CN103898063B - The screening method of semisolid screening culture medium and hybridoma - Google Patents
The screening method of semisolid screening culture medium and hybridoma Download PDFInfo
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- CN103898063B CN103898063B CN201210576356.0A CN201210576356A CN103898063B CN 103898063 B CN103898063 B CN 103898063B CN 201210576356 A CN201210576356 A CN 201210576356A CN 103898063 B CN103898063 B CN 103898063B
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Abstract
The invention discloses a kind of semisolid screening culture medium, every 40mL semisolid screening culture medium is become to be grouped into by following: 50 × HAT stock solution of 0.8mL, the foetal calf serum of 10mL, the 100 × penicillin of 0.4mL and the dual anti-solution of Streptomycin sulphate, the L-glutaminate solution of the 1mol/L of 0.16mL, the beta-mercaptoethanol solution of the 1mmol/L of 0.5mL, the anti-solution of rabbit against murine two of 100 × isothiocyanic acid element mark of 0.4mL and the methylcellulose gum semisolid medium of surplus.The invention also discloses a kind of screening method adopting the hybridoma of above-mentioned semisolid screening culture medium, fused cell is colony growth on semisolid medium, separated from one another, each clone is formed by individual cells, the hybridomas grew avoiding not secretory antibody affects the growth of the hybridoma of secretory antibody very soon, compared with traditional limiting dilution assay, screening efficiency is higher.
Description
Technical field
The present invention relates to field of immunology, particularly relate to a kind of semisolid screening culture medium and use the screening method of hybridoma of this semisolid screening culture medium.
Background technology
The ultimate principle of monoclonal antibody technique be by secretory antibody but can not long-term cultivation B cell with can the myeloma cell of long-term cultivation in vitro hybridize, screen the hybridoma that obtains and can have again the characteristic of myeloma cell's infinite multiplication by secretory antibody, the antibody bred by a hybridoma, secreted is called monoclonal antibody, and it is only for an epitope.During cytogamy, B cell separates from the spleen of the animal of antigen immune, and myeloma cell is through mutagenesis and screens the defective type that obtains, and itself can not secretory antibody; And be the defective type of enzyme hypoxanthine guanine nucleotidyl transferase (HGPRT) and thymidine kinase (TK), can not survival in the screening culture medium (HAT) containing xanthoglobulin (hypoxanthineH), aminopterin (aminopterin, A), thymus pyrimidine (thymidineT).In cell mass after fusion, the splenocyte do not merged and the splenocyte mutually merged, can not cultured continuously, can only survive in the medium several days, and the myeloma cell of the myeloma cell of not merging and fusion for want of HGPRT can not survive in HAT substratum, the hybridoma only having myeloma cell and splenocyte to be formed can be survived in HAT substratum, so hybridoma can normally growth and breeding and being selected.
In a spleen, the ratio that the cell of energy secretory antibody accounts for is little, and the cell of more than 90% can not secretory antibody.Even if carried out artificial immunization, the cell proportion secreting the antibody of the antigen for artificial immunization has also been very low, and the fusion of two cells is random, therefore, although the spleen of a successful immunization can reach 2 × 10
8more than individual cell, but the quantity that can obtain target hybridoma after cytogamy is also very limited, and how screening and cloning goes out these hybridomas secreting object antibody is very crucial problems.
The cloning of hybridoma is workload the best part in monoclonal antibody preparation process, the traditional cloning method in laboratory is limiting dilution assay, this method needs repeatedly to carry out limiting dilution and obtains Monoclonal hybridomas, consuming time longer, before cloning, the hybridoma of some not secretory antibodies often grows very fast, the growth affecting the hybridoma of secretory antibody easily causes positive colony to be lost, and screening efficiency is lower.
Summary of the invention
Based on this, be necessary to provide the screening method of semisolid screening culture medium that a kind of screening efficiency is higher and hybridoma.
A kind of semisolid screening culture medium, semisolid screening culture medium described in every 40mL is become to be grouped into by following: 50 × HAT stock solution of 0.8mL, the foetal calf serum of 10mL, the 100 × penicillin of 0.4mL and the dual anti-solution of Streptomycin sulphate, the L-glutaminate solution of the 1mol/L of 0.16mL, the beta-mercaptoethanol solution of the 1mmol/L of 0.5mL, the anti-solution of rabbit against murine two of 100 × isothiocyanic acid element mark of 0.4mL and the methylcellulose gum semisolid medium of surplus;
Described methylcellulose gum semisolid medium is by being that 2 × 1640 substratum of 1:1 and the Methyl cellulose solution of 4% fully mix and obtains according to volume ratio.
In one embodiment, described 2 × 1640 substratum are by operating preparation as follows: by 1640 culture medium dry powders, the 1gNaHCO of 10.4g
3, 0.055g Sodium.alpha.-ketopropionate adds the abundant stirring and dissolving of ultrapure water of 500mL, then adjust ph is 7.0 ~ 7.4, degerming finally by 0.22 μm of membrane filtration, obtains described 2 × 1640 substratum.
In one embodiment, the Methyl cellulose solution of described 4% is by operating preparation as follows: take 20g methylcellulose gum and be dissolved in 500mL ultrapure water, be stirred to solution thickness at 4 DEG C homogeneous and do not have white particle, last autoclaving obtains the Methyl cellulose solution of described 4%.
A screening method for hybridoma, comprises the steps:
Step one, be that the Methyl cellulose solution of 2 × 1640 substratum and 4% fully mixes by 1:1 according to volume ratio, obtain methylcellulose gum semisolid medium;
Step 2, preparation semisolid screening culture medium, semisolid screening culture medium described in every 40mL is become to be grouped into by following: the methylcellulose gum semisolid medium that 50 × HAT stock solution of 0.8mL, the foetal calf serum of 10mL, the 100 × penicillin of 0.4mL and the dual anti-solution of Streptomycin sulphate, the L-glutaminate solution of the 1mol/L of 0.16mL, the beta-mercaptoethanol solution of the 1mmol/L of 0.5mL, the anti-solution of rabbit against murine two of 100 × isothiocyanic acid element mark of 0.4mL and the step one of surplus obtain;
Step 3, lymphocyte and mouse myeloma are carried out cytogamy after, cell after merging is placed in 37 DEG C of preculture 16 ~ 24h, then the cell after preculture is joined in the semisolid screening culture medium that step 2 obtains, add Prostatropin and hybridoma cell fusion Cloning Factor simultaneously, the concentration of Prostatropin is made to be 5ng/ml, the concentration of hybridoma cell fusion Cloning Factor is 15ul/ml, pour in plate after 37 DEG C of standing bubble removings side by side, be placed in 37 DEG C, 5%CO
2incubator cultivate;
The cell that step 4, step 3 obtain is at 37 DEG C, 5%CO
2incubator cultivate 7 ~ 14 days, naked eyes are grown when cloning colony as seen in described plate, under fluorescent microscope to around have hyperfluorescenceZeng Yongminggaoyingguang signal positive cell group mark respectively, then chosen by the positive colony marked under the microscope, described positive colony is described hybridoma.
In one embodiment, in step one, described 2 × 1640 substratum are by operating preparation as follows: by 1640 culture medium dry powders, the 1gNaHCO of 10.4g
3, 0.055g Sodium.alpha.-ketopropionate adds the abundant stirring and dissolving of ultrapure water of 500mL, then adjust ph is 7.0 ~ 7.4, degerming finally by 0.22 μm of membrane filtration, obtains described 2 × 1640 substratum.
In one embodiment, in step, the Methyl cellulose solution of described 4% is by operating preparation as follows: take 20g methylcellulose gum and be dissolved in 500mL ultrapure water, and be stirred to solution thickness at 4 DEG C homogeneous and do not have white particle, last autoclaving obtains the Methyl cellulose solution of described 4%.
In one embodiment, the antigen described in step 2 is can the soluble proteins of coupling isothiocyanic acid element.
In one embodiment, step 4 be also included in described the positive colony marked is chosen after, to the step that described positive colony confirms, comprise following operation: adopt the method for liquid culture to cultivate in 96 orifice plates the positive colony chosen, one, every hole clone, after cell to cover with at the bottom of hole 1/2 ~ 2/3, draw nutrient solution and carry out antibody characteristic qualification.
The screening method of this hybridoma adopts semisolid screening culture medium, fused cell is colony growth on semisolid medium, separated from one another, each clone is formed by individual cells, the hybridomas grew avoiding not secretory antibody affects the growth of the hybridoma of secretory antibody very soon, compared with traditional limiting dilution assay, screening efficiency is higher.
Accompanying drawing explanation
Fig. 1 is the schema of the screening method of the hybridoma of an embodiment.
Embodiment
For enabling above-mentioned purpose of the present invention, feature and advantage become apparent more, are described in detail the specific embodiment of the present invention below in conjunction with accompanying drawing.Set forth a lot of detail in the following description so that fully understand the present invention.But the present invention can be much different from alternate manner described here to implement, those skilled in the art can when without prejudice to doing similar improvement when intension of the present invention, therefore the present invention is by the restriction of following public concrete enforcement.
The semisolid screening culture medium of one embodiment, wherein, semisolid screening culture medium described in every 40mL is become to be grouped into by following: 50 × HAT stock solution of 0.8mL, the foetal calf serum of 10mL, the 100 × penicillin of 0.4mL and the dual anti-solution of Streptomycin sulphate, the L-glutaminate solution of the 1mol/L of 0.16mL, the beta-mercaptoethanol solution of the 1mmol/L of 0.5mL, the anti-solution of rabbit against murine two of 100 × isothiocyanic acid element mark of 0.4mL and the methylcellulose gum semisolid medium of surplus.
Methylcellulose gum semisolid medium is by being that 2 × 1640 substratum of 1:1 and the Methyl cellulose solution of 4% fully mix and obtains according to volume ratio.
2 × 1640 substratum are by operating preparation as follows: by 1640 culture medium dry powders, the 1gNaHCO of 10.4g
3, 0.055g Sodium.alpha.-ketopropionate adds the abundant stirring and dissolving of ultrapure water of 500mL, is then 7.0 ~ 7.4 by hydrochloric acid or sodium hydroxide adjust ph, degerming finally by 0.22 μm of membrane filtration, obtains 2 × 1640 substratum.
The Methyl cellulose solution of 4% is by operating preparation as follows: take 20g methylcellulose gum and be dissolved in 500mL ultrapure water, and be stirred to solution thickness at 4 DEG C homogeneous and do not have white particle, last autoclaving obtains the Methyl cellulose solution of 4%.
The screening method of the hybridoma of one embodiment, as shown in Figure 1, step is as follows.
S10, be that the Methyl cellulose solution of 2 × 1640 substratum and 4% fully mixes by 1:1 according to volume ratio, obtain methylcellulose gum semisolid medium.
2 × 1640 substratum are by operating preparation as follows: by 1640 culture medium dry powders, the 1gNaHCO of 10.4g
3, 0.055g Sodium.alpha.-ketopropionate adds the abundant stirring and dissolving of ultrapure water of 500mL, is then 7.0 ~ 7.4 by hydrochloric acid or sodium hydroxide adjust ph, degerming finally by 0.22 μm of membrane filtration, obtains 2 × 1640 substratum.
1640 culture medium dry powder powder are purchased from Gibco company, and article No. is C31800-022.
The Methyl cellulose solution of 4% is by operating preparation as follows: take 20g methylcellulose gum and be dissolved in 500mL ultrapure water, and be stirred to solution thickness at 4 DEG C homogeneous and do not have white particle, last autoclaving obtains the Methyl cellulose solution of 4%.
Methylcellulose gum is purchased from sigma company, and article No. is M0512.
Be stirred to solution thickness at 4 DEG C homogeneous and do not have the operation of white particle to be: in 4 DEG C of refrigerators with magnetic stirring apparatus stirs spend the night homogeneous and there is no white particle to solution thickness.
Autoclaved operation can be: 121 DEG C of autoclaving 30min.The Methyl cellulose solution of sterilizing 4% can be preserved at 4 DEG C.
S20, preparation semisolid screening culture medium, every 40mL semisolid screening culture medium is become to be grouped into by following: the methylcellulose gum semisolid medium that 50 × HAT stock solution of 0.8mL, the foetal calf serum of 10mL, the 100 × penicillin of 0.4mL and the dual anti-solution of Streptomycin sulphate, the L-glutaminate solution of the 1mol/L of 0.16mL, the beta-mercaptoethanol solution of the 1mmol/L of 0.5mL, the anti-solution of rabbit against murine two of 100 × isothiocyanic acid element mark of 0.4mL and the S10 of surplus obtain.
50 × HAT stock solution is purchased from sigma company, and article No. is H0260.
Foetal calf serum is purchased from Hyclone company, and article No. is SV30087.
The working concentration of the anti-solution of rabbit against murine two that 100 × isothiocyanic acid element (fluoresceinisothiocyanate, FITC) marks is 1 ~ 10 μ g/mL, can preserve at-20 DEG C.
The rabbit against murine two of isothiocyanic acid element mark resists, and can directly be screened the cell clone of secretion specific antibody by antigen-antibody agglutination reaction.
The L-glutaminate solution of 1mol/L is by operating preparation as follows: take L-glutaminate 2.8g, add pure water 10mL and be heated to dissolve completely, filtration sterilization obtains the L-glutaminate solution of 1mol/L.
L-glutaminate available from Sigma, article No. is G8540.
The beta-mercaptoethanol solution of 1mmol/L is by operating preparation as follows: the beta-mercaptoethanol getting 70 μ L, adds in 100ml pure water, and filtration sterilization obtains the beta-mercaptoethanol solution of 1mmol/L ,-20 DEG C of preservations.
Beta-mercaptoethanol is purchased from Amresco company, and article No. is 0482.
In the dual anti-solution of 100 × penicillin and Streptomycin sulphate, the concentration of penicillin is 10000 units/mL, and the concentration of Streptomycin sulphate is 10000 μ g/ml.
The dual anti-solution of 100 × penicillin and Streptomycin sulphate is purchased from Hyclone company, and article No. is SV30010.
S30, lymphocyte and mouse myeloma are carried out cytogamy after, cell after merging is placed in 37 DEG C of preculture 16 ~ 24h, then the cell after preculture is joined in the semisolid screening culture medium that S20 obtains, add Prostatropin and hybridoma cell fusion Cloning Factor simultaneously, the concentration of Prostatropin is made to be 5ng/ml, the concentration of hybridoma cell fusion Cloning Factor is 15ul/ml, pour in plate after 37 DEG C of standing bubble removings side by side, be placed in 37 DEG C, 5%CO
2incubator cultivate.
Plate can select the plate of diameter 3.5cm, each plate packing 2mL substratum.
Prostatropin is purchased from Roche company, and article No. is 11363735001.
Hybridoma cell fusion Cloning Factor (HybridomaFusionandCloningSupplement, HFCS) is purchased from Roche company, and article No. is 11363735001.
The cell that S40, S30 obtain is at 37 DEG C, 5%CO
2incubator cultivate 10 ~ 14 days, naked eyes are grown when cloning colony as seen in plate, under fluorescent microscope to around have hyperfluorescenceZeng Yongminggaoyingguang signal positive cell group mark respectively, then chosen by the positive colony marked under the microscope, described positive colony is described hybridoma.
The operation chosen by the positive colony marked can select aseptic micropipet to complete.
Step S40 also comprises the step confirmed positive colony, comprise following operation: adopt the method for liquid culture to cultivate in 96 orifice plates the positive colony chosen, one, every hole clone, after cell to cover with at the bottom of hole 1/2 ~ 2/3, draws nutrient solution and carries out antibody characteristic qualification.
The methods such as nutrient solution ELISA can be got confirm positive colony, select the positive colony after confirming to carry out enlarged culturing, frozen and identification and analysis.
The screening method of this hybridoma, fused cell is in colony growth on semisolid medium, and separated from one another, each clone is formed by individual cells, does not therefore need repeatedly to clone to obtain single antibody.Add fluorescent mark two in substratum to resist, a step realizes clone and the positive-selecting of hybridoma, and workload is little, consuming time short; The ability of emiocytosis antibody can be judged according to fluorescence intensity, thus obtain the cell strain of hypersecretion antibody; Add B-cell stimulating factor and hybridoma cell fusion Cloning Factor in semisolid medium, ensure that the nutritional need of hybridoma, avoid the troublesome operation using feeder cell, greatly reduce risk of pollution.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (5)
1. a semisolid screening culture medium, it is characterized in that, semisolid screening culture medium described in every 40mL is become to be grouped into by following: 50 × HAT stock solution of 0.8mL, the foetal calf serum of 10mL, the 100 × penicillin of 0.4mL and the dual anti-solution of Streptomycin sulphate, the L-glutaminate solution of the 1mol/L of 0.16mL, the beta-mercaptoethanol solution of the 1mmol/L of 0.5mL, the anti-solution of rabbit against murine two of 100 × isothiocyanic acid element mark of 0.4mL and the methylcellulose gum semisolid medium of surplus;
Described methylcellulose gum semisolid medium is by being that 2 × 1640 substratum of 1:1 and the Methyl cellulose solution of 4% fully mix and obtains according to volume ratio;
Described 2 × 1640 substratum are by operating preparation as follows: by 1640 culture medium dry powders, the 1gNaHCO of 10.4g
3, 0.055g Sodium.alpha.-ketopropionate adds the abundant stirring and dissolving of ultrapure water of 500mL, then adjust ph is 7.0 ~ 7.4, degerming finally by 0.22 μm of membrane filtration, obtains described 2 × 1640 substratum.
2. semisolid screening culture medium according to claim 1, it is characterized in that, the Methyl cellulose solution of described 4% is by operating preparation as follows: take 20g methylcellulose gum and be dissolved in 500mL ultrapure water, be stirred to solution thickness at 4 DEG C homogeneous and do not have white particle, last autoclaving obtains the Methyl cellulose solution of described 4%.
3. a screening method for hybridoma, is characterized in that, comprises the steps:
Step one, be that the Methyl cellulose solution of 2 × 1640 substratum and 4% fully mixes by 1:1 according to volume ratio, obtain methylcellulose gum semisolid medium;
Step 2, preparation semisolid screening culture medium, semisolid screening culture medium described in every 40mL is become to be grouped into by following: the methylcellulose gum semisolid medium that 50 × HAT stock solution of 0.8mL, the foetal calf serum of 10mL, the 100 × penicillin of 0.4mL and the dual anti-solution of Streptomycin sulphate, the L-glutaminate solution of the 1mol/L of 0.16mL, the beta-mercaptoethanol solution of the 1mmol/L of 0.5mL, the anti-solution of rabbit against murine two of 100 × isothiocyanic acid element mark of 0.4mL and the step one of surplus obtain;
Step 3, lymphocyte and mouse myeloma are carried out cytogamy after, cell after merging is placed in 37 DEG C of preculture 16 ~ 24h, then the cell after preculture is joined in the semisolid screening culture medium that step 2 obtains, add Prostatropin and hybridoma cell fusion Cloning Factor simultaneously, the concentration of Prostatropin is made to be 5ng/ml, the concentration of hybridoma cell fusion Cloning Factor is 15ul/ml, pour in plate after 37 DEG C of standing bubble removings side by side, be placed in 37 DEG C, 5%CO
2incubator cultivate;
The cell that step 4, step 3 obtain is at 37 DEG C, 5%CO
2incubator cultivate 7 ~ 14 days, naked eyes are grown when cloning colony as seen in described plate, under fluorescent microscope to around have hyperfluorescenceZeng Yongminggaoyingguang signal positive cell group mark respectively, then chosen by the positive colony marked under the microscope, described positive colony is described hybridoma;
In step one, described 2 × 1640 substratum are by operating preparation as follows: by 1640 culture medium dry powders, the 1gNaHCO of 10.4g
3, 0.055g Sodium.alpha.-ketopropionate adds the abundant stirring and dissolving of ultrapure water of 500mL, then adjust ph is 7.0 ~ 7.4, degerming finally by 0.22 μm of membrane filtration, obtains described 2 × 1640 substratum;
Rabbit against murine two described in step 2 resists for can the soluble proteins of coupling isothiocyanic acid element.
4. the screening method of hybridoma according to claim 3, it is characterized in that, in step, the Methyl cellulose solution of described 4% is by operating preparation as follows: take 20g methylcellulose gum and be dissolved in 500mL ultrapure water, be stirred to solution thickness at 4 DEG C homogeneous and do not have white particle, last autoclaving obtains the Methyl cellulose solution of described 4%.
5. the screening method of hybridoma according to claim 3, it is characterized in that, step 4 be also included in described the positive colony marked is chosen after, to the step that described positive colony confirms, comprise following operation: adopt the method for liquid culture to cultivate in 96 orifice plates the positive colony chosen, one, every hole clone, after cell to cover with at the bottom of hole 1/2 ~ 2/3, draws nutrient solution and carries out antibody characteristic qualification.
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| CN104404082A (en) * | 2014-11-19 | 2015-03-11 | 上海美百瑞生物医药技术有限公司 | Efficient screening method of exogenous protein expression cell strain |
| CN107764789A (en) * | 2017-09-27 | 2018-03-06 | 成都微康生物科技有限公司 | Semisolid screening culture medium and its preparation method and application |
| CN108441472A (en) * | 2018-03-16 | 2018-08-24 | 河南赛诺特生物技术有限公司 | A method of efficiently separating single antigen-specific b cells |
| CN111808825B (en) * | 2020-09-14 | 2020-12-25 | 上海奥浦迈生物科技股份有限公司 | Selective medium for screening hybridoma cells and application |
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