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Hybridoma cell line with anti-coagulation factor VIII monoclonal antibody

A hybridoma cell line, monoclonal antibody technology, applied in genetic engineering, plant genetic improvement, biochemical equipment and methods, etc.

Active Publication Date: 2019-04-12
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current preparation of factor VIII in our country is still crudely obtained by cryoprecipitation, the specific activity is mostly lower than 100IU / mg, and the activity recovery rate is about 6%.

Method used

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  • Hybridoma cell line with anti-coagulation factor VIII monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0036] 1) Preparation of immune antigen:

[0037] Dissolve 1mg of human coagulation factor VIII in 0.25mL DMSO solution, add EDC, shake vigorously at room temperature for 2 hours in the dark, react overnight at 4°C, slowly add the above solution to the BSA solution, and shake for 2 hours in the dark at room temperature, the reaction product at 0.05molL -1 The conjugate of coagulation factor VIII and carrier protein BSA was obtained by dialysis in PBS buffer solution for 72 hours in the dark.

[0038]2) Immunization of mice:

[0039] Using human blood coagulation factor VIII-BSA conjugate as immune antigen, immunize 7-week-old female Balb / c mice, and mix it with complete Freund's adjuvant at a volume ratio of 1:1 at an immune dose of 100 μg / mouse, Emulsify to water-in-oil state, inject the emulsified complete antigen subcutaneously at multiple points on the back of the neck to immunize Balb / c mice, and after 2 weeks, emulsify with the same antigen and an equal volume of Freun...

Embodiment

[0050] 1. Materials and instruments

[0051] 1.1 Cell lines, tissue samples and animals

[0052] a. Myeloma cells (SP2 / 0): derived from ATCC (American Type Culture Collection).

[0053] b. Balb / c mice: purchased from the animal platform of Tsinghua University.

[0054] 1.2 Main reagents

[0055] a. Freund's complete adjuvant, Freund's incomplete adjuvant, HAT, HT, TMB, PEG (molecular weight 4000), EDC: purchased from Sigma;

[0056] b. HRP-labeled goat anti-mouse IgG: purchased from Gibco;

[0057] c. RPMI1640 medium: purchased from HyClone Company;

[0058] d. Acetate cellulose membrane (NC): purchased from Hybond company;

[0059] e. Fetal bovine serum: purchased from PAN company;

[0060] f. Mouse monoclonal antibody subclass typing kit: purchased from Thermo Branch;

[0061] g. Coating buffer: 0.795 g of sodium carbonate and 1.465 g of sodium bicarbonate were added to every 500 ml of deionized water, and the pH value was 9.6.

[0062] h. Substrate buffer solution: ...

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Abstract

The invention relates to a hybridoma cell strain for secreting an anti-human-coagulation factor VIII monoclonal antibody and a preparation method of the hybridoma cell strain. A conjugate obtained after bovine serum albumin (BSA) and a human coagulation factor VIII are coupled serves as an antigen immune Balb / c mouse, spleen cells of the mouse and SP2 / 0 myeloma cells are taken for cell fusion, an HAT culture medium is used for selective culture, and the hybridoma cell strain for stably secreting the anti-human-coagulation factor VIII monoclonal antibody is finally screened out through a limiting dilution method via multiple times of cloning. The hybridoma cell strain prepared through the method is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC C2015196.

Description

technical field [0001] The invention relates to a hybridoma cell strain and a preparation method thereof, in particular to a hybridoma cell strain of anti-human coagulation factor VIII monoclonal antibody and a preparation method thereof. Background technique [0002] Coagulation factors are an important protein component involved in the coagulation process. Its physiological function is to be activated when the blood vessel is bleeding, and stick together with platelets to plug the leak on the blood vessel. This process is called coagulation. Among the hemophilia patients, 80% of the patients are hemophilia A, that is, hemophilia A, and these patients mainly lack the coagulation factor VIII possessed by normal people. Hemophilia A is a lifelong disease, and there is still no cure at present, and it can only be treated by transfusion of human blood coagulation factor VIII replacement products. [0003] Coagulation factor VIII has a complex molecular structure and a molecu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/06C12N5/20
Inventor 张明徽
Owner TSINGHUA UNIV