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Hybridoma cell line producing monoclonal antibody against human d-dimer, preparation method and application

A hybridoma cell line and monoclonal antibody technology, applied in the field of biomedical engineering, can solve problems affecting research and development, occupation, etc.

Active Publication Date: 2016-01-20
CHONGQING TANSHENG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the antibodies used in D-dimer detection are all from abroad, and there is no report and sales of related antibodies in China, which objectively affects the research and development of domestic companies in D-dimer detection reagents, and to a certain extent leads to the current domestic The D-dimer detection reagent market is basically occupied by European and American companies

Method used

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  • Hybridoma cell line producing monoclonal antibody against human d-dimer, preparation method and application
  • Hybridoma cell line producing monoclonal antibody against human d-dimer, preparation method and application
  • Hybridoma cell line producing monoclonal antibody against human d-dimer, preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of anti-D-dimer monoclonal antibody

[0037] 1. Preparation of D-dimer monoclonal antibody

[0038] The purified D-dimer was emulsified with an equal amount of Freund's complete adjuvant, and the antigen emulsification was carried out by double syringes. After the emulsification was completed, five Balb / c mice aged about 8 weeks were given basic immunization (100 μg / ml) by multi-point subcutaneous injection in limbs and intraperitoneal injection. Two weeks later, the D-dimer was emulsified with the same amount of Freund's incomplete adjuvant, and the mice were boosted by subcutaneous injection at multiple points in the limbs and intraperitoneal injection (100 μg / ml); One booster immunization was performed in the same way, and the mice were sacrificed 7 days later to take out the mouse spleen, and the spleen cells were subjected to cell fusion.

[0039] The fusion process is to mix spleen cells and myeloma cells (Sp2 / 0) at a ratio of 8:1, and us...

Embodiment 2

[0056] Example 2 Detection of D-dimer by double antibody sandwich ELISA

[0057] 1. HRP-labeled antibody

[0058] This method is based on NaIO ４ The sugar molecules on the surface of HRP are first oxidized into aldehyde groups, and then combined with the amino groups of the antibody protein. The yield of the obtained enzyme-labeled antibody is high. The specific steps are as follows:

[0059] 1) Preparation of HRP enzyme: Weigh 10 mg of HRP enzyme, add 2 mL of pure water to make 5 mg / ml of HRP liquid, calculate by adding 34 μL per mg of HRP enzyme, add 340 μL of NaIO 4 , placed in the dark at 4°C, after 1 hour, add 250 μL of ethylene glycol according to the amount of 250 μL per mg of HRP enzyme, place in the dark at 4°C, transfer to the dialysis bag after 30 minutes, and use 1mM acetate buffer (pH4.0~4.4) The dialysis was performed overnight, and the medium was changed at least twice during the period, all of which were required to be protected from light.

[0060] 2) A...

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Abstract

The invention relates to a hybridoma cell line of a monoclonal antibody against human D-dimer, a preparation method and application thereof. In the present invention, the purified D-dimer is used as an immunogen to immunize animal Balb / c mice, and the monoclonal antibody capable of stably secreting anti-D-dimer is obtained by cloning and screening by ELISA method through hybridoma technology through multiple cell fusions hybridoma cell lines. Monoclonal antibody production by mouse ascites method. And use the prepared anti-D-dimer monoclonal antibody as capture antibody and detection antibody, use D-dimer as detection antigen, carry out antibody pairing test, obtain the antibody pairing that can detect natural D-dimer and use This established a double-antibody sandwich ELISA method for the detection of D-dimer. Hybridoma cell lines secrete corresponding monoclonal antibodies, which can be matched and detected in natural serum, and a double-antibody sandwich ELISA method for detecting D-dimer has been preliminarily established.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to a hybridoma cell line and a monoclonal antibody against human D-dimer produced therefrom. Background technique [0002] D-dimer (D-dimer) is a specific degradation product produced by fibrin monomer cross-linked by activated factor XIII and then hydrolyzed by plasmin. Specifically, under the action of thrombin, FactorXIIIa (transglutaminase) and calcium ions, fibrinogen in plasma removes fibrinopeptide A and B, exposes new binding sites, and spontaneously aggregates to form non- Covalently bonded fibrin bundles; under the action of FactorXIIIa, the fibrin bundles form γ-γ isopeptide bonds, and the polymerized form is stabilized by covalent bonds, which is called cross-linked fibrin, which is the basic of thrombus Skeleton; under the action of plasmin, cross-linked fibrin is degraded into cross-linked fibrin degradation products of different sizes. The cross-linke...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/68C12R1/91
Inventor 赵忠颢王晓东李金芳
Owner CHONGQING TANSHENG TECH
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