CN1257874A - Process for preparing antiglobulin of human lymphocyte - Google Patents
Process for preparing antiglobulin of human lymphocyte Download PDFInfo
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- CN1257874A CN1257874A CN 98125622 CN98125622A CN1257874A CN 1257874 A CN1257874 A CN 1257874A CN 98125622 CN98125622 CN 98125622 CN 98125622 A CN98125622 A CN 98125622A CN 1257874 A CN1257874 A CN 1257874A
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Abstract
A process for preparing the antiglobuline of human lymphocyte includes such steps as collecting human thymus gland, or the liquid drain from ductus thoracicus, or the T lymphocyte in human-blood as antigen, immunizing rabbit or horse, separating immune serum, diluting with buffering agent or physiological saline, adding the aldehydized human thromobocyte, red cell and placental tissue, removing hybrid antibody, depositing in alcohol and purifying with ion exchange or n-caprylic acid and ammonium sulfate. Its advantages are high purity (more than 95%), high effect on removing hybrid antibody, low toxic by-effect and high curative effect.
Description
The present invention relates to a kind of process for preparing antiglobulin of human lymphocyte, belong to the preparation method of biotechnological formulation.
The AHLG is a kind of biotechnological formulation, be used for the treatment of the reaction of acute aplastic anemia and histoorgan transplant rejection clinically, because human immunity mechanism and immune dysfunction, the lymphocytic participation of T has great role, it not only discharges toxic substance, also stimulate some cytokines of secretion of other cell simultaneously, and organ transplantation owing to joining reasons such as type is improper, causes immune dysfunction, existing AHLG, because preparation method's imperfection, it is bad to purify, and assorted antibody is many, cause result of treatment bad, toxic side effect is big.
Purpose of the present invention is the shortcoming and deficiency in order to overcome above-mentioned prior art just, and a kind of simple and effective AHLG's preparation method is provided, thus improved purity, reduced toxic side effect, better the performance equivalent action improves result of treatment.
The objective of the invention is that technical scheme realizes by arriving down:
Process for preparing antiglobulin of human lymphocyte is characterized in that it is undertaken by following step:
A) adopting T lymphocyte liquid in people's thymus gland or thoracil duct drainage liquid or the human blood is that antigen and Fu Shi Freund's complete adjuvant thorough mixing carry out fundamental immunity rabbit or horse, again with Freund thorough mixing booster immunization rabbit or horse 2-4 time, the immunizing potency of gathering blood measuring serum reaches at 1: 400 o'clock, stop immunity, and separate qualified immune serum;
B) in the qualified immune serum of (a) with phosphate buffered saline buffer or with physiological saline dilution 1-3 doubly after, its concentration that adds hydroformylation is 10
5-10
8Cell/ml human blood platelets, red corpuscle and placenta tissue mixing 3-4 hour, centrifugal antiplatelet, anti erythrocyte antibody and other the assorted antibody of removing in the blood plasma is collected supernatant liquor, promptly obtains elementary AHLG's serum;
C) adding-5 ℃ of ethanol then in elementary AHLG's serum of (b) item precipitates, making its ultimate density is 19%, left standstill after the stirring 1-2 hour, centrifugation, collecting precipitation, tail is abandoned supernatant liquor, to precipitate again with the dissolving of DEAE level pad and make suspension, and then suspension be joined the DEAE ion exchange column and carry out chromatography, wash with damping fluid again, the back sodium chloride solution gradient elution of 0.1-1.0M till no sphaeroprotein, collect the sphaeroprotein peak,, promptly obtain AHLG's liquid of purifying through desalination and concentration by ultrafiltration, add Sterile Filtration behind the excipient again, collect filtrate;
D) at last filtrate packing or freeze-drying are finished product.
Elementary AHLG's serum, also available 0.06M, PH=4.0 acetate buffer solution add n-caprylic acid after diluting one times, and making its ultimate density is 0.2-1.0%, stir centrifuging and taking supernatant after 1 hour, tail is abandoned precipitation, adds ammonium sulfate again in supernatant liquor, and final saturation ratio is 35-45%, stirred 1-2 hour, centrifugation, collecting precipitation, tail is abandoned supernatant liquor, to precipitate again and use dissolved in distilled water, through ultrafiltration desalination, the concentrated AHLG's liquid that obtains purifying.
Elementary AHLG's serum also can add-5 ℃ of ethanol earlier and precipitate, and making its ultimate density is 19%, stirring was left standstill 1-2 hour, centrifugation, collecting precipitation will precipitate with the damping fluid dissolving again and make suspension, add sulfuric acid to saturation ratio again and reach 35-45%, stir centrifugation in 1-2 hour, collecting precipitation, tail is abandoned supernatant liquor, again with the dissolved in distilled water precipitation, through ultrafiltration desalination, the concentrated AHLG's liquid that obtains purifying.
Immune serum and hydroformylation human blood platelets, red corpuscle consumption volume ratio are 10: 1-10: 2, with the placenta tissue consumption be weight and volume ratio 10: 2-10: 5; Excipient is a glycine.
Preparation method of the present invention, adopt immune purification process, make in the immune serum human blood platelets by hydroformylation, red corpuscle and placenta tissue can better absorb the corresponding assorted antibody in the serum, obtain elementary AHLG's serum, the employing several different methods is purified, preparation method of the present invention is selected more flexibly, and suit measures to local conditions, use ethanol sedimentation, purify by ion-exchange, also available n-caprylic acid, ammonium sulfate two-step precipitation are purified, also available ethanol sedimentation is purified with ammonium sulfate precipitation again, all gets and effect preferably.
According to preparation method of the present invention, AHLG's product of preparation has all reached index preferably through quality examination and clinical observation, and its result is as follows:
A) quality inspection standard is:
1) anti-human erythrocyte TPPA: agglutination titer was less than 1: 32.
2) anti human platelet TPPA: agglutination titer was less than 1: 4.
3) anti-human plasma TPPA: the two expansions of immunity can't precipitation line.
4) efficacy determinations: the inverse that suppresses the high dilution of garland more than 25% was represented, greater than 1: 512.
5) sphaeroprotein purity is greater than 95%.
B) face not observation:
Through three tame hospitals acute aplastic anemia, histoorgan transplant rejection reaction treatment are observed, the treatment acute aplastic anemia, therapeutic dose 200-300mg/ day, the efficient 55-70% that reaches, therapeutic dose 100mg/ day, can prevent and treat the rejection of organ pipe transplanting, in whole therapeutic process, not see tangible toxic side effects.
Owing to take technique scheme, make the technology of the present invention compared with the prior art have following advantage and effect:
A) preparation method of the present invention can effectively remove impurity elimination antibody, reduces toxic side effect to greatest extent, has therefore improved result of treatment, has obtained obvious effects through clinical observation;
B) product purity can reach more than 95%, the height of tiring, and quality is up to state standards;
C) preparation method is easy, effective, suitability is strong, selectivity is high.
Embodiment 1
T lymphocyte liquid is that antigen and Fu Shi Freund's complete adjuvant thorough mixing carry out the fundamental immunity rabbit one time in the employing people thymus gland, again with twice of freund 's incomplete adjuvant thorough mixing booster immunization rabbit, when reaching 1: 400 when tiring, stop immunity, gather blood, and separating immune serum, get immune serum 1000ml, add the long-pending phosphate buffered saline buffer of monoploid then, concentration is 0.02M, PH=7.2, adding concentration again is 105 cells/ml hydroformylation human blood platelets 100ml, red corpuscle 100ml and placenta tissue 20g mixed 3 hours, the centrifugal antiplatelet of removing in the blood plasma, anti erythrocyte antibody and other assorted antibody, collect supernatant liquor, obtain elementary AHLG's serum, adding-5 ℃ of ethanol again in above-mentioned serum gradually stirs, making its ultimate density is 19%, left standstill 1 hour, centrifugal collecting precipitation, making suspension 100ml with DEAE damping fluid dissolution precipitation again joins the DEAE ion exchange column and carries out chromatography, wash with damping fluid, till no sphaeroprotein, use 0.5M sodium-chlor gradient elution at last, collect the sphaeroprotein peak value, through the ultra-fine filter ultrafiltration, desalination, concentrate the back and add the 0.22u film sterilization filter filtration sterilization of glycine excipient, collect filtrate and be AHLG's finished fluid, be qualified product through packing or freeze-drying, its purity is 96%.
Embodiment 2
T lymphocyte liquid is that antigen and Fu Shi Freund's complete adjuvant thorough mixing carry out the fundamental immunity rabbit one time in the employing thoracil duct drainage, again with freund 's incomplete adjuvant mixing booster immunization rabbit three times, when reaching 1: 400 when tiring, stop immunity, gather blood and separating immune serum, get immune serum 1000ml, add one times physiological saline then, adding concentration again is 10
8Cell/ml hydroformylation human blood platelets 200ml, red corpuscle 200ml and placenta tissue 30g mixed 4 hours, centrifugation, collecting supernatant liquor makes elementary anti-from the lymphocyte serum globulin, then with after one times of the 0.06M PH=4.0 acetate buffer solution dilution, add n-caprylic acid, ultimate density is 0.7%, stir centrifuging and taking supernatant after 1 hour, adding its saturation ratio of ammonium sulfate more gradually is 35%, stirs 1.5 hours, the centrifugation collecting precipitation, tail is abandoned supernatant liquor, will precipitate and use dissolved in distilled water, uses the ultra-fine filter desalination, add the 0.22u film degerming device filtration sterilization of glycine excipient, collect filtrate and be AHLG's finished fluid, be qualified product through being distributed into freeze-drying, its purity is 95%.
Embodiment 3
Adopting human blood T lymphocyte is antigen, press the adjuvant fundamental immunity horse 1 time of embodiment 1 then, and booster immunization horse 4 times is gathered blood and extracts immune serum, gets immune serum 1000ml, and the physiological saline that doubles then adds 10 after diluting again
10Cell/ml hydroformylation human blood platelets, each 150ml of red corpuscle and placenta tissue 50g mixed 3.5 hours, and supernatant liquor is collected in centrifugation, and adds-5 ℃ of ethanol, and making its ultimate density is 19%, left standstill 1.5 hours, and supernatant liquor is collected in centrifugation.Be elementary AHLG's serum, to precipitate with the phosphate buffered saline buffer dissolving again, and add ammonium sulfate again, its saturation ratio is 45%, stirred 1.5 hours, centrifugation, the collecting precipitation dissolved in distilled water is through ultra-fine filter desalination, concentrated, adding glycine excipient, with the filtration sterilization of 0.22u film sterilization filter, collect filtrate and be anti-human lymphocyte border protein liquid, be qualified product through packing or freeze-drying, purity is 95.5%.
Claims (5)
1. process for preparing antiglobulin of human lymphocyte is characterized in that it is undertaken by following step:
A) adopting T lymphocyte liquid in people's thymus gland or thoracil duct drainage liquid or the human blood is that antigen and Fu Shi Freund's complete adjuvant thorough mixing carry out fundamental immunity rabbit or horse, again with Freund thorough mixing booster immunization rabbit or horse 2-4 time, the immunizing potency of gathering blood measuring serum reaches at 1: 400 o'clock, stop immunity, and separate qualified immune serum;
B) in the qualified immune serum of (a) with phosphate buffered saline buffer or with physiological saline dilution 1-3 doubly after, its concentration that adds hydroformylation is 10
5-10
8Cell/ml human blood platelets, red corpuscle and placenta tissue mixing 3-4 hour, centrifugal antiplatelet, anti erythrocyte antibody and other the assorted antibody of removing in the blood plasma is collected supernatant liquor, promptly obtains elementary AHLG's serum;
C) adding-5 ℃ of ethanol then in elementary AHLG's serum of (b) item precipitates, making its ultimate density is 19%, left standstill after the stirring 1-2 hour, centrifugation, collecting precipitation, tail is abandoned supernatant liquor, to precipitate again with the dissolving of DEAE level pad and make suspension, and then suspension be joined the DEAE ion exchange column and carry out chromatography, wash with damping fluid again, the back sodium chloride solution gradient elution of 0.1-1.0M till no sphaeroprotein, collect the sphaeroprotein peak,, promptly obtain AHLG's liquid of purifying through desalination and concentration by ultrafiltration, add Sterile Filtration behind the excipient again, collect filtrate;
D) at last filtrate packing or freeze-drying are finished product.
2. preparation method according to claim 1, it is characterized in that described elementary AHLG's serum, also available 0.06M, PH=4.0 acetate buffer solution add n-caprylic acid after diluting one times, making its ultimate density is 0.2-1.0%, stir centrifuging and taking supernatant after 1 hour, tail is abandoned precipitation, in supernatant liquor, add ammonium sulfate again, final saturation ratio is 35-45%, stirs centrifugation 1-2 hour, collecting precipitation, tail is abandoned supernatant liquor, will precipitate and use dissolved in distilled water, through ultrafiltration desalination, the concentrated AHLG's liquid that obtains purifying.
3. the preparation method who states according to claim 1 art, it is characterized in that described elementary AHLG's serum, also can add-5 ℃ of ethanol earlier precipitates, making its ultimate density is 19%, stirring was left standstill 1-2 hour, centrifugation, collecting precipitation, to precipitate again with the damping fluid dissolving and make suspension, and add sulfuric acid to saturation ratio again and reach 35-45%, stir centrifugation in 1-2 hour, collecting precipitation, tail is abandoned supernatant liquor, again with the dissolved in distilled water precipitation, through ultrafiltration desalination, the concentrated AHLG's liquid that obtains purifying.
4. according to claim 1 or 2 or 3 described methods, it is characterized in that described immune serum and hydroformylation human blood platelets, red corpuscle consumption volume ratio are 10: 1-10: 2, with the placenta tissue consumption be weight and volume ratio 10: 2-10: 5.
5. according to claim 1 or 2 or 3 described methods, it is characterized in that described excipient is a glycine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB981256228A CN1151174C (en) | 1998-12-21 | 1998-12-21 | Process for preparing antiglobulin of human lymphocyte |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB981256228A CN1151174C (en) | 1998-12-21 | 1998-12-21 | Process for preparing antiglobulin of human lymphocyte |
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|---|---|
| CN1257874A true CN1257874A (en) | 2000-06-28 |
| CN1151174C CN1151174C (en) | 2004-05-26 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB981256228A Expired - Fee Related CN1151174C (en) | 1998-12-21 | 1998-12-21 | Process for preparing antiglobulin of human lymphocyte |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1922207B (en) * | 2004-02-27 | 2012-06-06 | 奥克特珐玛股份有限公司 | A method of providing a purified, virus safe antibody preparation |
| CN104271600A (en) * | 2012-05-14 | 2015-01-07 | 诺和诺德A/S（股份有限公司） | Stabilised protein solutions |
| CN104530227A (en) * | 2014-11-24 | 2015-04-22 | 广西大学 | Anti-neuritis cell antibody preparation technology |
| CN115232783A (en) * | 2022-08-03 | 2022-10-25 | 武汉中生毓晋生物医药有限责任公司 | Preparation method of anti-human T cell pig immunoglobulin and hydroformylation placenta |
-
1998
- 1998-12-21 CN CNB981256228A patent/CN1151174C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1922207B (en) * | 2004-02-27 | 2012-06-06 | 奥克特珐玛股份有限公司 | A method of providing a purified, virus safe antibody preparation |
| CN104271600A (en) * | 2012-05-14 | 2015-01-07 | 诺和诺德A/S（股份有限公司） | Stabilised protein solutions |
| CN104271600B (en) * | 2012-05-14 | 2018-09-14 | 诺和诺德股份有限公司 | Stabilized protein solution |
| US11466051B2 (en) | 2012-05-14 | 2022-10-11 | Novo Nordisk A/S | Stabilised protein solutions |
| CN104530227A (en) * | 2014-11-24 | 2015-04-22 | 广西大学 | Anti-neuritis cell antibody preparation technology |
| CN115232783A (en) * | 2022-08-03 | 2022-10-25 | 武汉中生毓晋生物医药有限责任公司 | Preparation method of anti-human T cell pig immunoglobulin and hydroformylation placenta |
| CN115232783B (en) * | 2022-08-03 | 2023-03-10 | 武汉中生毓晋生物医药有限责任公司 | Preparation method of anti-human T cell pig immunoglobulin and hydroformylation placenta |
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| Publication number | Publication date |
|---|---|
| CN1151174C (en) | 2004-05-26 |
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