WO2021081880A1 - 一种杂交瘤细胞株及其用途 - Google Patents

一种杂交瘤细胞株及其用途 Download PDF

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WO2021081880A1
WO2021081880A1 PCT/CN2019/114658 CN2019114658W WO2021081880A1 WO 2021081880 A1 WO2021081880 A1 WO 2021081880A1 CN 2019114658 W CN2019114658 W CN 2019114658W WO 2021081880 A1 WO2021081880 A1 WO 2021081880A1
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antibody
antigen
binding portion
monoclonal antibody
detection
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PCT/CN2019/114658
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万晓春
李俊鑫
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中国科学院深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

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  • the invention relates to the field of immunology, in particular to a hybridoma cell line 1C12/1G12 and its use.
  • the hybridoma cell line 1C12/1G12 that secretes the anti-CD317 monoclonal antibody is prepared by hybridoma technology, and the anti-CD317 monoclonal antibody 1C12/1G12 produced by it can be used for western blotting, flow cytometry, immunohistochemistry and ELISA.
  • CD317 namely Cluster of Differentiation 317 (Cluster of Differentiation 317), also known as Bone Marrow Stromal Cell Antigen 2 (Bone Marrow Stromal Cell Antigen 2), Tetherin or HM1.24, is a terminally differentiated B cell discovered by monoclonal antibodies in 1994 Surface marker proteins may play an important role in the development of B cells. Because it is highly expressed in multiple myeloma cells, it is regarded as myeloma by tumor immunologists
  • CD317 is not only expressed on the surface of bone marrow stromal cells, mature B cells and myeloma cells, but also highly expressed in lung cancer, breast cancer and other cancer cells. It was first identified as an interferon-inducible host antiviral factor in 2008. Since then, more and more scientists have joined the exploration in this field. After nearly ten years of research, the structure, antiviral and immune properties of CD317 have been elaborated, and some new functions such as participation in tumor progression and restraining the release of exosomes have been discovered one after another. The research enthusiasm remains the same.
  • the anti-CD317 monoclonal antibody prepared by this technology specifically targets CD317 molecules that bind to human and murine cells, and can be applied to experiments such as western blotting, flow cytometry, immunohistochemistry, and ELISA, and is a powerful tool for studying the function of CD317.
  • One aspect of the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds CD317.
  • the monoclonal antibody or antigen-binding portion thereof is deposited in the Chinese Type Culture Collection (CCTCC) with a deposit CCTCC NO: C2019156 hybridoma cell line 1C12/1G12 was produced.
  • CTCC Chinese Type Culture Collection
  • Another aspect of the present invention provides a hybridoma cell line whose preservation information is the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: C2019156.
  • CTCC China Center for Type Culture Collection
  • compositions comprising the aforementioned monoclonal antibody or antigen-binding portion.
  • Another aspect of the present invention provides the use of the isolated anti-CD317 monoclonal antibody or its antigen-binding portion of the present invention to detect the presence or expression level of CD317 in a sample, or to prepare a kit or a detection reagent Purpose: The kit or detection reagent is used to detect the presence or expression level of CD317 in a sample.
  • the sample includes, but is not limited to, a blood sample, a tissue sample, and a cell sample.
  • the antibody or antigen binding portion thereof may further include a detectable label.
  • methods for detecting the presence or expression level of CD317 in a sample include ELISA detection methods, Western blot detection methods, flow cytometry detection methods, and immunohistochemical detection methods.
  • the kit includes an ELISA detection kit or a Western blot detection kit
  • the detection reagent includes a flow cytometry detection reagent and an immunohistochemical detection reagent.
  • Another aspect of the present invention provides the use of the isolated anti-CD317 monoclonal antibody or antigen binding portion thereof in ELISA detection.
  • Another aspect of the present invention provides the application of the isolated anti-CD317 monoclonal antibody or antigen binding portion thereof in Western blot detection.
  • Another aspect of the present invention provides the use of the isolated anti-CD317 monoclonal antibody or its antigen binding portion of the present invention in flow cytometric detection.
  • Another aspect of the present invention provides the use of the isolated anti-CD317 monoclonal antibody or antigen binding portion thereof in immunohistochemical detection.
  • an ELISA detection kit which includes the isolated anti-CD317 monoclonal antibody of the present invention or an antigen-binding portion thereof.
  • the antibody or antigen-binding portion thereof may also include a detectable label.
  • the kit may also include a second antibody that specifically recognizes the antibody or antigen binding portion thereof; the second antibody binding portion may also include a detectable label.
  • Another aspect of the present invention provides a Western blot detection kit, which includes the isolated anti-CD317 monoclonal antibody or antigen binding portion thereof according to the present invention.
  • the antibody or antigen-binding portion thereof may also include a detectable label.
  • the kit may also include a second antibody that specifically recognizes the antibody or antigen binding portion thereof; the second antibody binding portion may also include a detectable label.
  • Another aspect of the present invention provides a flow cytometry detection reagent, which includes the isolated anti-CD317 monoclonal antibody or antigen binding portion thereof according to the present invention.
  • the antibody or antigen-binding portion thereof may also include a detectable label.
  • the cell detection reagent may also include a second antibody that specifically recognizes the antibody or antigen binding portion thereof; the second antibody binding portion may also include a detectable label.
  • an immunohistochemical detection reagent which includes the isolated anti-CD317 monoclonal antibody or antigen binding portion thereof according to the present invention.
  • the antibody or antigen-binding portion thereof may also include a detectable label.
  • the cell detection reagent may also include a second antibody that specifically recognizes the antibody or antigen binding portion thereof; the second antibody binding portion may also include a detectable label.
  • the term “antigen-binding portion” of an antibody refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen (for example, CD317) to which the antibody binds, and compete with the intact antibody against the antigen.
  • the specific binding See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. It can be used by recombinant DNA technology. Or through the enzymatic or chemical cleavage of the whole antibody to produce the antigen-binding portion.
  • the antigen-binding portion includes Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, Single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and such polypeptides, which contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • CDR complementarity determining region
  • the antigen-binding portion of the antibody is a single-chain antibody (e.g., scFv), in which the VL and VH domains pair to form a monovalent molecule by pairing a linker that enables it to be produced as a single polypeptide chain (see, e.g., Bird et al. , Science 242: 423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)).
  • scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having an amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448).
  • Other linkers that can be used in the present invention include those described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996) , Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • antibodies are diabodies, ie, diabodies, in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short is used to allow pairing between the two domains of the same chain , Thereby forcing the domain to pair with the complementary domain of the other chain and create two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), And Poljak RJ et al., Structure 2: 1121-1123 (1994)).
  • hybridomas As used herein, the terms “hybridoma”, “hybridoma cell line” and “hybridoma cell line” are used interchangeably, and when referring to the terms “hybridoma”, “hybridoma cell line” and “hybridoma cell line” In the case of “tumor cell lines”, it also includes subclones and progeny cells of hybridomas.
  • the present invention relates to the following biological materials that have been deposited in the Chinese Type Culture Collection (CCTCC, Wuhan, Wuhan University, China):
  • Hybridoma cell line 1C12/1G12 (referred to as 1C12/1G12 in this article), its preservation number is CCTCC NO: C2019156, and the preservation time is August 07, 2019.
  • Figure 1 shows the application of 1C12/1G12 antibody to the flow cytometric detection of CD317 expression in human Hela cells.
  • Figure 2 shows the application of 1C12/1G12 antibody in an ELISA experiment.
  • Figure 3 shows the application of 1C12/1G12 antibody in Western blot experiments.
  • Figure 4 shows the application of 1C12/1G12 antibody in immunohistochemistry experiments.
  • the left image is a slice of tonsil tissue
  • the middle image is a lung tissue slice
  • the right image is a parathyroid tissue slice.
  • the concentration of 1C12/1G12 antibody is 0.25ug/ml.
  • the results showed that CD317 was positive in tonsils and lung tissues, and CD317 was not expressed in parathyroid glands.
  • the hybridoma method (first proposed by Kohler et al., Nature, 256:495 (1975)).
  • Use CD317 protein antigen purchased from Beijing Yiqiao Shenzhou Company
  • use Freund's complete adjuvant to emulsify the antigen for the first immunization
  • use Freund's incomplete adjuvant for the second immunization.
  • the emulsifying antigen is injected subcutaneously at 5-6 points, and the amount of antigen injected per mouse is ⁇ 100ug.
  • Ten days after the third immunization a small amount of blood was collected from the tail of the mice for serum titer ELISA test.
  • mice with high antibody titer >1:100000 were selected for the fourth immunization, and the antigen protein was injected into the abdominal cavity. , Each mouse was injected with 100ug.
  • the CD317 antibody-secreting hybridoma cells were screened by ELISA method, and subcloned by the limiting dilution method. The ELISA method was screened to obtain the CD317 antibody-secreting monoclonal hybridoma cell line 1C12/1G12. Freeze storage to preserve seeds.
  • the 1C12/1G12 antibody can bind to the CD317 protein on the surface of Hela cells ( Figure 1) and is used in flow cytometry.
  • Each well of the microtiter plate is coated with 100ng CD317 protein, and coated overnight at 4°C. After blocking and washing the plate, add 2 times diluted 1C12/1G12 antibody, the highest concentration is 40ug/ml, the lowest concentration is 19.5ng/ml, Well 100ul, incubate at 37°C for 1h. Wash the plate 5 times with PBST, add 1:5000 diluted HRP-goat anti-mouse IgG antibody (purchased from Biyuntian), and incubate at 37°C for 45 min. Wash the plate 5 times with PBST, add 100ul TMB to each well, react for 3min at room temperature and avoid light, add 50ul stop solution to each well to stop, and measure OD450 with a microplate reader.
  • 1C12/1G12 can be used for ELISA detection applications.
  • 1C12/1G12 can be used for Western blot detection applications.
  • Antigen heat retrieval Soak the slices in a pH 9.0 antigen retrieval solution (10mM Tris, 1mM EDTA solution, 0.05% Tween 20, pH 9.0) In, 95,, 5min.
  • the bio-layer interferometry (BLI) principle of the Octet K2 instrument (PALL ForteBio) is used to detect the affinity (KD value) of the antibody.
  • the antibody was diluted with SD buffer (1 ⁇ PBS, 0.05% BSA, 0.002% Tween 20).
  • SD buffer (1 ⁇ PBS, 0.05% BSA, 0.002% Tween 20).
  • Protein A sensor or avidin sensor For computer operation, use Protein A sensor or avidin sensor to couple antibodies, and then incubate antigens of various concentrations. All binding data were collected at 30°C.
  • the reaction program consists of four steps: antibody coupling sensor; baseline acquisition 60s; binding antigen, calculating Kon value; dissociating antigen or antibody, calculating Koff value.
  • the ratio of Kon and Koff is the affinity KD value of the antibody.

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Abstract

本发明涉及一种杂交瘤细胞株及其用途，具体涉及一种杂交瘤细胞株1C12/1G12及其用途。用杂交瘤技术制备分泌抗CD317单克隆抗体的杂交瘤细胞株1C12/1G12，其产生的抗CD317单克隆抗体1C12/1G12可以用于免疫印迹，流式细胞技术、免疫组化和ELISA。

Description

一种杂交瘤细胞株及其用途 技术领域
本发明涉及免疫学领域，具体涉及一种杂交瘤细胞株1C12/1G12及其用途。用杂交瘤技术制备分泌抗CD317单克隆抗体的杂交瘤细胞株1C12/1G12，其产生的抗CD317单克隆抗体1C12/1G12可以用于免疫印迹，流式细胞技术、免疫组化和ELISA。
背景技术
CD317，即分化抗原簇317(Cluster of Differentiation 317)，又叫骨髓基质细胞抗原2(Bone Marrow Stromal Cell Antigen 2)、Tetherin或HM1.24，是1994年通过单克隆抗体发现的终末分化B细胞表面标志蛋白，可能在B细胞的发育过程中发挥重要作用。因其在多发性骨髓瘤细胞中高表达，被肿瘤免疫学家当作骨髓瘤
免疫疗法的靶向抗原，随之开展诸多靶向治疗相关的研究。期间发现CD317除了表达在骨髓基质细胞、成熟B细胞以及骨髓瘤细胞表面之外，在肺癌、乳腺癌等多种癌细胞中高表达。2008年首次被鉴定为干扰素诱导型宿主抗病毒因子，此后越来越多的科学家加入到该领域的探索中。经过近十年的研究，目前已经阐述了CD317结构、抗病毒及免疫特性等问题，也陆续发现了一些诸如参与肿瘤进展、束缚外泌体释放等新功能，研究热度不减当年。
作为生物学研究领域应用最广泛的工具，抗体产品的地位毋庸置疑。而抗体的特异性和应用范围长久以来困扰着抗体产业，也是整个生物学研究领域的极大危机。抗体产品糟糕的质量会直接导致实验结果的误差，以及研究结果无法被重复和再现。研究项目的进展往往会因为抗体带来的问题停滞不前，由此造成的损失也是惊人的。据2011年的数据统计，仅在美国，平均每年就有3.5亿美元被浪费在这些无效抗体上。而在世界范围内，每年竟有8亿美元被浪费，占到了全球科研抗体总开销的50％。
发明内容
本技术制备的抗CD317单克隆抗体特异性靶向结合人和鼠细胞的CD317分子，可以应用于免疫印迹，流式细胞技术、免疫组化和ELISA等实验，是研究CD317功能的有力工具。
本发明一个方面，提供了一种分离的特异性结合CD317的单克隆抗体或其抗原结合部分，所述单克隆抗体或其抗原结合部分由保藏在中国典型培养物保藏中心(CCTCC)，具有保藏号CCTCC NO：C2019156的杂交瘤细胞系1C12/1G12产生。
本发明另一个方面提供一种杂交瘤细胞系，其保藏信息为中国典型培养物保藏中心(CCTCC)，保藏号CCTCC NO：C2019156。
本发明另一个方面提供了一种组合物，包含前述的单克隆抗体或抗原结合部分。
本发明再一个方面提供了本发明所述的分离的抗CD317单克隆抗体或或其抗原结合部分在检测CD317在样品中的存在或其表达水平的用途，或者用于制备试剂盒或检测试剂的用途，所述试剂盒或检测试剂用于检测CD317在样品中的存在或其表达水平。
在本发明的技术方案中，所述样品包括但不限于，血液样品、组织样品和细胞样品。
在本发明的技术方案中，所述抗体或其抗原结合部分还可包括可检测的标记。
在本发明的技术方案中，检测CD317在样品中的存在或其表达水平的方法包括ELISA检测方法、Western blot检测方法、流式细胞检测方法、免疫组化检测方法。
在本发明的技术方案中，所述试剂盒包括ELISA检测试剂盒或Western blot检测试剂盒，所述检测试剂包括流式细胞检测试剂、免疫组化检测试剂。
本发明再一个方面提供了本发明所述的分离的抗CD317单克隆抗体或其抗原结合部分在ELISA检测中的应用。
本发明再一个方面提供了本发明所述的分离的抗CD317单克隆抗体或其抗原结合部分在Western blot检测中的应用。
本发明再一个方面提供了本发明所述的分离的抗CD317单克隆抗体或其抗原结合部分在流式细胞检测中的应用。
本发明再一个方面提供了本发明所述的分离的抗CD317单克隆抗体或其抗原结合部分在免疫组化检测中的应用。
本发明再一个方面提供了ELISA检测试剂盒，其包括本发明所述的分离的抗CD317单克隆抗体或与或其抗原结合部分。优选地，所述抗体或其抗原结合部分还可包括可检测的标记。所述试剂盒还可包含第二抗体，其特异性识别所述抗体或其抗原结合部分；所述第二抗体结合部分还可包括可检测的标记。
本发明再一个方面提供Western blot检测试剂盒，其包括本发明所述的分离的抗CD317单克隆抗体或其抗原结合部分。优选地，所述抗体或其抗原结合部分还可包括可检测的标记。所述试剂盒还可包含第二抗体，其特异性识别所述抗体或其抗原结合部分；所述第二抗体结合部分还可包括可检测的标记。
本发明再一个方面提供了流式细胞检测试剂，其包括本发明所述的分离的抗CD317单克隆抗体或其抗原结合部分。优选地，所述抗体或其抗原结合部分还可包括可检测的标记。所述细胞检测试剂还可包含第二抗体，其特异性识别所述抗体或其抗原结合部分；所述第二抗体结合部分还可包括可检测的标记。
本发明再一个方面提供了免疫组化检测试剂，其包括本发明所述的分离的抗CD317单克隆抗体或其抗原结合部分。优选地，所述抗体或其抗原结合部分还可包括可检测的标记。所述细胞检测试剂还可包含第二抗体，其特异性识别所述抗体或其抗原结合部分；所述第二抗体结合部分还可包括可检测的标记。
在本发明中，术语抗体的“抗原结合部分”是指全长抗体的一个或多个部分，所述部分保持结合抗体所结合的相同抗原(例如，CD317)的能力，与完整抗体竞争对抗原的特异性结合。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原结合部分。在一些情况下，抗原结合部分包括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)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。
在一些情况下，抗体的抗原结合部分是单链抗体(例如，scFv)，其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如，Bird等人，Science 242：423-426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85：5879-5883(1988))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90：6444-6448)。可用于本发明的其它接头包括由Alfthan等人(1995)，Protein Eng.8：725-731，Choi等人(2001)，Eur.J.Immunol.31：94-106，Hu等人(1996)，Cancer Res.56：3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293：41-56和Roovers等人(2001)，Cancer Immunol.描述的那些。
在一些情况下，抗体是双抗体，即，双价抗体，其中VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如，Holliger P.等人，Proc.Natl.Acad.Sci.USA 90：6444-6448(1993)，和Poljak R.J.等人，Structure 2：1121-1123(1994))。
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C3和C16单克隆抗体)获得抗体的抗原结合部分(例如，上述抗体片段)，并且以与对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部分。
如本文中所使用的，术语“杂交瘤”、“杂交瘤细胞系”和“杂交瘤细胞株”可互换使用，并且当提及术语“杂交瘤”、“杂交瘤细胞系”和“杂交瘤细胞株”时，其还包括杂交 瘤的亚克隆和后代细胞。
关于生物材料保藏的说明
本发明涉及下列已在中国典型培养物保藏中心(CCTCC，中国，武汉，武汉大学)进行保藏的生物材料：
杂交瘤细胞株1C12/1G12(在本文中简称为1C12/1G12)，其保藏号为CCTCC NO:C2019156，保藏时间为2019年08月07日。
附图说明
图1为1C12/1G12抗体应用在流式检测人Hela细胞CD317的表达。
图2为1C12/1G12抗体应用在ELISA实验。
图3为1C12/1G12抗体应用在免疫印迹(Western blot)实验。1：一抗是PBS，2：一抗是1C12/1G12抗体，3：一抗是稀释10万倍的免疫小鼠血清。
图4为1C12/1G12抗体应用在免疫组化实验。左图是扁桃体组织切片，中间图是肺组织切片，右图是甲状旁腺组织切片。1C12/1G12抗体浓度为0.25ug/ml。结果显示扁桃体和肺组织CD317表达阳性，甲状旁腺不表达CD317。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例对本发明的技术方案进行以下详细说明，应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1 CD317单克隆抗体的生产
采用杂交瘤法(由Kohler等，Nature,256:495(1975)首先提出)。用CD317蛋白抗原(购自北京义翘神州公司)免疫雌性BALB/c小鼠(6周龄)，首次免疫使用弗氏完全佐剂进行乳化抗原，第二次免疫开始，使用弗氏不完全佐剂乳化抗原，皮下5～6点注射，每只小鼠注射的抗原量为～100ug。在第3次免疫后10天，对小鼠剪尾采集少量血液进行血清效价ELISA检测，选择抗体滴度高(>1:100000)的小鼠进行加强第4次免疫，通过腹腔注射抗原蛋白，每只小鼠注射100ug。第4次免疫后3～5天，处死小鼠取其脾细胞与SP2/0细胞融合，通过HAT培养基培养获得稳定的杂交瘤细胞。通过ELISA法筛选得到能分泌CD317抗体的杂交瘤细胞，通过有限稀释的方法进行亚克隆，ELISA法筛选得到能分泌CD317抗体的单克隆杂 交瘤细胞株1C12/1G12，通过逐级扩大培养，液氮冻存保种。
腹水抗体制备：雌性BALB/c小鼠(8周龄)腹腔注射弗氏不完全佐剂，每只小鼠注射0.5ml，3～5天后腹腔注射处于对数生长期的杂交瘤细胞，每只小鼠注射1～5×10 5个细胞(0.5ml)，注射杂交瘤细胞～11天后处死小鼠，获得腹水。3000rpm，4℃离心10min，去除沉淀，用10倍体积1×PB溶液稀释腹水，混匀后过0.45μm滤膜。通过Protein G(Protein G Sepharose 4 Fast Flow,GE Healthcare)亲和纯化腹水，得到纯化的CD317抗体蛋白。
实施例2 CD317单克隆抗体的流式检测应用
1)收集Hela，吹散计数，分装5个离心管，每管3×10 5个细胞，500g离心5min，去上清，分别加入1ml PBS，500g离心5min，去上清。
2)加入PBS或5ug/ml 1C12/1G12抗体，放入冰上，孵育，30min。
3)各加入1ml PBS+5％FBS，500g离心5min，去上清，各加入100ul DMEM培养液+1ul PE goat anti-mouse lgG(minimal x-reactivity)antibody(biolegend,405307)，冰上孵育20min。
4)各加入1ml PBS+5％FBS，500g离心5min，去上清。
5)每管加入300ul PBS+5％FBS，重悬，使用贝克曼流式细胞仪(Beckman CytoFLEX CM)检测。
1C12/1G12抗体能与Hela细胞(图1)表面的CD317蛋白结合，应用于流式细胞检测。
实施例3 CD317单克隆抗体的ELISA检测应用
酶标板每孔包被100ng CD317蛋白，4℃包被过夜，经封闭洗板后，加入2倍比稀释的1C12/1G12抗体，最高浓度为40ug/ml，最低浓度为19.5ng/ml，每孔100ul，37度孵育1h。PBST洗板5次，加入1:5000稀释的HRP-羊抗小鼠IgG抗体(购自碧云天)，37℃孵育45min。PBST洗板5次，每孔加入100ul TMB，室温避光反应3min，每孔加入50ul终止液终止，酶标仪测OD450。
如图2所示，1C12/1G12能用于ELISA检测应用。
实施例4 CD317单克隆抗体的Western blot检测应用
CD317蛋白加5×loading buffer混合后，煮沸变性6min，跑SDS-PAGE，每孔上样20ul，～50ng蛋白/孔，400mA转膜1h，5％脱脂奶粉室温封闭1h，加入PBS或2ug/ml 1C12/1G12抗体或稀释10万倍的免疫小鼠血清室温孵育1h，PBST洗3遍后加入1:5000稀释羊抗鼠IgG二抗，室温孵育45min，PBST洗3遍后加入Western HRP底物(密理博，WBLUR0500)显影。
如图3所示，1C12/1G12能用于Western blot检测应用。
实施例5 CD317单克隆抗体的免疫组化应用
1.切片常规脱蜡至水。
2.抗原热修复(热引导的抗原决定簇修复，Heat Induced Epitope Retrieval，HIER)：将切片浸泡在pH9.0的抗原修复溶液(10mM Tris,1mM EDTA溶液，0.05％吐温20,pH 9.0)中，95,，5min。
3.为了降低内源性过氧化物酶造成的非特异性背景染色，将切片放在Hydrogen Peroxide Block(Abcam)中孵育10-15分钟。
4.PBST洗5min/2次。
5.加Ultra V Block(Thermo)，在室温下孵育5分钟以封闭非特异性的背景染色。
6.PBST洗5min/2次。
7.加1C12/1G12抗体工作液37℃2小时。
8.PBST洗5min/2次。
9.加Primary Antibody Enhancer(Thermo)，在室温下孵育20分钟。
10.PBST洗5min/2次。
11.加HRP Polymer(酶标二抗，购自碧云天)，在室温下孵育30分钟。
12.PBST洗5min/2次。
13.向1ml DAB Plus Substrate(Thermo)中滴加1-2滴DAB Plus Chromogen(Thermo)，混匀后滴加到切片上，孵育15分钟。
14.自来水充分冲洗，复染，脱水，透明，封片
结果如图4所示，1C12/1G12能用于免疫组化。
实施例5 抗体结合抗原的亲和力检测
利用Octet K2仪器(PALL ForteBio)的生物膜层反射光干涉(bio-layer interferometry，BLI)原理检测抗体的亲和力(KD值)。用SD缓冲液(1×PBS，0.05％BSA，0.002％吐温20)稀释抗体。上机操作，利用Protein A传感器或亲和素传感器偶联抗体，然后孵育各种浓度的抗原。所有结合数据都在30℃收集。反应程序由四步组成：抗体偶联传感器；基线采集60s；结合抗原，计算Kon值；解离抗原或抗体，计算Koff值。Kon和Koff的比值为抗体的亲和力KD值。
结果显示：1C12/1G12抗体的亲和力KD为9.16×10 -11M，比抗CD317单抗6H2H5(KD＝3.83×10 -10M，申请号201711314176.4)和抗CD317单抗1C12C6(KD＝5.69×10 -9M，申请号201610554612.4)的亲和力更强。

Claims (15)

  1. 一种分离的特异性结合CD317的单克隆抗体或其抗原结合部分，所述单克隆抗体或其抗原结合部分由保藏在中国典型培养物保藏中心(CCTCC)，具有保藏号CCTCC NO：C2019156的杂交瘤细胞系1C12/1G12产生。
  2. 一种杂交瘤细胞系，其保藏信息为中国典型培养物保藏中心(CCTCC)，保藏号CCTCC NO：C2019156。
  3. 一种组合物，包含权利要求1所述的单克隆抗体或其抗原结合部分。
  4. 权利要求1所述的单克隆抗体或其抗原结合部分在检测CD317在样品中的存在或其表达水平的用途。
  5. 根据权利要求4所述的用途，检测CD317在样品中的存在或其表达水平的方法包括ELISA检测方法、Western blot检测方法、流式细胞检测方法、免疫组化检测方法。
  6. 权利要求1所述的单克隆抗体或抗原结合部分在用于制备检测CD317试剂盒或检测试剂的用途，所述试剂盒或检测试剂用于检测CD317在样品中的存在或其表达水平。
  7. 根据权利要求6所述的用途，所述试剂盒为ELISA检测试剂盒或Western blot检测试剂盒，所述检测试剂为流式细胞检测试剂或免疫组化检测试剂。
  8. 一种试剂盒，包括权利要求1所述的分离的抗CD317单克隆抗体或其抗原结合部分。
  9. 根据权利要求8所述的试剂盒，所述抗体或其抗原结合部分还包括可检测的标记。
  10. 根据权利要求8-9任一项所述的试剂盒，所述细胞检测试剂还包含第二抗体，其特异性识别权利要求1所述的单克隆所述单克隆抗体或其抗原结合部分。
  11. 根据权利要求8-10任一项所述的试剂盒，所述试剂盒为ELISA检测试剂盒或Western blot检测试剂盒。
  12. 一种检测试剂，其包括权利要求1所述的分离的抗CD317单克隆抗体或其抗原结合部分。
  13. 根据权利要求12所述的检测试剂，所述抗体或其抗原结合部分还包括可检测的标记。
  14. 根据权利要求12-13任一项所述的检测试剂，所述细胞检测试剂还包含第二抗体，其特异性识别所述权利要求1所述的单克隆抗体或其抗原结合部分。
  15. 根据权利要求12-14任一项所述的检测试剂，所述检测试剂为免疫组化检测试剂或流式细胞检测试剂。
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