Hybridomas cell strain of anti-human macroglobulin monoclonal antibody

A hybridoma cell line and monoclonal antibody technology, applied in the field of hybridoma cell line and its preparation, can solve the problems of low activity and recovery rate

Active Publication Date: 2016-02-24
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation of macroglobulin in my country is still crudely obtained by cryoprecipitation, and the activity and recovery rate are relatively low.

Method used

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  • Hybridomas cell strain of anti-human macroglobulin monoclonal antibody

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Experimental program
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Effect test

preparation example Construction

[0036] 1) Preparation of immune antigen:

[0037] Dissolve 1mg of human macroglobulin 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 macroglobulin and carrier protein BSA was obtained by dialysis in PBS buffer solution for 72 hours in the dark.

[0038] 2) Immunization of mice:

[0039]Use human macroglobulin-BSA conjugate as the immune antigen to immunize 7-week-old female Balb / c mice, and mix it with Freund's complete adjuvant at a volume ratio of 1:1 at an immunization 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 Freund's incomplete adjuvant, 1...

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 hybridomas cell strain secreting anti-human macroglobulin monoclonal antibody and a preparation method thereof. The preparation method comprises using a conjugate obtaining by performing coupling on bovine serum albumin (BSA) and human macroglobulin 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, performing multiple cloning by using a limiting dilution process, and finally screening and obtaining the hybridomas cell strain stably secreting anti-human macroglobulin monoclonal antibody. The prepared hybridomas cell strain is preserved in China Center for Type Culture Collection with the preservation number of CCTCC C2015190.

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 macroglobulin monoclonal antibody and a preparation method thereof. Background technique [0002] Macroglobulin (macroglobulin) is the serum molecular weight > 4 × 10 5 A general term for a class of serum globulins. The research is clearer is α2-macroglobulin, the molecular weight is about 7.25×10 5 , which is a protease inhibitor. [0003] Macroglobulin is mostly produced by monocytes. It is a biologically active macromolecular glycoprotein in the blood. In addition to its anti-radiation damage effect, it can also participate in immune regulation. It has been proven that macroglobulin can inhibit various immune responses in vivo and in vitro, and has a close relationship with chronic liver and kidney diseases. [0004] The detection of macroglobulin in urine can be used to judge whether it is selective, thus ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C12N15/06C07K16/38C12R1/91
Inventor 张明徽
Owner TSINGHUA UNIV
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