WO1996001277A1 - PROCEDE ET KIT D'IMMUNODOSAGE D'ANTIGENES Fas SOLUBLES - Google Patents

PROCEDE ET KIT D'IMMUNODOSAGE D'ANTIGENES Fas SOLUBLES Download PDF

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WO1996001277A1
WO1996001277A1 PCT/JP1995/000349 JP9500349W WO9601277A1 WO 1996001277 A1 WO1996001277 A1 WO 1996001277A1 JP 9500349 W JP9500349 W JP 9500349W WO 9601277 A1 WO9601277 A1 WO 9601277A1
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PCT/JP1995/000349
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Japanese (ja)
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Shin Yonehara
Jun Noguchi
Takahisa Hachiya
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Medical & Biological Laboratories Co., Ltd.
Japan Tabacco, Inc.
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Application filed by Medical & Biological Laboratories Co., Ltd., Japan Tabacco, Inc. filed Critical Medical & Biological Laboratories Co., Ltd.
Publication of WO1996001277A1 publication Critical patent/WO1996001277A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons

Definitions

  • the present invention relates to a kit for immunological measurement of a soluble Fas antigen, a method for immunological measurement of a soluble Fas antigen, the kit for the measurement, a chimeric soluble Fas antigen that can be used in the measurement method, and a DNA encoding the same.
  • the present invention relates to an expression vector containing the DNA, a transformed cell transformed with the expression vector, and a method for producing a chimeric soluble Fas antigen by culturing the cell.
  • the present invention relates to a monoclonal antibody reactive with the chimeric soluble Fs antigen, and a fused cell producing the monoclonal antibody.
  • apoptotic bodies In the case of necrosis, the permeability of cell membranes increases at an early stage, organelles such as nuclear mitochondria swell, and eventually lysosomes are destroyed, and cells are destroyed by released proteases etc. I do.
  • organelles such as nuclear mitochondria swell, and eventually lysosomes are destroyed, and cells are destroyed by released proteases etc. I do.
  • apoptosis no major change in the structure of mitochondrial perisome is observed, and chromosomes condense in the nucleus and cytoplasm shrink in the early stages.
  • the nucleus may be fragmented into several parts, or a bubble-like structure may form on the cell surface, after which the cell may be divided into minicells called apoptotic bodies.
  • Passive death which is caused by external causes, so to speak, is mainly caused by necrosis, whereas preprogrammed cell death seen during development, differentiation or tissue turnover is thought to be caused by apoptosis.
  • the protein present on the surface of living cells which is thought to be closely related to the control of cell death called apoptosis or programmed cell death, is called the Fas antigen and penetrates the lipid bilayer of the cell membrane once or several times. It is a transmembrane protein composed of three main regions: an extracellular region (soluble region), a transmembrane region and a cytoplasmic region.
  • the Fas antigen has a structure very similar to tumor necrosis factor (TNF) receptor and nerve growth factor (NGF), and a new type that causes cell death by receiving an external signal.
  • TNF tumor necrosis factor
  • NGF nerve growth factor
  • Fas antigen is related, and attention has been paid to the relationship between the expression of the Fas antigen and diseases such as autoimmune diseases.
  • Fa s antigen j a Fa s antigen that is soluble in blood, serum, plasma, and body fluids such as urine, saliva, and bone marrow.
  • measuring the amount of soluble Fa s antigen in body fluids such as serum is important for predicting, diagnosing and treating various diseases that may be related to the dynamics of Fa s antigen such as autoimmune diseases. It is useful for establishing and determining the effects of treatment, and there is a keen need to establish the measurement method and measurement reagents (kits).
  • Methods for detecting and measuring soluble Fas antigens in body fluids such as serum include the Western blot method (WB method), radioimmunoassay (Radioimmunoassay ⁇ RIA), and enzyme immunoassay.
  • the method Enzyme Immunoassay: EIA method or Enzyme-linked Immunosolbent Assay: ELISA method
  • the W.B. method has high sensitivity and high specificity, but lacks quantitativeness, and is not suitable for processing a large number of samples at once.
  • the RIA method uses radioactive substances as labeling substances, it has the disadvantage that it can only be used by qualified persons in controlled facilities.
  • the EIA method or the ELISA method does not have the above-mentioned problems and can be measured easily and with high sensitivity, but until now, soluble Fas antigens in body fluids such as serum using such a method have been used. A simple measurement method with high accuracy and sufficient sensitivity is not known.
  • a calibration method is needed to confirm the sensitivity or accuracy of the measurement method and to indirectly determine the soluble Fas antigen content from the measured values.
  • a purified soluble Fas antigen As a standard substance for measuring the amount of soluble Fs antigen, it is desirable to use a purified protein product containing at least the Fs antigen soluble region (extracellular region) soluble in serum or the like.
  • a DNA sequence encoding a soluble region (extracellular region) of a desired transmembrane protein is converted to an immunoglobulin heavy chain (A method of expressing a recombinant chimeric soluble protein using a DNA sequence linked to a DNA sequence encoding a constant region (Hc chain) of the H chain is known (Nature. Vol. 337, 525). -531. 1989. Capon et al., J. Exp. Med., Linsley et al., International Patent Application Publication. W093 / 00431, International Patent Application Publication W091 / 10438, etc.).
  • the expression efficiency of the target protein is extremely low, and the Fc region of the expressed recombinant quinula-soluble protein has an ability to bind to immunoglobulin Fc receptor pig.
  • major drawbacks such as the inability to accurately test and measure the function and biological activity of the target protein region.
  • mouse hematopoietic cells express a transmembrane protein called AIC2A in their cell membrane.
  • AIC2A is an iS subunit that constitutes a receptor for mouse IL-13 (hereinafter, also referred to as "mouse IL-13 receptor yS subunit AIC2AJ”), and forms a heterodimer with various subunits.
  • mouse IL-13 receptor yS subunit AIC2AJ As a high-affinity receptor for mouse IL-13 [The EMBO Journal. Vol. 5, No. 5, pp. 1875-1884. 1992, Haruya Kahiko et al., Blood. No. 79. No. 4 Vol. 80, No. 1, pp.
  • mouse IL-13 receptor / 3 subunit AIC2A is a transmembrane protein. It is noteworthy that, despite being a protein, its extracellular domain protein is efficiently expressed in cells of higher animals using the cDNA sequence encoding the region.
  • An object of the present invention is to provide an immunoassay kit for measuring a soluble Fas antigen in a body fluid such as serum which could not be measured conventionally, and an immunoassay method for a soluble Fas antigen. That is.
  • Another object of the present invention is to provide a chimeric soluble Fas antigen that can be used in the kit and method for immunoassay of the soluble Fas antigen, a DNA encoding the same, and an expression containing the DNA.
  • An object of the present invention is to provide a vector, a transformed cell transformed with the vector, and a method for producing a chimeric soluble Fas antigen by culturing the cell.
  • an object of the present invention is to provide a monoclonal antibody having reactivity with the chimeric soluble Fs antigen, and a fused cell producing the monoclonal antibody.
  • the present inventors have proposed a highly accurate, highly sensitive and simple immunoassay method for a soluble Fas antigen in a body fluid such as serum using an ELISA method (or an EIA method). After extensive research on immunoassay kits, it was found that a plurality of anti-Fas monoclonal antibodies produced by the present inventors differed from soluble Fas antigen and intact Fas antigen in body fluids such as serum, respectively. It was found that it has reactivity.
  • Fas antigen used for producing a monoclonal antibody against the Fas antigen. It is possible to easily and efficiently express the extracellular region of the Fas antigen, a transmembrane protein, at the mouth of invit using a gene recombination technique as a soluble activator while maintaining its intact three-dimensional structure.
  • the chimera as a chimeric protein between the soluble region of the Fas antigen and the extracellular region of mouse IL-13 receptor / 5 subunit AIC2A prepared by the present inventors.
  • Soluble in body fluids such as serum can be obtained by using a sandwich ELISA using a soluble Fas antigen as a standard substance and one or more monoclonal antibodies arbitrarily selected from the plurality of anti-Fas monoclonal antibodies.
  • the present inventors have found that the Fas antigen can be easily measured with high accuracy and high sensitivity, and have completed the present invention.
  • a first aspect of the present invention relates to a solid-phased anti-Fas monoclonal antibody obtained by binding an anti-Fas monoclonal antibody capable of specifically binding to a soluble Fas antigen to an insoluble support, and a standard substance.
  • This is a measurement kit for immunologically measuring a soluble Fas antigen, which comprises a soluble Fas antigen.
  • a measurement for immunologically measuring a soluble Fas antigen which further comprises a labeled second anti-Fas monoclonal antibody capable of specifically binding to the soluble Fas antigen. Kit.
  • the soluble Fas antigen as a standard substance is composed of the extracellular region of human Fas antigen or mouse Fas antigen and the extracellular region of mouse IL-13 receptor S subunit AIC2A or immunoglobulin weight.
  • a kit for immunologically measuring a soluble Fas antigen which is characterized as being a chimeric soluble Fas antigen comprising a constant region of the chain, 2) an anti-Fas monoclonal antibody bound to an insoluble support Is a monoclonal antibody produced from a hybridoma selected from the group consisting of hybridoma clones VB3, CBE, WB3, ZB4, UB2, AX6, JAE and CHI1.
  • Second anti-Fas monoclonal antibody consists of hybridoma clones VB3, CBE, WB 3. ZB4, UB2, AX6, JAE and CHI1 From the group Soluble F the as antigen immunologically measuring kit for measuring to Toku ⁇ that from the barrel High Priestess dormer is a monoclonal antibody which is produced or, 4) labeled second anti F the as monochromator Immunizing a soluble Fas antigen characterized in that the ronal antibody is a monoclonal antibody labeled with a labeling substance selected from the group consisting of peroxidase, 8-D-galactosidase, microperoxidase, alkaline phosphatase, and biotin. This is a measurement kit for performing a biological measurement.
  • a second aspect of the present invention is a method for immunologically measuring a soluble Fs antigen, which comprises the following steps a) to c) and d) to f).
  • a step of quantifying a soluble F ss antigen contained in the sample from the calibration curve is a step of quantifying a soluble F ss antigen contained in the sample from the calibration curve.
  • the soluble Fas antigen as the standard substance is composed of the extracellular region of human Fas antigen or mouse Fas antigen and the extracellular region of mouse IL-13 receptor 3 subunit AIC2A or the immunoglobulin heavy chain.
  • a chimeric soluble Fas antigen comprising a constant region, and a method for immunologically measuring a soluble Fas antigen
  • 2) an anti-Fas monoclonal antibody bound to an insoluble support comprising a hybridoma clone VB3, CBE, WB3, ZB4, UB2, AX6, JAE and CH11 Immunoassay method for soluble Fas antigen characterized by being a monoclonal antibody produced from a hybridoma selected from the group consisting of CH11, 3) Second Anti-F As a monoclonal antibody, wherein the monoclonal antibody is a monoclonal antibody produced from a hybridoma selected from the group consisting of hybridoma clones VB3, CBE, WB3, Z
  • a method for immunoassay of Fas antigen or 4) the second labeled anti-Fas monoclonal antibody is selected from the group consisting of peroxidase, yS-D-galactosidase, microperoxysidase, lipophosphatase, and biotin.
  • This is an immunoassay method for a soluble Fas antigen, which is a monoclonal antibody labeled with a selected labeling substance.
  • the third aspect of the present invention is a chimeric soluble Fas antigen comprising the extracellular region of human or mouse Fas antigen and the extracellular region of mouse IL13 receptor A-subunit AIC2A.
  • Specific examples include a chimeric soluble Fas antigen having the amino acid sequence shown in SEQ ID NO: 11 or 19 in the sequence listing.
  • a fourth aspect of the present invention is a DNA encoding the chimeric soluble Fas antigen.
  • Specific examples include a DNA having a DNA sequence shown in SEQ ID NO: 10 or 18 in the sequence listing.
  • a fifth aspect of the present invention is an expression vector containing a DNA encoding the chimeric soluble Fas antigen.
  • Specific examples include an expression vector containing the DNA sequence shown in SEQ ID NO: 10 or 18 in the sequence listing.
  • a sixth aspect of the present invention is a transformed cell transformed with the expression vector. Specifically, a cell line identified by International Accession No. FERM BP-4436 or 4437 can be mentioned.
  • a seventh aspect of the present invention is a method for producing a chimeric soluble Fas antigen by culturing the transformed cell. More specifically, there is provided a method for producing a chimeric soluble Fs antigen, which comprises culturing the transformed cells in a medium and obtaining proteins produced in the culture supernatant.
  • An eighth aspect of the present invention is a fused cell obtained by cell fusion between an antibody-producing cell obtained from a mammal immunized with the chimeric soluble Fas antigen and a mammalian myeloma cell line. Specifically, International Deposit No. FERM BP—4394, 4667 Or the cell line identified in 4756.
  • a ninth aspect of the present invention is a monoclonal antibody reactive with the chimeric soluble Fs antigen, specifically, a monoclonal antibody produced by the fused cells.
  • FIG. 1 is a diagram showing a calibration curve in a Sandwich ELISA method using a chimeric soluble human Fas antigen as a standard substance (standard solution).
  • the vertical axis represents the absorbance, and the horizontal axis represents the concentration (ng / nil) of the standard chimeric soluble human Fas antigen.
  • FIG. 2 is a diagram showing a calibration curve in the Sandwich ELISA method using a chimeric soluble human Fas antigen as a standard substance (standard solution).
  • the vertical axis represents the absorbance, and the horizontal axis represents the concentration (ngZml) of the standard chimeric soluble human Fas antigen.
  • FIG. 3 is a restriction map of plasmid pCEV4.
  • FIG. 4 is a restriction map of plasmid pME18S.
  • FIG. 5 is a construction process diagram of plasmid pME18S / hFas ⁇ EXT.
  • Figure 6 is a construction process diagram of the plasmid pMEl8ShFas ⁇ EXT—AIC2A ⁇ EXT.
  • Figure 7 shows SDS-polyacrylamide gel electrophoresis for confirming the expression of hFas ⁇ EXT-AlC2A ⁇ EXT (chimeric soluble human Fas antigen) in the culture supernatant.
  • FIG. 7 shows SDS-polyacrylamide gel electrophoresis for confirming the expression of hFas ⁇ EXT-AlC2A ⁇ EXT (chimeric soluble human Fas antigen) in the culture supernatant.
  • Figure 8 shows cell death of Fa s antigen-expressing cells induced by anti-human Fa s antibody CHI 1 by apoptosis by soluble h Fa s -AIC 2A protein (Qin) and soluble AI C2A protein ( ⁇ ). It is a figure which shows the suppression effect.
  • the vertical axis represents the viable cell rate (%), and the horizontal axis represents the dilution rate.
  • FIG. 9 is a construction process diagram of plasmid pME18S / mFas ⁇ EXT.
  • FIG. 10 is a process diagram of the construction of plasmid pME18SmFas ⁇ EXT—AIC2A ⁇ EXT.
  • FIG. 11 shows SDS-polyacrylamide gel electrophoresis analysis to confirm the expression of mFas'EXT-AIC2A ⁇ EXT (chimeric soluble mouse Fas antigen) in the culture supernatant.
  • FIG. FIG. 12 shows rat anti-mouse Fas monoclonal antibodies RMF2, RF6, RMF9 and RMF13 (each indicated by a dotted line) and control monoclonal antibodies, anti-rat IgG or anti-rat IgM ( Each is shown by a solid line.)
  • Is a diagram showing the reactivity to L5178Y that does not express Fas antigen and L5178Y that overexpresses mouse Fas antigen.
  • the vertical axis represents the number of cells, and the horizontal axis represents the amount of the monoclonal antibody bound to the cell surface expressed by the fluorescence intensity.
  • FIG. 13 shows hamster anti-mouse Fas monoclonal antibodies RK-8, SK-8, C6-1 and P4-4 (each indicated by a dotted line) and a control monoclonal antibody, anti-hamster IgG (each a solid line).
  • Fig. 4 shows the reactivity of L5178Y not expressing Fas antigen and L5178Y overexpressing mouse Fas antigen.
  • the vertical axis represents the number of cells, and the horizontal axis represents the amount of the monoclonal antibody bound to the cell surface represented by the fluorescence intensity.
  • Fas antigen J in the present invention means, unless otherwise specified, a mammalian Fas antigen such as human, mouse, rat, guinea pig, hamster, rabbit, dog or monkey, and preferably human Mouse or rat Fas antigen, particularly preferably human Fas antigen.
  • the immobilized anti-Fas monoclonal antibody J bound to the insoluble support J It is used to be supported on the support by adsorption or chemical bonding.
  • water such as plastic represented by polystyrene resin, polycarbonate resin, silicone resin or nylon resin, glass, etc. It is made of an insoluble substance and has an internal volume for adding a sample such as serum, etc. Any known material can be used as long as such a material is used.
  • a plastic plate having a large number of wells such as a 96-well microplate
  • a rate is used.
  • immobilized anti-Fas monoclonal antibody j means an anti-Fas monoclonal antibody that is supported on the “insoluble support” by physical adsorption or chemical bonding. By reacting a sample such as serum with the “immobilized anti-Fas monoclonal antibody”, the soluble Fas antigen contained in the sample is trapped by the present antibody.
  • the “labeling substance” used for labeling j is peroxidase, 1D-galactosidase, micropel Enzymes such as oxidase or alkaline phosphatase;] I, 3 I, radioisotopes such as C or tritium, and antibodies or antigens such as biotin. Examples of commonly used labeling substances are exemplified. “When using piotin as the labeling substance j, a method of labeling with piotin and reacting with avidin peroxidase is used. According to this method, measurement can be performed with higher sensitivity and sensitivity.
  • an enzyme such as peroxidase, ⁇ -D-galactosidase, microperoxidase, or calcium phosphatase, or biotin.
  • the “monoclonal antibody” means an anti-Fas monoclonal antibody labeled with a labeling substance as described above.
  • the soluble anti-Fas antigen trapped by the immobilized anti-Fas monoclonal antibody described above is allowed to react with the second anti-Fas monoclonal antibody J, which has been labeled, and specifically bound to the solid anti-Fas monoclonal antibody.
  • the second anti-Fas monoclonal antibody J may be the same monoclonal antibody as the above-mentioned ⁇ the immobilized anti-Fas monoclonal antibody or may be a different monoclonal antibody, but is preferably used.
  • Is a monoclonal that can specifically bind to an antigenic determinant at a position different from the antigenic determinant of the soluble Fas antigen to which the immobilized anti-Fas monoclonal antibody binds.
  • the second anti-Fas monoclonal antibody labeled with the immobilized anti-Fas monoclonal antibody usually recognizes different antigenic determinants of the soluble Fas antigen. If two identical antigenic determinants are present, they may recognize the same antigenic determinant. In this case, one of the determinants binds to the immobilized anti-Fas monoclonal antibody and the other one binds to the labeled second anti-Fas monoclonal antibody.
  • the anti-Fas monoclonal antibody used as the “immobilized anti-Fas monoclonal antibody” or the “second anti-Fas monoclonal antibody” in the measurement kit and the measurement method of the present invention includes at least a bodily fluid such as serum. Any monoclonal antibody that can specifically bind to the soluble Fas antigen can be used. Specifically, for example, International Immunology. Vol. 6, No. 12, pp. 1849-1856, 1994, Journal of Experimental Medicine, Vol. 169, 1747-1756, 1989 and Cell, Vol. 66, 233.
  • IgM anti-Fas monoclonal antibody produced from CHI1 (IgM) and the like.
  • the measurement kit of the present invention contains at least a solid-phased anti-Fas monoclonal antibody bound to an insoluble support and a soluble Fas antigen as a standard substance, and preferably a labeled second anti-Fs It is characterized in that it contains a monoclonal antibody.
  • the ELISA method E
  • the ISA method) or the ELISA method using the sandwich method (E1A method) can be used to measure soluble Fas antigen in body fluids such as serum. It is possible. However, when measurement is performed by the ELISA method using a competitive method, it is necessary to obtain a large amount of purified Fas antigen that retains the intact structure of soluble Fas antigen present in blood. Obtaining large quantities of antigen is laborious. Therefore, it is preferable to use the sandwich ELISA method in the measurement of the soluble Fs antigen by the ELISA method.
  • the sandwich ELISA method of the present invention can be performed according to the purpose and conditions of measurement.
  • the soluble Fas antigen is measured by combining at least two appropriately selected anti-Fas monoclonal antibodies and setting up a sandwich ELISA method. Specifically, it is characterized by using a solid-phased anti-Fas monoclonal antibody and a labeled second anti-Fas monoclonal antibody.
  • the immobilized anti-Fas monoclonal antibody and the labeled second anti-Fas monoclonal antibody may be the same anti-Fas monoclonal antibody or different anti-Fas monoclonal antibodies.
  • a combination of two anti-Fas monoclonal antibodies that recognize and bind to different antigenic determinants of the soluble Fas antigen. If the same anti-Fas monoclonal antibody is used, two identical antigenic determinants are present at different positions on the soluble Fas antigen, and each monoclonal antibody binds to each of the two antigenic determinants. It means to do.
  • IgG One or two hybridomas selected from CBE (IgG), JAE (IgM), AX6 (IgM), CHI1 (IgM), etc. are produced respectively.
  • One or two anti-Fas monoclonal antibodies can be immobilized or labeled before use.
  • An example of a preferred combination includes, as a monoclonal antibody for preparing an immobilized anti-Fas monoclonal antibody, an anti-Fas monoclonal antibody obtained from clone CBE, and a labeled second anti-Fas monoclonal antibody.
  • Anti-Fas monoclonal antibodies obtained from clone VB3 can be used.
  • the “chimeric soluble Fas antigen” of the present invention can be used as a standard substance for immunoassays and kits for immunoassay for soluble Fas antigens, or as an immunoassay used for the production of monoclonal antibodies against Fa sitogen. is useful as a sensitizing antigen c the ⁇ chimeric soluble F the as antigens J is Kimeratanpa the soluble region and murine IL- 3 Resebu evening one Sabuyunitto AIC 2 a of F the as antigen using Yadenko engineering technique Or the soluble region of the Fas antigen and the constantity of the immunoglobulin heavy chain (H chain) It can be produced and used as a chimeric protein with a region (constant region: Fc region). Preferably, it is a chimeric protein of the soluble region of the Fas antigen and mouse IL-13 receptor / 3-subunit AIC2A, which is another subject of the present invention.
  • This chimeric protein of the soluble region of the Fas antigen and the mouse IL-13 receptor 3 subunit A1C2A contains an amino acid sequence substantially constituting the extracellular region of the Fas antigen downstream of the expression promoter.
  • An amino acid sequence that substantially constitutes the extracellular region of the encoding DNA and mouse 1L-1 / 3 receptor / 3 subunit AIC 2A An expression vector constructed by introducing a chimeric DNA consisting of the encoding DNA It can be obtained by transforming the host cell, culturing the resulting transformed cells in a medium, and producing the protein produced in the culture supernatant by simple hybridization. Specifically, it can be manufactured as follows.
  • Total RNA is prepared from cells capable of producing a Fs antigen, and a cDNA library is prepared using these as a type II.
  • the cells used here can be arbitrarily selected. If the cells produce human Fas antigen, for example, human T tumor cell line KT3, if mouse Fas antigen, mouse macrophage BAM 3 can be used.
  • a clone containing the cDNA encoding the Fs antigen is selected from the cDNA library, and a full-length cDNA encoding the Fs antigen is obtained from the clone. From the full-length cDNA, a cDNA fragment encoding an amino acid sequence substantially constituting the extracellular region of the Fs antigen is excised and introduced into an appropriate plasmid.
  • a cDNA fragment encoding an amino acid sequence substantially constituting the extracellular region of the mouse interleukin-3 receptor subunit AIC2A or the constant region Fc of the immunoglobulin H chain is prepared in the same manner as described above.
  • a cDNA fragment encoding a substantially constitutive amino acid sequence is obtained.
  • a mouse interleukin-13 receptor 3 subunits AIC2A, with or without an inserted DNA sequence, downstream of a cDNA fragment encoding an amino acid sequence substantially constituting the extracellular region of the Fas antigen Substantially the extracellular area of A Fas antigen opened with a restriction enzyme to introduce a cDNA fragment encoding the constituent amino acid sequence or a cDNA fragment encoding the amino acid sequence substantially constituting the constant region Fc of the immunoglobulin H chain Said plasmid into which a cDNA fragment encoding an amino acid sequence substantially constituting the extracellular region of said mouse has been introduced, and said extracellular region of said mouse interleukin-13 receptor 1; 3 subunit AIC2A A cDNA fragment encoding an amino acid sequence that constitutes an amino acid sequence or a cDNA fragment that encodes an amino acid sequence that substantially constitutes the constant region Fc of the immunoglobulin H chain.
  • each DNA fragment is subjected to DNA fragmentation using a DNA ligation kit in a conventional manner.
  • the ligated to obtain Burasumi Dobe Kuta one chimeric protein with F the as antigens and AIC 2 A is unloaded capable braking produced.
  • a suitable host cell is transformed with the plasmid vector, the transformed cell is cultured in a medium, and a chimeric soluble Fs antigen as a chimeric protein is collected from the culture medium.
  • the expression vector used in the production of the chimeric soluble Fas antigen may be selected from plasmids commonly used in test research or the industrial field, depending on the host cell to be cultivated, and according to the purpose. Can be selected and used. For example, pME18S, pCEV4, pEF-BOS, and PHSAPR-1 can be used.
  • a DNA sequence encoding a novel signal peptide, and a promoter which is a DNA sequence essential for the initiation of transcription of this DNA into messenger RNA (mRNA) by RNA polymerase, are co- DNA sequence encoding a terminator, which is a DNA sequence essential for the transcription termination control, and SD sequence (Shine-Dalgarno sequence) encoding a base sequence on mRNA that is a ribosome binding site.
  • the DNA sequence has been introduced, and depending on the purpose, for example, a DNA sequence encoding a DNA replication origin essential for replication of this vector in a host cell, mRNA of DNA, etc. DNA sequence that encodes an enhancer, which is a DNA sequence for improving the transcription efficiency to DNA, and a polyadenylic acid signal sequence (poly A signal), which is a DNA sequence that improves the stability of mRNA. Such DNA sequences can be introduced.
  • the expression promoter can be selected from promoters commonly used in test research or the industrial field, and can be used according to the host cell to be transformed and depending on the purpose.
  • SV40 promoter LTR promoter, SRa promoter, EF-1 ⁇ promoter, 3 actin promoter, imnoglobulin promoter and the like can be used.
  • the host cell depending on the expression vector and the purpose, depending on the purpose, there are test studies, natural cells or artificially established recombinant cells, which are commonly used in the industrial field. It can be used by selecting from animal cells that are not limited to. For example, mouse-derived cells (COP, L, C127, Sp2 / 0, L5178Y and NS-1 etc.), rat-derived cells, hamster-derived cells (BHK and CH0, etc.), monkey-derived cells (COS and COS 3. COS7, CV1 and Ve1 o, etc.) and human-derived cells (Hela, cells derived from diploid fibroblasts, myeloma cells and Nama wa), etc., and preferably L51. 78Y, COS 1 cells, COS 3 cells, COS 7 cells, cells derived from human diploid fibroblasts and myeloma cells
  • the selection of the transformed cells can be performed using a conventional cell selection technique by imparting one or more properties such as drug resistance, temperature sensitivity, auxotrophy or radiation mutability to the transformed cells.
  • the selection marker DNA used in the present invention is usually used in test research or in the industrial field.
  • Such selectable marker DNA can be selected and used.
  • ampicillin (Amp) resistant DNA tetracycline (Tc) resistant DNA, thymidine kinase (Tk) resistant DNA, 6-thioguanine (HGP RT) resistant DNA, neomycin (Neo) resistant DNA and hygromycin Drug-resistant DNA such as resistant DNA can be used.
  • the obtained culture is fractionated by filtration, centrifugation, or the like. It can be purified according to the method. That is, for example, methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, methods using molecular weight such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and ion exchange chromatography.
  • solubility such as salting out and solvent precipitation
  • dialysis dialysis
  • ultrafiltration methods using molecular weight such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and ion exchange chromatography.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • Hydroxyl abatite mouth chromatography A method using first-class charge, a method using specific affinity such as affinity chromatography, a method using difference in hydrophobicity such as reversed-phase high-performance liquid chromatography, etc.
  • a method utilizing isoelectric point such as point electrophoresis may be used.
  • the “substantially constituting amino acid sequence” means the extracellular region of the Fas antigen or the mouse IL-13 receptor; the extracellular region of the 3-subunit AIC2A in vivo. Means an amino acid sequence containing at least the minimum amino acid sequence necessary to maintain the function or biological activity, and deletion, modification or addition of N-terminal or C-terminal amino acids is allowed according to the purpose.
  • the monoclonal antibody of the present invention specifically, an anti-Fas monoclonal antibody used for a solid-phased anti-Fas monoclonal antibody or a labeled anti-Fas monoclonal antibody, and a monoclonal antibody against the chimeric soluble Fas antigen.
  • the null antibody can be produced by a known general method for producing a monoclonal antibody.
  • a hybridoma fused cell
  • a fused cell is prepared from antibody ⁇ living cells and myeloma cells (myeloma cells), and the hybridoma is cloned.
  • Chimeric soluble Fas antigen as an immunizing antigen can be immunized with mice, rats, guinea pigs, hamsters, or egrets. Mammals, preferably mice, rats or hamsters. Immunization is carried out by subcutaneously, intramuscularly, intravenously, footpad II or intraperitoneally injecting or transplanting the immunogen into these mammals one to several times.
  • animals are immunized 1 to 4 times about every 1 to 2 weeks after the first immunization, and finally immunized about 1 to 4 weeks later, and immunized about 3 to 5 days after the final immunization
  • Antibody-producing cells are obtained from
  • the preparation of a hybridoma that secretes a monoclonal antibody can be performed according to the method of Koehler and Mirushi Utain (Nature, 256, 495-497. 1975) and a modification method analogous thereto.
  • the monoclonal antibody in the present invention is preferably a mouse of the same species as an antibody-producing cell contained in the spleen, lymph node, bone marrow, tonsil, etc., preferably spleen, obtained from the animal immunized as described above, Culturing a fusion cell (hypridoma) obtained by fusion with a mammal such as a rat, guinea pig, hamster, rabbit, human or the like, more preferably mouse, rat or human myeloma cell line (myeloma cell) Prepared by Cultivation can be performed in vitro or in vivo in a mouse, rat, guinea pig, hamster, or egret, etc., preferably in a mouse or rat, more preferably in ascites of a mouse, and the like. Can be obtained from ascites.
  • myeloma cells used for cell fusion include myeloma derived from mouse P3 / X63-AG8, P3 / NSI / l-Ag4-l (abbreviated as NS-1), P3 / X63-Ag8.
  • UU SP2 / 0-Agl4, FO or BW5147 rat-derived myeloma 210RCY3_Ag.2.3.
  • Screening of fused cell clones that produce monoclonal antibodies is performed by culturing the fused cells, for example, in a microtiter plate, and determining the reactivity of the culture supernatant, which shows growth, with the antigen, for example, RIA (radiation). Immunoassay) or enzyme immunoassay such as ELISA.
  • Monoclonal antibody purification and isolation are performed by purifying blood or ascites containing the monoclonal antibody, which is obtained by the above-described method, by ion exchange chromatography (0 £ 8 £ or 0 £ 52, etc.) It can be performed by subjecting to affinity column chromatography such as a immunoglobulin column or a protein A column.
  • the monoclonal antibody in the present invention specifically, an anti-Fas monoclonal antibody used for a solid-phased anti-Fas monoclonal antibody or a labeled anti-Fas monoclonal antibody, and a monoclonal antibody against the chimeric soluble Fas antigen are: Includes monoclonal antibodies belonging to any of the immunoglobulin classes of IgG, Ig. IgA, IgD and IgE.
  • the transformed cells or antibody-producing hybrids of the present invention are IgG or IgM.
  • the transformed cells or fused cells are grown, maintained, and preserved in accordance with various conditions such as the characteristics of the cell type to be cultured, the purpose of the test and research, and the culturing method.
  • Any nutrient medium derived from a known nutrient medium or a known basal medium, such as those used to produce antibodies or antibodies It is possible to implement a medium Yore, Te.
  • a basic medium for example, Ham 'F12 medium, low calcium medium such as MCDB 153 medium or low calcium MEM medium and MCDB 104 medium, MEM medium, D-MEM medium, RPMI 1640 medium, ASF104 medium, RD medium Medium, such as a high calcium medium or a Fisher's medium.
  • the basic medium may be, for example, serum, hormone, cytokin, and / or An inorganic or organic substance can be contained.
  • the immunoassay kit for a soluble Fas antigen and the immunoassay method for a soluble Fas antigen according to the present invention include an immunoassay kit for a soluble Fas antigen in a body fluid such as serum which has not yet been established. It provides the first immunological measurement method. Using the measurement kit and the measurement method, serum and other body fluids of patients suffering from autoimmune diseases such as rheumatoid (Rheumatoid Arthritis; RA) or SLE, which have been difficult to measure until now. It is possible to measure the amount of soluble Fas antigen easily, with high accuracy and high sensitivity.
  • the dynamics of the Fas antigen such as the autoimmune disease is involved, foreseeing, diagnosing, establishing a treatment policy, and judging the effect of the treatment.
  • the present measurement kit and measurement method are extremely useful clinically. Furthermore, the measurement kit and the measurement method of the present invention lack quantification and are unsuitable for processing a large amount of samples at a time.
  • the stamp mouth method or a specific qualified person for using radioactive materials Is simpler, more accurate and more sensitive than radioimmunoassay (RIA), which can only be performed in a specific controlled facility. In addition, a large number of samples can be measured at one time. Is possible.
  • the method for producing a chimeric soluble Fas antigen of the present invention is a method for producing a soluble region of a transmembrane protein, such as a not-yet-established Fas antigen, as a soluble active substance in vitro in a simple and large amount. Is the first to offer. By using this production method, it is possible to produce the soluble region of the Fas antigen, which has been very difficult to obtain until now, as a soluble active substance simply and in large quantities at the in-vitro mouth.
  • the chimeric soluble Fas antigen thus produced is required in a method for measuring a soluble Fas antigen such as a kit for immunological measurement of a soluble Fas antigen of the present invention and an immunological measurement method. Very useful as a purification standard (standard). Furthermore, the chimeric soluble Fas antigen is extremely useful not only as a standard substance but also as an immunizing antigen for producing a monoclonal antibody against a soluble region of the Fas antigen which is important as a ligand recognition site of the Fas antigen. It is.
  • the embodiments of the present invention will be described more specifically with reference to Examples, but it is needless to say that the present invention is not limited to the embodiments described below.
  • the soluble human Fas antigen used as a standard was a chimeric soluble human Fas antigen and was prepared as follows.
  • the transformed cells JM109 prepared according to Example 7 described below (accession number FER BP-4436, deposited on October 6, 1999, at the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry ) Using Gene Pulser (Bio-Rad Co., Ltd.) using Brasmid pMBl 8S / hFas.EXT-A ⁇ C2A.EXT and Brasmid PMAMneo (Clontech Co., Ltd.) Then, mouse lymphoma cells L5178Y (ATCC CRL1722) were co-transformed by electroporation.
  • the cells were cultured in a medium containing the antibiotic G418, and a G418-resistant clone was selected to obtain a chimeric soluble human Fast antigen-expressing clone.
  • the clone was mass-cultured using a 10% Poma serum-containing Fischer medium, and the culture supernatant was collected and concentrated, and then subjected to column chromatography using Q Sepharose (Pharmacia Co., Ltd.) as a standard substance. Were obtained chimeric soluble human Fas antigens.
  • a Ba1 bZc female mouse was immunized with human diploid male fibroblast FS-7 expressing human Fas antigen.
  • the sensitized mouse was laparotomized, the spleen was excised, ground on a stainless steel mesh in serum-free RPMI 1640 medium, and the spleen cell solution was centrifuged (1500 rpm for 7 minutes). The centrifugation residue was collected and suspended in a bloodless RPMI 1640 culture solution. Furthermore, the cells were washed twice with a serum-free RPMI 1640 culture solution to obtain antibody-producing mouse spleen cells.
  • Mouse myeloma cells NS- 1 a (ATCC TIB18), at 37, 5% C0 2 under incubated in 1 0% FCS and 50 U / m 1 kanamycin-containing AS F- 1 04 medium (Ajinomoto (Ltd.)) did.
  • ⁇ 2-3> Preparation of anti-human Fa s monoclonal antibody-producing hybridomas
  • Mouse-free myeloma cells NS-1 cells washed with serum-free RPMI 1640 medium and mouse spleen cells prepared in ⁇ 2-1> were mixed. The RPMI 1640 solution was centrifuged (1000 rpm for 10 minutes), and the centrifuged residue was collected. Hertzenberg, L. (Herzenberg, L. ⁇ ⁇ ) et al. (Selected Methods in Cellular Immunology. 351, 1980) ), And multiple colonies of hybridomas were obtained.
  • the hybridoma clone CHI1 (2X10, pcs.) was cultured in serum-free ASF 104 medium (Ajinomoto Co., Inc.) at 37'C for 5 days, and the obtained culture supernatant was used for omega cell (Philtron Co., Ltd.). )). The supernatant was collected and supplied to a hydroxylapatite column (Asahi Optical Co., Ltd.). It was eluted with sodium phosphate (pH 7.4, 10-400 mM) using an FPLC system (Pharmacia 7 Chemical Co., Ltd.).
  • the eluted fraction was collected, subjected to SDS-PAGE, and purified to a purity of 95% or more (see Journal of Experimental Medicine, Vol. 16, pp. 1747-1756, 1989).
  • Antibodies can be purchased from the Institute of Medical Biology, Inc.
  • a fragment containing the human Fas antigen cDNA obtained by digesting the plasmid pF58 containing the cDNA encoding the human Fas antigen with XhoI was used as an expression vector pEF—BOS (Nucleic acid research, 18, p. 5322, 1990) using a BstXI adapter to obtain an expression plasmid pEFF58.
  • V sp I digested was 2 5 GZml of Peff 5 8 Fragment 2-5 / was digested with the E c oR I £ g / ml OpMAn eo ( Clontech (Ltd.)) fragment, 1 1 0 7 (0.8 ml) of mouse T lymphoma WR19L cells (ATCC TIB52) were co-transformed by electoporation.
  • the cells were cultured in a medium containing the antibiotic G418 to select G418 resistant clones.
  • the obtained G418 resistant clone was analyzed by a flow cytometer and an ultradilution method to clone a transformant cell WR19L12a overexpressing human Fas antigen (CELL, Vol. 66, 233-243, 1991).
  • WR19L12a cells were homogenized and centrifuged to obtain a plasma membrane surface, which was used as an immunizing antigen.
  • the B1bZc mouse was immunized with the plasma membrane fraction of WR19L12a, a human Fast antigen-overexpressing transformed cell, and antibody-producing mouse spleen cells were obtained in the same manner as described above.
  • hybridoma clones were obtained in the same manner as described above, and seven anti-human Fas monoclonal antibody-producing hybridoma clones, ZB 4 (IgG), UB2 (IgG), VB3 (IgG), CBE (IgG), WB3 (IgG), AX6 (IgM) and JAE (Ig) .
  • anti-human F a s monoclonal antibodies can be purchased from Institute for Medical Biology, Inc.
  • Anti-Fas monoclones obtained as described above from Hypri-Doma clone CBE The IgG fraction of the lonal antibody was dissolved in a 0.1 M phosphate buffer (pH 7.4, containing 0.1% sodium azide) to adjust the concentration to 0.005 mg / ml. This solution was added to each well of a 96-well microplate (Maxi Soap, manufactured by Nunc Corporation) in a quantity of 1001 and allowed to stand at 4'C for about 18 hours to bind the antibody to the microplate. .
  • a kit for measuring soluble Fas antigen was prepared by combining the microplate-type immobilized anti-human Fas monoclonal antibody and the peroxidase-labeled anti-Fas monoclonal antibody prepared in Examples 3 and 4, respectively.
  • the standard substance chimeric soluble human Fas antigen was measured to prepare a calibration curve, and the soluble Fas antigen in the human serum sample was actually measured.
  • the standard chimeric soluble human Fas antigen prepared in Example 1 was similarly diluted 50-fold with PBS to obtain a standard solution.
  • the concentration of the soluble Fas antigen in the sample was determined by multiplying the quantitative value of the diluted sample by a dilution factor.
  • microplate type reagent immobilized monoclonal antibody
  • the sample or standard solution prepared in ⁇ 5-1> was dispensed at a concentration of 100 l Z ⁇ ⁇ .
  • a bottle in which PBS alone was dispensed in 1001 wells was blinded.
  • the reaction was allowed to stand at room temperature for 1 hour to react. After the completion of the reaction, each well was washed three times with 5% PBS, and the PBS was removed well.
  • the peroxidase-labeled anti-Fas monoclonal antibody prepared in Example 4 was concentrated at a concentration of 250 to 1,000 times with 1 OmM sodium phosphate buffer (pH 8.0) supplemented with 0.1% BSA and 0.15 M NaCl. Diluted. The diluted solution was dispensed at a concentration of 100 ⁇ Z ⁇ l into a microplate that had been subjected to an antigen-antibody reaction with ⁇ > 2>, and allowed to stand for 1 hour to react. After the completion of the reaction, each well was washed three times with PBS, and the buffer was removed well.
  • Tetramethylbenzidine dihydrochloride (Sigma) was dissolved in 1 OmM citrate buffer (pH 6.8) to prepare a 1.6 mM tetramethylbenzidine dihydrochloride solution. Hydrogen peroxide was diluted with 1 OmM citrate buffer (pH 6.8) to prepare a 1 OmM hydrogen peroxide solution. Equal amounts of the 1.6 mM tetramethylbenzidine dihydrochloride solution and 1 OmM hydrogen peroxide solution were mixed to obtain an enzyme substrate solution. The substrate solution is dispensed at 100 ° C / degree S in microplates reacted with peroxidase-labeled anti-Fas monoclonal antibody in ⁇ 5-3>, and left at room temperature for 10 to 20 minutes And reacted. Next, 1.5N phosphoric acid was dispensed to each gel at a concentration of 100 w 1 Z gel, and the enzyme activity of peroxidase was stopped by lowering the pH.
  • the degree of color development generated in each microplate plate was detected by measuring the absorbance at a wavelength of 450 nm using an absorptiometer (PR-A4, manufactured by BANDOSHI Co., Ltd.).
  • concentration of the soluble Fas antigen in the sample was derived from a calibration curve prepared by performing measurement as described above using a standard solution based on the detected absorbance.
  • Table 1 shows the measurement results using the standard solution, and Fig. 1 shows a calibration curve based on the measurement results.
  • Table 2 shows the measurement results of soluble Fs antigen in serum samples of healthy subjects, SLE patients, and RA patients derived based on the calibration di.
  • Table 1 Standard soluble Fs antigen concentration (ng / ml) Absorbance (A450nm)
  • Anti-Fas monoclonal antibody IgG fraction obtained from Hypri-Doma clone CBE as described in Example 2-8> was added to 0.1 M phosphate buffer (pH 7.4, containing 0.1% sodium azide). And adjusted to a concentration of 0.01 mg / ml. Add 100 ⁇ l of this solution to each well of a 96-well microplate (manufactured by Nunc Co., Ltd.) and let stand for about 18 hours at 4 to bind the antibody to the microplate. I let it.
  • the IgG fraction of the anti-Fas monoclonal antibody obtained from the hybridoma clone VB3 as described in Example ⁇ 2-8> was passed through 0.1 l acetate buffer (pH 4.2). After the analysis, the protein concentration was adjusted to 5 mg / ml, 3% of the total protein amount of vepsin was added, and the mixture was stirred at 37 overnight to carry out the reaction. The reaction was stopped by adding 2 M Tris-HCl buffer (pH 8.0) all at once, and then ultragel equilibrated with 0.1 M phosphate buffer (pH 6.5) containing 0.2 M sodium chloride. Gel filtration was performed using an Ac A44 (manufactured by LKB) column (2.0 ⁇ 60 cm) to obtain 2.5 mg of F (ab ′) 2 fraction.
  • microplate-immobilized anti-human Fas monoclonal antibody and the peroxidase-labeled anti-Fas monoclonal antibody prepared in ⁇ 6-1> and ⁇ 6-2> above were combined to form a kit for measuring soluble Fas antigen.
  • the kit the standard substance chimeric soluble Fas antigen was measured to prepare a standard, and the soluble Fas antigen in the human blood sample was actually measured.
  • a sample was prepared from serum obtained from peripheral blood of each of healthy subjects (200), SLE patients (50) and RA patients (50) in the same manner as in Example 5
  • a standard solution was prepared from the standard chimeric soluble human Fas antigen prepared in Example 1.
  • an anti-progenitor antibody reaction was performed with the microplate-type reagent (immobilized monoclonal antibody) prepared in ⁇ 6-1> in the same manner as in Example 5 ⁇ 5-2>. .
  • the peroxidase-labeled anti-Fas monoclonal antibody prepared in ⁇ 6_2> was bound to the conjugate of the microplate-type reagent (the immobilized monoclonal antibody) and the soluble Fas antigen in the sample or standard solution.
  • the degree of color development in each well of the microplate was detected by measuring the absorbance at a wavelength of 492 nm using an absorbance meter (manufactured by Tosoichi Co., Ltd., MPR-A4).
  • concentration of the soluble F ss antigen in the sample was derived from a calibration curve prepared based on the detected absorbance by performing measurement as described above using a standard solution.
  • Table 3 shows the results of measurement using the standard solution
  • FIG. 2 shows a calibration curve based on the measurement results.
  • Table 4 shows the measurement results of soluble Fs antigen in serum samples of healthy subjects, 51 ⁇ £ patients and 118 patients derived based on the calibration curve.
  • soluble Fas antigen was detected in the sera of healthy subjects, SLE patients and RA patients, and significantly higher concentrations of soluble Fa antigens were detected in SLE and RA patients than in healthy subjects. It can be seen that s antigen was detected, and particularly in SLE patients, a remarkably high concentration of soluble Fa s antigen was detected.
  • the concentration of soluble Fas antigen in body fluids of healthy persons and patients can be easily measured by implementing the kit for immunological measurement of soluble Fas antigen and the immunological measuring method of the present invention at clinical sites. be able to.
  • Example 6 In place of the peroxidase-labeled anti-Fas monoclonal antibody prepared in ⁇ 6-2> of Example 4 or Example 6, an anti-Fas monoclonal antibody prepared with piotin prepared as described below was used.
  • the soluble Fas antigen can be measured in the same manner as in Examples 5 and 6.
  • the IgG fraction of the anti-Fas monoclonal antibody obtained from the hybridoma clone VB3 as described in Example 2-8> was dissolved in 0.11 ⁇ ⁇ -acid buffer (158.5) and the IgG concentration was Was adjusted to 5 mgZm1.
  • NHS-LC-B I OT IN similarly prepared by dissolving the IgG solution in 0. IMiJt acid buffer (pH 8.5) to prepare 25 mg nom 1 )
  • To the IgG 1 0 mg was added, and the mixture was stirred at room temperature for 4 hours using a stirrer to prepare a biotin-labeled anti-Fas monoclonal antibody.
  • the enzyme peroxidase, galactosidase, etc.
  • avidin peroxidase, galactosidase, etc.
  • the immobilized anti-Fas monoclonal antibody is used.
  • Form a complex of mono-soluble anti-Fas anti-Piotin-labeled anti-Fas monoclonal antibody and avidin-labeled enzyme and measure the color of the chromogenic substrate in the same manner as in Examples 5 and 6 to determine whether it is in the sample or in the standard solution. The concentration of soluble Fas antigen was measured.
  • the transformed cells JM109 used in the preparation of the standard chimeric soluble human Fas antigen in Example 1 were prepared as follows.
  • the cell line used in this example was prepared in advance.
  • Human T tumor cell line KT3 (available from Dr. Shimizu, Kanazawa Medical University): Contains 10% fetal calf serum (FCS) and 5 ng / 1 human recombinant IL-16 (Ajinomoto) Cultured in RPMI 1640 medium.
  • 'Monkey cell line COS 7 (ATCC CRL 1651): cultured in DMEM medium containing 10% FCS.
  • mice anti-human Fas antibody CH11 (IgM) used in this example was prepared in advance in the same manner as in Example 2 (Yonehara et al., J. Exp. Med .. Vol. 169, 1747- 1756 p. 1989) 0
  • Plasmid pCEV4 (Science, 247: 324-327; see 1990, Itou et al.): Plasmid pcDSRc Mol. Cell Biol., 2: 161, 1982, Okayama et al.
  • DNA was inserted into the NdeI cleavage site and PstI cleavage site of pcDSRa, and the Notl cleavage site in the stuffer (XbaI cleavage fragment) was removed by filling in, and the NotI linker was removed.
  • the plasmid PCEV4 was constructed by inserting it into the I cleavage site.
  • Brasmid pME18S (available from Dr. Kazuo Maruyama, The University of Tokyo): A cDNA cloning vector suitable for expression in animal cells constructed using PCEV4 as the basic skeleton.
  • RNA was prepared from KT3 by the guanidine isothionate / acid phenol method (Anal. Biochem., Vol. 162, pp. 156-159, 1987, Chomczynski and Sacchi et al.), And poly (A ) RNA was selected by o 1 igo (d T) cellulose column chromatography. Random hexamer oligonucleotide (pdN6) ( Using M-MLV RNAase H "reverse transcriptase as a primer with BRL (manufactured by) and oligo (dT) (BRL (manufactured by)), double-stranded cDNA was synthesized from the RNA (Cell, No. 1).
  • the DNA sequence of the B st X I non-palindromic adapter used is shown in SEQ ID NO: 1.
  • the filtered cells were plated on the previously prepared banning dish (54). Leave at room temperature for 2 to 3 hours to allow the cells to adhere to the dish, and then wash gently three times with PBS / EDTA / NaN 3 (2 ml) containing 5% FCS to remove cells that do not bind to the dish .
  • Plasmid DNA was prepared from the COS cells bound to the dish. That is, a 0.6% 303 solution (0.4 ml) containing 1 OmI [ED chohachi] was added to each dish and left at room temperature for 20 minutes.
  • ⁇ 8-1 -F> Obtain full-length hF as cDNA
  • the colony hybridization method using the 0.5 kb Xhol-BamHI cleavage fragment at the 5 'end of pF3 obtained in ⁇ 8-1-1 E> Ten cDNA clones were obtained from the cDNA library created in D>. Restriction enzyme mapping confirmed that these clones contained a 1.8-2.6 kb insert with the same restriction map.
  • the longest clone, pF58 consists of 2534 bp, starting from the 3 'terminal polyadenylation signal sequence (ATT AAA), the translation initiation codon located at bases 195 to 197.
  • ATT AAA 3 'terminal polyadenylation signal sequence
  • a 2555 bp DNA containing a full-length Fas antigen cDNA extracted as a XhoI digestion fragment from pF58 green was incorporated into a mammalian expression plasmid PCEV4 using a Bst XI adapter to construct a plasmid pCEV4ZhFas.
  • the cDNA sequence encoding the full-length hFas and the corresponding amino acid sequence are shown in SEQ ID NOs: 2 and 3, respectively.
  • the pCEV4 hFas constructed in ⁇ 8-1-1 F> was digested with XhoI, and the full-length hFas cDNA was extracted as an XhoI digestion fragment (2555 bp). Also, pME18S was digested with XhoI, and a portion of the stuffer was removed as an XhoI digestion fragment. The pME18S fragment was treated with alkaline phosphatase (BAP) (E. coli C75; Takara Shuzo Co., Ltd.) to remove the phosphate group at the 5 'end of the cut surface.
  • BAP alkaline phosphatase
  • the plasmid pME18SZhFas was constructed by ligating the full-length hFascDNA to the XhoI-cleaved fragment (2555 bp) and the BAP-treated pME18S fragment using a DNA ligation kit (Takara Shuzo).
  • pME 18 SZhF as is digested with Bg1 II and PstI, and transmembrane of hFas
  • the cDNA region corresponding to the cloning region and the subsequent intracellular region was removed.
  • the blunt ends were then blunted using the DNA blunting kit (Takara Shuzo), the pME18S fragment was self-ligated, and the plasmid pME18 containing only the cDNA encoding hFas ⁇ EXT was introduced.
  • S / hFa s ⁇ EXT was constructed.
  • Brasmid pME18S / hFas ⁇ EXT construction process is shown in Fig. 5, and the cDNA sequence encoding the extracellular region of hFas (hFas ⁇ EXT) and the corresponding amino acid sequence are shown in SEQ ID NOs: 4 and 4, respectively. 5
  • the cell line used in this example was prepared in advance.
  • Mouse obese cell line MC / 9 (ATCC CL 8306): Proliferated in RPMI 1640 medium containing 100 U / m1 recombinant mouse IL-13 and 10% FCS.
  • Monkey cell line COS 7 (ATCC CRL 1651): Propagated in DMEM medium containing 10% FCS.
  • the mouse anti-AIC2 antibody (IgM) used in this example was prepared in advance according to the report of Yonehara et al. (Int. Immunol. Vol. 2, No. 2, pp. 143-150, 1990).
  • a double-stranded cDNA was synthesized from p01y (A) RNA selected from MCZ9 in the same manner as described in ⁇ 8-1-1 D>, and ligated to Bst XI non-palindromic adapter. CDNAs longer than 1.5 kb were separated by electrophoresis and ligated to the BstXI cut pCEV4 vector fragment. The vector was transformed into E. coli DH5 ⁇ by the electroporation method to prepare a cDNA library derived from mouse mast cell MCZ9 (Science, Vol. 247, 324-327, 1990, see Itto et al.). ). ⁇ 8-3-E> Acquisition of AIC 2 A cDNA fragment
  • a panning dish was prepared by coating a goat anti-rat IgM antibody (Cappel) as a secondary antibody in the same manner as described in ⁇ 8-11).
  • Cappel rat anti-Aic 2 antibody
  • 24 plasmid clones transformed with COS 7 were obtained, and each clone was analyzed. After confirming that it had a 6 kb AI C2A cDNA fragment (insertion sequence), a brassmid clone pAI C2-2 was selected for use as a probe for the next step (Science. Vol. 247, pp. 324-327). , 1990, see Itou et al.).
  • DNA containing the full-length AIC2AcDNA extracted from the PAI C2--26 clone as an XhoI-cleaved fragment was incorporated into the mammalian expression plasmid pCEV4 using the BstXI adapter to construct the plasmid PCEV4ZA IC2A to construct the full-length AIC2.
  • the cDNA sequence encoding A, the corresponding amino acid sequence, the cDNA sequence encoding the extracellular region of AIC2A (AIC2A • EXT), and the corresponding amino acid sequence are shown in SEQ ID NOs: 6 to 9, respectively.
  • the plasmid pME18SZhFasEXT constructed in ⁇ 8-2> was digested with NotI, linearized, and the blunt end was blunted using a DNA blunt kit, and the phosphate group at the 5 'end of the cut surface was digested. Was treated with BAP to remove.
  • DralU-cleaved AIC 2A • EXTC DNA fragment was introduced into the NotI-cleaved pME 18 S / hFas • EXT brassmid fragment previously prepared, and the Brassmid pME 18 S / hF as ⁇ EXT-A IC 2 A ⁇ EXT was constructed.
  • Figure 6 shows the construction process of pME 18 S / hF as-EXT-A IC 2AEXT, and the cDNA sequence encoding hF as EXT-A IC 2AEXT and the corresponding amino acid sequence. These are shown in SEQ ID NOs: 10 and 11, respectively.
  • Plasmid pME18S / hFas-EXT-AIC2A.EXT constructed in ⁇ 8-4> was transformed into E. coli cell line JM109, and colonies were analyzed to select transformed cells.
  • the transformed E. coli cell strain JM109 was deposited internationally with the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry (Accession number: FERM BP-4436, date of deposit: October 6, 1993). ).
  • the soluble hFas-AIC2A protein was tested for cell death due to apoptosis of Fas antigen-expressing cells induced by anti-human Fas antibody. The effect of the suppression was investigated.
  • the cells were subjected to MTT assay (Immunology Letters, Vol. 19, pp. 261-268, 1988), and the viable cell ratio (%, average of three measured values) was measured using a microplate reader (0. D. 595-655). ) was measured.
  • soluble AIC 2A obtained by using pME18S / AIC2A ⁇ EXTT reconstructed from PCEV4ZA IC2A constructed in 8-3-F> was converted to RPMI1640 containing 1056FCS.
  • Control experiments were performed in the same manner as described above, using solutions diluted to 1/8, 1/16, 1/32, 1Z64 and 1/128 with the culture solution.
  • Cell death due to apoptosis of the Fas antigen-expressing cells induced by the anti-human Fas antibody was significantly suppressed in a highly correlated manner by the addition of the soluble hFas-AIC2A protein obtained in this example. Therefore, it was confirmed that the chimeric soluble protein obtained in this example had an intact function as a soluble human Fas antigen.
  • the cell strain used in this example was prepared in advance.
  • RNA from BAM3 treated with LPS (lipolivosaccharide) by the guanidinoisothiocyanate phenol method (Anal. Biochem., Vol. 162, pp. 6-159; see 1987, Chomczynski and Sacchi et al.).
  • LPS lasine-phosphate
  • oligo (dT) cellulose column chromatography A double-stranded cDNA was synthesized from the RNA using oligo (dT) as a primer (Nature, Vol. 319, p. 415, 1986, Nagayu et al.).
  • the double-stranded cDNA was methylated with EcoR I methylase, ligated with EcoR I linker, digested with EcoR I, and subjected to agarose gel electrophoresis (low temperature gel) to obtain a cDNA longer than 70 Obp. These were ligated to an Ig 11 vector (Stratagene (manufactured by Stratagene)). , Respectively which the vector into E. C 01 i Y 1090 by electroporation volley Chillon method was transformed to prepare a mouse macrophage BAM3 derived cDNA library (3. 2 X 1 0 ⁇ plaques).
  • the longest fragment of the 0.5 to 1.5 kb cDNA fragment obtained by digesting the cDNA obtained from the clone with EcoRI was used as the brassmid pB1uescript KS (+) ( The plasmid was subcloned into Stratagene (manufactured by Stratagene) to construct a plasmid pMF1.
  • clone pMFI was composed of 1479 bp, 3 ′ terminal polyadenylation signal sequence (ATT AAA), translation initiation codon (ATG ) And a translation stop codon (TAG) located at bases 1031 to 1033, confirming that clone pMF1 has full-length mFasc DNA. J. I Set unol., 148: 1274-1279; 1992, see Fukunaga et al.).
  • the cDNA sequence encoding full-length mFas and the corresponding amino acid sequence are shown in SEQ ID NOs: 12 and 13, respectively.
  • the pME18S constructed in ⁇ 8-1-1C> was digested with EcoRI and XbaI to prepare a pME18S fragment.
  • the pMF1 constructed at 9> 11C> was digested with EcoRI and F0kI to obtain a cDNA fragment encoding the extracellular region of mFas.
  • a linker (ligated) DNA was prepared using a DNA synthesizer (DNA synthesizer).
  • DNA synthesizer DNA synthesizer
  • phospho-DNA sequence is shown in SEQ ID NOs: 14 and 15, and Brasmid pME.
  • Fig. 9 shows the construction steps of 18 S / mF as and EXT, and the cDNA sequence encoding mF as ⁇ EXT and the corresponding amino acid sequence are shown in SEQ ID NOs: 16 and 17, respectively.
  • the plasmid p CEV4ZA constructed in ⁇ 8-3-1-F> was digested with Drain to extract cDNA encoding AIC2AEXT, and the fragment was digested using a DNA-end blunting kit. The cut end was blunted and treated with BAP to remove the phosphate group at the 5 'end of the cut surface.
  • the linker DNA sequence of the plasmid pME18S / mFas.EXT constructed in 9-2> was digested with DraIII, and the DNA ligation kit was used to cut the Dralll-cut AIC2AEXT.
  • the cDNA fragment was ligated to the DrAIII cleavage linker DNA to construct the plasmid pME18S / mFas-EXT-AIC2A.EXT.
  • Figure 10 shows the construction process of ME 18 S / mF as and EXT-A IC 2 A and EXT.
  • the cDNA sequence and the corresponding amino acid sequence encoding mF as-EXT-A IC 2 A and EXT are respectively shown. These are shown in SEQ ID NOs: 18 and 19.
  • the serum-free culture obtained by culturing in serum-free D-MEM medium for 12 hours is centrifuged and centrifuged. Collected.
  • the obtained centrifuged supernatant was analyzed by SDS-polyacrylamide gel electrophoresis, and as a result, it was confirmed that the obtained soluble protein was obtained as a soluble mFas-AIC2A protein.
  • the centrifuged supernatant was subjected to column chromatography using Q Sepharose to purify a soluble protein comprising an extracellular region of mFas and an extracellular region of AIC2A. The results are shown in FIG.
  • mice myeloma Tsumugi ⁇ NS- 1 (ATCC TIB18), 37 '(:, 53 ⁇ 4C0 2 below, were cultured in 1 0% FCS and 5 OUZml kanamycin ASF- 1 04 medium (Ajinomoto (Ltd.)).
  • Each of the hybridomas was cultured in a serum-free ASF104 culture medium, and the culture supernatant was centrifuged (8000 rpm for 20 minutes). The centrifuged supernatant was recovered, and each monoclonal antibody was purified by column chromatography using Protein G Sepharose (Pharmacia).
  • rat monoclonal antibody subclass identification kit (Zymed)
  • RMF2 was IgG1
  • RMF6 was IgG2a
  • RMF9 was IgG2a
  • RMF13 was
  • mice myeloma cells NS- 1 ATCC TIB18
  • 37, under 5% C0 2, 1 0% FCS and 50 U / m 1 kanamycin-containing ASF- 1 04 medium (Ajinomoto
  • the reactivity was determined by introducing the mouse lymphoma cell L5178Y (ATCC CRL1722) which does not express the Fas antigen and the full-length mouse Fas cDNA obtained by 9-1-1-C> by gene engineering. It was confirmed by comparing the reactivity to the L5178Y cells overexpressing the mouse Fas antigen.
  • the mouse Fa s antigen overexpressing L5178 Y cells were prepared as follows.
  • the plasmid pMF1 containing the full-length mouse Fas cDNA obtained in ⁇ 9-11C> was digested with EcoR1, and the cut ends were digested using a DNA end blunting kit (Takara Shuzo Co., Ltd.). Was smoothed to obtain a DNA fragment containing cDNA encoding full-length mouse Fas cDNA.
  • the expression vector pME183 described above in ⁇ 8-1-1C> was digested with 110 I, and the cut ends were blunted using a DNA end blunting kit (Takara Shuzo Co., Ltd.). The l8S fragment was obtained.
  • the pMF1 fragment having the full-length mouse Fas cDNA and the pME18S fragment were ligated using a DNA ligation kit (Takara Shuzo Co., Ltd.) to obtain a plasmid pME18S / mFas having the full-length mouse Fas cDNA. It was constructed.
  • This pME18S / mFas was digested with PvuI by electroporation (290 V) using Gene Pulser (manufactured by Bio-Rad) to obtain pME18S / mF.
  • a mouse lymphoma cell L5178Y (ATCCCRL1722) was co-transformed with the as fragment and a plasmid p MAMneo (Clontech) fragment obtained by digestion with EcoRI.
  • the transformed cells were cultured in a medium containing the antibiotic G418 to select for G418-resistant clones and by limiting dilution to obtain mouse Fas antigen-overexpressing L5178Y cells.
  • Mouse lymphoma cells L5178 Y not expressing the Fas antigen or the suspension of the mouse Fs antigen overexpressing L5178 YB vesicle were added to the suspension obtained in Examples 10 and 11, respectively.
  • PBS containing the anti-mouse Fas monoclonal antibody (20 PL g / m 1) was added, and the mixture was allowed to react on water for 1 hour.
  • PBS containing FITC-labeled anti-rat IgG, FITC-labeled anti-hamster IgG or FITC-labeled anti-rat IgM each 20 gZml was added and reacted for 1 hour. After washing each cell with PBS (with 4), the reactivity was analyzed by cytofluorimetry.
  • anti-rat IgG As a control, anti-rat IgG, anti-hamster IgG and anti-rat IgM (Pharraingen) were similarly measured.
  • each of the obtained monoclonal antibodies was a monoclonal antibody having specific reactivity to mouse Fas antigen.
  • Sequence type SEQUENCE TYPE: nucleic acid
  • Sequence type SEQUENCE TYPE: nucleic acid
  • Sequence type SEQUENCE TYPE: nucleic acid
  • GAC CAG CAA ATG GCA CTC ACA TGG GGG CTG TGC TAC ATG GCA CTG GTG 226 Asp Gin Gin Met Ala Leu Thr Trp Gly Leu Cys Tyr Met Ala Leu Val
  • GAG AAA CTC ATG TGG TCA GAG TGC CCG TCA TCC CAC CGC TGT GTG CCC 466 Glu Lys Leu Met Trp Ser Glu Cys Pro Ser Ser His Arg Cys Val Pro
  • AGC CCC TCT GGG GAT CAT TTC CTG CTG GAA TGG AGT GTA TCT CTT GGG 658 Ser Pro Ser Gly Asp His Phe Leu Leu Glu Trp Ser Val Ser Leu Gly
  • AGC ATC TAT GCT GCC CGT GTG CGC ACT CGG CTG TCC GCG GGT TCA
  • AGC 850 Ser lie Tyr Ala Ala Arg Val Arg Thr Arg Leu Ser Ala Gly Ser Ser
  • GAG GAG AAA TGC TCT CCG GTG GTG AAG GAG CCG GAG GCC AGC GTC TAC 1090 Glu Glu Lys Cys Ser Pro Val Val Lys Glu Pro Gin Ala Ser Val Tyr
  • AAG CCC ATC TCT GAC TAC CAC GGG ATC TGG AGC GAG TGG AGC AAT GAG 1474 Lys Pro lie Ser Asp Tyr ASP Gly He Trp Ser Glu Trp Ser Asn Glu
  • GCC CAA AGC CCC AGT CTC TGT CTT AAG CTG CCC AGG GTC CCC TCT GGA 2434 Ala Gin Ser Pro Ser Leu Cys Leu Lys Leu Pro Arg Val Pro Ser Gly
  • Sequence type SEQUENCE TYPE: nucleic acid
  • ATC AGC CCC TCT GGG GAT CAT TTC CTG CTG GAA TGG AGT GTA TCT CTT 480 l ie Ser Pro Ser Gly Asp His Phe Leu Leu Glu Trp Ser Val Ser Leu
  • Sequence type SEQUENCE TYPE: nucleic acid
  • AAG ACT GTT ACT ACA GTT GAG ACT CAG AAC 144 Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gin Asn
  • TGT AAA CCA AAC TTT TTT TGT AAC TCT ACT GTA TGT GAA CAC TGT GAC 432 Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp 115 120 125
  • AAG GAG CCG GAG GCC AGC GTC TAC ACC CGC TAC CGC TGC ACT CTA CCT 1392 Lys Glu Pro Gin Ala Ser Val Tyr Thr Arg Tyr Arg Cys Ser Leu Pro
  • Gin Gly Leu lie Asn Met Thr Leu Leu Tyr His Gin Leu Asp Lys lie
  • Pro Pro lie Leu Asn Gin Thr Lys Asn Arg Asp Ser Tyr Ser Leu His
  • Trp Glu Thr Gin Lys lie Pro Lys Tyr lie Asp His Thr Phe Gin Val
  • Ser Tyr Cys Ala Arg Val Arg Val Lys Pro lie Ser As Tyr Asp Gly 545 550 555 560 lie Trp.
  • Ser Glu Trp Ser Asn Glu Tyr Thr Trp Thr Thr Asp Trp Val
  • Sequence type SEQUENCE TYPE: nucleic acid
  • AAA AGG AGA CAG GAT GAC CCT GAA TCT AGA ACC TCC AGT CGT GAA ACC 682 Lys Arg Arg Gin Asp Asp Pro Glu Ser Arg Thr Ser Ser Arg Glu Thr

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Abstract

L'invention concerne un kit et un procédé d'immunodosage d'antigènes Fas solubles présents dans un fluide corporel tel que du sérum, et permettant de déterminer aisément, avec une grande précision et sensibilité, la quantité d'antigènes Fas solubles présents dans le fluide corporel d'un patient atteint de maladies autoimmunes telles que le rhumatisme ou SLE, ce qui a été difficile à déterminer jusqu'à présent, ce kit et ce procédé permettant également de doser en une seule fois une grande quantité d'échantillon. L'invention concerne également un antigène Fas soluble comme substance active soluble et qui est très utile comme substance étalon pure utilisée dans ce procédé et ce kit et également en tant qu'antigène d'immunisation utilisé pour produire des anticorps monoclonaux contre la région soluble d'un antigène Fas.
PCT/JP1995/000349 1994-07-06 1995-03-03 PROCEDE ET KIT D'IMMUNODOSAGE D'ANTIGENES Fas SOLUBLES WO1996001277A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP6/154706 1994-07-06
JP15470694 1994-07-06
JP2563795A JPH0875745A (ja) 1994-07-06 1995-02-14 可溶性Ｆａｓ抗原の免疫学的測定方法及びその測定用キット
JP7/25637 1995-02-14

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WO1996001277A1 true WO1996001277A1 (fr) 1996-01-18

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PCT/JP1995/000349 WO1996001277A1 (fr) 1994-07-06 1995-03-03 PROCEDE ET KIT D'IMMUNODOSAGE D'ANTIGENES Fas SOLUBLES

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JP (1) JPH0875745A (fr)
WO (1) WO1996001277A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0729300A4 (fr) * 1993-11-15 1998-08-05 Lxr Biotechnology Inc Nouvelle proteine fas et procedes d'utilisation
CN104459100A (zh) * 2014-11-25 2015-03-25 成都威尔诺生物科技有限公司 一种抗sm-rnp抗体试剂盒
EP3474016A1 (fr) 2008-12-10 2019-04-24 Joslin Diabetes Center, Inc. Procédés de diagnostic et de prédiction d'une maladie rénale

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CELL, Vol. 75, (December 1993), TAKASHI SUDA et al., "Molecular Cloning and Expression of the Fas Ligand, a Novel Member of the Tumor Necrosis Factor Family", pages 1169-1178. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 267, No. 15, (May 1992), ALEXANDER OEHM et al., "Purification and Molecular Cloning of the APO-1 Cell Surface Antigen, A Member of the Tumor Necrosis Factor/Nerve Growth Factor Receptor Superfamily", pages 10709-10715. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 267, No. 2, (January 1992), HUEY-MEI WANG et al., "Structure of Mouse Interleukin 3 (IL-3) Binding Protein (AIC2A)", pages 979-983. *
JOURNAL OF LEUKOCYTE BIOLOGY, (1993), BRENDAN CLASSON et al., "Expression of a Soluble Form of the Fas Antigen", Suppl. page 63. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 91, (May 1994), SHINO HANABUCHI et al., "Fas and Its Ligand in a General Mechanism of T-Cell-Mediated Cytotoxicity", pages 4930-4934. *
SCIENCE, Vol. 263, (March 1994), JIANHUA CHENG et al., "Protection from Fas-Mediated Apoptosis by a Soluble Form of the Fas Molecule", pages 1759-1762. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0729300A4 (fr) * 1993-11-15 1998-08-05 Lxr Biotechnology Inc Nouvelle proteine fas et procedes d'utilisation
EP3474016A1 (fr) 2008-12-10 2019-04-24 Joslin Diabetes Center, Inc. Procédés de diagnostic et de prédiction d'une maladie rénale
CN104459100A (zh) * 2014-11-25 2015-03-25 成都威尔诺生物科技有限公司 一种抗sm-rnp抗体试剂盒

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