CN101508976B - Culture medium for preparing hybridoma cell clone of monoclone antibody and preparation method - Google Patents
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- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 description 1
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Abstract
本发明提供了一种制备单克隆抗体的杂交瘤细胞克隆用培养基及其制备方法，按常规方法配制RPMI 1640完全培养基，然后分别与一定比例的BHK-21、CRFK、Hela等细胞的无细胞培养上清混合，完全培养基与每种细胞上清的体积比为(7～1)∶1。本发明的优点在于：使用本发明所提供的杂交瘤细胞克隆用培养基，可完全省略在常规杂交瘤细胞克隆过程中以BALB/c小鼠腹腔巨噬细胞作为饲养细胞的环节，大大简化了克隆操作，并使被克隆细胞的生长速度明显提高。The invention provides a hybridoma cell cloning culture medium for preparing monoclonal antibodies and a preparation method thereof. The RPMI 1640 complete medium is prepared according to a conventional method, and then mixed with a certain proportion of BHK-21, CRFK, Hela and other cells without The cell culture supernatants were mixed, and the volume ratio of the complete medium to each cell supernatant was (7-1):1. The advantages of the present invention are: using the medium for hybridoma cell cloning provided by the present invention can completely omit the link of using BALB/c mouse peritoneal macrophages as feeder cells in the conventional hybridoma cell cloning process, which greatly simplifies Cloning operation, and the growth rate of the cloned cells is significantly increased.
Description
Technical field
The invention belongs to biological technical field, especially provide a kind of friendship oncocyte for preparing monoclonal antibody to clone with substratum and preparation method.
Background technology
Monoclonal antibody is used widely in the affinity purification and the oncotherapy field of immunology detection, target protein with the specificity and the sensitivity of its height.In the Monoclonal Antibody process, the positive hybridoma cell hole is used 1640 perfect mediums and adopted limiting dilution assay to clone, clone cell poor growth under this condition at present.For promoting the fast breeding of individual cells, before the clone, at first prepare the peritoneal macrophage suspension of normal BALB/c usually, then with 2 * 10
4～2 * 10
5Cells/well is added to be waited to clone in the plate.This process need prepares before cell clone, and the peritoneal macrophage quantity variance of different operating person preparation is bigger, and is subjected to the animal individual differentia influence, is unfavorable for the stdn of the process of cloning, and has also increased the probability that pollutes simultaneously.
Summary of the invention
The invention provides a kind of hybridoma cell clone substratum for preparing monoclonal antibody, overcome the complicated processes of conventional hybridization oncocyte clone process of preparing peritoneal macrophage.
The present invention also provides a kind of preparation method who prepares the hybridoma cell clone of monoclonal antibody with substratum, is applicable to the production of monoclonal antibody.
The hybridoma cell clone substratum of the monoclonal antibody of the present invention preparation, mainly by following raw material by volume portion rate form:
7～1 parts of 1640 perfect mediums, 1 part of passage cell supernatant;
Described passage cell supernatant is the cell log growth nutrient solution in early stage, by the acellular supernatant liquor of centrifugal acquisition.Passage cell is selected from: a kind of in BHK-21, CRFK, the Hela continuous cell line.
The preparation technology of substratum of the present invention may further comprise the steps:
1) preparation of 1640 serum free mediums
With RPMI 1640, Hepes, NaHCO
3Use deionized water dissolving, adjust pH to 7.0 back constant volume to 1000 milliliter, 0.22 μ m membrane filtration degerming, aseptic subpackaged;
2) two anti-preparations
Preparation contains the two anti-solution of penicillin 1,000,000 units, Streptomycin sulphate 1,000,000 units;
3) preparation of 1640 perfect mediums
Get 1640 serum free mediums of 79 milliliters of steps 1, add 20 milliliters of foetal calf serums, 1 milliliter of two anti-solution;
4) passage cell culture medium preparation
Get 1640 serum free mediums of 89 milliliters of steps 1, add 10 milliliters of foetal calf serums, 1 milliliter of two anti-solution;
5) collection of the cultivation of passage cell and supernatant
The passage cell supernatant is the cell log nutrient solution in growth early stage, and by the acellular supernatant liquor of centrifugal acquisition, passage cell is selected from: a kind of in BHK-21, CRFK, the Hela continuous cell line;
6) the hybridoma cell clone substratum of preparation monoclonal antibody
With the passage cell supernatant of 1640 perfect mediums of step 3 preparation and step 5 (7～1) by volume: 1 mixes, and the pH value of mixed culture medium is transferred to 7.2, promptly obtain the hybridoma cell clone substratum of preparation monoclonal antibody of the present invention, respectively called after BHK-1640, CRFK-1640, Hela-1640.
The test example
Use the prepared substratum of the present invention and respectively SP2/0 cell and anti-rabies virus monoclonal antibody secretory cell are cloned, and compare with conventional cloning process.
Operation steps is as follows:
1.BALB/c the preparation of peritoneal macrophage suspension: extract healthy BALB/c mouse one branch hole ball, treat that agonal stage that blood will flow to end draws neck to put to death, be soaked in 15min in 75% the ethanol solution, in Bechtop, prepare peritoneal macrophage suspension then according to a conventional method with complete 1640 substratum dilution, and with 2 * 10
5The density of/ml joins in 96 well culture plates, every hole 100 μ l.
2. substratum of the present invention is added to Tissue Culture Plate: BHK21, CRFK, the isocellular acellular culture supernatant of Hela are mixed prepared hybridoma cell clone substratum with complete 1640 substratum (by volume) at 1: 3, amount with every hole 100 μ l is added in 96 well culture plates, and every kind of substratum is established 32 repeating holes.
3. the SP2/0 cell that will be in logarithmic phase is diluted to 10/ml with 1640 perfect mediums with different hybridoma cell clone cultures respectively with the anti-rabies virus monoclonal antibody secretory cell, join in the culture plate that contains same medium then respectively, every hole 100 μ l put under 37 ℃ of 5% CO2 condition and cultivate.100 μ l same medium are changed in every hole during respectively at the 6th day and the 9th day, detect the tiring of clone cell hole supernatant of clone's size and secretion anti-rabies virus monoclonal antibody during by the 12nd day.So that clone cell growth porocyte colony diameter (mean number ± standard deviation) and antibody titer (OD to be arranged under the same culture conditions
450) as basis for estimation.
The comparison of table 1. the present invention and existing substratum
As seen in Table 1, when clone cell grows into the 12nd day, no matter be the SP2/0 or the cell of secrete monoclonal antibody, use the formed cell colony diameter of substratum not only greater than the formed colony diameter of conventional substratum with 3 kinds of different hybridoma cell clones, and secreted antibody titer also is higher than secreted the tiring of conventional substratum in the supernatant.Find more also that further 3 kinds of hybridoma cell clones are with all being better than CRFK-1640 and Hela-1640 substratum aspect cell colony diameter and the antibody titer to contain the BHK21-1640 substratum in the substratum.
Positively effect of the present invention is: a kind of substratum and collocation method thereof of simplifying the hybridoma cell clone process is provided, show that by the clonogenic assay of SP2/0 cell substratum provided by the present invention can replace existing substratum fully with the hybridoma of secretion anti-rabies virus.When hybridoma being cloned, not only saved the complicated processes of conventional hybridization oncocyte clone process of preparing peritoneal macrophage, and clonal growth speed and secreted antibody titer also are improved significantly with limiting dilution assay.
Another advantage of the present invention is greatly to have eliminated that individual and human factor helps the stdn of the process of cloning to the influence that clone's process is produced because of mouse, and easy and simple to handle, so has the potential Commercial Prospect.
Embodiment
For the ease of understanding the present invention, especially exemplified by following examples.Its effect is understood that it is to explaination of the present invention but not to any type of restriction of the present invention.
Embodiment 1
The hybridoma cell clone preparation technology of substratum may further comprise the steps:
1) preparation of 1640 serum free mediums
With RPMI 1640 (dry powder) (production of GIBCO company) 10.4 grams, 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES, the worker is given birth in Shanghai, analytical pure) 3.574 grams, NaHCO3 (Beijing Chemical Plant, analytical pure) 2 900 milliliters of deionized waters of gram, adjust pH to 7.0 back constant volume to 1000 milliliter, 0.22 the degerming of μ m membrane filtration is aseptic subpackaged stand-by;
2) 100 times concentrate two anti-preparations
Penicillin 1,000,000 units, Streptomycin sulphate 1,000,000 units are dissolved in the 100ml deionized water successively, with 0.22 μ m membrane filtration degerming, make two anti-solution, ℃ preservation of packing bottle postposition-20 is stand-by;
3) preparation of 1640 perfect mediums
Get 1640 serum free mediums of 79 milliliters of steps 1 preparation, add 20 milliliters of foetal calf serums (productions of GIBCO company), two resist 1 milliliter stand-by;
4) passage cell culture medium preparation
Get 1640 serum free mediums of 89 milliliters of steps 1 preparation, add successively 10 milliliters of foetal calf serums, two anti-1 milliliter stand-by;
5) monolayer cell Digestive system (10 * stock solution) configuration
Get bovine trypsin 2.5 grams, sodium-chlor 40 grams, Repone K 2.0 grams, glucose 5.0 grams, NaHCO
32.9 gram, EDTA 1.0 gram are dissolved in 450 milliliter three successively and heat up in a steamer in the water, treat that pancreatin dissolves fully after, add 1% phenol redly, and add three and heat up in a steamer water to 500 milliliter, degerming behind the 0.22 μ m membrane filtration ,-20 ℃ of preservations are stand-by;
6) collection of the cultivation of passage cell and supernatant
(1) collection of the cultivation of BHK-21 cell and supernatant
Take out BHK-21 cell freezing pipe from liquid nitrogen container, drop into quick-thawing in 37 ℃ of water-baths immediately, the cold pipe of depositing of jog all melted it in 1 minute.Centrifugal 5 minutes of 200g/min, supernatant is abandoned in aseptic suction in Bechtop, after suspending with an amount of passage cell nutrient solution, is inoculated in the Tissue Culture Flask, puts in 37 ℃ of constant incubators and cultivates.
Behind about 48hr, when cell fission propagation has been covered with the culturing bottle bottom, pour out outmoded nutrient solution in the culturing bottle, embathe cell 2 times with 1 times monolayer cell Digestive system 2ml at every turn, add 0.5ml monolayer cell Digestive system tiling culturing bottle bottom at last, leave standstill after 2-5 minute and observe.Change is big when the intercellular substance, and the body of flapping gently this moment makes cell detachment, adds the passage cell nutrient solution then, regulates cell density to 5 * 10
5/ ml divides flask culture.Aseptic collecting cell supernatant behind the 24h, the centrifugal 10min of 800g, the fresh passage cell nutrient solution of adding equal volume in former culturing bottle simultaneously, collect to cultivate the supernatant of 12h, the same centrifugal after, the supernatant that secondary is collected merges, foetal calf serum concentration is adjusted into 20%, and-20 ℃ frozen standby.
(2) collection of the cultivation of CRFK cell and supernatant
Take out CRFK cell freezing pipe from liquid nitrogen, put into 37 ℃ of water-bath quick-thawings immediately, 200g/min is centrifugal 5 minutes then, and supernatant is abandoned in aseptic suction, with being inoculated in the Tissue Culture Flask after the dilution of passage cell substratum, cultivates in 37 ℃ of constant incubators.
When cell fission propagation has been covered with the culturing bottle bottom, pour out outmoded nutrient solution in the culturing bottle, disperse with the monolayer cell Digestive system, adjusting cell density with fresh passage cell substratum then is 5 * 10
5Divide flask culture behind the/ml.Aseptic collecting cell supernatant behind the 24h, the centrifugal 10min of 800g, the fresh passage cell substratum of adding equal volume in former culturing bottle simultaneously, collect to cultivate the supernatant of 12h once more, the same centrifugal after, the supernatant that secondary is collected merges, serum-concentration is adjusted into 20%, and-20 ℃ frozen standby.
(3) collection of the cultivation of Hela and supernatant
Take out Hela cell freezing pipe from liquid nitrogen container, put into 37 ℃ of water-bath quick-thawings immediately, the jog freeze pipe all melted it in 1 minute.Centrifugal 5 minutes of 200g/min, supernatant is abandoned in aseptic suction in Bechtop, after suspending with an amount of passage cell nutrient solution, is inoculated in the Tissue Culture Flask, puts in 37 ℃ of constant incubators and cultivates.
Behind about 48hr, when cell fission propagation has been covered with the culturing bottle bottom, pour out outmoded nutrient solution in the culturing bottle, the monolayer cell Digestive system with 1 times of 3ml embathes cell 3 times at every turn, add 0.5ml monolayer cell Digestive system tiling culturing bottle bottom at last, leave standstill after 2-5 minute and observe.After the intercellular substance becomes big, and the body of flapping gently this moment makes cell detachment, regulate cell density to 5 * 10 with the passage cell substratum
5Divide flask culture behind the/ml.Aseptic collecting cell supernatant behind the 24h, the centrifugal 10min of 800g, the fresh passage cell substratum of adding equal volume in former culturing bottle simultaneously, collect to cultivate the supernatant of 12h once more, the same centrifugal after, the supernatant that secondary is collected merges, serum-concentration is adjusted into 20%, and-20 ℃ frozen standby.
7) the hybridoma cell clone substratum of preparation monoclonal antibody
With the passage cell supernatant of 1640 perfect mediums and step 6 preparation (7～1) by volume: 1 mixed, and the pH value of mixed culture medium is transferred to 7.2, promptly obtain the different hybridoma cell clone substratum of preparation monoclonal antibody of the present invention.Respectively according to source called after BHK-1640, CRFK-1640, the Hela-1640 of supernatant adding.
Embodiment 2
Contain of the comparison of the hybridoma cell clone of different ratios passage cell supernatant with substratum and conventional substratum
1. with testing routine step 1 preparation BALB/c peritoneal macrophage suspension and being added in 96 orifice plates.
2. the hybridoma cell clone of different ratios is with the configuration of substratum and be added in 96 orifice plates substratum that will be disposed by embodiment step 7, is added in 96 well culture plates with the amount of every hole 100 μ l, and every kind of substratum is established 32 repeating holes.
3. the substratum that disposed of Application Example step 7 will be in SP2/0 cell dilution to the 10/ml of logarithmic phase, join respectively then in the culture plate that contains same medium, and every hole 100 μ l put under 37 ℃ of 5%CO2 conditions and cultivate.100 μ l same medium are changed, formed cell colony diameter under the more different culture medium condition during by the 12nd day in every hole during respectively at the 6th day and the 9th day.
As seen in Table 2, different substratum clones' SP2/0 cell, when growing into the 12nd day, the blending ratio of cleer and peaceful 1640 perfect mediums is 1 on the passage cell: when (1～7), formed cell colony diameter is all obviously greater than the formed colony diameter of conventional substratum.Further more also find, when cleer and peaceful 1640 perfect mediums on the passage cell by 1: when mix (3～5), the colony diameter of the formed colony diameter of cell during greater than other ratios.When blending ratio is identical, contain the formed colony diameter of the hybridoma cell clone substratum maximum of BHK21 cell conditioned medium.
The formed colony diameter of SP2/0 cell under the different culture medium condition of table 2.
Claims (1)
1. hybridoma cell clone substratum for preparing monoclonal antibody, mainly by following raw material by volume portion rate form:
7～1 parts of 1640 perfect mediums, 1 part of passage cell supernatant;
Described passage cell supernatant is the cell log growth acellular supernatant liquor in early stage, and passage cell is selected from:
A kind of in BHK-21, CRFK, the Hela continuous cell line;
The hybridoma cell clone of described preparation monoclonal antibody may further comprise the steps with the preparation process of substratum:
1) preparation of 1640 serum free mediums
With RPMI 1640, Hepes, NaHCO
3Use deionized water dissolving, adjust pH to 7.0, packing after the degerming;
2) two anti-preparations
Penicillin 1,000,000 units, Streptomycin sulphate 1,000,000 units are dissolved in the 100ml deionized water successively, with 0.22 μ m membrane filtration degerming, make two anti-solution, ℃ preservation of packing bottle postposition-20 is stand-by;
3) preparation of 1640 perfect mediums
Get 1640 serum free mediums of 79 milliliters of step 1), add 20 milliliters of foetal calf serums, 1 milliliter of two anti-solution;
4) passage cell culture medium preparation
Get 1640 serum free mediums of 89 milliliters of step 1), add 10 milliliters of foetal calf serums, 1 milliliter of two anti-solution;
5) collection of passage cell supernatant
The passage cell supernatant is the cell log nutrient solution in growth early stage, and by the acellular supernatant liquor of centrifugal acquisition, passage cell is selected from: a kind of in BHK-21, CRFK, the Hela continuous cell line;
6) the hybridoma cell clone substratum of preparation monoclonal antibody
The passage cell supernatant that step 3) is prepared 1640 perfect mediums and step 5 is (7～1) by volume: 1 mixed, and the pH value of mixed-culture medium is transferred to 7.2, promptly obtain substratum.
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| CN103865879A (en) * | 2012-12-17 | 2014-06-18 | 深圳先进技术研究院 | Culture medium for promoting hybridoma growth and preparation method thereof |
| CN103865880A (en) * | 2012-12-17 | 2014-06-18 | 深圳先进技术研究院 | Culture medium for promoting hybridoma growth and preparation method thereof |
| CN108239624B (en) * | 2016-12-26 | 2021-12-03 | 云南省畜牧兽医科学院 | Hybridoma cell liquid culture medium free of feeder cells |
| CN113493774B (en) * | 2020-03-20 | 2024-01-16 | 南京松天盛科生物科技有限公司 | Culture medium for improving hybridoma cell fusion rate |
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| CN1229851A (en) * | 1999-03-02 | 1999-09-29 | 华东理工大学 | Hybridizing tumour cell non-serum culture medium |
| CN101195817A (en) * | 2007-12-28 | 2008-06-11 | 天津百若克医药生物技术有限责任公司 | Hybrid tumor cell amplification culture medium and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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