CN1763096A - Animal composite immune globulin production method - Google Patents
Animal composite immune globulin production method Download PDFInfo
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- CN1763096A CN1763096A CN 200510047201 CN200510047201A CN1763096A CN 1763096 A CN1763096 A CN 1763096A CN 200510047201 CN200510047201 CN 200510047201 CN 200510047201 A CN200510047201 A CN 200510047201A CN 1763096 A CN1763096 A CN 1763096A
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Abstract
The production process of composite immune globulin for animal includes the following steps: preparing immune blood serum and packing immune blood serum at -20 deg.c inside storing container; separating immune blood serum to extract immune globulin IgG, IgA and IgM; purifying immune globulin IgG, IgA and IgM; mixing immune globulin IgG, IgA and IgM in the ratio of 8-10 to 1 to 1-2; and nanofiltering. The composite immune globulin thus produced includes three kinds of immune globulin, IgG, IgA and IgM, and has three kinds of immune functions, high immune effect of preventing animal viral diseases and easy absorption.
Description
Technical field:
The present invention relates to the production method of a kind of animal with production method, the especially animal composite immune globulin of immunoglobulin (Ig).
Background technology:
Zoonosis toxicity disease is a great problem of puzzlement aquaculture always, and is annual because the loss that zoonosis toxicity disease causes is startling.Except with the antiviral medicine, adopt virus-specific hyper-immune serum treatment zoonosis toxicity disease on the present veterinary clinic more.At present, the production method of virus-specific hyper-immune serum is to carry out according to the following steps:
1. measure a certain amount of animal serum, under slow condition of stirring, slowly add isopyknic 80% supersaturation ammonium sulfate, whenever add one and add second again after dropping in mixing, being put into 4 ℃ after stirring left standstill 1-2 hour, take out the back with 3000～5000 rev/mins centrifugal 15～20 minutes, abandon supernatant, use and the isopyknic physiological saline solution precipitation of initial serum;
2. under slow condition of stirring, slowly add isopyknic 80% supersaturation ammonium sulfate, be put into 4 ℃ after stirring and left standstill 1-2 hour, centrifugal 15～20 minutes after taking out with 3000～5000 rev/mins, abandon supernatant, use and the isopyknic physiological saline solution precipitation of initial serum;
3. repeating step 2;
4. solution is adorned dialysis tubing, dialysed 48 hours, and changed 3～4 times dialyzates (distilled water or physiological saline or PBS damping fluid) every day for 4 ℃;
5. the good immunoglobulin (Ig) of will dialysing is packed in the container, freezing preservation.
Existing production method exists following shortcoming:
1. to extract mainly be the IgG immunoglobulin (Ig) to this method, and immunological species is single, can not reach the ideal immune effect;
2. ammonium sulfate easily remains in the product, and product purity is not high, and the injection back is difficult for absorbing.
Summary of the invention:
The present invention is that the existing in prior technology immunoglobulin (Ig) is single in order to solve, immune effect is paid no attention to and thought of the low technical problem of product purity, and a kind of production method with three kinds of immunoglobulin (Ig)s, animal composite immune globulin that purity is high is provided.
Technical solution of the present invention is:
A kind of production method of animal composite immune globulin comprises the steps:
A. prepare immune serum, immune serum is sub-packed in the container be stored in-20 ℃ standby;
B. use immune serum separation and Extraction immunoglobulin IgG, IgA and IgM;
C. purifying immunoglobulin IgG, IgA and IgM;
D. three kinds of immunoglobulin IgGs, IgA, IgM are pressed 8～10: 1: 1～2 mixed;
E. use nano level aperture membrane filtration.
Described concrete steps with immune serum separation and Extraction immunoglobulin IgG, IgA and IgM are as follows:
A. the water of immune serum with 3 times of volumes is mixed, transfer pH value to 7.7, the temperature of solution is reduced to 0 ℃;
B. under stirring fast, add-20 ℃ of precooled ethanol, make it produce precipitation, centrifugal collecting precipitation;
C. precipitation is suspended from the physiological saline of 25 times of volumes, adds the acetic acid solution of 50mmol/L, make it form precipitation, centrifugation precipitation, supernatant;
D. supernatant is transferred PH to 7.4, add-20 ℃ of precooled ethanol, make it produce precipitation, centrifugal collecting precipitation is IgG;
E. the c step being obtained precipitation is suspended in 0 ℃ of water, the acetic acid solution that adds 50mmol/L is transferred PH to 5.1, get supernatant after centrifugal, adjust ionic strength to 0.075～0.01, pH5.5, adding is chilled to-20 ℃ ethanol in advance then, makes it produce precipitation, and centrifugal back gained is precipitated as the mixture of IgA and IgM;
F. mixture is dissolved in the phosphate buffered saline buffer of 2mmol/L, pH6.0, dialysis is 3 times under 4 ℃ of conditions;
G. centrifugal, the gained precipitation is IgM, and supernatant is IgA.
The concrete steps of described purifying immunoglobulin IgG, IgA and IgM are as follows:
(2) IgG purification:
1. material
Albumin A-agar gel;
The CL4B damping fluid:
A.0.05M Tris/0.15M NaCl, pH8.6 includes 0.02% sodium azide;
B.0.05M phosphoric acid salt/0.15M NaCl, pH7.0;
C.0.05M Citrate trianion/0.15M NaCl, ph5.5;
D.0.05M acetate/0.15M NaCl, pH4.3;
E.0.05M glycine/0.15M NaCl, pH2.3;
2. working method
A. take by weighing albumin A-sepharose CL4B, the pillar bottom of packing into after expanding with buffer A is blocked with nylon wire group;
B. use rare sodium hydroxide adjustment, treat the antibody-solutions pH to 8.6 of affinity chromatography;
C. with chromatography column on 50 milligrams of antibody samples of content, treat that sample enters the post bed after, monitor elutriant with the albumen of buffer A elution of bound and with ultraviolet photometer;
D. use buffer B, C, D, the continuous wash-out of E, collect isolate and mixed immediately, dialyse again and concentrate, promptly get the IgG of purifying.
(2) purifying IgM
1. material
2mmol/l phosphate buffered saline buffer pH6.0; Tris damping fluid-TBS; Sepharose6B; Adorn post with TBS.
2. working method
A. the extract with IgM is added on the Sepharose6B post, uses the TBS wash-out under the room temperature, collects eluate, monitors absorbance value under the 280nm wavelength;
B. first peak is the IgM that contains pentamer, and a small peak is macromolecular condensation product subsequently.
(3) purifying IgA
1. material
Sephadex G200 is with 0.1M phosphate buffered saline buffer ApH6.8 balance dress post, 100 centimetres of column lengths, 3.2 centimetres of internal diameters; The 0.01M phosphate buffer soln B that contains 0.1MNaCl, pH7.5; The 0.01M phosphate buffer soln C that contains 0.25MNaCl, pH7.5; The DEAE-Mierocrystalline cellulose, 30 milliliters of wet deposition volume chromatography column dress posts.
2. working method
A. with the IgA extract through Sephadex G200 pillar buffer A wash-out, collect 1 pipe for per 10 milliliters, under the 280nm wavelength, monitor absorption value, after a spot of foreign matter, first main peaks is the IgA dimer;
B. the IgA that collects is dialysed with buffer B, and adorns post with same damping fluid equilibrated ion-exchanger,
C. with behind 60 milliliters of the buffer B wash-outs with damping fluid C150 milliliter wash-out, collect a pipe for per 5 milliliters, application of sample is used same buffer solution elution again; Under 280nm, monitor absorption value, collect IgA.
Include IgA, IgM and three kinds of immunoglobulin (Ig)s of IgG by the composite immune globulin that the present invention produced, can possess three kinds of immunologic functions of IgA, IgM and IgG simultaneously, good immune effect has solved the puzzlement problem of zoonosis toxicity disease to aquaculture; Remove three kinds of other albumen of the ultrawhite nothing of immune globulin in the product, be difficult for the phenomenon that absorbs after the injection that can avoid because ammonium sulfate etc. remains in the product to be caused.
Embodiment:
A. prepare immune serum
(1). select the susceptible isogenic animal for use, use a certain amount of immunogen immune, immune time is specifically formulated according to different animals, different immunogen;
(2). the separation of immune serum: the container that blood will be housed was placed on room temperature about 1 hour, and it is fully solidified, and put 4 ℃ then and spent the night;
(3). the clear and bright serum of sucking-off places in the bottle; If contain hemocyte or clot in the serum, with 3000g centrifugal 20 minutes, collect the clear and bright serum in top;
(4). immune serum is sub-packed in the appropriate containers be stored in-20 ℃ standby.
B. use immune serum separation and Extraction immunoglobulin IgG, IgA and IgM
(1). the water of serum with 3 times of volumes is mixed, transfer pH value to 7.7, put in-5 ℃～0 ℃ low temperature tank (aqueous ethanolic solution of interior Sheng 20%) or the ice-water bath, the temperature of solution is reduced to 0 ℃;
(2). under stirring fast, adding-20 ℃ of precooled ethanol to final concentrations is 20%, and remains in the ice bath, makes it produce precipitation, contains polytype immunoglobulin (Ig) in the precipitation, and 4000 change 30 minutes centrifugal collecting precipitations;
(3). precipitation is suspended from the physiological saline of 25 times of volumes, the acetic acid solution that adds 50mmol/L is transferred about PH to 5.1, makes it form precipitation, and with 4000 rev/mins rotating speed centrifugal 30 minutes, contain IgA and IgM in the precipitation, contain IgG in the supernatant;
(4). supernatant is transferred about PH to 7.4, and adding-20 ℃ of precooled ethanol to final concentrations is 25%, and remains in the water-bath, makes it produce precipitation, and centrifugal collecting precipitation is IgG;
(5). institute in (3) is obtained precipitation be suspended in 0 ℃ of water, add the acetic acid solution accent PH to 5.1 of 50mmol/L, get supernatant, adjustment ionic strength to 0.075～0.01, PH5.5 after centrifugal.Adding ethanol to the final concentration that is chilled to-20 ℃ in advance then is 10%, and remains in the ice bath, and centrifugal back gained precipitation is mainly IgA, IgM.
(6) mixture is dissolved in the phosphate buffered saline buffer pH6.0 of 2mmol/L, 4 ℃ of dialysis 3 times down.
(7) centrifugal, the gained precipitation is IgM, and IgA is then in supernatant.
C. purifying immunoglobulin IgG, purification IgA, IgM
(1) purifying of IgG: albumin A-agar gel affinity chromatography
1. material
Albumin A-agar gel CL4B (Protein A-Sepharose CL4B Sweden phamacia company product) damping fluid:
A. get 6.06 gram Tris and add 8.76 gram NaCL and be dissolved in 800 ml waters, transfer pH8.6 with HCl, add sodium azide liquid and water to 1000 milliliter, make 0.05M Tris/0.15M NaCL, pH8.6 includes the buffer A of 0.02% sodium azide.
B. get 2.17 gram Na
2HPO
4Add 1.35 gram NaH
2PO
4Add 8.76 gram NaCL and be dissolved in successively in 1000 ml waters, pH7.0 makes 0.05M phosphoric acid salt/0.15M NaCL, the buffer B of pH7.0.
C. get 2.68 gram citric acids and (containing a part water) add 10.96 gram trisodium citrates (containing two molecular waters) and add 8.76 gram NaCL and add 1000 milliliters in water, pH5.5 makes 0.05M Citrate trianion/0.15M NaCL, the damping fluid C. of ph5.5
D. get 6.8 gram sodium-acetates and add 8.76 gram NaCL and add 800 milliliters in water, add acetic acid and transfer the pH4.3 water to add to 1000 milliliters, make 0.05M acetate/0.15M NaCL, the damping fluid D of pH4.3
E. get 5.6 the gram glycine add 8.76 the gram NaCL add 800 milliliters in water, transfer pH2.3 with strong acid, add water to 1000 milliliters, make 0.05M glycine/0.15M NaCL, the damping fluid E. of pH2.3
2. working method
A. taking by weighing the pillar bottom of packing into after albumin A-sepharose CL4B expands with buffer A blocks with nylon wire group.
B. treat the antibody-solutions pH to 8.6 of affinity chromatography with rare sodium hydroxide adjustment.
C., chromatography column on the about 50 milligrams of antibody samples of content falls, treat that sample enters the post bed after, with the albumen (monitoring elutriant) of buffer A elution of bound with ultraviolet photometer.
D. use buffer B, C, D, the continuous wash-out of E,, the fraction that collect to be fit to is neutralized immediately, dialyses and concentrates.
E. the regeneration of post, D washes post with damping fluid, and then can finish the regeneration of post with buffer A balance pillar.
(2) purifying of IgM
1. material
2mM phosphate buffered saline buffer pH6.0; Tris damping fluid (TBS); Sepharose6B; Adorn post with TBS, column chromatography device.
2. working method
A. the extract with IgM is added on the Sepharose6B post, uses the TBS wash-out under the room temperature, collects eluate, monitors absorbance value under the 280nm wavelength;
B. first peak is the IgM that contains pentamer, and a small peak is macromolecular condensation product subsequently.
(3) IgA purifying
1. material
Sephadex G200, with 0.1M phosphate buffered saline buffer ApH6.8 balance dress post, 100 centimetres of column lengths, interior through 3.2 centimetres; 0.1MNaCl 0.01M phosphate buffer soln B, pH7.5; 0.25MNaCl 0.01M phosphate buffer soln C, pH7.5; The DEAE-Mierocrystalline cellulose, 30 milliliters of wet deposition volume chromatography column dress posts.
2. working method
A. with extracting solution through Sephadex G200 pillar with pH6.8 0.1M phosphate buffered saline buffer wash-out, collect 1 pipe for per 10 milliliters, under the 280nm wavelength, monitor absorption value, at a spot of foreign matter (cohesion albumen) afterwards, first main peaks is the IgA dimer;
B. the IgA that collects is dialysed with buffered soln A, and adorn post with same damping fluid equilibrated ion-exchanger, application of sample is used same buffer solution elution again;
C. during the about 60 milliliters of damping fluids of wash-out, then the absorption value of 280nm wavelength is returned baseline, and the damping fluid C150 milliliter wash-out of usefulness is collected a pipe, monitor absorption value under 280nm for per 5 milliliters then;
D. the free secretory component damping fluid that can be begun is washed off, and IgA is then gone out by the buffer solution elution of higher concentration salt.
Assay, work in-process inspection, inspection after construction
Protein content determination: ultraviolet absorption method, the formula below utilizing are calculated protein content unit and are mg/ml:Pr=(1.45*OD
280-0.74*OD
260)
D. three kinds of immunoglobulin IgGs, IgA, IgM are pressed 8～10: 1: 1～2 mixed;
E. use nano level aperture membrane filtration.
The work in-process calibrating: liquid preparation should be done physico-chemical examination and pyrogen test in Sterile Filtration with after removing virus, and samples and do sterility test, and the result meets the requirements.
Inspection after construction: the every batch of finished product of sampling is done comprehensively quality examination such as physical examination, chemical assay, protein content, heat stability test, sterility test, pyrogen test and proof test respectively, and the result meets the requirements.
Claims (3)
1. the production method of an animal composite immune globulin comprises the steps:
A. prepare immune serum, immune serum is sub-packed in the container be stored in-20 ℃ standby;
B. use immune serum separation and Extraction immunoglobulin IgG, IgA and IgM;
C. purifying immunoglobulin IgG, IgA and IgM;
D. three kinds of immunoglobulin IgGs, IgA, IgM are pressed 8～10: 1: 1～2 mixed;
E. use nano level aperture membrane filtration.
2. the production method of animal composite immune globulin according to claim 1 is characterized in that: described concrete steps with immune serum separation and Extraction immunoglobulin IgG, IgA and IgM are as follows:
A. the water of immune serum with 3 times of volumes is mixed, transfer pH value to 7.7, the temperature of solution is reduced to 0 ℃;
B. under stirring fast, add-20 ℃ of precooled ethanol, make it produce precipitation, centrifugal collecting precipitation;
C. precipitation is suspended from the physiological saline of 25 times of volumes, adds the acetic acid solution of 50mmol/L, make it form precipitation, centrifugation precipitation, supernatant;
D. supernatant is transferred PH to 7.4, add-20 ℃ of precooled ethanol, make it produce precipitation, centrifugal collecting precipitation is IgG;
E. the c step being obtained precipitation is suspended in 0 ℃ of water, the acetic acid solution that adds 50mmol/L is transferred PH to 5.1, get supernatant after centrifugal, adjust ionic strength to 0.075～0.01, pH5.5, adding is chilled to-20 ℃ ethanol in advance then, makes it produce precipitation, and centrifugal back gained is precipitated as the mixture of IgA and IgM;
F. mixture is dissolved in the phosphate buffered saline buffer of 2mmol/L, pH6.0, dialysis is 3 times under 4 ℃ of conditions;
G. centrifugal, the gained precipitation is IgM, and supernatant is IgA.
3. the production method of animal composite immune globulin according to claim 2, the concrete steps of described purifying immunoglobulin IgG, IgA and IgM are as follows:
(1) IgG purification:
1. material
Albumin A-agar gel;
The CL4B damping fluid:
A.0.05M Tris/0.15M NaCl, pH8.6 includes 0.02% sodium azide;
B.0.05M phosphoric acid salt/0.15M NaCl, pH7.0;
C.0.05M Citrate trianion/0.15M NaCl, ph5.5;
D.0.05M acetate/0.15M NaCl, pH4.3;
E.0.05M glycine/0.15M NaCl, pH2.3;
2. working method
A. take by weighing albumin A-sepharose CL4B, the pillar bottom of packing into after expanding with buffer A is blocked with nylon wire group;
B. use rare sodium hydroxide adjustment, treat the antibody-solutions pH to 8.6 of affinity chromatography;
C. with chromatography column on 50 milligrams of antibody samples of content, treat that sample enters the post bed after, monitor elutriant with the albumen of buffer A elution of bound and with ultraviolet photometer;
D. use buffer B, C, D, the continuous wash-out of E, collect isolate and mixed immediately, dialyse again and concentrate, promptly get the IgG of purifying.
(2) purifying IgM
1. material
2mmol/l phosphate buffered saline buffer pH6.0; Tris damping fluid-TBS; Sepharose6B; Adorn post with TBS.
2. working method
A. the extract with IgM is added on the Sepharose6B post, uses the TBS wash-out under the room temperature, collects eluate, monitors absorbance value under the 280nm wavelength;
B. first peak is the IgM that contains pentamer, and a small peak is macromolecular condensation product subsequently.
(3) purifying IgA
1. material
Sephadex G200 is with 0.1M phosphate buffered saline buffer ApH6.8 balance dress post, 100 centimetres of column lengths, 3.2 centimetres of internal diameters; The 0.01M phosphate buffer soln B that contains 0.1MNaCl, pH7.5; The 0.01M phosphate buffer soln C that contains 0.25MNaCl, pH7.5; The DEAE-Mierocrystalline cellulose, 30 milliliters of wet deposition volume chromatography column dress posts.
2. working method
A. with the IgA extract through Sephadex G200 pillar buffer A wash-out, collect 1 pipe for per 10 milliliters, under the 280nm wavelength, monitor absorption value, after a spot of foreign matter, first main peaks is the IgA dimer;
B. the IgA that collects is dialysed with buffer B, and adorn post with same damping fluid equilibrated ion-exchanger, application of sample is used same buffer solution elution again;
C. with behind 60 milliliters of the buffer B wash-outs with damping fluid C150 milliliter wash-out, collect a pipe for per 5 milliliters, under 280nm, monitor absorption value, collect IgA.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100472018A CN100339386C (en) | 2005-09-12 | 2005-09-12 | Animal composite immune globulin production method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100472018A CN100339386C (en) | 2005-09-12 | 2005-09-12 | Animal composite immune globulin production method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1763096A true CN1763096A (en) | 2006-04-26 |
| CN100339386C CN100339386C (en) | 2007-09-26 |
Family
ID=36747435
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103197053A (en) * | 2013-03-15 | 2013-07-10 | 上海市血液中心 | Anti-IgA antibody detection kit |
| CN105126100A (en) * | 2015-09-23 | 2015-12-09 | 成都蓉生药业有限责任公司 | IgM-rich human immunoglobulin preparation and preparation method thereof |
| CN105950576A (en) * | 2016-05-26 | 2016-09-21 | 成都远睿生物技术有限公司 | Method for extracting multiple proteins from bovine blood |
| CN111166879A (en) * | 2019-12-04 | 2020-05-19 | 昆明白马制药有限公司 | Pig immunoglobulin oral liquid |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6955917B2 (en) * | 1997-06-20 | 2005-10-18 | Bayer Healthcare Llc | Chromatographic method for high yield purification and viral inactivation of antibodies |
| US5886154A (en) * | 1997-06-20 | 1999-03-23 | Lebing; Wytold R. | Chromatographic method for high yield purification and viral inactivation of antibodies |
-
2005
- 2005-09-12 CN CNB2005100472018A patent/CN100339386C/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103197053A (en) * | 2013-03-15 | 2013-07-10 | 上海市血液中心 | Anti-IgA antibody detection kit |
| CN103197053B (en) * | 2013-03-15 | 2015-02-18 | 上海市血液中心 | Anti-IgA antibody detection kit |
| CN105126100A (en) * | 2015-09-23 | 2015-12-09 | 成都蓉生药业有限责任公司 | IgM-rich human immunoglobulin preparation and preparation method thereof |
| CN105126100B (en) * | 2015-09-23 | 2021-01-26 | 成都蓉生药业有限责任公司 | IgM-rich human immunoglobulin preparation and preparation method thereof |
| CN105950576A (en) * | 2016-05-26 | 2016-09-21 | 成都远睿生物技术有限公司 | Method for extracting multiple proteins from bovine blood |
| CN111166879A (en) * | 2019-12-04 | 2020-05-19 | 昆明白马制药有限公司 | Pig immunoglobulin oral liquid |
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| Publication number | Publication date |
|---|---|
| CN100339386C (en) | 2007-09-26 |
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Effective date of registration: 20200603 Address after: 116000 No.9 Yingxu Road, Ganjingzi District, Dalian City, Liaoning Province Co-patentee after: JIANGSU SANYI BIOENGINEERING Co.,Ltd. Patentee after: DALIAN SANYI ANIMAL DRUG Co.,Ltd. Co-patentee after: SHANDONG HEZE SANYI BIO-ENGINEERING Co.,Ltd. Address before: 116036, No. 9, Xu Xu Road, Dalian hi tech park, Ganjingzi District, Liaoning Patentee before: DALIAN SANYI ANIMAL DRUG Co.,Ltd. |
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