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Anti-macroglobulin monoclonal antibody hybridoma cell line

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

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

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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  • Anti-macroglobulin monoclonal antibody hybridoma cell line

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Experimental program
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Effect test

preparation example Construction

[0036] 1) Preparation of immune antigen:

[0037] Dissolve 1 mg of human macroglobulin in 0.25 mL DMSO solution, add EDC, shake vigorously for 2 hours at room temperature and avoid light, and react overnight at 4°C. Add the above solution slowly and dropwise to the BSA solution and shake for 2 hours at room temperature away from light. In 0.05molL -1 After 72 hours of dialysis against light in PBS buffer, a conjugate of macroglobulin and carrier protein BSA was obtained.

[0038] 2) Mouse immunity:

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

Embodiment

[0050] 1. Materials and instruments

[0051] 1.1 Cell lines, tissue specimens and animals

[0052] a. Myeloma cells (SP2 / 0): 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;

[0058] d. Cellulose acetate membrane (NC): purchased from Hybond;

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

[0060] f. Murine monoclonal antibody subtype typing kit: purchased from Thermo Branch;

[0061] g. Coating buffer: 0.795g sodium carbonate and 1.465g sodium bicarbonate are added to each 500ml deionized water, and the pH is 9.6.

[0062] h. Substrate buffer: Add 9.2g of disodium hydrogen phosphate dodecahydrate and 2.55g of citric aci...

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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 line and a preparation method thereof, in particular to a hybridoma cell line of an anti-human macroglobulin monoclonal antibody and a preparation method thereof. Background technique [0002] Macroglobulin is the molecular weight in serum> 4×10 5 A collective term for a class of serum globulins. The research is clearer is α2-macroglobulin, the molecular weight is about 7.25×10 5 , It is a protease inhibitor. [0003] Macroglobulin is mostly produced by monocytes. It is a macromolecular glycoprotein with biological activity in the blood. In addition to anti-radiation damage, it can also participate in immune regulation. It has been confirmed that macroglobulin can inhibit a variety of immune responses in vivo and in vitro, and is closely related to chronic liver and kidney diseases. [0004] The detection of macroglobulin in the urine can be used to determine whether it is selective, thereby providing a basis for t...

Claims

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Application Information

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