WO2023063331A1 - 抗３'，４'－ジデヒドロ－３'－デオキシシチジン抗体 - Google Patents

抗３'，４'－ジデヒドロ－３'－デオキシシチジン抗体 Download PDF

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WO2023063331A1
WO2023063331A1 PCT/JP2022/037956 JP2022037956W WO2023063331A1 WO 2023063331 A1 WO2023063331 A1 WO 2023063331A1 JP 2022037956 W JP2022037956 W JP 2022037956W WO 2023063331 A1 WO2023063331 A1 WO 2023063331A1
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ddhc
antibody
derived
maleimide
biological sample
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真一 秋山
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Tokai National Higher Education and Research System NUC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • 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
    • G01N33/531—Production of immunochemical test materials

Definitions

  • the present invention provides an antibody that specifically binds to 3',4'-Didehydro-3'-deoxycytidine (hereinafter also referred to as "ddhC”) (sometimes referred to as “anti-ddhC antibody” or “this antibody”); method for detecting ddhC derived from a biological sample using an anti-ddhC antibody; kit for detecting ddhC derived from a biological sample containing an anti-ddhC antibody ; etc.
  • ddhC 3',4'-Didehydro-3'-deoxycytidine
  • SLE Systemic lupus erythematosus
  • systemic inflammation such as fever, general malaise, and damage to various organs, including joints, nervous system, blood, skin, kidneys, gastrointestinal tract, and lungs, may occur at once or over time.
  • SLE repeats remission and exacerbation, and often takes a chronic course.
  • the type, course, and degree of symptoms of SLE vary from patient to patient, making it more difficult to diagnose than other diseases.
  • Renal damage caused by SLE is called lupus nephritis, and whether or not lupus nephritis develops and severity of lupus nephritis symptoms at the time of initiation of treatment are factors that influence the life prognosis of SLE patients. Since delay in diagnosis of SLE leads to deterioration of prognosis, SLE is a disease for which early diagnosis and early treatment are important. Currently, the diagnosis of SLE is made by comprehensively judging many factors, including the presence or absence of SLE-related symptoms and the presence or absence of antinuclear antibodies in the blood. does not exist. Therefore, there is still a need for new detection methods that lead to early diagnosis of SLE.
  • nephrotic syndrome is a general term for kidney diseases characterized by massive proteinuria and hypoproteinemia, and its causative diseases are diverse, including primary glomerular disease, collagen disease, and metabolic disease.
  • the age at which it is afflicted ranges widely from children to the elderly.
  • the diagnostic criteria for adult nephrotic syndrome are (i) urinary protein of 3.5 g or more per day, and (ii) serum albumin concentration of 3.0 g/dL or less. Two points are defined as essential conditions, and (iii) presence of edema and (iv) dyslipidemia are also widely used as additional reference conditions.
  • nephrotic syndrome there are various types of nephrotic syndrome. Glomerulosclerosis (FSGS), lupus nephritis (LN), IgA nephropathy (IgA), amyloid nephropathy (RA) and the like. Accurate diagnosis of the type of nephrotic syndrome is clinically very important, because different treatment methods are applied depending on the type of nephrotic syndrome.
  • renal biopsy is necessary for the definitive diagnosis of nephrotic syndrome, as well as renal diseases such as nephritis and nephrotic syndrome.
  • renal biopsy is a highly invasive examination with a risk of bleeding, renal biopsy cannot be performed in patients whose general condition has deteriorated or who are elderly in many cases. For example, diabetic nephropathy and nephrosclerosis are often diagnosed by clinical speculation without renal biopsy.
  • a renal biopsy cannot be performed, there is a problem that it becomes difficult to diagnose a disease, grasp the pathophysiology, and select an appropriate treatment method. Therefore, a method capable of determining renal disease and its disease type with high accuracy and less invasiveness without performing highly invasive renal biopsy has a very high clinical value.
  • Patent Document 1 For example, a method of using urinary human megalin as a renal injury marker (Patent Document 1) and Patent Document 2 describe a combination of specific metabolites such as creatinine and aspartic acid in blood as markers for distinguishing diabetic nephropathy. has been reported (Patent Document 2).
  • systemic lupus erythematosus preferably lupus nephritis
  • urinary ddhC concentration as an index
  • An object of the present invention is to provide an antibody that specifically binds to 3',4'-didehydro-3'-deoxycytidine (ddhC), a method for specifically detecting ddhC derived from a biological sample, and a method for specifically detecting ddhC derived from a biological sample.
  • the object is to provide a kit or the like for the purposeful detection.
  • the nitrogen atom of the cytidine base of ddhC is condensed with a maleimide-containing carboxylic acid to prepare a maleimide-labeled ddhC, which is prepared by binding KLH (Keyhole Limpet Hemocyanin) through the maleimide group of the maleimide-labeled ddhC.
  • KLH Keyhole Limpet Hemocyanin
  • the present invention is as follows. [1] an antibody that specifically binds to 3',4'-didehydro-3'-deoxycytidine; [2] 3',4'-didehydro-3'-, comprising detecting 3',4'-didehydro-3'-deoxycytidine derived from a biological sample using the antibody of [1] above A method for detecting deoxycytidine (hereinafter sometimes referred to as "this detection method"). [3] The detection method according to [2] above, wherein the biological sample is urine.
  • a polynucleotide encoding an anti-ddhC antibody (hereinafter referred to as "anti-ddhC antibody polynucleotide”); a vector comprising a promoter and an anti-ddhC antibody polynucleotide operably linked downstream of the promoter (hereinafter referred to as "anti-ddhC antibody expression vector”); host cells into which an anti-ddhC antibody expression vector has been introduced (hereinafter referred to as "anti-ddhC antibody-expressing host cells”); A hybridoma that produces an anti-ddhC antibody (hereinafter referred to as "anti-ddhC antibody-producing hybridoma"); method of manufacturing; can be mentioned.
  • Such a production method further includes a step of preparing a cell clone that produces an anti-ddhC antibody using cell fusion technology, and a step of screening by ELISA using maleimide-labeled ddhC bound to a carrier protein or labeling substance. things are preferred.
  • the subject antibody is an antibody that specifically binds to ddhC derived from a biological sample.
  • ddhC derived from a biological sample can be specifically detected, and in particular, ddhC in urine can be time-effective and cost-effective compared to the conventional method of detecting ddhC using a mass spectrometer.
  • QOL Quality of Life
  • FIG. 2 shows the results of measurement of urine ddhC by antigen-competitive ELISA using an anti-ddhC mouse monoclonal antibody (M01).
  • the subject antibody is 3′,4′-didehydro-3′-deoxycytidine (ddhC), an antibody that specifically binds to a compound of the formula: means an antibody that recognizes and binds to ddhC (preferably urinary ddhC) by a highly specific antigen-antibody recognition mechanism.
  • ddhC 3′,4′-didehydro-3′-deoxycytidine
  • the present antibody includes human-derived antibodies; mixture of dozens of antibodies), monoclonal antibody; chimeric antibody or humanized antibody in which a partial region (e.g., constant region) of an antibody is replaced with a region derived from a different species, obtained by digesting a monoclonal antibody with pepsin F(ab') 2 antibody fragment, Fab' antibody fragment obtained by reducing F(ab') 2 antibody fragment, antibody fragment such as Fab obtained by papain digestion of monoclonal antibody, antibody heavy (H) chain scFv (single-chain antibody) in which the variable region and the antibody light (H) chain variable region are linked by an amino acid bridge;
  • H antibody heavy
  • H single-chain antibody
  • the antibody of interest is preferably isolated.
  • isolated means that the antibody is removed from the environment in which it originally exists by artificial manipulation, or is expressed in an environment different from the environment in which the antibody originally exists, and the antibody originally exists. It means that it exists in a state different from the state in which it is.
  • an “isolated antibody” refers to an antibody derived from a certain individual, which is not subjected to external manipulation (artificial manipulation), and which is in the body of the individual or in tissues or body fluids (blood) derived from the body. , plasma, serum, etc.).
  • the antibody of interest is preferably an antibody produced from an organism or cell produced by artificial manipulation (for example, an antibody produced from a hybridoma).
  • Such "antibodies produced from engineered organisms or cells” do not include antibodies produced from naturally occurring organisms or B-cells (that have not been engineered).
  • the subject antibody usually has an H chain complementarity determining region (CDR) 1, an H chain CDR2, and an H chain CDR3, and an L chain CDR1, an L chain CDR2, and an L chain CDR3, and usually each of these CDRs 1-3 Linked to the amino (N)- and carboxyl (C)-termini of the regions are framework regions (FR).
  • CDRs specificity to ddhC may be generated by bringing the CDRs closer to each other when the H chain and L chain of the present antibody form a three-dimensional structure.
  • the H chain CDR1 in this antibody is usually located at positions H26-32 according to Chothia numbering.
  • the H chain CDR2 in the present antibody is usually numbered by Chothia (see document "Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917"), H52, H52A, and H53-56. exist in position.
  • the heavy chain CDR3 in the antibody of interest is generally located at positions H95-99, H101, and H102, numbered according to Chothia.
  • the L chain CDR1 in the present antibody is usually located at positions L24 to 34 according to Chothia numbering.
  • the light chain CDR2 in the antibody of interest is generally located at positions L50-56 according to Chothia numbering.
  • the L chain CDR3 in the present antibody is usually present at positions L89-97 according to Chothia numbering.
  • the promoter in the anti-ddhC antibody expression vector may be any region that initiates transcription of the mRNA encoded by the anti-ddhC antibody polynucleotide located downstream of the promoter, and the promoter usually includes a transcription start site (TSS). .
  • TSS transcription start site
  • the promoter and anti-ddhC antibody expression vector in the anti-ddhC antibody expression vector can be appropriately selected according to the type of host cell (or host organism) to be introduced.
  • anti-ddhC antibody expression vectors include, for example, vectors such as YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), or vectors derived from such vectors.
  • promoters include promoters of glycolytic genes such as hexose kinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock protein promoter, and MF ⁇ 1 promoter. , CUP1 promoter, and the like.
  • the subject vectors include, for example, pcDNAI, pcDM8 ( Funakoshi Co., Ltd.), pAGE107 (JP-A-3-22979; Cytotechnology, 3,133, (1990)), pAS3-3 (JP-A-2-227075), pCDM8 (Nature, 329,840, (1987)), pcDNAI/ Vectors such as Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 (J.Biochemistry, 101, 1307 (1987)), pAGE210, and those derived from such vectors can be mentioned. Examples include the cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter
  • CMV cytomegalovirus
  • the subject vector includes, for example, a recombinant baculovirus production method
  • vectors such as pVL1392, pVL1393, pBlueBacIII (both manufactured by Invitrogen) or those derived from such vectors can be mentioned
  • promoters include, for example, polyhedrin promoter, p10 promoter etc. can be mentioned.
  • expression vectors include, for example, Ti plasmids, tobacco mosaic virus vectors, etc. or those derived from such vectors.
  • promoters include cauliflower mosaic virus (CaMV) 35S promoter, rice actin 1 promoter, and the like.
  • anti-ddhC antibody expression vector in order to further increase the gene expression efficiency, those further containing the nucleotide sequence of the enhancer region or ribosome binding site (RBS; ribosome binding site).
  • drug resistance genes for example, spectinomycin resistance gene, chloramphenicol resistance gene, tetracycline resistance gene, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, hygromycin resistance gene, blasticidin resistance gene, geneticin resistance gene, etc.
  • the enhancer region is usually placed upstream from the promoter and the RBS is usually placed between the promoter and the anti-ddhC antibody polynucleotide.
  • the nucleotide sequence of the anti-ddhC antibody polynucleotide incorporated into the anti-ddhC antibody expression vector may be codon-optimized according to the host cell for expression.
  • An anti-ddhC antibody expression vector can be produced by a known method using gene recombination technology.
  • the biological species of the anti-ddhC antibody-expressing host cells may be those in which the mRNA of the anti-ddhC antibody polynucleotide is transcribed and the anti-ddhC antibody protein is expressed. , mammals (eg, humans, mice, rats, monkeys, etc.), and insects (eg, Spodoptera frugiperda, Trichoplusiani, etc.).
  • Anti-ddhC antibody-producing hybridomas may be cells (fused cells) obtained by fusing two or more cells (preferably mammalian cells) that produce anti-ddhC antibodies, and produce anti-ddhC antibodies.
  • a fusion cell of a B-cell capable of proliferating and a cell having proliferative potential eg, myeloma cell is preferred.
  • Anti-ddhC antibody-expressing host cells can be obtained by introducing (transfecting) an anti-ddhC antibody expression vector into host cells by a method suitable for the type of host cell.
  • the method for introducing the anti-ddhC antibody expression vector into the yeast may be any method that introduces DNA into the yeast. )), the spheroplast method (Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)), the lithium acetate method (J. can.
  • the method for introducing the anti-ddhC antibody expression vector into mammalian cells may be any method that introduces DNA into mammalian cells. Cytotechnology, 3, 133 (1990)), calcium phosphate method (JP-A-2-227075), lipofection method (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)). .
  • methods for introducing an anti-ddhC antibody expression vector into insect cells include, for example, "Current Protocols in Molecular Biology", “Baculovirus Expression Vectors, A Laboratory Manual, W.H. Freeman and Company, New York (1992)", “Bio/Technology, 6, 47 (1988)", etc., co-transfection of the subject vector (transfer vector) and baculovirus-derived genomic DNA into the insect cells and methods for producing recombinant baculoviruses. Examples of such cotransfection methods include the calcium phosphate method (JP-A-2-227075) and the lipofection method (Proc. Natl. Acad. Sci. U.S.A., 84, 7413 (1987)). .
  • methods for introducing the anti-ddhC antibody expression vector into the plant cells include, for example, methods using Agrobacterium (JP-A-59-140885, JP-A-59-140885, JP-A-59-140885, Japanese Patent No. 60-70080), electroporation method (Japanese Patent Laid-Open No. 60-251887), method using a particle gun (gene gun) (Japanese Patent No. 2606856, Japanese Patent No. 2517813), etc. can be mentioned.
  • the present antibody can be obtained by culturing the anti-ddhC antibody-expressing host cells obtained by the method described above in a culture medium suitable for the host cells.
  • chimeric antibodies can be produced based on the technique described in JP-A-2005-245337.
  • transgenic animals such as mice, cattle, goats, sheep, chickens, and pigs into which an anti-ddhC antibody polynucleotide (anti-ddhC antibody expression vector) is incorporated are produced using transgenic animal production technology, and such transgenic animals Antibodies derived from anti-ddhC antibody polynucleotides can also be produced in large amounts from blood, milk, etc.
  • ddhC prepared by condensing the nitrogen atom of the cytidine base of ddhC with a maleimide-containing carboxylic acid to prepare maleimide-labeled ddhC, and binding KLH (Keyhole Limpet Hemocyanin) via the maleimide group of the maleimide-labeled ddhC - KLH (antigen) is immunized to non-human animals (e.g., mice, rats), cell fusion technology is used to prepare cell clones that produce antibodies against ddhC, carrier proteins or maleimide labels bound to labeling substances
  • Anti-ddhC antibody-producing hybridomas and culture supernatants containing anti-ddhC antibodies can be obtained by screening by ELISA using ddhC. The subject antibody can be separated and purified from such a culture supernatant using a known antibody purification technique.
  • the carrier protein a wide range of natural or synthetic macromolecular proteins that are commonly used for the preparation of antigens can be used.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • OVA ovalbumin
  • horse serum albumin human serum albumin
  • sheep serum albumin rabbit serum albumin
  • animal albumins such as ovalbumin, bovine serum globulin
  • Animal globulins such as horse serum globulin, human serum globulin, sheep serum globulin, rabbit serum globulin, egg globulin, animal thyroglobulin such as bovine thyroglobulin, horse thyroglobulin, human thyroglobulin, sheep thyroglobulin, rabbit thyroglobulin
  • animal hemoglobins such as bovine hemoglobin, horse hemoglobin, human hemoglobin, sheep hemoglobin and rabbit hemoglobin, animal hemocyanins, polylysine, polyglutamic acid, lysine
  • the present detection method may be a method comprising a step of detecting ddhC derived from a biological sample using the present antibody, and specific detection methods include immunohistochemical staining using the present antibody, ELISA. (Enzyme-linked immunosorbent assay) method, RIA (Radioimmunoassay) method, Western blotting method, and the like.
  • the detection kit is a kit containing the antibody or a label thereof limited to the use of "detecting ddhC derived from a biological sample", and such a kit generally includes Components, such as carriers, pH buffers, stabilizers, as well as package inserts, such as instructions for use, instructions for detecting ddhC from biological samples, etc., are usually included.
  • labeling substance includes, for example, peroxidase (eg, HRP [Horseradish Peroxidase]), alkaline phosphatase, ⁇ -D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase , enzymes such as malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase, fluorescence such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelate, dansyl chloride or tetramethylrhodamine isothiocyanate Substances, Green Fluorescence Protein (GFP), Cyan Fluorescence Protein (CFP), Blue Fluorescence Protein (BFP), Yellow Fluorescence Protein (YFP), Red Fluorescence Protein ( red fluorescence protein (RF
  • the biological sample may be a sample derived from a living body such as a subject (e.g., systemic lupus erythematosus patient) containing ddhC, and non-humidal samples such as tissues, cells, organs, blood, urine, saliva, etc.
  • a subject e.g., systemic lupus erythematosus patient
  • non-humidal samples such as tissues, cells, organs, blood, urine, saliva, etc.
  • blood plasma, serum
  • urine or tissue is preferred.
  • Example 1 Acquisition of anti-ddhC antibody-producing hybridoma clones
  • ddhC was discovered as a biomarker for detecting systemic lupus erythematosus (Patent Document 3). Therefore, an attempt was made to produce an anti-ddhC antibody.
  • Material 1-1 ddhC ddhC was commissioned and synthesized by FUJIFILM Wako Pure Chemical Industries, Ltd. based on N-acetylcytidine (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) as a starting material.
  • Method 2-1 Preparation of antigen-sensitized mouse or rat-derived lymphocytes
  • An antigen for immunization (ddhC-KLH) is mixed with an adjuvant (Freund's Complete Adjuvant [manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.]) to emulsify
  • Antigen for immunization (200 ⁇ g/100 ⁇ L) was prepared and immunized by injection into 3 BALB/c mice and 2 WKY rats each. Twenty-one days after immunization, secondary lymphoid tissues were excised from each mouse and rat, erythrocytes were hemolyzed, and lymphocytes used for cell fusion were prepared according to a standard method.
  • Hybridoma Lymphocytes and myeloma cells collected from mice and rats were fused in the presence of 50% polyethylene glycol according to a standard method to obtain hybridomas.
  • the hybridomas were dispersed in HAT medium and cultured in four 96-well plates. Seven days after the cell fusion, the medium was replaced with a new HT medium, the culture was continued, and 9 days after the cell fusion, the hybridoma culture supernatant was collected from each well. Cell culture was performed in a CO 2 incubator (5% CO 2 /20% O 2 , 95% humidity, 37° C.).
  • test substance immunodeficiency virus
  • normal mouse or rat serum normal mouse or rat serum as a control; or hybridoma culture supernatant
  • 50 ⁇ L of the test substance was added to each well and incubated at room temperature for 60 minutes.
  • 50 ⁇ L of a solution containing an HRP-conjugated anti-mouse IgG-Fc antibody or anti-rat IgG-Fc antibody was added and incubated at room temperature for 30 minutes.
  • HRP substrate OPDA (12-oxo-Phytodienoic acid) (manufactured by Thermo Scientific) was added, and the mixture was placed in a dark room at room temperature for 30 minutes. incubated. Thereafter, 50 ⁇ L of 1 M H 3 PO 4 solution was added to each well, and color development at a wavelength of 492 nm was measured using a plate reader (Infinite F50R, manufactured by Tecan).
  • Anti-mouse IgG-Fc antibody or anti-rat IgG-Fc antibody (2.5 ⁇ g/mL, 50 ⁇ L) is coated on each well of a 96-well plate, and a test substance (immune mouse or rat and normal mouse or rat serum; or hybridoma culture supernatant) and 50 ⁇ L of a solution containing an antigen for antibody activity measurement (HRP-labeled ddhC) were added and incubated at room temperature for 60 minutes. After each well was washed three times with a PBS-Tween solution, 50 ⁇ L of OPDA was added and incubated in a dark room at room temperature for 30 minutes. Then, 50 ⁇ L of 1 M H 3 PO 4 solution was added to each well, and color development at a wavelength of 492 nm was measured using a plate reader.
  • Antigen-competitive ELISA method A test substance (immune mouse or rat serum , and normal mouse or rat serum; or hybridoma culture supernatant) with 50 ⁇ L of various concentrations (0, 0.01 ⁇ g/mL, 0.1 ⁇ g/mL, 1 ⁇ g/mL, 10 ⁇ g/mL, and 100 ⁇ g/mL). 50 ⁇ L of maleimide-labeled ddhC and 50 ⁇ L of 100 ⁇ g/mL HRP-labeled ddhC were added and incubated with shaking for 30 minutes at room temperature.
  • Hybridoma clones that were positive in the primary screening were expanded and cultured for several days, and then the culture supernatant of the hybridomas was collected from each well and subjected to the three types of ELISA methods described above. was used to perform secondary screening of anti-ddhC antibody-producing hybridoma clones.
  • 6 mouse-derived clones and 6 rat-derived hybridoma clones were obtained as positive hybridoma clones in all of the above three ELISA methods.
  • cloning was performed by the limiting dilution method to obtain monoclonal hybridoma clones.
  • 6 mouse-derived clones and 5 rat-derived hybridoma clones were obtained as anti-ddhC antibody-producing hybridoma clones.
  • Example 2 Measurement of urinary ddhC by competitive ELISA method using anti-ddhC antibody.
  • Method 1-1 Purification of Anti-ddhC Antibody Among the 5 mouse-derived anti-ddhC antibody-producing hybridoma clones obtained, the culture supernatant of 1 clone (M01 strain) was affinity purified according to a standard method to obtain a purified antibody (anti-ddhC mouse monoclonal An antibody [anti-ddhC mouse monoclonal antibody [M01]]) was prepared.
  • Antigen-competitive ELISA method A solution was prepared by adding maleimide-labeled ddhC to a predetermined concentration (0, 1, 10, 50, 100 ⁇ g/mL) in healthy human urine. Coated with mouse IgG-Fc antibody (2 ⁇ g / mL, 50 ⁇ L), blocked with 200 ⁇ L of blocking solution (Blocking One, manufactured by Nacalai), washed 3 times with PBS-Tween solution, anti-ddhC mouse Monoclonal antibody (M01) (2 ⁇ g/mL, 50 ⁇ L), urine containing ddhC at the predetermined concentration, and 50 ⁇ L of 100 ⁇ g/mL HRP-labeled ddhC were added and incubated with shaking at room temperature for 30 minutes.
  • M01 anti-ddhC mouse Monoclonal antibody
  • the present invention contributes to early detection and early treatment of diseases associated with increased or decreased ddhC, such as systemic lupus erythematosus.

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WO2026027878A1 (en) 2024-07-30 2026-02-05 Imperial College Innovations Limited Detection of viral infections using ddhc-specific aptamers

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WO2009107384A1 (ja) * 2008-02-29 2009-09-03 国立大学法人名古屋大学 急性腎障害及び予後推定用バイオマーカー並びにその用途
WO2013183596A1 (ja) * 2012-06-06 2013-12-12 国立大学法人名古屋大学 腎疾患のバイオマーカー及びその用途
WO2020138260A1 (ja) * 2018-12-26 2020-07-02 学校法人慶應義塾 全身性エリテマトーデスの検出方法

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
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