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Anti-MG7-Ag monoclonal antibody and application thereof

A monoclonal antibody, mg7-ag technology, applied in the field of immunology, can solve the problems of high price, difficult long-term guarantee of quality and stability, difficulty in adapting to prospective clinical research, etc., 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, most of the existing kits are limited by the fact that key reagents such as detection antibodies cannot be produced by themselves, which is not only expensive, but also difficult to guarantee the quality and stability for a long time; some kits are also limited by the use of polyclonal antibodies, and the supply of polyclonal antibodies is limited. There are large batch-to-batch differences, and such kits are difficult to meet the needs of large-scale and long-term prospective clinical research

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation and Identification of Anti-MG7-Ag Monoclonal Antibody MGd1Ab

[0035] 1. Immunity and cell fusion:

[0036] Monoclonal antibody MGd1-Ab was obtained by immunizing Balb / c mice with purified CEACAM5 protein expressed in eukaryotic cells.

[0037] The specific method is as follows:

[0038] 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.

[0039] Screening of ...

Embodiment 2

[0045] Example 2 Preparation and Purification of Ascites of Anti-MG7-Ag Monoclonal Antibody

[0046] 1. Preparation of ascites: 10-week-old male BALB / c mice (purchased from Beijing Weitong Lihua) were intraperitoneally injected with 0.5ml 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.

[0047] 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), elution was carried out with acidic eluent (100mmol / LGlycin-HCl) at pH 2.6, and the OD 280 For com...

Embodiment 3

[0048] Example 3 Using MGd1-Ab immunohistochemical method to detect the expression of MG7-Ag in tissue chips

[0049] 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;

[0050] 2. Freshly prepared 3% H 2 o 2 10min at room temperature; then wash 3 times with PBS, 5min each time;

[0051] 3. Block with 10% normal sheep serum for 30 minutes at room temperature;

[0052] 4. Carefully aspirate and discard the blocking solution without washing. Add appropriate concentration of MGd1-Ab to the slices respectively and incubate overnight at 4°C. The slices were then washed 3 times with PBS;

[0053] 5. Add HRP-labeled goat anti-mouse IgG working solution dropwise on the slice, and incubate at 37°C for 1 hour;

[0054] 6. Wash the slices twice with PBS, soak the slices in the substrate ...

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Abstract

The invention provides an anti-MG7-Ag monoclonal antibody and an application thereof. The monoclonal antibody is an anti-MG7-Ag monoclonal antibody MGd1-Ab. The invention also provides a hybridoma cell line secreting the anti-MG7-Ag monoclonal antibody MGd1-Ab. The invention also provides a kit comprising the anti-MG7-Ag monoclonal antibody MGd1-Ab, and an application of the kit for detecting gastric cancer antigen MG7-Ag and an application of the kit for detecting expression level of the gastric cancer antigen MG7-Ag in a clinical tissue sample. The anti-MG7-Ag monoclonal antibody provided bythe invention has uniform texture and high specificity. The detection kit of the present invention can regulate sensitivity and detection range according to applications, thereby detecting expressionlevel of the gastric cancer antigen MG7-Ag in 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): C07K16/30C12N5/20G01N33/577G01N33/574C12R1/91
CPCC07K16/30C07K2317/32C07K2317/35G01N33/57407G01N33/57419G01N33/57446G01N33/57484G01N33/577G01N2800/52
Inventor 聂勇战吴开春樊代明赵青川
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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