Method for preparing antihuman recombinant tissue factor monoclonal antibody

A technology of monoclonal antibody and tissue factor, which is applied in the biological field to achieve the effects of stable quality, increased yield and high specificity

Active Publication Date: 2010-06-02
山西省生物研究院有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there is no commercial anti-TF monoclonal antibody in China.

Method used

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  • Method for preparing antihuman recombinant tissue factor monoclonal antibody
  • Method for preparing antihuman recombinant tissue factor monoclonal antibody
  • Method for preparing antihuman recombinant tissue factor monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Preparation of anti-human recombinant tissue factor monoclonal antibody

[0035] 1. Immunogen

[0036] Select 200510012414.7 patent to construct and express human recombinant tissue factor (rhTF 243 ) as an immunogen.

[0037] 2. Immunization of mice

[0038] Basic immunity: take rhTF stored in Tris-HCl buffer 243 (1mg / ml) was diluted to 120μg / ml, fully emulsified and mixed with Freund's complete adjuvant or Freund's incomplete adjuvant according to the volume ratio of 1:1, disinfected the abdomen of BALB / c mice with alcohol cotton balls, and selected 5 The mice were immunized by subcutaneous injection, and the injection volume was 0.5ml / only. Immunization volume 0.5ml, 30μg rhTF 243 / only, after the injection, disinfect the injection site with alcohol cotton balls to prevent infection.

[0039] Booster immunization: two weeks later, take rhTF stored in buffer 243 Dilute to 60 μg / ml, fully emulsify and mix with Freund’s complete adjuvant or Freund’s incomplete a...

Embodiment 2

[0056] Preparation of Anti-human Recombinant Tissue Factor Monoclonal Antibody Ascites with Different Inoculation Density

[0057] 1. Preparation of hybridoma cells

[0058] The anti-human recombinant tissue monoclonal antibody hybridomas obtained in Example 1 were selected and conventionally expanded and cultivated in DMEM high-glucose cell culture medium before use.

[0059] 2. Selection and pretreatment of experimental animals

[0060] Select 14-week-old, female, multiparous BALB / c mice, use liquid paraffin as a sensitizer, and sensitize one week before inoculating hybridoma cell lines, 0.5ml / mouse.

[0061] 3. Inoculation of hybridoma cell lines

[0062] The hybridoma cell line was subcultured through tissue culture flask cells, and the cells were collected after the cells grew to the logarithmic phase (1000rpm, 6min, centrifuged to remove the supernatant), and the adjusted cells were resuspended in serum-free basal medium (avoiding the introduction of bovine antibody) ...

Embodiment 3

[0075] Preparation of anti-human recombinant tissue factor monoclonal antibody ascites in different treatment groups

[0076] The anti-human recombinant tissue monoclonal antibody hybridomas obtained in Example 1 were selected and conventionally expanded and cultivated for use.

[0077] Select 21 14-week-old BALB / c mice purchased from the same batch, including 7 female, male and female multiparous mice, and divide them into 3 groups. Sensitize with liquid paraffin one week before hybridoma cell line inoculation, 0.5ml / only. One week later, the same density (5×10 5 cells / ml) of the hybridoma cell suspension, and the ascites was collected when the mice were dying.

[0078] The results of ascites production are shown in Table 2.

[0079] Table 2 Ascites production and antibody titer of mice in different treatment groups

[0080]

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Abstract

The invention discloses a method for preparing an antihuman recombinant tissue factor monoclonal antibody. The method comprises the following steps: combining a truncated human recombinant tissue factor obtained by purification after prokaryotic expression with a freund adjuvant immunized mouse; fusing mouse spleen cells with myeloma cells; selectively cultivating and screening an HAT selective medium; after the subcloning cultivation is subjected to limiting dilution, selecting target hybridoma cells by an ELISA method, performing massive proliferation, and injecting into an abdominal cavity of a syngeneic mouse to prepare ascites; and obtaining the antihuman recombinant tissue factor monoclonal antibody which has excellent potency and higher yield. The antihuman recombinant tissue factor monoclonal antibody prepared by the method of the invention has the advantages of high sensitivity and strong specificity, can be used for immunoaffinity purification of the human recombinant tissue factor, and also can be used for research and development of anticoagulant medicaments and medicaments for treating tumors.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a preparation method of anti-human recombinant tissue factor monoclonal antibody. Background technique [0002] Human tissue factor (Tissue Factor, TF) is a glycoprotein with a molecular weight of 47KD, containing 263 amino acid residues, and is a transmembrane receptor protein. TF is the receptor and cofactor of coagulation factor VII, and is the initiating protein of extrinsic coagulation pathway. The TF protein molecule is divided into three parts, 21 amino acid residues in the intracellular region, 23 amino acid residues in the transmembrane part, which is hydrophobic, and 219 amino acid residues in the extracellular part, which is the amino terminal and has sugar chains. [0003] Tissue factor is the most important initiating factor in physiological coagulation. It is the main component of clinical prothrombin time detection reagents and the main factor affecting the determinatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28
Inventor 赵邑赵峰梅潘薇薇何永吉齐延红梁雅丽卢晓霞
Owner 山西省生物研究院有限公司
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