoassay (CLIA method) using a chemiluminescent compound as a label, electrochemiluminescent immunoassay (ECLEIA method) using an electrochemiluminescent substance as a label, using a fluorescent substance as a label Examples include, but are not limited to, fluorescence immunoassay, immunochromatography, Western blotting, and immunoblotting. Examples of the enzyme immunoassay include ELISA, CLEIA (chemiluminescent enzyme immunoassay), and bioluminescent enzyme immunoassay; examples of the bioluminescent enzyme immunoassay include the BLEIA (registered trademark) method. can be mentioned.

When applying a labeled immunoassay as an immunological detection method, at least one of the antigen-capturing antibody (immobilized antibody) immobilized (bound) to a solid phase and the detection antibody is the monoclonal antibody of the present invention. If desired, the monoclonal antibody of the present invention may be labeled by binding a labeling substance to directly detect full-length free AIM, or the monoclonal antibody of the present invention may be labeled without binding a labeling substance. Full-length free AIM may be indirectly detected using a secondary antibody to which a substance is bound.

In a labeled immunoassay, when the monoclonal antibody of the present invention is used as at least one of an antigen-capturing antibody (immobilized antibody) immobilized (bound) on a solid phase and a labeled antibody (detection antibody), the other one is The antibody may be any antibody capable of binding to AIM (anti-AIM antibody), and antibodies other than the monoclonal antibody of the present invention may also be used. The other antibody may be a monoclonal antibody or a polyclonal antibody. Preferably, the monoclonal antibody that is combined with the monoclonal antibody of the invention does not compete in binding with full-length free AIM (ie, recognizes a different epitope).

In a labeled immunoassay, when the monoclonal antibody of the present invention is used as an antigen-capturing antibody and another antibody is used as a detection antibody, the other antibody can be labeled and used. Furthermore, if the other antibody is not labeled, a labeled secondary antibody or the like may be similarly used.

Here, the "secondary antibody" is an antibody that shows reactivity with an antibody (primary antibody) that directly binds to an antigen. For example, when a mouse antibody is used as the primary antibody, an anti-mouse IgG antibody can be used as the secondary antibody. Labeled secondary antibodies that can be used for antibodies derived from various species such as rabbits, goats, and mice are commercially available, and an appropriate secondary antibody can be selected depending on the species from which the primary antibody is derived. and can be used. Instead of the secondary antibody, it is also possible to use protein G, protein A, etc. to which a labeling substance is bound.

Therefore, as described above, in addition to the method of directly detecting full-length free AIM using the monoclonal antibody of the present invention bound to a labeling substance, there is a method in which the monoclonal antibody of the present invention is not bound to a labeling substance and the labeling substance is not bound to the monoclonal antibody of the present invention. An indirect detection method using a bound secondary antibody or the like can also be used.

The labeling substance is not particularly limited as long as it can be detected by binding to an antibody, but examples include enzymes (such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), firefly luciferase, etc.), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC), etc.), fluorescent proteins (e.g., allophycocyanin (APC) and phycoerythrin (R-PE)), and radioactive substances such as ¹²⁵ I. Isotopes, magnetic particles, latex particles (e.g. polystyrene, styrene-butadiene copolymer, etc.), metal colloid particles (e.g. gold, silver, copper, iron, platinum, palladium, etc., or mixtures thereof), avidin, biotin Examples include.

When an enzyme is used as a labeling substance, various detections can be performed depending on the substrate by adding a peroxide and/or a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, or the like as a substrate.

For example, when horseradish peroxidase (HRP) is used as an enzyme, o-phenylenediamine (coloring), tetramethylbenzidine (TMBZ) (coloring), luminol (chemiluminescence), etc. are used as substrates along with hydrogen peroxide. Alternatively, when alkaline phosphatase (ALP) is used as the enzyme, its substrate may be p-nitrophenyl phosphate (color development), AMPPD (registered trademark) (chemiluminescence), or the like.

When firefly luciferase is used as an enzyme, firefly luciferin or the like may be used as a substrate together with ATP, and the biotinylated luciferase described in Japanese Patent No. 3466765 is used as an enzyme, and the biotinylated luciferase described in Japanese Patent No. 4379644 or Japanese Patent No. 4503724 is used as a substrate. Luciferin described in the publication may be used.

Furthermore, when the enzyme binds to streptavidin and reacts with the substrate to generate fluorescence, luminescence, or color, the label may be biotin.

As a method for binding the antibody and the labeling substance, a known method can be used, for example, a biotin-avidin system can also be used. In this method, for example, a biotinylated antibody is treated with an avidinized labeling substance, and the interaction between biotin and avidin is utilized to bind the labeling substance to the antibody.

Further, as a method for immunologically detecting full-length free AIM, a non-competitive assay method including a sandwich method or a competitive assay method can be used as the measurement principle.

In addition, as a method for immunologically detecting full-length free AIM in a sample, a heterogeneous method (e.g., sandwich method) that performs B/F separation using an insoluble carrier, etc. can also be used. It is also possible to measure by a homogeneous method (for example, immunoagglutination method) that does not involve.

In the sandwich method, full-length free AIM is captured with an antigen-capturing antibody immobilized (bound) on a solid phase, recognized by a detection antibody bound to a labeling substance, and after B/F separation (washing), the labeling substance itself is Alternatively, full-length free AIM in the sample is detected by adding a substrate for a labeling substance such as an enzyme and causing color development. As the principle for detecting full-length free AIM in the method of the present invention, a sandwich method is preferable because a highly sensitive detection system can be constructed.

In addition, as in the immunochromatography method, full-length free AIM is recognized with a detection antibody bound to a labeling substance, and while B/F separation is performed, full-length free AIM is recognized with an antigen-capturing antibody immobilized (bound) on a solid phase. The labeling substance may be captured and detected depending on the type of labeling substance.

In addition, as in the BLEIA method, an antigen-capturing antibody is bound to magnetic particles, the antibody is reacted with full-length free AIM in the sample, and after B/F separation, it is reacted with a biotinylated detection antibody. After F separation, an immunoreaction is performed using avidin labeled with luciferase, and after B/F separation, luciferin is added, and the enzymatic activity of the luciferase complex is detected by bioluminescence to detect full-length free AIM in the sample. It's okay.

In addition, as in the immunoaggregation method, insoluble carrier particles (solid phase) to which the monoclonal antibody of the present invention is immobilized (bound) are used in a liquid phase to form an immune complex between the insoluble carrier particles and full-length free AIM. Utilizing the property of insoluble carrier particles aggregating upon formation, aggregation of insoluble carrier particles may be detected by measuring turbidity, visual inspection, or measuring absorbance. The immunoaggregation method is preferred in the method of the present invention because it does not require a B/F separation step before detecting full-length free AIM and has the advantage that full-length free AIM can be detected simply and quickly.

Examples of the insoluble carrier (solid phase) include polystyrene, polycarbonate, polyvinyltoluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, latex, gelatin, agarose, nitrocellulose, Sepharose, glass, metal, ceramics, or magnetic material. Insoluble carriers in the form of particles, plates, test pieces, etc. made of a material such as carbohydrate can be used. In particular, metal or magnetic particles can be used as the insoluble carrier particles, but latex particles are preferred, and polystyrene latex is generally used.

Antibodies can be immobilized on the surface of a solid phase by known techniques, such as physical adsorption or chemical bonding. The capture antibody may be directly immobilized on the solid phase, or may be immobilized indirectly. For example, the capture antibody can be indirectly immobilized on the solid phase by immobilizing a substance that binds to the capture antibody on the solid phase and binding the capture antibody to the substance. Examples of the substance that binds to the capture antibody include, but are not limited to, the above-mentioned secondary antibodies, protein G, protein A, and the like. Furthermore, when the capture antibody is biotinylated, an avidinized solid phase can be used.

In the method of the present invention, when full-length free AIM is detected using a combination of a monoclonal antibody specific for full-length AIM and a monoclonal antibody specific for free AIM, one is used as an antigen-capturing antibody, and the other is used as an antigen-capturing antibody. may be used as a detection antibody.

Quantitation of full-length free AIM from the obtained measured values can generally be performed by comparison with measured values from standard samples. In this case, for example, the full-length free AIM in the sample is quantified by examining where the actual measured value is located on a standard curve (calibration curve) created based on the measured value of the standard sample. be able to.

The present invention also provides a test agent for AIM-related diseases comprising (a) a monoclonal antibody specific for full-length free AIM or (b) a combination of a monoclonal antibody specific for full-length AIM and a monoclonal antibody specific for free AIM. provide. The present invention also provides a test agent for AIM-related diseases, which includes an antibody for immunologically detecting full-length free AIM in a human-derived sample, preferably in human urine. The antibodies include the above-mentioned monoclonal antibody specific for full-length free AIM (the first embodiment of the antibody), and a combination of the above-mentioned monoclonal antibody specific for full-length AIM and a monoclonal antibody specific for free AIM (the first embodiment of the antibody). Second aspect).

As described above, the monoclonal antibody contained in the test agent of the present invention may be bound to a labeling substance, or may be bound to a solid phase. Examples of the solid phase include the above-mentioned insoluble carriers, and for example, when the detection principle is a sandwich method such as a sandwich ELISA method, examples thereof include plates, fibrous substances, particles, etc. to which the monoclonal antibody of the present invention is bound. When immunochromatography is used as the detection principle, an immunochromatography device in which the monoclonal antibody of the present invention is bound to an insoluble carrier particle contained in a labeled reagent zone (in the case of a detection antibody) or a detection zone (in the case of a capture antibody) can be used. It will be done. Furthermore, when the immunoagglutination method is used as the detection principle, examples include insoluble carrier particles, such as latex particles, to which the monoclonal antibody of the present invention is bound.

In addition to the antibody component, the test agent of the present invention may contain other components such as sterile water, physiological saline, a buffer, and a preservative, if necessary.

The present invention also provides a test kit for AIM-related diseases, which includes the above-described test agent of the present invention. The kit of the present invention can be further combined with standard samples (reagents containing full-length free AIM at various concentrations), control reagents, sample dilution solutions, dilution cartridges, washing solutions, and the like, as necessary. When using an enzyme label for detection, substrates, reaction termination solutions, etc. necessary for detecting the label can be included. When detecting full-length free AIM indirectly, a labeled substance that binds to the primary antibody (secondary antibody, protein A, etc.) can be included. Furthermore, when the monoclonal antibody of the present invention is biotinylated, an avidinized label can be included. The kit of the present invention can further include instructions for using the kit.

The present invention provides a method for immunologically detecting full-length free AIM in a sample, which comprises contacting the sample with a monoclonal antibody specific for full-length free AIM (the first embodiment of the antibody) described above. There are no particular limitations on the sample as long as the sample can contain full-length free AIM.

Furthermore, the present invention provides a composition for immunologically analyzing full-length free AIM in a sample, comprising a monoclonal antibody (first embodiment of the antibody) specific for the above-mentioned full-length free AIM, A solid phase is provided for immunologically analyzing full-length free AIM in a sample to which a monoclonal antibody specific for AIM (the first embodiment of the antibody) is bound. The solid phase may be any of the solid phases mentioned above, for example, plates such as plastic plates, fibrous substances such as nitrocellulose, particles such as magnetic particles, latex particles, colloidal gold particles, etc., and there are no particular limitations. . Further provided is a kit for immunologically analyzing full-length free AIM in a sample, which includes the composition or solid phase.

The embodiments described above are described to facilitate understanding of the present invention, and are not described to limit the present invention. Furthermore, each element disclosed above is not limited to the above content, but is intended to include all design changes, equivalents, and equivalent methods that fall within the technical scope of the present invention. For example, the antibodies used in the method of the present invention and the antibodies contained in the test reagent and kit of the present invention are not limited to monoclonal antibodies, but also include polyclonal antibodies.

[Reference Example 1] Preparation of anti-AIM antibody Mice were immunized with human recombinant AIM (hereinafter referred to as rAIM) (30-50 μg) as an antigen 3-6 times at 3-week intervals. The immunized splenocytes and mouse myeloma cells were fused, and the fused cells were cloned by limited dilution. After cloning the cells, they were screened by ELISA using an immobilized immunogen to obtain cells that produced antibodies that reacted with rAIM. Cells in which antibody production was confirmed were cultured, and the antibodies produced in the culture supernatant were purified using protein A or protein G. ) was obtained.

[Reference Example 2] Production of rAIM and rSmall AIM rAIM was produced by the method described in the literature (Seikagaku Vol. 84, No. 7, Purification of Functional rAIM Protein, pp. 588-591, 2012), and the full-length AIM (hereinafter also referred to as full-length AIM). Recombinant Small AIM (rSmall AIM) is based on the literature (Seikagaku Vol. 84, No. 7, Functional rAIM protein Purification, pp. 588-591, 2012) and used as Small AIM (hereinafter also referred to as Small AIM).

[Example 1] Specificity analysis of antibodies against free AIM A rabbit polyclonal anti-AIM antibody obtained by immunizing a rabbit with rAIM and obtained by a conventional method and each monoclonal antibody obtained in Reference Example 1 were used as solid-phase antibodies. Reactivity with free AIM or IgM-bound AIM obtained by the following method was confirmed.

(1) Sample The measurement sample was prepared by dissolving a serum specimen in a buffer solution (0.05M phosphate buffer: pH 7.0, sodium chloride: 0.3M), injecting it into a column, and gel-filtering it under the following conditions. Chromatographic fractionation could be performed.

Gel filtration chromatography conditions Equipment used: SHIMADZU SPD-20AV
Column: PHENOMENEX (registered trademark) SEC-3000
Mobile phase: 0.05M phosphate buffer, 0.3M NaCl, pH 7.0
Flow rate: 0.5mL/min
Fraction: 0.5mL/fraction

A fraction with an elution time of 10 to 17 minutes (eluate volume: 5 to 8.5 mL) was quantified using an AIM measurement kit (manufactured by IBL) to prepare a sample of 20 ng/mL of IgM-bound AIM. Similarly, samples with 20 ng/mL of free AIM were prepared from fractions with elution times of 19-23 minutes (eluate volumes of 9.5-11.5 mL).

(2) Measurement method After dispensing the solid-phase antibody (clone 1, clone 3, clone 4, or clone 81) into each well of a 96-well microplate and immobilizing it, wash it and add 1% BSA. The solution was added and left at room temperature for 2 hours. The plate was washed, 50 μL of the sample was added, and the reaction was allowed to proceed at room temperature for 1 hour. Next, the solution in the well was removed by suction, and after washing, 50 μL of rabbit polyclonal anti-AIM antibody was added and reacted at room temperature for 1 hour. Further, the solution in the well was removed by suction, and after washing, 50 μL of anti-rabbit IgG-HRP was added and allowed to react at room temperature for 1 hour. Further, the solution in the well was removed by suction, and after washing, 50 μL of hydrogen peroxide solution and o-phenylenediamine-containing buffer solution were added and reacted, and then 50 μL of 2N sulfuric acid was added as a reaction stop solution. Measurement was performed using a microplate reader at a measurement wavelength of 492/650 nm.

(3) Measurement Results For each antibody, Table 1 shows the ratio (in %) of the reactivity of IgM-bound AIM to the reactivity of free AIM, using the measured value of free AIM as a reference (100). Antibody clone 3, antibody clone 4, and antibody clone 1 were found to be specific for free AIM.

It was confirmed by sandwich ELISA that the polyclonal antibody used in this example reacts with peptides at positions 20-128, 124-240, and 236-337 of AIM.

[Example 2] Antibody specificity analysis for full-length AIM and Small AIM A rabbit polyclonal anti-AIM antibody obtained by immunizing a rabbit with rAIM and obtained by a conventional method and each monoclonal antibody obtained in Reference Example 1 were used as solid phase antibodies. The reactivity with the full-length AIM or Small AIM obtained by the method of Reference Example 2 was measured using the following methods.

(1) Sample The measurement sample was obtained by adjusting the full-length AIM or Small AIM obtained by the method of Reference Example 2 to 20 ng/mL with PBS.

(2) Measurement method The reactivity of each antibody was measured using the same measurement method as in Example 1, except for the measurement sample.

For each antibody, Table 2 shows the ratio (in %) of the reactivity of Small AIM to the reactivity of full-length AIM, with the measured value of full-length AIM as the standard (100). Antibody clone 3 and antibody clone 4 were shown to be full-length AIM-specific. On the other hand, antibody clone 1 and antibody clone 81 reacted with Small AIM.

It was confirmed by sandwich ELISA that the polyclonal antibody used in this example reacts with peptides at positions 20-128, 124-240, and 236-337 of AIM.

[Example 3] Measurement of full-length AIM by sandwich ELISA method The full-length AIM obtained in Reference Example 2 was fractionated by gel filtration chromatography according to elution time (eluate amount), and reactivity was measured.

(1) Sample The full-length AIM obtained in Reference Example 2 was fractionated by gel filtration chromatography in the same manner as in Example 1, and the elution time was 8 to 28 minutes (eluate volume: 4-14 mL). Each fraction was used as a sample.

(2) Measurement method The AIM concentration of the obtained sample was measured using Human AIM/CD5L Assay Kit (IBL, #27265).

(3) Measurement results The measurement results are shown in Figure 1. It was confirmed that the full-length AIM obtained in Reference Example 2 had a main peak in the fractionated sample around 20 minutes (10 mL).

[Example 4] Specificity analysis for free AIM by bioluminescent enzyme immunoassay (hereinafter referred to as BLEIA method) (1) Sample The measurement sample was a serum specimen mixed with a buffer solution (0.05M phosphate buffer: pH 7. 0.0, sodium chloride: 0.3M) and injected into a column to perform fractionation by gel filtration chromatography under the following conditions.

Gel filtration chromatography conditions Equipment used: SHIMADZU SPD-20AV
Column: PHENOMENEX (registered trademark) SEC-3000
Mobile phase: 0.05M phosphate buffer, 0.3M NaCl, pH 7.0
Flow rate: 0.5mL/min
Fraction: 0.5mL/fraction
Fractions of every 0.5 mL with an elution time of 9 minutes to 25 minutes (eluate volume 4.5-12.5 mL) were used as samples.

(2) Specificity analysis for free AIM Using a fractionated sample of a serum sample, clone 1, clone 3, clone 4, and clone 81 obtained in Reference Example 1 were combined as shown in Table 3 below, and the BLEIA method was used. Measurements were made with.

(3) Measurement method (BLEIA method)
This was carried out using the method described in JP-A-10-239314 (biotin is bound to both the antibody and the enzyme). In detail, various antibody-immobilized magnetic particles obtained by immobilizing various solid-phase antibodies on magnetic particles, a biotin-labeled antibody (clone 81) obtained by mixing a labeling antibody (clone 81) and a biotinylation reagent, and streptavidin-biotinylated luciferase. Then, for each antibody combination of Reagents 1 to 3, 80 μL of biotin-labeled antibody solution, 100 μL of sample, and 20 μL of antibody-immobilized magnetic particles (1.5 mg/mL) were mixed and reacted at 37°C for 15 minutes. Ta. Furthermore, 500 μL of BL cleaning solution (Eiken Chemical) was added to the reaction solution containing the magnetic particles, and the BL cleaning solution was removed. Subsequently, 80 μL of streptavidin-biotinylated luciferase was added and reacted at 37° C. for 15 minutes. 500 μL of BL cleaning solution (Eiken Chemical) was added to the reaction solution containing the magnetic particles, and the cleaning solution was removed. Next, 50 μL of BL Luminescence Reagent 1 from the BL Luminescence Reagent Set (Eiken Chemical) and 50 μL of BL Luminescence Substrate Solution, which is a substrate solution for luciferase (luciferin solution), were added, and the luminescence intensity was measured using a fully automatic biochemiluminescence immunoassay device BLEIA- 1200, and the AIM concentration in the sample was calculated.

(4) Measurement of total AIM (ELISA method)
AIM concentration was measured using a commercially available kit Human AIM/CD5L Assay Kit (IBL, #27265) (sandwich ELISA method) (comparative example).

(5) Measurement results The measurement results are shown in Figure 2. For all of Reagent 1, Reagent 2, and Reagent 3, only the peak of the fractionated sample with an elution time (flow rate) of around 20 minutes (10 mL) was confirmed, and it was found that the peak was specific to free AIM.

[Example 5] Specificity analysis for full-length AIM (1) Sample Full-length AIM or Small AIM obtained by the method described in Reference Example 2 was diluted with PBS to give 1.25, 2.5, 5, 10, and 20 ng, respectively. /mL.

(2) Measurement method (BLEIA method)
The luminescence value was measured in the same manner as in Example 4 (BLEIA method) except for the measurement sample.

(3) Measurement results The measurement results are shown in Figure 3. Concentration-dependent reactions with AIM were observed for all reagents. Reagent 1 also showed a concentration-dependent reaction in Small AIM. On the other hand, Reagent 2 and Reagent 3 were found to have low reactivity to Small AIM and were specific to full-length free AIM.

[Example 6] Specificity analysis for full-length AIM and Small AIM (1) Sample Samples were prepared by diluting full-length AIM or Small AIM obtained by the method described in Reference Example 2 with PBS (the amount of AIM in each sample was For concentrations, see Table 4 below).

(2) Measurement method (BLEIA method)
The measurement was performed in the same manner as in Example 4 (BLEIA method) except for the measurement sample.

(3) Measurement results Table 4 shows the measurement results.

The results of measuring the sample with Reagent 1 showed a tendency consistent with the total amount of full-length AIM and small AIM. On the other hand, Reagent 2 and Reagent 3 were not affected by Small AIM and had a concentration consistent with that of full-length AIM. These results revealed that Reagent 2 and Reagent 3 can quantitatively measure full-length AIM in a sample containing both Small AIM and full-length AIM.

[Example 7] Measurement of urine specimens (1) Sample Five urine specimens were measured as samples. No dilution of the specimen was performed.

(2) Measurement method (BLEIA method)
The measurement was performed in the same manner as in Example 4 (BLEIA method), except for the measurement sample and the use of the antibody combination of reagents 1 and 2.

(3) Measurement results The measurement results are shown in Table 5.

The measured values of Reagent 1 and Reagent 2 were different. Since small AIM and full-length free AIM are present in patient urine samples, reagent 1, which measures both small AIM and full-length free AIM, gives a high measured value, while reagent 2, which measures only full-length free AIM, gives a high measured value. It is thought that these measured values deviate from each other.

[Example 8] Measurement of serum samples (1) Sample Five serum samples were measured as samples. The specimen was diluted 500 times with a solution containing 1% BSA.

(2) Measurement method (ELISA method)
The obtained sample was measured by the following sandwich ELISA method using the antibody combination of Reagents 1 and 2 shown in Example 4.

First, 50 μL of solid-phase antibodies were dispensed into each well of a 96-well microplate, and then washed to immobilize each solid-phase antibody. After washing, a solution containing 1% BSA was added and left at room temperature for 2 hours. Further, the labeling antibody was biotinylated and labeled using the biotin labeling reagent Biotin (AC5) 2Slufo-osu (Dojindo Laboratories). The antibody immobilized plate was washed, 50 μL of the standard solution or sample was added, and the reaction was allowed to proceed at room temperature for 1 hour. Subsequently, the solution in the well was removed by suction, and after washing, 50 μL of biotin-labeled antibody was added and reacted at room temperature for 1 hour. Further, the solution in the well was removed by suction, and after washing, 50 μL of streptavidin-HRP was added and reacted at room temperature for 1 hour. Further, the solution in the well was removed by suction and washed, and 50 μL of a buffer solution containing hydrogen peroxide and o-phenylenediamine was added to cause a reaction, and then 50 μL of 2N sulfuric acid was added as a reaction stop solution. Measurement was performed using a microplate reader at a measurement wavelength of 492/650 nm.

(3) Measurement results Table 6 shows the measurement results.

The measured values of Reagent 1 and Reagent 2 were in agreement. This is because most of the free AIM present in serum samples is full-length free AIM, so the measured values of reagent 1, which measures both small AIM and full-length free AIM, and reagent 2, which measures only full-length free AIM, match. It is thought that this was done.

[Example 9] Measurement of AIM in serum (1) Sample 10 μL of EzApply (AE-1430, ATTO) was added to 0.5 μL of human serum sample or 10 ng of full-length AIM and Small AIM prepared in Reference Example 2, and the mixture was heated at 95°C. A sample was prepared by heating for 10 minutes.

(2) Measurement method (Western blotting)
After adding a sample to a polyacrylamide gel and performing electrophoresis, the proteins in the gel were transferred to a PVDF membrane. This was shaken in 5% skim milk/PBS at 25°C for 1 hour. Subsequently, after washing four times with PBS-T, the plate was reacted overnight at 4°C in a solution containing a commercially available polyclonal anti-AIM antibody (CD5L Antibody, NBP1-76700, NOVUS BIOLOGICALS). Furthermore, after washing four times using PBS-T, the mixture was reacted in a TIDY Blot WESTERN BLOT DETECTION REAGENT:HRP (BIO-RAD) solution at 25° C. for 1 hour. Finally, after washing four times with PBS-T, a chemiluminescence detection reagent was used to react for 1 minute, and chemiluminescence was detected with a CCD camera.

(3) Measurement results The measurement results are shown in Figure 4. In the human serum sample, a full-length AIM band was observed, but a small AIM band was not observed, making it clear that small AIM was hardly present in the serum. Since most of the AIM present in the serum sample was full-length AIM, in Example 8, the measured values of Reagent 1, which measured both Small AIM and full-length free AIM, and Reagent 2, which measured only full-length free AIM, were in agreement. it is conceivable that.

[Example 10] Epitope mapping of anti-AIM antibodies Antibody clone 1, clone 3, clone 4, and clone 81 used in reagents 1 to 3 shown in Example 4 were analyzed for epitopes.

(1) Preparation of AIM deletion mutant An expression vector (pcDNA3.3) for the full-length AIM protein of SEQ ID NO: 1 or each deletion mutant was prepared (Table 7), transfected into HEK293T cells, and the culture supernatant was collected. Mutants A to D and rAIM (full-length AIM) were obtained.

(2) Immunoprecipitation Antibodies clone 1, clone 3, clone 4, and clone 81 were each reacted with DYNABEADS (registered trademark) magnetic beads protein G (Invitrogen) for 10 minutes. After washing once, 300 μL of the collected culture supernatant was added. As NC (negative control), the culture supernatant of HEK293T cells into which the vector had not been introduced was added in the same manner. The reaction mixture was allowed to react at room temperature for 60 minutes, and washed three times with a washing solution. The washing solution was removed, 30 μL of EzApply (AE-1430, ATTO) was added, and the mixture was heated at 95° C. for 10 minutes to prepare a sample for Western blotting.

(3) Western Blot After adding 20 μL of the sample to a polyacrylamide gel and performing electrophoresis, the proteins in the gel were transferred to a PVDF membrane. This was shaken in 5% skim milk/PBS at 25°C for 1 hour. Subsequently, after washing four times with PBS-T, the plate was reacted overnight at 4°C in a solution containing a commercially available polyclonal anti-AIM antibody (CD5L Antibody, NBP1-76700, NOVUS BIOLOGICALS). Furthermore, after washing four times using PBS-T, the mixture was reacted in a TIDY Blot WESTERN BLOT DETECTION REAGENT:HRP (BIO-RAD) solution at 25° C. for 1 hour. Finally, after washing four times with PBS-T, a chemiluminescence detection reagent was used to react for 1 minute, and chemiluminescence was detected with a CCD camera.

The results are shown in FIG. Antibody clone 3 and antibody clone 4 did not react with mutants A to D, but reacted with rAIM. From this, it was revealed that antibody clone 3 and antibody clone 4 react with a part of the SRCR3 domain in AIM (positions 263 to 347, particularly positions 295 to 347 of AIM shown in SEQ ID NO: 1). On the other hand, antibody clone 81 was found to react with the SRCR1 domain since it reacted with mutants A to C and rAIM. Clone 1, which is a free AIM-specific antibody, did not react with variant A, but reacted with variant B, variant C, and rAIM, indicating that it reacts with the SRCR2 domain.

[Example 11] Measurement of urine specimens from patients with acute kidney injury (AKI) (1) Samples For patients who underwent cardiac large vessel surgery using cardiopulmonary bypass, preoperatively, immediately postoperatively, 3 hours postoperatively, and postoperatively. Urine was collected after 6 hours, 1 day, 2 days, 3 days, 4 days, 5 days, and 6 days.

According to the KDIGO criteria, 83 urine specimens clinically diagnosed as having developed AKI or not having developed AKI were used. There were 17 patients who developed AKI (AKI) and 66 patients who did not develop AKI (non-AKI).

(2) Measurement of AIM AIM was measured in the same manner as in Example 4 (BLEIA method), except that a collected urine specimen was used and a combination of antibodies from Reagents 1 and 2 was used.

(3) Measurement of urine creatinine The collected urine specimen was sampled using an automatic clinical chemistry analyzer JCA-BM6070 (JEOL Ltd.) using Exdia (registered trademark) XL'Eiken'CRE-V (Eiken Chemical Co., Ltd.). The creatinine concentration in the sample was calculated. The measured AIM concentration was corrected by the creatinine concentration to obtain an AIM creatinine correction value.

(4) Change in median urinary AIM concentration FIG. 6 shows the change in the median urinary AIM creatinine correction value in AKI patients (AKI) and non-AKI patients.

Increased concentrations of both free AIM and full-length free AIM were observed postoperatively in patients who developed AKI. An increase in the concentration of free AIM was observed even in non-symptomatic subjects, but no obvious increase in the concentration of full-length free AIM was observed. By eliminating the influence of Small AIM, which also exists in healthy human urine, the difference between non-AKI and AKI becomes clear, so the full-length free AIM measurement system is more advantageous in diagnosing AKI than the free AIM measurement system. It turned out to be.

(5) Median ratio of AKI patients (AKI) and non-AKI patients Median value of urinary free AIM or full-length free AIM creatinine-corrected value in AKI patients and non-AKI patients, and the median value Table 8 shows the ratio of those who developed AKI and those who did not develop AKI.

The median ratio of AKI/non-AKI was clearly larger for full-length free AIM, indicating that measurement of full-length free AIM (Reagent 2) is more advantageous in diagnosing AKI than measurement of the total amount of AIM (Reagent 1). It turns out that there is something.

(6) Comparison of AKI patients (AKI) and non-AKI patients by U test FIG. 7 shows a comparison of AIM creatinine correction values in urine before surgery and 3 hours after surgery. Mann-Whitney U test was used for statistical analysis.

Before surgery, no significant difference was observed in both free AIM and full-length free AIM between patients who developed AKI and those who did not. Three hours after surgery, there was no significant difference in free AIM between those who developed AKI and those who did not, but full-length free AIM showed a significantly higher value in those who developed AKI (p<0.05). . It was found that full-length free AIM is not affected by small AIM, which is also present in the urine of healthy people, and is advantageous for diagnosis.

(7) Changes in urinary AIM concentration in each case Figure 8 shows changes in AIM creatinine correction values in 5 non-symptomatic patients.

Although there are cases in which free AIM levels increase postoperatively even in non-symptomatic subjects, no clear postoperative concentration increase was observed in full-length free AIMs. In non-symptomatic subjects, it was thought that the free AIM concentration increased due to the increase in the Small AIM concentration after surgery. On the other hand, since full-length free AIM is not affected by the increase in concentration of Small AIM in non-symptomatic patients, it is thought that no increase in concentration was observed after surgery.

(8) Changes in urinary AIM concentration in each case Figure 9 shows changes in AIM creatinine correction values in four cases of AKI patients.

Postoperative increases in concentration of both free AIM and full-length free AIM were observed in patients who developed AKI.

According to the present invention, by using full-length free AIM as an index, it is possible to test AIM-related diseases with high accuracy, which can greatly contribute to the diagnosis of AIM-related diseases.

Claims (16)

A method for testing AIM-related diseases, the method comprising immunologically detecting full-length free AIM in human urine using the antibody described in (a) or (b) below.
(a) Monoclonal antibody specific for full-length free AIM (b) Combination of monoclonal antibody specific for full-length AIM and monoclonal antibody specific for free AIM 2. The method of claim 1, wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 263 to 347 of human AIM. 2. The method of claim 1, wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 295 to 347 of human AIM. The method according to any one of claims 1 to 3, wherein the AIM-related disease is a renal disease. A test agent for AIM-related diseases, comprising the antibody described in (a) or (b) below, for immunologically detecting full-length free AIM in human urine.
(a) Monoclonal antibody specific for full-length free AIM (b) Combination of monoclonal antibody specific for full-length AIM and monoclonal antibody specific for free AIM The test agent according to claim 5, wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 263 to 347 of human AIM. The test agent according to claim 5, wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 295 to 347 of human AIM. A test kit for AIM-related diseases, comprising the test drug according to any one of claims 5 to 7. The test agent according to any one of claims 5 to 7, or the test kit according to claim 8, wherein the AIM-related disease is a kidney disease. A method for testing an AIM-related disease, comprising the step of detecting full-length free AIM. 11. The method for testing an AIM-related disease according to claim 10, which comprises immunologically detecting full-length free AIM using a monoclonal antibody specific for full-length free AIM. The testing method according to claim 11, wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 263 to 347 of human AIM. The testing method according to claim 11, wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 295 to 347 of human AIM. A test agent for an AIM-related disease comprising (a) a monoclonal antibody specific for full-length free AIM or (b) a combination of a monoclonal antibody specific for full-length AIM and a monoclonal antibody specific for free AIM. The test agent according to claim 14, wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 263 to 347 of human AIM. The test agent according to claim 14, wherein the monoclonal antibody specific for full-length free AIM is a monoclonal antibody that binds to positions 295 to 347 of human AIM.