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Anti-MG7-Ag monoclonal antibody of and application thereof

A monoclonal antibody, mg7-ag technology, applied in the field of immunology, can solve problems affecting diagnosis and therapeutic applications, and achieve high affinity and antigen specificity

Active Publication Date: 2020-01-14
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although it has been confirmed that CEACAM5 is involved in the occurrence and development of various epithelial tumors such as gastric cancer, especially the function of CEACAM5 is related to its high degree of glycosylation, there is no antibody against the glycosylation modification of this protein, which seriously affects the development of this protein. The application of molecules in the diagnosis and treatment of gastric cancer and other tumors

Method used

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  • Anti-MG7-Ag monoclonal antibody of and application thereof
  • Anti-MG7-Ag monoclonal antibody of and application thereof
  • Anti-MG7-Ag monoclonal antibody of and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation and Identification of Anti-MG7-Ag Monoclonal Antibody CEA37-Ab

[0034] 1. Immunity and cell fusion:

[0035] Monoclonal antibody CEA37-Ab was obtained by immunizing Balb / c mice with eukaryotically expressed and purified CEACAM5 protein.

[0036] The specific method is as follows:

[0037] Three six-week-old Balb / c mice (purchased from Beijing Weitong Lihua) were immunized each time, and the second immunization was carried out at an interval of 4 weeks. The intraperitoneal immunization was boosted 3 days before the fusion, and the antibody titer of the mice was determined, and the antibody titer was selected. The splenocytes of the mouse with the highest valence were used for cell fusion: the fusion agent was 50% PEG4000, and the ratio of SP2 / 0 to splenocytes was 1:10 for fusion, and RPMI-1640 culture medium containing HAT was added, placed in a 96-well plate 5%CO 2 , and cultured in a 37°C constant temperature incubator.

[0038] Screening o...

Embodiment 2

[0044] Example 2 Preparation and Purification of Ascites of Anti-MG7-Ag Monoclonal Antibody

[0045] 1. Preparation of ascites: 10-week-old male BALB / c mice (purchased from Beijing Weitong Lihua) were intraperitoneally injected with 0.5 mL of liquid paraffin, and 10 days later, each mouse was intraperitoneally injected with 1X106 hybridoma cell suspension washed with normal saline. The solution was collected after ascites accumulated in mice, and stored at -20°C after aliquoting.

[0046] 2. Purification: Use ProteinG column (purchased from GenScript, L00209) for purification, collect 10 mL of each ascites, centrifuge at 3000 rpm for 10 min, absorb the supernatant, dilute 5 times with equilibrium buffer, filter with a 0.45 μm filter, and use a protein purifier (purchased from AKTAPurifier) ​​for purification, after washing away impurity proteins with equilibration buffer (PBS), elute with acidic eluent (100mmol / LGlycin-HCl) at pH 2.6, and collect OD 280 For components >0.5,...

Embodiment 3

[0047] Example 3 Using CEA37-Ab Immunohistochemical Method to Detect the Expression of MG7-Ag in Tissue Chips

[0048] 1. Human gastric cancer tissue microarrays (surgical specimens confirmed by pathology at Xijing Hospital of Fourth Military Medical University) were selected. Dewax with xylene to gradient alcohol dehydration, wash with PBS twice, 5min each time;

[0049] 2. Freshly prepared 3% H 2 o 2 10min at room temperature; then wash 3 times with PBS, 5min each time;

[0050] 3. Block with 10% normal sheep serum for 30 minutes at room temperature;

[0051] 4. Carefully aspirate and discard the blocking solution without washing, add appropriate concentration of CEA37-Ab to the slices respectively, and incubate overnight at 4°C. The slices were then washed 3 times with PBS;

[0052] 5. Add HRP-labeled goat anti-mouse IgG working solution dropwise on the slice, and incubate at 37°C for 1 hour;

[0053] 6. Wash the slices twice with PBS, soak the slices in the substra...

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Abstract

The invention provides a MG7-Ag monoclonal antibody and application thereof. The monoclonal antibody is an anti-MG7-Ag monoclonal antibody CEA37-Ab. The invention further provides a hybridoma cell line which secretes the anti-MG7-Ag monoclonal antibody CEA37-Ab. The invention further provides a kit comprising the anti-MG7-Ag monoclonal antibody CEA37-Ab and application of the kit to detection of gastric cancer antigen MG7-Ag and application to detection of the expression level of the gastric cancer antigen MG7-Ag in a clinical tissue sample. The anti-MG7-Ag monoclonal antibody has a uniform texture and high specificity. The detection kit can adjust and control the sensitivity and detection range according to the application, and the expression level of the gastric cancer antigen MG7-Ag canbe detected with high sensitivity.

Description

technical field [0001] The invention belongs to the field of immunology, and relates to an anti-MG7-Ag monoclonal antibody and its use, more specifically to the use of a kit containing the antibody in detecting the level of MG7-Ag in a sample, and a kit containing the antibody Use in auxiliary diagnosis of tumors, monitoring of tumors and prognosis of tumor patients. Background technique [0002] Tumor protein markers are certain substances produced and released by tumor cells, which often exist in the form of metabolites such as antigens, enzymes, proteins, or peptide hormones in the patient's tumor cell tissue or in the body fluids and excretions of the host. The immune profile can identify or diagnose a tumor. Tumor protein markers are mainly used clinically for the discovery of primary tumors, the screening of high-risk groups for tumors, the differential diagnosis of benign and malignant tumors, the judgment of tumor development, the observation and evaluation of tumor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/32C12N5/20G01N33/574G01N33/577
CPCC07K16/32G01N33/57407G01N33/57419G01N33/57446G01N33/577
Inventor 聂勇战吴开春樊代明赵青川
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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