Hybridoma cell line with anti-coagulation factor vii polyclonal antibody

A technology of hybridoma cell line and polyclonal antibody, applied in the field of hybridoma cell line and its preparation

Active Publication Date: 2019-02-22
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the development of human plasma coagulation factor VII polyclonal antibody, and there is no high-purity human coagulation factor VII product. The human coagulation factor VII detection reagents used are basically purchased from abroad. The coagulation factor VII detection reagent must first prepare the human coagulation factor VII polylon antibody

Method used

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  • Hybridoma cell line with anti-coagulation factor vii polyclonal antibody
  • Hybridoma cell line with anti-coagulation factor vii polyclonal antibody

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Experimental program
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Effect test

preparation example Construction

[0035] 1) Preparation of immune antigen:

[0036] Dissolve 1mg of human coagulation factor VII in 0.25mL DMSO solution, add EDC, shake vigorously at room temperature for 2 hours in the dark, and react overnight at 4°C, slowly add the above solution to the BSA solution, shake at room temperature for 2 hours, and the reaction product at 0.05molL -1 The conjugate of human coagulation factor VII and carrier protein BSA was obtained by dialysis in PBS buffer solution for 72 hours in the dark.

[0037] 2) Immunization of mice:

[0038] Using human coagulation factor VII-BSA conjugate as immune antigen, immunize 7-week-old female Balb / c mice at an immunization dose of 100 μg / mouse, mix it with Freund's complete adjuvant in a volume ratio of 1:1, 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 Freund's inco...

Embodiment

[0047] 1. Materials and instruments

[0048] 1.1 Cell lines, tissue samples and animals

[0049] a. Myeloma cells (SP2 / 0): derived from ATCC (American Type Culture Collection).

[0050] b. Balb / c mice: purchased from the animal platform of Tsinghua University.

[0051] 1.2 Main reagents

[0052] a. Freund's complete adjuvant, Freund's incomplete adjuvant, HAT, HT, TMB, PEG (molecular weight 4000), EDC: purchased from Sigma;

[0053] b. HRP-labeled goat anti-mouse IgG: purchased from Gibco;

[0054] c. RPMI1640 medium: purchased from HyClone Company;

[0055] d. Acetate cellulose membrane (NC): purchased from Hybond company;

[0056] e. Fetal bovine serum: purchased from PAN company;

[0057] f. Mouse polyclonal antibody subclass typing kit: purchased from Thermo Branch;

[0058] 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.

[0059] h. Substrate buffer solution: ...

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Abstract

The invention relates to a hybridomas cell strain secreting anti-human coagulation factor VII polyclonal antibody and a preparation method thereof. The preparation method comprises using a conjugate obtaining by performing coupling on bovine serum albumin (BSA) and human coagulation factor VII as an antigen for immunizing Balb / c mouse, getting spleen cell of the mouse and performing cell fusion with SP2 / 0 myeloma cell, performing selective culture by using HAT medium, selecting fusion cell of the mouse spleen cell and the mouse myeloma cell SP2 / 0, and finally identifying the hybridomas cell strain secreting anti-human coagulation factor VII polyclonal antibody through ELISA. The prepared hybridomas cell strain is preserved in China Center for Type Culture Collection with the preservation number of CCTCC C2015195.

Description

technical field [0001] The present invention relates to a hybridoma cell strain and a preparation method thereof, in particular to a hybridoma cell strain of anti-human coagulation factor VII polyclonal antibody and a preparation method thereof. Background technique [0002] Human coagulation factor VII is a vitamin K-dependent single-chain glycoprotein synthesized by hepatocytes, and is the initiator of the exogenous coagulation pathway in the body. The concentration of human coagulation factor VII in plasma is only (0.5-2) mg / L, and the half-life is 6-8 hours. Whether it is primary or secondary coagulation factor VII deficiency in the human body, it may cause bleeding. Even life-threatening. Studies have confirmed that human coagulation factor VII not only plays an important role in physiological hemostasis, but also its plasma level of high and low gene polymorphisms is also related to the occurrence of some diseases. Human coagulation factor VII polyclonal antibody is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/06C12N5/20C12R1/91
Inventor 张明徽
Owner TSINGHUA UNIV
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