CN110684107B

CN110684107B - Monoclonal antibody (MGd1) against MG7-Ag and use thereof

Info

Publication number: CN110684107B
Application number: CN201810736466.6A
Authority: CN
Inventors: 聂勇战; 吴开春; 樊代明; 赵青川
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2018-07-06
Filing date: 2018-07-06
Publication date: 2021-03-23
Anticipated expiration: 2038-07-06
Also published as: CN110684107A

Abstract

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.

Description

Monoclonal antibody (MGd1) against MG7-Ag and use thereof

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 form of metabolites such as antigens, enzymes, proteins or peptide hormones in tumor cell tissues of patients or in host body fluids and exclusions, and can identify or diagnose tumors according to biochemical or immunological characteristics. 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 tumor development degree, observing and evaluating the tumor treatment effect, predicting tumor recurrence and prognosis 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 which is simple and convenient to operate, high in sensitivity, low in cost and suitable for large-batch MG7-Ag detection. In addition, the anti-MG 7-Ag monoclonal antibody can also be used for immunoblotting, immunoprecipitation, immunohistochemistry, enzyme-linked immunosorbent assay and chemiluminescence, 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, immunochemiluminescence assay, 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.

EXAMPLE 1 preparation and characterization of anti-MG 7-Ag monoclonal antibody MGd1Ab

1. Immunization and cell fusion:

the eukaryotic expression purified CEACAM5 protein is used for immunizing Balb/c mice to prepare the monoclonal antibody MGd 1-Ab.

The specific method comprises the following steps:

3 Balb/c mice (purchased from Beijing Wittingle) with the age of six weeks are immunized, the second immunization is carried out at an interval of 4 weeks, 3 days before the fusion, the abdominal cavity is used for strengthening the immunization, the antibody titer of the mice is measured, and spleen cells of the mice with the highest antibody titer are selected for cell fusion: fusing agent 50% PEG4000, SP2/0 and splenocyte at a ratio of 1:10, adding HAT-containing RPMI-1640 culture solution, and placing 5% CO in 96-well plate₂And culturing in a constant temperature incubator at 37 ℃.

Screening of positive hybridoma cells: positive cell clones are screened by an immunohistochemical contrast method or/and an ELISA method of the gastric cancer tissues/the tissues beside the cancer respectively, and finally a positive hybridoma MGd1-A is screened.

2. Cloning and subcloning: and (3) selecting hybridoma cells with high positive values by adopting a limiting dilution method for cloning and subcloning until the positive rate reaches 100%.

3. Subclass identification: the concentrated cell culture supernatant was subjected to immunodiffusion with goat anti-mouse IgG1, 2a, 2b, IgG3, and IgM antiserum (Shanghai Biochemical research institute) to determine the IgG subclass of the hybridoma. The subclass of MGd1-Ab mAb is IgG1, respectively.

4. And (3) specific analysis: adding MGd1-Ab diluted by different times into a micropore plate coated by gastric cancer antigen MG7-Ag respectively, adding normal mouse IgG antibody with the same concentration as a negative control, and incubating for 1 hour at room temperature; wash reaction wells 5 times with PBST (0.05% Tween); then, HRP-labeled goat anti-mouse IgG anti-B was added, and the mixture was developed at room temperature for 15 minutes, and stop buffer (2MH2S04) was added. The absorbance at A450 was measured with a microplate reader. The results are shown in FIG. 1A, where MG7-Ag specifically binds MGd1-Ab and no specific binding to normal mouse IgG.

5. And (3) sensitivity comparison: adding MGd1-Ab diluted by 100 times, 1000 times, 10000 times, 100000 times and 1000000 times, 10000000 times and a purchased commercial gastric cancer CEA antibody CD66E (eBioscience, anti-human CD66E, E12720-103 and 100ug) into a microporous plate coated by gastric cancer antigen MG7-Ag, and incubating for 1 hour at room temperature; wash reaction wells 5 times with PBST (0.05% Tween); then, a secondary goat anti-mouse HRP-labeled antibody was added thereto, and the mixture was developed at room temperature for 15 minutes, followed by addition of stop buffer (2MH2S 04). The results are shown in FIG. 1B.

From the results, it can be seen that: the MGd1 antibody has higher sensitivity than commercial CD66e, and can be diluted 10000000 times to undergo color reaction with MG7-Ag, namely the antibody titer of the antibody, and the titer of CD66e is 1000000

EXAMPLE 2 preparation and purification of ascites fluid by anti-MG 7-Ag monoclonal antibody

1. Preparing ascites: 10 weeks old male BALB/c mice (purchased from Beijing Wittingerihua) were intraperitoneally injected with 0.5ml of liquid paraffin, 10 days later, each mouse was intraperitoneally injected with 1X106 hybridoma cell suspension washed with physiological saline, collected after ascites accumulation, and stored at-20 ℃.

2. And (3) purification: purifying with ProteinG column (from Chrysomy, L00209), collecting ascites each L0mL, centrifuging at 3000rpm for 10min, collecting supernatant, diluting with balance Buffer by 5 times, filtering with 0.45 μm filter, purifying with protein purifier (from AKTApurifier), washing off impurity proteins with balance Buffer (PBS), eluting with acidic eluate (100mmol/LGlycin-HCl) of pH2.6, collecting OD₂₈₀>0.5 fraction, finally adding 100 μ l of 2M Tris. cl neutralizing solution to each ml for neutralization, subpackaging the purified antibody for freezing and storing at-20 ℃, taking 4 μ 1 for 12% SDS-PAGE gel electrophoresis and identifying the purity. As a result, referring to FIG. 2, the purity of the purified MGd1-Ab was high (above 90%).

Example 3 detection of MG7-Ag in tissue chips Using MGd1-Ab immunohistochemistryExpression of

1. Human gastric cancer tissue chips (pathologically confirmed surgical specimens at the Cijing Hospital, fourth university of military medical science) were selected. Dewaxing with xylene until gradient alcohol dehydration, washing with PBS for 2 times and 5 min/time;

2. freshly prepared 3% H₂O₂Room temperature for 10 min; then washing with PBS for 3 times and 5 min/time;

3. sealing 10% normal sheep serum at room temperature for 30 min;

4. carefully remove the blocking solution by aspiration, do not wash, drop the appropriate concentration of MGd1-Ab onto each section, and incubate overnight at 4 ℃. The sections were then washed 3 times with PBS;

5. dropping a goat anti-mouse IgG working solution marked by HRP on the section, and incubating for 1 hour at 37 ℃;

6. washing the slices with PBS for 2 times, soaking the slices in a DAB solution serving as a substrate for developing for 20 minutes;

7. the color development was stopped with PBS and the sections were counterstained with hematoxylin. The sections were differentiated with 1% hydrochloric acid alcohol, dehydrated with gradient alcohol, xylene transparent, and then covered with a cover glass on the tissue sections for microscopic observation.

Results referring to FIG. 3, MG7-Ag expression in gastric cancer tissues was detected using MGd 1-Ab. The results show that the gastric cancer tissue has high expression of MG7-Ag, and the paracarcinoma normal tissue of the gastric cancer does not express MG 7-Ag. Commercial CD66E antibody (eBioscience, anti-humanCD66E, E12720-103,100ug) recognized the same class of antigen, but staining in gastric cancer tissue was not ideal and paracancerous with non-specific staining.

Example 4 Detection of MG7-Ag in KATOIII cells from gastric carcinoma Using MGd1-Ab immunoblotting (WesternBlot) Expression of

Detection of KATOIII cells (purchased from ATCC, HTB-103) by immunoblotting Using MGd1-Ab as a Primary antibody^TM) Expression of gastric carcinoma-associated antigen MG 7-Ag:

1. cell lysate RIPA lyses human gastric cancer cell line KATOIII cells, extracts total cell protein and carries out SDS-PAGE electrophoresis, and the loading amount of each hole protein is 50 mu g;

2. transferring the electrophoresis product to a nitrocellulose membrane, stabilizing the pressure for 100V, and keeping the time for 1 h;

3. sealing with 5% skimmed milk powder for 1h, washing with TBST for 3 times;

4. antibody 1. mu.g/mL (MGd1-Ab) was incubated overnight at 4 ℃;

5. TBST is washed for 3 times at room temperature for 10 min/time;

6. adding HRP-labeled goat anti-mouse IgG (1: 3000, diluted with 5% skimmed milk powder-TBST), incubating at room temperature for 2h, and shaking and washing with TBST for 3 times, 10 min/time;

7. developing, fixing, drying and storing.

Results referring to FIG. 4, MGd1-Ab was used for the WesternBlot assay of MG7-Ag expression in gastric carcinoma KATOIII cells.

Example 5 identification of antigen MG7-Ag Using MGd1-Ab

Identification of MG7-Ag was performed using Co-immunoprecipitation (Co-IP) in combination with LC-MS/MS mass spectrometry. The process and the results of identifying MG7-Ag are shown in FIG. 5.

1. Co-immunoprecipitation (co-IP)

KATOIII cell lysate 1ml +3ml IP Lysis Buffer, mixing (here 50ul is separated and reserved as input), adding Protein A/G80. mu.l, turning reaction at 4 ℃ for 1h, centrifuging at 1000G 4 ℃ for 5min, and taking supernatant (pre-clearing); mu.g of antibody (MGd1-Ab, control IgG antibody) per 1mL of sample,

turning and reacting for 1h at 4 ℃; adding 40 mul Protein A/G, and turning over at 4 ℃ for reaction overnight; centrifuging at 1000rpm at 4 deg.C for 5min, sucking off the supernatant, adding PBS 1ml, mixing, centrifuging, sucking off the supernatant, washing repeatedly for 4 times, adding 40ul Loading Buffer, boiling for 10min, centrifuging, performing 10% SDS-PAGE electrophoresis, and performing gel staining with Coomassie brilliant blue and silver nitrate respectively;

LC-MS/MS mass spectrometry:

according to the result of gel staining, protein bands of the antibody group and the control group are compared, differential protein bands cut from the gel are placed in an Eppendorf tube, and the proteins are subjected to treatments such as decolorization, reduction, alkylation, enzymolysis, extraction, desalination and the like.

Mass spectrometry was performed on a Thermo Scientific QE LC-MS mass spectrometer, and the chromatographically eluted peptide fragments were subjected to LC mass spectrometry and tandem mass spectrometry (MS/MS) after entering the mass spectrometry ion source, and the proteins were identified by querying the database using the combination of the deduced sequence and the peptide fragment mass. The LC-MS/MS mass spectrum results after MGd1-Ab co-immunoprecipitation are shown in FIGS. 6a-c, and the peptide fragment is identified as CEACAM5 by mass spectrum.

Glycosylation validation

As shown in FIG. 5, the anti-CEA antibody (abcam, Ab4539) and MGd1-Ab both recognized eukaryotic CEA-expressing 293T cells, and after deglycosylation treatment of eukaryotic CEA-expressing 293T cells, the anti-CEA antibody still recognized non-glycosylated CEA, while MGd1-Ab did not recognize non-glycosylated CEA;

the anti-CEA antibody can react with the prokaryotic expression of CEA, while the MGd1-Ab does not react with the prokaryotic expression of CEA, and the MGd1-Ab recognizes the antigen as glycosylated CEACAM 5.

Example 6 specific expression of MG7-Ag in gastric cancer tissue

The MGd1-Ab is used for immunohistochemical detection, and 11 normal tissues and cells such as normal gastric mucosa, colon, esophagus, lung, liver, pancreas, skin, ovary, smooth muscle, red blood cells, lymphocytes and the like are found to be negative; very few intestinal metaplastic cells in the gastric mucosa were positive by 1.7% (2/118 cases, clinical specimen of digestive disease hospital of first subsidiary hospital of military medical university of air force); the single-layer epidermal cells of the free edge of the intestinal cavity surface in the embryo colon mucosa are colored to different degrees, and the positive rate is 28.6-57.1%; the antibody is negative in liver cancer, pancreatic cancer, eye cancer, skin cancer and ovarian cancer, the reaction positive rate in tissues of stomach cancer, colon cancer and esophagus cancer is higher than that of other tumors, and the reaction positive rate in tissues of stomach cancer is 77.9 percent (127/163, clinical samples of digestive diseases hospital of first subsidiary hospital of military medical university) is higher than that of other tumors, thus prompting that MG7-Ag is specifically expressed in tissues of stomach cancer.

Example 7 comparison of Positive detection rates of MG7-Ag and other gastric cancer antigens

The immunohistochemical detection of MG7-Ag, CA19-9, CA72-4, CEA and CA125 tumor markers on the same tissue chip series section of 59 cases of gastric cancer (clinical samples of digestive disease hospital in first subsidiary hospital of air force military medical university) showed that the positive rate of MG7-Ag in gastric cancer was 72.9% higher than that of other tumor markers (CA 19-952.5%, CA 72-430.5%, CEA 50.8% and CA 12513.6%).

325 cases of tissue chips detect the expression of MG7-Ag and CEA in gastric cancer tissues, the result is shown in Table 1, the positive rate (69.8%) of MG7-Ag expression is obviously higher than CEA (58.2%), and the expression intensity of MG7-Ag in tissues is higher than CEA,

the positive rate (56.9%) of "+ +") is also obviously higher than that of CEA (35.1%), which indicates that MG7-Ag has better sensitivity than CEA in the aspect of gastric cancer diagnosis and is possibly a potential marker for gastric cancer diagnosis.

TABLE 1 expression intensity of MG7-Ag and CEA in gastric cancer tissue

Example 8 killing of gastric cancer cells by MGd1-Ab

Cell killing assay: gastric cancer cells SGC7901, AGS and GES (immortalized gastric mucosal epithelial cells) (source ATCC cell bank) in logarithmic growth phase were respectively inoculated into 96-well plates (each well was 5X 10)³One), normally culturing for 48 h; each cell was assigned MGd1-Ab group, mouse anti-tubercular antibody control group and blank control group, respectively, MGd1-Ab and anti-tubercular antibody were diluted to 400. mu.g/ml, 200. mu.g/ml, 100. mu.g/ml, 50. mu.g/ml and 25. mu.g/ml with RPMI-1640 culture medium containing 1.5% fetal bovine serum. Washing cells once, adding culture solution containing antibody with different concentrations, and placing in CO₂The cells are continuously cultured in the incubator, the state of the cells is observed under an inverted microscope, the MGd1-Ab with different concentrations is added into the gastric cancer cells for 48 to 72 hours, and the cells can be found to have obvious apoptosis characteristics such as poor refractivity, weak adherence and the like even die from 48 hours under the microscope. FIG. 8 shows typical behavior of SGC7901 after MGd1-Ab treatment when MGd1-Ab concentration reaches 100 μ g/ml; 72h MTT method detection proves that the survival gastric cancer cells SGC7901 and AGS are obviously reduced (P)<0.05), the antituberculous antibody control has no such killing effect, and any concentration of MGd1-Ab has no obvious killing effect on the immortalized gastric mucosal epithelial cells GESAnd (4) acting.

Claims

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 CCTCC NO: 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.

5. The device of claim 4, wherein the bodily fluid is ascites.

6. 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.

7. The device of claim 6, wherein the malignancy is gastric, colon, or esophageal cancer.

8. The device of any one of claims 3-7, wherein the device is a kit.

9. The device of any one of claims 3-7, wherein 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.

10. 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.

11. The use of claim 10, wherein the detection uses a method selected from one or more of immunoblotting, immunoprecipitation, immunohistochemistry, enzyme-linked immunosorbent assay, or immunochemiluminometry.