CN101633920A - Preparation method of monoclonal antibody of resisting treeshrew IgG - Google Patents
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Abstract
The invention relates to a preparation method of a monoclonal antibody of resisting treeshrew IgG, which specifically relates to the field of a monoclonal antibody and separation and purification of treeshrew IgG protein and belongs to the technical field of biology. The preparation method comprises the preparation steps: separating and purifying treeshrew IgG; immunizing a BALB/C rat; combining hybridoma; screening the hybridoma; cloning the hybridoma; preparing ascites of the monoclonal antibody; detecting the valence of the ascites of the monoclonal antibody; and preparing the monoclonal antibody.
Description
Technical field:
The present invention relates to the preparation method of monoclonal antibody of resisting treeshrew IgG, particularly, relate to the monoclonal antibody field, relate to the proteic isolation and purification of IgG of tree shrew, belong to biological technical field.
Background technology:
Tree shrew (Tupaia belangeri, tree shrew) is a kind of small-sized mammalian that is similar to squirrel, it is small, growth and breeding is fast, be easy to catch, raise and train and breed, cost is low, its metabolism and gross anatomy are than the more approaching mankind of rodent, along with the minimizing gradually of domestic and international non-human primates resource and the development trend of laboratory animal miniaturization, tree shrew is as the possible animal model of the human relative disease of research, be subjected to extensive concern, now be applied to virus, neural, psychology, diabetes, cerebral ischemia, blood vessel, tumour, the research of aspects such as parasite.At present confirmed that tree shrew can be comprised hsv, rotavirus, hepatitis A virus (HAV), hepatitis B virus etc. by the multiple virus infection relevant with human diseases.
Existing Many researchers has been done a lot of work the checking tree shrew aspect the hepatitis C animal model, the tree shrew primary hepatocyte is external can be infected by HCV and support virus replication, but also HCV infection of live body tree shrew.Hepatitis C virus acceptor research aspect, sequential analysis show the I of B family type scavenger receptor (SR-BI) of tree shrew and the human homology that height is arranged.Therefore tree shrew might replace chimpanzee to become the cheapness of hepatitis C research, the small animal model that is easy to get, thereby accelerates the exploitation of HCV medicine and the research of vaccine.But because the present shortage of domestic and international two anti-and tree shrew antibody detection methods, seriously limited with tree shrew as the correlated virus of object learn, immunologic research.
Summary of the invention:
The object of the present invention is to provide the method for IgG of tree shrew isolation and purification, for the preparation monoclonal antibody of resisting treeshrew IgG is prepared antigen, the isolation and purification that comprises IgG of tree shrew, improvement to traditional monoclonal antibody technique, preparation and screening resisting treeshrew IgG monoclonal hybridoma, the preparation of monoclonal antibody of resisting treeshrew IgG etc., overcome in the prior art two anti-be the shortage of monoclonal antibody of resisting treeshrew IgG.Also provide the preparation method of the hybridoma cell strain of monoclonal antibody of resisting treeshrew IgG, to solve the technological deficiency that lacks the hybridoma cell strain of monoclonal antibody of resisting treeshrew IgG in the prior art.
Above-mentioned purpose of the present invention is to be achieved by following technical scheme:
The preparation method of monoclonal antibody of resisting treeshrew IgG comprises that isolation and purification, immune balb/c mice, fusion hybridoma, screening hybridoma, hybridoma cell cloneization, preparation odd contradictive hydroperitoneum, the detection odd contradictive hydroperitoneum of IgG of tree shrew tired, the preparation process of monoclonal antibody.
The isolation and purification step of IgG of tree shrew is to get tree shrew serum, separate with sad-ammonium sulfate precipitation method, with Sephacry-S200 gel-filtration chromatography and DEAE-Sephadex A-50 ion exchange chromatography purifying, with SDS-PAGE electrophoresis detection separation and purification result.
The immune balb/c mice step is that IgG of tree shrew is mixed with the equivalent Freund's complete adjuvant, whirlpool concussion emulsification half an hour, the complete collare back and belly of emulsification thigh ditch subcutaneous injection 6-8 BALB/C mice in age in week, exempt from carrying out two with method after IgG of tree shrew and the emulsification of equivalent Freund's incomplete adjuvant after two weeks, two exempt from two weeks afterwards to carry out three with IgG of tree shrew and the injection of equivalent Freund's incomplete adjuvant emulsification pneumoretroperitoneum exempts from, carry out after two weeks again exempting from the same four the exempting from of step with three, four exempt to recall stimulation after two weeks, 3-4 extracting spleen cell after week merges with myeloma cell SP2/0.
Screening hybridoma step is to cultivate with HAT substratum selectivity, detects positive hybridoma cell with indirect elisa method.
The hybridoma cell clone step is to adopt limiting dilution assay to carry out cloning, detect by screening hybridoma step again, the positive colony cell clone again and in liquid nitrogen frozen part, repeat 4 time cloningizations, positive up to 100%.
Preparation odd contradictive hydroperitoneum step is the BALB/C mice of handling through whiteruss, the positive hybridoma cell 1 * 10 that the intraperitoneal injection purifying is good
6Individual, through an about week, treat that mouse web portion expands the back and extracts ascites, it is standby to get supernatant after centrifugal.
The preparation process of monoclonal antibody is with salting-out process odd contradictive hydroperitoneum to be carried out preliminary purification, monoclonal antibody is made being further purified of chromatography; SDS-PAGE detects purity, and the content of antibody detects antibody titer with determined by ultraviolet spectrophotometry commonly used with indirect ELISA.
The technical scheme that the present invention is concrete:
The isolation and purification of IgG of tree shrew is to adopt sad-ammonium sulfate precipitation method that tree shrew serum is carried out initial gross separation earlier, carry out purifying with Sephacry-S200 gel-filtration chromatography and DEAE-Sephadex A-50 ion exchange chromatography then, with the isolation and purification result of SDS-PAGE electrophoresis detection IgG of tree shrew.The IgG of tree shrew of purifying is mixed with the equivalent Freund's complete adjuvant, whirlpool concussion emulsification half an hour, the complete collare back and belly of emulsification thigh ditch subcutaneous injection 6-8 BALB/C mice in age in week, exempt from carrying out two with method after IgG of tree shrew and the emulsification of equivalent Freund's incomplete adjuvant after two weeks, two exempt from two weeks afterwards to carry out three with IgG of tree shrew and the injection of equivalent Freund's incomplete adjuvant emulsification pneumoretroperitoneum exempts from, carry out four again after two weeks and exempt from (exempting from) with three, four exempt to recall stimulation after two weeks, 3-4 after week extracting spleen cell and the SP2/0 cell that is in logarithmic phase merge with 50% PEG1500.Cultivate with HAT substratum selectivity, detect positive hybridoma cell with indirect elisa method.Clone positive hybridoma cell with limiting dilution assay.The cell inoculation that purifying is good is gone into the BALB/C mice abdominal cavity and is produced ascites collection ascites, and indirect ELISA detects ascites and tires.With salting-out process odd contradictive hydroperitoneum is carried out preliminary purification, monoclonal antibody is made being further purified of chromatography.SDS-PAGE detects purity, and the content with determined by ultraviolet spectrophotometry antibody commonly used detects antibody titer with indirect ELISA.
Positively effect of the present invention is: having prepared highly purified IgG of tree shrew is antigen, prepared the hybridoma cell strain that produces monoclonal antibody of resisting treeshrew IgG, prepared monoclonal antibody of resisting treeshrew IgG, for the tree shrew immunology detection provides antibody, for tree shrew is laid a good foundation as the research of aspects such as correlated virus of object, immunology.
Description of drawings:
Fig. 1 be tree shrew serum sad-ammonium sulfate extraction thing Sephacryl-S200 column chromatography figure;
Fig. 2 is DEAE-Sephadex A-50 anion-exchange chromatography figure;
Fig. 3 is each step purified product SDS-PAGE figure;
(14.4kD～94kD) 2: sad-ammonium sulfate precipitation dialysis back 3: cross behind the S200 post the second peak 4:S200, second peak and cross first peak 5 behind the A50 ion column: the tree shrew whole serum for 1:Marker;
Fig. 4 is protein standard relative mobility and logarithm molecular weight standard curve;
The positive hybridoma TM1 figure of Fig. 5;
Fig. 6 is monoclonal antibody subgroup identification indirect ELISA reaction result figure.
Embodiment:
Below in conjunction with accompanying drawing, further specify essentiality content of the present invention with embodiments of the invention, but do not limit the present invention with this.
Embodiment 1:
The isolation and purification of IgG of tree shrew:
The isolation and purification of IgG of tree shrew, tree shrew serum adopts sad-ammonium sulfate precipitation method to carry out initial gross separation earlier, carry out purifying with Sephacry-S200 gel-filtration chromatography and DEAE-Sephadex A-50 ion exchange chromatography, with SDS-PAGE electrophoresis detection separation and purification result.
(1) preparation of tree shrew serum:
Adopt disconnected neck blood taking method, blood is room temperature or 37 ℃ of following placements 1 hour, then 4 ℃ of refrigerator overnight.Collect supernatant, 12000rpm, centrifugal 10min.The serum that obtains is frozen immediately in cryogenic refrigerator, and is standby.
(2) isolation and purification of IgG of tree shrew:
1. sad-ammonium sulfate precipitation method:
Get 1 part of serum and add 4 parts of 0.06mol/l pH 4.8 acetate buffer solutions dilutions, add sad (25 μ l/ml), the following edged of room temperature stirs, and continues 30min; The centrifugal 20min of 10000rpm gets supernatant, adds isopyknic PBS, transfers pH to 7.4 with 1M NaOH; (0.277g solid ammonium sulfate/ml) make its final concentration be about 45%, 4 ℃ of stirring 30min down leaves standstill 1～2h, precipitation occurs to add ammonium sulfate again; With centrifugal 10000rpm, 30min.Abandon supernatant, throw out is dissolved in PBS, 4 ℃ of dialysed overnight, during change liquid 3～4 times.With above-mentioned protein crude extract, vacuum lyophilization ,-70 ℃ are frozen standby.
2.Sephacryl-S200 gel permeation chromatography:
Tree shrew serum through the extract behind sad-ammonium sulfate precipitation with column volume 1% on sample, behind the Sephacryl-S200 column chromatography, 2 albumen elution peak F1 appear, F2 sees Fig. 1.Electrophoresis detection shows that first peak F1 is the foreign protein peak, and the second peak F2 is target protein peak (IgG).Collect the second peak albumen and carry out the SDS-PAGE analysis, the result shows that relative molecular mass is all removed greater than the foreign protein of IgG, but still has some small molecular weights or the foreign protein suitable with the target protein molecular weight not to remove.
3.DEAE-Sephadex A-50 ion exchange chromatography:
The second protein peak F with the Sephacryl-S200 post
2Cross the A-50 ion column.With Tris-HCl (0.05M, pH8.0) balance pillar, cross 2 column volumes earlier, albumen and ion-exchanger are fully adsorbed, and then with damping fluid Tris-HCl (0.05mol/1, pH8.0) and contain 3 column volumes of same buffer linear elution of 1MNaCl, collect before the wash-out respectively and the liquid behind the wash-out, detect its absorbancy under 280nm.The result shows, does not contain albumen in the collection liquid before the linear elution, and Fig. 2 is the tomographic map behind the wash-out, and first peak F1 is a target protein.
4. the preservation of sample:
The albumen that will obtain through twice chromatographic adds polyoxyethylene glycol (molecular weight 6000) under 4 ℃ of the dialysis tubings of packing into, concentrates the back and measures protein content, concentrating sample is divided into small packages 0.02-0.05ml ,-20 ℃ of preservations.
(3) purity of protein is measured:
In sepn process, isolating intermediate product and end product-IgG are kept sample respectively, the discontinuous SDS-polyacrylamide gel electrophoresis with 15% detects, to judge its composition and purity.
Cross the protein peak F of Sephacryl-S200 post gained through the protein crude extract administration of saltouing
2Electrophoretic band mainly contain 6, the 1st and the 2nd has three between 45～35kD between 45～66.2kD, the 6th near 24kD, what protein concentration was higher is the 2nd band and the 6th band, is the target protein that we want.Electrophorogram (Fig. 3) shows that albumen has obtained purifying really.We concentrated the DEAE-SephadexA-50 post once more with albumen again on this basis, and as can be seen from the figure the purity of target protein is higher, rarely seen heavy chain immunoglobulin, light chain place two bands among the figure.
(4) mensuration of molecular weight:
According to serum immune globulin SDS-PAGE electrophoretogram behind Sephacryl-S200 and DEAE-Sephadex A-50 anion-exchange chromatography purifying, measure the distance of each protein band migration.Migration distance with tetrabromophenol sulfonphthalein is a standard 1, calculates relative migration distance Rf.Calculate the logarithm of the molecular weight of protein standard, and make typical curve equation (see Table 1 and table 2).
Because the tree shrew serum IgG is under SDS and 2 mercapto ethanol effect, covalent disulfide bonds is reduced in the molecule, is dissociated into the subunit form, and monomeric IgG is disassembled is H, L chain subunit.Rarely seen molecular weight is about two protein bands of 53kD and 28kD in the electrophorogram (Fig. 3), represents heavy chain (H chain) and the light chain (L chain) of IgG respectively, shows that the tree shrew serum IgG purity that obtains by purifying is higher.Because the IgG molecule all is made up of one four chain unit, is respectively two identical heavy chains and light chain, the molecular weight of deducibility tree shrew serum IgG is about 160kD thus.
Table 1 protein standard migration distance and relative mobility and logarithm molecular weight
Table 2 testing protein migration distance and relative mobility
The typical curve equation is:
y＝-0.9936x+5.1972 R
2＝0.9841
Y is the logarithm (logM of molecular weight
W); X is relative mobility (Rf)
Heavy chain x
1=0.471 substitution Equation for Calculating gets y
1=4.729
Light chain x
2=0.747 substitution Equation for Calculating gets y
2=4.545
Molecular weight M
W=10
y
Heavy chain: 53.5kd light chain: 28.4kD
IgG
MW＝(53.5+28.4)×2≈160kD
Embodiment 2:
The foundation of resisting treeshrew IgG hybridoma cell strain and MONOCLONAL ANTIBODIES SPECIFIC FOR thereof:
(1) immune animal:
Four immune balb/c mices of IgG of tree shrew with separation and purification, immunity for the first time mixes IgG of tree shrew with the equivalent Freund's complete adjuvant, whirlpool concussion emulsification half an hour, the complete collare back and belly of emulsification thigh ditch subcutaneous injection BALB/C mice in 6～8 age in week, exempt from carrying out two with method after IgG of tree shrew and the emulsification of equivalent Freund's incomplete adjuvant after two weeks, two exempt from two weeks afterwards to carry out three with IgG of tree shrew and the injection of equivalent Freund's incomplete adjuvant emulsification pneumoretroperitoneum exempts from, carry out four again after two weeks and exempt from (exempting from) with three, one to four injection volume of exempting from only is 100 μ g/, injection 0.3～0.4ml.Four exempt to recall stimulation after two weeks, and tail vein injection IgG of tree shrew 50 μ g/ only.Indirect ELISA method detects the serum titer after the immunity.
(2) preparation of hybridoma cell strain and screening:
Fetching to recall stimulates 3～4 days mouse spleen cell and SP2/0 myeloma cell to merge with 50% PEG1500 according to a conventional method.Cultivate with the RPMI-1640 that contains 1 * HAT and 10%FBS.Merge and begin to occur fused cell after three days, 158 are the fusion hole in 480 holes, and fusion rate is 32.9%.Detect monoclonal antibody of resisting treeshrew IgG with indirect elisa method, detect 110 holes, wherein positive hole is 2, and positive rate is 1.8%.Through four limited dilution clonings, obtain the cell strain of a strain stably excreting monoclonal antibody of resisting treeshrew IgG at last, be labeled as TM1.
(3) preparation of ascites and tiring and the evaluation of antibody subclass:
With the positive hybridoma cell strain with 1 * 10
6The female BALB/C mice of an individual/abdominal injection about presensitized 10 ages in week, after 7～10 days, gather mouse ascites, detect ascites with indirect elisa method and tire, do contrast with myeloma cell line SP2/0 ascites, bag is 10 μ g/ml by the antigenic concentration of IgG of tree shrew.
The result: SP2/0 ascites and antigen are reactionless, and it is 1.28 * 10 that monoclonal antibody is tired
6According to the reaction of monoclonal antibody subgroup identification test kit explanation carrying out indirect ELISA, result (Fig. 6) shows that excretory antibody belongs to the IgG1 subclass.
Embodiment 3:
Purification of Monoclonal Antibodies:
1. salting-out process carries out the preliminary purification of antibody:
1 part of ascites and be put on the mixture of ice and water with the abundant mixing of volume physiological saline is put in and drips 2 part of 100% saturated ammonium sulphate on the magnetic stirring apparatus again, and limit edged concussion mixing is final concentration 50% saturated ammonium sulphate and precipitates, and 4 ℃ are spent the night.High speed centrifugation 10000rpm, 4 ℃, 15min.Abandon supernatant and floating matter, and precipitation is dissolved in the physiological saline of 2 times of ascites original volume amounts.Adding simultaneously the long-pending saturated ammonium sulphate of monoploid again is final concentration 33%, and operation steps is with 50% saturated ammonium sulphate, precipitation then 4 ℃ spend the night high speed centrifugation 10000rpm, 4 ℃, 15min.Abandon supernatant and floating matter, and precipitation is dissolved in the physiological saline of ascites original volume amount.The dialysis tubing of packing into and anticipating places dialysis buffer liquid 0.01M, pH7.4 PBS, among about 1000ml, and 4 ℃, to stir, ammonium sulfate is removed in dialysis.Change liquid 3-4 every day, was generally 3 days, monitors NH with nessler reagent
4 +Separate out situation, when no ammonium ion is separated out, stop dialysis.
2. chromatography is further purified:
Monoclonal antibody through preliminary purification is further purified with Q sepharose Fast Flow displacement chromatography technology again.Level pad is the Tris-HCl damping fluid of 20mM pH7.5, chromatography column XK16/20, Pharmacia AKTA FPLC chromatographic system.Adorn post to specifications, level pad balance chromatography column, flow velocity 5mL/min.Level pad with 3 times of volumes behind the last sample carries out wash-out, and with the linear gradient elution bonded antibody protein that NaCl increases progressively, linear gradient is 0-1.0MNaCl, 10 times of column volumes.A
280Detect the albumen of collecting wash-out.SDS-PAGE detects purity, and the content with determined by ultraviolet spectrophotometry antibody commonly used detects antibody titer with indirect ELISA.
Claims (8)
1, the preparation method of monoclonal antibody of resisting treeshrew IgG comprises that isolation and purification, immune balb/c mice, fusion hybridoma, screening hybridoma, hybridoma cell cloneization, preparation odd contradictive hydroperitoneum, the detection odd contradictive hydroperitoneum of IgG of tree shrew tired, the preparation process of monoclonal antibody.
2. preparation method according to claim 1, it is characterized in that: the isolation and purification step of IgG of tree shrew is to get tree shrew serum, separate with sad-ammonium sulfate precipitation method, with Sephacry-S200 gel-filtration chromatography and DEAE-Sephadex A-50 ion exchange chromatography purifying, with SDS-PAGE electrophoresis detection separation and purification result.
3. preparation method according to claim 1, it is characterized in that: the immune balb/c mice step is that IgG of tree shrew is mixed with the equivalent Freund's complete adjuvant, whirlpool concussion emulsification half an hour, the complete collare back and belly of emulsification thigh ditch subcutaneous injection 6-8 BALB/C mice in age in week, exempt from carrying out two with method after IgG of tree shrew and the emulsification of equivalent Freund's incomplete adjuvant after two weeks, two exempt from two weeks afterwards to carry out three with IgG of tree shrew and the injection of equivalent Freund's incomplete adjuvant emulsification pneumoretroperitoneum exempts from, carry out after two weeks again exempting from the same four the exempting from of step with three, four exempt to recall stimulation after two weeks, 3-4 extracting spleen cell after week merges with myeloma cell SP2/0.
4. preparation method according to claim 1 is characterized in that: screening hybridoma step is to cultivate with HAT substratum selectivity, detects positive hybridoma cell with indirect elisa method.
5. preparation method according to claim 1, it is characterized in that: the hybridoma cell clone step is to adopt limiting dilution assay to carry out cloning, detect by screening hybridoma step again, the positive colony cell is cloned and frozen part in liquid nitrogen again, repeat 4 time cloningizations, positive up to 100%.
6. preparation method according to claim 1 is characterized in that: preparation odd contradictive hydroperitoneum step is the BALB/C mice of handling through whiteruss, the positive hybridoma cell 1 * 10 that the intraperitoneal injection purifying is good
6Individual, through an about week, treat that mouse web portion expands the back and extracts ascites, it is standby to get supernatant after centrifugal.
7. preparation method according to claim 1 is characterized in that: the preparation process of monoclonal antibody is with salting-out process odd contradictive hydroperitoneum to be carried out preliminary purification, monoclonal antibody is made being further purified of chromatography; SDS-PAGE detects purity, and the content of antibody detects antibody titer with determined by ultraviolet spectrophotometry commonly used with indirect ELISA.
8, preparation method according to claim 1, the isolation and purification step that it is characterized in that IgG of tree shrew is to get tree shrew serum, separate with sad-ammonium sulfate precipitation method, with Sephacry-S200 gel-filtration chromatography and DEAE-Sephadex A-50 ion exchange chromatography purifying, with SDS-PAGE electrophoresis detection separation and purification result; The immune balb/c mice step is that IgG of tree shrew is mixed with the equivalent Freund's complete adjuvant, whirlpool concussion emulsification half an hour, the complete collare back and belly of emulsification thigh ditch subcutaneous injection 6-8 BALB/C mice in age in week, exempt from carrying out two with method after IgG of tree shrew and the emulsification of equivalent Freund's incomplete adjuvant after two weeks, two exempt from two weeks afterwards to carry out three with IgG of tree shrew and the injection of equivalent Freund's incomplete adjuvant emulsification pneumoretroperitoneum exempts from, carry out after two weeks again exempting from the same four the exempting from of step with three, four exempt to recall stimulation after two weeks, 3-4 extracting spleen cell after week merges with myeloma cell SP2/0; Screening hybridoma step is to cultivate with HAT substratum selectivity, detects positive hybridoma cell with indirect elisa method; The hybridoma cell clone step is to adopt limiting dilution assay to carry out cloning, detect by screening hybridoma step again, the positive colony cell clone again and in liquid nitrogen frozen part, repeat 4 time cloningizations, positive up to 100%; Preparation odd contradictive hydroperitoneum step is the BALB/C mice of handling through whiteruss, the positive hybridoma cell 1 * 10 that the intraperitoneal injection purifying is good
6Individual, through an about week, treat that mouse web portion expands the back and extracts ascites, it is standby to get supernatant after centrifugal; The preparation process of monoclonal antibody is with salting-out process odd contradictive hydroperitoneum to be carried out preliminary purification, monoclonal antibody is made being further purified of chromatography; SDS-PAGE detects purity, and the content of antibody detects antibody titer with determined by ultraviolet spectrophotometry commonly used with indirect ELISA.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101923093A (en) * | 2010-07-13 | 2010-12-22 | 昆明理工大学 | Tri-antibody sandwich ELISA detection method of IgG of tree shrew |
| CN108753734A (en) * | 2018-05-23 | 2018-11-06 | 中国医学科学院医学生物学研究所 | Anti-treeing Shrew CD8 molecule monoclonal antibodies and hybridoma cell strain and the application for secreting the antibody |
| CN110256554A (en) * | 2019-06-27 | 2019-09-20 | 贵州盛世康生物科技有限公司 | The preparation method of immunoglobulin subclass antibodies |
-
2009
- 2009-08-24 CN CN200910094864A patent/CN101633920A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101923093A (en) * | 2010-07-13 | 2010-12-22 | 昆明理工大学 | Tri-antibody sandwich ELISA detection method of IgG of tree shrew |
| CN101923093B (en) * | 2010-07-13 | 2013-12-18 | 昆明理工大学 | Tri-antibody sandwich ELISA detection method of IgG of tree shrew |
| CN108753734A (en) * | 2018-05-23 | 2018-11-06 | 中国医学科学院医学生物学研究所 | Anti-treeing Shrew CD8 molecule monoclonal antibodies and hybridoma cell strain and the application for secreting the antibody |
| CN110256554A (en) * | 2019-06-27 | 2019-09-20 | 贵州盛世康生物科技有限公司 | The preparation method of immunoglobulin subclass antibodies |
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