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RANKL-TNF sample region mouse monoclonal antibody and its preparation method and use

A monoclonal antibody, mouse-derived technology, applied in biochemical equipment and methods, antibodies, microorganism-based methods, etc., can solve the problems of high price, increase the pain and economic pressure of RA patients, inhibit differentiation and alleviate bone loss. , the effect of slowing down the progress

Inactive Publication Date: 2013-12-11
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

TNF-α antagonists are more expensive, RANKL monoclonal antibodies have not yet been marketed in China, and the combination of drugs increases the pain and economic pressure of RA patients

Method used

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  • RANKL-TNF sample region mouse monoclonal antibody and its preparation method and use
  • RANKL-TNF sample region mouse monoclonal antibody and its preparation method and use
  • RANKL-TNF sample region mouse monoclonal antibody and its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 animal immunization

[0031] RANKL-TNF fusion protein immunized 6-8w female BALB / c mice (SPF grade), divided into protein immunization group and control group according to body weight, and subcutaneously immunized mice at multiple points in the abdomen (recorded as the 0th day after the first immunization, respectively on 21d, 21d, 42d, 63d booster immunization), each mouse immunization dose is 100μg (200μl). For the initial immunization, the immunogen was emulsified with an equal amount of Complete Freud’s adjuvant (CFA), and for booster immunization, the immunogen was emulsified with an equal amount of Incomplete Freud’s adjuvant (Incomplete Freud’s adjuvant, IFA). One week after each immunization, blood was collected from the orbital venous plexus, the serum was separated, and the level of neutralizing antibodies produced in the mice was detected by indirect ELISA. The specific operation is to coat the ELISA plate with RANKL-TNF fusion protein (5 μg / ml)...

Embodiment 2

[0032] Example 2 Preparation of RANKL-TNF-like region monoclonal antibody

[0033] On the third day after the shock immunization, the mouse with the highest antibody titer was selected, sacrificed by cervical dislocation, the spleen of the mouse was removed under a sterile environment, the connective tissue was separated, the spleen was cut into pieces, and the spleen cells were obtained by grinding with a 200-mesh cell grinder. liquid. The myeloma cell line SP2 / 0 was resuscitated one week in advance and cultured in RPMI1640 medium containing 20% ​​fetal bovine serum (FBS). After harvesting the myeloma cells, count the myeloma cells and splenocytes respectively, mix the splenocytes and myeloma cells at a ratio of 5:1, centrifuge at 4°C, 1000rpm / min, 10min, remove the cell supernatant, flick At the bottom of the centrifuge tube, break up the cell pellet, add 1ml of 50% MW4000 polyethylene glycol (PEG) drop by drop while stirring and mixing within 1min, then stir the cell pelle...

Embodiment 3

[0034] Example 3 Subclonal Screening and Identification of Monoclonal Cell Lines

[0035] 5-7 days after cell fusion, observe the state of the cells in each well under the microscope. The clear cells arranged in piles under the microscope are the successfully fused hybridoma cells. Coat the ELISA plate with RANKL-TNF fusion protein (5 μg / ml), overnight at 4°C, and block with 5% PBST skimmed milk powder at room temperature for 4 hours, take 50 μl of the cell supernatant for indirect ELISA detection, and dilute the HRP-labeled secondary antibody at 1:20,000. Select to produce high-titer antibodies (such as figure 1 The titer shown in B is 10 4 ) cells were subcloned.

[0036] Mouse peritoneal macrophages were prepared the day before subcloning as feeder cells. The specific method is as follows: after 6-8w BALB / c (SPF grade) cervical dislocation, 75% ethanol soaked and disinfected, cut open the abdominal skin and peritoneum, clamped the peritoneum with tweezers, cut a small mo...

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Abstract

The invention relates to a RANKL-TNF sample region mouse monoclonal antibody and its preparation method and use. The RANKL-TNF sample region mouse monoclonal antibody is extracted from a RANKL-TNF sample region mouse monoclonal antibody hybridoma cell strain and has the preservation number of CGMCC NO.6853. The RANKL-TNF sample region mouse monoclonal antibody and its related reagents can effectively neutralize TNF-alpha in vitro thereby inhibiting cell apoptosis caused by TNF-alpha, and neutralize RANKL in vitro thereby inhibiting osteoclast generation. The RANKL-TNF sample region mouse monoclonal antibody can effectively relieve an acute inflammation degree, improve bone mass of a mouse suffering from osteoporosis, improve a cancellous bone trabecula microstructure, and effectively improve osteoporosis symptoms.

Description

technical field [0001] The present invention relates to the field of biological preparations, in particular to a RANKL-TNF-like region mouse-derived monoclonal antibody and its preparation method and application. Background technique [0002] Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. RA has two main pathological features, manifested as chronic synovial inflammation and subsequent destruction of articular cartilage and bone, which eventually lead to joint deformity and disability in patients. In 2006, the National Bureau of Statistics conducted the second survey on the disabled and found that the disability rate of rheumatoid arthritis accounted for 79.4% of the physical disabilities caused by the disease, resulting in the loss of labor force and seriously affecting the national health level and national economic development. Therefore, the study New formulations are of great significance in the treatment of RA. [0003] Chronic progressive systemi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/24A61K39/395A61P19/02A61P19/08A61P19/10A61P29/00C12R1/91
Inventor 赵文明钱红燕袁慧慧
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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