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Rapid and high-flux rabbit polyclonal antibody in-vitro production method

A polyclonal antibody and production method technology, applied in the field of antibody preparation, can solve the problems of unstable quality and large product batch differences, and achieve the effects of large-scale and stable production, shortened production cycle, and low unit cost

Inactive Publication Date: 2021-02-19
JINGJIE PTM BIOLAB HANGZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And because of the limitations of rabbit health and lifespan, multi-antibody products often need to re-immunize new rabbits, resulting in large batch differences and unstable quality.

Method used

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  • Rapid and high-flux rabbit polyclonal antibody in-vitro production method
  • Rapid and high-flux rabbit polyclonal antibody in-vitro production method
  • Rapid and high-flux rabbit polyclonal antibody in-vitro production method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 The rapid and high-throughput rabbit polyclonal antibody production method provided by the present invention in vitro

[0054] The rapid and large-throughput rabbit polyclonal antibody production method in vitro provided in this example is shown in the flow chart of the invention ( figure 1 ), including the following steps:

[0055] Step 1): Peripheral blood mononuclear cell suspension obtained from immunized rabbits

[0056] In this embodiment, the peripheral blood mononuclear cell suspension is preferably obtained from the rabbit after the third immunization, and the preparation method of the peripheral blood mononuclear cell suspension is as follows:

[0057] a) Add 20 mL of lymphocyte separation medium Ficoll (Tianjin Haoyang, Cat#: LTS1077) into a 50 mL centrifuge tube;

[0058] b) Take 10mL of anticoagulated rabbit venous blood after the third immunization and 10mL of sterile DPBS (Gibco, Cat#: D8537-500ML) and mix thoroughly according to 1:1, and slow...

Embodiment 2

[0079] The optimization of embodiment 2 B lymphocyte culture medium

[0080] The method of optimizing the B lymphocyte culture medium is as follows: use the traditional B lymphocyte culture medium and optimized multi-group culture medium with different components, take 10ml each into a plate, insert the antigen-specific B lymphocytes obtained in step 2, and the cells The density was controlled at 0.01M / ml, cultured continuously for 15 days, and the cell growth curve was drawn by counting every 2 days.

[0081] Among them, the traditional B lymphocyte culture medium is the control group, and the optimized culture medium with different components is the test group:

[0082] Control group: B cell conventional medium Formula: RPMI1640 (Gibco, Cat#: C11875500BT) + CD40L (abcam, Cat#: ab179625) + 20ng / ml IL-6 (Noveprotein, Cat#: CG39) + 10% FBS (Hyclone , Cat#: SV30208);

[0083] Test group 1: RPMI1640 (Gibco, Cat#: C11875500BT) + CD40L (abcam, Cat#: ab179625) + 20ng / ml IL-6 (Nove...

Embodiment 3

[0091] Embodiment 3 and the comparison of traditional method

[0092] In this example, according to the in vitro production method of rabbit polyclonal antibody provided in Example 1 (wherein the B lymphocyte culture medium adopts the test group 4 optimized in Example 2), and the traditional production method, the in vitro production effect of rabbit polyclonal antibody is tested. Comparison, wherein the traditional production method is the collection of rabbit serum after the fourth immunization, the results are shown in Table 1, and the product quality obtained by WB detection, the results are as follows Figure 5 As shown, where channel 1 is the product purified by the method provided in Example 1, channel 2 is the product purified by traditional serum, channel 3 is the negative control, and channel 4 is the positive control.

[0093] Table 1 Comparison of the effect of the method provided in Example 1 and the traditional method for in vitro production of rabbit polyclonal an...

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Abstract

The invention provides a rapid and high-flux rabbit polyclonal antibody in-vitro production method,. According to the method, a B lymphocyte separation technology is mainly adopted to realize rapid, large-scale and stable production of rabbit polyclonal antibodies through in-vitro amplification culture, a B lymphocyte culture medium is optimized, tryptone is adopted, and through cooperation with blank rabbit serum, the B lymphocyte culture medium is prepared. The differentiation and proliferation amount of B lymphocytes are greatly improved, and a basis of cell quantity is provided for in-vitro production of the rabbit polyclonal antibodies. Through the method provided by the invention, the rabbits can be immediately subjected to blood sampling for production after being immunized for thethird time, the production cycle of products is greatly shortened, and the yield of the obtained rabbit polyclonal antibodies is 30 times or above of that of serum with the same volume in the traditional method. The method has the advantages of short cycle, high yield, low unit cost, stable quality and the like, and is suitable for large-scale production and application.

Description

technical field [0001] The present invention relates to the field of antibody preparation, specifically, the present invention relates to a rapid and large-throughput rabbit polyclonal antibody production method in vitro. Background technique [0002] Rabbit polyclonal antibody refers to the antibody produced by the rabbit's immune system due to the stimulation of antigen. The antigen is often composed of multiple antigenic determinants, and a rabbit B lymphocyte can only be stimulated by one antigenic determinant of the antigen to produce a corresponding specific antibody, which is called a monoclonal antibody. (Monoclonal Antibody). Therefore, a variety of antigenic determinants stimulate the immune system of rabbits, and correspondingly produce a variety of specific monoclonal antibodies, and these monoclonal antibodies are mixed together to form polyclonal antibodies. Antibodies produced by the rabbit immune system have the advantages of strong affinity, good specifici...

Claims

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Application Information

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IPC IPC(8): C07K16/00C07K1/22C12N5/0781
CPCC07K16/00C07K2317/14C12N5/0635C12N2500/84C12N2501/2306C12N2501/52
Inventor 魏力章红芳程仲毅潘红阳
Owner JINGJIE PTM BIOLAB HANGZHOU CO LTD
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