WO2014073653A1 - 腎炎の病変部位の検査方法およびそのための試薬 - Google Patents
腎炎の病変部位の検査方法およびそのための試薬 Download PDFInfo
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- WO2014073653A1 WO2014073653A1 PCT/JP2013/080296 JP2013080296W WO2014073653A1 WO 2014073653 A1 WO2014073653 A1 WO 2014073653A1 JP 2013080296 W JP2013080296 W JP 2013080296W WO 2014073653 A1 WO2014073653 A1 WO 2014073653A1
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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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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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
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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
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4727—Calcium binding proteins, e.g. calmodulin
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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
Definitions
- the present invention is for determining a lesion site of nephritis characterized by comparing monomer FSP1 concentration and multimeric FSP1 concentration contained in a biological sample derived from a subject with Fibroblast-specific protein 1 (hereinafter FSP1).
- FSP1 Fibroblast-specific protein 1
- the present invention relates to an inspection method and a reagent for performing the inspection.
- FSP1 is a protein identified as a marker specific to fibroblasts (Non-patent Document 1), and encodes a filament-bound S100A4 protein having an anti-EF hand domain (Non-patent Document 2).
- Non-patent Document 1 the active glomerular disease such as meniscal glomerulonephritis
- the present inventors showed increased expression of FSP1 in podocytes and meniscus cells (Non-patent Document 3), and patients with meniscal glomerulonephritis
- urinary FSP1 can be a biomarker of the disease (Non-patent Document 4).
- urinary FSP1 is a positive indicator of disease activity because it shows a positive correlation with the frequency of glomeruli with cellular meniscus formation.
- An object of the present invention is to provide a method for determining a lesion site of nephritis and a reagent for performing the determination.
- FSP1 can be detected in biological samples of patients with interstitial kidney disease, and that the FSP1 is dominant in multimeric form.
- FSP1 found in biological samples of patients with crescent-forming glomerulonephritis was also found to be dominant in monomeric form.
- this invention consists of the following.
- An inspection method for determining a lesion site of nephritis comprising the following steps: (1) a step of measuring any two or more of total FSP1, concentration of monomeric FSP1, and multimeric FSP1 in a biological sample derived from a subject; (2) A step of comparing the monomer FSP1 concentration and the multimeric FSP1 concentration in the biological sample derived from the subject.
- the test method according to [1] or [2], wherein the measurement of the total FSP1 concentration, the monomeric FSP1 concentration, and the multimeric FSP1 concentration is measured by an immunochemical measurement method.
- the reagent according to [5], wherein the lesion site of nephritis to be determined is glomerulus or stromal tissue.
- test method of the present invention and the reagent therefor can quickly and easily determine the lesion site of a nephritic patient or a subject suspected of having nephritis.
- Sequence corresponding to amino acid numbers 7 to 18 of the sequence 4: Sequence corresponding to amino acid numbers 10 to 21 of the amino acid sequence of SEQ ID NO: 2, 5: Amino acid numbers 68 to 79 of the amino acid sequence of SEQ ID NO: 2 6: sequence corresponding to amino acid numbers 71 to 82 of the amino acid sequence of SEQ ID NO: 2, 7: sequence corresponding to amino acid numbers 74 to 85 of the amino acid sequence of SEQ ID NO: 2, 8: sequence A sequence corresponding to amino acid numbers 77 to 88 of the amino acid sequence of No. 2; 9: a sequence corresponding to amino acid numbers 80 to 92 of the amino acid sequence of SEQ ID No.
- F1 represents F1-2 monoclonal antibody.
- I11 represents the I11-23 monoclonal antibody.
- Each lane was electrophoresed with a human fibroblast extract, a mouse fibroblast extract, and a human epithelial cell extract.
- the F1-2 antibody recognizes the N-terminus with low molecular homology between human FSP1 and mouse FSP1, and the I11 antibody recognizes the EF hand domain with high molecular homology between human FSP1 and mouse FSP1.
- the recognition site of I13 antibody is considered to be the same as F1-2 antibody.
- the present invention provides an inspection method for determining a lesion site of nephritis using a biological sample derived from a subject.
- the subject to which the test method of the present invention can be applied is not particularly limited, and examples thereof include a patient who can be clinically determined as a nephritis patient but whose lesion site cannot be identified, or a subject suspected of developing nephritis. It is done.
- the lesion sites of nephritis determined by the examination method of the present invention include glomeruli (ie, glomerular lesions) and stromal tissues (ie, stromal lesions).
- the glomerular lesion determined in the present invention is a glomerular lesion having a characteristic that the monomer FSP1 is contained in the biological sample, and a highly active glomerular lesion is more suitable for the determination.
- the activity of glomerular lesions is high means a state in which the formation of the meniscus in the glomeruli, the proliferation of mesangial cells, or the infiltration of T cells or macrophages into the glomeruli is observed.
- glomerular lesions include meniscus glomerulonephritis, IgA nephropathy, lupus nephritis, purpura nephritis and the like.
- the interstitial lesion determined in the present specification is an interstitial lesion having characteristics including multimeric FSP1 in a biological sample, and a highly active interstitial lesion is more suitable for determination.
- high activity of stromal lesions refers to a state in which infiltration into the stroma of T cells or macrophages is observed.
- interstitial lesions include interstitial nephritis and renal interstitial fibrosis.
- the biological sample used in the test method of the present invention is not particularly limited as long as it is collected from the subject.
- urine such as urine can be easily collected from the living body, or biological tissue can be used as the biological sample.
- a kidney etc. are mentioned.
- urine is used as the biological sample, for example, natural urine, catheter urine and the like can be mentioned.
- urine samples suspected of urinary tract infection and gross hematuria are preferably excluded.
- a urine sample may be used for a test
- the sample may be collected from a subject immediately before the examination is performed, or may be collected in advance and stored frozen.
- the inspection method for determining a lesion site of nephritis includes the following steps.
- the total FSP1 concentration refers to the concentration of FSP1 protein containing monomeric FSP1 and multimeric FSP1 in a biological sample.
- the method for measuring the total FSP1 concentration, monomeric FSP1 concentration and multimeric FSP1 concentration of the present invention is not particularly limited, and the amount of antigen in a biological sample, antibody corresponding to the amount of antigen, and antibody-antigen complex Any measurement method may be used as long as it is a measurement method that detects the amount of the substance by chemical or physical means.
- gel electrophoresis eg, SDS-PAGE, NATIVE-PAGE, two-dimensional gel electrophoresis, etc.
- various separation and purification methods eg, ion exchange chromatography, hydrophobic chromatography, Gel filtration chromatography, affinity chromatography, reverse phase chromatography, isoelectric focusing, capillary electrophoresis, etc.
- ionization methods eg, electron impact ionization, field desorption, secondary ionization, fast atom collisions
- MALDI matrix-assisted laser desorption ionization
- electrospray ionization method etc.
- mass spectrometer eg, double-focusing mass spectrometer, quadrupole analyzer, time-of-flight mass spectrometer, Fourier transform mass
- an analyzer or an ion cyclotron mass spectrometer it is also preferably performed by an immunochemical measurement method.
- FSP1 detected in a sample takes a monomeric form or a multimeric form (may include a dimer or higher multimeric form).
- SEQ ID NO: 2 As a monomeric form of FSP1, SEQ ID NO: 2 And a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 is about 70% or more, preferably about 80% or more, more preferably about 90%, with the amino acid sequence represented by SEQ ID NO: 2.
- the amino acid sequences having homology of about 95% or more, most preferably about 98% or more are particularly preferable.
- the FSP1 of the present invention is preferably a protein having the amino acid sequence represented by SEQ ID NO: 2, that is, human FSP1.
- an FSP1-specific antibody is used when an immunochemical assay is used.
- FSP1-specific antibodies used in the present invention include antibodies that recognize both monomeric FSP1 and multimeric FSP1 from biological samples, antibodies that recognize only multimeric FSP1, and antibodies that recognize only monomeric FSP1. Can be mentioned.
- antibodies that recognize both monomeric FSP1 and multimeric FSP1 are two types of combinations that recognize different epitopes, as described in the immunochemical assay method described below. Can be used. Therefore, it is preferable that the epitope recognized by each antibody is at a position distant from FSP1.
- an antibody that recognizes the EF hand calcium binding domain of FSP1 as an epitope preferably an antibody that recognizes the amino acid sequence represented by SEQ ID NO: 4 as an epitope, and the N-terminal amino acid sequence of FSP1 as an epitope
- An antibody that recognizes preferably an antibody that recognizes the amino acid sequence represented by SEQ ID NO: 3 as an epitope.
- the FSP1-specific antibody of the present invention can be produced by a known method using an appropriate immunogen.
- the immunogen include FSP1 full-length protein or a partial peptide thereof.
- the full-length protein of FSP1 used as an immunogen is a protein as described above.
- the FSP1 partial peptide used as an immunogen is a peptide comprising a partial amino acid sequence of the same or substantially the same amino acid sequence as SEQ ID NO: 2.
- the FSP1 partial peptide used as an immunogen may be a peptide containing the N-terminal amino acid sequence of FSP1 or the EF hand calcium binding domain of FSP1.
- An example of the N-terminal amino acid sequence of FSP1 is the same or substantially the same amino acid sequence as the sequence corresponding to amino acid numbers 1 to 12 (SEQ ID NO: 3) in the amino acid sequence represented by SEQ ID NO: 2.
- the same or substantially the same as the sequence corresponding to amino acid numbers 80 to 92 (SEQ ID NO: 4) in the amino acid sequence represented by SEQ ID NO: 2 Is a peptide sequence comprising the amino acid sequence of
- FSP1 used as an immunogen can be produced from mammalian cells or tissues by a known protein purification method. Specifically, by homogenizing mammalian tissues or cells, and separating and purifying soluble fraction and / or nuclear fraction by chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography, etc. FSP1 can be manufactured.
- FSP1 or a partial peptide thereof used as an immunogen can also be produced according to a known peptide synthesis method.
- the peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method.
- the target protein can be produced by removing the protecting group.
- the FSP1-specific antibody used in the present invention includes those produced in animal blood, those produced in hybridomas, and produced in hosts transformed with expression vectors containing antibody genes by genetic engineering techniques. And those produced in cells containing antibody genes to be screened by the phage display method, human antibodies obtained directly from transgenic mice producing human antibodies, and the like. FSP1-specific antibodies can be produced by methods known to those skilled in the art.
- FSP1 is administered to a mammal at a site where antibody production is possible by administration, or together with a carrier and a diluent.
- the FSP1 used as an immunogen should be a complex that is cross-linked to a carrier protein such as partial peptide (if necessary, bovine serum albumin, KLH (Keyhole Limpet Hemocyanin) even if it is the full-length protein of FSP1 described above. May also be possible.
- Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Examples of mammals to be used include monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep and goats, and mice and rats are preferably used.
- Monoclonal antibody-producing hybridomas can be prepared by fusing the cells with myeloma cells.
- hybridoma culture supernatant is added to a solid phase (eg, microplate) on which FSP1 is adsorbed directly or with a carrier, and then radioactive substances and enzymes are added.
- a solid phase eg, microplate
- Anti-immunoglobulin antibodies labeled with the above (anti-mouse immunoglobulin antibodies are used when cells used for cell fusion are mice) or protein A, and a method for detecting monoclonal antibodies bound to a solid phase, anti-immunoglobulin Examples include a method in which a hybridoma culture supernatant is added to a solid phase on which an antibody or protein A is adsorbed, FSP1 labeled with a radioactive substance or an enzyme is added, and a monoclonal antibody bound to the solid phase is detected.
- Separation and purification of monoclonal antibodies can be carried out in the same way as in the separation and purification of ordinary polyclonal antibodies (eg, salting out, alcohol precipitation, isoelectric precipitation, electrophoresis, ion exchanger (eg, DEAE) adsorption / desorption method, ultracentrifugation, gel filtration, antigen-binding solid phase or specific purification method to obtain antibody by dissociating the binding using active adsorbent such as protein A or protein G]
- active adsorbent such as protein A or protein G
- the total FSP1 concentration, monomeric FSP1 concentration and multimeric FSP1 concentration in a biological sample derived from a subject can be measured by an immunochemical measurement method, respectively. . Specifically, for example, it can be carried out as follows.
- Sandwich ELISA method, immunochromatography method, agglutination method The test solution containing a biological sample is reacted with the immobilized FSP1-specific antibody (primary reaction), and the same or different from the immobilized FSP1-specific antibody. After quantifying the labeled FSP1-specific antibody (secondary reaction), the amount of total FSP1, multimeric FSP1 or monomeric FSP1 in the test solution is measured by measuring the activity of the label. Can do.
- the immobilized FSP1-specific antibody may be an antibody that recognizes both monomeric FSP1 and multimeric FSP1, an antibody that recognizes only multimeric FSP1, or an antibody that recognizes only monomeric FSP1.
- a microplate well, a nitrocellulose membrane, latex beads, magnetic beads, or the like can be used as the solid phase carrier.
- the solid phase method may be physically adsorbed, or may be oriented by chemical bonding or the like. Further, the solid phase may be preliminarily performed before the primary reaction, or may be performed after the formation of the sandwich complex.
- the labeled FSP1-specific antibody is used by binding a label capable of detecting the antigen-antibody reaction product.
- the labeled antibody may be the same or different from the immobilized FSP1-specific antibody.
- a radioisotope, a gold colloid, an enzyme, a fluorescent substance, a luminescent substance, an enzyme substrate, or the like can be used.
- the FSP1-specific antibody used for the secondary reaction is also immobilized on latex beads or magnetic beads.
- the sandwich complex may be aggregated and the turbidity of the aggregate itself may be used for detection.
- a sandwich complex composed of multimeric FSP1 and / or monomeric FSP1 and FSP1-specific antibodies in a biological sample is selected by a step of removing non-specifically reacted substances and unreacted antibodies.
- the removal method may be a washing step using a buffer containing a surfactant, a separation step using magnets or electrochemical properties, or a separation step based on a difference in mobility such as molecular sieve or chromatography. Any method may be used as long as only the body remains specifically.
- the immune complex can be measured by using any of the above detection means.
- the amount of immune complex is determined by, for example, absorbance, fluorescence intensity, luminescence intensity, etc., but may be separately quantified by comparison with the amount of immune complex indicated by FSP1 of known concentration, or simply the intensity You may compare qualitatively only.
- the FSP1 detected by the above assay is an antibody in which the immobilized FSP1-specific antibody and the labeled FSP1-specific antibody recognize both different monomeric FSP1 and multimeric FSP1.
- one antibody is an antibody that recognizes the EF hand calcium-binding domain of FSP1 as an epitope and the other antibody is an antibody that recognizes the N-terminal amino acid sequence of FSP1 as an epitope
- multimers in biological samples Both FSP1 and monomeric FSP1 can be detected.
- only the multimeric FSP1 can be obtained by using the same antibody as the immobilized FSP1-specific antibody and the labeled FSP1-specific antibody (for example, an antibody that recognizes the EF hand calcium binding domain of FSP1 as an epitope). Detection and monomer FSP1 can be calculated. Alternatively, multimeric FSP1 or monomeric FSP1 can be directly detected by using an antibody that recognizes only multimeric FSP1 or an antibody that recognizes only monomeric FSP1 as a solid-phased FSP1-specific antibody.
- the measurement method based on the above sandwich ELISA method is a competition in which only one or both FSP1-specific antibodies recognize only one type of multimeric FSP1 or monomeric FSP1.
- a measurement method based on the measurement principle may be used.
- FSP1 or its equivalent antigenic substance (partial peptide of FSP1) is labeled with a radioisotope, colloidal gold, enzyme, fluorescent substance, luminescent substance or enzyme substrate, etc. as an antigen, microplate well, nitrocellulose
- a measurement system for FSP1 may be constructed by adding ordinary technical considerations to those skilled in the art to the normal conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like. For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Continue Radioimmunoassay” (published in Kodansha, 1974), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), edited by Eiji Ishikawa et al.
- urinary FSP1 is dominant in monomeric form in patients with crescent-forming glomerulonephritis. Moreover, urinary FSP1 shows a positive correlation with the frequency of appearance of glomeruli in which cellular meniscus formation is observed. On the other hand, in patients with interstitial nephritis or renal interstitial fibrosis, urinary FSP1 is dominant in the multimeric form. Therefore, the testing method for determining the lesion site of nephritis is such a positive between monomeric FSP1 concentration and glomerular lesion prevalence, or multimeric FSP1 concentration and interstitial lesion prevalence. Based on correlation.
- a correlation diagram between the monomeric FSP1 concentration and multimeric FSP1 concentration and the presence or absence of glomerular and interstitial lesions should be prepared in advance, and the concentrations of monomeric FSP1 and multimeric FSP1 in subjects You may compare with a correlation diagram.
- the concentration comparison is preferably performed based on the presence or absence of a significant difference.
- the lesion site of nephritis is glomerular
- the multimeric FSP1 concentration is higher than the monomeric FSP1 concentration (more than 51% of the sum of the monomeric FSP1 concentration and the multimeric FSP1 concentration)
- the lesion site of nephritis approximates the concentration in a biological sample of a patient with glomerular or interstitial tissue, it is judged that the lesion site of nephritis is likely to be glomerular or interstitial tissue Can do.
- the present invention extends to a test reagent for determining a lesion site of nephritis.
- the reagent of the present invention is not particularly limited as long as it is a reagent for simply carrying out the above-described inspection method of the present invention.
- the reagent for testing contains an FSP1-specific antibody.
- the FSP1 antibody include an antibody that recognizes both monomer FSP1 and multimeric FSP1, an antibody that recognizes only multimeric FSP1, and an antibody that recognizes only monomer FSP1. It is preferable to have.
- Examples of the FSP1-specific antibody in the present invention include the same antibodies as those described in the above test method.
- the FSP1-specific antibody of the present invention can be provided in a state dissolved in water or a suitable buffer.
- the FSP1-specific antibody of the present invention can be provided in a state immobilized on an appropriate solid phase.
- the solid phase include, but are not limited to, a microplate well, a nitrocellulose membrane, latex beads, and magnetic beads.
- the FSP1-specific antibody of the present invention can be provided in a state of being previously labeled with the above-mentioned labeling substance, or can be provided separately from the labeling substance, and can be used after labeling.
- the reagent of the present invention is another substance necessary for the reaction for measuring the monomeric FSP1 concentration or the multimeric FSP1 concentration in addition to the antibody, and the reaction is adversely affected by storing in the coexisting state. It may further contain non-substances. Alternatively, the reagent may be provided with a separate reagent containing other substances necessary in the reaction to detect FSP1.
- the other substance for detecting FSP1 includes a reaction buffer, a blocking solution, an enzyme substrate, a nonspecific reaction inhibitor, a preservative, or a surfactant.
- FSP1 expression vector was prepared by inserting the full-length human FSP1 gene into a pET-49b (+) vector (Novagen) having a GST tag sequence and a His tag sequence.
- BL21DE3 competent cells were transformed with the vector, and the protein was inducibly expressed using isopropyl- ⁇ -D-thiogalactopyranoside (Takara Bio Inc.).
- the expressed fusion protein was purified by column chromatography using HisTrap HP column (GE Healthcare), then GST tag and His tag were cleaved using human rhinovirus 3C protease (Novagen), and purified recombinant human FSP1 (rFSP1 ).
- rFSP1 50 ⁇ g / 250 ⁇ l was emulsified in an equal volume of CFA (Difco Laboratories) and used as the antigen.
- CFA Cosmetic Food and Drugs
- antigens were administered intraperitoneally to BALB / c mice (female, 7 weeks old) (CLEA Japan Inc.), and similar immunizations were carried out 2 weeks, 4 weeks and 6 weeks later.
- the antigen was boosted in the tail vein without adjuvant.
- the spleen was removed from the mouse and dissociated through a 100-mesh steel net.
- Dissociated splenocytes (2 ⁇ 10 8 ) were fused with the same number of myeloma cells in the presence of 50% polyethylene glycol (Roche Applied Science).
- the fused cells were suspended in a selective growth medium containing 5% Briclone (Archport), seeded in a 96-well culture plate (2 ⁇ 10 5 cells / well), and cultured while periodically changing the medium.
- the supernatant of the well containing hybridoma colonies was screened for the presence of specific antibodies using direct ELISA, and hybridoma cells derived from positive wells were cloned twice by limited dilution.
- Each clone was cultured in medium and the antibody-rich supernatant was concentrated by ammonium sulfate precipitation, dialyzed against PBS and stored at -80 ° C. Supernatants were collected from 484 wells having hybridoma cells, and antibody detection was attempted. Using direct ELISA and native PAGE, five antibodies were found to bind to rFSP1. Among these five clones, two antibodies (F1-2 and I11-23) having high titer and binding to rFSP1 were selected. Isotype analysis (AbD Serotec) revealed that F1-2 belongs to the IgG2a ( ⁇ ) subclass and I11-23 belongs to the IgG1 ( ⁇ ) subclass.
- F1-2 is N-terminal of FSP1 (amino acid number 1 of the amino acid sequence represented by SEQ ID NO: 2).
- -11 recognizes the sequence corresponding to the twelfth position; SEQ ID NO: 3
- I11-23 corresponds to the EF hand calcium binding domain (sequence corresponding to amino acid numbers 80 to 92 in the amino acid sequence represented by SEQ ID NO: 2). It was found to recognize SEQ ID NO: 4) (FIG. 1).
- F1-2 recognized the N-terminus of FSP1
- I11-23 recognized the EF hand calcium binding domain (FIG. 2).
- Example 2 Measurement of urinary FSP1 using a sandwich ELISA method on a sample derived from a patient with renal disease 2 menstrual glomerulonephritis patients, 1 patient with interstitial nephritis, 1 patient with renal interstitial fibrosis Urine samples were collected from the urine. Urine samples were collected three times independently from one of the two meniscal glomerulonephritis patients and the interstitial nephritis patient. Urine samples were also collected from membranous nephropathy and nephrosclerosis, where glomerular lesions are less active. The amount of FSP1 in the urine sample was measured using a sandwich ELISA method.
- the I11-23 antibody purified from the ascites antibody was immobilized on each well of a 96-well microplate at a solid phase concentration (1.25 ⁇ g / mL), and 100 ⁇ L of a sample obtained by diluting the collected urine specimen 4 times was brought into contact with the well. It was. After a primary reaction at room temperature for 1 hour, the wells were washed three times. Next, F1-2 antibody and I11-23 antibody purified from ascites antibody were labeled with HRP, and 100 ⁇ L of labeled F1-2 antibody diluted 2000 times and 100 ⁇ L of labeled I11-23 antibody diluted 4000 times were contacted with the wells, respectively. I let you.
- test method of the present invention and the reagent therefor can quickly and easily determine the lesion site of a nephritic patient or a subject suspected of having nephritis.
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Abstract
Description
しかし、生体試料中FSP1と腎疾患の関連性が実証されているのは半月体形成性糸球体腎炎に留まり、腎間質性疾患と生体試料中FSP1との関わりは未だ報告がない。また、腎炎患者における病変部位を簡便に判定する方法も臨床現場において依然として希求されている。
本発明者らは、これらの知見に基づいてさらに研究を重ねた結果、本発明を完成するに至った。
[1]以下の工程を含むことを特徴とする、腎炎の病変部位を判定するための検査方法:
(1)被験者由来の生体試料における総FSP1濃度、単量体FSP1濃度および多量体FSP1濃度のうちいずれか2つ以上を測定する工程、
(2)被験者由来の生体試料における単量体FSP1濃度および多量体FSP1濃度を比較する工程。
[2]生体試料が尿試料であることを特徴とする、[1]に記載の検査方法。
[3]総FSP1濃度、単量体FSP1濃度および多量体FSP1濃度の測定が、免疫化学的測定法によって測定されることを特徴とする、[1]または[2]に記載の検査方法。
[4]判定される腎炎の病変部位が、糸球体または間質組織である[1]~[3]のいずれか1つに記載の検査方法。
[5]FSP1特異的抗体を、少なくとも1種類含む、腎炎の病変部位を判定するための試薬。
[6]判定される腎炎の病変部位が、糸球体または間質組織である[5]に記載の試薬。
従って、本発明の腎炎の病変部位を判定するための検査方法は、以下の工程を含むことを特徴とする。
(1)被験者由来の生体試料における総FSP1濃度、単量体FSP1濃度および多量体FSP1濃度のうちいずれか2つ以上を測定する工程、
(2)被験者由来の生体試料における単量体FSP1濃度および多量体FSP1濃度を比較する工程。
本発明において総FSP1濃度とは、生体試料における単量体FSP1および多量体FSP1を含むFSP1タンパク質の濃度をいう。
ペプチド合成法は、例えば、固相合成法、液相合成法のいずれであってもよい。免疫原として用いられるFSP1を構成し得る部分ペプチドもしくはアミノ酸と残余部分とを縮合し、生成物が保護基を有する場合は保護基を脱離することにより目的とするタンパク質を製造することができる。
FSP1特異的抗体は当業者に公知の方法によって作製することができる。
ハイブリドーマの作製に際しては、抗原を免疫された哺乳動物、例えば、マウスから抗体価の認められた個体を選択し最終免疫の2~5日後に脾臓又はリンパ節を採取し、それらに含まれる抗体産生細胞を骨髄腫細胞と融合させることにより、モノクローナル抗体産生ハイブリドーマを調製することができる。
固相化したFSP1特異的抗体に生体試料を含む被験液を反応させ(1次反応)、さらに固相化したFSP1特異的抗体と同一または別異の標識化したFSP1特異的抗体を反応させ(2次反応)た後、標識体の活性を測定することにより被験液中の総FSP1量、多量体FSP1量または単量体FSP1量を定量することができる。
上記サンドイッチELISA法等を測定原理とする測定法は、FSP1特異的抗体の一方または両方が1種類の多量体FSP1または単量体FSP1のみを認識する抗体のみを用いることによる競合法を測定原理とする測定法であってもよい。この場合、抗原としてFSP1またはそれと同等の抗原性を有する物質(FSP1の部分ペプチド)を、ラジオアイソトープ、金コロイド、酵素、蛍光物質、発光物質あるいは酵素基質等により標識し、マイクロプレートウェル、ニトロセルロース膜、ラテックスビーズ、磁気ビーズなどに固相化された多量体FSP1のみを認識する抗体または単量体FSP1のみを認識する抗体に対して、生物試料中のFSP1と競合させることにより、生物試料中のFSP1を測定することができる。
上記サンドイッチELISA法または競合法を用いた測定法において、多量体FSP1のみを認識する抗体または単量体FSP1のみを認識する抗体等を用い、予め生体試料中の多量体FSP1もしくは単量体FSP1のいずれか一方を該抗体等で吸収、除去する工程を経た後に、FSP1の測定を行なうことにより、除去されなかったFSP1のみを測定することもできる。
GSTタグ配列およびHisタグ配列を有するpET-49b(+)ベクター(Novagen)に全長ヒトFSP1遺伝子を挿入することによってFSP1発現ベクターを調製した。BL21DE3コンピテント細胞を該ベクターで形質転換し、イソプロピル-β-D-チオガラクトピラノシド(Takara Bio Inc.)を用いてタンパク質を誘導発現させた。発現した融合タンパク質をHisTrap HP カラム(GE Healthcare)を用いて、カラムクロマトグラフィーによって精製後、ヒトライノウイルス3Cプロテアーゼ(Novagen)を用いてGSTタグおよびHisタグを切断し、精製組換えヒトFSP1(rFSP1)を取得した。rFSP1(50μg/250μl)を等量のCFA(Difco Laboratories)中で乳化し、抗原として用いた。
モノクローナル抗体を生じさせるため、BALB/cマウス(雌、7週齢)(CLEA Japan Inc.)に抗原を腹腔内投与し、同様の免疫を2週間、4週間、6週間後に行った。4回目の免疫から1週間後、アジュバント無しで抗原を尾静脈中に追加接種した。追加接種から3日後、マウスから脾臓を摘出し、100-メッシュスチール網を通して、解離させた。解離した脾細胞(2x108)を、50%ポリエチレングリコール(Roche Applied Science)の存在下、同数のミエローマ細胞と融合させた。融合細胞を5%Briclone(Archport)を含む選択増殖培地中に懸濁し、96ウェル培養プレート(2x105細胞/ウェル)に播いて、定期的に培地を交換しながら培養した。ハイブリドーマコロニーを含むウェルの上清に対してダイレクトELISAを用いて特定の抗体が存在するかスクリーニングに供し、陽性ウェル由来のハイブリドーマ細胞を限定希釈によって2回クローニングした。各クローンを培地中で培養し、抗体豊富な上清を硫酸アンモニウム沈殿によって濃縮し、PBSで透析し、-80℃で保存した。
ハイブリドーマ細胞を持つ484ウェルから上清を回収し、抗体の検出を試みた。ダイレクトELISAとnative PAGEを用いて、5つの抗体が、rFSP1に結合することを見出した。これら5つのクローンのうち、高力価を有する、rFSP1に結合する2種類の抗体(F1-2およびI11-23)を選抜した。アイソタイプ解析(AbD Serotec)では、F1-2はIgG2a(κ)サブクラスに属し、I11-23 はIgG1(κ)サブクラスに属することが判った。PepSpots(JPT Peptide Technologies)を用いたエピトープマッピングでは、これら2つのモノクローナル抗体は異なるエピトープを認識し、F1-2はFSP1のN末端(配列番号:2で表されるアミノ酸配列のうち、アミノ酸番号1~12番目に該当する配列;配列番号:3)を認識し、I11-23 はEF handカルシウム結合ドメイン(配列番号:2で表されるアミノ酸配列のうち、アミノ酸番号80~92番目に該当する配列;配列番号:4)を認識することがわかった(図1)。また、ヒトFSP1とマウスFSP1を用いたエピトープ結合実験でも、F1-2はFSP1のN末端を認識し、I11-23 はEF handカルシウム結合ドメインを認識した(図2)。
半月体形成性糸球体腎炎患者2名、間質性腎炎患者1名、腎間質線維化患者1名から尿検体を採取した。半月体形成性糸球体腎炎患者2名のうちの1名および間質性腎炎患者からは独立に3回ずつ尿検体を採取した。また、糸球体病変の活動性が低い膜性腎症と腎硬化症からも尿検体を採取した。尿検体中のFSP1の量をサンドイッチELISA法を用いて測定した。腹水抗体から精製したI11-23抗体を固相化濃度(1.25μg/mL)で96穴マイクロプレートの各ウェルに固相化し、採取した尿検体を4倍希釈した試料100μLを該ウェルに接触させた。室温で1時間、1次反応させた後、ウェルを3回洗浄した。次に腹水抗体から精製したF1-2抗体およびI11-23抗体をそれぞれHRP標識し、2000倍希釈した標識化F1-2抗体100μLおよび4000倍希釈した標識化I11-23抗体100μLをウェルにそれぞれ接触させた。室温で1時間、2次反応させた後、ウェルを3回洗浄した。基質液100μLをウェルに接触させ、37℃で30分間反応させた。反応停止液100μLをウェルに接触させ、反応を停止させた後、反応液の吸光度を測定した。予め上記の方法によって各濃度FSP1(0,1,2,4,8,16,32,64ng/mL) 100μLを標準タンパク質溶液として吸光度とFSP1濃度の標準曲線を作成し、該標準曲線を基に得られた各検体の吸光度から各FSP1濃度(ng/mL)を推定し、併せてその差を算出した(表1)。また、活動性の高い糸球体腎炎である半月体形成性糸球体腎炎(A1-3、D)および活動性の高い間質性腎炎である間質性腎炎、腎間質線維化(B1-3、C)については、多量体FSP1濃度と単量体FSP1濃度の割合をグラフとして表示した(図3)。
同一のエピトープを認識する固相化I11-23抗体と標識化I11-23抗体によってFSP1が検出されるという結果は、間質性病変である間質性腎炎および腎間質線維化患者の尿中では多量体FSP1が存在し、また単量体FSP1よりも存在比が高いことから、それがドミナントであることを示唆している。一方、異なるエピトープを認識する固相化I11-23抗体と標識化F1-2抗体によってFSP1が検出されるという結果は、糸球体病変である半月体形成性糸球体腎炎患者の尿中では単量体FSP1が存在し、また多量体FSP1よりも存在比が高いことから、それがドミナントであることを示唆している。従って、尿検体中のFSP1が多量体としてドミナントであるか単量体としてドミナントであるかが、腎炎の病変部位と相関することを確認できた。
Claims (6)
- 以下の工程を含むことを特徴とする、腎炎の病変部位を判定するための検査方法:
(1)被験者由来の生体試料における総FSP1濃度、単量体FSP1濃度および多量体FSP1濃度のうちいずれか2つ以上を測定する工程、
(2)被験者由来の生体試料における単量体FSP1濃度および多量体FSP1濃度を比較する工程。 - 生体試料が尿試料であることを特徴とする、請求項1に記載の検査方法。
- 総FSP1濃度、単量体FSP1濃度および多量体FSP1濃度の測定が、免疫化学的測定法によって測定されることを特徴とする、請求項1または2に記載の検査方法。
- 判定される腎炎の病変部位が、糸球体または間質組織である請求項1~3のいずれか1項に記載の検査方法。
- FSP1特異的抗体を、少なくとも1種類含む、腎炎の病変部位を判定するための試薬。
- 判定される腎炎の病変部位が、糸球体または間質組織である請求項5に記載の試薬。
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Non-Patent Citations (18)
Title |
---|
"METHODS IN ENZYMOLOGY", vol. 121, ACADEMIC PRESS, article "Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)" |
"Methods in ENZYMOLOGY", vol. 70, article "Immunochemical Techniques" |
"METHODS IN ENZYMOLOGY", vol. 73, article "Immunochemical Techniques" |
"METHODS IN ENZYMOLOGY", vol. 74 |
"METHODS IN ENZYMOLOGY", vol. 84, article "Immunochemical Techniques (Part D: Selected Immunoassays)" |
"METHODS IN ENZYMOLOGY", vol. 92, article "Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)" |
EIJI ISHIKAWA ET AL.: "Enzyme Immunoasssay", 1978, IGAKU-SHOIN |
EIJI ISHIKAWA ET AL.: "Enzyme Immunoasssay(2nd edition)", 1982, IGAKU-SHOIN |
EIJI ISHIKAWA ET AL.: "Enzyme Immunoasssay(3rd edition)", 1987, IGAKU-SHOIN |
HIROSHI IRIE: "Radioimmunoassay", 1974, KODANSHA |
HIROSHI IRIE: "Supplementary volume of Radioimmunoassay", 1979, KODANSHA |
IWANO ET AL., JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY, vol. 23, 2012, pages 209 - 214 |
IWANO M ET AL.: "Urinary FSP1 is a biomarker of crescentic GN.", J AM SOC NEPHROL., vol. 23, no. 2, February 2012 (2012-02-01), pages 209 - 214, XP055249529 * |
OKADA ET AL., THE AMERICAN PHYSIOLOGICAL SOCIETY, vol. 273, 1997, pages F563 - 574 |
STRUTS ET AL., THE JOURNAL OF CELL BIOLOGY, vol. 130, 1995, pages 393 - 405 |
YAMAGUCHI ET AL., AMERICAN JOURNAL OF KIDNEY DISEASES, vol. 54, 2009, pages 653 - 664 |
YOSHIHARU NISHITANI ET AL.: "Hito Jin Seiken Soshiki deno FSP1 no Kyokuzai to Kanshitsu Byohen tono Kanren", THE JAPANESE JOURNAL OF NEPHROLOGY, vol. 43, no. 3, 25 April 2001 (2001-04-25), pages 246, XP008178994 * |
YUKINARI YAMAGUCHI ET AL.: "Atarashii Biomarker to shite no Nyochu FSP1 no Rinshoteki Igi", THE JAPANESE JOURNAL OF NEPHROLOGY, vol. 52, no. 3, 25 May 2010 (2010-05-25), pages 305, XP008178993 * |
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