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Monoclonal antibody of blood coagulation factor IX

A monoclonal antibody and blood coagulation factor technology, which is applied in the direction of coagulation/fibrinolytic factors, anticoagulation factor immunoglobulin, peptide preparation methods, etc., can solve the problems of impossibility, low safety of blood transfusion, susceptibility to infection, etc.

Active Publication Date: 2013-11-20
浙江耶大生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is limited by the source of blood and cannot be realized when blood is scarce
In addition, the safety of blood transfusion is relatively low, and it is easy to be infected with many blood-borne diseases such as hepatitis A, hepatitis B, hepatitis C, and AIDS. Therefore, it is necessary to purify blood coagulation factors in plasma or express them through genetic engineering.

Method used

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  • Monoclonal antibody of blood coagulation factor IX
  • Monoclonal antibody of blood coagulation factor IX
  • Monoclonal antibody of blood coagulation factor IX

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 Balb / c mouse immunization and splenocyte isolation

[0042] 1. Materials

[0043] Human plasma-derived FIX protein (Prospec Company), 5-week-old female Balb / c mice.

[0044] 2. Methods and results

[0045] For the first immunization, human plasma-derived FIX protein was mixed with an equal volume of Freund's complete adjuvant, and 5-week-old female Balb / c mice were injected intraperitoneally, 100 μg / mouse; after the third week, the second immunization was performed, and FIX protein was mixed with The same amount of Freund's incomplete adjuvant was mixed and injected intraperitoneally, 100 μg / mouse; after the fifth week, the third immunization was performed, this time without adjuvant, and each mouse was injected intraperitoneally with 100 μg FIX protein.

[0046] 3-5 days after the third immunization, the mice were extirpated by enucleation of eyeballs and bloodletting. Take out the mouse spleen by aseptic operation, put it in a sterilized plate, wash it...

Embodiment 2

[0047] Example 2 Fusion of mouse lymphocytes and myeloma cells and screening of hybridoma cells

[0048] 1. Materials

[0049] Mouse myeloma cell Sp2 / 0 was preserved in our laboratory, OPTI-MEM, HAT medium and fetal bovine serum were purchased from Gibco, PEG was purchased from Sigma Company, BCA protein quantification kit was purchased from Piece Company, HRP-labeled goat anti-mouse The secondary antibody was purchased from Zhongshan Jinqiao Company.

[0050] 2. Methods and results

[0051] One week before fusion, resuscitate mouse myeloma cells Sp2 / 0 at 37°C, 5% CO 2 The cells were cultured in an incubator, and the cells were passaged once 3 days before fusion. On the day of fusion, harvest myeloma cells, count, and put 5.0×10 7 Myeloma cells were washed twice with serum-free medium for later use.

[0052] Spleen cells equivalent to 1 / 2 spleen of the mouse were mixed with myeloma cells, centrifuged at 1300 rpm for 5 min, the supernatant was discarded, and removed as cle...

Embodiment 3

[0057] Example 3 Using immunoblotting to detect the specificity of antigen-antibody binding

[0058] 1. Materials

[0059] DAB substrate was purchased from Piece Company, and skimmed milk powder was purchased from BD Company.

[0060] 2. Methods and results

[0061] Mix 100μg of FIX protein with SDS-PAGE5×loading buffer evenly, boil at 100°C for 10min, load the sample, electrophoresis, electrotransfer to NC membrane, block with 5% skimmed milk powder at room temperature for 2h, and press the above six monoclonal antibodies with 5% skimmed milk powder respectively. Diluted at 1:1000, hybridized at room temperature for 1 h, and washed thoroughly with 0.05% PBST. HRP-goat anti-mouse secondary antibody was diluted 1:2000 with 5% skimmed milk powder, hybridized at room temperature for 1 hour, washed thoroughly with 0.05% PBST, added DAB substrate, and the results were as follows figure 1 , except the monoclonal antibody 7G2-A2 (the third lane) which has non-specific binding, all...

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PUM

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Abstract

The invention discloses a monoclonal antibody of a blood coagulation factor IX. The antibody comprises a light chain and a heavy chain, wherein the variable region amino acid sequence of the light chain is represented by SEQ ID NO.3, and the variable region amino acid sequence of the heavy chain is represented by SEQ ID NO.4. The invention also discloses a preparation method of the antibody. The antibody can specifically identify the blood coagulation factor IX. The invention further discloses a method for purifying the blood coagulation factor IX from blood plasma through utilizing the antibody. The blood coagulation factor IX can be conveniently and efficiently purified from the blood plasma through utilizing the antibody, so the infection risks of the diseases comprising hepatitis A, the hepatitis B, AIDS and the like caused by the patient blood transfusion supplementation of the blood coagulation factor IX are reduced.

Description

technical field [0001] The present invention relates to an antibody, in particular to an anti-coagulation factor IX monoclonal antibody. Background technique [0002] Coagulation factors are various protein components involved in the blood clotting process. Their physiological role is to be activated when blood vessels bleed, stick together with platelets and plug the leaks in blood vessels. This process is called coagulation. They are partially produced by the liver and can be inhibited by coumarin. For unified naming, the World Health Organization uses Roman numerals in the order in which they were discovered, including coagulation factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII, XIII, etc., factor Coagulation factors discovered after XIII, after years of verification, are considered to have no decisive impact on coagulation function, and are no longer included in the number of coagulation factors. Among them, coagulation factor IX (Factor IX, FIX) is also ca...

Claims

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Application Information

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IPC IPC(8): C07K16/36G01N33/68G01N33/577C07K14/745C07K1/22
Inventor 焦永军张黎曾晓燕郭喜玲史智扬崔仑标周明浩
Owner 浙江耶大生物医药有限公司
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