阅读说明：本技术 抗MG7-Ag的单克隆抗体及其用途 (anti-MG 7-Ag monoclonal antibody and application thereof ) 是由 聂勇战 吴开春 樊代明 赵青川 于 2018-07-06 设计创作，主要内容包括：本发明提供了一种抗MG7-Ag的单克隆抗体及其用途。所述单克隆抗体为抗MG7-Ag的单克隆抗体MGd1-Ab。本发明还提供了分泌所述抗MG7-Ag的单克隆抗体MGd1-Ab的杂交瘤细胞株。本发明还提供了包含所述抗MG7-Ag的单克隆抗体MGd1-Ab的试剂盒,以及所述试剂盒用于检测胃癌抗原MG7-Ag的用途；和用于在临床组织样本中检测胃癌抗原MG7-Ag表达水平的用途。本发明提供的抗MG7-Ag的单克隆抗体质地均一、特异性强。本发明的检测试剂盒可根据用途进行灵敏度和检测范围调控,从而高度敏感地检测胃癌抗原MG7-Ag表达水平。(The invention provides a monoclonal antibody for resisting MG7-Ag and application thereof. The monoclonal antibody is a monoclonal antibody MGd1-Ab of MG 7-Ag. The invention also provides a hybridoma cell strain secreting the anti-MG 7-Ag monoclonal antibody MGd 1-Ab. The invention also provides a kit containing the anti-MG 7-Ag monoclonal antibody MGd1-Ab, and application of the kit in detecting gastric cancer antigen MG 7-Ag; and for detecting the expression level of gastric cancer antigen MG7-Ag in clinical tissue samples. The monoclonal antibody of the invention for resisting MG7-Ag is homogeneous and has strong specificity. The detection kit can regulate and control the sensitivity and the detection range according to the application, thereby detecting the expression level of the gastric cancer antigen MG7-Ag with high sensitivity.)

1. An anti-MG 7-Ag monoclonal antibody MGd1-Ab, wherein the monoclonal antibody MGd1-Ab is secreted by a hybridoma cell strain MGd1 with the preservation number of CCTCCNO: C2016130.

2. A hybridoma cell strain MGd1 with the preservation number of CCTCC NO: C2016130.

3. A device for detecting the presence and/or level of MG7-Ag in a sample, the device comprising the anti-MG 7-Ag monoclonal antibody MGd1-Ab of claim 1.

4. The device of claim 3, wherein the sample is a tissue, blood, or bodily fluid of a subject; preferably, the body fluid is ascites fluid.

5. The device of claim 4, wherein the subject is a patient with a malignant tumor or a high risk group suffering from a malignant tumor;

preferably, the malignant tumor is gastric cancer, colon cancer or esophageal cancer.

6. The device of any one of claims 3-5, wherein the device is a kit;

preferably, the device is a kit for aiding in the diagnosis of a tumor, monitoring a tumor, and/or prognosis of a patient with a tumor.

7. Use of the anti-MG 7-Ag monoclonal antibody MGd1-Ab of claim 1 in the preparation of a reagent or kit for detecting the presence and/or level of MG7-Ag in a sample.

8. The use of claim 7, wherein the detection uses an immunological method; preferably, the immunological method is selected from one or more of immunoblotting, immunoprecipitation, immunohistochemistry, enzyme-linked immunosorbent assay or immunochemiluminometry.

Technical Field

The invention belongs to the field of immunology, and relates to an anti-MG 7-Ag monoclonal antibody and an application thereof, in particular to an application of a kit containing the antibody in detecting the level of MG7-Ag in a sample, and an application of the kit containing the antibody in auxiliary diagnosis of tumors, monitoring of tumors and prognosis of tumor patients.

Background

Tumor protein markers are substances produced and released by tumor cells, often exist in the tumor cell tissues of patients or in host body fluids and effluents in the form of metabolites such as antigens, enzymes, proteins or peptide hormones, and can identify or diagnose tumors according to biochemical or immunological properties. The tumor protein marker is mainly used for finding primary tumors, screening high risk groups of tumors, carrying out differential diagnosis on benign and malignant tumors, judging the development degree of the tumors, observing and evaluating the treatment effect of the tumors, predicting the recurrence and prognosis of the tumors and the like in clinic. In recent years, new tumor markers are continuously discovered, and detection means are continuously perfected and improved, so that the sensitivity and accuracy of the tumor markers are continuously improved, and a more exact and sufficient basis is provided for early diagnosis and early treatment of tumor patients.

The incidence of malignant tumors has continued to rise worldwide in recent years. Stomach cancer is one of the most common cancers worldwide, new cases of stomach cancer live at the first global level every year in China at present, the morbidity and the mortality are more than 2 times of the average level in the world, and about 1 Chinese die of stomach cancer every 2-3 minutes. The mortality rate of gastric cancer in China is now second only to lung cancer, and is the second largest cancer killer. Because the gastric cancer is mostly hidden, no obvious symptoms exist in the early stage, patients often arrive at the late stage when seeing a doctor, and the prognosis of a plurality of patients with similar clinical traditional pathology and TNM stage performance is greatly different, reflecting the complexity of the occurrence and development of the gastric cancer. At present, an ideal gastric cancer tumor marker for diagnosis or prognosis is not available. For example, carcinoembryonic antigen (CEA) which is used as a marker of digestive tract tumor is increased in some non-tumor diseases such as gastritis, liver cirrhosis and ulcerative colitis for decades, and the specificity is not ideal. Therefore, the search for tumor markers and/or tumor antigens related to the occurrence and development of gastric cancer is of great significance to clinical diagnosis, disease development monitoring and prognosis judgment of gastric cancer, and also has great significance to the research on the molecular mechanism of tumor occurrence.

MG7-Ag is an antigen recognized by a gastric cancer monoclonal antibody successfully prepared by taking a poorly differentiated gastric cancer cell line as an immunogen, and researches show that: the positive expression rate of MG7-Ag is obviously increased in the process of the evolution from normal gastric mucosa to chronic gastritis to intraepithelial neoplasia (P < 0.001). Meanwhile, positive expression of MG7-Ag is found to be positively correlated with the invasion depth of gastric cancer (P <0.001), and then survival stratification analysis is carried out on MG7-Ag, and the positive rate of MG7-Ag is obviously higher (P <0.05) compared with traditional tumor antigens such as CEA, CA199, CA125 and CA72-4, wherein the survival stratification analysis shows that the positive rate is correlated with the prognosis of patients with advanced clinical stages (III and IV) (P is 0.033). The research shows that the MG7-Ag has obvious expression increase in precancerous lesion, which indicates that the MG7-Ag is probably a key regulatory molecule of the gastric cancer generation process; MG7-Ag has better sensitivity in gastric cancer diagnosis; in addition, MG7-Ag may be an independent index for judging the prognosis of patients with advanced clinical stage.

The above studies indicate that MG7-Ag is closely related to cell proliferation, transformation, and malignant transformation, and plays an important role in the development and progression of tumors. However, the prior art quantitative methods for detecting MG7-Ag are mainly competitive Radioimmunoassay (RIA) using polyclonal antibodies and enzyme-linked immunosorbent assay (ELISA) using a double antibody sandwich assay format. The ELISA method has high sensitivity, strong specificity, stable labeling reagent and no radiation pollution and harm of the RIA method, thereby having wide application. However, most of the existing kits are limited in that key reagents such as detection antibodies cannot be produced by themselves, so that the price is high, and the quality and the stability are difficult to guarantee for a long time; some kits are also limited by the use of polyclonal antibodies, which are available in limited quantities and vary widely from batch to batch, and are difficult to adapt to the need for large batches of prospective clinical studies over a long period of time.

Based on this, an anti-MG 7-Ag monoclonal antibody with strong specificity and high sensitivity is urgently needed so as to contribute to clinical diagnosis and prognosis judgment of gastric cancer and guide clinical treatment.

Disclosure of Invention

Therefore, in order to overcome the defects of the prior art, the invention aims to provide an anti-MG 7-Ag monoclonal antibody which is uniform in physique, good in stability and capable of being produced in large scale for a long time; the invention also provides a kit for detecting the anti-MG 7-Ag, which is simple and convenient to operate, high in sensitivity, low in cost and suitable for mass production. In addition, the anti-MG 7-Ag monoclonal antibody can also be used for immunoblotting, immunoprecipitation, immunohistochemistry, enzyme-linked immunosorbent assay or immunochemiluminescence, and can be used for preparing medicines for treating MG7-Ag positive tumors.

In one aspect, the invention provides an anti-MG 7-Ag monoclonal antibody MGd1-Ab, wherein the monoclonal antibody MGd1-Ab is secreted by a hybridoma cell strain MGd1 with the preservation number of CCTCC NO: C2016130.

On the other hand, the invention provides a hybridoma cell strain MGd1 with the preservation number of CCTCC NO: C2016130.

Wherein the preservation number of the hybridoma cell strain MGd1 is CCTCC NO: C2016130 (preservation date: 2016, 6 and 23 days, preservation number is CCTCC NO: C201613, preservation place: China center for type culture Collection, address: eight path 299 # Wuhan university school in Wuchang district of Wuhan, Hubei province (first attached small opposite side of Wuhan university), preservation center of Wuhan university postal code: 430072);

an anti-MG 7-Ag monoclonal antibody MGd1-Ab according to the invention, wherein the subtype of the monoclonal antibody is identified as IgG 1.

In yet another aspect, the invention provides a device for detecting the presence and/or level of MG7-Ag in a sample, the device comprising the anti-MG 7-Ag monoclonal antibody MGd1-Ab of the invention.

Preferably, the sample is a tissue, blood or body fluid of the subject; preferably, the bodily fluid is ascites;

preferably, the subject is a patient with a malignant tumor or a high risk group suffering from a malignant tumor; more preferably, the malignant tumor is gastric cancer, colon cancer or esophageal cancer.

Preferably, the device is a kit; more preferably, the device is a kit for aiding in the diagnosis of a tumor, monitoring a tumor, and/or prognosis of a patient with a tumor.

The invention provides the use of the monoclonal antibody MGd1-Ab in the preparation of a reagent or kit for detecting the presence and/or level of MG7-Ag in a sample. The detection method uses an immunization method; more preferably, the immunological method is selected from the group consisting of immunoblotting, immunoprecipitation, immunohistochemistry, enzyme-linked immunosorbent assay, immunochemiluminometry, and the like.

In still another aspect, the present invention provides a detection reagent comprising the monoclonal antibody of the present invention. The detection reagent may be prepared by any method known to those skilled in the art, for example, by mixing the monoclonal antibody of the present invention with a suitable carrier, such as water, saline, a buffer, etc. The detection reagent can be used for detecting the expression level of MG7-Ag protein in a clinical sample, and predicting the metastasis tendency of early tumors and the prognosis of patients; the kit can also be used for detecting the expression of MG7-Ag protein in serum of an early tumor patient, can dynamically detect the change of the MG7-Ag protein level in the tumor progression process, and provides a reference basis for a clinician to formulate an individualized treatment scheme.

In one embodiment of the present invention, the applicant immunized Balb/c mice with eukaryotic expression of purified CEACAM5 protein to obtain hybridoma strain MGd1-Ab secreting monoclonal antibody against gastric cancer. Positive cell clones were screened by immunohistochemical comparison of gastric cancer/paracarcinoma tissues or/and ELISA, respectively. The inventor obtains multiple positive cell clones through multiple tests. The monoclonal antibodies secreted by the positive clones are analyzed and identified, and the CCTCC NO of the monoclonal antibody is C2016130, and the MG7-Ag monoclonal antibody MGd1-Ab secreted by the monoclonal antibody has higher sensitivity and specificity, can identify gastric cancer antigen MG7-Ag protein, can be used for immunological detection without limitation to immunohistochemistry, immunoblotting, immunoprecipitation, enzyme-linked immunosorbent assay, immunochemiluminescence and the like, and can be used for detection of the expression level of MG7-Ag protein in clinical samples.

Compared with the prior art, the hybridoma cell line has stronger capability of secreting the monoclonal antibody and can stably secrete a large amount of the monoclonal antibody. The inventor finds that although the prior art has proved that MG7-Ag (example 4 proves that MG7-Ag is glycosylated CEACAM5, MG7-Ag is a general term for glycosylated CEACAM5 in the application and is distinguished from the antibody aiming at CEACAM5 antigen on the market) is involved in the occurrence and development of various epithelial tumors such as gastric cancer, especially the function of MG7-Ag is related to the high glycosylation, and at present, no antibody modified by glycosylation of the protein is available, thus seriously affecting the application of the molecule in diagnosis and treatment of tumors such as gastric cancer. The monoclonal antibody of the invention can be specifically combined with glycosylated human CEACAM5 antigen, and has higher affinity and antigen specificity with glycosylated CEACAM 5.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1A shows the specific identification of MGd1-Ab of the present invention (ELISA plate coated with MG7-Ag protein, normal murine IgG antibody negative control);

FIG. 1B is a graph showing the sensitivity of the reaction of the MGd1-Ab antibody of the present invention and the commercial antibody CD66e with antigen

FIG. 2 is an SDS-PAGE analysis of purified MGd1-Ab, from which it can be seen that the purified MGd1-Ab is highly pure;

FIG. 3 is a photograph showing that MG7-Ag in a tissue chip is detected by MGd1-Ab immunohistochemistry, indicating that MG7-Ag expression in gastric cancer tissue can be detected by MGd1-Ab, and the result shows that MG7-Ag is highly expressed in gastric cancer tissue, but MG7-Ag is not expressed in paragastric cancer normal tissue;

FIG. 4 shows that MGd1-Ab can be used for Western Blot detection of MG7-Ag expression in gastric cancer KATOIII cells by using MGd1-Ab immunoblotting to detect MG7-Ag expression in gastric cancer cells;

FIG. 5 is a flow chart for identifying MG7-Ag using MGd1-Ab using Co-immunoprecipitation (Co-IP) and LC-MS/MS mass spectrometry in combination;

FIGS. 6a-c are CEACAM5 peptide fragment diagrams of Co-immunoprecipitation (Co-IP) and LC-MS/MS mass spectrometry combined with identification of MG 7-Ag;

FIG. 7 shows the positive rate of MG7-Ag in gastric cancer using MGd1-Ab and other tumor markers.

FIG. 8 shows that MGdl-Ab kills gastric cancer cells SGC7901 (cell state, MTT).

Biological preservation information

Wherein the preservation number of the hybridoma cell strain MGd1 is CCTCC NO: C2016130, the preservation date is: 2016, 6 and 23 days, with a preservation number of CCTCC NO: C2016130, depository: china center for type culture Collection, Address: in the Wuhan university school of Wuhan 299 in Wuchang district of Wuhan city, Hubei province (the first small facing attached to Wuhan university), the Wuhan university Collection center is postcode: 430072);

Detailed Description

In order to clearly understand the essence of the present invention, the present invention will be described in further detail with reference to the following embodiments and accompanying drawings, but the present invention is not limited thereto.

Unless otherwise indicated, the reagents used in the following examples are analytical grade reagents and are commercially available from a regular channel.