WO2017022315A1 - 腎疾患の検査方法 - Google Patents
腎疾患の検査方法 Download PDFInfo
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- WO2017022315A1 WO2017022315A1 PCT/JP2016/066227 JP2016066227W WO2017022315A1 WO 2017022315 A1 WO2017022315 A1 WO 2017022315A1 JP 2016066227 W JP2016066227 W JP 2016066227W WO 2017022315 A1 WO2017022315 A1 WO 2017022315A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/56—Staging of a disease; Further complications associated with the disease
Definitions
- the present invention typically relates to a method for examining a renal disease or a method for assisting diagnosis. Specifically, the present invention relates to a method for examining or diagnosing renal disease, which comprises detecting free AIM in a biological sample of a subject. The present invention further relates to a kit for examining or diagnosing renal disease.
- AIM apoptosis inhibitor of macrophage; also referred to as CD5L, api6, Sp ⁇
- CD5L apoptosis inhibitor of macrophage
- api6 Sp ⁇ apoptosis inhibitor of macrophage
- AIM has a structure in which scavenger receptor cysteine-rich (SRCR) domains, which are specific sequences containing many cysteine residues, are connected in tandem, and each cysteine residue is disulfide bonded to each other within each domain. By doing so, it is considered to have a compact spherical three-dimensional structure.
- SRCR scavenger receptor cysteine-rich
- AIM is a sticky protein, and various molecules have been reported as binding partners. For example, it is known to have the ability to agglutinate bacteria by recognizing pathogen-associated molecular patterns (PAMPs) of bacteria and fungi such as lipoteichoic acid (LTA) and lipopolysaccharide (LPS).
- PAMPs pathogen-associated molecular patterns
- LTA lipoteichoic acid
- LPS lipopolysaccharide
- Patent Document 2 there are many cells in the body that bind AIM to the surface or take it up into the cell, and in addition to uptake into the macrophage itself, which is a production cell, in adipocytes, endocytosis via the scavenger receptor CD36. Has been reported to induce lipolysis (Non-patent Document 3).
- Non-Patent Document 4 AIM binds to IgM in blood.
- AIM is stable in the blood without being excreted in the urine, so that the binding to IgM is deeply involved, and almost all AIM is bound to IgM in the blood. It has been reported that it hardly exists as a monomer (Non-patent Documents 5 and 6).
- Patent Document 1 discloses a method for diagnosing or testing an AIM-related disease including measuring the AIM concentration in a sample collected from a subject, and examples of the related disease include kidney disease. ing.
- Non-Patent Document 7 describes the correlation between the expression of AIM in macrophages in the kidney tissue of SHRsp rats and the number of macrophages infiltrating, suggesting that the expression of AIM is important for the progression of nephrosclerosis.
- Non-Patent Document 8 describes that a lack of blood AIM significantly increases the risk of progression from a renal disorder event to chronic renal failure.
- Non-Patent Document 9 in the process of examining the role of AIM in the onset / progression process of renal injury, in unilateral ureter ligation model and renal ischemia reperfusion model, in AIM-deficient mice, compared to wild-type mice, It is described that renal tissue damage accompanied by tubular necrosis is significantly enhanced.
- An object of the present invention is to provide a method for detecting a renal disease or an auxiliary method for diagnosing a renal disease, and a kit that can be used for these methods, which are excellent in sensitivity and specificity.
- the inventors of the present invention conducted extensive research to solve the above-mentioned problems, and found that free AIM was significantly increased in biological samples of subjects with renal diseases. In blood, it was thought that free AIM could hardly be confirmed by nature, so it is surprising that a significant increase in free AIM was confirmed in the blood of subjects with renal disease.
- the present invention is based on the above findings and includes the following features.
- the present invention relates to a method for examining renal disease, comprising a step of detecting or quantifying free AIM in a biological sample derived from a subject.
- the present invention can be a method for assisting in the diagnosis of renal disease, comprising a step of detecting or quantifying free AIM in a biological sample derived from a subject.
- the present invention can be a method for detecting or quantifying free AIM in a biological sample derived from a subject in order to examine renal disease.
- the biological sample can be a body fluid.
- the body fluid may be selected from the group consisting of serum, plasma, whole blood and urine.
- the method of the present invention is characterized in that the detection or quantification is by immunoassay.
- the detection or quantification may be performed by bringing a biological sample into contact with an antibody that binds to free AIM.
- the antibody that binds to free AIM is an antibody that specifically binds to free AIM.
- the detection or quantification may be measurement by ELISA.
- the detection or quantification is performed without exposing a blood sample selected from serum, plasma or whole blood to protein denaturing conditions and to reducing conditions. It is characterized by.
- the present invention also relates to a kit for examining or diagnosing renal disease, which comprises an antibody that binds to free AIM.
- the antibody that binds to free AIM is an antibody that specifically binds to free AIM.
- the kidney disease can be acute kidney injury or chronic kidney disease.
- the chronic kidney disease is chronic nephritis, chronic renal failure, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, nephropathy associated with collagen disease or IgM nephropathy. Can be.
- a method for detecting a renal disease or a method for assisting in the diagnosis of a renal disease there are provided a method for detecting a renal disease or a method for assisting in the diagnosis of a renal disease, and a kit that can be used for these methods.
- FIG. 1 is an electrophoretogram showing the results of detecting free AIM and IgM-bound AIM using anti-AIM antibodies in the sera of healthy individuals and acute kidney injury (AKI) patients.
- FIG. 2 shows an ELISA analysis in which the free AIM concentration in urine of 5 healthy subjects, 5 AKI patients (Acute), and 5 patients recovered from AKI (Recovery) was detected using a human AIM monoclonal antibody, respectively. The results are shown.
- FIG. 3 is an electrophoretogram showing the results of detecting free AIM and IgM-bound AIM using anti-AIM antibodies in the serum of patients with chronic kidney injury (CKD).
- FIG. 4 shows the results of detecting free AIM using the Human AIM ELISA kit (CY-8080; CircuLex) in the sera of healthy individuals and chronic kidney injury (CKD) patients.
- the method for examining renal disease includes a step of detecting or quantifying free AIM in a biological sample derived from a subject.
- AIM is a secreted protein with a molecular weight of about 50 kDa that is specifically produced by tissue macrophages, and is known to be expressed in humans and many mammals and birds other than humans.
- AIM is stably present in blood without being excreted in urine, it is known that most of it forms a complex with IgM and hardly exists as a monomer. (Non-Patent Documents 5 and 6).
- Non-patent Document 8 it has been reported that the lack of AIM in blood is associated with the risk of progression to chronic renal failure.
- free AIM refers to a monomeric AIM that does not form a complex with other binding partners of AIM, particularly IgM, in a biological sample.
- the present invention relates to a method for assisting diagnosis of renal disease, comprising a step of detecting or quantifying free AIM in a biological sample derived from a subject.
- “examination of renal disease” or “assisting diagnosis of renal disease” means examining a sample collected from a subject in order to obtain information necessary for diagnosis. Therefore, the method of the present invention can be implemented, for example, by an inspection company.
- the present invention relates to a method for detecting or quantifying free AIM in a biological sample derived from a subject in order to examine renal disease.
- the subject is not particularly limited, and examples thereof include a subject who has a risk of developing or suspected of developing kidney disease.
- Subjects also include subjects who have already developed renal disease, in which case, for the purpose of determining the prognosis of such subject or for the purpose of determining the effects of a particular renal disease treatment.
- the present invention can be implemented.
- a subject typically refers to a human but can also include other animals including, for example, other primates, rodents, dogs, cats, horses, sheep, pigs, and the like.
- renal diseases include both acute kidney injury and chronic kidney disease.
- chronic kidney disease include, but are not limited to, chronic nephritis, chronic renal failure, nephrotic syndrome, diabetic nephropathy, nephrosclerosis, IgA nephropathy, hypertensive nephropathy, collagen disease Examples include nephropathy and IgM nephropathy.
- the biological sample derived from the subject is not particularly limited as long as it can be collected from the subject, and can be, for example, a tissue or a body fluid.
- Tissues include, but are not limited to, ovary, uterus, breast, thyroid, brain, esophagus, tongue, lung, pancreas, stomach, small intestine, duodenum, large intestine, bladder, kidney, liver, prostate, gallbladder, pharynx, muscle, bone And the like, but not limited to.
- the body fluid include, but are not limited to, blood such as serum, plasma or whole blood, lymph fluid, tissue fluid, body cavity fluid, digestive fluid, runny nose, urine and the like.
- the bodily fluid may be a bodily fluid collected from a subject, or may be obtained by subjecting the collected bodily fluid to a treatment such as dilution or concentration that is normally performed.
- a treatment such as dilution or concentration that is normally performed.
- the person who collects and prepares the biological sample derived from the subject used in the present invention may be the same person as the person who performs the process of the present invention, or may be a different person.
- the biological sample derived from the subject used in the present invention may be collected or prepared at the time of carrying out the present invention, or may be collected or prepared and stored in advance.
- the method for detecting or quantifying free AIM is not particularly limited as long as it can distinguish and detect free AIM and AIM that forms a complex with another binding partner (for example, IgM) (hereinafter also referred to as complex AIM).
- a commonly used protein detection method can be applied.
- Such a protein detection method is preferably a method capable of quantitatively or semi-quantitatively measuring free AIM. Examples of such a method include, but are not limited to, a method using an antibody that binds to free AIM, ion exchange chromatography, mass spectrometry, and the like.
- the apparatus used for detecting free AIM is not particularly limited, and can be appropriately selected according to the method of detecting or measuring free AIM.
- HPLC equipment mass spectrometry equipment (mass spectrometry), electrophoresis equipment (capillary electrophoresis apparatus, etc.), fully automatic or semiautomatic enzyme immunoassay equipment, cell washer, fully automatic or semiautomatic chemiluminescence immunoassay equipment
- Luminescence measuring device fully automatic or semi-automatic electrochemiluminescence immunoassay device, optical measuring device, plate reader, CCD camera, fully automatic or semi-automatic fluorescence immunoassay device, fluorescence measuring device, fully automatic or semi-automatic radioimmunoassay device, liquid scintillation A counter, a coulter counter, a surface plasmon measuring device, a blotting device, a densitometer, etc.
- Luminescence measuring device fully automatic or semi-automatic electrochemiluminescence immunoassay device, optical measuring device, plate reader,
- an antibody that binds to free AIM also referred to herein as “anti-free AIM antibody”
- an antibody that binds to free AIM it is preferred to use the immunoassay used.
- the anti-free AIM antibody is not particularly limited as long as it can recognize and bind to AIM.
- the anti-free AIM antibody can be prepared according to a known method for both monoclonal antibodies and polyclonal antibodies.
- monoclonal antibodies are isolated from non-human mammals immunized with free AIM or free AIM fragment, and this is fused with myeloma cells to produce a hybridoma, and the antibody produced by this hybridoma is purified. Can be obtained.
- Polyclonal antibodies can also be obtained from the sera of animals immunized with free AIM or free AIM fragments.
- the free AIM fragment is a partial peptide of free AIM, and the anti-free AIM fragment antibody recognizes free AIM.
- the immunogen include primates such as humans and monkeys, rodents such as rats and mice, free AIM or free AIM fragments such as dogs, cats, horses, sheep, and pigs. It is not limited.
- antibody refers to a full-length immunoglobulin molecule that is naturally occurring or produced by a gene recombination technique, or an immunologically active fragment of an immunoglobulin molecule such as an antibody fragment.
- antibodies of the present invention include any type, class, subclass, eg, IgG, IgE, IgM, IgD, IgA, IgY, IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 and IgA 2 and the like are included. These antibodies can be prepared using conventional techniques, and may be polyclonal antibodies or monoclonal antibodies.
- antibody fragments include F (ab ′) 2 , F (ab) 2 , Fab ′, Fab, Fv, scFv, and the like.
- An antibody fragment can be prepared, for example, by a gene recombination technique using a nucleic acid encoding the antibody fragment, or can be prepared by cleaving a full-length antibody with an enzyme.
- the anti-free AIM antibody is preferably a monoclonal antibody.
- examples of anti-free AIM antibodies include, for example, AIM-CL-6 (Accession Number: NITE BP-1092) and AIM-CL-7 deposited at the Patent Microorganism Depository Center, National Institute for Product Evaluation and Technology. (Accession number: NITE BP-1093).
- the anti-free AIM antibody used in the present invention is preferably an antibody that specifically binds to free AIM.
- “Specific binding” refers to binding that is different from non-specific interaction, and in the present invention, it further has a higher binding affinity for free AIM than for AIM complexed with IgM. It refers to an antibody having sex.
- “specific binding” is, for example, at least about 10 ⁇ 4 M, or at least about 10 ⁇ 5 M, or at least about 10 ⁇ 6 M, or at least about 10 ⁇ 7 for free AIM.
- an antibody having a Kd of M, or at least about 10 ⁇ 8 M, or at least about 10 ⁇ 9 M, or at least about 10 ⁇ 10 M, or at least about 10 ⁇ 11 M, or at least about 10 ⁇ 12 M, or more can be indicated by
- “specific binding” means binding to free AIM without substantially binding to other polypeptides different from free AIM, including AIM complexed with IgM. To do.
- an immunoassay in the present invention a method generally used by those skilled in the art can be used. However, a method or conditions under which free AIM and complex AIM are detected or quantified should be selected. .
- a biological sample other than urine for example, a blood sample selected from serum, plasma or whole blood is used
- free AIM is combined with free AIM upon detection or quantification using an anti-free AIM antibody. It is preferable to fractionate the biological sample based on the molecular weight as necessary so that the body AIM can be distinguished and detected. It is also preferable to perform detection or quantification using an anti-free AIM antibody after separating free AIM from the complex AIM.
- the biological sample is not exposed to protein denaturing conditions or reducing conditions that eliminate the binding state of AIM bound to IgM to form a complex.
- protein denaturing or reducing conditions are well known to those skilled in the art as methods or conditions in which the quaternary structure of the protein is destroyed.
- the immunoassay uses a detectably labeled anti-free AIM antibody or a detectably labeled anti-free AIM antibody (secondary antibody).
- enzymes such as peroxidase and alkaline phosphatase
- radioactive substances such as 125 I, 131 I, 35 S, and 3 H
- fluorescein isothiocyanate rhodamine, dansyl chloride, phycoerythrin, tetra
- fluorescent substance such as methylrhodamine isothiocyanate, a near-infrared fluorescent material, or a CLIA method
- an antibody labeled with a luminescent substance such as luciferase, luciferin, or aequorin is used.
- antibodies labeled with nanoparticles such as colloidal gold and quantum dots can also be detected.
- free AIM antibody can also be detected and measured by labeling an anti-free AIM antibody with biotin and binding avidin or streptavidin labeled with an enzyme or the like.
- an ELISA method using an enzyme label is preferable because it can measure a target simply and rapidly.
- a sandwich method can be used.
- An anti-free AIM antibody is immobilized on a solid phase carrier, and an appropriately treated biological sample is added and reacted, and then an anti-free AIM antibody that recognizes another epitope labeled with an enzyme is added and reacted. After washing, the free AIM concentration can be determined by reacting with the enzyme substrate, causing color development, and measuring the absorbance.
- an unlabeled free AIM antibody primary antibody
- an antibody against the unlabeled antibody secondary antibody
- An enzyme label may be further added.
- DAB 3,3′-diaminobenzidine
- TMB 3,3′5,5′-tetramethylbenzidine
- OPD o-phenylenediamine
- NPP p-nitropheny phosphate
- an agglutination method is also preferable as a method that can easily detect a trace amount of protein.
- the aggregation method include a latex aggregation method in which an antibody is bound to latex particles.
- the concentration of the antigen can be determined by irradiating the sample with near-infrared light and quantifying the aggregate by measuring the absorbance (turbidimetric method) or measuring the scattered light (Nippon method).
- kits capable of specifically detecting free AIM can be used for detecting or quantifying free AIM.
- a kit for example, a commercially available kit can be mentioned, and it can also be carried out by using it according to the product instruction of the kit.
- Examples of such a kit include, but are not limited to, Human AIM ELISA kit (CY-8080; Circulex).
- the method of the present invention may further include a step of determining that it is a renal disease based on the amount of free AIM in the biological sample derived from the subject.
- the amount of free AIM in the present invention is not particularly limited as long as it is a concentration of the free AIM contained in the subject-derived biological sample, or a quantitative value or a semi-quantitative value corresponding thereto.
- the amount of free AIM in the present invention includes a measured value obtained by directly measuring the detected amount of the free AIM, a measured value obtained by indirectly measuring the detected amount of the free AIM through detection of a label, and the like. be able to.
- the amount of free AIM in the biological sample derived from the subject is compared with, for example, the amount of free AIM in the biological sample of a healthy subject serving as a control as a reference value.
- the amount of free AIM in the biological sample is large, it can be determined that the disease is kidney disease.
- a healthy person as a control means an individual who has been previously known not to develop kidney disease.
- a large amount of free AIM means that the amount of free AIM derived from the subject is larger than the reference value (cut-off value) set to distinguish healthy people from patients with renal disease.
- the reference value can be set, for example, by subjecting the amount of free AIM in a biological sample of a renal disease patient and the amount of free AIM in a biological sample of a healthy body group as a control to ROC analysis or the like.
- the ROC analysis is, for example, an analysis method capable of evaluating the detection ability and diagnostic ability of a disease examination method, and is described in the Journal of the Japan Society for Clinical Laboratory Automation “Diagnosis Utility Evaluation Manual for Clinical Examination” Ver. 1.3 (2004.9.1), Vol. 29 Suppl. 1 (Vol. 154) (issued September 1, 2004).
- the reference value can be determined as a value obtained by adding a value obtained by doubling or tripleting the standard deviation to the average value of the detection level of a healthy person, and further, sensitivity (detection rate) / specificity (false), for example.
- the value can be determined as appropriate so as to satisfy a low balance of the positive rate).
- kits according to the present invention is a kit for performing the above-described method for examining a renal disease according to the present invention, and includes an anti-free AIM antibody.
- the anti-free AIM antibody is an antibody that specifically binds to free AIM.
- the test or diagnostic aid kit includes reagents and devices necessary for measuring the free AIM concentration in a biological sample by immunoassay using an antigen-antibody reaction between free AIM and anti-free AIM antibody.
- the kit of the present invention is for measuring free AIM concentration by sandwich ELISA, microtiter plate; anti-free AIM antibody for capture; anti-free AIM antibody labeled with alkaline phosphatase or peroxidase; And an alkaline phosphatase substrate (such as NPP) or a peroxidase substrate (such as DAB, TMB, microtiter plate OPD).
- the capture antibody and the labeled antibody recognize different epitopes.
- a capture antibody is immobilized on a microtiter plate, and a biological sample which has been appropriately processed and diluted is added thereto, followed by incubation, and the sample is removed and washed.
- the labeled antibody is added and then incubated, and the substrate is added to cause color development.
- the free AIM concentration can be determined by measuring color development using a microtiter plate reader or the like.
- the kit of the present invention is for measuring free AIM concentration by a sandwich ELISA method using a secondary antibody, a microtiter plate; an anti-free AIM antibody for capture; free AIM antibody; secondary antibodies include antibodies against anti-free AIM antibody labeled with alkaline phosphatase or peroxidase; and alkaline phosphatase (NPP, etc.) or peroxidase substrate (DAB, TMB, OPD, etc.).
- the capture antibody and the primary antibody recognize different epitopes.
- a capture antibody is immobilized on a microtiter plate, and a biological sample that has been appropriately treated and diluted is added thereto, followed by incubation, and the sample is removed and washed. Subsequently, the primary antibody is added for incubation and washing, and the enzyme-labeled secondary antibody is further added for incubation, and then the substrate is added for color development.
- the free AIM concentration can be determined by measuring color development using a microtiter plate reader or the like.
- the reaction is amplified and the detection sensitivity can be increased.
- kit of the present invention further contains necessary buffer solution, enzyme reaction stop solution, microplate and the like.
- Labeled antibody is not limited to antibodies labeled with an enzyme, radioactive (125 I, 131 I, 35 S, 3 H , etc.), fluorescent substance (fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine iso It may be an antibody labeled with thiocyanate, near-infrared fluorescent material, etc.), luminescent substance (luciferase, luciferin, aequorin, etc.), nanoparticles (gold colloid, quantum dots), etc.
- a biotinylated antibody can be used as a labeled antibody, and labeled avidin or streptavidin can be added to the kit.
- Still another embodiment of the diagnostic kit of the present invention includes one for measuring free AIM concentration by latex agglutination.
- This kit contains anti-free AIM antibody-sensitized latex, a biological sample and anti-free AIM antibody are mixed, and agglomerates are quantified by an optical method. It is also preferred that the kit contains an agglutination plate that visualizes the agglutination reaction.
- Still another embodiment of the diagnostic kit of the present invention includes one for measuring free AIM concentration by electrochemiluminescence.
- This kit contains a carrier carrying an anti-free AIM antibody, adds a biological sample that has been appropriately treated and diluted, and incubates it, and then removes and cleans the sample. Next, an electrochemiluminescent substance (ruthenium or the like) labeled antibody is added and incubated, and then light is emitted by applying electric energy on the electrode.
- the free AIM concentration is quantified by measuring the amount of luminescence.
- Example 1 Production of mouse anti-human AIM monoclonal antibody ⁇ Animal sensitization> Full length human rAIM (2 mg / ml) as an antigen was mixed with an equal amount of TiterMax Gold (G-1 Funakoshi) to prepare an emulsion.
- two Balb / c mice (Charles River Co., Ltd.), 8 week-old females, were administered 50 ⁇ L to the sole of the hind paw. Two weeks later, the same administration was carried out, and after another two weeks or more, 50 ⁇ g of the antigen solution was administered to the hind foot sole to prepare for cell fusion after 3 days.
- Mouse P3U1 was used for myeloma cells, and for growth culture, a medium in which glutamine and pyruvic acid were added to RPMI1640 (11875-119 GIBCO) and FBS (S1560 BWT) was added to 10% was used. As antibiotics, penicillin and streptomycin were added in appropriate amounts.
- ⁇ Cell fusion> Popliteal lymph nodes were aseptically removed from mice subjected to cardiac blood collection under anesthesia, placed in a # 200 mesh beaker, and the cell suspension was adjusted while pushing with a silicon rod. The cells were subjected to centrifugal washing twice with RPMI 1640 and then counted. The myeloma cells in the logarithmic growth phase were collected by centrifugation, washed, adjusted so that the ratio of lymphocytes to myeloma cells was 5: 1, and mixed centrifugation was performed. Cell fusion was performed using PEG1500 (783641 Roche).
- the medium used was a myeloma cell culture medium supplemented with HAT supplement (21060-017 GIBCO) to a FBS concentration of 15%.
- Example 2 Expression of serum free AIM in sera of patients with acute kidney injury AIM in sera of 3 healthy subjects and 6 patients with acute kidney injury (AKI) was examined by Western blot using an anti-AIM antibody.
- the serum was subjected to SDS-polyacrylamide gel electrophoresis under non-reducing conditions, the protein was transferred to a PVDF membrane (Immobilon, Millipore), and the mouse anti-human AIM monoclonal antibody prepared in Example 1 at 4 ° C., overnight A primary antibody reaction was performed.
- HRP-conjugated anti-mouse IgG antibody was used as the secondary antibody, and Luminata Forte Western HRP Substrate (Millipore) was used as a detection reagent, and signal was detected with Image Quant LAS 4000 (GE Healthcare).
- AIM bound to IgM with a molecular weight of> 600 kDa was detected in healthy human serum, but free AIM band with molecular weight ⁇ 40 kDa was detected in the serum of 6 AKI patients in addition to the band of IgM-bound AIM. (FIG. 1).
- Example 3 Increase in free AIM in urine of patients with acute kidney injury
- the ELISA for quantifying AIM was used to measure the free AIM concentration in the urine of healthy and AKI patients.
- the mouse anti-human AIM monoclonal antibody prepared in Example 1 was immobilized on a plate and patient urine was added. After washing, HRP-labeled mouse anti-human AIM monoclonal antibody was reacted and developed with HRP substrate. Since only free AIM is present in urine, the urinary AIM detected by this ELISA is free AIM.
- Free AIM concentration in urine was measured in 5 healthy subjects, 5 AKI patients (Acute), and 5 patients recovered from AKI (Recovery).
- Statistical analysis used Man-Whitney U test. As a result, the free AIM concentration in urine was significantly higher in AKI patients than in healthy individuals (p ⁇ 0.001). Moreover, it was shown that the urine free AIM density
- Example 4 Expression of serum free AIM in sera of chronic kidney disease patients
- Anti-AIM antibodies according to the method described in Example 2 were used for AIM in sera of 3 healthy subjects and 13 chronic kidney disease (CKD) patients. It examined by the Western blot.
- CKD patient serum a free AIM band with a molecular weight of ⁇ 40 kDa was detected in addition to an IgM-bound AIM band with a molecular weight of> 600 kDa (FIG. 3).
- CKD patients in this method included patients with chronic nephritis (chronic glomerulonephritis), diabetic nephropathy, IgA nephropathy, and nephrosclerosis.
- Example 5 Free AIM concentration in serum of CKD patients Using a Human AIM ELISA kit (CircuLex CY-8080) for quantifying free AIM, the free AIM concentration in the serum of 20 healthy individuals and 15 CKD patients was measured. The measurement was carried out according to the instruction manual for the kit. Statistical analysis used Man-Whitney U test. As a result, the free AIM concentration was significantly higher in CKD patients than in healthy individuals (FIG. 4) (p ⁇ 0.001).
- renal diseases can be tested with excellent sensitivity and specificity.
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Abstract
Description
free AIMフラグメントは、free AIMの部分ペプチドであり、抗free AIMフラグメント抗体は、free AIMを認識する。また、免疫原としては、例えば、ヒトやサルなどの霊長類、ラットやマウスなどのげっ歯類、イヌ、ネコ、ウマ、ヒツジ、ブタなどのfree AIMまたはfree AIMフラグメントが挙げられるが、これらに限定されない。
洗浄後、酵素基質と反応、発色させ、吸光度を測定することにより、free AIM濃度を求めることができる。また、固相担体に固定した抗free AIM抗体と生体試料中のfree AIMを反応させた後、非標識free AIM抗体(一次抗体)を添加し、この非標識抗体に対する抗体(二次抗体)を酵素標識してさらに添加してもよい。
また、基準値は、例えば、健常人の検出レベルの平均値に、標準偏差を2倍あるいは3倍した値を加算した値として定めることもでき、更に、感度(検出率)・特異性(偽陽性率の低さ)をバランスよく満たす値に適宜定めることができる。
捕獲抗体と標識抗体は、異なるエピトープを認識する。
このようなキットの場合、まず、マイクロタイタープレートに捕獲抗体を固定し、ここに適宜処理し希釈した生体試料を添加した後インキュベートし、試料を除去して洗浄する。次に、標識抗体を添加した後インキュベートし、基質を加えて発色させる。マイクロタイタープレートリーダー等を用いて発色を測定することにより、free AIM濃度を求めることができる。
捕獲抗体と一次抗体は、異なるエピトープを認識する。
このようなキットでは、まず、マイクロタイタープレートに捕獲抗体を固定し、ここに適宜処理し希釈した生体試料を添加した後インキュベートし、試料を除去して洗浄する。続いて、一次抗体を添加してインキュベートおよび洗浄を行い、さらに酵素標識した二次抗体を添加してインキュベートを行った後、基質を加えて発色させる。マイクロタイタープレートリーダー等を用いて発色を測定することにより、free AIM濃度を求めることができる。二次抗体を用いることにより、反応が増幅され検出感度を高めることができる。
本発明の診断用キットのさらに別の態様として、電気化学発光法によってfree AIM濃度を測定するためのものも挙げられる。このキットは、抗free AIM抗体を担持する担体を含み、適宜処理し希釈した生体試料を添加してインキュベート後、試料を除去して洗浄する。次に電気化学発光物質(ルテニウム等)標識抗体を加えてインキュベート後、電極上で電気エネルギーを与えて発光させる。この発光量を計測することによりfree AIM濃度を定量する。
<動物感作>
抗原として全長ヒトrAIM(2mg/ml)を等量のTiterMaxGold(G-1フナコシ)と混合しエマルジョンを作製した。免疫動物にはBalb/cマウス(チャールズリバー(株))8週齢のメス2匹を用い、後ろ足底部へ50μLを投与した。2週間後に同様の投与を行い、更に2週間以上をおいて抗原溶液50μgを後ろ足底部へ投与し3日後の細胞融合に備えた。
ミエローマ細胞にはマウスP3U1を用い、増殖培養には、RPMI1640(11875-119 GIBCO)にグルタミンとピルビン酸を加えFBS(S1560 BWT社)を10%になるように添加した培地を用いた。抗生物質としてはペニシリン、ストレプトマイシンを適量加えた。
麻酔下にて心臓採血を行ったマウスから無菌的に膝窩リンパ節を摘出し、#200メッシュ付ビーカーにのせ、シリコン棒で押しながら細胞浮遊液を調整した。細胞はRPMI1640にて2回の遠心洗浄を行った後、細胞数をカウントした。対数増殖期の状態のミエローマ細胞を遠心により集め洗浄後、リンパ細胞とミエローマ細胞の比率が5対1となるように調整し、混合遠心を行った。細胞融合はPEG1500(783641 ロシュ)を用いて行った。すなわち、細胞ペレットへ1mLのPEG液を3分間かけて反応させ、その後、段階的に希釈を行い遠心にて洗浄した後、培地を加え96ウェルプレート15枚へ200μLずつ入れ、1週間の培養を行った。培地にはミエローマ細胞用培地にHATサプリメント(21060-017 GIBCO)を加え、FBS濃度を15%にしたものを用いた。
凍結保存された融合細胞を解凍し、増殖培養を行った後、1週間以上前に0.5mlのプリスタン(42-002 コスモバイオ)を腹腔内投与したヌードマウス(BALB/cAJcl-nu/nu 日本クレア)の腹腔へ1×107個を投与し、およそ2週間後に4~12mlの腹水を得た。遠心処理にて固形物を除去した後、凍結保存を行った。
健常人3例、急性腎障害(AKI)患者6例の血清におけるAIMを、抗AIM抗体を用いたウエスタンブロットにて検討した。血清を非還元条件下でSDS-ポリアクリルアミドゲル電気泳動を行い、蛋白質をPVDF膜(Immobilon,ミリポア社)に転写し、実施例1で作製したマウス抗ヒトAIMモノクローナル抗体と4℃、overnightにて一次抗体反応を行った。二次抗体にHRP結合抗マウスIgG抗体、検出試薬としてLuminata Forte Western HRP Substrate (ミリポア社)を用い、シグナルの検出はImage Quant LAS 4000 (GE Healthcare)にて行った。
AIMを定量するELISA を用いて、健常人とAKI患者の尿中のfree AIM濃度を測定した。AIM ELISAでは、実施例1で作製されたマウス抗ヒトAIMモノクローナル抗体をプレートに固相化し、患者尿を添加した。洗浄後、HRP標識マウス抗ヒトAIMモノクローナル抗体を反応させ、HRP基質によって発色させた。尿中ではfree AIMのみが存在することから、本ELISAによって検出される尿中AIMは、free AIMである。
健常人5例、AKI患者(Acute)5例、AKIから回復した患者(Recovery)5例の尿中のfree AIM濃度を測定した。統計解析はMan-Whittney U testを用いた。その結果、尿中のfree AIM濃度は、健常人と比べ、AKI患者で有意に高かった(p<0.001)。また、AKIからの回復に伴い、尿中free AIM濃度が低下することが示された(図2)。
健常人3例、慢性腎臓病(CKD)患者13例の血清におけるAIMを、実施例2に記載の方法による抗AIM抗体を用いたウエスタンブロットにて検討した。CKD患者血清では、分子量>600 kDa のIgM結合AIMのバンドに加え、分子量<40kDaのfree AIMのバンドが検出された(図3)。本方法におけるCKD患者には、慢性腎炎(慢性糸球体腎炎)、糖尿病性腎症、IgA腎症、腎硬化症の患者が含まれていた。
free AIMを定量するHuman AIM ELISA kit(CircuLex社CY-8080)を用い、健常人20例、CKD患者15例の血清中のfree AIM濃度を測定した。測定はキットの取り扱い手順書に従って実施した。統計解析はMan-Whittney U testを用いた。その結果、free AIM濃度は、健常人と比べ、CKD患者で有意に高かった(図4)(p<0.001)。
Claims (15)
- 腎疾患の検査方法であって、
被験体に由来する生体試料中のfree AIMを検出または定量する工程を含む、
方法。 - 腎疾患の診断を補助する方法であって、
被験体に由来する生体試料中のfree AIMを検出または定量する工程を含む、
方法。 - 腎疾患を検査するために、被験体に由来する生体試料中のfree AIMを検出または定量する方法。
- 請求項1~3のいずれか1項に記載の方法であって、
前記生体試料が、血清、血漿、全血および尿からなる群から選択される、
方法。 - 請求項1~4のいずれか1項に記載の方法であって、
前記腎疾患が、急性腎障害又は慢性腎臓病である、
方法。 - 請求項5に記載の方法であって、
前記慢性腎臓病が、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症又はIgM腎症である、
方法。 - 請求項1~6のいずれか1項に記載の方法であって、
前記検出または定量は、イムノアッセイによるものである、
方法。 - 請求項7に記載の方法であって、
前記検出または定量は、生体試料とfree AIMに結合する抗体とを接触させることにより行う、
方法。 - 請求項8に記載の方法であって、
前記検出または定量は、free AIMに特異的に結合する抗体により行う、
方法。 - 請求項8または請求項9に記載の方法であって、
前記検出または定量は、ELISAによる測定である、
方法。 - 請求項7~10のいずれか1項に記載の方法であって、
前記検出または定量は、血清、血漿または全血から選択される血液試料をタンパク質変性条件に曝露することなく、かつ、還元条件に曝露することなく行うことを特徴とする、
方法。 - free AIMに結合する抗体を含む、腎疾患の検査または診断補助用キット。
- 請求項12に記載のキットであって、
前記抗体が、free AIMに特異的に結合する抗体である、
キット。 - 請求項12または請求項13に記載のキットであって、
前記腎疾患が、急性腎障害または慢性腎臓病である、
キット。 - 請求項14に記載のキットであって、
前記慢性腎臓病が、慢性腎炎、慢性腎不全、ネフローゼ症候群、糖尿病性腎症、腎硬化症、IgA腎症、高血圧性腎症、膠原病に伴う腎症またはIgM腎症である、
キット。
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