WO2017215637A1 - Human endometrial cancer marker, antibody, and application of antibody - Google Patents

Human endometrial cancer marker, antibody, and application of antibody Download PDF

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WO2017215637A1
WO2017215637A1 PCT/CN2017/088501 CN2017088501W WO2017215637A1 WO 2017215637 A1 WO2017215637 A1 WO 2017215637A1 CN 2017088501 W CN2017088501 W CN 2017088501W WO 2017215637 A1 WO2017215637 A1 WO 2017215637A1
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endometrial cancer
antibody
em2d9
human
human endometrial
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PCT/CN2017/088501
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李翀
史桂芝
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李翀
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    • 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
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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 belongs to the field of tumor immunology.
  • the present invention relates to a novel endometrial cancer tumor marker GAB31, and a monoclonal antibody EM2D9 against GAB31. Cell and histological levels demonstrate that EM2D9 specifically recognizes human endometrial cancer cells and human endometrial cancer tissues.
  • the invention also relates to a method for detecting human endometrial cancer by immunomagnetic beads.
  • the antigen detected by the detection method is the novel endometrial cancer marker GAB31 of human in the present invention, and the detection antibody is the monoclonal antibody EM2D9 against human endometrial cancer GAB31 in the present invention.
  • Endometrial cancer is one of the most common malignant tumors in the female reproductive system, and its incidence rate is the highest in the female reproductive system malignant tumors in Europe and the United States. Compared with European and American countries, the incidence of endometrial cancer in developing countries is lower, but the mortality rate is higher. As the lives and eating habits of Chinese people tend to be westernized, especially in economically developed areas, the incidence of endometrial cancer is increasing year by year. Currently, it ranks second in female reproductive system malignancies in China, second only to cervical cancer. The age of onset is getting younger. The cause of endometrial cancer is still unclear.
  • endometrial cancer Although early diagnosis of endometrial cancer includes transvaginal ultrasound, endometrial biopsy, diagnostic curettage, endometrial cytology, and many other methods, screening for endometrial cancer in asymptomatic general populations and at risk groups The method has not yet formed a unified guide or recommendation in the industry. Therefore, exploring the strategy of endometrial cancer screening is a hotspot in the research of endometrial cancer in the medical field. At present, the commonly used diagnostic methods for endometrial cancer are used for screening strategies, and all have more or less defects.
  • Transvaginal ultrasound can not determine the boundary value of endometrial thickness in endometrial lesions; endometrial biopsy is more or less uncomfortable, and a considerable proportion of subjects are missed because of insufficient tissue; diagnostic curettage It is an invasive operation. It is obviously inappropriate to include a screening strategy, and it is not suitable for non-developed areas because of the medical level. The cost of hysteroscopy is high, and the diagnostic value alone is limited.
  • the characteristics of endometrial cytology are the most Close to screening strategies, however, there is currently no uniform cytology standard. It can be seen that the screening strategy for endometrial cancer still needs further exploration.
  • CD44 is involved in the invasion and metastasis of endometrial cancer cells.
  • CD44 belongs to the cell multifunctional adhesion molecule and is a transmembrane glycoprotein encoded by a single gene, which is widely present in a variety of cells and tissues.
  • CD44 has 10 variant exons, so it can form a variety of isomers by selective shearing and splicing, and bind to the corresponding ligands on the cell surface, which can regulate various physiological and pathological processes of cells.
  • CD44 is highly expressed in a variety of tumors and is closely related to the growth, invasion and metastasis of tumor cells and the homing of hematopoietic stem cells in the tumor environment. It can be used as a marker for cancer stem cells or as a tumor screen.
  • the biological function and expression regulation mode of CD44 vary according to different conditions, such as tumor type and growth conditions. Many Factors can affect its expression, such as estrogen receptors, transcription factors and so on. Abnormal glycosylation of CD44 can lead to changes in the malignancy of tumor cells, and to explore the abnormal modification of CD44 in endometrial cancer, which can provide a new target for screening, diagnosis, prognosis and treatment of endometrial cancer. It will be of great significance for the diagnosis and treatment of endometrial cancer.
  • the present invention extracts total protein immunized mice from fresh human endometrial cancer tissues to prepare hybridoma cell lines.
  • An antibody EM2D9 capable of highly specific binding to human endometrial cancer tissues was screened by ELISA, and the antibody belongs to the IgG1 subclass. Immunohistochemistry confirmed that EM2D9 antibody had a strong positive reaction with human endometrial cancer tissue, but no cross-reaction with human normal endometrial tissue.
  • the present invention identifies by immunoprecipitation combined with mass spectrometry and sugar chip results that the antigen recognized by the EM2D9 antibody is an abnormally glycosylated CD44 which is localized on the cell membrane and has an epitope of: Galb1 ⁇ 3 (Neu5Aca2 ⁇ 6) GlcNAcb1 ⁇ 4Galb1 ⁇ 4Glcb is a new endometrial cancer marker.
  • the invention couples the EM2D9 antibody into the nano magnetic particle, prepares an anti-endometrial cancer immunomagnetic beads (EM2D9-MB), captures and enriches the detached endometrial cancer cells, and stains with the pathological smear of Wright's Giemsa. Microscopic examination under the microscope.
  • the kit is suitable for early screening, prognosis monitoring and pathological diagnosis of tumor patients.
  • the innovation of the invention lies in: (1) screening and preparing a monoclonal antibody EM2D9 against human endometrial cancer, which has a strong positive reaction with human endometrial cancer tissue, and human normal endometrial tissue No cross-reactivity; (2) revealed that the epitope recognized by EM2D9 antibody is Galb1 ⁇ 3 (Neu5Aca2 ⁇ 6) GlcNAcb1 ⁇ 4Galb1 ⁇ 4Glcb, which is a new endometrial cancer marker; (3) developed an EM2D9 based Highly sensitive antibody magnetic beads method for detecting human endometrial cancer.
  • the present invention provides a novel human endometrial cancer tumor marker, abnormally glycosylated CD44, designated GAB31.
  • GAB31 a novel human endometrial cancer tumor marker, abnormally glycosylated CD44, designated GAB31.
  • a hybridoma cell line producing a monoclonal antibody against GAB31 was obtained, and the hybridoma cell line secreted the monoclonal antibody EM2D9, and the epitope recognized by the monoclonal antibody EM2D9 was Galb1 ⁇ 3 (Neu5Aca2 ⁇ 6) GlcNAcb1 ⁇ 4Galb1 ⁇ 4Glcb. .
  • the monoclonal antibody has a strong positive reaction with human endometrial cancer tissue and no cross-reaction with human normal endometrial tissue.
  • the invention also provides an immunomagnetic beads (EM2D9-MB) diagnostic method based on monoclonal antibody EM2D9 for diagnosis of in vitro endometrial cancer.
  • the present invention provides an aberrantly glycosylated human endometrial cancer tumor marker GAB31 which is aberrantly glycosylated CD44 characterized by a glycostructure Galb1 ⁇ 3 comprising an epitope on CD44 ( Neu5Aca2 ⁇ 6) GlcNAcb1 ⁇ 4Galb1 ⁇ 4Glcb.
  • amino acid sequence of CD44 is set forth in SEQ ID No: 1.
  • Another object of the present invention is to provide an antibody against the human endometrial cancer marker GAB31 of the present invention which is capable of specifically recognizing the glycostructure Galb1 ⁇ 3 (Neu5Aca2 ⁇ 6) GlcNAcb1 ⁇ 4Galb1 as an epitope.
  • the antibody is a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody.
  • Another object of the present invention is to provide a kit for detecting human endometrial cancer comprising the above-described antibody EM2D9 of the present invention.
  • the antibody is monoclonal antibody EM2D9 against human endometrial cancer GAB31, which is secreted by a hybridoma cell line with accession number CGMCC No. 11796.
  • Another object of the present invention is to provide a conjugate comprising the anti-human endometrial cancer GAB31 conjugated with a substance selected from the group consisting of biomarkers, antitumor drugs, toxins and radioactive agents .
  • Another object of the present invention is to provide a kit for detecting or treating human endometrial cancer comprising the above-described antibody of the present invention.
  • the detection is carried out by coupling the nano-magnetic particles with the monoclonal antibody EM2D9, preferably by coupling the EM2D9 antibody to the nano-magnetic particles to prepare for anti-endometrial cancer immunity.
  • Magnetic beads (EM2D9 ⁇ MB).
  • the sample to be tested is a uterine scraping containing human endometrial cancer exfoliated cells.
  • Another object of the present invention is to provide a hybridoma cell line secreting monoclonal antibody EM2D9 against human endometrial cancer GAB31, which has a accession number of CGMCC No. 11796.
  • FIG. 1 Immunohistochemical map of EM2D9 mAb to human endometrial cancer tissue sections.
  • FIG. 1 Immunohistochemical map of EM2D9 mAb to human normal endometrial tissue sections.
  • mice purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • mice were immunized with fresh human endometrial cancer tissue protein homogenate at a dose of 20 ug total protein.
  • Mice were immunized by intraperitoneal injection. The mice were re-immunized two weeks later. After the serum titer of the mouse reached the requirement, the immunization was boosted once, and the spleen of the mouse was taken 3 days later to prepare a suspension of lymphocytes to prepare for cell fusion.
  • Resusciting myeloma cell Sp2/0 ATCC CRL ⁇ 1772), 8-AG (8-azaguanine) was screened to maintain cell sensitivity to HAT.
  • the lymphocyte suspension prepared in step 1) is fused with myeloma cells, and the specific method is referred to the "Guide to the Editing Immunology Experiment" ((United States) JE Science Roots (USA) DH Margulis, etc. Science Press, published in January 2009).
  • the fused cell suspension is added to a culture medium containing feeder cells.
  • HAT selection medium purchased from Invitrogen; HAT ie H: Hypoxanthine hypoxanthine, A: Aminopterin methotrexate, T: Thymidine thymidine was selectively cultured.
  • the antibody-secreting hybridoma cell strain was determined by an ELISA method. The specific method is: extracting total protein of endometrial cancer tissue, coating with 0.05 mol/L carbonate buffer (pH 9.6) at 4 ° C overnight, and adding 10% BSA at 37 ° C for 3 hours. The cells were washed 3 times with PBST, then 100 ul of the supernatant to be tested was added, and incubated at 37 ° C for 1 h. After washing 3 times, an anti-mouse horseradish peroxidase-labeled secondary antibody IgG-HRP (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) was added and incubated at 37 ° C for 1 h.
  • IgG-HRP purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.
  • TMB purchased from Beijing Zhongshang Jinqiao Biotechnology Co., Ltd.
  • 50 ul of the stop solution was added for color development, and after standing at room temperature for 5 min, 50 ul of the stop solution was added.
  • the OD value at a wavelength of 450 nm was read with a microplate reader. An OD value greater than 2 times the OD value of the negative control was considered positive.
  • Cloning and cryopreservation of hybridomas The selected positive hybridoma cells were cloned and cultured by limiting dilution method. After three rounds of cloning culture, hybridoma cell clones with high titer monoclonal antibodies were screened for expanded culture.
  • a positive hybridoma cell line obtained in the present invention is a monoclonal hybridoma cell line against human endometrial cancer, and the hybridoma cell line is deposited on the Chinese Microbial Culture Collection Management Committee on December 23, 2015. Center (CGMCC, China, Beijing), the deposit number is CGMCC No.11796.
  • the above-described hybridoma cell strain stably stabilizing the monoclonal antibody EM2D9 (Accession No. CGMCC No. 11796) was expanded and cultured, and the cell culture supernatant was collected. Affinity chromatography of monoclonal antibody EM2D9 was performed using Protein G. The simple procedure was to first equilibrate the Protein G affinity chromatography column (purchased from "GE") with PBS; then, the cell culture containing EM2D9 monoclonal antibody was supernatantd.
  • Protein G affinity chromatography column then wash the column with PBS until the OD value of the washing solution flowing out of the column is close to zero; elute the column with 0.2 M glycine-HCL solution (pH 2.8), and collect the eluent in a separate tube. The OD value of each collection tube was measured. The eluate containing EM2D9 mAb was dialyzed against PBS and frozen at -20 °C.
  • Human endometrial cancer tissues and human normal endometrial tissues were sectioned using the EM2D9 monoclonal antibody prepared in Example 1, and the results are shown in Figs. The results showed that human endometrial cancer tissues showed a positive reaction after immunohistochemical staining by EM2D9 monoclonal antibody (Fig. 1), while human normal endometrial tissue showed a negative reaction after immunohistochemical staining with EM2D9 monoclonal antibody. figure 2).
  • Human endometrial cancer tissues, human normal endometrial tissues and other tissues were detected by immunohistochemistry using the EM2D9 monoclonal antibody prepared in Example 1, and the results are shown in Table 1. The results showed that EM2D9 antibody had a strong positive reaction with human endometrial cancer tissue, but no cross-reaction with human normal endometrial tissue and other tissues.
  • Total protein extraction 50mg human endometrial cancer tissue, after cutting and grinding into homogenate, add 1ml of three decontamination lysate, lyse at 4 °C for 5 minutes, centrifuge at 12000 rpm for 20 minutes, take the supernatant, which is human uterus Membrane cancer tissue total protein.
  • EM2D9 monoclonal antibody specifically recognizes human endometrial cancer tissue, it indicates that the antigen is abnormally glycosylated CD44, and its epitope Galb1 ⁇ 3 (Neu5Aca2 ⁇ 6) GlcNAcb1 ⁇ 4Galb1 ⁇ 4Glcb is only expressed in human uterus.
  • Membrane cancer tissue cells Since EM2D9 monoclonal antibody specifically recognizes human endometrial cancer tissue, it indicates that the antigen is abnormally glycosylated CD44, and its epitope Galb1 ⁇ 3 (Neu5Aca2 ⁇ 6) GlcNAcb1 ⁇ 4Galb1 ⁇ 4Glcb is only expressed in human uterus.
  • Membrane cancer tissue cells since EM2D9 monoclonal antibody specifically recognizes human endometrial cancer tissue, it indicates that the antigen is abnormally glycosylated CD44, and its epitope Galb1 ⁇ 3 (Neu5Aca2 ⁇ 6) GlcNAcb1 ⁇ 4Galb
  • EM2D9-MB anti-endometrial cancer immunomagnetic beads
  • the human endometrial cancer immunodiagnostic reagent of the present invention has the following positive effects compared with the prior art: (1) high sensitivity, and the sensitivity of the method is high due to the enrichment of the conjugated monoclonal antibody EM2D9 immunomagnetic particles.
  • the conventional exfoliative cytology detection method (2) strong specificity, because the method is coupled with EM2D9 antibody that specifically binds to endometrial cancer tissue cells, it can specifically recognize endometrial cancer cells; (3 It is convenient and quick, and saves patients' medical expenses. Because of its high sensitivity and strong specificity, the detection rate of endometrial cancer patients is high, avoiding repeated diagnosis and saving of patients.

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Abstract

A new human endometrial cancer tumor marker, named GAB31. The tumor marker is abnormally glycosylated CD44, which contains a saccharide structure of Galb1-3(Neu5Aca2-6)GlcNAcb1-4Galb1-4Glcb of epitope. Also provided is an antibody which specifically recognizes the antigen, and it is found through immunomagnetic bead experiments on clinical samples that the EM2D9-MB positive detection rate of a detecting method using the antibody to recognize the antigen GAB31 is obviously higher than that of a conventional cast-off cell microscopic examination method.

Description

一种人子宫内膜癌的标志物、抗体及其应用A marker, antibody and application thereof for human endometrial cancer 技术领域Technical field
本发明属于肿瘤免疫学领域。本发明涉及一种新型子宫内膜癌肿瘤标志物GAB31，以及抗GAB31的单克隆抗体EM2D9。细胞和组织学水平证明：EM2D9特异性识别人子宫内膜癌细胞及人子宫内膜癌组织。本发明还涉及一个免疫磁珠法检测人子宫内膜癌的方法。该检测方法所检测的抗原为本发明中的人的子宫内膜癌新型标记物GAB31，检测抗体为本发明中抗人子宫内膜癌GAB31的单克隆抗体EM2D9。The invention belongs to the field of tumor immunology. The present invention relates to a novel endometrial cancer tumor marker GAB31, and a monoclonal antibody EM2D9 against GAB31. Cell and histological levels demonstrate that EM2D9 specifically recognizes human endometrial cancer cells and human endometrial cancer tissues. The invention also relates to a method for detecting human endometrial cancer by immunomagnetic beads. The antigen detected by the detection method is the novel endometrial cancer marker GAB31 of human in the present invention, and the detection antibody is the monoclonal antibody EM2D9 against human endometrial cancer GAB31 in the present invention.
背景技术Background technique
子宫内膜癌是女性生殖***最常见的恶性肿瘤之一，其发病率高居欧美国家女性生殖***恶性肿瘤首位。相对于欧美国家，发展中国家子宫内膜癌发病率较低，但死亡率较高。随着我国百姓的生活及饮食***的原因，非发达地区无法普及；宫腔镜检查费用高昂，且单独运用诊断价值有限；子宫内膜细胞学的特点最接近筛查策略，但是，当前缺乏统一的细胞学标准。可见，子宫内膜癌的筛查策略仍需要进一步的探究。Endometrial cancer is one of the most common malignant tumors in the female reproductive system, and its incidence rate is the highest in the female reproductive system malignant tumors in Europe and the United States. Compared with European and American countries, the incidence of endometrial cancer in developing countries is lower, but the mortality rate is higher. As the lives and eating habits of Chinese people tend to be westernized, especially in economically developed areas, the incidence of endometrial cancer is increasing year by year. Currently, it ranks second in female reproductive system malignancies in China, second only to cervical cancer. The age of onset is getting younger. The cause of endometrial cancer is still unclear. Although early diagnosis of endometrial cancer includes transvaginal ultrasound, endometrial biopsy, diagnostic curettage, endometrial cytology, and many other methods, screening for endometrial cancer in asymptomatic general populations and at risk groups The method has not yet formed a unified guide or recommendation in the industry. Therefore, exploring the strategy of endometrial cancer screening is a hotspot in the research of endometrial cancer in the medical field. At present, the commonly used diagnostic methods for endometrial cancer are used for screening strategies, and all have more or less defects. Transvaginal ultrasound can not determine the boundary value of endometrial thickness in endometrial lesions; endometrial biopsy is more or less uncomfortable, and a considerable proportion of subjects are missed because of insufficient tissue; diagnostic curettage It is an invasive operation. It is obviously inappropriate to include a screening strategy, and it is not suitable for non-developed areas because of the medical level. The cost of hysteroscopy is high, and the diagnostic value alone is limited. The characteristics of endometrial cytology are the most Close to screening strategies, however, there is currently no uniform cytology standard. It can be seen that the screening strategy for endometrial cancer still needs further exploration.
近年来，肿瘤标志物由于其较高的特异性和灵敏度的特点，对肿瘤的筛查、诊断、分类、预后监测以及治疗提供相当大的指导意义。有研究表明CD44参与子宫内膜癌细胞的侵袭和转移。CD44属于细胞多功能粘附分子,是单基因编码的一种跨膜糖蛋白,其广泛存在于多种细胞和组织。CD44有10个变异外显子，因而其可以通过选择性剪切、拼接形成多种异构体,结合细胞表面相应配体,进而能够调节细胞的多种生理、病理过程。研究表明，CD44分子在多种肿瘤中高表达,并与肿瘤细胞的生长、侵袭、转移能力以及肿瘤环境中的造血干细胞的归巢密切相关,并可作为肿瘤干细胞的标志物,或可作为肿瘤筛查、预后判断及治疗的靶点。CD44的生物学功能及表达调控模式因不同条件而异，比如肿瘤种类、生长条件。多 种因素可影响其表达,如***受体、转录因子等。CD44的异常糖基化修饰可导致肿瘤细胞恶性程度的改变，探究子宫内膜癌中CD44的异常修饰，可为子宫内膜癌的筛查、诊断、预后判断及治疗提供一种新的靶点，对于子宫内膜癌的诊断和治疗将具有极其重要的意义。In recent years, due to its high specificity and sensitivity, tumor markers provide considerable guidance for tumor screening, diagnosis, classification, prognosis monitoring and treatment. Studies have shown that CD44 is involved in the invasion and metastasis of endometrial cancer cells. CD44 belongs to the cell multifunctional adhesion molecule and is a transmembrane glycoprotein encoded by a single gene, which is widely present in a variety of cells and tissues. CD44 has 10 variant exons, so it can form a variety of isomers by selective shearing and splicing, and bind to the corresponding ligands on the cell surface, which can regulate various physiological and pathological processes of cells. Studies have shown that CD44 is highly expressed in a variety of tumors and is closely related to the growth, invasion and metastasis of tumor cells and the homing of hematopoietic stem cells in the tumor environment. It can be used as a marker for cancer stem cells or as a tumor screen. The target of investigation, prognosis and treatment. The biological function and expression regulation mode of CD44 vary according to different conditions, such as tumor type and growth conditions. many Factors can affect its expression, such as estrogen receptors, transcription factors and so on. Abnormal glycosylation of CD44 can lead to changes in the malignancy of tumor cells, and to explore the abnormal modification of CD44 in endometrial cancer, which can provide a new target for screening, diagnosis, prognosis and treatment of endometrial cancer. It will be of great significance for the diagnosis and treatment of endometrial cancer.
发明内容Summary of the invention
本发明从新鲜的人子宫内膜癌组织中提取总蛋白免疫小鼠，制备杂交瘤细胞株。用ELISA的方法筛选到一株能与人子宫内膜癌组织高度特异结合的抗体EM2D9，该抗体属于IgG1亚类。免疫组织化学证实，EM2D9抗体与人子宫内膜癌组织呈强阳性反应，而与人正常子宫内膜组织无交叉反应。The present invention extracts total protein immunized mice from fresh human endometrial cancer tissues to prepare hybridoma cell lines. An antibody EM2D9 capable of highly specific binding to human endometrial cancer tissues was screened by ELISA, and the antibody belongs to the IgG1 subclass. Immunohistochemistry confirmed that EM2D9 antibody had a strong positive reaction with human endometrial cancer tissue, but no cross-reaction with human normal endometrial tissue.
本发明通过免疫沉淀结合质谱与糖芯片结果鉴定，EM2D9抗体识别的抗原是一种异常糖基化的CD44，其定位于细胞膜上，其表位为：Galb1‐3(Neu5Aca2‐6)GlcNAcb1‐4Galb1‐4Glcb属于全新的子宫内膜癌标志物。The present invention identifies by immunoprecipitation combined with mass spectrometry and sugar chip results that the antigen recognized by the EM2D9 antibody is an abnormally glycosylated CD44 which is localized on the cell membrane and has an epitope of: Galb1‐3 (Neu5Aca2‐6) GlcNAcb1‐4Galb1 ‐4Glcb is a new endometrial cancer marker.
本发明将EM2D9抗体偶联纳米磁颗粒，制备成抗子宫内膜癌免疫磁珠(EM2D9‐MB)，捕获并富集脱落的子宫内膜癌细胞，经瑞氏吉姆萨病理涂片染色，在显微镜下进行镜检诊断。该试剂盒适用于肿瘤病人的早期筛查、预后监测及病理辅助诊断。The invention couples the EM2D9 antibody into the nano magnetic particle, prepares an anti-endometrial cancer immunomagnetic beads (EM2D9-MB), captures and enriches the detached endometrial cancer cells, and stains with the pathological smear of Wright's Giemsa. Microscopic examination under the microscope. The kit is suitable for early screening, prognosis monitoring and pathological diagnosis of tumor patients.
本发明的创新性在于：(1)筛选并制备了一种抗人子宫内膜癌的单克隆抗体EM2D9，该抗体与人子宫内膜癌组织呈强阳性反应，而与人正常子宫内膜组织无交叉反应；(2)揭示了EM2D9抗体识别的抗原表位为Galb1‐3(Neu5Aca2‐6)GlcNAcb1‐4Galb1‐4Glcb，为全新的子宫内膜癌标志物；(3)开发了一种基于EM2D9抗体的高灵敏度的用于检测人子宫内膜癌的免疫磁珠方法。The innovation of the invention lies in: (1) screening and preparing a monoclonal antibody EM2D9 against human endometrial cancer, which has a strong positive reaction with human endometrial cancer tissue, and human normal endometrial tissue No cross-reactivity; (2) revealed that the epitope recognized by EM2D9 antibody is Galb1‐3 (Neu5Aca2‐6) GlcNAcb1‐4Galb1‐4Glcb, which is a new endometrial cancer marker; (3) developed an EM2D9 based Highly sensitive antibody magnetic beads method for detecting human endometrial cancer.
发明详述Detailed description of the invention
本发明提供了一种全新的人子宫内膜癌肿瘤标志物—异常糖基化的CD44，命名为GAB31。并获得了产生抗GAB31的单克隆抗体的杂交瘤细胞株，该杂交瘤细胞株分泌单克隆抗体EM2D9，单克隆抗体EM2D9识别的抗原表位为Galb1‐3(Neu5Aca2‐6)GlcNAcb1‐4Galb1‐4Glcb。该单克隆抗体与人子宫内膜癌组织呈强阳性反应，而与人正常子宫内膜组织无交叉反应。本发明还提供了基于单克隆抗体EM2D9的体外子宫内膜癌诊断的免疫磁珠(EM2D9‐MB)诊断方法。The present invention provides a novel human endometrial cancer tumor marker, abnormally glycosylated CD44, designated GAB31. A hybridoma cell line producing a monoclonal antibody against GAB31 was obtained, and the hybridoma cell line secreted the monoclonal antibody EM2D9, and the epitope recognized by the monoclonal antibody EM2D9 was Galb1‐3 (Neu5Aca2‐6) GlcNAcb1‐4Galb1‐4Glcb. . The monoclonal antibody has a strong positive reaction with human endometrial cancer tissue and no cross-reaction with human normal endometrial tissue. The invention also provides an immunomagnetic beads (EM2D9-MB) diagnostic method based on monoclonal antibody EM2D9 for diagnosis of in vitro endometrial cancer.
具体地，本发明提供了一种异常糖基化的人子宫内膜癌肿瘤标志物GAB31，其为异常糖基化的CD44，其特征是在CD44上包含抗原表位的糖结构Galb1‐3(Neu5Aca2‐6)GlcNAcb1‐4Galb1‐4Glcb。In particular, the present invention provides an aberrantly glycosylated human endometrial cancer tumor marker GAB31 which is aberrantly glycosylated CD44 characterized by a glycostructure Galb1‐3 comprising an epitope on CD44 ( Neu5Aca2‐6) GlcNAcb1‐4Galb1‐4Glcb.
在一个优选方案中，所述CD44的氨基酸序列如SEQ ID No:1所示。 In a preferred embodiment, the amino acid sequence of CD44 is set forth in SEQ ID No: 1.
本发明的另一个目的是提供针对本发明所述的人子宫内膜癌标志物GAB31的抗体，其能够特异性识别所述作为抗原表位的糖结构Galb1‐3(Neu5Aca2‐6)GlcNAcb1‐4Galb1‐4Glcb，所述抗体为多克隆抗体或单克隆抗体，优选单克隆抗体。Another object of the present invention is to provide an antibody against the human endometrial cancer marker GAB31 of the present invention which is capable of specifically recognizing the glycostructure Galb1‐3 (Neu5Aca2‐6) GlcNAcb1‐4Galb1 as an epitope. ‐4Glcb, the antibody is a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody.
本发明的另一个目的是提供一种用于检测人子宫内膜癌的试剂盒，其包含本发明上述的抗体EM2D9。一个优选实施方案中，所述的抗体是抗人子宫内膜癌GAB31的单克隆抗体EM2D9，该单克隆抗体由保藏号为CGMCC No.11796的杂交瘤细胞株分泌。Another object of the present invention is to provide a kit for detecting human endometrial cancer comprising the above-described antibody EM2D9 of the present invention. In a preferred embodiment, the antibody is monoclonal antibody EM2D9 against human endometrial cancer GAB31, which is secreted by a hybridoma cell line with accession number CGMCC No. 11796.
本发明的另一个目的是提供一种缀合物，其中包含所述抗人子宫内膜癌GAB31与选自以下组分组成的物质缀合：生物标记物、抗肿瘤药物、毒素和放射性活性剂。Another object of the present invention is to provide a conjugate comprising the anti-human endometrial cancer GAB31 conjugated with a substance selected from the group consisting of biomarkers, antitumor drugs, toxins and radioactive agents .
本发明的另一个目的是提供一种用于检测或治疗人子宫内膜癌的试剂盒，其中包含本发明上述的抗体。Another object of the present invention is to provide a kit for detecting or treating human endometrial cancer comprising the above-described antibody of the present invention.
在一个优选实施方案中，在所述的试剂盒中，所述的检测是通过单克隆抗体EM2D9偶联纳米磁颗粒进行，优选将EM2D9抗体偶联纳米磁颗粒，制备成抗子宫内膜癌免疫磁珠(EM2D9‐MB)。在一个优选实施方案中，所述待测样品是含人子宫内膜癌脱落细胞宫腔刮取物。In a preferred embodiment, in the kit, the detection is carried out by coupling the nano-magnetic particles with the monoclonal antibody EM2D9, preferably by coupling the EM2D9 antibody to the nano-magnetic particles to prepare for anti-endometrial cancer immunity. Magnetic beads (EM2D9‐MB). In a preferred embodiment, the sample to be tested is a uterine scraping containing human endometrial cancer exfoliated cells.
本发明的另一个目的是提供分泌抗人子宫内膜癌GAB31的单克隆抗体EM2D9的杂交瘤细胞株，其保藏号为CGMCC No.11796。Another object of the present invention is to provide a hybridoma cell line secreting monoclonal antibody EM2D9 against human endometrial cancer GAB31, which has a accession number of CGMCC No. 11796.
附图说明DRAWINGS
图1.EM2D9单抗对人子宫内膜癌组织切片的免疫组化图。Figure 1. Immunohistochemical map of EM2D9 mAb to human endometrial cancer tissue sections.
图2.EM2D9单抗对人正常子宫内膜组织切片的免疫组化图。Figure 2. Immunohistochemical map of EM2D9 mAb to human normal endometrial tissue sections.
图3.糖芯片检测结果。Figure 3. Sugar chip test results.
具体实施方式detailed description
下文将参考实施例详细描述本发明，所述实施例仅是意图举例说明本发明，而不是意图限制本发明的范围。本发明的范围由后附的权力要求具体限定。The invention is described in detail below with reference to the embodiments, which are intended to illustrate the invention, but not to limit the scope of the invention. The scope of the invention is specifically defined by the appended claims.
实施例1：EM2D9单克隆抗体的制备和纯化Example 1: Preparation and purification of EM2D9 monoclonal antibody
(1)杂交瘤的制备(1) Preparation of hybridoma
1)动物免疫及细胞培养：采用新鲜的人子宫内膜癌组织蛋白匀浆液免疫Balb/C小鼠(购买自北京维通利华实验动物技术有限公司)，以每只20ug总蛋白的剂量进行腹腔注射方式免疫小鼠。两周后对小鼠再次免疫。小鼠血清效价达到要求后，加强免疫一次，3天后取小鼠脾脏，制备成淋巴细胞悬液，准备细胞融合。复苏骨髓瘤细胞Sp2/0(ATCC CRL‐1772), 并用8‐AG(8氮杂鸟嘌呤)筛选以维持细胞对HAT的敏感性。1) Animal immunization and cell culture: Balb/C mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were immunized with fresh human endometrial cancer tissue protein homogenate at a dose of 20 ug total protein. Mice were immunized by intraperitoneal injection. The mice were re-immunized two weeks later. After the serum titer of the mouse reached the requirement, the immunization was boosted once, and the spleen of the mouse was taken 3 days later to prepare a suspension of lymphocytes to prepare for cell fusion. Resusciting myeloma cell Sp2/0 (ATCC CRL‐1772), 8-AG (8-azaguanine) was screened to maintain cell sensitivity to HAT.
2)细胞融合：将步骤1)制备的淋巴细胞悬液与骨髓瘤细胞进行融合，具体方法参照《精编免疫学实验指南》((美国)J.E.科学根(美国)D.H.马古利斯等，科学出版社，2009年1月出版)。将融合后细胞悬液加入含有饲养细胞培养基中培养。融合24小时后，加HAT选择培养基(购自Invitrogen公司；HAT即H:Hypoxanthine次黄嘌呤，A:Aminopterin甲氨喋呤，T:Thymidine胸腺嘧啶核苷)进行选择性培养。2) Cell fusion: The lymphocyte suspension prepared in step 1) is fused with myeloma cells, and the specific method is referred to the "Guide to the Editing Immunology Experiment" ((United States) JE Science Roots (USA) DH Margulis, etc. Science Press, published in January 2009). The fused cell suspension is added to a culture medium containing feeder cells. After 24 hours of fusion, HAT selection medium (purchased from Invitrogen; HAT ie H: Hypoxanthine hypoxanthine, A: Aminopterin methotrexate, T: Thymidine thymidine) was selectively cultured.
3)抗体检测：通过ELISA方法确定分泌抗体的杂交瘤细胞株。具体方法是：提取子宫内膜癌组织总蛋白，用0.05mol/L碳酸盐缓冲液(pH9.6)4℃包被过夜，加入10％BSA 37℃封闭3小时。PBST洗涤3次，然后再加入100ul待检上清，37℃孵育1h。洗涤3次，加入抗鼠的辣根过氧化物酶标记的二抗IgG‐HRP(购自北京中杉金桥生物技术有限公司)，37℃孵育1h。洗涤3次，加入50ul TMB(购自北京中杉金桥生物技术有限公司)显色，室温静置5min后，加入50ul终止液。用酶标仪读取波长为450nm的OD值。OD值大于阴性对照OD值的2倍以上的视为阳性。3) Antibody detection: The antibody-secreting hybridoma cell strain was determined by an ELISA method. The specific method is: extracting total protein of endometrial cancer tissue, coating with 0.05 mol/L carbonate buffer (pH 9.6) at 4 ° C overnight, and adding 10% BSA at 37 ° C for 3 hours. The cells were washed 3 times with PBST, then 100 ul of the supernatant to be tested was added, and incubated at 37 ° C for 1 h. After washing 3 times, an anti-mouse horseradish peroxidase-labeled secondary antibody IgG-HRP (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) was added and incubated at 37 ° C for 1 h. After washing 3 times, 50 ul of TMB (purchased from Beijing Zhongshang Jinqiao Biotechnology Co., Ltd.) was added for color development, and after standing at room temperature for 5 min, 50 ul of the stop solution was added. The OD value at a wavelength of 450 nm was read with a microplate reader. An OD value greater than 2 times the OD value of the negative control was considered positive.
3)杂交瘤的克隆化和冻存：使用有限稀释法将筛选出的阳性杂交瘤细胞进行克隆化培养。经过3轮的克隆化培养，筛选出高效价单克隆抗体的杂交瘤细胞克隆扩大培养。3) Cloning and cryopreservation of hybridomas: The selected positive hybridoma cells were cloned and cultured by limiting dilution method. After three rounds of cloning culture, hybridoma cell clones with high titer monoclonal antibodies were screened for expanded culture.
本发明中获得的一种阳性杂交瘤细胞株为抗人子宫内膜癌的单克隆杂交瘤细胞株，该杂交瘤细胞株于2015年12月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC，中国，北京)，保藏号为CGMCC No.11796。A positive hybridoma cell line obtained in the present invention is a monoclonal hybridoma cell line against human endometrial cancer, and the hybridoma cell line is deposited on the Chinese Microbial Culture Collection Management Committee on December 23, 2015. Center (CGMCC, China, Beijing), the deposit number is CGMCC No.11796.
(2)EM2D9单抗的制备和纯化(2) Preparation and purification of EM2D9 monoclonal antibody
将上述稳定分泌单克隆抗体EM2D9的杂交瘤细胞株(保藏号CGMCC No.11796)扩大培养，并收集细胞培养上清。采用Protein G对单抗EM2D9进行亲和层析纯化，简单步骤是：首先用PBS平衡Protein G亲和层析柱(购自“GE”公司)；然后将含EM2D9单抗的细胞培养上清过Protein G亲和层析柱；随后用PBS洗涤层析柱，直到流出柱子的洗涤液的OD值接近零；使用0.2M的甘氨酸‐HCL溶液(PH2.8)洗脱柱子，分管收集洗脱液，测定各收集管的OD值。含EM2D9单抗的洗脱液经PBS透析浓缩后‐20℃冻存。The above-described hybridoma cell strain stably stabilizing the monoclonal antibody EM2D9 (Accession No. CGMCC No. 11796) was expanded and cultured, and the cell culture supernatant was collected. Affinity chromatography of monoclonal antibody EM2D9 was performed using Protein G. The simple procedure was to first equilibrate the Protein G affinity chromatography column (purchased from "GE") with PBS; then, the cell culture containing EM2D9 monoclonal antibody was supernatantd. Protein G affinity chromatography column; then wash the column with PBS until the OD value of the washing solution flowing out of the column is close to zero; elute the column with 0.2 M glycine-HCL solution (pH 2.8), and collect the eluent in a separate tube. The OD value of each collection tube was measured. The eluate containing EM2D9 mAb was dialyzed against PBS and frozen at -20 °C.
实施例2：EM2D9单克隆抗体的鉴定Example 2: Identification of EM2D9 monoclonal antibody
使用实施例1中制备的EM2D9单抗对人子宫内膜癌组织和人正常子宫内膜组织切片，结果如图1、2所示。结果表明，人子宫内膜癌组织经EM2D9单抗免疫组化染色显色后呈现阳性反应(图1)，而人正常子宫内膜组织经EM2D9单抗免疫组化染色显色后呈现阴性反应(图2)。 Human endometrial cancer tissues and human normal endometrial tissues were sectioned using the EM2D9 monoclonal antibody prepared in Example 1, and the results are shown in Figs. The results showed that human endometrial cancer tissues showed a positive reaction after immunohistochemical staining by EM2D9 monoclonal antibody (Fig. 1), while human normal endometrial tissue showed a negative reaction after immunohistochemical staining with EM2D9 monoclonal antibody. figure 2).
使用实施例1中制备的EM2D9单抗，免疫组化检测人子宫内膜癌组织、人正常子宫内膜组织和其它组织，结果如表1所示。结果表明，EM2D9抗体与人子宫内膜癌组织呈强阳性反应，而与人正常子宫内膜组织和其它组织无交叉反应。Human endometrial cancer tissues, human normal endometrial tissues and other tissues were detected by immunohistochemistry using the EM2D9 monoclonal antibody prepared in Example 1, and the results are shown in Table 1. The results showed that EM2D9 antibody had a strong positive reaction with human endometrial cancer tissue, but no cross-reaction with human normal endometrial tissue and other tissues.
表1Table 1
免疫组化检测抗人子宫内膜癌单抗EM2D9对多种组织的免疫反应Immunohistochemical detection of anti-human endometrial cancer monoclonal antibody EM2D9 immune response to various tissues
组织(癌组织和正常组织)Tissue (cancer tissue and normal tissue) EM2D9抗体EM2D9 antibody
人子宫内膜癌组织(Patient#1)Human endometrial cancer tissue (Patient#1) ++++++
人正常子宫内膜组织(Patient#1)Human normal endometrial tissue (Patient#1) －-
人子宫内膜癌组织(Patient#2)Human endometrial cancer tissue (Patient#2) ++++++
人正常子宫内膜组织(Patient#2)Human normal endometrial tissue (Patient#2) －-
人子宫内膜癌组织(Patient#3)Human endometrial cancer tissue (Patient#3) ++++
人正常子宫内膜组织(Patient#3)Human normal endometrial tissue (Patient#3) －-
人子宫内膜癌组织(Patient#4)Human endometrial cancer tissue (Patient#4) ++++
人正常子宫内膜组织(Patient#4)Human normal endometrial tissue (Patient#4) －-
人子宫内膜癌组织(Patient#5)Human endometrial cancer tissue (Patient#5) ++++++
人正常子宫内膜组织(Patient#5)Human normal endometrial tissue (Patient#5) －-
人肾癌组织Human renal cell carcinoma －-
人正常肾组织Normal kidney tissue －-
人胃癌组织Human gastric cancer tissue －-
人正常胃组织Human normal stomach tissue －-
人肝癌组织Human liver cancer tissue －-
人正常肝组织Human normal liver tissue －-
人乳腺癌组织Human breast cancer tissue －-
人正常乳腺组织Human normal breast tissue －-
人食管癌组织Human esophageal cancer tissue －-
人正常食管组织Normal esophageal tissue －-
人结肠癌组织Human colon cancer tissue －-
人正常结肠组织Human normal colon tissue －-
实施例3：GAB31抗原的制备Example 3: Preparation of GAB31 antigen
1)总蛋白提取：50mg人子宫内膜癌组织，剪碎研磨成匀浆后，加入1ml的三去污裂解液，4℃裂解5分钟，12000rpm离心20分钟，取上清，即为人子宫内膜癌组织总蛋白。1) Total protein extraction: 50mg human endometrial cancer tissue, after cutting and grinding into homogenate, add 1ml of three decontamination lysate, lyse at 4 °C for 5 minutes, centrifuge at 12000 rpm for 20 minutes, take the supernatant, which is human uterus Membrane cancer tissue total protein.
2)免疫沉淀：加入100ug EM2D9单抗，4℃孵育2h。2) Immunoprecipitation: 100 ug of EM2D9 mAb was added and incubated for 2 h at 4 °C.
3)抗原鉴定：加入50ul Protein G beads，4℃孵育2h。用PBS洗涤beads，然后用0.2M的甘氨酸‐HCL溶液(PH 2.8)洗脱beads，进行质谱分析。质谱结果如表2所示。质谱结果表明，EM2D9的抗原为CD44。经过分析EM2D9的糖心片数据(如图3所示)发现，EM2D9单抗识别的抗原表位为人CD44的糖链，该表位是Galb1‐3(Neu5Aca2‐6)GlcNAcb1‐4Galb1‐4Glcb。由于EM2D9单抗只特异性的识别人子宫内膜癌组织，表明该抗原为异常糖基化修饰的CD44，其表位Galb1‐3(Neu5Aca2‐6)GlcNAcb1‐4Galb1‐4Glcb只表达于人子宫内膜癌组织细胞。3) Antigen identification: 50ul Protein G beads were added and incubated for 2 h at 4 °C. The beads were washed with PBS, and then the beads were eluted with a 0.2 M glycine-HCL solution (pH 2.8) for mass spectrometry analysis. The mass spectrum results are shown in Table 2. Mass spectrometry results indicated that the antigen of EM2D9 was CD44. After analyzing the glycocardial data of EM2D9 (as shown in Figure 3), it was found that the epitope recognized by EM2D9 mAb was the sugar chain of human CD44, which was Galb1‐3 (Neu5Aca2‐6) GlcNAcb1‐4Galb1‐4Glcb. Since EM2D9 monoclonal antibody specifically recognizes human endometrial cancer tissue, it indicates that the antigen is abnormally glycosylated CD44, and its epitope Galb1‐3 (Neu5Aca2‐6) GlcNAcb1‐4Galb1‐4Glcb is only expressed in human uterus. Membrane cancer tissue cells.
表2Table 2
EM2D9抗原的质谱鉴定Mass Spectrometric Identification of EM2D9 Antigen
AccessionAccession MassMass ScoreScore
CD44HUMANCD44HUMAN 176389176389 217217
Albumin HUMANAlbumin HUMAN 4236742367 4343
Stratifin HUMANStratifin HUMAN 2381923819 3232
Actin,gamma 1HUMANActin, gamma 1HUMAN 1537615376 3232
Desmoglein 1HUMANDesmoglein 1HUMAN 3853238532 3030
Histone H2B HUMANHistone H2B HUMAN 7251372513 3030
Hornerin HUMANHornerin HUMAN 3128531285 2828
Keratin 13HUMANKeratin 13HUMAN 6352763527 2525
Keratin 15HUMANKeratin 15HUMAN 1972819728 2525
Keratin 6B HUMANKeratin 6B HUMAN 2438624386 2020
实施例4：人子宫内膜癌免疫诊断试剂的制备Example 4: Preparation of human endometrial cancer immunodiagnostic reagent
利用本发明中所制备的单抗EM2D9，本发明我们将EM2D9抗体偶联纳米磁颗粒，制备成抗子宫内膜癌免疫磁珠(EM2D9‐MB)。将其与宫腔刮取物混合，捕获并富集脱落的 子宫内膜癌细胞，经瑞氏吉姆萨病理染色，在显微镜下进行诊断。该试剂盒适用于肿瘤病人的早期筛查、预后监测及病理辅助诊断。实验证实本发明的人子宫内膜癌免疫诊断试剂与已有技术相比具有以下积极效果：(1)灵敏度高，由于偶联单抗EM2D9免疫磁颗粒的富集作用，该方法的灵敏度远高于常规的脱落细胞学的检测方法；(2)特异性强，由于该方法偶联了特异性结合子宫内膜癌组织细胞的EM2D9抗体，它能特异性的识别子宫内膜癌细胞；(3)方便快捷，节省患者就医费用，由于该方法灵敏度高、特异性强等特点，对子宫内膜癌患者的检出率高，避免患者反复就医确诊，节省费用。Using the monoclonal antibody EM2D9 prepared in the present invention, in the present invention, we coupled the EM2D9 antibody to nano magnetic particles to prepare an anti-endometrial cancer immunomagnetic beads (EM2D9-MB). Mix it with the uterine scraping, capture and enrich the shedding Endometrial cancer cells were stained with Wright's Giemsa pathology and diagnosed under a microscope. The kit is suitable for early screening, prognosis monitoring and pathological diagnosis of tumor patients. The experiment confirmed that the human endometrial cancer immunodiagnostic reagent of the present invention has the following positive effects compared with the prior art: (1) high sensitivity, and the sensitivity of the method is high due to the enrichment of the conjugated monoclonal antibody EM2D9 immunomagnetic particles. In the conventional exfoliative cytology detection method; (2) strong specificity, because the method is coupled with EM2D9 antibody that specifically binds to endometrial cancer tissue cells, it can specifically recognize endometrial cancer cells; (3 It is convenient and quick, and saves patients' medical expenses. Because of its high sensitivity and strong specificity, the detection rate of endometrial cancer patients is high, avoiding repeated diagnosis and saving of patients.
具体实验方法如下：The specific experimental methods are as follows:
1)宫腔刮取物采集：子宫内膜癌患者来自北京大学第三医院。1) Intrauterine scraping collection: Patients with endometrial cancer are from Peking University Third Hospital.
2)子宫内膜细胞的富集：将偶联EM2D9抗体的磁颗粒与患者的宫腔刮取物温孵育20min，然后使用磁力架将磁颗粒与患者的宫腔刮取物分离，从而富集子宫内膜癌细胞；2) Enrichment of endometrial cells: The magnetic particles coupled with the EM2D9 antibody were incubated with the patient's uterine scrapings for 20 min, and then the magnetic particles were separated from the patient's uterine scrapings using a magnetic stand to enrich Endometrial cancer cells;
3)瑞氏吉姆萨病理染色：将步骤2)富集得到的子宫内膜癌细胞进行瑞氏吉姆萨病理染色，并制备染色玻片用于镜检。3) Wright's Giemsa pathological staining: The endometrial cancer cells enriched in step 2) were stained with Wright's Giemsa pathology, and stained slides were prepared for microscopic examination.
4)镜检诊断：根据子宫内膜癌细胞的形态特征，判断病理染色结果。4) Microscopic diagnosis: According to the morphological characteristics of endometrial cancer cells, the pathological staining results were judged.
EM2D9‐MB法与常规涂片法阳性检出率比较结果，如表3所示。结果表明，采用EM2D9‐MB检测了来自北京大学第三医院32例子宫内膜癌患者的宫腔刮取物，EM2D9‐MB的阳性检出率为90.63％(29/32)，远高于常规脱落细胞涂片的阳性检出率43.75％(14/32)。因此，子宫内膜癌细胞特异表达的GAB31是一个很有应用前景的子宫内膜癌标志物，抗子宫内膜癌免疫磁珠(EM2D9‐MB)是一种简单高效的子宫内膜癌诊断试剂。 The comparison results of the positive detection rates of the EM2D9-MB method and the conventional smear method are shown in Table 3. The results showed that EM2D9‐MB was used to detect uterine scraping from 32 patients with endometrial cancer in Peking University Third Hospital. The positive detection rate of EM2D9‐MB was 90.63% (29/32), which was much higher than the conventional one. The positive detection rate of exfoliated cell smears was 43.75% (14/32). Therefore, GAB31, which is specifically expressed in endometrial cancer cells, is a promising endometrial cancer marker. Anti-endometrial cancer immunomagnetic beads (EM2D9-MB) is a simple and effective diagnostic tool for endometrial cancer. .
表3table 3
Patient No.Patient No. 常规涂片法Conventional smear method LC128‐MB法LC128‐MB method
#1#1 ++ ++
#2#2 －- ++
#3#3 －- ++
#4#4 ++ ++
#5#5 －- ++
#6#6 ++ ++
#7#7 －- ++
#8#8 －- ++
#9#9 －- －-
#10#10 ++ ++
#11#11 －- ++
#12#12 ++ －-
#13#13 －- ++
#14#14 ++ ++
#15#15 －- ++
#16#16 －- ++
#17#17 ++ ++
#18#18 ++ ++
#19#19 ++ ++
#20#20 －- ++
#21#twenty one －- ++
#22#twenty two －- ++
#23#twenty three ++ ++
#24#twenty four －- ++
#25#25 ++ ++
#26#26 －- ++
#27#27 －- ++
#28#28 ++ －-
#29#29 ++ ++
#30#30 －- ++
#31#31 ++ ++
#32#32 －- ++
合计total 43.75％(14/32)43.75% (14/32) 90.63％(29/32)90.63% (29/32)

Claims (9)

  1. 一种人子宫内膜癌肿瘤标志物GAB31，其为异常糖基化的CD44，所述异常糖基化的CD44是指CD44带有作为抗原表位的糖结构Galb1‐3(Neu5Aca2‐6)GlcNAcb1‐4Galb1‐4Glcb，命名为GAB31。A human endometrial cancer tumor marker GAB31, which is aberrantly glycosylated CD44, which refers to CD44 with a sugar structure Galb1‐3 (Neu5Aca2‐6) GlcNAcb1 as an epitope. ‐4Galb1‐4Glcb, named GAB31.
  2. 根据权利要求1所述的人子宫内膜癌肿瘤标志物GAB31，其中所述CD44的氨基酸序列如SEQ ID No:1所示。The human endometrial cancer tumor marker GAB31 according to claim 1, wherein the amino acid sequence of the CD44 is represented by SEQ ID No: 1.
  3. 一种针对根据权利要求1或2所述的人子宫内膜癌肿瘤标志物GAB31的抗体，其能够特异性识别所述作为抗原表位的糖结构Galb1‐3(Neu5Aca2‐6)GlcNAcb1‐4Galb1‐4Glcb，所述抗体为特异性抗人子宫内膜癌CD44的单克隆抗体EM2D9，该单克隆抗体有保藏号为CGMCC No.11796的杂交瘤细胞株分泌。An antibody against the human endometrial cancer tumor marker GAB31 according to claim 1 or 2, which is capable of specifically recognizing the sugar structure Galb1‐3 (Neu5Aca2‐6) GlcNAcb1‐4Galb1‐ as an antigenic epitope 4Glcb, the antibody is a monoclonal antibody EM2D9 specific for anti-human endometrial cancer CD44, which is secreted by a hybridoma cell line with the accession number CGMCC No. 11796.
  4. 根据权利要求1所述标志物或权利要求3所述的抗体用于制备诊断子宫内膜癌试剂的应用。Use of the marker of claim 1 or the antibody of claim 3 for the preparation of a diagnostic endometrial cancer agent.
  5. 一种缀合物，其中抗人子宫内膜癌CD44的抗体EM2D9与选自以下组分组成的缀合物：生物标记物、抗肿瘤药物、毒素、放射性活性剂、磁珠，所述抗体EM2D9为单克隆抗体，由保藏号为CGMCC No.11796的杂交瘤细胞株分泌。A conjugate, wherein the antibody EM2D9 against human endometrial cancer CD44 is conjugated with a component selected from the group consisting of a biomarker, an antitumor drug, a toxin, a radioactive agent, a magnetic bead, said antibody EM2D9 It is a monoclonal antibody secreted by a hybridoma cell line with the accession number CGMCC No. 11796.
  6. 一种用于检测或治疗人子宫内膜癌的试剂盒，其中包含根据权利要求1或3所述的抗原或抗体。A kit for detecting or treating human endometrial cancer, comprising the antigen or antibody according to claim 1 or 3.
  7. 根据权利要求6所述的试剂盒，其中所述检测是通过免疫磁珠法进行，所述免疫磁珠法是采用将权利要求3所述的抗体EM2D9偶联纳米磁颗粒，制备成抗子宫内膜癌免疫磁珠EM2D9‐MB。The kit according to claim 6, wherein said detecting is carried out by an immunomagnetic beads method, wherein said antibody EM2D9 according to claim 3 is coupled to nanomagnetic particles to prepare an anti-uterine Membrane cancer immunomagnetic beads EM2D9-MB.
  8. 根据权利要求6或7所述的试剂盒，其中待测样品是含子宫内膜癌脱落细胞的人宫腔刮取物。The kit according to claim 6 or 7, wherein the sample to be tested is a human uterine scraping containing endometrial cancer exfoliated cells.
  9. 分泌抗人子宫内膜癌CD44的抗体EM2D9的杂交瘤细胞株，其保藏号为CGMCC No.11796。 A hybridoma cell line secreting an antibody against human endometrial cancer CD44, EM2D9, having a accession number of CGMCC No. 11796.
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