CN102321720B - Method for producing pure human-derived monoclonal antibody by mixed cell culture - Google Patents
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Abstract
The invention relates to a method for producing a pure human-derived monoclonal antibody by mixed cell culture, which comprises the following steps: co-culturing T-cells, B-cells and human lymph node endothelial cells, and activating specific immunological cells with antigen to promote B-cell division and proliferation, thereby obtaining a monoclonal antibody cell strain with antigenic specificity; and obtaining the pure human-derived monoclonal antibody. The technique adopted by the invention is a mixed cell culture method, is simple and easy to implement and convenient to operate, settles a matter at one go, has a simple screening process, and saves substantial human and material resources; by using the invention, the production efficiency of the monoclonal antibody is greatly enhanced, and the yield of producing antibody from fusion cells is multiplied; and most cloning successfully implements the transformation from Type IgM to IgG, so that the production probability of the IgG type monoclonal antibody is greatly increased.
Description
Technical field
The invention belongs to immunoglobulin (Ig) preparation field, be specifically related to a kind of mixed cell culture and produce the method for pure human monoclonal antibody, the method is tied endotheliocyte by T cell, B cell, human lymphoid and is carried out co-cultivation, by specific immunocyte through antigen activates, promote B cell fission and increment, thereby obtain the cell strain of monoclonal antibody of antigen-specific, the pure human monoclonal antibody of reentrying.
Background technology
Monoclonal antibody pharmacy is biological technical field with the fastest developing speed in current bio-pharmaceuticals.Monoclonal antibody medicine research and development are at present produced and are roughly divided into 3 classes: 1) animal (comprising humanization mouse) direct immunization method; 2) gene library screening, comprises phage library and yeast expressed library, etc.; 3), other biological method.But these methods are difficult to produce pure humanized, the monoclonal antibody medicine of high adhesive force.
Antibody is the proteinoid by Mammals B Hemapoiesis, can specific identification antigen, be called as " biological missile ".The antibody with single antigen recognition characteristic is called as monoclonal antibody (abbreviation monoclonal antibody).Monoclonal antibody technological invention is eighties of last century seventies, and monoclonal antibody pharmacy originates in eighties of last century eighties.The basis of monoclonal antibody technology is that immune muroid animal obtains monoclonal cell strain, and the antibody of acquisition is mouse source antibody.But mouse source protein is not suitable as human medicine, therefore just expedite the emergence of humanization mouse technology.But what immune humanization mouse produced is people-mouse mould assembly antibody, still has its limitation as human medicine.Monoclonal antibody medicine how to produce pure people source is the new challenge of bio-pharmaceuticals.
The main immunocyte of human body is divided into T and B cell, and B cell is responsible for producing monoclonal antibody albumen, and T cell is responsible for providing B Growth of Cells to grow needed signal and cell growth factor.Vitro culture the directly concept of immune stimulatory cells produce monoclonal antibody product just have report (In vitro immunization of human B lymphocytes with cultured melanoma cells (SK-MEL 28) Zhang XM as far back as nineteen ninety; Borrebaeck CA, Human Antibodies Hybridomas. 1990; 1 (1): 42-6).The main body of its test design can simplified summary be external mixed culture T and B cell and import antigen.The difficult problem that cannot avoid of such test is that most immunocyte (B cell) can be dead in about 2 time-of-weeks, to such an extent as to test cannot go on.The solution of a compromise is to transform and transform immunocyte by EBV virus, to avoid necrocytosis.But the importing of EBV virus, belongs to the high-risk process of biological pollution, the harm of its generation is far longer than its positive effect; Only can carry out scientific experiment exploration, and can not be applied to bio-pharmaceuticals.On the whole, such success of the test rate is very low, possesses hardly operability.Therefore, a kind of productive rate of pharmaceutical industries needs is high, lifeless matter pollutes, technology simple to operate is prepared pure human monoclonal antibody cell strain, then produced pure human monoclonal antibody.
Summary of the invention
The invention provides a kind of mixed cell culture and produce the method for pure human monoclonal antibody, the method is tied endotheliocyte by T cell, B cell, human lymphoid and is carried out co-cultivation, by specific immunocyte through antigen activates, promote B cell fission and increment, thereby obtain the cell strain of monoclonal antibody of antigen-specific, the pure human monoclonal antibody of reentrying.
The present invention is achieved by the following technical solutions:
A kind of mixed cell culture is produced the method for pure human monoclonal antibody, T cell, B cell, human lymphoid are tied to endotheliocyte and carry out co-cultivation, by specific immunocyte through antigen activates, promote B cell fission and increment, thereby obtain the cell strain of monoclonal antibody of antigen-specific, the pure human monoclonal antibody of reentrying.
The method comprised with the next stage:
A, first stage: cells in vitro mixed culture and stimulation:
A. the voluntary blood donor of health that Hepatitis B virus vaccine was injected in screening, the blood sample that collection is the voluntary blood donor of health of immune positive reaction to Hepatitis B virus vaccine carries out separating-purifying processing, obtains human body CD-4 T cell;
B. described human body CD-4 T cell is carried out to activating reaction with Hepatitis B virus vaccine antigen small peptide, obtain the human body CD-4 T cell activating;
C. to A-a step in the voluntary blood donor's of health the blood sample of same hepatitis b vaccine immune positive reaction carry out separating-purifying processing, obtain human body B cell;
D. in vitro human lymphoid is tied to sample and carry out separating-purifying processing, obtain human lymphoid and tie endotheliocyte;
E. human body CD-4 T cell, human body B cell, the human lymphoid of described activation are tied to endotheliocyte dilution for mixed cell suspension, carry out mixed culture, and in culturing process, add antigen to stimulate, obtain the cell of maturation, propagation;
The time of described mixed culture is 3 weeks;
The method of described stimulation is:
Described mixed cell suspension is joined in 6 porocyte culture plates by the amount in the 8 every holes of mL/, then to add ultimate density be the hepatitis B surface antigen HBsAg of 50-1000 ng/mL;
Be the opportunity of described stimulation: within the 1st day in described mixed culture, stimulate for the first time; Within the 8th day in described mixed culture, stimulate for the second time;
In described mixed cell suspension, the ratio that the CD-4 T cell of described human activin, human body B cell, human lymphoid tie the final concentration of endotheliocyte is 5:20:0.1;
B, subordinate phase: cytogamy:
The cell of described maturation, propagation and people are carried out to fusion treatment with myeloma cell, obtain merging hybridoma; Described fusion treatment adopts polyoxyethylene glycol induction;
E: cultivate after merging:
Carry out second day of cell cultures at described fused cell suspension in 37 DEG C, nutrient solution is changed to HT-RPMI1640 nutrient solution, proceed cell cultures;
F: specificity screening:
Carry out cell cultures 2-3 week at described fused cell suspension in 37 DEG C, the supernatant liquor of choosing in the cell hole of culture plate carries out enzyme-linked immunosorbent assay screening, detects IgG and IgM concentration in this supernatant liquor; Select the high cell hole of IgG concentration, carry out specificity screening; The fused cell that described specificity screening obtains positive findings is hybridoma; Described hybridoma is increased in a large number;
C, phase III: mono-clonal:
The hybridoma of above-mentioned amplification is carried out to mono-clonal, obtain monoclonal cell strain; The method of described mono-clonal is restricted gradient dilution;
D, fourth stage: positive colony checking:
Carry out after cell cultures 2-3 week in 37 DEG C in described monoclonal cell strain, the supernatant liquor of now choosing in the cell hole of culture plate carries out enzyme-linked immunosorbent assay screening, detects IgG and IgM concentration in nutrient solution; Select the high cell hole of IgG concentration, carry out specificity screening, obtain the monoclonal cell strain that is antigen-specific of positive findings;
E, five-stage: monoclonal antibody is produced:
After the monoclonal cell strain of described antigen-specific is increased again, carry out the production of extensive monoclonal antibody, then carry out the separating-purifying processing of monoclonal antibody, obtain described pure human monoclonal antibody.
The present invention has following beneficial effect compared to existing technology:
The technology that the present invention adopts is mixed cell culture method, simple, easy to operate, settles at one go, and screening process is simple, saves a large amount of manpower and materials;
Monoclonal antibody production efficiency of the present invention improves greatly, and the ratio that fused cell produces antibody is multiplied;
The present invention is sufficient condition evolution maturation because cell has in the incubation period that reaches 3 weeks, and therefore the present invention has larger assurance to obtain the ideal type IgG of monoclonal antibody medicine; Most clone of the present invention has successfully realized the conversion in type from IgM to IgG, produces IgG type monoclonal antibody probability and greatly improves;
In the present invention, because B cell is stimulating and differentiation, when ripe, there is good environment, likely only obtaining some high adhesive force monoclonal antibody products in the test period;
The present invention can produce the monoclonal antibody medicine of anti-plurality of antigens an external culture cycle, efficiency of research and development can be multiplied;
The present invention can directly develop 100% pure humanized monoclonal antibody product, needn't carry out conversion processing by complicated means.
Brief description of the drawings
In the B-f step of Fig. 1: embodiment 1, the IgG content in the supernatant liquor in the cell hole of the described culture plate that enzyme-linked immunosorbent assay (ELISA) is measured.
Embodiment
Embodiment 1
The present embodiment is the method that mixed cell culture is produced pure human monoclonal antibody cell strain, and described pure human monoclonal antibody is hbv antibody.
The method comprised with the next stage: cells in vitro mixed culture and stimulation, cytogamy, mono-clonal, specificity checking and monoclonal antibody are produced;
A, first stage: cells in vitro mixed culture and stimulation:
A. the voluntary blood donor of health that Hepatitis B virus vaccine was injected in screening, the blood sample that collection is the voluntary blood donor of health of immune positive reaction to Hepatitis B virus vaccine carries out separating-purifying processing, obtains human body CD-4 T cell;
Described separating-purifying adopts immunological magnetic bead sorting method, selects CD-4 magnetic bead monoclonal antibody, and described CD-4 magnetic bead monoclonal antibody is purchased from Miltenyi company, BD Biosciences company or Stem cell technologies company; The detailed step of sorting is referring to the description of product;
B. described human body CD-4 T cell is carried out to activating reaction with Hepatitis B virus vaccine antigen small peptide, obtain the human body CD-4 T cell activating;
Described Hepatitis B virus vaccine antigen small peptide is preS1 20-47aa, and aminoacid sequence is: WSPQAQGMLKTLPADPPPAST VRQSGRQ; Described preS1 20-47aa is synthetic by the sub-light of middle section or other companies;
The step of described activating reaction is:
Described human body CD-4 T cell is dispersed in RPMI1640 nutrient solution, reaches 2 × 10
6the final concentration of cells/ml, obtains cell suspending liquid; In described cell suspending liquid, add final concentration to be
hepatitis B virus vaccine antigen small peptide and concentration be 0.75-50ng/L, be preferably the interleukin II (IL-2) of 10 ng/mL, obtain suspending liquid A; Again the human body CD-4 T cell in suspending liquid A is cultivated; Described cultivation is at CO
2in cell culture incubator, carry out, incubation time is 72 hours, and temperature is 37
0c; Human body CD-4 T cell in above-mentioned suspending liquid A, by the amount in 8-10mL/ hole, joins in 6 porocyte culture plates, then puts into incubator;
C. to A-a step in the voluntary blood donor's of health the blood sample of same hepatitis b vaccine immune positive reaction carry out separating-purifying processing, obtain human body B cell;
Described separating-purifying adopts immunological magnetic bead sorting method, selects CD-20 magnetic bead monoclonal antibody, and described CD-20 magnetic bead monoclonal antibody is purchased from Miltenyi company or BD Biosciences company etc., and the detailed step of sorting is referring to the description of product;
D. in vitro human lymphoid is tied to sample and carry out separating-purifying processing, obtain human lymphoid and tie endotheliocyte
(being called for short FDC);
Described in vitro human lymphoid ties sample source in people's tonsil product of the excision of fresh collection: described people's tonsil product is cleaned with medical aseptic physiological saline, chopping, grind, remove by filter reticular tissue residue, obtain described in vitro human lymphoid and tie sample;
Described human lymphoid is tied to sample RPMI1640 nutrient solution and be uniformly mixed into single-cell suspension liquid, then carry out separating-purifying processing, obtain described human lymphoid and tie endotheliocyte;
Described separating-purifying adopts immunological magnetic bead sorting method, selects FDC1 magnetic bead monoclonal antibody, and described FDC1 magnetic bead monoclonal antibody is purchased from Miltenyi company, BD Biosciences company, or other suppliers, and the detailed step of sorting is referring to the description of product;
E. human body CD-4 T cell, human body B cell, the human lymphoid of described activation are tied to endotheliocyte dilution for mixed cell suspension, carry out mixed culture, and in culturing process, add antigen to stimulate, obtain the cell of maturation, propagation;
The time of described mixed culture is 3 weeks;
In described mixed culture, human body CD-4 T cell, human body B cell, the human lymphoid of described activation are tied to endotheliocyte RPMI1640 nutrient solution and carry out dilution process, obtain mixed cell suspension; In described mixed cell suspension, the ratio that the human body CD-4 T cell of described activation, human body B cell, human body are fawned on the final concentration of endotheliocyte is 5:20:0.1; The final concentration of the human body CD-4 T cell activating is 0.5 × 10
6cells/ml, the final concentration of human body B cell is 2 × 10
6cells/ml, the final concentration that human lymphoid ties endotheliocyte is 1 × 10
4cells/ml;
The method of described stimulation is:
Described mixed cell suspension is joined in 6 porocyte culture plates by the amount in the 8 every holes of mL/, then to add ultimate density be 50-1000 ng/mL, be preferably the hepatitis B surface antigen HBsAg of 500 ng/mL; Be the opportunity of described stimulation: within the 1st day in described mixed culture, stimulate for the first time; Within the 8th day in described mixed culture, stimulate for the second time;
The described mixed culture time is 3 weeks, changes weekly nutrient solution one time; The mode of changing nutrient solution is: the supernatant liquor in the cell hole in 6 described porocyte culture plates is migrated out to 3-5ml, be preferably 4mL, then add gently the fresh RPMI1640 cell culture fluid of equivalent, note not stirring cell;
B, subordinate phase: cytogamy:
The cell of described maturation, propagation and people are carried out to fusion treatment with myeloma cell, obtain merging hybridoma; Described fusion treatment adopts polyoxyethylene glycol induction;
Described fusion hybridoma has the function of normal B emiocytosis antibody, also has the characteristic of the unrestricted fissiparity of cancer cells;
The method of described fusion treatment is:
A. people prepares with myeloma cell:
6-8 days before fusion treatment starts takes out described people and carries out thawing processing with myeloma cell, then increases from liquid nitrogen or dry ice, obtains people's myeloma cell of the fusion use of a large amount of fresh amplifications;
The nutrient solution of described amplification processing is RPMI1640 nutrient solution; 1-2 days is processed in described amplification, and recovery cell in T-25 culturing bottle after 3 days is transferred to the cell of recovery T-75 culturing bottle Inner and cultivates; Be cultured to logarithmic phase, microscopy cell density is 40%-60%; After the cell cultures phase finishes, directly wash cell 3 times with RPMI1640 nutrient solution, carry out cell counting, density is 5-10 × 10
6cells/ml;
In the processing of described amplification, while carrying out centrifugal treating, rotating speed is preferably and is no more than 300g or 1000RPM, and the time is 5 minutes, and temperature is room temperature; Microscopy under the microscope at any time;
Described T-25, it is on sale that T-75 culturing bottle city looks like Corning company etc., uses and see the description of product; Described people uses myeloma cell (K6H6/B5) purchased from American Type Culture Collecti (ATCC);
B. polyoxyethylene glycol (PEG, polyethyleneglycol) solution is prepared: be that 50% polyglycol solution is preheated to 37 DEG C by concentration, obtain the polyglycol solution of preheating;
C. treat the preparation of fused cell: the people of the cell of described maturation, propagation and fusion use is mixed than 1:1 by quantity with myeloma cell, carry out centrifugal treating, abandon supernatant liquor, retain precipitation; With RPMI1640 nutrient solution, the cell mass in described precipitation is broken up again, obtained treating fused cell suspension, carry out cell cultures in 37 DEG C; In described centrifugal treating, rotating speed is 1000RPM, and the time is 5 minutes, and temperature is room temperature; Described centrifugal treating adopts 50ml centrifuge tube; The consumption of each fusion treatment is people's myeloma cell of the described fusion use in a T-75 culturing bottle;
D. cytogamy:
In step B-c, start in 2 minutes of cell cultures, to the described polyglycol solution for the treatment of to add in fused cell suspension the preheating described in 0.7-1ml, dropwise add, mix while adding; In ensuing 2 minutes, add again the polyglycol solution of 2ml preheating; In ensuing 2 minutes, slowly add again the polyglycol solution of 8 mL preheatings, obtain the preliminary cell merging; If microscopy be can't see a large amount of fusion Fine born of the same parents after ensuing 1 hour, prove failure of cytogamy;
The cell of described preliminary fusion is carried out to centrifugal treating, abandon supernatant liquor, retain precipitation; In described precipitation, add 30ml HAT-RPMI nutrient solution, obtain fused cell suspension; In described centrifugal treating, rotating speed is 1000RPM, and the time is 5 minutes, and temperature is room temperature; In this step, centrifugal force can not be excessive, in order to avoid the fragile cell compacting of just having merged; If cannot suspend merging Fine born of the same parents easily and equably, prove failure of cytogamy;
The formula of described HAT-RPMI nutrient solution is: on the basis of RPMI1640 nutrient solution, add that 20% FCS, 5% Glutamine, 1:100 doubly dilute 100 × concentrated mycillin mixed solution and 1:100 doubly dilute 100 × concentrated HAT liquid; All reagent is the special product of cell cultures purchased from Invitrogen company above.
Described fused cell suspension is carried out to cell cultures in 37 DEG C; After 30 minutes, described fused cell suspension is divided and is filled to cell cultures dish, continue at 37 DEG C and carry out cell cultures; Described cell cultures dish is the 96 flat cell cultures dishes in hole, adds 50-100 μ l(3-4 to drip in every hole) described fused cell suspension;
E: cultivate after merging:
Carry out second day of cell cultures at described fused cell suspension in 37 DEG C, nutrient solution is changed to HT-RPMI1640 nutrient solution, proceed cell cultures;
The formula of described HT-RPMI nutrient solution is: on the basis of RPMI1640 nutrient solution, add that 20% FCS, 5% Glutamine, 1:100 doubly dilute 100 × concentrated mycillin mixed solution and 1:100 doubly dilute 100 × concentrated HT liquid; All compositions are the special product of cell cultures purchased from Invitrogen company.
In the process of operation, attention action is soft, must not stir cell, and cell cultures dish is not placed of a specified duration in incubator outside;
At described fused cell suspension in 37 DEG C of microscopies that carry out for second day or the 3rd day that carry out cell cultures, visible a large amount of cell dimers, approximately every hole 25-50, this is because the first division after cytogamy; As lose a large amount of cell dimers, prove failure of cytogamy;
Carry out behind the 3rd day of cell cultures in 37 DEG C at described fused cell suspension, a large amount of cells can be dead rapidly, and last every hole is remaining 1-2 stabilized cell group only; At described fused cell suspension, in 37 DEG C of approximately one week Inner that carries out cell cultures, microscopy can be seen cell heap; Carry out cell cultures after approximately 2 weeks at described fused cell suspension in 37 DEG C, cell culture fluid starts flavescence, shows that cell starts to secrete a large amount of antibody products;
F: specificity screening:
Carry out cell cultures 2-3 week at described fused cell suspension in 37 DEG C, be preferably after 2 weeks, the flavescence a little of nutrient solution color, the supernatant liquor of now choosing in the cell hole of culture plate in 25-50uL/ hole carries out enzyme-linked immunosorbent assay (ELISA) screening, detects IgG and IgM concentration in this supernatant liquor; Select the high cell hole of IgG concentration, carry out specificity screening; The fused cell that described specificity screening obtains positive findings is hybridoma; Described hybridoma is moved in 24 holes or 6 porocyte culture plates and increased in a large number;
As shown in Figure 1, Y-axis is with IgG content in this supernatant liquor of OD value representation; X-axis is in steps A-b, to add the effect of different IL-2 in cell cultures.As shown in the figure, A, in different cell type composite tests, only has many cells (human body CD-4 T cell (T), human body B cell (B), human lymphoid's endotheliocyte (F)) mixed culture could effectively produce a large amount of pure human monoclonal antibodies; And which kind of cell combination no matter other cultural methods adopt, the output of the pure human monoclonal antibody of generation is extremely low.B, in nutrient solution, add certain density interleukin II (IL-2), contribute to strengthen cell viability, increase the output of pure human monoclonal antibody.
Described specificity screening method can be Western blot (Western), immunoprecipitation method (immunoprecipitation) and enzyme-linked immunosorbent assay (ELISA), if can make the cell strain (Cell-line) of antigen at cell surface stably express, can utilize cell antigen surface expression cell strain to carry out the specificity screening (concrete grammar is shown in FACS operation instruction) of antibody in supernatant liquor with flow cytometer (company such as FACS, BD is on sale);
C, phase III: mono-clonal:
The hybridoma of above-mentioned amplification is carried out to mono-clonal, obtain monoclonal cell strain; The method of described mono-clonal is restricted gradient dilution;
The method of described restricted gradient dilution is: the hybridoma of above-mentioned amplification is carried out to cell counting, and then will be by 10,000 cells/mL dilution; Again based on this, according to 1:10 ratio, carry out gradient series dilution with 10% RPMI1640 cell culture fluid, concrete concentration is: 1000 cells/mL, 100 cells/mL, 10 cells/mL, 1 cell/mL and 0.1 cell/mL and 0.05 cell/mL, 0.01 cell/mL, every kind of concentration cell is prepared 20 mL;
The cell of above-mentioned each concentration is transferred in flat 96 porocyte culture plates and cultivated in 37 DEG C according to 200 μ l/ holes; Cultivate 3-4 week, obtain monoclonal cell strain; Now, under the microscope, in single hole, individual cells forms the cell heap of single cima shape through too much taking turns division growth;
D, fourth stage: positive colony checking:
Carry out, after cell cultures 2-3 week, being preferably 2 weeks in 37 DEG C in described monoclonal cell strain, now choose 25-50
supernatant liquor in the cell hole of the culture plate in L/ hole carries out enzyme-linked immunosorbent assay (ELISA) screening, detects IgG and IgM concentration in nutrient solution; Select the high cell hole of IgG concentration, carry out specificity screening, obtain the monoclonal cell strain that is antigen-specific of positive findings;
Described specificity screening method can be Western blot (Western), immunoprecipitation method (immunoprecipitation) and enzyme-linked immunosorbent assay (ELISA), if can make the cell strain (Cell-line) of antigen at cell surface stably express, can utilize cell antigen surface expression cell strain to carry out the specificity screening (concrete grammar is shown in FACS operation instruction) of antibody in supernatant liquor with flow cytometer (company such as FACS, BD is on sale);
Also can select the positive monoclonal cell strain of above-mentioned specificity screening result to carry out the test of antibody adhesion strength and (adopt Bia Core instrument, concrete grammar is referring to instrument service manual), from multiple positive colonies, select that antibody production is high, the monoclonal cell strain of strong adhesion, for the production of monoclonal antibody drug.
E, five-stage: monoclonal antibody is produced:
After the monoclonal cell strain of described antigen-specific is increased again, carry out the production of extensive monoclonal antibody, then carry out the separating-purifying processing of monoclonal antibody, obtain described pure human monoclonal antibody.
More than the method for the monoclonal cell strain of the antigen-specific described in amplification is conventional cell culture processes, and concrete operation step is:
First the monoclonal cell strain of described antigen-specific is grown in T-25 Tissue Culture Flask, in the time that cell density reaches approximately 60%, transfer in T-75 Tissue Culture Flask; Approximately, after 2-3 days, the cell of every bottle is divided and installed in 5 bottles of T-75, grow into before 2-3 days cells are covered with bottle, cell is distributed into 5-10 × 10
6cell/Guan Leng freezes in liquid nitrogen container;
More than the nutrient solution in amplification is RPMI1640 cell culture fluid;
Freezing method is ordinary method above, and concrete operation step is:
All cells are carried out to centrifugal treating with the centrifuge tube of 50cc and separate, then wash 3 times with RPMI1640 cell culture fluid, in above-mentioned centrifugal treating, rotating speed is 1500RPM, and the time is 5 minutes, and temperature is room temperature; Carry out again cell counting; Then according to 10 × 10
6the concentration of cell/mL mixes with refrigerating fulid, by 5-10 × 10
6in 1.5 ml of cell/pipe packing under aseptic condition or the special freeze pipe of cell of 2 ml; Again above-mentioned all freeze pipes that cell is housed are placed on to-20
0c refrigerator overnight, then moves on to above-mentioned all freeze pipes that cell is housed in liquid nitrogen container and preserves.
While needs, take out the above-mentioned freeze pipe that cell is housed and carry out the production of described extensive monoclonal antibody.
The step of the production of described extensive monoclonal antibody is:
A. thawing:
Take out the above-mentioned freeze pipe that cell is housed and be placed on ice, with 70% alcohol spray disinfectant;
Be placed on 37 with palm thawing or by the above-mentioned freeze pipe that cell is housed again
0c water bath rocks 1 minute gently, can see that liquid melts substantially;
Again the above-mentioned freeze pipe that cell is housed is carried out to centrifugal treating, obtain the monoclonal cell of antigen-specific; In described centrifugal treating, rotating speed is 1500RPM, and the time is 5 minutes, and temperature is 4
0c;
By the monoclonal cell of described antigen-specific with 37
0the RPMI1640 cell culture fluid of C preheating is washed cell 3 times; The above-mentioned cell of washing adopts centrifugal treating, and rotating speed is 1500RPM, and the time is 5 minutes;
B. cultivate:
The monoclonal cell that above-mentioned RPMI1640 cell culture fluid was washed to the antigen-specific of 3 times is transferred in a T-25 Tissue Culture Flask, 37
0c renewal cultivation 2-3 days;
Again the monoclonal cell of the antigen-specific after above-mentioned renewal cultivation is transferred in T-75 Tissue Culture Flask and cultivated 2-3 days, then transfer in T-175 Tissue Culture Flask and cultivate 2-3 days;
Carry out again cell counting, and the monoclonal cell of the antigen-specific after above cultivation is divided equally to amount reproduction in 5-10 T-175 Tissue Culture Flask and grown;
Etc. above-mentioned T-175 cell culture fluid yellowing, then the monoclonal cell centrifugation of the antigen-specific that cultivation is obtained, and abandon it, and retain the monoclonal cell culture supernatant of antigen-specific.
C. separation antibody:
The monoclonal cell culture supernatant of described antigen-specific is moved to 4
0c preserves; The monoclonal cell culture supernatant of the antigen-specific described in separating-purifying again, obtains described pure human monoclonal antibody.
Described separating and purifying method is ordinary method, is preferably albumin A or PROTEIN C particle chromatography column method.If condition license, also can be used fast protein liquid chromatography (Fast protein liquid chromatography, is abbreviated as FPLC).
All ingredients in the present embodiment and equipment are market conventional products, can buy and obtain in market.
Claims (1)
1. the method that mixed cell culture is produced pure human monoclonal antibody, it is characterized in that: the method is tied endotheliocyte by T cell, B cell, human lymphoid and carried out co-cultivation, by specific immunocyte through antigen activates, promote B cell fission and propagation, thereby obtain the cell strain of monoclonal antibody of antigen-specific, the pure human monoclonal antibody of reentrying;
Described pure human monoclonal antibody is hbv antibody;
The method comprised with the next stage:
A, first stage: cells in vitro mixed culture and stimulation:
A. the voluntary blood donor of health that Hepatitis B virus vaccine was injected in screening, the blood sample that collection is the voluntary blood donor of health of immune positive reaction to Hepatitis B virus vaccine carries out separating-purifying processing, obtains human body CD-4 T cell;
B. described human body CD-4 T cell is carried out to activating reaction with Hepatitis B virus vaccine antigen small peptide, obtain the human body CD-4 T cell activating;
The aminoacid sequence of described Hepatitis B virus vaccine antigen small peptide is: WSPQAQGMLKTLPADPPPAST VRQSGRQ;
C. to A-a step in the voluntary blood donor's of health the blood sample of same hepatitis b vaccine immune positive reaction carry out separating-purifying processing, obtain human body B cell;
D. in vitro human lymphoid is tied to sample and carry out separating-purifying processing, obtain human lymphoid and tie endotheliocyte;
Described human lymphoid ties endotheliocyte and derives from human body tonsil product, the dendritic cells,follicular FDC extracting from tonsil;
Described people's tonsil product is cleaned with medical aseptic physiological saline, and chopping, grinds, and removes by filter reticular tissue residue, obtains described in vitro human lymphoid and ties sample;
Described human lymphoid is tied to sample RPMI1640 nutrient solution and be uniformly mixed into single-cell suspension liquid, then carry out separating-purifying processing, obtain described human lymphoid and tie endotheliocyte;
E. human body CD-4 T cell, human body B cell, the human lymphoid of described activation are tied to endotheliocyte dilution for mixed cell suspension, carry out mixed culture, the ratio that the human body CD-4 T cell of described activation, human body B cell, human body are fawned on the final concentration of endotheliocyte is 5:20:0.1, and in culturing process, add antigen to stimulate, obtain the cell of maturation, propagation;
The method of described stimulation is:
Described mixed cell suspension is joined in 6 porocyte culture plates by the amount in the 8 every holes of mL/, then to add ultimate density be 50-1000 ng/mL; Be the opportunity of described stimulation: within the 1st day in described mixed culture, stimulate for the first time; Within the 8th day in described mixed culture, stimulate for the second time; The described mixed culture time is 3 weeks, changes weekly nutrient solution one time; The mode of changing nutrient solution is: the supernatant liquor in the cell hole in 6 described porocyte culture plates is migrated out to 3-5ml, then add gently the fresh RPMI1640 cell culture fluid of equivalent, note not stirring cell;
B, subordinate phase: cytogamy:
The cell of described maturation, propagation and people are carried out to fusion treatment with myeloma cell, obtain merging hybridoma; Described fusion treatment adopts polyoxyethylene glycol induction;
C, phase III: mono-clonal:
The hybridoma of above-mentioned amplification is carried out to mono-clonal, obtain monoclonal cell strain; The method of described mono-clonal is restricted gradient dilution;
D, fourth stage: positive colony checking:
Carry out after cell cultures 2-3 week in 37 DEG C in described monoclonal cell strain, the supernatant liquor of now choosing in the cell hole of culture plate carries out enzyme-linked immunosorbent assay screening, detects IgG and IgM concentration in nutrient solution; Select the high cell hole of IgG concentration, carry out specificity screening, obtain the monoclonal cell strain that is antigen-specific of positive findings;
E, five-stage: monoclonal antibody is produced:
After the monoclonal cell strain of described antigen-specific is increased again, carry out the production of extensive monoclonal antibody, then carry out the separating-purifying processing of monoclonal antibody, obtain described pure human monoclonal antibody.
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