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Method for preparing rabbit against human T lymphocytes immune serum

A lymphocyte and immune serum technology, applied in biochemical equipment and methods, extracellular fluid diseases, antibodies, etc., can solve problems such as difficulty in the source of raw materials, save manpower and material resources, expand production scale, and reduce production costs.

Inactive Publication Date: 2007-12-12
YUEYANG BOKANG BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to overcome the disadvantages of difficult raw material sources in the existing preparation methods, and provide a new preparation method of rabbit anti-human T lymphocyte immune serum

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Using human thymic T lymphocytes to immunize rabbits to prepare immune serum.

[0019] Collect aborted embryos over 6 months old, separate the thymus under aseptic conditions, wash the isolated thymus with Hnak’s buffer and gently grind to disperse the thymocytes and remove fat, collect and count the thymocytes, press 1×10 6 Inoculate per milliliter, add various cytokines that can induce thymocytes to differentiate into mature T lymphocytes in the medium. When more than 95% of the thymocytes differentiate into mature T lymphocytes, start collecting and counting the cells, and inoculate mature T lymphocytes In the peripheral blood medium containing various stimulating factors, the cell inoculation amount is 1×10 6 Each milliliter, culture and pass for a total of 6 generations, collect T lymphocytes to immunize New Zealand rabbits. The immunization is divided into 3 times, the immunization time is 70 days, and the three immunization doses are 5×10 7 , 1×10 8 , 1×10...

Embodiment 2

[0022] Example 2: Preparation of rabbit anti-human T lymphocyte immune serum using the method of the present invention.

[0023] 1. Before blood collection, test volunteer blood donors for HBV, HCV, AIDS, syphilis and other items. Use the CS-3000Plus Amicus machine produced by Baxter to collect lymphocytes, wash with Hank's buffer, and centrifuge at 1000rpm and 4℃ for 10min. Discard the supernatant, wash and centrifuge 3 times.

[0024] 2. Use Hank's buffer to mix lymphocytes into a cell suspension with a suitable concentration, add an appropriate amount of specific gravity adjustment solution and mix well, pour it into a test tube containing lymphatic separation solution, centrifuge at 2000rpm, 4℃ for 15min, centrifuge for a total of 2 to 4 times , Add Hank's solution and wash repeatedly, centrifuge at 1000rpm, 4℃ for 10min, until there is no red blood cell under microscope, and platelet is less than 10 / HP.

[0025] 3. Isolate T lymphocytes with mouse anti-human CD3 monoclonal an...

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PUM

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Abstract

The invention relates to a preparation method of rabbit anti-human T lymphocyte immune serum with cultured cell of T lymphocyte as immunogen and rabbit as immune animal. The immunization schedule comprises 1 fundamental immunity and 2booster immunizations, wherein complete adjuvant is added into antigen of fundamental immunity while no adjuvant is added in booster immnunization. The collected qualified blood serum precipitate has immunizing potency of 1:512, erythrocyte antibody valence below 1:128, and thrombocyte antibody valence below 1:8.The inventive method can reduce the production costs and enlarge scale of production through walking out the bottle neck of insufficient raw material of thymus.

Description

Technical field [0001] The invention belongs to the field of preparation methods of biological preparation raw materials, and specifically relates to a preparation method of anti-human T lymphocyte immune serum. Background technique [0002] Anti-human lymphocyte immunoglobulin (ATG) is a kind of biological immunosuppressant, which can activate complement after combining with the patient’s T lymphocytes, inhibit or remove T lymphocytes in the human body, and is mainly used clinically for rejection of tissue organ transplantation , Acute aplastic anemia, autoimmune hemolytic anemia, primary thrombocytopenic purpura and other malignant immune diseases, the market demand is not less than 2 million per year, and has a broad market prospect. [0003] At present, the domestic preparation method of ATG is: using human thymic lymphocytes as immunogens, immunizing pigs to obtain the anti-human lymphocyte immunoglobulin product raw material serum, which is purified and refined into pig ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/16A61K39/395C12N5/08A61P7/00A61P37/06
Inventor 毛智立
Owner YUEYANG BOKANG BIO TECH