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Treatment method of human peripheral blood T lymphocytes, immunogen preparation and application

A technology of lymphocytes and human peripheral blood, applied in the direction of anti-animal/human immunoglobulin, cell culture active agent, blood/immune system cells, etc., can solve the problems of complex antigen components, poor standardization of titer testing, thymus Obtaining lymphocytes is difficult and other problems, to achieve the effect of simplifying the production and purification process

Active Publication Date: 2021-06-25
武汉中生毓晋生物医药有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, it is difficult to obtain human thymus lymphocytes as raw materials, and the standardization of titer testing is poor
In addition, when thymocytes are routinely used as antigens, the antigen components are relatively complex, and mixed antibodies such as red blood cell antibodies, thymus tissue antibodies, and platelet antibodies will be produced during the immunization process, making the production and purification process of subsequent ALG products complicated.

Method used

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  • Treatment method of human peripheral blood T lymphocytes, immunogen preparation and application
  • Treatment method of human peripheral blood T lymphocytes, immunogen preparation and application
  • Treatment method of human peripheral blood T lymphocytes, immunogen preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Separation, cryopreservation and quality evaluation of lymphocytes

[0024] Such as figure 1 As shown, take the leukocyte filter disc (acquisition source: Wuhan Central Blood Station) and place it in a biological safety cabinet, backflush the leukocyte filter disc with 0.9% normal saline, and collect the backflush normal saline containing leukocytes in a sterile bottle .

[0025] Use a pipette to draw 15mL of lymphocyte separation solution (manufacturer model: Fujian Sany) into the separation tube (50mL size), and add 15-30mL of diluted blood samples. Centrifuge at 1200g for 10min. After centrifugation, the sample in the separation tube is stratified from top to bottom: plasma-buffy coat-separation fluid-compartment-separation fluid-erythrocyte sedimentation. The sample stratification is as follows figure 2 shown. Use a pipette to suck the buffy coat into a new sterile centrifuge tube (50mL), add PBS buffer (pH≈7.2-7.4) to 45mL, resuspend the washed cell s...

Embodiment 2

[0031] Example 2 Proliferation culture conditions and surface antigen identification of lymphocytes

[0032] This embodiment explores the influence of culture conditions on in vitro proliferation and culture of lymphocytes, and studies lymphocyte proliferation medium (culture environment: 37 ° C, 5% CO 2 incubator) for cell proliferation cycle, state, density, activity and identification of surface antigen molecules. The exploration of some test cases is shown in Table 2:

[0033] Table 2 Statistical Table of Proliferation and Culture Exploration Test

[0034]

[0035]

[0036] Among them, the culture conditions of the frozen cells in the test groups No. 2-6 are as follows: image 3 As shown, it shows that obvious lymphocyte aggregation can occur in 3 to 6 days, and the cell growth reaches a peak in 8 to 10 days of culture, and is in a plateau after 14 days (such as Figure 3-8 ), after 21 days, the cell viability detected by trypan blue staining was over 90%.

[003...

Embodiment 3

[0050] Example 3 Antigen preparation, pig immunization method and titer detection

[0051] The lymphocytes in the 2-6 test group that had been proliferated and cultured for 14 days were centrifuged at 1500 rpm for 5 minutes, collected, washed 2-3 times with 0.9% NaCl saline, and counted by a cell counter. The cell viability must be greater than 90%.

[0052] Prepare the amount of antigenic cells according to the weight of healthy pigs: 100 million proliferating lymphocytes for every 5-8 kg. Under aseptic conditions, take out the mineral oil adjuvant and the antigen and mix quickly according to the mass ratio of 1:1, then add 10 μL of molecular adjuvant according to the immune dose of each pig, mix well, shake for 10-20min, and form the oil-in-water antigen. Wherein, the formula for preparing the oil-in-antigen preparation is as follows in Table 5:

[0053] Table 5 The formula consumption table of different antigen preparations

[0054]

[0055] Multiple intramuscular inje...

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Abstract

The invention relates to a treatment method of human peripheral blood T lymphocyte, animmunogen preparation, a preparation method of antiserum, and application. T lymphocytes are separated and purified by using a waste leukocyte filter disc, multiplication culture is carried out, and antigen surface marker identification comparison and activity comparison are carried out on the separated T lymphocytes and the cultured T lymphocytes; the high-activity T lymphocytes subjected to multiplication culture are used as an immunogen, antiserum is prepared by using a healthy pig, the titer after immunization is evaluated by using results of an E rose ring inhibition experiment and a cytotoxicity experiment, and formulating an immunization program, so as to prepare a large amount of antiserum; and anti-plasma is collected for plasma separation, and the plasma separation is used for preparation of ALG products. According to the invention, the limitation problem that fresh whole blood needs to be collected every time in the titer verification of the anti-human T lymphocyte immune globulin preparation raw material and the titer verification of the finished product can be solved, and meanwhile, the bottleneck problem of the anti-human T lymphocyte immune globulin raw material can also be solved.

Description

technical field [0001] The invention relates to the technical field of blood products, in particular to a processing method for human peripheral blood T lymphocytes, preparation and application of immunogen preparations and antiserum. Background technique [0002] Anti-human lymphocyte immunoglobulin (ALG) is mainly used for the prevention and treatment of immune rejection in clinical organ transplantation, the prevention of graft-versus-host reaction in bone marrow transplantation, and the treatment of aplastic anemia and other diseases. [0003] Anti-human lymphocyte immunoglobulin products routinely use human thymus lymphocytes as immunogens. After immunizing pigs, the raw material serum of anti-human lymphocyte immune protein products is obtained, and then purified and refined to obtain pig anti-human lymphocyte immunoglobulin preparations. [0004] However, it is difficult to obtain human thymus lymphocytes as raw materials, and the standardization of titer testing is p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C07K16/06C07K16/18
CPCC12N5/0636C07K16/065C07K16/18C12N2501/2302C12N2501/105C12N2501/515
Inventor 邹浩勇张智薛红刚殷文曲王智程文静黄梦张颂邓龙兴余健洪紫兰
Owner 武汉中生毓晋生物医药有限责任公司
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