WO2020103691A1 - 针对人抗缪勒管激素的特异性抗体及其应用 - Google Patents

针对人抗缪勒管激素的特异性抗体及其应用

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Publication number
WO2020103691A1
WO2020103691A1 PCT/CN2019/115998 CN2019115998W WO2020103691A1 WO 2020103691 A1 WO2020103691 A1 WO 2020103691A1 CN 2019115998 W CN2019115998 W CN 2019115998W WO 2020103691 A1 WO2020103691 A1 WO 2020103691A1
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WIPO (PCT)
Prior art keywords
antibody
amino acid
antigen
müllerian hormone
sequence
Prior art date
Application number
PCT/CN2019/115998
Other languages
English (en)
French (fr)
Inventor
熊君辉
陈自敏
徐伟玲
王龙
柯起沈
李丽华
宋浏伟
孙旭东
葛胜祥
Original Assignee
厦门万泰凯瑞生物技术有限公司
厦门大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 厦门万泰凯瑞生物技术有限公司, 厦门大学 filed Critical 厦门万泰凯瑞生物技术有限公司
Priority to CA3120409A priority Critical patent/CA3120409A1/en
Priority to US17/295,361 priority patent/US20220146515A1/en
Priority to AU2019384259A priority patent/AU2019384259A1/en
Priority to KR1020217018475A priority patent/KR20210093961A/ko
Priority to EP19888178.1A priority patent/EP3885363A4/en
Priority to JP2021527973A priority patent/JP7200375B2/ja
Publication of WO2020103691A1 publication Critical patent/WO2020103691A1/zh

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Classifications

    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • the invention belongs to the field of biomedical testing, and mainly relates to a specific antibody against human anti-Müllerian hormone and its application.
  • Anti-Mullerian hormone also known as anti-Mullerian hormone, is a type of monoglycoprotein secreted by testicular immature support cells and granulosa cells of ovarian growth follicles, which belongs to transforming growth factor ⁇ The superfamily has the important functions of inhibiting the development of male Mullerian tubes and regulating the development of sex cells and gonads. In recent years, scholars from various countries have conducted extensive research on their role in the field of female reproduction. In women, AMH can regulate follicular development, reflect ovarian reserve function, predict ovarian response to superovulation, and have certain value in the study of reproductive endocrine diseases.
  • US Patent 7,897,350 describes a composition and method for detecting AMH in a sample in which the antibody binds to the mature region or C-terminal region of AMH.
  • European Patent EP2161579A describes a method for detecting or quantifying at least one biologically active form of AMH in a sample, such as a cut AMH or C-terminal AMH, and antibodies bound thereto.
  • WO2014204327A1 also discloses an antibody against the N-terminal region of AMH, wherein the N-terminal region refers to 1-451 residues of the human propeptide or 25-451 residues when the leader sequence is removed.
  • the antigen is detected using the above-mentioned anti-AMH antibody, there is a problem of poor detection stability and low sensitivity.
  • the first aspect of the present invention provides a monoclonal antibody antibody or antigen-binding fragment thereof against human anti-Müllerian hormone, said antibody or antigen-binding fragment thereof specifically binds to an epitope of human anti-Müllerian hormone, said The epitope is selected from epitopes included in the sequence spanning amino acid residues 26 to 85 of the anti-Müllerian hormone, epitopes included in the sequence spanning amino acids 38 to 45 of the anti-Müllerian hormone, The epitope included in the sequence spanning amino acid residues 40 to 46 of the anti-Müllerian hormone, the epitope included in the sequence spanning amino acid residues 37 to 44 of the anti-Müllerian hormone, included in The epitope in the sequence spanning amino acid residues 39 to 45 of the anti-Müllerian hormone, the epitope included in the sequence spanning amino acid residues 36 to 47 of the anti-Müllerian hormone, included in the spanning The epitope in the sequence of amino acid residues 37 to 46 of the
  • the monoclonal antibody of the present invention specifically recognizes the human anti-Müllerian hormone epitope and does not cross-react with anti-Müllerian hormones from other species, including goat anti-Müllerian hormone, owl anti-Müllerian hormone, bovine anti Mullerian hormone, horse anti-mullerian hormone, monkey anti-mullerian hormone, canine anti-mullerian hormone, deer anti-mullerian hormone, mouse anti-mullerian hormone, guinea pig anti-mullerian hormone, etc.
  • the antibodies of the invention are chimeric antibodies or humanized antibodies.
  • the antigen-binding fragments of the present invention are selected from scFv, Fab, Fab ', (Fab') 2 , Fv fragments, and diabody.
  • the present invention provides a monoclonal antibody antibody against human anti-Müllerian hormone, which specifically binds to a table contained in a sequence spanning amino acid residues 38 to 45 of the anti-Müllerian hormone Bit.
  • the antibody comprises the heavy chain CDR sequences CDR1, GFAFSTYD, CDR2, ISPGGGAT, CDR3, AGRRDYYGMDY as shown in SEQ ID NO: 1-3; and the light chain CDR sequence CDR1, QSLVHSNGNTY as shown in SEQ ID NO: 4-6, CDR2 KVS, CDR3 SQSTHVPWT.
  • the antibody comprises the heavy chain variable region sequence shown in SEQ ID NO: 7
  • the monoclonal antibody of the present invention is a monoclonal antibody secreted by the hybridoma cell line C9F2-2 deposited at the Chinese Type Culture Collection (Wuhan University, China) on August 19, 2018.
  • the deposit number of the hybridoma cell line is CCTCC NO: C2018183.
  • the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody of the present invention or an antigen-binding fragment thereof, or its heavy chain variable region and / or light chain variable region.
  • the isolated nucleic acid molecule encodes the antibody or antigen-binding fragment thereof, or its heavy chain variable region and / or light chain variable region.
  • the present invention provides a vector (eg, cloning vector or expression vector), which comprises the isolated nucleic acid molecule of the present invention.
  • the vectors of the present invention are, for example, plasmids, cosmids, bacteriophages and the like.
  • the vector is capable of expressing the antibody or antigen-binding fragment of the present invention in a subject (eg, mammal, eg, human).
  • the invention provides a host cell comprising the isolated nucleic acid molecule of the invention or the vector of the invention.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • the host cell of the invention is a mammalian cell.
  • the antibody or antigen-binding fragment of the present invention can specifically bind human anti-Müllerian hormone, and thus can be used to detect the presence or level of human anti-Müllerian hormone in a sample.
  • the present invention provides a kit comprising the antibody of the present invention or an antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment of the invention bears a detectable label.
  • the detectable label may be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. It is particularly preferred that such labels can be suitable for immunological detection (eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.).
  • Such labels include, but are not limited to, enzymes (eg, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.), radionuclides ( For example, 3 H, 125 I, 35 S, 14 C, or 32 P), fluorescent dyes (eg, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), algae Hemoglobin (PE), Texas Red, Rhodamine, quantum dots or cyanine dye derivatives (eg Cy7, Alexa 750)), luminescent substances (eg chemiluminescent substances such as acridine ester compounds), magnetic beads (E.g, ), Calorimetric markers such as colloidal gold or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) beads, and avidin (eg,
  • enzymes e
  • Patents that teach the use of this marker include, but are not limited to, US Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference).
  • the markers covered in the present invention can be detected by methods known in the art.
  • radioactive labels can be detected using photographic film or scintillation calculators
  • fluorescent labels can be detected using photodetectors to detect the emitted light.
  • Enzyme labels are generally detected by providing a substrate for the enzyme and detecting the reaction products generated by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored labels.
  • a detectable label as described above can be attached to the antibody or antigen-binding fragment of the present invention through linkers of different lengths to reduce potential steric hindrance.
  • the present invention provides a method for detecting the presence or level of human anti-Müllerian hormone in a sample, which includes the step of using the antibody or antigen-binding fragment thereof of the present invention.
  • the antibody or antigen-binding fragment of the invention also carries a detectable label.
  • the method further comprises using a detectably labeled reagent to detect the antibody or antigen-binding fragment of the present invention.
  • the method can be used for diagnostic purposes or non-diagnostic purposes (eg, the sample is a cell sample, not a sample from a patient).
  • the use of the antibody or antigen-binding fragment thereof of the present invention in the preparation of a kit for detecting the presence or level of human anti-Müllerian hormone in a sample is provided.
  • the present invention provides a method for detecting fragments of AMH in a biological sample derived from human fluid, the method comprising:
  • At least one of the antibodies or antigen-binding fragments thereof is directed against the epitope contained in the sequence of amino acid residues 37 to 46 of the anti-Müllerian hormone.
  • one antibody or antigen-binding fragment thereof is directed against an epitope contained in the sequence of amino acid residues 38 to 45 of anti-Müllerian hormone
  • the other antibody or antigen-binding fragment thereof is directed An epitope included in the sequence of amino acid residues 358 to 369 of the anti-Müllerian hormone, or an epitope included in the sequence of amino acid residues 491 to 502 of the anti-Müllerian hormone, or included in An epitope in the sequence of amino acid residues 260 to 271 of the anti-Müllerian hormone, or an epitope included in the sequence of amino acid residues 330 to 343 of the anti-Müllerian hormone.
  • At least one of the at least two antibodies or antigen-binding fragments thereof is immobilized on the surface of the solid phase.
  • at least one of the other antibody or antibodies is labeled, preferably by covalent attachment of chemiluminescent or fluorescent dyes.
  • the antibody or antigen-binding fragment thereof directed against the epitope contained in the sequence spanning amino acid residues 38 to 45 of the anti-Müllerian hormone is immobilized on the surface of the solid phase .
  • antibody refers to an immunoglobulin molecule that generally consists of two pairs of polypeptide chains (each pair has one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is composed of 3 domains (CH1, CH2 and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of a domain CL.
  • the constant domain does not directly participate in the binding of antibodies to antigens, but exhibits various effector functions, such as mediating immunoglobulins and host tissues or factors, including various cells of the immune system (eg, effector cells) and classical complement Combination of the first component of the system (C1q).
  • VH and VL regions can also be subdivided into regions with high denaturation (called complementarity determining regions (CDR)), with more conserved regions called framework regions (FR) interspersed therebetween.
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components.
  • the variable regions (VH and VL) of each heavy / light chain pair respectively form an antigen binding site.
  • the allocation of amino acids in each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917 ; The definition of Chothia et al. (1989) Nature 342: 878-883.
  • CDR complementarity determining region
  • the precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health, Service, National Institutes of Health) Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al.
  • the CDRs contained in the antibody or antigen-binding fragment of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibody or antigen-binding fragment of the invention are preferably determined by the Kabat, Chothia, or IMGT numbering system. In certain embodiments, the CDRs contained in the antibody or antigen-binding fragment of the invention are preferably determined by the Kabat numbering system.
  • framework region or "FR" residues refer to those amino acid residues in the antibody variable region other than CDR residues as defined above.
  • antibody is not limited by any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • antigen-binding fragment of an antibody refers to a polypeptide comprising a fragment of a full-length antibody, which retains the ability to specifically bind the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody It is also called “antigen-binding portion” for specific binding to antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated by reference in its entirety for all purposes. It can be obtained by recombinant DNA technology Or by enzymatic or chemical fragmentation of intact antibodies to produce antigen-binding fragments of antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab ', F (ab') 2 , Fd, Fv, dAb and complementarity determining regions (CDR) Fragments, single chain antibodies (eg, scFv), chimeric antibodies, diabody, linear antibody, Nanobody (technique from Domantis), domain antibody (technique from Ablynx), probody and such polypeptides , Which contains at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.
  • Antigen-binding fragments of antibodies can be obtained from a given antibody (eg, the antibody provided by the present invention) using conventional techniques known to those skilled in the art (eg, recombinant DNA technology or enzymatic or chemical fragmentation). ) And specifically screen the antibody for antigen-binding fragments in the same way as for intact antibodies.
  • a given antibody eg, the antibody provided by the present invention
  • conventional techniques known to those skilled in the art eg, recombinant DNA technology or enzymatic or chemical fragmentation.
  • antibody it includes not only whole antibodies but also antigen-binding fragments of the antibodies.
  • the terms “monoclonal antibody”, “monoclonal antibody”, “mAb” have the same meaning and are used interchangeably interchangeably, which refers to one from a group of highly homologous antibody molecules An antibody or a fragment of an antibody, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously.
  • the monoclonal antibody has high specificity for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies and usually contain at least 2 or more different antibodies, which usually recognize different epitopes on the antigen.
  • the modifier "monoclonal” only indicates that the antibody is characterized by being obtained from a highly homologous group of antibodies, and it cannot be understood that the antibody needs to be prepared by any specific method.
  • the monoclonal antibodies of the present invention can be prepared by a variety of techniques, such as hybridoma technology (see, for example, Kohler et al. Nature, 256: 495,1975), recombinant DNA technology (see, for example, US Patent Application 4,816,567), or bacteriophage Antibody library technology (see, for example, Clackson et al. Nature 352: 624-628, 1991, or Marks et al. J. Mol. Biol. 222: 581-597, 1991).
  • Antibodies can be purified by well-known techniques such as affinity chromatography using protein A or protein G. Subsequently or as an alternative, the specific antigen (the target molecule recognized by the antibody) or its epitope can be fixed on the column and the immunospecific antibody can be purified by immunoaffinity chromatography.
  • immunoaffinity chromatography For purification of immunoglobulins, for example, D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28) can be referred to.
  • the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets.
  • the strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K D ) of the interaction.
  • K D refers to a particular antibody - antigen interaction solutions dissociation equilibrium constant, which is used to describe the binding affinity between antibody and antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -9 M, such as less than about 10 -9 M, 10 -10 M, Affinities (K D ) of 10 -11 M or 10 -12 M or less bind the antigen.
  • the specific binding properties between the two molecules can be measured using methods well known in the art, for example, using surface plasmon resonance (SPR) in a BIACORE instrument.
  • the AMH antigen is diluted with carbonate buffer (20mmol / L CB, pH9.6) and coated on a polyvinyl chloride plate.
  • the pretreatment is to compare the antibody purified by the protein A column with the synthetic AMH truncated antigen sequence (each synthesized (See the last supplement for sequence) (100ng / ml antibody: 50ug / ml truncated antigen) Incubate for 30min, which is the test sample; the control sample is 100ng / ml antibody: PBS, incubate for 30min, and then incubate the test sample and The control sample was added to the AMH antigen plate and incubated for 30 minutes, the plate was washed 5 times, GAM-HRP (1/5000 dilution) was added for 30 minutes, the plate was washed 5 times, and the substrate was added for 15 minutes.
  • control sample OD value-test sample OD value (control sample OD value-test sample OD value) / control sample OD value
  • judgment standard is: more than 90% It shows a good competitive effect, showing that the antibody should recognize the epitope, the data is shown in Table 1 below.
  • the truncated antigen sequences are as follows:
  • the 5 monoclonal antibodies identified are highly reactive in full-length AMH and truncated antigen in 26-85.
  • the AMH antigen is diluted with carbonate buffer (20mmol / L CB, pH9.6) and coated on a polyvinyl chloride plate.
  • the pretreatment is to compare the antibody purified by the protein A column with the synthetic AMH truncated antigen sequence (each synthesized The sequence is shown in Table 3) (100ng / ml antibody: 50ug / ml truncated antigen) incubated for 30min, which is the test sample; the control sample is 100ng / ml antibody: PBS, incubated for 30min, and then the incubated test sample and The control sample was added to the AMH antigen plate and incubated for 30 minutes, the plate was washed 5 times, GAM-HRP (1/5000 dilution) was added for 30 minutes, the plate was washed 5 times, and the substrate was added for 15 minutes.
  • the AMH antigen is diluted with carbonate buffer (20mmol / L CB, pH9.6) and coated on the polyvinyl chloride plate.
  • Pretreatment of the antibody purified by the protein A column and the amino acid mutations of AMH36-47 of different humans synthesized The short peptides are incubated in proportion (100ng / ml antibody: 50ug / ml truncated antigen) for 30min, which is the test sample; the control sample is (100ng / ml antibody: PBS) for 30min, and then the test sample after incubation
  • the value is read at the wavelength of 450/620 in the microplate reader, and finally the calculated OD value is calculated and analyzed, the calculation formula is (control sample OD value-test sample OD value) / control sample OD value, the judgment standard is: less than 90%
  • the value indicates that the amino acid is a key amino acid, and the mutation has a great influence on the antibody response.
  • the data analysis is shown in Table 4 below.
  • the key position of C8H3-2 is 42.45;
  • the key influence site of 7D11-1 is 38.42.45;
  • the key position of C17B10 is 42.45.46;
  • the key position of G8B11 is 39.45;
  • the AMH antigen is diluted with carbonate buffer (20mmol / L CB, pH9.6) and coated on the polyvinyl chloride plate.
  • Pretreatment of the antibody purified by the protein A column and the amino acid mutations of AMH36-47 of different humans synthesized The short peptides were incubated in proportion (see Table 6 for their respective synthetic sequences) (100ng / ml antibody: 50ug / ml truncated antigen) for 30min, which was the test sample; the control sample was (100ng / ml antibody: PBS) for 30min, Then add the test sample and control sample after incubation to the AMH antigen plate and incubate for 30min, wash the plate 5 times, add GAM-HRP (1/5000 dilution) and incubate for 30min, wash the plate 5 times, add substrate to incubate 15min.
  • Monoclonal antibody is diluted with phosphate buffer (20mmol / LPB, pH7.4), coated on a polyvinyl chloride plate after dilution, and a monoclonal antibody is labeled with horseradish peroxidase (labeled monoclonal antibody is F10G7-2, Xiamen Wantai Kerry Biotechnology Co., Ltd., article number M3382; it specifically binds to amino acids 260 to 271 of the AMH epitope).
  • AMH quality control product dilution value (20ng / ml, 10ng / ml, 5ng / ml, 2.5ng / ml, 1.25ng / ml, 0.5ng / ml, 0.1ng / ml, 0.05ng / ml, 0.01ng / ml).
  • Sample sera, positive control and blank control were added to the corresponding wells 40ul, incubated at 37 ° for 40min, washed plate 5 times with F10G7-2-HRP (1/500 dilution) incubated for 40min, washed plate 5 times, added bottom The material was incubated for 15 minutes. The value is read under the wavelength of 450-620 in the microplate reader, and the data analysis is shown in Table 7 below.
  • R1 reagent (currently added system).
  • a second antibody monoclonal antibody F10G7-2, Xiamen Wantai Kerry Biotechnology Co., Ltd., article number M3382
  • the coating amount of the second antibody is 5 ⁇ 15ug / ml acridinium ester.
  • Tris buffer containing 0.1% ⁇ 0.5% bovine serum albumin at pH 8.0 ⁇ 9.0 to stop ⁇ 3h.
  • the pre-excitation solution is a 1% (w / v) hydrogen peroxide solution.
  • the excitation solution is a 1mol / L sodium hydroxide solution.
  • the detection method of the above AMH assay kit includes the following steps: 100ul of sample R1 reagents are added to the reaction wells in sequence at a volume ratio of 1: 1, and incubated for 15 to 20 minutes. After the incubation, use Wash with Tween 20 phosphate buffer, then add 20-25ul R2 reagent, incubate for 10-15min, after incubation, wash with Tween 20 phosphate buffer containing 0.05-0.08%, add pre-stimulation solution 100-200ul For pre-excitation. Remove the pre-excitation liquid, add 100-200ul of the excitation liquid to perform excitation and detection. Among which (100) samples were tested, the correlation is as follows:

Abstract

本发明属于生物医学检验领域，提供了针对人抗缪勒管激素的特异性抗体及其应用。

Description

针对人抗缪勒管激素的特异性抗体及其应用 技术领域
本发明属于生物医学检验领域，主要涉及针对人抗缪勒管激素的特异性抗体及其应用。
背景技术
抗缪勒管激素(anti-Mullerian hormone,AMH)，也称为抗苗勒管激素，是由睾丸未成熟支持细胞和卵巢生长卵泡的颗粒细胞分泌的一类单糖蛋白，属于转化生长因子β超家族，具有抑制雄性缪勒管发育、调节两性生殖细胞和性腺发育的重要功能。近年来，各国学者对其在女性生殖领域的作用进行了广泛的研究。在女性中，AMH可以调节卵泡发育、反映卵巢储备功能、预测卵巢对超排卵的反应性，对生殖内分泌疾病的研究有一定的价值。目前，关于免疫法检测AMH的记载有许多。例如，美国专利7,897,350记载了检测样本中AMH的组合物和方法，其中抗体结合AMH的成熟区或C端区。欧洲专利EP2161579A记载了用于检测或定量样本中至少一种AMH的生物活性形式，例如切断的AMH或C端AMH的方法以及与其结合的抗体。
另外，也有一些其他出版物中公开了抗AMH的N端区的抗体。例如，Hudson et al.(1990)J.Clin.Endocrin.Metab.70:16-22描述了一种免疫分析技术，其中两种单克隆抗体分别针对AMH的前体和N端区。Long et al.(2000)J.CIin.Endocrin.Metab.85:540-544描述了一种免疫分析技术，其也使用了两种单克隆抗体，一种结合AMH的N端表位，另一种结合AMH的C端区。此外，W02014204327A1中也公开了一种抗AMH的N端区的抗体，其中N端区指人前肽的1-451个残基或者在除去前导序列时为25-451个残基。然而，当利用上述抗AMH抗体检测抗原时，存在检测稳定性差且灵敏度低的问题。
因此，本领域需要开发针对人抗缪勒管激素的特异性抗体。
发明内容
本发明的第一个方面提供了针对人抗缪勒管激素的单克隆抗体抗体或其抗原结合片段，所述抗体或其抗原结合片段特异性结合人抗缪勒管激素的表位，所述表位选自包含在跨越抗缪勒管激素的第26至85位氨基酸残基的序列中的表位，包含在跨越抗 缪勒管激素的第38至45位氨基酸的序列中的表位，包含在跨越抗缪勒管激素的第40至46位氨基酸残基的序列中的表位，包含在跨越抗缪勒管激素的第37至44位氨基酸残基的序列中的表位，包含在跨越抗缪勒管激素的第39至45位氨基酸残基的序列中的表位，包含在跨越抗缪勒管激素的第36至47位氨基酸残基的序列中的表位，包含在跨越抗缪勒管激素的第37至46位氨基酸残基的序列中的表位。优选地，抗体或其抗原结合片段特异性结合包含在跨越抗缪勒管激素的第38至45位氨基酸残基的序列中的表位。
本发明的单克隆抗体特异性识别人抗缪勒管激素表位，不与其他物种来源的抗缪勒管激素产生交叉反应，包括山羊抗缪勒管激素，猫头鹰抗缪勒管激素，牛抗缪勒管激素，马抗缪勒管激素，猴子抗缪勒管激素，犬抗缪勒管激素，鹿抗缪勒管激素，小鼠抗缪勒管激素，豚鼠抗缪勒管激素等。在某些优选的实施方案中，本发明的抗体是嵌合抗体或人源化抗体。在某些优选的实施方案中，本发明的抗原结合片段选自scFv、Fab、Fab’、(Fab’) 2、Fv片段、双抗体(diabody)。
在一个具体实施方式中，本发明提供了针对人抗缪勒管激素的单克隆抗体抗体，其特异性结合包含在跨越抗缪勒管激素的第38至45位氨基酸残基的序列中的表位。
优选地，所述抗体包含分别如SEQ ID NO:1-3所示的重链CDR序列CDR1 GFAFSTYD，CDR2 ISPGGGAT，CDR3 AGRRDYYGMDY；和SEQ ID NO:4-6所示的轻链CDR序列CDR1 QSLVHSNGNTY，CDR2 KVS，CDR3 SQSTHVPWT。
更优选地，所述抗体包含如SEQ ID NO:7所示的重链可变区序列
EVQLVESGGGLVKPGGSLKLSCAASGFAFSTYDMSWVRQTPEKRLEWVAYISPGGGATYYPDTVKGRFTISRDNAKNTLYLQMTSLKSEDTAMYYCAGRRDYYGMDYWGQGTSVTVSS和SEQ ID NO:8所示的轻链可变区序列
Figure PCTCN2019115998-appb-000001
在优选的实施方式中，本发明的单克隆抗体是于2018年8月19日保藏于中国典型培养物保藏中心(中国，武汉，武汉大学)的杂交瘤细胞株C9F2-2分泌的单克隆抗体，该杂交瘤细胞株的保藏编号为CCTCC NO:C2018183。
在另一个方面，本发明提供了一种分离的核酸分子，其包含编码本发明的抗体或 其抗原结合片段，或其重链可变区和/或轻链可变区的核苷酸序列。在某些优选的实施方案中，所述分离的核酸分子编码本发明的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区。
在另一个方面，本发明提供了一种载体(例如克隆载体或表达载体)，其包含本发明的分离的核酸分子。在某些优选的实施方案中，本发明的载体是例如质粒，粘粒，噬菌体等。在某些优选的实施方案中，所述载体能够在受试者(例如哺乳动物，例如人)体内表达本发明的抗体或其抗原结合片段。
在另一个方面，本发明提供了一种宿主细胞，其包含本发明的分离的核酸分子或本发明的载体。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。在某些优选的实施方案中，本发明的宿主细胞是哺乳动物细胞。
本发明的抗体或其抗原结合片段能够特异性结合人抗缪勒管激素，从而可用于检测人抗缪勒管激素在样品中的存在或其水平。
因此，在另一个方面，本发明提供了一种试剂盒，其包括本发明的抗体或其抗原结合片段。在某些优选的实施方案中，本发明的抗体或其抗原结合片段带有可检测的标记。在本发明中，所述可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。特别优选的是，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。这类标记是本领域熟知的，包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如， 3H、 125I、 35S、 14C或 32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、发光物质(例如化学发光物质，如吖啶酯类化合物)、磁珠(例如，
Figure PCTCN2019115998-appb-000002
)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。教导该标记物的使用的专利包括，但不限于，美国专利3,817,837；3,850,752；3,939,350；3,996,345；4,277,437；4,275,149；及4,366,241(全部通过引用并入本文)。本发明中涵盖的标记物可通过本领域已知的方法检测。例如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测 通过酶对底物的作用产生的反应产物来检测，及测热标记物通过简单可视化着色标记物来检测。在某些实施方案中，可通过不同长度的接头将如上所述的可检测的标记连接至本发明的抗体或其抗原结合片段，以降低潜在的位阻。
在另一个方面，本发明提供了检测人抗缪勒管激素在样品中的存在或其水平的方法，其包括使用本发明的抗体或其抗原结合片段的步骤。在一个优选的实施方案中，本发明的抗体或其抗原结合片段还带有可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用带有可检测的标记的试剂来检测本发明的抗体或其抗原结合片段。所述方法可以用于诊断目的，或者非诊断目的(例如，所述样品是细胞样品，而非来自患者的样品)。
在另一个方面，提供了本发明的抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测人抗缪勒管激素在样品中的存在或其水平。
在具体的实施方案中，本发明提供了一种用于在源自人体液的生物样品中检测AMH其片段的方法，该方法包括：
将样品与针对抗缪勒管激素不同表位的至少两种抗体或其抗原结合片段相接触，以及定性或定量检测所述至少两种抗体与抗缪勒管激素或所述片段的结合，其中结合指示所述样品中抗缪勒管激素或所述片段存在或浓度；
其中至少一种抗体或其抗原结合片段针对包含在抗缪勒管激素的第37至46位氨基酸残基的序列中的表位。
在优选的实施方式中，其中一种抗体或其抗原结合片段针对包含在抗缪勒管激素的第38至45位氨基酸残基的序列中的表位，另一种抗体或其抗原结合片段针对包含在抗缪勒管激素的第358至369位氨基酸残基的序列中的表位，或包含在抗缪勒管激素的第491至502位氨基酸残基的序列中的表位，或包含在抗缪勒管激素的第260至271位氨基酸残基的序列中的表位，或包含在抗缪勒管激素的第330至343位氨基酸残基的序列中的表位。
在本发明的方法的优选实施方案中，所述至少两种抗体或其抗原结合片段中的至少一种被固定化在固相表面上。优选情况下，另一种或另一些抗体中的至少一种被标记，优选通过共价连接化学发光或荧光染料进行标记。
在本发明的方法的具体实施方案中，针对包含在跨越抗缪勒管激素的第38至45位氨基酸残基的序列中的表位的抗体或其抗原结合片段被固定化在固相表面上。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、生物化学、核酸化学、免疫学实验室等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。这些氨基酸残基的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol. 27:55-77,2003)。
在本发明中，本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat编号系统确定。
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、F(ab’) 2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)、线性抗体(linear antibody)、纳米抗体(技术来自Domantis)、结构域抗体(技术来自Ablynx)、probody和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“单克隆抗体”、“单抗”、“mAb”具有相同的含义且可互换使用可互换，其是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即，除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2 种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。此外，修饰语“单克隆”仅表明该抗体的特征为从高度同源的抗体群中获得，不能理解为需要通过任何特定方法来制备所述抗体。
本发明的单克隆抗体可以通过多种技术进行制备，例如杂交瘤技术(参见，例如Kohler等人.Nature,256:495,1975)，重组DNA技术(参见，例如美国专利申请4,816,567)，或噬菌体抗体库技术(参见，例如Clackson等.Nature352：624-628,1991，或Marks等.J.Mol.Biol.222：581-597,1991)。
抗体可通过公知的技术，例如使用蛋白A或蛋白G的亲和层析进行纯化。随后或作为替代，可将特异性抗原(该抗体识别的靶分子)或其抗原表位固定在柱上，并通过免疫亲合层析法来纯化免疫特异性抗体。免疫球蛋白的纯化可参考例如D.Wilkinson(The Scientist,published by The Scientist,Inc.,Philadelphia Pa.,Vol.14,No.8(Apr.17,2000),pp.25-28)。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(K D)表示。在本发明中，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于约10 -9M，例如小于约10 -9M、10 -10M、10 -11M或10 -12M或更小的亲和力(K D)结合该抗原。两分子间的特异性结合性质可使用本领域公知的方法进行测定，例如使用表面等离子体共振术(SPR)在BIACORE仪中测定。
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列实施例仅用于说明本发明，而不是对本发明的范围的限定。根据优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得可实施。
实施例1.针对人抗缪勒管激素的特异性抗体的产生
1.选择6只雌性BALB/c小鼠以AMH抗原乳化完全弗氏佐剂(CFA)(Sigma，St.Louis，MO)皮下免疫，两周后使用同样的抗原乳化不完全弗氏佐剂(IFA)(Sigma，St.Louis，MO)进行免疫，间隔2周继续免疫2针。
2.在最初免疫接种后6和8周以及在处死时从眼后毛细血管获得血样，所述处死是在9周后，并在同时取出脾脏。将脾细胞与SP2/0骨髓瘤细胞融合，CO 2培养箱培养7天，细胞上清换液，2天后，对产生杂交瘤细胞系检测。
3.间接法筛选，AMH抗原用碳酸缓冲液(20mmol/L CB pH 9.6)稀释后包被于聚氯乙烯板上，然后加入待检样品，最后加入HRP标记的羊抗鼠二抗(保存于20mmol/L PBS pH 7.4)中，筛选细胞系分泌与固定化的重组人类AMH(Anti-MüllerianHormone)结合抗体的能力。
4.细胞经过三次稳定克隆化，得到稳定细胞株，大量培养，冻存细胞保种，诱导小鼠产生腹水，腹水亲和柱纯化，得到纯度大于90％的5个单抗C8H3-2、C9F2-2、7D11-1、C17B10、G8B11，保存在20mmol PBS溶液中。单克隆抗体C9F2-2于2018年8月19日保藏于中国典型培养物保藏中心，杂交瘤细胞株C9F2-2的保藏编号为CCTCC NO:C2018183。
实施例2：
单抗评价与截短抗原鉴定：
1.AMH抗原用碳酸缓冲液(20mmol/L CB，pH 9.6)稀释后包被于聚氯乙烯板上，预处理将蛋白A柱纯化的抗体与合成的AMH截短抗原序列进行比例(各自合成序列见最后补充)(100ng/ml抗体：50ug/ml截短抗原)温育30min，即为检测样品；对照样品是100ng/ml抗体：PBS，温育30min，再将温育后的检测样品和对照样品加入到AMH抗原板内温育30min，清洗板5次，加入GAM-HRP(1/5000稀释)温育30min，清洗板5次，加入底物温育15min。酶标仪波长450/620下读值，最后将检测出来的OD值进行计算分析，计算公式为(对照样品OD值－检测样品OD值)/对照样品OD值，判定标准为：大于90％以上显示有良好的竞争效果，显示抗体应该识别该表位，数据如下表1所示。
Figure PCTCN2019115998-appb-000003
Figure PCTCN2019115998-appb-000004
各截短抗原序列如下所示：
26-85aa：
Figure PCTCN2019115998-appb-000005
86-185aa
Figure PCTCN2019115998-appb-000006
171-270aa
Figure PCTCN2019115998-appb-000007
256-355aa
Figure PCTCN2019115998-appb-000008
341-440aa
Figure PCTCN2019115998-appb-000009
426-525aa
Figure PCTCN2019115998-appb-000010
461-560aa
Figure PCTCN2019115998-appb-000011
2.经鉴定5个单抗在全长AMH反应性强，在26-85截短抗原反应性强。
实施例3：
单抗表位鉴定：
1.AMH抗原用碳酸缓冲液(20mmol/L CB，pH 9.6)稀释后包被于聚氯乙烯板上，预处理将蛋白A柱纯化的抗体与合成的AMH截短抗原序列进行比例(各自合成序列见表3)(100ng/ml抗体：50ug/ml截短抗原)温育30min，即为检测样品；对照样品是100ng/ml抗体：PBS，温育30min，再将温育后的检测样品和对照样品加入到AMH抗原板内温育30min，清洗板5次，加入GAM-HRP(1/5000稀释)温育30min，清洗板5次，加入底物温育15min。酶标仪波长450/620下读值，最后将检测出来的OD值进行计算分析，计算公式为(对照样品OD值－检测样品OD值)/对照样品OD值，判定标准为：大于90％以上显示有良好的竞争效果，显示抗体应该识别该表位，数据分析见下表2。
  C8H3-2 C9F2-2 7D11-1 C17B10 G8B11
21-35 23％ 10％ 20％ 2％ 29％
31-45 79％ 90％ 100％ 98％ 97％
41-55 100％ 68％ 49％ 10％ 100％
51-65 -23％ 9％ 6％ -9％ 2％
61-75 17％ 35％ 50％ 4％ 35％
71-85 27％ 21％ 21％ 4％ 36％
31-41 19％ 10％ 87％ -1％ 32％
31-42 18％ 16％ 81％ 30％ 33％
31-43 21％ 25％ 84％ 22％ 31％
31-44 13％ 38％ 99％ 18％ 32％
32-41 2％ 9％ 78％ -2％ 19％
33-41 26％ 9％ 70％ 9％ 37％
34-41 -1％ 3％ 66％ 18％ 23％
34-45 90％ 98％ 100％ 98％ 99％
35-45 94％ 97％ 100％ 98％ 99％
36-44 34％ 32％ 100％ 27％ 41％
36-45 91％ 94％ 100％ 98％ 99％
36-47 99％ 100％ 100％ 98％ 99％
37-43 9％ 57％ 83％ 37％ 18％
37-44 3％ 24％ 100％ 13％ 28％
37-45 93％ 91％ 100％ 98％ 100％
38-44 9％ 33％ 61％ 15％ 32％
38-45 87％ 92％ 78％ 99％ 98％
39-45 84％ 68％ 22％ 101％ 97％
40-45 38％ -14％ 2％ 4％ 81％
40-46 90％ -2％ 18％ 39％ 96％
40-47 97％ -19％ 4％ 40％ 100％
41-45 53％ 15％ 21％ 30％ 43％
41-46 82％ 15％ 29％ 29％ 56％
41-47 93％ 64％ 29％ 20％ 97％
41-48 98％ -1％ 14％ 12％ 100％
41-49 101％ 3％ 23％ 9％ 99％
41-50 99％ 65％ 62％ 18％ 99％
42-46 28％ -10％ 13％ -9％ 13％
42-47 86％ 6％ 19％ -4％ 80％
42-48 85％ 2％ 18％ 9％ 82％
42-49 90％ -8％ 7％ -20％ 87％
42-50 91％ 57％ 16％ -2％ 86％
43-50 8％ 17％ 52％ 3％ 31％
41-51 99％ 71％ 40％ 4％ 99％
42-51 93％ 60％ 30％ 6％ 83％
43-51 -9％ -10％ 3％ -4％ 4％
表3
名称 氨基酸序列
21－35 GTEALRAEEPAVGTS
31－45 AVGTSGLIFREDLDW
41－55 EDLDWPPGSPQEPLC
51－65 QEPLCLVALGGDSNG
61－75 GDSNGSSSPLRVVGA
71－85 RVVGALSAYEQAFLG
31-41 AVGTSGLIFRE
31-42 AVGTSGLIFRED
31-43 AVGTSGLIFREDL
31-44 AVGTSGLIFREDLD
32-41 VGTSGLIFRE
33-41 GTSGLIFRE
34-41 TSGLIFRE
34-45 TSGLIFREDLDW
35-45 SGLIFREDLDW
36-44 GLIFR EDLD
36-45 GLIFREDLDW
36-47 GLIFR EDLDW PP
37-43 LIFR EDL
37-44 LIFR EDLD
37-45 LIFREDLDW
38-44 IFR EDLD
38-45 IFREDLDW
39-45 FREDLDW
40-45 REDLDW
40-46 REDLDWP
40-47 REDLDWPP
41-45 EDLDW
41-46 EDLDWP
41-47 EDLDWPP
41-48 EDLDWPPG
41-49 EDLDWPPGS
41-50 EDLDWPPGSP
42-46 DLDWP
42-47 DLDWPP
42－48 DLDWPPG
42-49 DLDWPPGS
42-50 DLDWPPGSP
43-50 LDWPPGSP
41-51 EDLDWPPGSPQ
42-51 DLDWPPGSPQ
43-51 LDWPPGSPQ
1.通过实验数据分析单抗表位初步验证在C8H3-2(41-55)，C9F2-2(31-45),7D11-1(31-45),G8B11(31-45/41-55),C17B10(31-45)。
2.通过实验数据分析单抗表位验证在C8H3-2(40-46)，C9F2-2(38-45),7D11-1(37-44),G8B11(40-46/41-48/39-45),C17B10(39-45)。
实施例4
1.AMH抗原用碳酸缓冲液(20mmol/L CB，pH 9.6)稀释后包被于聚氯乙烯板上，预处理将蛋白A柱纯化的抗体与合成的不同人的AMH36-47各个氨基酸突变的短肽进行按比例(100ng/ml抗体：50ug/ml截短抗原)温育30min，即为检测样品；对照样品是(100ng/ml抗体：PBS)温育30min，再将温育后的检测样品和对照样品加入到AMH抗原板内温育30min，清洗板5次，加入GAM-HRP(1/5000稀释)温育30min，清洗板5次，加入底物温育15min。酶标仪波长450/620下读值，最后将检测出来的OD值进行计算分析，计算公式为(对照样品OD值－检测样品OD值)/对照样品OD值，判定标准为：低于90％的值显示该氨基酸为关键氨基酸，突变对于抗体的反应影响很大。数据分析见下表4。
突变的位置 突变后序列 C8H3-2 C9F2-2 7D11-1 C17B10 G8B11
36 ALIFREDLDWPP 100％ 100％ 101％ 99％ 99％
37 GAIFREDLDWPP 99％ 97％ 100％ 99％ 99％
38 GLAFREDLDWPP 100％ 96％ 84％ 100％ 100％
39 GLIAREDLDWPP 98％ 9％ 100％ 91％ 88％
40 GLIFAEDLDWPP 99％ 88％ 100％ 100％ 99％
41 GLIFRADLDWPP 98％ 7％ 99％ 90％ 99％
42 GLIFREALDWPP 18％ 98％ 69％ 48％ 96％
43 GLIFREDADWPP 99％ 94％ 100％ 100％ 100％
44 GLIFREDLAWPP 99％ 64％ 99％ 97％ 99％
45 GLIFREDLDAPP 27％ 100％ 12％ 24％ 29％
46 GLIFREDLDWAP 99％ 99％ 98％ 84％ 99％
47 GLIFREDLDWPA 99％ 99％ 99％ 99％ 99％
2.从分析的结果可知，
C8H3-2关键的影响位点是42.45位；
C9F2-2关键的影响位点是39.41.44位；
7D11-1关键的影响位点是38.42.45位；
C17B10关键的影响位点是42.45.46位；
G8B11关键的影响位点是39.45位；
与上面表位鉴定的结果相符合。
实施例5
单抗种属特异性评价：
1.AMH抗原用碳酸缓冲液(20mmol/L CB，pH 9.6)稀释后包被于聚氯乙烯板上，预处理将蛋白A柱纯化的抗体与合成的不同人的AMH36-47各个氨基酸突变的短肽进行按比例(各自合成序列见表6)(100ng/ml抗体：50ug/ml截短抗原)温育30min，即为检测样品；对照样品是(100ng/ml抗体：PBS)温育30min，再将温育后的检测样品和对照样品加入到AMH抗原板内温育30min，清洗板5次，加入GAM-HRP(1/5000稀释)温育30min，清洗板5次，加入底物温育15min。酶标仪波长450/620下读值，最后将检测出来的OD值进行计算分析，计算公式为(对照样品 OD值－检测样品OD值)/对照样品OD值，判定标准为：大于90％以上代表有交叉反应。数据分析见下表5。
不同物种的序列 C8H3 C9F2-2 7D11 17B10 G8B11
AMH3647－1 1％ -6％ 46％ 41％ 46％
AMH3647－2 77％ 11％ 4％ 85％ 99％
AMH3647－3 7％ 8％ -3％ -4％ 24％
AMH3647－4 13％ 22％ 99％ 93％ 86％
AMH3647－5 83％ 32％ 98％ 98％ 99％
AMH3647－6 12％ 3％ -17％ -8％ 10％
AMH3647－7 19％ 6％ -4％ 19％ 19％
AMH3647－8 13％ 8％ 62％ 33％ 48％
AMH3647－9 15％ -3％ 58％ 30％ 38％
AMH3647－10 19％ 8％ -6％ -3％ 31％
AMH3647－11 24％ 5％ 30％ 13％ 29％
AMH3647－12 41％ 5％ 29％ 16％ 48％
AMH3647－13 27％ 11％ 31％ 26％ 35％
AMH3647－14 28％ 29％ 42％ 29％ 86％
AMH3647－15 65％ 2％ -16％ 10％ 78％
AMH3647－16 30％ -2％ -18％ 2％ 100％
AMH3647－17 92％ 9％ -23％ 37％ 99％
AMH3647－18 42％ 19％ 56％ 64％ 95％
AMH3647－19 35％ -1％ -16％ 44％ 93％
AMH3647－20 21％ 3％ 59％ 61％ 32％
AMH3647－21 15％ 10％ 18％ -7％ 16％
AMH3647－22 75％ -9％ 60％ 20％ 82％
AMH3647－23 28％ -8％ 46％ 1％ 0％
AMH3647－24 94％ 19％ 55％ 99％ 99％
AMH3647－25 37％ -2％ 78％ 34％ 95％
AMH3647－26 93％ 61％ 42％ 99％ 99％
AMH3647－27 100％ 100％ 99％ 99％ 99％
表6
序列 种属 合成编号
ALIFQQDWDWPL 鹿 AMH3647－1
DLIFHEDWAWPP 河狸 AMH3647－2
GLIFFEDGIWPL 仓鼠 AMH3647－3
GLIFHEDWDWLP 狐猴 AMH3647－4
GLIFHEDWDWPP 冕狐猴 AMH3647－5
GLIFHKDWDWQP 大婴猴 AMH3647－6
GLIFHPDWDWQP 猫 AMH3647－7
GLIFHQDWDWPA 鼠耳蝠 AMH3647－8
GLIFHQDWDWSP 菊头蝠 AMH3647－9
GLIFHWDWNWPP 猪 AMH3647－10
GLIFLEDGIWPP 林鼠 AMH3647－11
GLIFLEDGLWPP 沙鼠 AMH3647－12
GLIFLEDGVWPP 大鼠 AMH3647－13
GLIFLQDGMWSP 小鼹形鼠 AMH3647－14
GLIFPEDQVWPP 豚鼠 AMH3647－15
GLIFPEDWVWPP 八齿鼠 AMH3647－16
GLIFPEDLDWPP 黑猩猩 AMH3647－17
GLIFRQDWDWPP 白犀 AMH3647－18
GLLFQPDWDWPP 犬 AMH3647－19
TLIFQQDWDWPL 山羊 AMH3647－20
TLIFQQGWDWPL 绵羊 AMH3647－21
GLIFLEDELWPP 小鼠 AMH3647－22
ALIFQQAWDWPL 牛 AMH3647－23
GLIFGEDVDWPP 猫头鹰 AMH3647－24
GLIFHQDWDWPP 马 AMH3647－25
GLIFREDLGWPP 猴子 AMH3647－26
GLIFREDLDWPP 人 AMH3647－27
2.通过实验数据分析，我们C9F2-2表位38-45的单抗在种属特异上与骆驼，猫头鹰，牛，猴子，，马，小鼠，以及犬、绵羊序列均无交叉。
实施例7
单抗配对酶免评价：
1.单克隆化抗体用磷酸盐缓冲液(20mmol/LPB，pH7.4)，稀释后包被于聚氯乙烯板上，一株单克隆抗体标记上辣根过氧化物酶(标记单抗为F10G7-2，厦门万泰凯瑞生物技术有限公司，货号M3382；其特异性结合AMH表位260至271位氨基酸)。AMH质控品用稀释液稀释值(20ng/ml，10ng/ml，5ng/ml，2.5ng/ml，1.25ng/ml，0.5ng/ml，0.1ng/ml，0.05ng/ml，0.01ng/ml)。样本血清，阳性对照，空白对照，分别加入相应孔中40ul，置37°温育40min，清洗板5次加入F10G7-2-HRP(1/500稀释)温育40min，清洗板5次，加入底物温育15min。酶标仪波长450-620下读值，数据分析见下表7。
背景值 C8H3-2 C17B10 G8B11 7D11-1 C9F2-2
2.75 2.80 2.19 1.30 1.50 1.14
3.39 4.25 2.72 1.50 1.90 1.53
6.92 7.98 6.31 3.11 4.51 3.05
9.4 10.26 8.19 3.91 4.65 3.62
0.935 0.91 0.18 0.24 0.47 0.23
1.72 3.00 2.22 0.92 1.60 0.90
2.85 5.22 4.49 1.30 3.11 1.86
1.56 1.77 0.90 0.40 0.81 0.45
0.751 0.73 -0.08 0.12 0.23 0.19
3.24 3.80 2.20 0.96 1.53 1.18
0.473 0.42 -0.04 0.05 0.23 0.15
2.28 2.10 1.45 0.62 1.04 0.78
6.66 7.23 5.27 3.05 3.05 2.87
0.351 0.37 -0.02 0.15 0.01 -0.01
0.717 0.57 -0.27 0.14 0.15 0.13
3.75 4.26 2.71 1.56 2.92 1.73
0.359 0.28 -0.21 0.07 0.22 0.08
1.89 2.37 0.79 0.91 1.21 1.00
1.05 1.05 -0.23 0.30 0.48 0.29
6.97 7.29 4.02 2.41 3.10 2.81
8.14 6.11 4.10 3.38 3.72 2.98
4.16 3.24 1.36 1.21 2.04 1.55
0.892 0.48 0.02 0.19 0.40 0.25
0.321 0.20 -0.29 0.01 -0.06 -0.03
2.52 2.48 1.07 0.92 1.09 0.79
8.16 6.77 4.44 2.72 3.18 2.57
6.72 6.64 3.60 2.54 2.92 2.32
4.9 4.49 1.80 1.32 1.81 1.36
2.1 1.47 -0.31 0.37 0.55 0.40
1.95 3.12 0.87 1.05 1.98 1.12
6.57 5.29 1.92 1.44 2.56 1.86
1.49 1.25 0.24 0.38 0.87 0.59
6.2 6.52 4.39 1.95 2.39 2.44
1.36 1.01 -0.04 0.28 0.39 0.46
2.86 2.22 1.06 1.06 0.64 1.05
9.84 9.97 7.23 4.54 2.64 3.83
11.17 8.81 6.00 3.91 2.83 3.69
2.28 2.86 0.89 0.11 0.85 1.03
           
r2 0.9164 0.8131 0.9212 0.7362 0.9386
2.通过酶免配对验证38份血清比较相关性，如上特异性单抗C9F2-2配对表现出较好相关性。
实施例8
单抗配对发光评价：
1.下述实施例中的AMH检测试剂盒，其特征在于：包括如下组分：M试剂，含有0.5～1mg/ml的包被了第一抗体的磁性微粒、其中第一抗体(C9F2-2)的包被量为20～80ug/ml磁性微粒，上述磁性微粒购自Thermo Fisher Scientific，该试剂制备方法为：将所述第一抗体与磁性微粒混合于pH＝5.0～6.0的2-吗啉乙磺酸缓冲液中，于25℃～37℃包被1～3h，再加入pH＝8.0～9.0的0.1％～0.5％牛血清白蛋白的磷酸盐缓冲液终止1～3h，将其中包被后的磁性微粒分离后分散于pH＝7.0～8.0的磷酸盐缓冲液中，再加入酪蛋白和牛血清白蛋白。R1试剂，(目前加的体系)。R2试剂，含有包被了第二抗体(单克隆抗体F10G7-2，厦门万泰凯瑞生物技术有限公司，货号M3382)的吖啶酯、0.5～1％酪蛋白和0.5～1％牛血清白蛋白，溶剂为pH＝7.0～8.0的磷酸盐缓冲液，其中第二抗体的包被量为5～15ug/ml吖啶酯，该试剂的制备方法为：将所述第二抗体与吖啶酯混合于pH＝8.0～9.0的磷酸盐缓冲液中，于25℃～37℃包被1～3h，再加入pH＝8.0～9.0的含0.1％～0.5％牛血清白蛋白的Tris缓冲液终止1～3h。得母液，将该母液用pH＝7.0～8.0的磷酸盐缓冲液稀释至1～500倍使用。预激发液，为1％(w/v)过氧化氢溶液。激发液，为1mol/L氢氧化钠溶液。
2.上述AMH测定试剂盒进行检测的方法，包括如下步骤：按1：1的体积比在反应孔中依次加入样本R1试剂各100ul，孵育15～20min，孵育结束后，用含0.05～0.08％的吐温20磷酸盐缓冲液洗涤，再加入20～25ul R2试剂，孵育10～15min，孵育结束后，用含0.05～0.08％的吐温20磷酸盐缓冲液洗涤，加入预激发液100～200ul，进行预激发。去除预激发液，加入激发液100～200ul，进行激发和检测。其中检测(100)份样本，相关性如下：
样本序号 背景值ng/ml 定值
1 6.27 6.83
2 4.75 4.73
3 6.53 7.29
4 1.75 1.46
5 7.23 8.49
6 3.75 4.22
7 1.15 0.87
8 1.43 1.48
9 3.05 3.11
10 2.66 2.51
11 2.79 3.18
12 1.35 1.27
13 1.05 1.04
14 3.39 3.56
15 2.52 3.33
16 5.66 6.36
17 1.21 1.22
18 5.53 6.05
19 3.35 3.17
20 12.53 16.34
21 0.449 0.46
22 1.14 1.23
23 3.72 4.13
24 1.28 1.04
25 3.07 4.57
26 0.83 0.92
27 4.33 5.15
28 3.91 4.94
29 2.21 2.47
30 2.29 2.37
31 1.93 2.16
32 1.4 1.18
33 0.866 0.58
34 0.324 0.34
35 1.47 1.65
36 1.49 1.35
37 3.49 3.44
38 2.27 2.17
39 0.693 0.57
40 4.08 3.07
41 3.37 5.15
42 1.51 1.61
43 4.54 5.42
44 1.34 1.54
45 4.97 6.41
46 1.59 1.84
47 4.21 4.75
48 3.73 3.60
49 1.89 1.70
50 9.22 10.53
51 6.72 8.87
52 4 6.31
53 4.8 7.47
54 3.46 3.38
55 4.65 3.81
56 0.38 0.34
57 5.59 6.60
58 2.83 4.77
59 0.088 0.11
60 4.99 6.78
61 2.23 2.86
62 4.24 4.69
63 3.83 4.22
64 2.79 2.52
65 3.09 2.76
66 0.754 0.69
67 0.775 0.73
68 2.67 2.94
69 1.73 1.66
70 1.08 1.39
71 4.16 4.57
72 5.56 7.72
73 2.19 1.90
74 3.97 3.39
75 4.29 3.95
76 2.95 2.88
77 8.34 9.02
78 7.47 5.70
79 2.63 2.39
80 0.5 0.78
81 4.1 3.22
82 1.53 1.48
83 0.363 0.37
84 6.94 8.43
85 0.805 1.75
86 2.24 2.77
87 0.561 0.61
88 5.9 7.86
89 1.76 2.13
90 1.11 1.10
91 3.09 3.03
92 1.53 2.08
93 2.35 2.97
94 3.1 3.54
95 1.95 1.81
96 2.25 2.64
97 5.19 6.40
98 1.63 0.68
99 3.22 2.99
100 1.28 1.48
3.通过发光试剂盒检测100份血清样本，和对照试剂相关性R2可以达到0.93。

Claims (20)

  1. 一种与人抗缪勒管激素特异性结合的单克隆抗体，其中所述抗体与保藏编号为CCTCC NO:C2018183的杂交瘤产生的抗体C9F2-2特异性结合相同的抗缪勒管激素表位。
  2. 一种与人抗缪勒管激素特异性结合的单克隆抗体，其中所述抗体包含保藏编号为CCTCC NO:C2018183的杂交瘤产生的抗体C9F2-2的轻链可变区互补决定区(CDR)和重链可变区CDR。
  3. 一种与人抗缪勒管激素特异性结合的单克隆抗体，其中所述抗体包含轻链可变区和重链可变区，所述轻链可变区包含具有SEQ ID NO：4的氨基酸序列的CDR1、具有SEQ ID NO：5的氨基酸序列的CDR2和具有SEQ ID NO：6的氨基酸序列的CDR3，所述重链可变区包含具有SEQ ID NO：1的氨基酸序列的CDR1、具有SEQ ID NO：2的氨基酸序列的CDR2和具有SEQ ID NO：3的氨基酸序列的CDR3。
  4. 权利要求3的单克隆抗体，其中所述抗体包含：(a)具有SEQ ID NO：8的氨基酸序列的轻链可变区和具有SEQ ID NO：7的氨基酸序列的重链可变区。
  5. 一种与人抗缪勒管激素特异性结合的单克隆抗体，其中所述抗体包含抗原结合区，所述抗原结合区来源于保藏编号为CCTCC NO:C2018183的杂交瘤产生的抗体C9F2-2的轻链和重链可变区。
  6. 权利要求1-5任一项的单克隆抗体，其是抗原结合片段、单链抗体、嵌合抗体、单价抗体、多特异性抗体、人抗体或Fab片段。
  7. 一种与人抗缪勒管激素特异性结合的单克隆抗体，其中所述抗体获自保藏编号为CCTCC NO:C2018183的杂交瘤。
  8. 针对人抗缪勒管激素的单克隆抗体，其特异性结合包含在跨越抗缪勒管激素的第38至45位氨基酸残基的序列中的表位。
  9. 保藏编号为CCTCC NO:C2018183的杂交瘤细胞株。
  10. 编码权利要求1-8中任一项的抗抗缪勒管激素抗体的重链可变区和/或轻链可变区的多核苷酸。
  11. 一种包括权利要求10的多核苷酸的载体。
  12. 一种包括权利要求11的多核苷酸或权利要求11的载体的宿主细胞。
  13. 检测人抗缪勒管激素在样品中的存在或其水平的方法，其包括使用权利要求1-8中任一项的抗体或其抗原结合片段的步骤。
  14. 权利要求1-8中任一项的抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测人抗缪勒管激素在样品中的存在或其水平。
  15. 一种用于检测生物样品中AMH或其片段的方法，该方法包括：
    将样品与针对抗缪勒管激素不同表位的至少两种抗体或其抗原结合片段相接触，以及定性或定量检测所述至少两种抗体与抗缪勒管激素或所述片段的结合，其中结合指示所述样品中抗缪勒管激素或所述片段存在或浓度；
    其中至少一种抗体或其抗原结合片段针对包含在抗缪勒管激素的第38至45位氨基酸残基的序列中的表位。
  16. 权利要求15的方法，其中一种抗体或其抗原结合片段针对包含在抗缪勒管激素的第38至45位氨基酸残基的序列中的表位，另一种抗体或其抗原结合片段针对包含在抗缪勒管激素的第358至369位氨基酸残基的序列中的表位，或包含在抗缪勒管激素的第491至502位氨基酸残基的序列中的表位，或包含在抗缪勒管激素的第260至271位氨基酸残基的序列中的表位，或包含在抗缪勒管激素的第330至343位氨基酸残基的序列中的表位。
  17. 权利要求15的方法，其中至少两种抗体或其抗原结合片段中的至少一种被固定化在固相表面上，优选地，另一种或另一些抗体中的至少一种被标记，优选通过共价连接化学发光或荧光染料进行标记。
  18. 权利要求15或16的方法，其中针对包含在跨越抗缪勒管激素的第38至45位氨基酸残基的序列中的表位的抗体或其抗原结合片段被固定化在固相表面上，并且另一种抗体或其抗原结合片段针对包含在抗缪勒管激素的第260至271位氨基酸残基的序列中的表位。
  19. 用于检测抗缪勒管激素在样品中的存在或其水平的试剂盒，其包含权利要求1-8任一项所述的抗体或其抗原结合片段，其作为双抗夹心酶联免疫检测的第一单克隆抗体即包被抗体；和作为双抗夹心酶联免疫检测的第二单克隆抗体即酶标抗体，其中所述包被抗体和所述酶标抗体分别针对抗缪勒管激素不同的抗原决定簇；
    优选地，其中所述酶标抗体针对包含在抗缪勒管激素的第260至271位氨基酸残基的序列中的表位。
  20. 根据权利要求19所述的试剂盒，其还包括以下中的一种或多种：酶联板、包 被缓冲液、封闭液、显色底物、终止液、抗缪勒管激素标准品。
PCT/CN2019/115998 2018-11-20 2019-11-06 针对人抗缪勒管激素的特异性抗体及其应用 WO2020103691A1 (zh)

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AU2019384259A AU2019384259A1 (en) 2018-11-20 2019-11-06 Specific antibody for AMH, and uses thereof
KR1020217018475A KR20210093961A (ko) 2018-11-20 2019-11-06 Amh에 대해 특이적인 항체 및 이의 용도
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