Anti-D-dimer monoclonal antibody and preparation method thereof

A monoclonal antibody and dimer technology, applied in the biological field, can solve the problems of unfavorable industrial production of anti-D-dimer antibody, poor stability between batches, complicated production process, etc., to achieve control stability and improve The effect of expression amount and production cost reduction

Active Publication Date: 2018-12-21
智享生物(苏州)有限公司
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AI Technical Summary

Problems solved by technology

[0005] At present, the preparation method of anti-D-dimer is usually expressed by hybridoma cells or obtained by extracting ascites of animals. This preparation method is not only complicated in production process but also high in cost, and the stability between batches is relatively low. Poor, not conducive to the industrial production of anti-D-dimer antibody

Method used

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  • Anti-D-dimer monoclonal antibody and preparation method thereof
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  • Anti-D-dimer monoclonal antibody and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1: Screening of anti-D-dimer monoclonal antibodies

[0023] 1. Animal immunity

[0024] Take female 6-week-old BALB / c mice, and inject pure D-dimer with a concentration of 6 mg / mL for the first immunization, and inject it into the abdominal cavity and under the armpit of the limbs, with a total amount of 1 mL; every 2 weeks, the same method is used to boost Immunize once and immunize three times in total. On the 4th day after the last immunization, blood was collected from the eyeballs of mice after three immunizations, and the serum was centrifuged to separate the mice with high titer by ELISA method to prepare for fusion.

[0025] 2. Preparation of hybridoma cell lines

[0026] Take the splenocytes of mice that have completed the immunization process and fuse them with mouse myeloma cell SP2 / 0, and prepare feeder cells one day before fusion; take mouse splenocytes under aseptic conditions during fusion, and mix them with SP2 / 0 cells at a ratio of 10:1 Mix, m...

Embodiment 2

[0036] Example 2: Anti-D-dimer monoclonal antibody hybridoma expression

[0037] Hybridoma cells were cultured at 0.6×10 with CD Hybridoma (Gibco) medium 6 The inoculation density of cells / mL was diluted into a 3L cell culture reactor, and the initial culture volume was 1L; the stirring speed was set to 200r / min; the pH was set to 7.00, DeadBand was set to 0.10, and the associated sodium bicarbonate solution and CO 2 pH adjustment was performed; the dissolved oxygen was set to 40% (dissolved oxygen in saturated air was 100%), and the culture temperature was set to 36.5°C. On the 3rd, 5th, 7th, 9th, and 11th day, the feeding medium was used for feeding, and the feeding volume of the feeding medium Feed B Plus (Gibco) was 3% of the initial culture volume.

[0038] The maximum cell density reaches 8×10 6 cells / mL( figure 1 ), the cell viability was 70% at the end of the culture ( figure 2 ), the final expression level of anti-D-dimer monoclonal antibody reached 406mg / L ( ima...

Embodiment 3

[0040] Embodiment 3: Anti-D-dimer monoclonal antibody sequence optimization

[0041] The sequence of the constant region of the anti-D-dimer monoclonal antibody is replaced with a highly expressed sequence. The light chain sequence of the optimized complete monoclonal antibody is shown as SEQ ID NO.5, and the heavy chain sequence is shown as SEQ ID NO.6 shown.

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Abstract

The invention discloses an anti-D-dimer monoclonal antibody and a preparation method thereof, and belongs to the technical field of biology. The anti-D-dimer monoclonal antibody is first prepared by means of hybridoma, the sequence of the obtained antibody is then optimized, the output of the anti-D-dimer monoclonal antibody is increased by 54 percent under the premise of keeping affinity unchanged, and the production cost is greatly reduced. Compared with the conventional hybridoma culture and mouse ascites extraction methods, the method disclosed by the invention not only greatly increases expression and reduces the production cost, but also can effectively control the stability of product batches.

Description

technical field [0001] The invention relates to an anti-D-dimer monoclonal antibody and a preparation method thereof, belonging to the field of biotechnology. Background technique [0002] D-dimer is the end product of cross-linked fibrin after the action of plasmin. In the coagulation process, after thrombin hydrolyzes fibrinogen, it releases fibrin peptide A (FPA) and peptide B (FPB) successively, and the rest is soluble fibrin monomer (SFM). SFM is transformed into fibrin, followed by blood coagulation. The process is completed after a series of cross-linking reactions. After that, the fibrin formed is stable and generally insoluble, but can be degraded by plasmin. Cross-linked fibrin is in fibrinolysis During the enzymatic degradation process, several polymers are gradually formed, among which D-dimer is one of its specific products, and its molecular weight is 184000-202000Da. In a pathological state, the dynamic balance of coagulation and fibrinolysis is disrupted, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/36C12N15/13C12N5/10G01N33/68G01N33/577
CPCC07K16/36G01N33/577G01N33/68G01N2333/75
Inventor 郁庆明刘金涛诸葛鑫
Owner 智享生物(苏州)有限公司
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