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WO2024071008A1 - Procédé permettant d'isoler une molécule d'antigène - Google Patents

Procédé permettant d'isoler une molécule d'antigène Download PDF

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Publication number
WO2024071008A1
WO2024071008A1 PCT/JP2023/034650 JP2023034650W WO2024071008A1 WO 2024071008 A1 WO2024071008 A1 WO 2024071008A1 JP 2023034650 W JP2023034650 W JP 2023034650W WO 2024071008 A1 WO2024071008 A1 WO 2024071008A1
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Prior art keywords
antigen
antibody
molecules
binding
column
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English (en)
Japanese (ja)
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歩 田口
雄一 阿部
泰秀 奥本
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Aichi Prefecture
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Aichi Prefecture
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • 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 relates to a method for isolating and detecting antigen molecules, and a method for assisting in the diagnosis of diseases such as cancer and autoimmune diseases.
  • antigen molecules whether self-antigens or non-self-antigens, form antigen-antibody complexes by binding with antibodies.
  • antigen-antibody complexes By analyzing antigen-antibody complexes present in the body, it may be possible to detect any changes in the body, such as disease.
  • autoantibodies that recognize cancer antigens appear in the blood at an extremely early stage in the carcinogenesis process.
  • Autoantibodies that recognize cancer antigens form antigen-antibody complexes by binding to cancer antigens in the body, and are known to spread not only in tumor tissue but also in body fluids such as blood. It is also thought that antibodies against antigens derived from pathogens in infectious diseases, and antibodies against autoantigens in autoimmune diseases, each form antigen-antibody complexes and spread in body fluids.
  • Non-Patent Document 1 describes a proteomic analysis targeting cancer antigens bound to immunoglobulins in plasma.
  • this proteomic analysis some of the antigen molecules were identified by mass spectrometry in antigen-antibody complexes recovered from plasma using beads bound to protein A or G.
  • this method only identified some of the antigen molecules contained in relatively large quantities, and there was a problem that trace amounts of antigen molecules could not be identified because they were blocked by the large amount of immunoglobulin molecules contaminating the sample.
  • the object of the present invention is to provide a method for efficiently isolating and detecting antigen molecules from antigen-antibody complexes in biological samples.
  • beads conjugated with antibody-binding proteins such as protein A or G are used to isolate antigen-antibody complexes from other biomolecules based on the binding between the antibody-binding proteins and immunoglobulins.
  • antibody-binding proteins such as protein A or G
  • immunoglobulins that are not bound to antigen molecules. Therefore, in the elution step using an acidic solvent, a large amount of immunoglobulins is eluted in addition to the antigens dissociated from the immunoglobulins.
  • the large amount of contaminating immunoglobulins is a major obstacle to identifying antigen molecules, but it is extremely difficult to selectively separate antigen molecules from antibody molecules.
  • the inventors of the present application focused on the possibility that there may be a difference between the binding strength between antigen molecules and antibody molecules, and between antibody molecules and antibody-binding molecules, that allows selective dissociation of only one of them, and came up with the idea that by utilizing this difference in binding strength, it may be possible to selectively separate antigen molecules from antigen-antibody complexes ( Figure 1).
  • the inventors bound the antigen-antibody complex to a column on which antibody-binding molecules were fixed, and then eluted the antigen-antibody complex from the column using an elution solution with a specific pH.
  • an elution solution with a specific pH
  • the inventors further performed pH gradient liquid chromatography to identify the optimal pH conditions for selectively separating antigen molecules from antibody molecules. As a result, they clarified extremely favorable elution conditions for dramatically reducing the amount of contaminating antibody molecules and efficiently detecting even trace amounts of antigen molecules, and achieved the identification of more than six times as many antigen molecules as with conventional methods.
  • a method for isolating antigen molecules from a liquid sample containing an antigen-antibody complex comprising the steps of: a binding step of adding the liquid sample to a column on which an antibody-binding protein is immobilized, thereby binding the antibody molecules in the antigen-antibody complex to the antibody-binding protein; and an elution step of adding a buffer solution to the column, thereby dissociating the antigen molecules in the antigen-antibody complex from the antibody molecules, and eluting an eluate containing the antigen molecules,
  • a method for detecting an antigen molecule from a liquid sample containing an antigen-antibody complex comprising the steps of: a binding step of adding the liquid sample to a column on which an antibody-binding protein is immobilized, thereby binding the antibody molecules in the antigen-antibody complex to the antibody-binding protein; an elution step of adding a buffer solution to the column to dissociate the antigen molecules in the antigen-antibody complex from the antibody molecules, thereby eluting an eluate containing the antigen molecules; and a detection step of detecting the antigen molecules in the eluate,
  • the elution step comprises adding the buffer solution to the column while decreasing the pH of the buffer solution continuously or stepwise.
  • the liquid sample is a body fluid, an intraperitoneal lavage fluid, a cell culture fluid, a cell lysate, or a tissue lysate.
  • the body fluid is selected from the group consisting of blood, serum, plasma, cerebrospinal fluid, urine, saliva, and ascites.
  • the body fluid or peritoneal lavage fluid is derived from a patient suffering from a disease selected from the group consisting of cancer, an autoimmune disease, a collagen disease, an infectious disease, an inflammatory disease, and an aging-related disease.
  • a method for isolating an antigen molecule from a liquid sample containing an antigen-antibody complex comprising the steps of: a binding step of contacting the liquid sample with a carrier having an antibody-binding protein immobilized thereon, thereby binding the antibody molecules in the antigen-antibody complex to the antibody-binding protein; a dissociation step of contacting a buffer solution with the carrier to dissociate the antigen molecules in the antigen-antibody complex from the antibody molecules, and a recovery step of recovering the dissociated antigen molecules,
  • the method wherein the pH of the buffer is between pH 4.5 and pH 7.4.
  • a method for assisting in cancer diagnosis comprising: a binding step of adding a body fluid or peritoneal lavage fluid derived from a subject or control body, which contains an antigen-antibody complex containing an autoantigen molecule, to a column on which an antibody-binding protein is immobilized, thereby binding the antibody molecules in the antigen-antibody complex to the antibody-binding protein; an elution step of adding a buffer solution to the column to dissociate the autoantigen molecules in the antigen-antibody complex from the antibody molecules, thereby eluting an eluate containing the autoantigen molecules; a detection step of detecting the autoantigen molecule in the eluate; and a determination step of comparing the autoantigen molecules detected in the subject and the control, and determining that the subject is suffering from cancer when the amount of the cancer antigen detected in the subject is greater than the amount of the cancer antigen detected in the control.
  • a method for assisting in the diagnosis of an autoimmune disease comprising the steps of: a binding step of adding a body fluid or peritoneal lavage fluid derived from a subject or control body, which contains an antigen-antibody complex containing an autoantigen molecule, to a column on which an antibody-binding protein is immobilized, thereby binding the antibody molecules in the antigen-antibody complex to the antibody-binding protein; an elution step of adding a buffer solution to the column to dissociate the autoantigen molecules in the antigen-antibody complex from the antibody molecules, thereby eluting an eluate containing the autoantigen molecules; a detection step of detecting the autoantigen molecule in the eluate; and a determination step of comparing the autoantigen molecules detected from the subject and the control, and determining that the subject is suffering from an autoimmune disease when the amount of the autoantigen associated with the autoimmune disease detected from the subject is greater than the amount of the autoantigen detected from the control.
  • the present invention provides a method for efficiently isolating and detecting antigen molecules from antigen-antibody complexes in a biological sample.
  • FIG. 1 is a diagram showing a schematic diagram of the difference between the binding strength between an antigen molecule and an antibody molecule (shown as “binding strength A” in the diagram) and the binding strength between an antibody molecule and an antibody-binding protein (shown as “binding strength B” in the diagram).
  • 2A shows the experimental procedure and results of Example 2.
  • Fig. 2A shows the experimental procedure of Example 2.
  • Fractions 1 to 3 are collected by elution under weakly acidic conditions (pH 4.5), and then fractions 4 to 5 are collected by elution under more acidic conditions (pH 2.8).
  • Fig. 2B shows the results of electrophoresis of fractions 1 to 5 on an acrylamide gel.
  • FIG. 1 shows the changes over time in the pH of the eluate and the amount of protein in the eluate (absorbance at 280 nm) in the pH gradient liquid chromatography of Example 2.
  • 4 shows the results of mass spectrometry analysis of proteins in each elution fraction in Example 2.
  • Fig. 4A shows the number of times antigen molecules were identified by mass spectrometry.
  • Fig. 4B shows the number of times antibody molecules were identified by mass spectrometry.
  • FIG. 1 shows the amount of each type of antigen molecule detected in each fraction (number of times identified by mass spectrometry) detected in Example 2.
  • FIG. 1 shows the amount of each type of antigen molecule detected in each fraction (number of times identified by mass spectrometry) detected in Example 2.
  • Example 1 shows the amount of each type of antibody molecule detected in each fraction (number of times identified by mass spectrometry) detected in Example 2.
  • This is a diagram showing the results of detecting immunoglobulin (Ig) and antigen molecules in peritoneal lavage fluid.
  • Figure 7A shows the results of measuring the amount of IgG in peritoneal lavage fluid from 16 cases of pancreatic cancer surgery by ELISA. The values of the plasma samples shown on the left side of the graph indicate the theoretical plasma IgG values in healthy subjects.
  • Figure 7B shows the results of staining proteins after electrophoresis for two fractions eluted at pH 4 and pH 2 by performing pH gradient liquid chromatography using peritoneal lavage fluid (one case) and plasma sample (control) as target samples.
  • the figure shows the results of mass spectrometry analysis of immunoglobulin (Ig)-related proteins and proteins other than Ig (antigens) in peritoneal washings from 12 cases of pancreatic cancer surgery, including 8 cases of no recurrence and 4 cases of recurrence.
  • the detection numbers shown in the figure indicate the types of proteins detected.
  • ⁇ Methods for isolating/detecting antigen molecules> there is provided a method for isolating an antigen molecule from a liquid sample containing an antigen-antibody complex (hereinafter often abbreviated as "antigen molecule isolation method”). In a further aspect of the present invention, there is provided a method for detecting an antigen molecule from a liquid sample containing an antigen-antibody complex (hereinafter often abbreviated as "antigen molecule detection method”).
  • antigen-antibody complex refers to a complex formed by the binding of an antigen molecule and an antibody molecule.
  • antigen molecule and antibody molecule that constitute the antigen-antibody complex, and examples include the antigen molecules and antibody molecules described below.
  • antibody molecule refers to a protein that exhibits immune responsiveness to an antigen.
  • antibody is not limited as long as it is capable of forming an antigen-antibody complex in a liquid sample such as a body fluid.
  • an antibody is an immunoglobulin that belongs to the immunoglobulin superfamily.
  • the immunoglobulin may be of any class, one example of which is IgG.
  • IgG may be of any subclass, such as IgG1, IgG2, IgG3, or IgG4.
  • an "antigen molecule” or “antigen” refers to any molecule to which an antibody binds.
  • the antigen molecule is not particularly limited as long as it can be bound by an antibody, and may be any of proteins or peptide fragments, sugar chains, lipids, etc.
  • the antigen may be of any origin and may be either an autoantigen or a non-autoantigen.
  • the autoantigen and examples of such include cancer antigens and antigens related to autoimmune diseases.
  • Non-autoantigens include antigens and allergens derived from pathogens. Examples of antigens derived from pathogens include viral antigens, bacterial antigens, and fungal antigens.
  • allergens include inhalant allergens (e.g., pollen), food allergens (e.g., eggs, milk, wheat and buckwheat, seafood such as shrimp), and contact allergens (e.g., feathers, metals).
  • the antigens that can be isolated/detected by the method of the present invention are antigens that can dissociate from antibodies at a higher pH than the antibody-binding proteins described below, and examples of such antigens include antigens that bind to antibodies with a dissociation constant greater than the dissociation constant between the antibodies and the antibody-binding proteins described below.
  • cancer antigen refers to an antigen that can distinguish cancer cells from normal cells. Cancer antigens are also called tumor antigens or tumor-associated antigens, and typically refer to biomolecules that are specifically and/or overexpressed in cancer cells. Cancer antigens are biomolecules such as proteins, glycans, or lipids, and are not limited to full-length proteins consisting of wild-type amino acid sequences, but also include peptide fragments and neoantigens.
  • cancer antigens include, but are not limited to, A33, BAGE, B cell maturation antigen (BCMA), Bcl-2, ⁇ -catenin, CA19-9, CA125, carboxy-anhydrase-IX (CAIX), CCR4, CD5, CD19, CD20, CD21, CD22, CD24, CD33, CD37, CD45, CD123, CD133, CEA, CEACAM5, c-Met, CS-1, cyclin B1, DAGE, EBNA, EGFR, EpCAM, ephrin B2, estrogen receptor, FAP, folate binding protein, GAGE, and G25.
  • BCMA B cell maturation antigen
  • Bcl-2 B cell maturation antigen
  • ⁇ -catenin CA19-9, CA125
  • CAIX carboxy-anhydrase-IX
  • CCR4 CD5, CD19, CD20, CD21, CD22, CD24, CD33, CD37, CD45, CD123, CD133, CEA, CEACAM
  • GD2 disialoganglioside
  • GM2 GM2, gp75, gp100 (Pmel 17), ERBB2 (HER-2/neu), HPV E6, HPV E7, IGF1R, L1-CAM, LRP, MAGE, MART, mesothelin, MUC (MUC1, MUC2, etc.), MUM-1-B, myc, NYESO-1, p53, PRAME, progesterone receptor, PSA, PSCA, PSMA, ras, RORl, survivin, SV40 T, tenascin, TNF- ⁇ , TSTA tyrosinase, VEGF, WT1, hTERT, and PAP.
  • autoantigen refers to an antigen consisting of any biological molecule that constitutes the self's tissues, cells, etc. Autoantigens may be proteins, sugar chains, lipids, etc. Note that when the term “autoantigen” is used simply in this specification, it broadly refers to any antigen molecule of self-origin that binds to an antibody in the animal's body to form, or has the potential to form, an antigen-antibody complex, and is not limited to autoantigens that cause autoimmune diseases.
  • an "antibody-binding protein” refers to a protein that can be used for antibody affinity purification, and is also called an antibody-adsorbing ligand.
  • Specific examples of antibody-binding proteins include protein G, protein A, and modified or recombinant versions thereof.
  • Protein G and protein A are antibody-binding proteins derived from Streptococcus pyogenes and Staphylococcus aureus, respectively.
  • Examples of the modified or recombinant versions include a fusion protein in which protein A and protein G are fused (hereinafter referred to as "protein A/G").
  • the antibody-binding protein can be selected based on the antibody molecule of the antigen-antibody complex that is the subject of the method of the present invention, and one that can specifically bind to the antibody.
  • the dissociation constant between the antibody and the antibody-binding protein is preferably 10 -7 M or less, and preferably has a high affinity of, for example, 10 -8 M or less, and more preferably 10 -9 M or less.
  • the dissociation constants of protein A, protein G, and protein A/G are 10 -10 M to 10 -11 M. If the dissociation constant is within the above range, the binding to the antibody can be maintained under weakly acidic conditions.
  • the antibody binding protein is preferably Protein G, Protein A, or Protein A/G.
  • the term "column” or “separation column” refers to a container used to separate components in a sample in column chromatography such as affinity chromatography.
  • the column is filled with a solid support such as a resin, matrix, beads, or gel to which an antibody-binding protein is bound.
  • a solid support such as a resin, matrix, beads, or gel to which an antibody-binding protein is bound.
  • the material of the solid support filled in the column and it may be, for example, agarose, sepharose, cellulose, glass, silica, polystyrene, collodion charcoal, polymethacrylate, zirconia, zeolite, vinyl polymer, polymethacrylate, polyacrylamide, polyacrylic acid, or dextran.
  • columns to which an antibody-binding protein is immobilized include protein A columns, protein G columns, and protein A/G columns. It is preferable to equilibrate the column and the carrier described below with an equilibration buffer before adding or contacting the liquid sample.
  • the "carrier" used to isolate the antigen-antibody complex is not limited as long as the antibody-binding protein is fixed to the carrier surface and can be recovered by a recovery method such as precipitation, aggregation, sedimentation, centrifugation, filtration, or magnetic force.
  • beads whose surface is coated with the antibody-binding protein can be used. Beads are particulate insoluble carriers, and their average particle size is, for example, 10 nm to 10 ⁇ m.
  • the material of the beads is not particularly limited, and may be a polymer, a magnetic material, silica, agarose, or sepharose. In particular, magnetic beads made of a magnetic material are preferably used because they are easy to recover or purify.
  • elution refers to the liquid that has passed through the column being discharged from the column.
  • Method of elution it may be, for example, elution based on gravity or elution based on pressure from outside the column.
  • the term "living body” refers to a cell, tissue, organ, or individual. It is not limited to, but includes, for example, individuals such as humans, livestock animals (cows, horses, sheep, goats, pigs, chickens, ostriches, etc.), race horses, pet animals (dogs, cats, rabbits, etc.), laboratory animals (mice, rats, guinea pigs, monkeys, marmosets, etc.), and wild and zoo animals (chimpanzees, wildcats, squirrels, eagles, flamingos, etc.), or cells, tissues, organs, or organs derived therefrom.
  • livestock animals cows, horses, sheep, goats, pigs, chickens, ostriches, etc.
  • race horses pet animals
  • pet animals dogs, cats, rabbits, etc.
  • laboratory animals mice, rats, guinea pigs, monkeys, marmosets, etc.
  • wild and zoo animals chim
  • a "liquid sample” is a sample that can be applied to the method for isolating an antigen molecule or the method for detecting an antigen molecule of the present invention, and can be added to a column or can come into contact with a carrier.
  • the sample contains an antigen-antibody complex
  • examples of the sample include liquids derived from living organisms such as body fluids, and liquid samples prepared from any biological sample.
  • Preferred examples of liquid samples include body fluids, peritoneal lavage fluids, cell culture fluids, cell homogenates, and tissue homogenates.
  • the liquid sample may be derived from a healthy subject or a patient with any disease. For example, it may be derived from a patient with cancer, an autoimmune disease, a collagen disease, an infectious disease, an inflammatory disease, or an aging-related disease.
  • the term "biological sample” refers to any sample derived from a living organism. Examples include tissues, cells, and body fluids. Examples of tissues include tissue homogenates, tissue sections, and biopsy tissues.
  • the tissues and cells include, for example, tissues and cells of a subject that are affected or potentially affected by a disease, as well as corresponding tissues and cells in a healthy subject, and may be, for example, tissues or cells derived from the brain, liver, spleen, heart, pancreas (including pancreatic islets), lung, esophagus, kidney, ovary, stomach, colon, prostate, breast (including mammary gland), muscle, thyroid, and gums.
  • the biological sample may be derived from a healthy subject or a patient with any disease.
  • the biological sample may be derived from a patient with cancer, an autoimmune disease, a collagen disease, an infectious disease, an inflammatory disease, or an aging-related disease.
  • body fluid examples include cerebrospinal fluid, interstitial fluid, blood (including serum, plasma, and interstitial fluid), lymphatic fluid, extracts of each tissue or cell, pleural fluid, sputum, tears, nasal discharge, saliva, urine, ascites, etc.
  • Blood may be serum or plasma prepared from blood.
  • blood, serum, plasma, cerebrospinal fluid, urine, saliva, and ascites are preferred because they are relatively easy to collect.
  • Body fluids may be derived from a healthy subject or a patient with any disease. For example, they may be derived from a patient with cancer, an autoimmune disease, a collagen disease, an infectious disease, an inflammatory disease, or an aging-related disease.
  • peritoneal lavage fluid refers to a liquid that is introduced into the peritoneal cavity during surgery such as tumor resection to wash the peritoneal cavity, and is then collected after washing.
  • peritoneal lavage fluid a liquid that is introduced into the peritoneal cavity during surgery such as tumor resection to wash the peritoneal cavity, and is then collected after washing.
  • saline is usually used.
  • Antigen molecules contained in peritoneal lavage fluid can be biomarkers related to cancer metastasis and prognosis.
  • cancer is not limited, but examples include adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma.
  • Specific types of cancer include, for example, malignant melanoma, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer (including colon cancer and rectal cancer), small intestine cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, stomach cancer, kidney cancer, liver cancer, pancreatic cancer, biliary tract cancer (including gallbladder cancer and bile duct cancer), brain tumor, head and neck cancer, mesothelioma, osteosarcoma, soft tissue sarcoma, glioma, pediatric tumors such as neuroblastoma, blood cancer, lymphoma, and myeloma.
  • blood cancers examples include leukemias (e.g., B-cell leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), lymphomas (e.g., non-Hodgkin's lymphoma), and myelomas (e.g., multiple myeloma).
  • leukemias e.g., B-cell leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia
  • lymphomas e.g., non-Hodgkin's lymphoma
  • myelomas e.g., multiple myeloma
  • autoimmune disease refers to a disease in which the immune system causes an inappropriate response against one's own cells, tissues, and/or organs, resulting in, for example, inflammation or damage.
  • autoimmune diseases include lupus (e.g., systemic lupus erythematosus), celiac disease, acute disseminated encephalomyelitis, acute motor axonal neuropathy, Addison's disease, adiposity (Dercum's disease), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, autoimmune pancreatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, Behcet's disease, Bickerstaff encephalitis, bullous pemphigoid, celiac disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy, cicatricial neuropathy, and inflammatory neuropathy.
  • lupus e
  • aging-related disease refers to a disease that can develop with aging. Specific examples include collagen disease, diabetes, frailty, dementia, Alzheimer's disease, chronic kidney disease, arteriosclerosis, etc.
  • the term "subject” refers to a living organism to which the method for isolating antigen molecules, the method for detecting antigen molecules, and the method for assisting in the diagnosis of disease of the present invention are applied.
  • organisms include humans (particularly referred to as “subjects” in this case), livestock animals (cattle, horses, sheep, goats, pigs, etc.), racehorses, pets (dogs, cats, rabbits, etc.), laboratory animals (mice, rats, guinea pigs, monkeys, marmosets, etc.), wild and zoo animals (chimpanzees, wildcats, squirrels, etc.), etc.
  • organisms such as humans or mammals that have or may develop diseases such as cancer or autoimmune diseases are included.
  • healthy individual refers to an individual not suffering from a specific disease, and preferably an individual not suffering from any disease.
  • healthy tissue is also included in the broad definition of healthy individuals. Therefore, not only at the individual level, but also at the tissue level, such as normal parts of tissue taken from a patient, if the condition is healthy, it will be called a healthy individual.
  • control refers to a healthy individual, that is, an individual not suffering from a specific disease (e.g., cancer or an autoimmune disease), preferably an individual not suffering from any disease.
  • the control may be one individual or multiple individuals.
  • detection as used in this specification can be replaced with inspection, measurement, determination, or determination assistance.
  • separation refers only to increasing the ratio of a target component compared to other components, and is not limited to completely separating a specific component from other components.
  • separation antigen molecules from antibody molecules means increasing the content ratio of antigen molecules compared to antibody molecules in a sample. For example, this refers to preparing a sample in which the ratio of antigen molecules to antibody molecules is increased compared to the sample before separation, or obtaining such a fraction.
  • the antigen molecule isolation method and antigen molecule detection method of the present invention have different steps depending on whether the antigen-antibody complex is bound to a column or to a carrier.
  • the isolation method when the antigen-antibody complex is bound to a column will be referred to as a "method of isolating antigen molecules using a column”
  • the isolation method when the antigen-antibody complex is bound to a carrier will be referred to as a "method of isolating antigen molecules using a carrier”.
  • the detection method when the antigen-antibody complex is bound to a column will be referred to as a "method of detecting antigen molecules using a column”
  • the detection method when the antigen-antibody complex is bound to a carrier will be referred to as a "method of detecting antigen molecules using a carrier”.
  • the method for isolating antigen molecules using the column of the present invention includes a binding step and an elution step as essential steps.
  • the method for detecting antigen molecules using the column of the present invention includes a binding step, an elution step, and a detection step as essential steps. The composition of each step will be described below.
  • the "binding step” is a step in which a liquid sample is added to a column to which an antibody-binding protein is immobilized, and the antibody molecules in the antigen-antibody complex are allowed to bind to the antibody-binding protein.
  • “adding" to the column can also be rephrased as loading or applying.
  • the liquid sample is added to the column, and as a result, the liquid sample can be caused to flow through the column.
  • the column is preferably pre-equilibrated to a pH at which the antigen-antibody complex can bind to the antibody-binding protein.
  • the liquid sample is preferably prepared and equilibrated to a pH at which the antigen-antibody complex can bind to the antibody-binding protein by, for example, mixing with a loading buffer before being added to the column.
  • the pH of the column and the liquid sample added to the column may be the pH at the start of elution of the elution buffer used in the elution step described below, or a higher pH.
  • the pH is 5.4 or higher, 6.4 or higher, 6.8 or higher, 7.4 or higher, or 8.0 or higher, and preferably a neutral pH (for example, pH 6.4 to pH 8.6, pH 6.8 to pH 8.0, or pH 7.4).
  • the components and buffers contained in the liquid sample for pH adjustment are not particularly limited, but examples include phosphate, citric acid, succinic acid, acetic acid, ammonium hydroxide, Tris, glycine, phosphoric acid, Good buffer, boric acid, sodium carbonate, HEPES, etc.
  • the “elution step” is a step of adding a buffer solution to the column to dissociate the antigen molecules in the antigen-antibody complex from the antibody molecules, and eluting an eluate containing the antigen molecules.
  • This step is characterized in that the buffer solution added to the column for elution (hereinafter referred to as “elution buffer solution”) is added to the column while continuously or stepwise decreasing the pH of the buffer solution.
  • “adding a buffer to a column while continuously decreasing the pH of the buffer” specifically means using a pH gradient as the elution buffer.
  • Methods for preparing a pH gradient are well known in the art, and can be prepared, for example, by mixing two or more buffers with different pH values (e.g., pH 4.0 and pH 7.4/pH 2.7 and pH 7.4) while continuously changing the mixing ratio.
  • “adding a buffer to the column while gradually decreasing the pH of the buffer” refers to adding an elution buffer having one or more pH values different from those in the binding step described above to the column. There are no limitations on the pH intervals at which the pH is gradually changed.
  • the pH range in which the pH is continuously or stepwise decreased in the elution step is within the range of pH 7.4 to pH 4.5.
  • the starting pH in the elution step is pH 7.4 or lower, and may be, for example, pH 7.3, pH 7.2, pH 7.1, pH 7.0, pH 6.9, pH 6.8, pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.3, pH 6.2, pH 6.1, pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH 5.2, pH 5.1, pH 5.0, pH 4.9, or pH 4.8 or lower.
  • the end point pH in the elution step is pH 4.5 or higher, and may be, for example, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, or pH 7.0 or higher.
  • Exemplary pH ranges include pH 7.4 to pH 6.5, pH 6.5 to pH 5.5, and/or pH 5.5 to pH 4.5, and the pH can be lowered continuously or stepwise from pH 7.4 to pH 4.5.
  • buffer solutions for elution include elution solutions containing buffer components such as Tris, Good buffer, acetate ions, citrate ions, succinate ions, phosphate ions, formate ions, propionate ions, glycine, ⁇ -aminobutyric acid, and lactic acid.
  • buffer components such as Tris, Good buffer, acetate ions, citrate ions, succinate ions, phosphate ions, formate ions, propionate ions, glycine, ⁇ -aminobutyric acid, and lactic acid.
  • surfactants Teween 20 and Triton-X100
  • urea guanidine
  • amino acids such as arginine and lysine.
  • the elution rate is not limited as long as the elution buffer passes through the column slowly, and may be, for example, 0.1 mL/min to 5 mL/min.
  • the amount of the elution buffer used in the elution step is usually 1 to 10 times, preferably 3 to 5 times, the column volume.
  • the elution step yields an eluate eluted from the column.
  • the eluate contains antigen molecules dissociated from antibody molecules, and since different antigen molecules are contained in the eluate based on the pH at the time of elution, it is preferable to obtain the eluate by dividing it into multiple fractions.
  • a fraction collector may be used for fractionation.
  • the “detection step” is a step of detecting antigen molecules in the eluate obtained in the above-mentioned elution step.
  • the method for detecting the antigen molecule in the detection step may be any known protein detection method, and is not particularly limited, but may include, for example, mass spectrometry or immunological detection.
  • mass spectrometry method There is no limitation to the mass spectrometry method, and a person skilled in the art may appropriately use the DDA method (Data Dependent Analysis), the DIA method (Data Independent Analysis), the PRM method (Parallel Reaction Monitoring), or the SRM method (Selection Reaction Monitoring).
  • immunological detection method refers to a method for detecting an antigen molecule of interest using an antibody or an antibody fragment that specifically binds to the antigen molecule of interest.
  • immunological detection methods include enzyme-linked immunosorbent assays (including ELISA and EIA), Western blotting, radioimmunoassay (RIA), immunoprecipitation, and flow cytometry.
  • ELISA and EIA enzyme-linked immunosorbent assays
  • RIA radioimmunoassay
  • flow cytometry mass spectrometry and immunological detection are both known techniques in the art. Therefore, specific measurement methods may be performed according to known methods. For example, the method described in Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York may be used as a reference.
  • the method for isolating antigen molecules using a column of the present invention and the method for detecting antigen molecules using a column can also include a preparation step and/or a washing step as a selection step in addition to the essential steps described above.
  • the "preparation process” refers to a process for preparing a liquid sample from a sample collected from a living organism.
  • a body fluid sample can be prepared by mixing with a loading buffer to a pH level at which the antigen-antibody complex can bind to the antibody-binding protein.
  • a tissue or cell sample can be prepared into a liquid sample by a crushing or solubilization process. By carrying out this process before the binding process described above, a liquid sample that can be added to a column can be prepared.
  • the "washing step” is a step in which components not bound to the antibody-binding protein, such as antigen-antibody complexes and contaminants other than antibodies, are eluted from the column after the binding step described above and before the elution step by adding a washing buffer to the column.
  • the washing buffer used in the washing step is a solution used to remove contaminants remaining in the column.
  • the washing buffer is preferably one that does not allow the antigen-antibody complex to flow out of the column, and for example, a buffer having the same pH as the loading buffer can be used.
  • the amount of washing buffer used in the washing step is usually 1 to 20 times the column volume, and preferably 3 to 5 times.
  • the number of washes is, for example, one or more times, and can be, for example, 1, 2, 3, or 4 times.
  • the method for isolating an antigen molecule using the carrier of the present invention includes a binding step, a dissociation step, and a recovery step as essential steps. Also, the method for detecting an antigen molecule using the carrier of the present invention includes a binding step, a dissociation step, a recovery step, and a detection step as essential steps. The composition of each step will be described below.
  • the "binding step” is a step of contacting a liquid sample with a carrier to which an antibody-binding protein is immobilized, thereby binding the antibody molecules in the antigen-antibody complex to the antibody-binding protein.
  • the carrier used in the binding step is preferably equilibrated in advance to a pH at which the antigen-antibody complex can bind to the antibody-binding protein.
  • the liquid sample is preferably adjusted and equilibrated to a pH at which the antigen-antibody complex can bind to the antibody-binding protein by mixing with a buffer solution or the like before contacting with the carrier.
  • the pH at which the liquid sample is brought into contact with the carrier in the binding step may be higher than the pH of the dissociation buffer used in the dissociation step described below, for example, pH 5.4 or higher, pH 6.4 or higher, pH 6.8 or higher, pH 7.4 or higher, or pH 8.0 or higher, and is preferably in the neutral range (for example, pH 6.4 to pH 8.6, pH 6.8 to pH 8.0, or pH 7.4).
  • the method for contacting the liquid sample with the carrier in the binding step can be mixed with the carrier and then allowed to stand or shake if necessary.
  • the carrier after the binding step can be recovered by sedimentation, aggregation, precipitation, filtration, centrifugation, magnetic force, or the like depending on the type of carrier.
  • components that are not bound to the antibody-binding protein such as antigen-antibody complexes and components other than antibodies, can be removed from the carrier.
  • the washing buffer used in the washing step is preferably one that does not dissociate the antigen-antibody complex bound to the carrier, and for example, a buffer having the same pH as the buffer used for the contact can be used.
  • the number of washes is, for example, one or more times, and can be, for example, one, two, three, or four times.
  • the "dissociation step” is a step of contacting a buffer solution with the carrier after the binding step to dissociate the antigen molecules in the antigen-antibody complex from the antibody molecules.
  • the pH of the buffer used in the dissociation step (hereinafter referred to as "dissociation buffer”) is pH 4.5 to pH 7.4.
  • Exemplary pHs include pH 5.0 to pH 7.0, pH 5.4 to pH 6.5, and pH 5.8 to pH 6.2. Further examples include pH 6.5 to pH 7.4, pH 5.5 to pH 6.4, and pH 4.5 to pH 5.4.
  • dissociation buffer examples include buffers containing buffer components such as Tris, Good buffer, acetate ions, citrate ions, succinate ions, phosphate ions, formate ions, propionate ions, glycine, ⁇ -aminobutyric acid, lactic acid, etc.
  • buffers containing buffer components such as Tris, Good buffer, acetate ions, citrate ions, succinate ions, phosphate ions, formate ions, propionate ions, glycine, ⁇ -aminobutyric acid, lactic acid, etc.
  • a surfactant Teween 20 or Triton-X100
  • urea urea
  • guanidine or amino acids such as arginine or lysine.
  • the "recovery step” is a step of recovering the dissociated antigen molecules after the dissociation step.
  • the recovery method may be any method capable of separating the antigen molecules dissociated in the dissociation buffer from the carrier, and may involve, for example, removing the carrier by precipitation, aggregation, sedimentation, filtration, centrifugation, or magnetic force depending on the type of carrier, and recovering the dissociation buffer containing the antigen molecules.
  • the antigen molecules recovered in the recovery step may be subjected to further purification, such as by cutting out the range corresponding to the desired size after electrophoresis such as SDS-PAGE using an acrylamide gel.
  • the method for isolating antigen molecules using the carrier of the present invention and the method for detecting antigen molecules using the carrier can also include a preparation step and/or a detection step as a selection step. These steps can be carried out in accordance with the method described above in "(Method for isolating/detecting antigen molecules using a column)," so a description thereof will be omitted here.
  • cancer diagnosis method a method for assisting in the diagnosis of cancer
  • autoimmune disease diagnosis method a method for assisting in the diagnosis of autoimmune disease
  • the cancer differentiation method of the present invention includes a binding step, an elution step, a detection step, and a determination step as essential steps, and includes a preparation step and/or a washing step as a selection step.
  • the autoimmune disease differentiation method of the present invention includes a binding step, an elution step, a detection step, and a determination step as essential steps, and includes a preparation step and/or a washing step as a selection step.
  • the binding step in the cancer differentiation method and autoimmune disease differentiation method of the present invention (hereinafter, the two methods are collectively referred to as the "differentiation method of the present invention") is similar to the binding step described in the above “(Method for isolating/detecting antigen molecules using a column)" except that a body fluid or peritoneal lavage fluid derived from a subject and a control body containing an antigen-antibody complex containing an autoantigen molecule is used as a liquid sample, so a detailed description thereof will be omitted here.
  • the elution step and detection step in the differentiation method of the present invention are similar to the elution step and detection step described in the above "(Method for isolating/detecting antigen molecules using a column)" except that an autoantigen molecule is eluted/detected as the antigen molecule, so a detailed description thereof will be omitted here.
  • the preparation step and washing step which are selection steps in the differentiation method of the present invention, are similar to the preparation step and washing step described in the "(Method for isolating/detecting antigen molecules using a column)", so a detailed description thereof will be omitted here.
  • the determination step is a step of comparing the autoantigen molecules detected from the subject and the control, and determining that the subject is suffering from cancer if the amount of the cancer antigen detected from the subject is greater than the amount of the cancer antigen detected from the control.
  • the determination step is a step of comparing the autoantigen molecules detected from the subject and the control, and determining that the subject is suffering from an autoimmune disease if the amount of autoantigens associated with the autoimmune disease detected from the subject is greater than the amount of autoantigens detected from the control.
  • autoantigens associated with autoimmune diseases refers to antigens that are targeted by the immune system when the immune system elicits an inappropriate response against self-cells, tissues, and/or organs in the above-mentioned autoimmune diseases. Many autoantigens associated with autoimmune diseases are known.
  • autoantigens associated with idiopathic thrombocytopenic purpura e.g., glycoprotein IIb/IIIa, glycoprotein Ib/IX
  • autoantigens associated with Graves' disease e.g., antithyroid stimulating hormone receptor TSHR
  • autoantigens associated with systemic lupus erythematosus SLE
  • nuclear antigens (Sm) such as non-histone nuclear protein antigens, PCNA
  • autoantigens associated with multiple sclerosis e.g., proteolipid protein PLP-1, myelin basic protein MBP, ⁇ -B-crystallin
  • autoantigens associated with type 1 diabetes e.g., insulin, glutamic acid decarboxylase 2, tyrosine phosphatase IA2/IA-2 ⁇ , Gl ima38, P52, GLUT-2
  • autoantigens associated with Guillain-Barre syndrome e.g., peripheral myelin protein I
  • the method of comparing the autoantigen molecules of the subject and the control is not limited.
  • a cutoff value for the amount of autoantigen molecules detected in the subject is determined based on the amount of autoantigen molecules detected in the control, and comparison is made based on the cutoff value.
  • a predetermined value is determined as the cutoff value, and if the measured value is equal to or greater than this value, it can be determined that the amount of autoantigen molecules in the subject is greater than the amount of autoantigen molecules in the control.
  • the cutoff value is a boundary value for comparing the amount of autoantigen molecules in a subject and a control.
  • the cutoff value can usually be calculated based on the disease incidence rate and the sensitivity and specificity calculated using a receiver operating characteristic curve (ROC curve). There are no particular limitations on the method for setting the cutoff value.
  • the amount of autoantigen molecules in a healthy subject not suffering from cancer or an autoimmune disease, or the average amount of autoantigen molecules in a group of healthy subjects can be set as a cutoff value, and when the amount of autoantigen molecules in a subject is higher than the cutoff value, it can be determined that the amount of autoantigen molecules in the subject is greater than the amount of autoantigen molecules in a control subject.
  • a cutoff value may be set to 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, or 6 times or more the amount of autoantigen molecules in healthy subjects not suffering from cancer or autoimmune disease, or the average amount of autoantigen molecules in a group of healthy subjects, and when the amount of autoantigen molecules in the subject is higher than the cutoff value, it may be determined that the amount of autoantigen molecules in the subject is greater than the amount of autoantigen molecules in the control subject.
  • the measured values obtained from the control group can be classified by percentile, and the percentile value used for the classification can be used as the cutoff value. For example, if the 95th percentile of the amount of autoantigen molecules obtained from the control group is used as the cutoff value, and the amount of autoantigen molecules in the subject is equal to or greater than the 95th percentile, it can be determined that the amount of autoantigen molecules in the subject is greater than the amount of autoantigen molecules in the control group.
  • the amount of autoantigen molecules in a control healthy subject differs from the amount of autoantigen molecules in a subject, and does not necessarily need to be measured each time. For example, if the amount of sample used in the measurement, the measurement method, and the measurement conditions are kept constant, the amount of autoantigen molecules from a control healthy subject that was previously measured can be reused.
  • autoantigen molecules are detected from a body fluid collected from a subject, thereby enabling rapid and easy diagnosis of cancer or an autoimmune disease.
  • a treatment plan e.g., type, dosage, administration interval, etc. of anticancer drug or autoimmune disease drug
  • at least one of drug therapy and radiation therapy can be performed.
  • the accuracy of disease diagnosis can also be improved by combining the diagnosis method of the present invention with other methods (e.g., X-ray photography; ultrasound (echo) examination; endoscopy; mammography; palpation; pelvic examination; rectal examination; CT examination; MRI examination; PET-CT examination and other imaging tests; blood tests; pathological tests such as cytology and tissue diagnosis; tumor marker tests; and/or genetic diagnosis).
  • other methods e.g., X-ray photography; ultrasound (echo) examination; endoscopy; mammography; palpation; pelvic examination; rectal examination; CT examination; MRI examination; PET-CT examination and other imaging tests; blood tests; pathological tests such as cytology and tissue diagnosis; tumor marker tests; and/or genetic diagnosis).
  • Example 1 Isolation of antigen molecules using a weakly acidic elution solution (the purpose) Normal body fluid samples derived from living organisms are rich in antibody molecules. Antibody molecules can be separated from other biomolecules using antibody-binding proteins such as protein A. On the other hand, antigen molecules in antigen-antibody complexes, which are formed by binding antigen molecules to antibody molecules, cannot be separated from antibody molecules using the same method.
  • proteins recovered from the gel on which fractions 1-3, 4, and 5 were electrophoresed were analyzed by mass spectrometry, and 347 types of proteins were identified.
  • mass spectrometry In the method described in the previous paper (Capello, M. et al., Nature Communications, 2019, 10:254.), more than half of the identified proteins were contaminating antibodies, but in this example, antigen proteins accounted for more than 70% of the identified proteins.
  • Example 2 High-precision isolation of antigen molecules based on pH gradient liquid chromatography (the purpose) After binding the antigen-antibody complex, in which the antigen molecule and the antibody molecule are bound, to Protein A, pH gradient liquid chromatography is applied to identify the optimal pH conditions for selectively separating only the antigen molecule. Furthermore, it is verified that the antigen molecules can be separated based on the difference in pH conditions under which the antigen molecule can dissociate from the antibody molecule.
  • a total of 20 mL of the elution solution was passed through the column while the ratio of the pH 2.7 buffer to the pH 7.4 buffer was continuously increased from 0% to 73%, and then 8 mL of the 73% mixture was passed through the column.
  • the flow rate was 1.0 mL/min.
  • the pH 2.7 elution solution was passed through the column to elute the remaining proteins.
  • the elution solution was collected as different fractions every 1.0 mL.
  • the protein contained in the eluate was monitored over time by measuring the absorbance at 220 nm (detection wavelength for peptide bonds)/280 nm (detection wavelength for aromatic amino acids).
  • FIG. 3 shows the time-dependent changes in the pH of the eluate and the amount of protein in the eluate (absorbance at 280 nm) in the pH gradient liquid chromatography.
  • the amount of protein in the eluate showed a small peak between pH 7.4 and pH 4.5, and then showed a large peak corresponding to the elution of antibody molecules between pH 7.4 and pH 4.0. This result suggested that antigen molecules were selectively eluted while antibody molecules remained bound to the column and were hardly eluted during the change in the eluate pH from 7.4 to 4.5.
  • Figures 4A and 4B show the number of times antigen molecules and antibody molecules were identified, respectively. Note that this number of identifications corresponds to the total number of proteins detected, regardless of the type of protein, and therefore this result does not represent the type of antigen molecule.
  • Antigen molecules were selectively eluted from fractions 10 to 26 in the pH gradient ( Figure 4A). In contrast, antibody molecules were hardly eluted in fractions before the start of the pH gradient (fractions 5-9) and fractions from the start to the end of the pH gradient (fractions 10-26), but were eluted in large quantities from fraction 27 onwards (Figure 4B).
  • Figures 5 and 6 show the amount of antigen molecules and antibody molecules detected in each fraction for each type of antigen molecule or antibody molecule. These results show that when the eluent changes from pH 7.4 to pH 4.5 (in the range from fraction 10 to fraction 26), antigen molecules are selectively eluted, while the majority of antibody molecules remain bound to the column and are not eluted. This demonstrates that this pH range is extremely advantageous for detecting antigen proteins that are present in very small amounts in blood, such as autoantigens and cancer-specific antigens.
  • Example 3 Isolation of antigen molecules from plasma of colon cancer patients (the purpose) The antigen molecules contained in the antigen-antibody molecule complex are isolated and identified using plasma collected and prepared from colorectal cancer patients.
  • the antigen separation method using an elution solution prepared at a specific weakly acidic pH succeeded in identifying more than twice as many antigen molecules as the conventional method.
  • the antigen separation method using a pH gradient, as described in Examples 2 and 3 of the present application achieved the identification of more than two to six times as many antigen molecules as the conventional method.
  • Example 4 Isolation/detection of antigen molecules from peritoneal lavage fluid (the purpose) The peritoneal washings of pancreatic cancer patients are used to isolate and identify antigen molecules contained in antigen-antibody complexes.
  • IgG immunoglobulin G
  • Figure 7A shows a graph of the theoretical plasma IgG values in healthy subjects as a control. It was revealed that the peritoneal washings of pancreatic cancer patients contained large amounts of IgG, similar to that found in plasma.

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Abstract

La présente invention aborde le problème de la fourniture d'un procédé dans lequel une molécule d'antigène est efficacement isolée d'un complexe antigène-anticorps, et détectée dans celui-ci, dans un échantillon biologique.　L'invention concerne un procédé qui isole une molécule d'antigène d'un échantillon liquide contenant un complexe antigène-anticorps et qui implique une étape de liaison pour ajouter l'échantillon liquide à une colonne à laquelle une protéine de liaison à l'anticorps est fixée, et pour lier une molécule d'anticorps dans le complexe antigène-anticorps à la protéine de liaison à l'anticorps, et une étape d'élution pour éluer un éluat qui comprend la molécule d'antigène par séparation de la molécule d'antigène dans le complexe antigène-anticorps de la molécule d'anticorps, ladite étape d'élution impliquant la diminution continue ou incrémentielle du pH de la solution tampon tout en ajoutant la solution tampon à la colonne.
PCT/JP2023/034650 2022-09-26 2023-09-25 Procédé permettant d'isoler une molécule d'antigène Ceased WO2024071008A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0772156A (ja) * 1993-06-21 1995-03-17 Kureha Chem Ind Co Ltd 癌悪性度の体外診断法
WO2000020442A1 (fr) * 1998-10-05 2000-04-13 Suzuki, Nobutaka Peptides antigenes specifiques aux tumeurs
WO2011078332A1 (fr) * 2009-12-25 2011-06-30 中外製薬株式会社 Procédé de modification de polypeptide pour purifier un multimère de polypeptide
JP2016538275A (ja) * 2013-11-04 2016-12-08 グレンマーク ファーマシューティカルズ， エセ．アー． Ｔ細胞再標的化ヘテロ二量体免疫グロブリン（ｈｅｔｅｒｏ−ｄｉｍｅｒｉｃ ｉｍｍｕｎｏｇｌｏｂｕｌｉｎ）の製造
JP2017070289A (ja) * 2011-03-25 2017-04-13 株式会社カネカ アフィニティー分離マトリックス用タンパク質

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0772156A (ja) * 1993-06-21 1995-03-17 Kureha Chem Ind Co Ltd 癌悪性度の体外診断法
WO2000020442A1 (fr) * 1998-10-05 2000-04-13 Suzuki, Nobutaka Peptides antigenes specifiques aux tumeurs
WO2011078332A1 (fr) * 2009-12-25 2011-06-30 中外製薬株式会社 Procédé de modification de polypeptide pour purifier un multimère de polypeptide
JP2017070289A (ja) * 2011-03-25 2017-04-13 株式会社カネカ アフィニティー分離マトリックス用タンパク質
JP2016538275A (ja) * 2013-11-04 2016-12-08 グレンマーク ファーマシューティカルズ， エセ．アー． Ｔ細胞再標的化ヘテロ二量体免疫グロブリン（ｈｅｔｅｒｏ−ｄｉｍｅｒｉｃ ｉｍｍｕｎｏｇｌｏｂｕｌｉｎ）の製造

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