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Processing method of human peripheral blood T lymphocytes and immunogen preparation and application

A lymphocyte and processing method technology, applied in the field of human peripheral blood T lymphocyte processing, can solve the problems of complex antigen composition, difficulty in obtaining thymic lymphocytes, and complex production and purification process of ALG products, and achieves a simplified production and purification process. Effect

Active Publication Date: 2022-05-17
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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  • Processing method of human peripheral blood T lymphocytes and immunogen preparation and application
  • Processing method of human peripheral blood T lymphocytes and immunogen preparation and application
  • Processing method of human peripheral blood T lymphocytes and immunogen preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0024] like 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 (specification 50mL), 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 layer into a new sterile centrifuge tube (specification 50mL), add PBS buffer (pH≈7.2-7.4) to 45mL, resus...

Embodiment 2

[0030] Example 2 Proliferation culture conditions and surface antigen identification of lymphocytes

[0031] This example 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:

[0032] Table 2 Statistical table of proliferation culture exploratory test

[0033]

[0034] 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 appear in 3 to 6 days, the cell growth reaches the peak in 8 to 10 days of culture, and reaches a plateau after 14 days, and the cell viability detected by trypan blue staining after 21 days reaches more than 90%.

[0035] It can be seen from the abo...

Embodiment 3

[0048] Example 3 Antigen preparation, pig immunization method and titer detection

[0049] Centrifuge the lymphocytes cultured for 14 days in the 2-6 test group at 1500rpm for 5min, collect, wash 2-3 times with 0.9% NaCl saline, and count the dead and alive with a cell counter. The cell viability must be greater than 90%.

[0050] Prepare the amount of antigenic cells according to the weight of healthy pigs: 100 million proliferating lymphocytes per 5-8 kg. Under aseptic conditions, take out the mineral oil adjuvant and the antigen and mix quickly at a mass ratio of 1:1, then add 10 μL molecular adjuvant according to the immune dose of each pig, mix well, and shake for 10-20 minutes to form the oil-in-water antigen. Wherein, the formula for preparing the oil-in-antigen preparation is as follows in Table 5:

[0051] Table 5 Formulation dosage table of different antigen preparations

[0052]

[0053] Multiple intramuscular injections were performed on both sides of the neck...

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Abstract

The present invention relates to a treatment method for human peripheral blood T lymphocytes, preparation and application of immunogen preparations and antiserum, in which discarded white blood cell filter discs are used to separate and purify T lymphocytes, and the T lymphocytes are proliferated and cultured, and the isolated T lymphocytes Compared with the cultured T lymphocytes for antigen surface marker identification and activity comparison; then the expanded and cultured highly active T lymphocytes were used as immunogens, and healthy pigs were used to prepare antiserum, and E rosette inhibition experiments and cell Toxicity test results to evaluate the potency after immunization, formulate immunization procedures, and then a large amount of antiserum can be prepared; then anti-plasma is collected for plasma separation and used for the preparation of ALG products. The present invention can solve the restriction problem that fresh whole blood needs to be collected each time in the potency test of raw materials for preparation of anti-human T lymphocyte immunoglobulin and the potency test of finished products, and can also solve the problem of the bottleneck of the raw material of anti-human T lymphocyte immunoglobulin .

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 Patents(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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