Human blood coagulation factor VIII resisting monoclonal antibody as well as preparation method and application thereof

A monoclonal antibody and human coagulation factor technology, applied in the field of genetic engineering, can solve the problems of unsatisfactory antibodies, difficult detection, lack of specificity, etc.

Inactive Publication Date: 2010-12-08
SUZHOU ZELGEN BIOPHARML
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing antibodies are still unsatisfactory. Due to the heterogeneity of FVIII molecules, the concentration in plasma is very small (0.1-0.2ug / ml)
Therefore, it is extremely difficult to detect, so there is still a lack of an effective method for the detection of human coagulation factor VIII with strong specificity.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Preparation of Monoclonal Antibody

[0045] (1) Immunized animals:

[0046] On the first day, antigen 50 μg / mouse, 0.25ml antigen + 0.25ml complete Freund’s adjuvant / mouse, mixer at 6000 rpm for 10 minutes, fully mixed, subcutaneous multi-point injection, 5 mice / antigen; day 28 , antigen 50μg / mouse, 0.25ml antigen + 0.25ml incomplete Freund's adjuvant / mouse, stirrer 6000 rpm for 10 minutes, fully mixed, subcutaneous multi-point injection, 5 mice / antigen; on the 60th day, antigen 50 μg / mouse, 0.25ml antigen + 0.25ml incomplete Freund’s adjuvant / mouse, stirrer 6000 rpm for 10 minutes, fully mixed, multi-point subcutaneous injection, 5 mice / antigen; on the 90th day, antigen 50μg / One mouse, 0.25ml antigen + 0.25ml incomplete Freund's adjuvant per mouse, mixer at 6000 rpm for 10 minutes, fully mixed, multi-point subcutaneous injection, 5 mice per antigen. Four days before fusion, 30 μg of antigen was injected intraperitoneally.

[0047] (2) Cell fusion:

[00...

Embodiment 2

[0109] Example 2: Purification and detection of antibodies

[0110] Ascites was prepared from 8 hybridoma cells and purified with commercially available Protein A. The operation method was referred to its instruction manual, and the purified monoclonal antibody was obtained as a result.

[0111] Binding specificity and affinity tests were carried out after the antibodies were purified, and the results showed that the specificity and affinity of the 8 mAbs were all very good, especially the specificity and affinity of the monoclonal antibody F8-3 was the best, which was higher than that of other general monoclonal antibodies (such as hybrid The monoclonal antibody produced by tumor cell H) was an order of magnitude higher.

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PUM

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Abstract

The invention provides a human blood coagulation factor VIII resisting monoclonal antibody as well as a preparation method and application thereof. The monoclonal antibody has the characteristic of strong specificity in combination with the human blood coagulation factor VIII antigen. The invention also provides a corresponding monoclonal antibody segment, immune conjugate and a detection kit for detecting the human blood coagulation factor VIII.

Description

technical field [0001] The invention relates to the field of genetic engineering. More specifically, the present invention relates to an anti-human coagulation factor VIII monoclonal antibody and its preparation method and use. Background technique [0002] Hemophilia is actually not a rare disease. It is an inherited bleeding disorder caused by a deficiency of clotting factors VIII or IX. It affects almost all men. The most common of these is hemophilia A caused by the deficiency or functional defect of blood coagulation factor VIII. The clinical manifestations are hemorrhage after joint or muscle trauma or spontaneous bleeding. If not treated in time, it will cause joint damage, disability, and severe bleeding can lead to death. . Coagulation factor VIII can be used to treat hemophilia A who lack plasma coagulation factor VIII. In the correction or prevention of bleeding, emergency or elective surgery, this product can temporarily replace the missing coagulation factor. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/36C07K19/00C12N5/20G01N33/577
Inventor 盛泽林
Owner SUZHOU ZELGEN BIOPHARML
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