CN101270380B - Screening method for hybrid tumor cell monoclonal preparation - Google Patents
Screening method for hybrid tumor cell monoclonal preparation Download PDFInfo
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Abstract
The invention relates to a method of realizing the screening and the monoclone preparation of hybridoma cell in one step. The method comprises that the semisolid medium of the monoclone of hybridoma cell is firstly prepared, the basic fibroblast growth factor of human is added into the fusion cell, then hydridoma fusion factor and clone factor are added and well-mixed, then the mixed material and the semisolid medium are gently well-mixed and packaged into plates and cultivated in the condition of 35 DEG C and CO2 with concentration of 5 percent; when visible clone colony grows in the plates, the clone is aspirated from the culture medium by a micropipettor and moved onto a cell culture plate for liquid scale-up culture, and one clone is moved in one hole; till the cell grows to half to two-third of the hole bottom, the culture supernatant is aspirated to do positive detection, and the positive hole is chosen to directly scale-up frozen and stored for further carrying out the characteristic identification of antibody or ascites production. The method of realizing the screening and the monoclone preparation of hybridoma cell in one step has the advantages of the simple operation, the short culture period, not requiring feeder cells and easily achieving the screening and the monoclone preparation of hybridoma cell after cell fusion in one step.
Description
Technical field
The present invention relates to a kind of method that can realize the cultivation preparation of filtering hybridoma and mono-clonal a step, belong to biological technical field.
Background technology
Since scientist Kohler in 1975 and Milstein created monoclonal antibody (McAb) technology, this technology development was perfect, and its application is also increasingly extensive.The cloning of fused cell is the steps necessary that obtains secretion monospecific antibody cell strain.At present.The method of fused cell cloning mainly contains following several:
Limiting dilution assay this be the present method that usually adopts of each laboratory.Namely after cytogamy, with the fused cell direct inoculation in containing HAT and selecting 96 or 24 orifice plates of substratum, through the detected positive hole of ELISA, within time as early as possible with its cell dilution to 4-8 cell/ml (being equivalent to 0.4-0.8 cell/every hole).From angle of statistics, this extent of dilution can make approximately that every hole, hole of 36% contains 1 cell, in the time of at the bottom of cell count grows to 10～50% holes, detects with ELISA, with detected positive hole clone more as stated above, until 90% above individual cells hole is the antibody-secreting positive.Limiting dilution assay is based on the Poisson regularity of distribution, and therefore cell counting is to obtain suitable dilution assurance exactly, could improve like this ratio of slender hilum.Owing to incessantly there being one to melt the platform Growth of Cells in every hole, squeezed by the hypertrophy of antibody-secreting negative cells by preventing positive colony, clone as early as possible positive cell very important, but this can cause necrocytosis or chromosome elimination unavoidably.Desirable result is according to above-mentioned extent of dilution, has in 20～50% hole Growth of Cells to occur.In addition, in liquid nutrient medium, fibroblastic growth is vigorous, also can hinder the growth of hybrid cell strain.Generally need at least 2～3 subclones with this method, because needs are cloned clone more repeatedly, the workload of cell cultures is large, complex operation, and cloning efficiency is low.Complex operation step, workload is large, length consuming time.
2. utilize the cell sorter (FACS) of fluorescence-activation: this method can select the positive cell of bouncing to carry out the cloning cultivation.But because the source and the quality that detect the required antigen coated fluorescence latex beads of positive cell are limit, also because the equipment of costliness is limit, this method fails to popularize.
3. soft agar semisolid medium method: due to agar fusing point higher (45～50 ℃), under room temperature, processing ease solidifies, and makes itself and cell be difficult for mixing, and cell distribution is uneven, and Growth of Cells is limited, and the clone selects difficulty and is difficult for detecting etc.
Summary of the invention
Technical problem to be solved by this invention is the deficiency that exists for above-mentioned prior art and provide a kind of simple to operate, and culture cycle is short, need not feeder cell, is easy to a step to realize the method for filtering hybridoma and monoclonal antibody preparation after cytogamy.
The present invention is that the technical scheme that the problem of the above-mentioned proposition of solution adopts is:
(1) 2 times of DMEM basic culture solutions of preparation: get
DulbeccoThe modified form culture medium dry powder is DMEM dry powder 10～15 grams, add 300～500 milliliters of pure water, add 3～5 gram sodium bicarbonates and abundant stirring and dissolving, regulate pH to 7.0～7.4 with 0.5～1.5 mole of every liter of hydrochloric acid and 0.5～1.5 mole of every liter of sodium hydroxide, filtration sterilization makes 2 times of DMEM basic culture solutions;
(2) preparation L-glutaminate mother liquor: take L-glutaminate 1～10 gram, add pure water 50-300 milliliter and be heated to dissolve fully, Sterile Filtration is prepared to such an extent that concentration is every liter of L-glutaminate mother liquor of 200 mmole, by 4 milliliters of packing of every pipe, and freezing preservation;
(3) preparation beta-mercaptoethanol mother liquor: get 50～80 microlitre beta-mercaptoethanols, join in the 50-120 ml pure water, filtration sterilization is prepared to get every liter of mother liquor of 10 mmole, freezing preservation;
(4) preparation methylated cellulose aqueous solution: take 0.6～2.4 gram 4000cp methylcellulose gum or Vltra tears, measure pure water, respectively 121～126 ℃ of sterilizations 30 minutes, measure 17.5～70 milliliters of pure water of having sterilized under aseptic condition and join gently in methylcellulose gum, stirred 20～24 hours under 2～8 ℃ of cold condition;
(5) HAT semisolid medium preparation: 15～60 milliliters of 2 times of DMEM basic culture solutions that add step (1) preparation in the methylated cellulose aqueous solution of step (4) preparation, 0.25～1 milliliter, the L-glutaminate mother liquor that adds step (2) preparation, 0.5～2 milliliter, the beta-mercaptoethanol mother liquor that adds step (3) preparation, add again: 10～40 milliliters of foetal calf serums, 0.5～2 milliliter of 100 times of commercially available MEM non-essential amino acid, 0.5～2 milliliter of the 10000 every ml penicillins of unit and every milliliter of Streptomycin sulphate mixed aqueous solution of 10000 micrograms, add 1～4 milliliter of 50 times of commercially available xanthoglobulin-aminopterin-induced syndromes-thymus pyrimidine solution (being commercially available 50 times of HAT solution), 2～8 ℃ were stirred 3～6 hours, namely obtain the HAT semisolid medium, it is also the hybrid tumor cell monoclonal semisolid medium,
(6) cell after the fusion was placed 35～38 ℃ of precultures 16～24 hours, then with 5～20 milliliters of 2 times of DMEM basic culture solution mixings that are preheated to step (1) preparation of 35～38 ℃, add mankind's Prostatropin (bFGF) 200-800 nanogram, adding hybridoma to merge and clone the factor is 0.5～2 milliliter of HFCS again, mixing, again with the HAT semisolid medium of step (5) preparation mixing gently, 35～38 ℃ of standing 10～30 minutes bubble removings side by side minute install to plate and 5% CO under 37 ℃
2Cultivate under condition;
(7) cultivated 9～14 days, plate or culture plate must not be taken out the incubator observation therebetween, treat to have grown in ware macroscopic clone's colony, to clone from this substratum with micropipet and draw, and move to Tissue Culture Plate and adopt the liquid amplification culture, every hole moves into 1 clone, at the bottom of cell grows to full hole 1/2～2/3 o'clock, draw culture supernatant and carry out positive detection, select positive hole directly to amplify frozen, further carry out antibody CHARACTERISTICS IDENTIFICATION or ascites production.
Press such scheme, described semisolid medium uses in a short time after preparation is completed and can be placed in 2～8 ℃ of preservations, if in long-time need not, need freezing preservation.
Press such scheme, described plate is diameter 35mm plate or 6 porocyte culture plates.
Fused cell of the present invention also can adopt the positive cell that needs subclone, is used for the hybrid tumor cell monoclonal semisolid medium and need not preculture.
HAT of the present invention need adopt xanthoglobulin-thymus pyrimidine HT to replace when carrying out the cell subclone.
Advantage of the present invention is: 1. media flow is large but be unlikely and affect cell and form the clone, can not solidify during operation, and is complete with cytomixis, selects convenience.2. fused cell is the colony growth, and is separated from one another, do not produce each other interference phenomenon, do not mix.3. each clone is formed by individual cells, does not therefore need repeatedly to clone to obtain single antibody secreting cell strain.4. clone the number showed increased, pick-up rate is high.Use this method, Single cell fusion (10
8Individual mouse boosting cell and 10
7Individual mouse myeloma SP2/O cell) altogether can obtain 900～1500 clones; And use limiting dilution assay, and reach the clone of same quantity, approximately need inoculate 230 96 orifice plates and could realize.Thereby greatly having alleviated the cell cultures workload, also significantly shorten experimental period.5. the hybridoma cell strain stability that obtains is strong.6. save the troublesome operation of preparation feeder cell, greatly reduced risk of pollution.7. effectively avoided being suppressed because fibroblastic growth makes the growth of hybridoma.
Embodiment
Embodiment 1
(1) 2 times of DMEM basic culture solution preparation: get DMEM 13.4 grams, add 500 milliliters of pure water, add 3.8 gram sodium bicarbonates and abundant stirring and dissolving, transfer pH to 7.2 with 1 mole of every liter of hydrochloric acid and 1 mole of every liter of sodium hydroxide, filtration sterilization makes 2 times of DMEM basic culture solutions.
(2) L-glutaminate mother liquor preparation: take L-glutaminate (L-Glutamine, Invitrogen) 5.8 grams, add 200 milliliters of pure water, be heated to 50 ℃ and make dissolving fully, Sterile Filtration, obtain every liter of L-glutaminate mother liquor of 200 mmole, by 4 milliliters of packing of every pipe, put-20 ℃ of preservations.
(3) beta-mercaptoethanol mother liquor preparation: get 70 microlitre beta-mercaptoethanols, add in 100 ml pure waters, filtration sterilization obtains every liter of beta-mercaptoethanol mother liquor of 10 mmole ,-20 ℃ of preservations.
(4) methylated cellulose aqueous solution preparation: get 25 milliliters of 4000cp methylcellulose gum 0.6 gram and ultrapure waters and sterilized 30 minutes in 121～126 ℃, get 12.5 milliliters of pure water of having sterilized under aseptic condition and join gently in methylcellulose gum, 4 ℃ of cold condition lower magnetic forces stirred 24 hours.
(5) HAT semisolid medium preparation: 16 milliliters of 2 times of DMEM basic culture solutions that add step (1) preparation in the final solution of step (4), 0.27 milliliter, the L-glutaminate mother liquor of step (2) preparation, 0.5 milliliter, the beta-mercaptoethanol mother liquor of step (3) preparation, 12 milliliters of foetal calf serums, 0.5 milliliter of MEM non-essential amino acid, the 10000 every ml penicillins of unit add 0.5 milliliter of every milliliter of Streptomycin sulphate mixed aqueous solution of 10000 microgram, 1 milliliter of commercially available 50 times of HAT solution, 4 ℃ of magnetic agitation 4 hours.
(6) merge rear cell and placed 37 ℃ of precultures 18 hours, then with 5 milliliters of whole solution mixings that are preheated to step 1 preparation of 37 ℃, add mankind's Prostatropin (bFGF) 200 nanograms, 0.5 milliliter of HFCS, after mixing, mix 37 ℃ of standing 20 minutes bubble removings side by side with the HAT semisolid medium of step 5 preparation, be sub-packed in plate, be placed on 37 ℃, 5% CO
2Cultivate under condition.
(7) cultivated 9～14 days, plate or culture plate must not be taken out the incubator observation therebetween, treat to grow in ware macroscopic clone's colony, will clone from this substratum with micropipet and draw, move to 96 porocyte culture plate liquid amplification culture, every hole moves into 1 clone.At the bottom of growing to full hole 1/2～2/3 o'clock, draw culture supernatant and carry out positive detection, select positive hole directly to amplify frozen, further carry out antibody CHARACTERISTICS IDENTIFICATION or ascites production.
Embodiment 2
(1) 2 times of DMEM basic culture solution preparation: get DMEM 13.4 grams, add 500 milliliters of pure water, add 3.8 gram sodium bicarbonates and abundant stirring and dissolving, transfer pH to 7.4 with 1 mole of every liter of hydrochloric acid and 1 mole of every liter of sodium hydroxide, filtration sterilization makes 2 times of DMEM basic culture solutions.
(2) L-glutaminate mother liquor preparation: take L-glutaminate (L-Glutamine, Invitrogen) 2.9 grams, add 100 milliliters of pure water, be heated to 52 ℃ and make dissolving fully, Sterile Filtration, obtain every liter of L-glutaminate mother liquor of 200 mmole, by 4 milliliters of packing of every pipe, put-20 ℃ of preservations.
(3) beta-mercaptoethanol mother liquor preparation: get 63 microlitre beta-mercaptoethanols, add in 90 ml water solution, filtration sterilization obtains every liter of beta-mercaptoethanol mother liquor of 10 mmole ,-20 ℃ of preservations.
(4) methylated cellulose aqueous solution preparation: get 25 milliliters of 4000cp methylcellulose gum 0.6 gram and pure water and sterilized 30 minutes in 121～126 ℃, get 12.5 milliliters of pure water of having sterilized under aseptic condition and join gently in methylcellulose gum, 4 ℃ of cold condition lower magnetic forces stirred 24 hours.
(5) HAT semisolid medium preparation: 15 milliliters of 2 times of DMEM basic culture solutions that add step (1) preparation in the final solution of step (4), 0.25 milliliter, the L-glutaminate mother liquor of step (2) preparation, 0.5 milliliter, the beta-mercaptoethanol mother liquor of step (3) preparation, 10 milliliters of foetal calf serums, 1 milliliter of MEM non-essential amino acid, the 10000 every ml penicillins of unit add 0.5 milliliter of every milliliter of Streptomycin sulphate mixed aqueous solution of 10000 microgram, 2 milliliters of commercially available 50 times of HAT solution, 4 ℃ of magnetic agitation 4 hours.
(6) merge rear cell and placed 37 ℃ of precultures 21 hours, then be preheated to the whole solution mixing of step 1 preparation of 37 ℃ with 10mL, add mankind's Prostatropin (bFGF) 400 nanograms, 1 milliliter of HFCS, after mixing, mix 37 ℃ of standing 20 minutes bubble removings side by side with the HAT semisolid medium of step 5 preparation, be sub-packed in plate, be placed on 37 ℃, 5% CO
2Cultivate under condition.
(7) cultivated 9～14 days, plate or culture plate must not be taken out the incubator observation therebetween, treat to grow in ware macroscopic clone's colony, will clone from this substratum with micropipet and draw, move to 96 porocyte culture plate liquid amplification culture, every hole moves into 1 clone.At the bottom of growing to full hole 1/2～2/3 o'clock, draw culture supernatant and carry out positive detection, select positive hole directly to amplify frozen, further carry out antibody CHARACTERISTICS IDENTIFICATION or ascites production.
Embodiment 3
(1) 2 times of DMEM basic culture solution preparation: get DMEM 13.4 grams, add 500 milliliters of pure water, add 3.8 gram sodium bicarbonates and abundant stirring and dissolving, transfer pH to 7.2 with 1 mole of every liter of hydrochloric acid and 1 mole of every liter of sodium hydroxide, filtration sterilization makes 2 times of DMEM basic culture solutions.
(2) L-glutaminate mother liquor preparation: take L-glutaminate (L-Glutamine, Invitrogen) 8.7 grams, add 150 milliliters of pure water, be heated to 50 ℃ and make dissolving fully, Sterile Filtration, obtain every liter of L-glutaminate mother liquor of 200 mmole, by 4 milliliters of packing of every pipe, put-20 ℃ of preservations.
(3) beta-mercaptoethanol mother liquor preparation: get 77 microlitre beta-mercaptoethanols, add in 110 ml water solution, filtration sterilization obtains every liter of beta-mercaptoethanol mother liquor of 10 mmole ,-20 ℃ of preservations.
(4) methylated cellulose aqueous solution preparation: get 25 milliliters of 4000cp methylcellulose gum 0.9 gram and pure water and sterilized 30 minutes in 121～126 ℃, get 12.5 milliliters of pure water of having sterilized under aseptic condition and join gently in methylcellulose gum, 4 ℃ of cold condition lower magnetic forces stirred 24 hours.
(5) HAT semisolid medium preparation: 16 milliliters of 2 times of DMEM basic culture solutions that add step (1) preparation in the final solution of step (4), 0.3 milliliter, the L-glutaminate mother liquor of step (2) preparation, 0.6 milliliter, the beta-mercaptoethanol mother liquor of step (3) preparation, 18 milliliters of foetal calf serums, 0.8 milliliter of MEM non-essential amino acid, the 10000 every ml penicillins of unit add 0.5 milliliter of every milliliter of Streptomycin sulphate mixed aqueous solution of 10000 microgram, 1 milliliter of commercially available 50 times of HAT solution, 4 ℃ of magnetic agitation 4 hours.
(6) get the hybridoma that needs subclone and be preheated to 5 milliliters the whole solution mixings that the step 1 of 37 ℃ is prepared, add mankind's Prostatropin (bFGF) 200 nanograms, 0.5 milliliter of HFCS, after mixing, mix with the HAT semisolid medium of step 5 preparation, 37 ℃ of standing 20 minutes bubble removings side by side are sub-packed in plate, are placed on 37 ℃, 5% CO
2Cultivate under condition.
(7) cultivated 9～14 days, plate or culture plate must not be taken out the incubator observation therebetween, treat to grow in ware macroscopic clone's colony, to clone from this substratum with micropipet and draw, and move to 96 porocyte culture plate liquid amplification culture, every hole moves into 1 clone, at the bottom of growing to full hole 1/2～2/3 o'clock, draw culture supernatant and carry out positive detection, select positive hole directly to amplify frozen, further carry out antibody CHARACTERISTICS IDENTIFICATION or ascites production.
Claims (2)
1. the screening method of a hybrid tumor cell monoclonal preparation, is characterized in that
(1) 2 times of DMEM basic culture solutions of preparation: getting Dulbecco modified form culture medium dry powder is DMEM dry powder 10～15 grams, add 300～500 milliliters of pure water, add 3～5 gram sodium bicarbonates and abundant stirring and dissolving, regulate pH to 7.0～7.4 with 0.5～1.5 mole of every liter of hydrochloric acid and 0.5～1.5 mole of every liter of sodium hydroxide, filtration sterilization makes 2 times of DMEM basic culture solutions;
(2) preparation L-glutaminate mother liquor: take L-glutaminate 1～10 gram, add pure water 50-300 milliliter and be heated to dissolve fully, Sterile Filtration is prepared to such an extent that concentration is every liter of L-glutaminate mother liquor of 200 mmole, by 4 milliliters of packing of every pipe, and freezing preservation;
(3) preparation beta-mercaptoethanol mother liquor: get 50～80 microlitre beta-mercaptoethanols, join in the 50-120 ml pure water, filtration sterilization is prepared to get every liter of mother liquor of 10 mmole, freezing preservation;
(4) preparation methylated cellulose aqueous solution: take 0.6～2.4 gram 4000cp methylcellulose gum or Vltra tears, measure pure water, respectively 121～126 ℃ of sterilizations 30 minutes, measure 17.5～70 milliliters of pure water of having sterilized under aseptic condition and join in methylcellulose gum, stirred 20～24 hours under 2～8 ℃ of cold condition;
(5) HAT semisolid medium preparation: 15～60 milliliters of 2 times of DMEM basic culture solutions that add step (1) preparation in the methylated cellulose aqueous solution of step (4) preparation, 0.25～1 milliliter, the L-glutaminate mother liquor that adds step (2) preparation, 0.5～2 milliliter, the beta-mercaptoethanol mother liquor that adds step (3) preparation, add again: 10～40 milliliters of foetal calf serums, 0.5～2 milliliter of 100 times of commercially available MEM non-essential amino acid, 0.5～2 milliliter of the 10000 every ml penicillins of unit and every milliliter of Streptomycin sulphate mixed aqueous solution of 10000 micrograms, add 50 times of commercially available xanthoglobulin-aminopterin-induced syndromes-1～4 milliliter of thymus pyrimidine solution, 2～8 ℃ were stirred 3～6 hours, namely obtain the HAT semisolid medium, it is also the hybrid tumor cell monoclonal semisolid medium,
(6) cell after the fusion was placed 35～38 ℃ of precultures 16～24 hours, then with 5～20 milliliters of 2 times of DMEM basic culture solution mixings that are preheated to step (1) preparation of 35～38 ℃, add mankind's HBGH-2 00-800 nanogram, add again hybridoma to merge and clone's factor is HFCS0.5～2 milliliter, mixing, again with the HAT semisolid medium of step (5) preparation mixing gently, 35～38 ℃ of standing 10～30 minutes bubble removings side by side minute install to plate and 5% CO under 37 ℃
2Cultivate under condition;
(7) cultivated 9～14 days, plate or culture plate must not be taken out the incubator observation therebetween, treat to have grown in ware macroscopic clone's colony, to clone from this substratum with micropipet and draw, and move to Tissue Culture Plate and adopt the liquid amplification culture, every hole moves into 1 clone, at the bottom of cell grows to full hole 1/2～2/3 o'clock, draw culture supernatant and carry out positive detection, select positive hole directly to amplify frozen, further carry out antibody CHARACTERISTICS IDENTIFICATION or ascites production.
2. by the screening method of hybrid tumor cell monoclonal preparation claimed in claim 1, it is characterized in that described plate is diameter 35mm plate or 6 porocyte culture plates.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810047640 CN101270380B (en) | 2008-05-08 | 2008-05-08 | Screening method for hybrid tumor cell monoclonal preparation |
Applications Claiming Priority (1)
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| CN103898060B (en) * | 2012-12-25 | 2015-12-23 | 深圳先进技术研究院 | The screening method of HAT semisolid screening culture medium and hybridoma |
| CN103898061B (en) * | 2012-12-26 | 2016-04-13 | 深圳先进技术研究院 | Semisolid medium and preparation method thereof and filtering hybridoma method |
| CN103898063B (en) * | 2012-12-26 | 2016-01-27 | 深圳先进技术研究院 | The screening method of semisolid screening culture medium and hybridoma |
| CN103898062B (en) * | 2012-12-26 | 2016-04-13 | 深圳先进技术研究院 | Latex semisolid medium and preparation method thereof and filtering hybridoma method |
| CN103278631B (en) * | 2013-04-03 | 2014-04-09 | 中国农业科学院油料作物研究所 | Aflatoxin B1 flow lag immunization time distinguishing fluorescence rapid-detection kit and application thereof |
| CN110205301A (en) * | 2019-06-14 | 2019-09-06 | 扬州大学 | A kind of method of hybridoma cell clone |
| CN111926019B (en) * | 2020-10-16 | 2020-12-25 | 和元生物技术（上海）股份有限公司 | H1 promoter with enhanced affinity for Pol II and Pol III |
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| CN1425774A (en) * | 2002-12-31 | 2003-06-25 | 中国科学院水生生物研究所 | Specific anti-round myxobolus monoclonal anti-body and its preparing method |
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| 《抗肠出血性大肠杆菌O157∶H7 EspA单克隆抗体的制备及其特性鉴定》;余抒等;《细胞与分子免疫学杂志》;20071231;第23卷(第7期);657－659 * |
| 余抒等.《抗肠出血性大肠杆菌O157∶H7 EspA单克隆抗体的制备及其特性鉴定》.《细胞与分子免疫学杂志》.2007,第23卷(第7期),657－659. |
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