JP2024039982A - How to assess the risk of developing acute kidney injury - Google Patents

Abstract

[Problem] To provide a method for evaluating the risk of developing acute kidney injury. SOLUTION: A method for evaluating the risk of developing acute kidney injury, including the step of detecting free AIM and/or full-length free AIM in a blood sample collected from a subject. [Selection diagram] None

Description

The present invention relates to a method for evaluating the risk of developing acute kidney injury using free AIM in blood and/or full-length free AIM as an indicator, and a reagent or kit for evaluating the risk of developing acute kidney injury. The present invention also relates to a biomarker for assessing the risk of developing acute kidney injury, including free AIM in the blood and/or full-length free AIM.

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, it is known to have the ability to bind to fungal pathogen-associated molecular patterns (PAMPs) such as LTA (lipoteichoic acid) and LPS (lipopolysaccharide) and agglutinate bacteria (Non-patent Document 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).

AIM is also known to bind to IgM in blood. In recent years, it has been reported that the binding of AIM to IgM is important for AIM to remain stable in blood without being excreted in urine. It has also been reported that in serum samples, most AIM exists as IgM-bound AIM, and is rarely present as a monomer (Non-Patent Documents 4 and 5). On the other hand, it has been revealed that AIM dissociates from IgM and becomes activated at the onset of disease, promoting the healing of the disease (Non-Patent Document 4). Free AIM dissociated from IgM in blood passes through the filtration membrane of the glomerulus and migrates to the proximal tubule. Free AIM adheres to dead cell masses in the tubule and promotes the phagocytosis of surrounding cells, contributing to the healing of acute kidney injury, at which time free AIM is excreted in urine. In fact, it has been reported that a significant increase in blood free AIM and urinary AIM concentrations is observed in patients with acute kidney injury (Non-Patent Document 6).

Thus, it has been suggested that there is a correlation between free AIM in blood and AIM in urine after the onset of acute kidney injury and acute kidney injury after onset. However, it is not clear whether AIM correlates with before the onset of acute kidney injury, that is, whether the onset of the disease can be predicted using AIM as an index. Furthermore, it is not clear what form of AIM (IgM-bound AIM, free AIM, etc.) is correlated with the risk of developing acute kidney injury.

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 that is highly correlated with the risk of developing acute kidney injury, and to use this as an indicator to develop acute kidney injury. The goal is to predict whether or not it will happen.

In order to solve the above problems, the present inventors produced monoclonal antibodies capable of identifying each form of AIM, and further constructed a detection system for various forms of AIM using them.

Note that, as forms of AIM, IgM-bound AIM and free AIM have been known for some time. However, as shown in Figure 1, the present inventors have developed free AIM through the production of the monoclonal antibody, and have further developed full-length free AIM and free AIM whose molecular weight has been reduced by truncating the C-terminus ( This makes it possible to detect two types of AIM (Small AIM).

Then, using the detection system, the concentration of free AIM (full-length free AIM and small AIM) and the concentration of full-length free AIM in preoperative blood samples (serum) in patients with and without acute kidney injury (AKI) were determined. The concentration, as well as the concentration of total AIM (total concentration of IgM-bound AIM and free AIM, also referred to as "total AIM") was measured. Furthermore, based on these measured values, these ratios are calculated as concentration of free AIM/total amount of AIM (hereinafter also referred to as free AIM/total amount of AIM), concentration of full-length free AIM/total amount of AIM (hereinafter also referred to as full-length free AIM/total amount of AIM). ) was calculated. As a result, the concentrations of free AIM, full-length free AIM, and their ratio were significantly higher in patients with AKI than in patients without AKI even before surgery. Therefore, the concentration of free AIM, the concentration of full-length free AIM, the total amount of free AIM/AIM, and the total amount of full-length free AIM/AIM (hereinafter also collectively referred to as "the amount of free AIM in serum") in serum are important indicators of the onset of AKI. It was shown to be useful for risk assessment.

Furthermore, existing urinary AKI-related markers and blood renal function markers were also evaluated for their ability to predict the risk of developing AKI, and compared with those of the amount of free AIM in serum. As a result, it was revealed that the amount of free AIM in serum exhibits superior predictive ability for the onset of AKI compared to urinary AKI-related markers.

Furthermore, the relationship between renal function and prediction of the onset of AKI was analyzed using known blood renal function markers and the amount of free AIM in serum. As a result, the correlation between the blood renal function marker and the amount of free AIM in serum was weak and varied widely. This suggests that the amount of free AIM in serum can reflect a different pathological condition than the blood renal function marker. Furthermore, it has been revealed that even in cases where the blood renal function marker shows false negatives or false positives, the onset of AKI can be accurately predicted by detecting the amount of free AIM in the serum. Furthermore, as a result of comparing subjects with chronic kidney disease patients with decreased renal function, the amount of free AIM in the serum showed superior predictive ability for the onset of AKI than the blood renal function marker. Therefore, the amount of free AIM in serum is a novel AKI onset prediction marker independent of renal function markers, and has been shown to have excellent predictive ability.

The inventors also discovered that the onset of AKI can be predicted with higher accuracy by combining the amount of free AIM in serum with other clinical information (clinical factors) regarding renal function, leading to the completion of the present invention. .

That is, the present invention provides the following aspects.

[1] A method for evaluating the risk of developing acute kidney injury, comprising the step of detecting free AIM and/or full-length free AIM in a blood sample collected from a subject.

[2] Method for assessing the risk of developing acute kidney injury, including the following steps (1) and (2) Step (1): Detecting free AIM and/or full-length free AIM in a blood sample collected from a subject Step (2): Free AIM and/or full-length free AIM detected in step (1),
The acute How to assess the risk of developing kidney damage.

[3] The method according to [1] or [2], wherein the subject is a human before undergoing surgical treatment.

[4] The free AIM and/or full-length free AIM is the ratio of the concentration of free AIM to the concentration of total AIM and/or the ratio of the concentration of full-length free AIM to the concentration of total AIM in the blood sample. , the method according to any one of [1] to [3].

[5] The method according to any one of [1] to [4], wherein the detection of free AIM and/or full-length free AIM is performed by an immunoassay.

[6] A reagent or kit for evaluating the risk of developing acute kidney injury, including a substance for detecting free AIM and/or full-length free AIM.

[7] The reagent or kit according to [6], wherein the substance for detecting free AIM and/or full-length free AIM is an antibody specific to free AIM and/or an antibody specific to full-length free AIM. .

[8] A biomarker for evaluating the risk of developing acute kidney injury, including free AIM and/or full-length free AIM in the blood.

According to the present invention, by using free AIM and/or full-length free AIM (concentration of free AIM, concentration of full-length free AIM, total amount of free AIM/AIM, total amount of full-length free AIM/AIM, etc.) in a blood sample as an index, , it becomes possible to evaluate the risk of developing acute kidney injury (AKI). Furthermore, according to the present invention, it is possible to predict the onset of AKI by reflecting it as a risk factor different from known blood renal function markers. Furthermore, when the subject is a chronic kidney disease patient, the present invention can predict the onset of AKI with higher accuracy than the blood renal function marker. Furthermore, in the present invention, by combining other clinical information (clinical factors) regarding renal function, it is also possible to predict the onset of AKI with higher accuracy.

It is a figure showing various forms of AIM. FIG. 2 is a diagram showing the results of analyzing a serum sample fractionated by gel filtration chromatography using an ELISA method using an AIM polyclonal antibody and an anti-AIM monoclonal antibody (clone 81). A diagram showing the results of analyzing serum samples fractionated by gel filtration chromatography by the BLEIA method using two types of anti-AIM monoclonal antibodies (Reagent 1: Clone 1 and Clone 81, Reagent 2: Clone 3 and Clone 81) It is. FIG. 2 is a diagram showing the results of analyzing a dilution series of full-length AIM and Small AIM by the BLEIA method using the reagent 1 or reagent 2. Concentration of free AIM, concentration of full-length free AIM, free AIM/total amount of AIM, full-length free AIM in preoperative serum of patients who developed AKI ("AKI" in the figure) or those who did not develop AKI ("non-AKI" in the figure) It is a figure which shows the average value of /AIM total amount. FIG. 2 is a diagram showing changes over time in the concentration (average value) of free AIM in the preoperative and postoperative serum of patients who developed AKI ("AKI" in the figure) or those who did not develop AKI ("non-AKI" in the figure). . This is a diagram showing changes over time in the concentration (average value) of full-length free AIM in the preoperative and postoperative serum of patients who developed AKI ("AKI" in the figure) or those who did not develop AKI ("non-AKI" in the figure). be. This is a diagram showing changes over time in the free AIM/total amount of AIM (average value) in the preoperative and postoperative serum of patients who developed AKI ("AKI" in the figure) or those who did not develop AKI ("non-AKI" in the figure). be. Diagram showing changes over time in the total amount of full-length free AIM/AIM (average value) in preoperative and postoperative serum of patients who developed AKI ("AKI" in the figure) or those who did not develop AKI ("non-AKI" in the figure) It is. This is a diagram showing the results of an analysis of the correlation between the concentration of blood renal function marker (sCr) and the concentration of free AIM, the concentration of full-length free AIM, the total amount of free AIM/AIM, or the total amount of full-length free AIM/AIM in serum. be. This is a diagram showing the results of an analysis of the correlation between the concentration of blood renal function marker (eGFR) and the concentration of free AIM, the concentration of full-length free AIM, the total amount of free AIM/AIM, or the total amount of full-length free AIM/AIM in serum. be.

The present invention relates to a method for evaluating the risk of developing acute kidney injury, which includes the step of detecting free AIM and/or full-length free AIM in a blood sample collected from a subject.

"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.

In the method of the present invention, "detection" includes not only the detection of the presence or absence of free AIM and/or full-length free AIM, but also the detection (for example, quantification) of the extent of the presence. More specifically, as shown in the Examples below, the concentration of free AIM in the blood sample, the concentration of full-length free AIM in the blood sample, the ratio of the concentration of free AIM to the total AIM concentration (total amount of AIM) in the blood sample, A suitable example is the ratio of the concentration of full-length free AIM to the total amount of AIM in a blood sample. Among these, it is more preferable to detect the ratio of the concentration of full-length free AIM to the total amount of AIM in a blood sample from the viewpoint of higher prediction accuracy for the onset of acute kidney injury. Note that "total AIM" includes both IgM-bound AIM and free AIM.

In the present invention, the "subject" is not particularly limited as long as it is a human, and may be a man or a woman. In addition, individuals of any age, including children, young people, middle-aged people, and the elderly, may be treated, but from the perspective of the risk of developing acute kidney injury, surgical procedures such as cardiovascular surgery, drug administration, and trauma are recommended. Suitable examples include humans before (scheduled to undergo) an event such as, a person undergoing or immediately after undergoing an event, and a person after undergoing an event.

Further, the "blood sample" collected from such a subject may be any sample containing blood or its components, and includes, for example, whole blood, serum, and plasma. Blood can be collected from a subject by methods known to those skilled in the art. For example, blood (whole blood) can be collected by collecting blood using a syringe or the like. Serum is a portion of whole blood from which blood cells and specific blood coagulation factors have been removed, and can be obtained, for example, as a supernatant after coagulating whole blood. Plasma is the part of whole blood from which blood cells have been removed, for example, by centrifugation under conditions that do not cause whole blood to clot (e.g., in the presence of a chelating agent (such as EDTA), an anticoagulant such as sodium citrate, and heparin). It can be obtained as the supernatant when subjected to Further, when the blood sample is used in the method of the present invention, it may be appropriately prepared in a form suitable for the method (for example, a protein solution extracted from the blood sample, etc.). Those skilled in the art can select and prepare a known method by considering the type and condition of the blood sample.

There is no particular restriction on the timing of collecting a blood sample from a subject, but for example, it may be before and/or after the occurrence of the event, which is a relatively short period of time before and after the occurrence of the event. It is preferable. A "relatively short period of time" means, for example, within 2 weeks before or after the event, preferably within 2 days before or after the event, more preferably 6 hours before or after the event. within 3 hours, most preferably within 3 hours before or after undergoing said event.

"Acute renal failure (AKI)", which is the subject of evaluation in the present invention, refers to a condition in which kidney function rapidly declines over a period of several hours to several days, and also includes the loss of body fluids associated with the decline in kidney function. It may also include water retention (water overflow), electrolyte imbalance (hyperkalemia, etc.), toxin accumulation (azotemia), etc. More specifically, for example, conditions that satisfy the following KDIGO criteria (AKI clinical practice guidelines 2016) (1) to (3) include (1) Serum creatinine level increases by 0.3 mg/dL or more within 48 hours;
(2) The basal serum creatinine value increases by 1.5 times or more within 7 days.
(3) Urine output remains below 0.5 mL/kg/hour for 6 hours or more.

In the method of the present invention, detection of various forms of AIM can be carried out by using immunological methods using antibodies, as shown in the Examples below, but especially if free AIM and full-length free AIM can be detected and quantified. It is not limited.

Examples of antibodies for immunologically detecting free AIM include antibodies specific for free AIM. In the present invention, "specific for free AIM" means that there is substantially no cross-reactivity with IgM-bound AIM, that is, the reactivity with IgM-bound AIM is sufficiently lower than the reactivity with free AIM. Sufficiently low means that the ratio of reactivity with IgM-bound AIM to reactivity with free AIM is 20% or less, preferably 10% or less, more preferably 6% or less, even more preferably 5% or less. .

The reactivity to these various forms of AIM can be evaluated, for example, by a sandwich ELISA method using a monoclonal antibody to be evaluated. There are no particular restrictions on the antibody to be combined with the monoclonal antibody to be evaluated in the sandwich ELISA method, as long as it can recognize all forms of AIM, including IgM-bound AIM, full-length free AIM, and small AIM. Antibody: Human CD5L Affinity Purified Polyclonal Ab (manufactured by R&D, product code: AF2797)) or a monoclonal antibody may be used. 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). In the sandwich ELISA method, for example, first, a plate (immobilized plate) on which a monoclonal antibody to be evaluated is immobilized is prepared, and a sample containing the target form of AIM (in this case, free) is placed on the immobilized plate. A sample containing 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, and after washing, finally, the signal intensity of the label is measured. When horseradish peroxidase (HRP) is used as a label, the signal can be measured using a microplate reader after adding a chromogenic substrate. As a result of the measurement, for example, if the reactivity to free AIM is 100 and the reactivity to IgM-bound AIM is 4, the monoclonal antibody to be evaluated has a reactivity to IgM-bound AIM that is lower than that to free AIM. 4%, it can be determined to be a free AIM-specific antibody.

A preferred embodiment of the free AIM-specific antibody of the present invention is a monoclonal antibody that does not bind to the polypeptide consisting of positions 1 to 229 of AIM, but binds to the polypeptide consisting of positions 1 to 259 of AIM. The recognition site of the monoclonal antibody is typically from the C-terminal region of the SRCR2 domain (positions 138 to 238) to the N-terminal region of the SRCR3 domain (positions 244 to 346) of AIM. A particularly preferred monoclonal antibody is a monoclonal antibody that recognizes the region from positions 230 to 259 of AIM, more preferably the region from positions 230 to 244.

A first embodiment of an antibody for immunologically detecting full-length free AIM includes an antibody specific for 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 various forms of AIM can be evaluated, for example, as described above, by a sandwich ELISA method using a monoclonal antibody to be evaluated. There are no particular restrictions on the antibody to be combined with the monoclonal antibody to be evaluated in the sandwich ELISA method, as long as it can recognize all forms of AIM, including IgM-bound AIM, full-length free AIM, and small AIM. Antibody: Human CD5L Affinity Purified Polyclonal Ab (manufactured by R&D, product code: AF2797)) or a monoclonal antibody may be used. 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).

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.

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 antibody for immunologically detecting total AIM may be any antibody that reacts with all of IgM-bound AIM, full-length free AIM, and Small AIM, and total AIM can be detected by combining these antibodies. be able to. It may be a monoclonal antibody or a polyclonal antibody. For example, for anti-AIM polyclonal antibodies obtained by immunization with AIM and conventional methods, fractionated samples of IgM-bound AIM, recombinant full-length free AIM, It can be obtained by screening for antibodies that react with recombinant Small AIM.

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 against the AIM is produced. The monoclonal antibodies produced by the selected hybridomas are analyzed for specificity to various forms of AIM and epitope analysis to identify clones that produce monoclonal antibodies having the above-mentioned characteristics. .

More specifically, a monoclonal antibody that specifically binds to free AIM uses a partial peptide of a specific region of AIM (for example, from the C-terminal region of the SRCR2 domain to the N-terminal region of the SRCR3 domain) as an immunogen. By using, it is possible to manufacture efficiently.

A monoclonal antibody that specifically binds to full-length free AIM is prepared by immunizing with AIM or a fragment containing positions 263 to 347, preferably 295 to 347, of SEQ ID NO: 1. By selecting a monoclonal antibody that binds to position 347, preferably positions 295 to 347, it 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 non-immunized animals. 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 preferably they originate 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 the 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). ,2000,OXFORD UNIVERSITY PRESS,Vandamme A M. et al., Eur. J. Biochem. 192:767-775, 1990). In expressing 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 we use transgenic animal production technology to create a transgenic animal (cow, goat, sheep, pig, etc.) into which an antibody gene has been inserted, 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.

Examples of methods for immunologically detecting free AIM, full-length free AIM, or total AIM (hereinafter also collectively referred to as "free AIM, etc.") include immunoassays using antibodies labeled with labeling substances (labeled immunoassays). This is an immunoassay method that uses a labeled detection antibody or a labeled antibody against the detection antibody. 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) on a solid phase and the detection antibody is specific to the above-mentioned free AIM, etc. Any antibody that specifically binds to the above-mentioned free AIM, etc. may be bound to the antibody and labeled with a labeling substance to directly detect the free AIM, etc.; Alternatively, free AIM or the like may be indirectly detected using a secondary antibody to which a labeling substance is bound, without binding the labeling substance to the antibody that binds to the antibody.

In a labeled immunoassay, an antibody that specifically binds to the above-mentioned free AIM, etc. 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). In this case, the other antibody may be any antibody capable of binding to AIM (anti-AIM antibody), and antibodies other than the above-mentioned antibodies that specifically bind to free AIM etc. can also be used. The other antibody may be a monoclonal antibody or a polyclonal antibody. It is preferable that the monoclonal antibody to be combined with the antibody that specifically binds to the above-mentioned free AIM etc. does not compete in binding with the free AIM etc. (ie, recognizes a different epitope).

In a labeled immunoassay, when an antibody that specifically binds to the above-mentioned free AIM or the like 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. bound to a labeling substance.

Therefore, as described above, in addition to the method of directly detecting free AIM, etc. using an antibody that specifically binds to the above-mentioned free AIM, etc. to which a labeling substance is bound, an antibody that specifically binds to the above-mentioned free AIM, etc. It is also possible to use a method of indirect detection using a secondary antibody or the like to which a labeling substance is bound, without binding a labeling substance to the labeling substance.

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)), fluorescent proteins (allophycocyanin (APC) and phycoerythrin (R-PE)), and radioactive isotopes such as ^125I . , magnetic particles, latex particles (e.g., polystyrene, styrene-butadiene copolymer), metal colloid particles (e.g., gold, silver, copper, iron, platinum, palladium, etc., or mixtures thereof), avidin, biotin, etc. It will be done.

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.

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

In the sandwich method, free AIM, etc. 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, free AIM or the like in the sample is detected by adding a substrate for a labeling substance such as an enzyme and causing color development. As a principle for detecting free AIM and the like 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 case of immunochromatography, free AIM, etc. is recognized with a detection antibody bound to a labeling substance, and while B/F separation is performed, free AIM, etc. 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 free AIM, etc. 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, and free AIM etc. in the sample is detected. It's okay.

In addition, as in the immunoagglutination method, insoluble carrier particles (solid phase) to which an antibody that specifically binds to the above-mentioned free AIM, etc. is immobilized (bound) are used in a liquid phase, and the insoluble carrier particles and free AIM, etc. The aggregation of insoluble carrier particles may be detected by turbidity measurement, visual observation, or absorbance measurement, taking advantage of the property of insoluble carrier particles to aggregate due to the formation of an immune complex with. The immunoaggregation method is preferred in the method of the present invention because it does not require a B/F separation step before detecting free AIM, etc., 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.

Quantification of free AIM and the like from the obtained measured values can generally be performed by comparison with measured values from standard samples. In this case, for example, free AIM etc. in the sample can be quantified by checking where the actual measured values are located on a standard curve (calibration curve) created based on the measured values of standard samples. be able to.

Furthermore, the amount of free AIM or full-length free AIM detected in this manner may be not only an absolute amount but also a relative amount. Examples of the relative amount include protein amount ratio (so-called numerical value expressed in arbitrary units (AU)) based on the measurement method or measurement device used for detection. Further, as the relative amount, for example, a value calculated based on the amount of another protein may be used. Such "other proteins" include, for example, total AIM, as shown in the Examples below. Reference proteins can also be exemplified. The "reference protein" according to the present invention may be any protein that stably exists in a blood sample and has a small difference in amount between different blood samples, such as an endogenous control (internal standard) protein. More specific examples include β-actin, α-tubulin, COX-4, GAPDH, lamin B1, PCNA, TBP, and VDCA1/Porin.

In the method of the present invention, the risk of developing AKI may be evaluated by comparing the amount of free AIM or full-length free AIM detected in this way with a reference amount of the same protein. That is, the method of the present invention can take the following embodiments.
(1) detecting the amount of free AIM and/or full-length free AIM in the blood sample collected from the subject;
(2) comparing the amount of free AIM and/or full-length free AIM detected in step (1) with a reference amount of free AIM and/or full-length free AIM, respectively;
(3) As a result of the comparison in step (2), if the amount of free AIM and/or full-length free AIM detected in step (1) is higher than the corresponding reference amount, the subject has acute kidney injury. A method for evaluating the risk of developing acute kidney injury, the method comprising the step of determining that the disorder will develop.

More specific embodiments include the following.
(1) detecting the concentration of free AIM and/or full-length free AIM in the blood sample collected from the subject;
(2) comparing the concentration of free AIM and/or full-length free AIM detected in step (1) with a reference concentration of free AIM and/or full-length free AIM, respectively;
(3) As a result of the comparison in step (2), if the concentration of free AIM and/or full-length free AIM detected in step (1) is higher than the corresponding reference concentration, the subject has acute kidney injury. A method for evaluating the risk of developing acute kidney injury, the method comprising the step of determining that the disorder will develop.

Further, the following may be mentioned as more specific embodiments.
(1) detecting the ratio of the concentration of free AIM to the concentration of total AIM and/or the ratio of the concentration of full-length free AIM to the concentration of total AIM in the blood sample collected from the subject;
(2) The ratio of the concentration of free AIM to the concentration of total AIM detected in step (1) and/or the ratio of the concentration of full-length free AIM to the concentration of total AIM, respectively. and/or a reference ratio of the concentration of full-length free AIM to the concentration of total AIM;
(3) As a result of the comparison in step (2), the ratio of the concentration of free AIM to the concentration of total AIM detected in step (1), and/or the ratio of the concentration of full-length free AIM to the concentration of total AIM detected in step (1) is determined. , determining that the subject will develop acute kidney injury if the ratio is higher than the corresponding reference ratio.

There is no particular restriction on the "reference amount (reference concentration, reference ratio, etc.)" of free AIM or full-length free AIM to be compared, and a person skilled in the art can, for example, determine the amount when using the above detection method etc. By using this as a standard, it can be set as a so-called cutoff value (threshold value) that can be used to determine whether or not acute kidney injury will develop.

More specifically, the reference amount may be, for example, a value determined by comparing the amount of free AIM or full-length free AIM in a human group that develops acute kidney injury and a human group that does not (e.g., acute kidney injury). The amount of free AIM or full-length free AIM (median, mean, or lower limit) in the group of people who develop the disorder and that (median, mean, or upper limit) in the group of people who did not develop acute kidney injury. ). Moreover, those skilled in the art can appropriately select a statistical analysis method suitable for the above-mentioned detection method to make the setting. Examples of statistical analysis methods include receiver operating characteristic analysis (ROC analysis), t-test, analysis of variance (ANOVA), Kruskal-Wallis test, Wilcoxon test, Mann-Whitney test, odds ratio, hazard ratio, Fisher's Examples include exact test, classification tree and decision tree analysis (CART analysis). Moreover, normalized data or standardized and normalized data can also be used for comparison.

Further, as a more specific example of setting in this way, as shown in the examples below, the reference amount (reference concentration) of free AIM concentration is preferably 700 to 1000 ng/mL, more preferably 800 ng/mL. ~900 ng/mL, more preferably about 850 ng/mL (particularly 849.10 ng/mL). The reference amount (reference concentration) of full-length free AIM concentration is preferably 800 to 1300 ng/mL, more preferably 900 to 1100 ng/mL, and even more preferably about 1000 ng/mL (particularly 1002.10 ng/mL). The reference amount (reference ratio) of free AIM/total AIM is preferably 0.10 to 0.20, more preferably 0.12 to 0.18 (especially 0.13). The reference amount (reference ratio) of full-length free AIM/total AIM is preferably 0.12 to 0.18, more preferably 0.13 to 0.17 (especially 0.16).

In addition, prediction of such onset is usually performed by a doctor (including a person who has received instructions from a doctor), but the data regarding free AIM and/or full-length free AIM mentioned above are important in determining whether or not treatment by a doctor is necessary and its timing. This is useful for diagnosis, including judgments such as Therefore, the method of the present invention provides a method for collecting data on free AIM and/or full-length free AIM for the purpose of disease risk assessment (diagnosis) by a doctor, a method for presenting the data to a doctor, a method for collecting data on free AIM and/or full-length free AIM, and a method for presenting data on free AIM and/or full-length free AIM. It can also be described as a method for comparing and analyzing the amount of AIM with each corresponding reference amount, and a method for assisting doctors in predicting the onset of acute kidney injury.

Although the preferred embodiments of the present invention have been described above, the present invention is not limited to the above embodiments. For example, as shown in the examples below, by combining not only the above-mentioned free AIM or full-length free AIM with other clinical information (clinical factors), it is also possible to predict the onset of AKI with higher accuracy. . Therefore, the present invention can also take the following aspects.
A method for evaluating the risk of developing acute kidney injury using free AIM or full-length free AIM in a blood sample collected from a subject and clinical factors as indicators.

As a more specific example, a method for assessing the risk of developing acute kidney injury includes the following steps (1) and (2) Step (1): Free AIM and/or total length in a blood sample collected from a subject Step (2) of detecting free AIM: Free AIM and/or full-length free AIM detected in step (1),
At least one clinical factor selected from the group consisting of physical characteristic factors, surgery-related factors, renal function-related factors, background diseases, and drug information in the subject is used as an index to determine the risk of developing acute kidney injury. An example is the step of evaluating.

In the present invention, "clinical factors" refer to clinical information other than free AIM in the blood and/or full-length free AIM, such as physical characteristic factors, surgery-related factors, renal function-related factors, background diseases, Examples include drug information.

"Physical characteristic factors" means the physical characteristics of the subject at the time of the occurrence of the above event, specifically, age, weight, euroSCORE II value (mortality risk score for cardiac surgery), gender, height, Examples include surface area, presence or absence of hepatitis B infection, etc.

"Surgery-related factors", when the above-mentioned event is a surgical operation, means matters related to the operation, such as the target disease of the operation and the surgical method. More specifically, the "diseases targeted for surgery" include TAA (thoracic aortic aneurysm), TR (tricuspid regurgitation), MR (mitral regurgitation), af (atrial fibrillation), and AS (aortic regurgitation). Examples include valvular stenosis), AsR (aortic stenosis and regurgitation), AP (angina pectoris), and AAE (aortic annulus ectasia). More specifically, the "surgical procedures" include AAR (ascending aortic replacement), TAR (total arch aortic graft replacement), maze (maze surgery), AVR (aortic valve replacement), and LAA Closure (left atrial appendage closure), MVP/TAP, PAR (partial arch aortic graft replacement), TAP (tricuspid valvuloplasty), MVP (mitral valvuloplasty), on pump beating CABG (coronary artery bypass using cardiopulmonary bypass) surgery), MICS (minimally invasive cardiac surgery), etc.

The term "renal function-related factor" refers to an index representing the function of the subject's kidney at the time of occurrence of the above event, and includes, for example, a renal function marker and a urinary biomarker. More specifically, the "kidney function marker" includes the stage of chronic kidney disease, glomerular filtration rate (eGFR), and serum creatinine. The "stage of chronic kidney disease" refers to the severity of chronic kidney disease as evaluated by CGA classification based on cause (C), renal function (GFR: G), and proteinuria (albuminuria: A). be. "Glomerular filtration rate" is a value calculated from the serum creatinine (Cr) value, age, and gender using the following formula.
Estimated GFR in men (mL/min/1.73 m ² ) = 194 x Cr ^-1.094 x age ^{- 0.287}
Estimated GFR in women (mL/min/1.73 m ² ) = 194 x Cr ^-1.094 x age ^{- 0.287} x 0.739.
Cr: Serum Cr value (mg/dL).
More specifically, the "urinary biomarker" includes the urinary concentration (μg/g·Cr) of TIMP-2, α1-m, NGAL, L-FABP, and KIM-1.

"Background disease" means a disease that the subject is suffering from at the time of the occurrence of the above event, such as heart failure, cAf (chronic atrial fibrillation), HTN (hypertension), PH (pulmonary hypertension), HL ( dyslipidemia), COPD (chronic obstructive pulmonary disease), and DM (diabetes mellitus). Note that "hypertension" refers to a condition in which the systolic blood pressure is 140 mmHg or more or the diastolic blood pressure is 90 mmHg or more in repeated measurements.

"Drug information" means the drugs that the subject was taking or being administered to the subject before the above event occurred, such as antihypertensive drugs, statins, antiuric acids, spironolactone, Beta blockers. , furosemide.

In the method of the present invention, the risk of developing acute kidney injury is evaluated using each of free AIM and/or full-length free AIM in a blood sample and the above-mentioned clinical factors as indicators, and then these results are taken into consideration. may ultimately predict the onset of acute kidney injury (for example, if the risk of developing acute kidney injury is assessed to be high based on both free AIM and/or full-length free AIM in the blood sample and the above clinical factors) As a result, a final assessment can be made that the subject is likely to develop acute kidney injury).

In addition, in the method of the present invention, as shown in Examples below, the onset of disease is determined based on an index (score, etc.) obtained by combining free AIM and/or full-length free AIM in a blood sample and the above clinical factors. Risks can also be assessed. As a specific example, each measurement value is calculated by entering the prediction formula below, and if the obtained score is higher than each cutoff value, the subject can be evaluated as likely to develop acute kidney injury. can.
Score = (AIM measurement value) x (free AIM coefficient) + (measurement values of clinical factors to be combined, etc.) x (clinical information coefficient) + constant term.For each coefficient, constant term, and cutoff value, see below. See Tables 6 and 7.

In addition, as clinical factors to be combined with free AIM in a blood sample and/or full-length free AIM, clinical factors that result in an AUC of 0.75 or more are preferable, such as age, thoracic aortic aneurysm, total arch These include partial aortic graft replacement, chronic kidney disease stage, glomerular filtration rate, serum creatinine, TIMP-2, chronic atrial fibrillation, hypertension, and dyslipidemia. Furthermore, the number of clinical factors in combination with free AIM and/or full-length free AIM in a blood sample is not limited to 1, but may be 2 or more, 5 or more, 10 or more, 15 or more, 20 or more, 30 or more. It's okay.

The present invention also provides a reagent for evaluating the risk of developing acute kidney injury, which includes a substance for detecting free AIM and/or full-length free AIM. Such substances include antibodies that specifically bind to the above-mentioned free AIM and the like.

As described above, the antibody contained in the reagent 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 above-mentioned antibodies are bound. When immunochromatography is used as the detection principle, an immunochromatographic device may be used in which the antibody 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). Furthermore, when the immunoagglutination method is used as the detection principle, examples include insoluble carrier particles to which the above-mentioned antibodies are bound, such as latex particles.

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

The present invention also provides a kit for evaluating the risk of developing acute kidney injury, which includes the above-mentioned reagent. The kit of the present invention can further include standard samples (reagents containing free AIM and the like 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 free AIM or the like indirectly, a labeled substance that binds to the primary antibody (secondary antibody, protein A, etc.) can be included. Furthermore, when the above antibody is biotinylated, an avidinized label can be included. The kit of the present invention can further include instructions for using the kit.

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 reagents and kits of the present invention are not limited to monoclonal antibodies, but also include polyclonal antibodies.

EXAMPLES Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples.

First, a measurement system for specifically detecting the amount of various forms of AIM was constructed by using combinations of antibodies listed in Table 1 below, as shown below, and their effectiveness was verified.

The anti-AIM polyclonal antibody is Human CD5L Affinity Purified Polyclonal Ab (manufactured by R&D Co., Ltd., product code: AF2797).

In addition, 1, 3, and 81 in the table indicate anti-AIM monoclonal antibody clone 1, clone 3, and clone 81, respectively, which were produced by the following method.

<Preparation of anti-AIM antibody>

Mice were immunized with human recombinant AIM (hereinafter referred to as rAIM) (30 to 50 μg) as an antigen 3 to 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 to obtain clone 1, clone 3, or clone 81.

Although not shown in the figure, clone 1 is an antibody against free AIM (full-length free AIM and Small AIM) that reacts with the SRCR2 domain of AIM. Regarding Clone 1, it has been confirmed that the ratio of reactivity with IgM-bound AIM to free AIM is 10% or less. Clone 3 is an antibody against full-length free AIM that reacts with a portion of the SRCR3 domain in AIM (positions 263-347, particularly positions 295-347 of AIM shown in SEQ ID NO: 1). Regarding clone 3, the ratio of the reactivity with IgM-bound AIM to the reactivity with free AIM is 10% or less, and the ratio of the reactivity with Small AIM to the reactivity with full-length AIM is 10% or less. It has been confirmed that there is. Clone 81 is an antibody against AIM (IgM-bound AIM and free AIM) that reacts with the SRCR1 domain of AIM.

<Sample preparation>
For the measurement sample, a serum sample was dissolved in a buffer solution (0.05M phosphate buffer: pH 7.0, sodium chloride: 0.3M), added to a column, and separated by gel filtration chromatography under the following conditions. I was able to do the painting.

Gel filtration chromatography conditions Equipment used: SHIMADZU SPD-20AV
Column: Phenomenex 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.

Using the fractionated sample of the serum specimen obtained as described above, the total amount of AIM was measured by ELISA using combinations of antibodies as shown in Table 1.

<Measurement of total AIM amount (ELISA method)>
First, 50 μL of a solid phase antibody (anti-AIM polyclonal antibody; manufactured by R&D Co., Ltd., AF2797) was dispensed into each well of a 96-well microplate, and then washed to immobilize the solid phase antibody. After washing, a 1% BSA solution was added and left at room temperature for 2 hours. Furthermore, the labeling antibody (clone 81) was biotinylated and labeled using the biotin labeling reagent Biotin (AC5) 2Slufo-osu (Dojindo Laboratories). 100 μL of the standard solution or sample was added to the immobilized plate and allowed to react at room temperature for 90 minutes. Subsequently, the solution in the well was removed by suction, and after washing, 100 μL of biotin-labeled antibody was added and reacted at room temperature for 90 minutes. Furthermore, 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 90 minutes. After removing the solution in the well by suction and washing, 100 μL of o-phenylenediamine-containing substrate solution was added as a chromogenic substrate and reacted. After adding 100 μL of 2N sulfuric acid as a reaction stop solution, a microplate reader was used. Measurement was performed at a measurement wavelength of 492/650 nm.

As a result, as shown in Figure 2, the ELISA method detects both a peak derived from IgM-bound AIM (eluate volume around 6 mL) and a peak derived from free AIM (eluate volume around 10 mL). It was confirmed that the total amount of AIM can be measured by using the anti-AIM polyclonal antibody and the anti-AIM monoclonal antibody together.

In addition, using the fractionated sample of the serum sample obtained as described above, measurements were performed using the following bioluminescent enzyme immunoassay method (BLEIA method) using a combination of antibodies as described in Table 1. .

<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 types of antibody-immobilized magnetic particles in which various solid-phase antibodies are immobilized on magnetic particles, various biotin-labeled antibodies obtained by mixing various labeling antibodies and biotinylated reagents, and streptavidin-biotinylated Luciferase was produced. Then, for each combination of clone 1 or 3 and clone 81, 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. I let it happen. 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 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. The results obtained are shown in FIG.

As a result, as shown in Figure 3, in the analysis using the BLEIA method described above, regardless of whether Reagent 1 or Reagent 2 was used, only the peak of the fractionated sample with an elution time (eluate volume) of around 20 minutes (10 mL) was detected. was detected, confirming that the amount of free AIM can be specifically measured.

Next, it was confirmed by the method shown below that the measurement system using the reagents shown in Table 1 was specific for full-length free AIM.

<Sample preparation>
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 used 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. These AIM recombinant proteins were diluted with PBS and adjusted to concentrations of 20, 10, 5, 2.5, and 1.25 ng/mL, respectively, to prepare a dilution series.

These dilution series were then analyzed by the above BLEIA method using Reagent 1 or Reagent 2 shown in Table 1. As a result, as shown in FIG. 4, both Reagent 1 and Reagent 2 showed concentration-dependent reactivity toward full-length AIM. Furthermore, a concentration-dependent response to rSmall AIM was also observed in Reagent 1. On the other hand, Reagent 2 had low reactivity to rSmall AIM. From these results and the results shown in FIG. 3, it was confirmed that Reagent 1 reacts with free AIM (full-length free AIM and Small AIM), while Reagent 2 specifically reacts with full-length free AIM.

Using the detection systems for various forms of AIM that have been confirmed to be effective as described above, blood samples from patients who underwent surgical procedures were analyzed as shown below, and the risk of developing acute kidney injury was determined. Relevance was assessed.

<Sample>
For patients who underwent cardiovascular surgery using a cardiopulmonary bypass, blood samples were taken before surgery, immediately after surgery, and 6 hours, 1 day, 2 days, and 3 days after surgery to obtain serum. Note that "preoperative" refers to the period from 2 weeks to just before the start of surgery. 102 patient samples diagnosed as AKI patients or non-patients according to the KDIGO criteria were used. There were 37 AKI patients and 65 non-AKI patients.

For the specimens (samples) thus obtained, various forms of AIM were detected by sandwich ELISA using combinations of antibodies as shown in Table 1 above. Note that the total amount of AIM was measured by the method described in <Measurement of total AIM amount (ELISA method)> above.

<Measurement of free AIM, full-length free AIM (ELISA method)>
First, 50 μL of a solid phase antibody (anti-AIM monoclonal antibody; clone 1 or clone 3) was dispensed into each well of a 96-well microplate, and then washed to immobilize each solid phase antibody. After washing, a 1% BSA solution was added and left at room temperature for 2 hours. Furthermore, the labeling antibody (clone 81) was biotinylated and labeled using the biotin labeling reagent Biotin (AC5) 2Slufo-osu (Dojindo Laboratories). 50 μL of the standard solution or sample was added to the immobilized plate and allowed to react 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. Furthermore, 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. After removing the solution in the well by suction and washing, 50 μL of o-phenylenediamine-containing substrate solution was added as a coloring substrate and reacted. After adding 50 μL of 2N sulfuric acid as a reaction stop solution, a microplate reader was used. Measurement was performed at a measurement wavelength of 492/650 nm.

<Calculation of free AIM/total amount of AIM, total length free AIM/total amount of AIM>
Concentration ratios were calculated using the amounts of AIM of various forms detected by each of the above ELISA methods. Specifically, the concentration of free AIM or full-length free AIM was divided by the concentration of the total amount of AIM to calculate the total amount of free AIM/AIM or the total amount of full-length free AIM/AIM.

[Example 1] Measurement of preoperative serum free AIM Concentration of free AIM, concentration of full-length free AIM, and free AIM/AIM in preoperative serum of subjects who developed AKI (37 cases) and subjects who did not develop AKI (65 cases) The average values of total amount and full-length free AIM/total amount of AIM are shown in FIG. Error bars indicate SD. Significant differences were tested using the Mann-Whitney U test.

As shown in Figure 5, the concentration of free AIM, the concentration of full-length free AIM, the total amount of free AIM/AIM, and the total amount of full-length free AIM/AIM in serum was significantly lower in patients with AKI than in patients without AKI. Significantly high values were observed. Therefore, it was shown that the amount of free AIM in serum is useful for evaluating the risk of developing AKI.

[Example 2] Postoperative progress measurement of free AIM Concentration of free AIM, concentration of full-length free AIM, and total amount of free AIM/AIM in preoperative serum of patients who developed AKI (37 cases) and those who did not develop AKI (65 cases) Figures 6 to 9 show the changes in the average value of full-length free AIM/total AIM from before surgery to 3 days after surgery. Error bars indicate SE.

As shown in Figures 6 to 9, patients who develop AKI have high concentrations of free AIM, concentration of full-length free AIM, total amount of free AIM/AIM, and total amount of full-length free AIM/AIM in serum both before and after surgery. Indicated.

The above results showed that the amount of free AIM in serum is useful for evaluating the risk of developing AKI.

[Comparative Example 1] Predicting AKI onset risk of urinary AKI-related markers <Sample>
Preoperative urine samples were collected from patients who underwent cardiac large vessel surgery using cardiopulmonary bypass. A total of 102 patient samples diagnosed as having developed AKI or not having developed AKI according to the KDIGO criteria were used. There were 37 patients who developed AKI (AKI) and 65 patients who did not develop AKI (non-AKI).

<Measurement of urine creatinine>
Using the collected urine specimen as a sample, the creatinine (Cr ) The concentration was calculated.

<Measurement of urinary AKI-related marker concentration>
Urinary NGAL, L-FABP, TIMP-2, IGFBP7, and KIM-1 were selected as urinary AKI-related markers. These concentrations were measured using the following commercially available measurement reagents.

NGAL: Human Lipocalin-2/NGAL DuoSet ELISA (R&D Systems),
L-FABP: High Sensitivity Human L-FABP ELISA Kit (CMIC),
TIMP-2: Human TIMP-2 DuoSet ELISA (R&D Systems),
IGFBP7: ELISA kit for insulin-like growth factor binding protein 7 (Cloud-Clone),
KIM-1: Human TIM-1/KIM-1/HAVCR DuoSet ELISA (R&D Systems).

However, o-phenylenediamine (FUJIFILM Wako Pure Chemical) was used for color development of NGAL, TIMP-2, and KIM-1, and 2N H ₂ SO ₄ was added to stop the reaction. The absorbance of each well was measured using a microplate reader Sunrise Rainbow RC (Tecan) at a wavelength of 492 nm.

<Calculation of onset risk prediction ability>
For each marker, an ROC curve was calculated comparing those who developed AKI and those who did not develop AKI, and the area under the curve (AUC) was calculated. The point closest to the upper left corner of the ROC curve was set as the cutoff value, and sensitivity and specificity were calculated. The results obtained are shown in Table 2 below.

[Example 3] Predicting risk of onset of blood renal function markers (sCr, eGFR) and amount of free AIM in serum <Sample>
Preoperative blood was collected and serum was obtained from patients who underwent cardiovascular surgery using cardiopulmonary bypass. A total of 102 patient samples diagnosed as having developed AKI or not having developed AKI according to the KDIGO criteria were used. There were 37 patients who developed AKI (AKI) and 65 patients who did not develop AKI (non-AKI).

<Measurement of serum creatinine (sCr)>
Serum creatinine was measured using "Cerotech" CRE-CL for creatinine measurement (manufactured by Serotec Co., Ltd.) according to the manufacturer's protocol.

<Calculation of eGFR>
eGFR was calculated according to the following formula.
Male eGFR (ml/min/1.73m ² ) = 194 x Cr ^-1.094 x age ^{- 0.287}
Female eGFR (ml/min/1.73 m ² ) = 194 × Cr ^−1.094 × age ^−0.287 × 0.739.

<Calculation of onset risk prediction ability>
For each marker, an ROC curve was calculated comparing those who developed AKI and those who did not develop AKI, and the area under the curve (AUC) was calculated. The point closest to the upper left corner of the ROC curve was set as the cutoff value, and sensitivity and specificity were calculated. The results obtained are shown in Table 3 below.

As shown in Table 3, the concentration of free AIM, the concentration of full-length free AIM, the total amount of free AIM/AIM, and the total amount of full-length free AIM/AIM in serum showed the same ability to predict the onset risk as serum creatinine and eGFR. Particularly in AUC, full-length free AIM/total amount of AIM was high.

As mentioned above, as a result of evaluating the ability of urinary AKI-related markers and blood renal function markers to predict the onset of AKI, as shown in Table 2, the amount of free AIM in serum was compared with that of urinary AKI-related markers. It was revealed that the method showed excellent predictive ability for the onset of AKI. Furthermore, as shown in Table 3, it was shown that detecting the amount of free AIM in serum has predictive ability equivalent to that of known blood renal function markers (sCr, eGFR).

[Example 4] Blood renal function markers (sCr, eGFR) and serum AIM risk prediction ability <AKI onset risk diagnosis using blood renal function markers and serum AIM>
Each case was diagnosed based on the cutoff value of each marker calculated in Example 3. The results of 8 of them are shown in Table 4.

There were cases in which the onset of AKI could be predicted using the concentration of free AIM, the concentration of full-length free AIM, the total amount of free AIM/AIM, the total amount of full-length free AIM/AIM, serum creatinine, and eGFR in serum. (#2, #10). On the other hand, even in cases where serum creatinine and eGFR showed false negatives and false positives, there were cases in which the onset of AKI could be accurately predicted by detecting the amount of free AIM in the serum (#52, #54 , #55, #59, #63, #79).

[Reference Example 1] Correlation between blood renal function markers and free AIM Blood renal function markers (sCr, eGFR) measured in Example 3 and free AIM amount in serum measured in Example 1 in each patient specimen The results of analyzing the relationship between the two are shown in FIGS. 10 and 11. As a result, as shown in FIGS. 10 and 11, the amount of free AIM in serum had a weak correlation with sCr and eGFR, and had large variations.

The above results suggested that renal function markers and the amount of free AIM in serum are both useful for predicting the risk of developing AKI, but they may reflect different pathological conditions.

[Example 5] Comparison of blood renal function markers (sCr, eGFR) and free AIM amount in serum in predicting onset risk in CKD patients <Sample>
For patients who had CKD (chronic kidney disease) as an underlying disease and underwent cardiovascular surgery using cardiopulmonary bypass, preoperative blood was collected, serum was obtained, and renal function markers and free serum levels were determined. The amount of AIM was measured. In addition, 55 patient samples were used that were diagnosed as having developed AKI or not having developed AKI according to the KDIGO criteria. There were 28 patients who developed AKI (AKI) and 27 patients who did not develop AKI (non-AKI).

<Calculation of predictive ability of onset risk>
For each marker, an ROC curve was calculated comparing those who developed AKI and those who did not develop AKI, and the area under the curve (AUC) was calculated. The point closest to the upper left corner of the ROC curve was set as the cutoff value, and sensitivity and specificity were calculated. The results obtained are shown in Table 5.

As shown in Table 5, the concentration of free AIM, the concentration of full-length free AIM, the total amount of free AIM/AIM, and the total amount of full-length free AIM/AIM in serum were found to be excellent in the development of AKI even in the patient group with CKD as an underlying disease. It showed predictive ability. Furthermore, the amount of free AIM in these serums showed better predictive ability than serum creatinine and eGFR.

[Example 6] AKI onset risk diagnosis by combining serum free AIM and clinical information <Calculation of onset risk prediction ability by combining serum free AIM and clinical information>
A multiple logistic regression analysis was performed for the patient of Example 1, combining the free AIM concentration in the serum before surgery and clinical information one item at a time, and the following optimal prediction formula was created and a score was calculated.
Score = (measured value of serum free AIM) x (coefficient a) + (measured value of combined items, etc.) x (coefficient b) + constant term An ROC curve is drawn from the obtained score of each case and the area under the curve (AUC ) was calculated. In addition, statistical software (StatFlex V7) was used for statistical analysis. The results obtained are shown in Tables 6 and 7 below.

Regarding "gender," the analysis was performed by inputting 0 for men and 1 for women into the above prediction formula. Regarding the "surgical target disease", the diseased patients were set as 1 and the unaffected patients were set as 0, and these were entered into the above prediction formula and analyzed. Regarding "surgical procedure", the analysis was performed by inputting the patients who underwent each surgical procedure as 1 and the patients who did not undergo it as 0. Regarding the "chronic kidney disease stage", enter the stage at the time of surgery as 0 for G1 patients, 1 for G2 patients, 2 for G3a patients, 3 for G3b patients, and 4 for G4 patients, and perform analysis. went. Regarding the "background disease", patients suffering from each disease at the time of surgery were entered as 1, and patients not suffering from the disease were entered as 0, and used for analysis. Regarding "drug information", patients who were administered each drug before surgery were entered as 1, and patients who were not administered were entered as 0, and used for analysis. For other items, analysis was performed by inputting each measured value itself.

As shown in Tables 6 and 7, the predictive ability of preoperative clinical information was improved by combining it with clinical information such as renal function, physical characteristics, and disease of the person undergoing surgery. In this way, it has become clear that the onset of AKI can be predicted with high accuracy by combining serum AIM with at least one or more clinical information. It should be noted that eGFR is an index that combines the three items of serum creatinine, age, and gender as mentioned above, but as shown in Table 3, serum creatinine alone is used as an index in predicting the onset of AKI. The result is about the same as the case where AUC is 0.71. On the other hand, regarding free AIM in serum, as shown in Table 6, when combined with age, the AUC was 0.76, and even when combined with gender, the AUC was 0.74, indicating a significant improvement. In this way, it has been revealed that the combination of free AIM in serum and other clinical information exhibits excellent predictive ability in the development of AKI, even compared to eGFR, which uses multiple factors as an index. Therefore, it is thought that full-length free AIM in serum also exhibits excellent predictive ability when combined with other clinical information.

As explained above, according to the present invention, it is possible to predict the onset of acute kidney injury. Therefore, the present invention enables early therapeutic intervention before the onset of acute kidney injury, and is therefore useful in the medical field related to the disease.

Claims (8)

A method for evaluating the risk of developing acute kidney injury, comprising the step of detecting free AIM and/or full-length free AIM in a blood sample collected from a subject. A method for evaluating the risk of developing acute kidney injury, including the following steps (1) and (2). Step (1): Detecting free AIM and/or full-length free AIM in a blood sample collected from a subject. Step (2): Free AIM and/or full-length free AIM detected in step (1),
The acute The process of evaluating the risk of developing kidney damage. 3. The method according to claim 1, wherein the subject is a human before undergoing surgical treatment. 4. The free AIM and/or full-length free AIM is a ratio of the concentration of free AIM to the concentration of total AIM and/or a ratio of the concentration of full-length free AIM to the concentration of total AIM in the blood sample. The method described in 1 or 2. 3. The method according to claim 1 or 2, wherein the detection of free AIM and/or full-length free AIM is performed by immunoassay. A reagent or kit for evaluating the risk of developing acute kidney injury, comprising a substance for detecting free AIM and/or full-length free AIM. The reagent or kit according to claim 6, wherein the substance for detecting free AIM and/or full-length free AIM is an antibody specific for free AIM and/or an antibody specific for full-length free AIM. A biomarker for evaluating the risk of developing acute kidney injury, including free AIM and/or full-length free AIM in the blood.