CN101084923A - Method for preparing rabbit against human T lymphocytes immune serum - Google Patents
Method for preparing rabbit against human T lymphocytes immune serum Download PDFInfo
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- CN101084923A CN101084923A CNA2007100351864A CN200710035186A CN101084923A CN 101084923 A CN101084923 A CN 101084923A CN A2007100351864 A CNA2007100351864 A CN A2007100351864A CN 200710035186 A CN200710035186 A CN 200710035186A CN 101084923 A CN101084923 A CN 101084923A
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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
The invention belongs to biological preparation method for preparing raw material field, be specifically related to the preparation method of anti-human T lymphocytes immune serum.
Background technology
Zetbulin (ATG) is a kind of biological immunosuppressant agent, can be with after patient T lymphocyte combines, activating complement, suppress or remove T lymphocyte in the human body, be mainly used in the treatment of the reaction of histoorgan transplant rejection, acute aplastic anemia, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura and other pernicious immunological diseases clinically, market demand is not less than 2,000,000 every year, has vast market prospect.
At present, the preparation method of domestic ATG is: end user's thymic lymphocytes are made immunogen, obtain Zetbulin's goods raw material serum behind the immune swine, this raw material becomes pig Zetbulin preparation after technology such as purify, refining.People's thymic lymphocytes that this method adopts are taken from the dead baby of hospital's induced labor more than 6 months, along with the reform of state plan child-bearing policy and perfect, the raising of people's family planning consciousness, improper childbirth situation reduces gradually, the cell source is quite difficult, and insufficient raw material has become the bottleneck that the limit production scale enlarges.The present main dependence on import of domestic antiangiogenic immune globulin products, and the market price of external product is 4000 yuan/, surpasses more than the twice of domestic like product price.Therefore, no matter still consider that from the development angle of Chinese biological industry all should greatly develop the independent brand product, and address this problem, matter of utmost importance is to solve the problem of immune raw material sources difficulty from the angle of domestic patient treatment cost.
Summary of the invention
The objective of the invention is to overcome the shortcoming of the raw material sources difficulty that existing preparation method exists, a kind of preparation method of new rabbit against human T lymphocytes immune serum is provided.
For achieving the above object, method of the present invention comprises:
1, separator cell is adopted human peripheral T lymphocyte;
2,0.85% normal saline washs centrifugal 3 times and removes platelet;
3, Hank ' s buffer dilution is adopted density centrifugal 2-3 time;
4, remove bone-marrow-derived lymphocyte with mouse anti human CD3 monoclonal antibody immunity magnetic strain method, be separated to the T lymphocyte;
5, lymphocytic the going down to posterity of T cultivated 5-6 generation;
6, collect t subset lymphocyte count and check motility rate;
7, branch carries out animal immune three times, blood sampling and separation of serum;
8, press the detection method coherent detection of " three ones of Pharmacopoeias of People's Republic of China ".
The invention has the advantages that:
1, uses the bottleneck that method of the present invention can be walked out the thymus insufficient raw material, expand the scale of production.
2, periphery blood T lymphocyte is cultivated the immunogen of preparation, and needed immunity amount is lower than the immunity amount of thymocyte cell when carrying out immunity, can significantly reduce production costs.
3, the T lymphocyte yield of application method purification of the present invention reaches more than 87%, purity reaches more than 95%, suppressing experiment etc. with its rabbit against human T lymphocytes immune serum of making antigen preparation at cell toxicant, rosettes tires and measures, the result has all surpassed required the tiring of reaching of " three ones of Pharmacopoeias of People's Republic of China " regulation, and in assorted detection of antibodies such as anti-human erythrocyte antibody and anti human platelet antibody, the result all is lower than desired level in the Chinese biological goods rules, this method has been saved lot of manpower and material resources, greatly reduces production cost.
The specific embodiment
Embodiment one: adopt people's thymus T lymphocyte immunity rabbit to prepare immune serum.
Collect the embryo that miscarries more than 6 months, carry out thymus under the aseptic condition and separate, grind gently after thymus Hnak ' the s buffer that will the exsomatize washing, disperse thymocyte cell and remove fat, collect thymocyte cell and counting, by 1 * 10
6Every milliliter of inoculation, adding in the culture medium to induce thymocyte cell to be divided into the lymphocytic various cytokines of mature T, begin collecting cell and counting when treating to be divided into the mature T lymphocyte more than 95% in the thymocyte cell, the mature T lymphocyte is inoculated in the peripheral blood culture medium that contains various stimulating factors, and the cell inoculation amount is 1 * 10
6Every milliliter, cultivate and go down to posterity, passed for 6 generations altogether, collect T lymphocyte immunity new zealand rabbit, immunity divides to be carried out for 3 times, and the immunity time is 70 days, and three times immunizing dose is respectively 5 * 10
7, 1 * 10
8, 1 * 10
8Individual cell is gathered immune serum and is carried out cellulotoxic experiment, Flos Rosae Rugosae experiment and anti erythrocyte antibody titre, the detection of antiplatelet antibody titre, and testing result sees Table 1.The animal serum that acquisition testing is qualified carries out the detection of above index again after the mixing, it is ℃ frozen standby to detect qualified samples-20.
The immune serum of table 1 conventional method preparation detects index
| Project | Index | Project | Index |
| Flos Rosae Rugosae forms and suppresses experiment | More than 1: 512 | Outward appearance | Weak yellow liquid, no haemolysis |
| Cell toxicant suppresses experiment | More than 1: 512 | Protein content | Greater than 95% |
| Human red blood cell antibody | Be lower than 1: 128 | HBsAg | Negative |
| Human blood platelets antibody | Be lower than 1: 8 | HIV-1 antibody | Negative |
| Undue toxicity's experiment | The reaction of animal Non Apparent Abnormality, weight increase | HIV-2 antibody | Negative |
| The thermal source experiment | Up to specification | HCV | Negative |
| Human plasma protein fraction antibody | Negative | Syphilis | Negative |
Embodiment two: adopt the inventive method to carry out the preparation of rabbit against human T lymphocytes immune serum.
1, before the blood sampling aspiration blood donor is carried out the detection of projects such as HBV, HCV, AIDS, syphilis, the CS-3000Plus Amicus machine that adopts the U.S. hundred special companies to produce is gathered lymphocyte, use Hank ' s buffer washing back 1000rpm, 4 ℃ of centrifugal 10min, abandon supernatant, wash altogether centrifugal 3 times.
2, use Hank ' s buffer lymphocyte to be made into the cell suspension of suitable concn, pour in the test tube that the lymph separating medium is housed after adding an amount of proportion regulator solution mixing, 2000rpm, 4 ℃ of centrifugal 15min, centrifugal 2～4 times altogether, add Hank ' s liquid cyclic washing, 1000rmp, 4 ℃ of centrifugal 10min do not have erythrocyte to microscopy, and platelet＜10/HP.
3, with the lymphocytic separation of mouse anti human CD3 monoclonal antibody immunity magnetic strain carrying out T: be fixed on the support after will filling in the syringe autoclaving of nylon hair, the cell culture fluid of pouring 37 ℃ into cleans 2～3 nylon hairs, shuts valve; Will be diluted to suitable concentration, about 5.00 * 10 with the culture fluid of heating in advance by isolated cells liquid
7Individual cell/ml pours cell suspension in the syringe into, makes it not have nylon hair post, covers syringe, 37 ℃ of incubation 45min to 1h; Open end opening, slowly release (1/min), be collected in the centrifuge tube is centrifugally promptly obtained required T lymphocyte.
4, the T cell is made into 1 * 10
7The cell suspension inoculation of/ml is in RPMI-1640 culture medium (50ml/ bottle), and every bottle contains 1ml T cell suspension, in 37 ℃, 5%CO
2, saturated humidity CO
2Cultivated 72 hours in the incubator.
5, change culture medium, add γ IL-2, condition of culture is the same, the visual cell growing state every 3-5 days in suitable ratio with passage, cultivate 3 all left and right sides collecting cells, with the centrifugal 10min of RPMI-1640,1000r/min, count after washing 3～5 times.The result shows that the T lymphocyte yield of purification reaches more than 87%, and purity reaches more than 95%.
6, adopt new zealand rabbit as immune animal, immune programme for children is: fundamental immunity adopts the mode of intramuscular injection, adds Freund's complete adjuvant in the antigen, and immunizing dose is 3 * 10
7Individual cell; Booster immunization adopts the mode of ear vein injection, does not add any adjuvant in the antigen, and the dosage of twice booster immunization is respectively 4 * 10
7With 3 * 10
7Individual cell.Behind the booster immunization after 8-14 days heart take a blood sample in the blood bag, the centrifugal 15min of 3000rpm, taking out serum on plasma-separating clip is human lymphocyte immune globulin antibody raw material ,-30 ℃ of preservations.
7, do the detection of E rosettes inhibition test, cytotoxicity test, human red blood cell TPPA, human blood platelets TPPA, abnormal toxicity test, pyrogenic test, human plasma protein fraction TPPA and other conventional causal organisms by the detection method of " three ones of Pharmacopoeias of People's Republic of China ".Testing result sees Table 2.The animal serum that acquisition testing is qualified carries out the detection of above index again after the mixing, it is ℃ frozen standby to detect qualified samples-20.
The immune serum of table 2 the present invention preparation detects index
| Project | Index | Project | Index |
| Flos Rosae Rugosae forms and suppresses experiment | More than 1: 512 | Outward appearance | Weak yellow liquid, no haemolysis |
| Cell toxicant suppresses experiment | More than 1: 512 | Protein content | Greater than 95% |
| Human red blood cell antibody | Be lower than 1: 128 | HBsAg | Negative |
| Human blood platelets antibody | Be lower than 1: 8 | HIV-1 antibody | Negative |
| Undue toxicity's experiment | The reaction of animal Non Apparent Abnormality, weight increase | HIV-2 antibody | Negative |
| The thermal source experiment | Up to specification | HCV | Negative |
| Human plasma protein fraction antibody | Negative | Syphilis | Negative |
The technology of the present invention is accomplished scale production; the antilymphocyte immune serum detects through users such as Beijing Biological Product Inst., Wuhan Biological Products Inst., Beijing Tiantan Biological Products Co.ltd, south China biological engineering company limited and middle safe pharmaceutcal corporation, Ltds; meet their production requirement fully, the raw blood plasma quality is highly commended by customers deeply.
Claims (3)
1, a kind of preparation method of rabbit against human T lymphocytes immune serum, it is characterized in that adopting human peripheral T lymphocyte cultured cell as immunogen, rabbit is an immune animal, and immune programme for children is: fundamental immunity adopts the mode of intramuscular injection, add Freund's complete adjuvant in the antigen, immunizing dose is 3 * 10
7Individual cell; Booster immunization adopts the mode of ear vein injection, does not add any adjuvant in the antigen, and the dosage of twice booster immunization is respectively 4 * 10
7With 3 * 10
7Individual cell.
2, the preparation method of rabbit against human T lymphocytes immune serum according to claim 1 is characterized in that the preparation method of human peripheral T lymphocyte cell is:
1) separator cell is adopted human peripheral T lymphocyte;
2) 0.85% normal saline washs centrifugal 3 times and removes platelet;
3) Hank ' s buffer dilution is adopted density centrifugal 2-3 time;
4) remove bone-marrow-derived lymphocyte with mouse anti human CD3 monoclonal antibody immunity magnetic strain method, be separated to the T lymphocyte;
5) lymphocytic the going down to posterity of T cultivated 5-6 generation;
6) collect t subset lymphocyte count and check motility rate.
3, the lymphocytic preparation method of human peripheral T according to claim 2 is characterized in that in the immune magnetic strain separating step, the employing condition is 37 ℃ of incubation 45～60min.
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| CNA2007100351864A CN101084923A (en) | 2007-06-22 | 2007-06-22 | Method for preparing rabbit against human T lymphocytes immune serum |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117069836A (en) * | 2023-09-22 | 2023-11-17 | 武汉中生毓晋生物医药有限责任公司 | Preparation method of anti-human T cell rabbit immunoglobulin |
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- 2007-06-22 CN CNA2007100351864A patent/CN101084923A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117069836A (en) * | 2023-09-22 | 2023-11-17 | 武汉中生毓晋生物医药有限责任公司 | Preparation method of anti-human T cell rabbit immunoglobulin |
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