CN109422808A - The preparation method of monoclonal antibody - Google Patents
The preparation method of monoclonal antibody Download PDFInfo
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- CN109422808A CN109422808A CN201710772030.8A CN201710772030A CN109422808A CN 109422808 A CN109422808 A CN 109422808A CN 201710772030 A CN201710772030 A CN 201710772030A CN 109422808 A CN109422808 A CN 109422808A
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
- C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
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Abstract
The present invention provides the preparation methods of the monoclonal antibody in technical field of biology, the following steps are included: (1) takes 8 week old female mice 10, eye socket blood sampling obtains negative serum, use OTA-BSA that mouse is immunized as immunogene, when head exempts from, 2 mg of immunogene is taken, preparation antigen concentration is 1mg Freund's complete adjuvant antigen, fully emulsified water-in-oil emulsion processed, is immunized mouse；Two are carried out after being spaced 2 weeks to exempt from, and mouse is immunized with incomplete Freund's adjuvant antigen；The last time booster immunization diluted immune stoste of PBS；(2) it prepares serum and screens immune serum, select myeloma cell line, recovery and culture to myeloma cell；(3) feeder cells are prepared and prepare immune mouse spleen cell suspension, carry out the fusion of cell；(4) hybridoma is screened, positive hybridoma cell is cloned, hybridoma is frozen and recovered；(5) monoclonal antibody is largely prepared；The immunity of confrontation OTA can be improved in the present invention.
Description
Technical field
The invention belongs to technical field of biology, the in particular to preparation method of monoclonal antibody.
Background technique
It is a variety of that OTA is widely present in Cereals class, raisins, cocoa, chocolate, coffee, grape wine, condiment, dairy products etc.
It in food and feed, can be entered in humans and animals body by food chain, there is cumulative toxicity.The pollution of OTA is more in feed
Seriously, animal takes in ota by contaminated feed of searching for food.Largely pass through the study found that nonruminant takes in intracorporal OTA
Blood is transferred to kidney, and being proximally and distally absorbed in alimentary canal and renal tubule, and OTA is divided repeatedly by hepato-enteric circulation
It secretes and absorbs, OTA is converted to avirulent metabolite OT α by the microbes positioned at enteron aisle；And enter blood OTA with
HAS combines closely, highly stable in animal body, is not easy to be metabolized degradation, therefore the kidney of animal food, especially pig,
Liver, muscle, blood etc. often have OTA detection.It is anti-for there may be OTA, needing to prepare monoclonal in human body or animal body
Body.
Summary of the invention
For the defects in the prior art, it is an object of the invention to overcome the technology of the above-mentioned pollution of OTA in the prior art
Problem, provides the preparation method of monoclonal antibody, and the immunity of human body or animal confrontation OTA can be improved in the present invention.
The object of the present invention is achieved like this: the preparation method of monoclonal antibody, comprising the following steps:
(1) 8 week old Balb/c female mice Shu 0 is taken, observation one week before being immunized, and eye socket blood sampling obtains negative serum, uses OTA-
BSA is immunized mouse as immunogene, and immunizing dose is 100 μ g/.When head exempts from, 2 mg of immunogene is taken, it is dense to prepare antigen
Degree is the Freund's complete adjuvant antigen 1 ml of 1 mgtnri, and fully emulsified Water-In-Oil (W/0) emulsion processed carries out Balb/c mouse
It is immune；Two are carried out after being spaced 2 weeks to exempt from, and mouse is immunized with incomplete Freund's adjuvant antigen 1 ml；Reinforced exempting from interval of 2 weeks
Epidemic disease 1 time, altogether plus exempt from 4 times, the last time booster immunization diluted immune stoste of PBS；
(2) it prepares immune serum and screens immune serum, myeloma cell line is selected, to the myeloma cell of selection
Recovery and culture；
(3) feeder cells are prepared and prepare immune mouse spleen cell suspension, carry out the fusion of cell；
(4) hybridoma is screened, positive hybridoma cell is cloned, hybridoma is frozen and recovered；
(5) monoclonal antibody is largely prepared.
As a further improvement of the present invention, in step (2), the method for preparing immune mouse resisting anteserum, specifically are as follows: three
10 days after exempting from, mouse is carried out thin in eye socket;Take a blood sample 0.2 ml, is stored at room temperature Ih, 4 °C of refrigerator overnights, 3000 rpm centrifugation
After lOmin, collects upper serum and dispensed, saved backup in 4 °C；Antiserum is screened, specifically are as follows: carry out serum detection
When, first determine the best effort concentration of coating antigen, antibody with indirect ELISA square matrix titration, then with indirect competitive ELISA method
Detect the specificity of antibody.The mouse extracting spleen cell that antiserum titre is high, inhibitory effect is good is selected to prepare hybridoma according to result
Cell selects SP2/0 myeloma cell line, recovers and cultivate to myeloma cell, specifically includes the following steps:
(201) the SP2/0 myeloma cell frozen is taken out from liquid nitrogen container, is put into rapidly in 37 °C of water-baths, to melt completely
Afterwards, it is slowly added to the endless full nutrient solution cell of 8 mL to it in super-clean bench and gently vibrates to mix hook-shaped at suspension and move to 15 mL
In centrifuge tube, subsequent lOOOrpm is centrifuged 5min；(202) it discards supernatant, DEME is slowly added into cell precipitation and is cultivated completely
Liquid, side edged mix gently, and then move into cell bottle sedimentation cell and DEME complete culture solution is added, be placed in CO2Culture
37 DEG C of cultures in case；
(203) myeloma cell after recovery is cultivated using the complete culture solution containing 20 % calf serums, selects logarithm raw
Long-term cell (viable count should reach 90%) is passed on, and is gently blown down cell with pipette before fusion, is collected in 50
In mL centrifuge tube, lOOOrpm is centrifuged 5min, cannots be used up full culture medium (serum-free) and cleans cell 2 times.Then full culture is cannotd be used up
Base weight is outstanding, and Trypan Blue is spare after counting.
As a further improvement of the present invention, in step (3), prepare feeder cells specifically includes the following steps:
(301) Balb/c mouse cervical vertebra de- 0 is put to death, is soaked in 75 % ethyl alcohol after about 5 min, move into super-clean bench；
(302) mouse web portion is fixed on dissecting pan upwards, wipes mouse web portion with cotton ball soaked in alcohol, lifts abdomen skin with tweezers
Skin cuts off an osculum (attention is sure not to break peritonaeum) with eye scissors, then tears skin, sufficiently exposes peritonaeum, uses alcohol swab
Ball sterilizes peritonaeum；
(303) the endless full nutrient solution of 3 ~ 5mL is drawn with syringe and inject mouse peritoneal, gently rub 2 min of abdominal cavity with cotton balls, then with infusing
Intraperitoneal liquid is sucked out in emitter, injects in centrifuge tube, is repeated several times to 10 mL of suction；
(304) gained liquid 1000rpm is centrifuged 5 min, abandons supernatant, with being counted after a small amount of culture solution suspension cell, is added
Appropriate HAT culture solution to cell concentration divides after being 2X105/mL to be laid in 96 porocyte culture plates, 100 holes nL/；
Prepare immune mouse spleen cell suspension the following steps are included:
(305) mouse cervical vertebra is taken off into white execution, be soaked in 75% ethyl alcohol after about 5 min, move into super-clean bench；
(306) mouse web portion is fixed on dissecting pan upwards, spleen can be seen after peeling off left abdomen skin, cut with eye scissors
Peritonaeum is opened, sterile taking-up spleen is put into the plate for having filled the endless full nutrient solution of 5-10 mL and cleans, and dials and go spleen all
The connective tissue enclosed；
(307) spleen is moved into the plate that another fills the endless full nutrient solution of 10mL, with syringe needle the one of spleen
Several apertures are pricked at end, and call action emitter draws other end inserting needle of the endless system-wide nutrient solution from spleen, until rinsing spleen to whiting, are allowed
Splenocyte is completely into culture solution Cao；
(308) spleen cell suspension is transferred in 50 mL centrifuge tubes, adds endless full nutrient solution to 30 mL, bubble hooks.1000
Rpm is centrifuged 5 min, abandons supernatant, then cannots be used up full nutrient solution and clean cell 1 ~ 2 time；
(309) cell is resuspended with the endless full nutrient solution of 10mL, blows and beats mixed sentence, it is spare after cell count；
Cell fusion specifically includes the following steps:
(3010) draw respectively containing 1X108 splenocyte and containing 2X107 myeloma cell cell suspension (two kinds of cell numbers it
Than being mixed in 50 mL centrifuge tubes for 5:1), endless full nutrient solution is added to 30 mL, 1000 rpm and is centrifuged 5min, abandons supernatant,
Centrifuge tube is inverted, is blotted residual liquid with blotting paper；
(3011) centrifuge tube tube bottom is flicked with hand, keeps precipitating loose uniformly in the pasty state；
(3012) right hand holds 50% PEG that pipette draws 37 °C of 0.7mL preheatings, left hand uniform rotation centrifuge tube, along tube wall
It is slowly added into, and controls and added in Imin；Then cell suspension is slowly sucked in pipette (time control 30s with
It is interior), after standing 30 s, then cell suspension is slowly blown back into centrifuge tube to (time controls within 30 s)；
(3013) the endless full nutrient solution of 25 mL is added in 5min immediately, terminates reaction, the specific speed that is added is, in the 1st min
In power Jie 1 mL, the 2nd min plus 4 mL, remaining 20 mL are added in 3 min, and when liquid feeding should gently rotate centrifuge tube；
(3014) 800rpm is centrifuged 7 min immediately, abandons supernatant, 10 mL HAT culture solutions is added, gently pressure-vaccum makes sedimentation cell suspend；
(3015) cell suspension is transferred in sterile petri dish, adding into HAT culture solution to total volume is 80 mL；
(3016) cell suspension is separately added into 8 pieces to be covered in 96 porocyte culture plates of feeder cells, 100 holes nL/ are placed in
37°C、5 %CO2Incubator in cultivate；
(3017) cell growth status is observed and recorded daily, partly changes liquid, 5- after fusion with HAT culture solution within the 3rd day after fusion
Liquid is changed entirely with HT culture solution within 6 days, supernatant detection is sucked out when length to hole floor space 1/5 ~ 1/4.
As a further improvement of the present invention, in step (4), the method for cloning positive hybridoma cell specifically:
(401) clone prepares word the previous day and supports cell, preparation method such as step (3)；
(402) positive hybridoma cell wait clone is blown and beaten after hooking repeatedly, carries out cell count；
(403) it according to count results, is carried out suitably being diluted to 100/mL with HT culture solution, each clone cell does 3 dilutions
Degree, respectively 20/mL, 10/mL, 5/mL, point kind is in 96 porocyte culture plates, and every hole l00 μ L, i.e. number of cells points
Not Wei 2/hole, 1/hole, 0.5/hole, each dilution at least 24 holes；
(404) culture plate is placed in 37 °C, incubator culture 7-10 days of 5% CO2, when clone cell is long to the 1/ of hole floor space
When 4 ~ 1/3, ELISA detection is carried out to its supernatant；
(405) it takes positive colony to expand culture, freeze, while continuing to be cloned into positive rate up to 100% by upper method；
(406) by finally positive rate up to 100% cell expansion culture and freezes a part twice;It is remaining to be used to produce ascites, it makes
Standby monoclonal antibody；
Hybridoma is frozen and recovered, specifically includes the following steps:
(407) freezing preceding 12 ~ 36h and changing liquid makes hybridoma be in logarithmic growth phase, cell viability preferably when freeze and can mention
High viability.The cell that preparation freezes is blown down from bottle wall, is moved into centrifuge tube, 1000 rpm are centrifuged 5 min, abandon supernatant.
Cell is suspended (IxlO6/mL) with frozen stock solution, is transferred in cryopreservation tube, is put into 4 °C of refrigerators after Ih after mixing, move into-
80 °C of refrigerator overnights are then moved into liquid nitrogen credit and are saved；
(408) cell cryopreservation tube for needing to take out preparation recovery from liquid nitrogen container when recovery, puts into 37 DEG C of water-baths rapidly, freezes
After content all melts in pipe, 1000 rpm are centrifuged 5 min, abandon supernatant, and complete culture solution is added, is transferred to Tissue Culture Flask
In, 37 °C are placed in, is cultivated in the constant incubator of 5%C02, liquid is changed after staying overnight, continues to cultivate spare；
(409) it recovers after Cryopreservation of Hybridoma Cells 3 months, after continuous passage 10 times, detects cell supernatant, identify miscellaneous
Hand over the stability of oncocyte secretion monoclonal antibody specific.
As a further improvement of the present invention, in step (5), a large amount of methods for preparing monoclonal antibodies, specifically include with
Lower step:
(1) health Balb/c female mice is selected, 1 ~ 2 week before being inoculated with hybridoma, first to injection 0.5mL in mouse peritoneal
Sterile stone pays for oil, can be used in pretreated mouse 2 ~ 3 months；
(2) well-grown positive hybridoma cell is collected, 1000 rpm are centrifuged 5 mim and abandon supernatant, and cell is resuspended in not exclusively
In culture solution, cell number is adjusted to 1 ~ 2*106/mL, every mouse peritoneal injects 0.5 mL cell suspension；
(3) after inoculating cell 7-12 days, it is seen that mouse web portion obviously expands, and sterilizes mouse part skin, connects 8 with 5niL syringe
Number syringe needle is pierced into abdominal cavity and extracts ascites, and generally collectable 2-5 mL can be extracted after ascites regeneration aggregation, again every 1-3 days pumpings
It takes 1 time, can generally extract 3 ~ 5 times；
(4) the ascites 3000rpm collected is centrifuged 10 min, collects faint yellow supernatant, dispenses, -80 DEG C save backup.
Specific embodiment
The preparation method of monoclonal antibody, comprising the following steps:
(1) 8 week old Balb/c female mices 10 are taken, observation one week before being immunized, and eye socket blood sampling obtains negative serum, uses OTA-
BSA is immunized mouse as immunogene, and immunizing dose is 100 μ g/.When head exempts from, 2 mg of immunogene is taken, it is dense to prepare antigen
Degree is the Freund's complete adjuvant antigen 1 ml of 1 mgtnri, and fully emulsified Water-In-Oil (W/0) emulsion processed carries out Balb/c mouse
It is immune；Two are carried out after being spaced 2 weeks to exempt from, and mouse is immunized with incomplete Freund's adjuvant antigen 1 ml；Reinforced exempting from interval of 2 weeks
Epidemic disease 1 time, altogether plus exempt from 4 times, the last time booster immunization diluted immune stoste of PBS；
(2) it prepares immune serum and screens immune serum, myeloma cell line is selected, to the myeloma cell of selection
Recovery and culture；
(3) feeder cells are prepared and prepare immune mouse spleen cell suspension, carry out the fusion of cell；
(4) hybridoma is screened, positive hybridoma cell is cloned, hybridoma is frozen and recovered；
(5) monoclonal antibody is largely prepared.
In step (2), the method for preparing immune mouse resisting anteserum, specifically are as follows: three exempt from 10 days afterwards, carry out eye socket to mouse
It is interior thin；Take a blood sample 0.2 ml, is stored at room temperature Ih, and 4 °C of refrigerator overnights after 3000 rpm are centrifuged l0min, are collected upper serum and carried out
Packing, saves backup in 4 °C；Antiserum is screened, specifically are as follows: when carrying out serum detection, first titrated with indirect ELISA square matrix
Method determines the best effort concentration of coating antigen, antibody, then the specificity with indirect competitive ELISA method detection antibody.According to knot
The mouse extracting spleen cell that fruit selection antiserum titre is high, inhibitory effect is good prepares hybridoma, selects SP2/0 myeloma cell
System, recovers to myeloma cell and cultivates, specifically includes the following steps:
(201) the SP2/0 myeloma cell frozen is taken out from liquid nitrogen container, is put into rapidly in 37 °C of water-baths, to melt completely
Afterwards, it is slowly added to the endless full nutrient solution cell of 8 mL to it in super-clean bench and gently vibrates to mix hook-shaped at suspension and move to 15 mL
In centrifuge tube, subsequent l000rpm is centrifuged 5min；(202) it discards supernatant, DEME is slowly added into cell precipitation and is cultivated completely
Liquid, side edged mix gently, and then move into cell bottle sedimentation cell and DEME complete culture solution is added, be placed in CO2Culture
37 DEG C of cultures in case；
(203) myeloma cell after recovery is cultivated using the complete culture solution containing 20 % calf serums, selects logarithm raw
Long-term cell (viable count should reach 90%) is passed on, and is gently blown down cell with pipette before fusion, is collected in 50
In mL centrifuge tube, lOOOrpm is centrifuged 5min, cannots be used up full culture medium (serum-free) and cleans cell 2 times.Then full culture is cannotd be used up
Base weight is outstanding, and Trypan Blue is spare after counting.
In step (3), prepare feeder cells specifically includes the following steps:
(301) Balb/c mouse cervical vertebra de- 0 is put to death, is soaked in 75 % ethyl alcohol after about 5 min, move into super-clean bench；
(302) mouse web portion is fixed on dissecting pan upwards, wipes mouse web portion with cotton ball soaked in alcohol, lifts abdomen skin with tweezers
Skin cuts off an osculum (attention is sure not to break peritonaeum) with eye scissors, then tears skin, sufficiently exposes peritonaeum, uses alcohol swab
Ball sterilizes peritonaeum；
(303) the endless full nutrient solution of 3 ~ 5mL is drawn with syringe and inject mouse peritoneal, gently rub 2 min of abdominal cavity with cotton balls, then with infusing
Intraperitoneal liquid is sucked out in emitter, injects in centrifuge tube, is repeated several times to 10 mL of suction；
(304) gained liquid 1000rpm is centrifuged 5 min, abandons supernatant, with being counted after a small amount of culture solution suspension cell, is added
Appropriate HAT culture solution to cell concentration divides after being 2X105/mL to be laid in 96 porocyte culture plates, 100 holes nL/；
Prepare immune mouse spleen cell suspension the following steps are included:
(305) mouse cervical vertebra is taken off into white execution, be soaked in 75% ethyl alcohol after about 5min, move into super-clean bench；
(306) mouse web portion is fixed on dissecting pan upwards, spleen can be seen after peeling off left abdomen skin, cut with eye scissors
Peritonaeum is opened, sterile taking-up spleen is put into the plate for having filled the endless full nutrient solution of 5-10 mL and cleans, and dials and go spleen all
The connective tissue enclosed；
(307) spleen is moved into the plate that another fills the endless full nutrient solution of 10mL, with syringe needle the one of spleen
Several apertures are pricked at end, and call action emitter draws other end inserting needle of the endless system-wide nutrient solution from spleen, until rinsing spleen to whiting, are allowed
Splenocyte is completely into culture solution Cao；
(308) spleen cell suspension is transferred in 50mL centrifuge tube, adds endless full nutrient solution to 30 mL, bubble hooks.1000 rpm
5 min are centrifuged, supernatant is abandoned, then cannots be used up full nutrient solution and cleans cell 1 ~ 2 time；
(309) cell is resuspended with the endless full nutrient solution of 10mL, blows and beats mixed sentence, it is spare after cell count；
Cell fusion specifically includes the following steps:
(3010) draw respectively containing 1X108 splenocyte and containing 2X107 myeloma cell cell suspension (two kinds of cell numbers it
Than being mixed in 50 mL centrifuge tubes for 5:1), endless full nutrient solution is added to 30 mL, 1000 rpm and is centrifuged 5min, abandons supernatant,
Centrifuge tube is inverted, is blotted residual liquid with blotting paper；
(3011) centrifuge tube tube bottom is flicked with hand, keeps precipitating loose uniformly in the pasty state；
(3012) right hand holds 50% PEG that pipette draws 37 °C of 0.7mL preheatings, left hand uniform rotation centrifuge tube, along tube wall
It is slowly added into, and controls and added in Imin；Then cell suspension is slowly sucked in pipette (time control 30s with
It is interior), after standing 30 s, then cell suspension is slowly blown back into centrifuge tube to (time controls within 30 s)；
(3013) the endless full nutrient solution of 25 mL is added in 5min immediately, terminates reaction, the specific speed that is added is, in the 1st min
Add 1 mL, in the 2nd min plus 4 mL, remaining 20 mL are added in 3 min, and when liquid feeding should gently rotate centrifuge tube；
(3014) 800rpm is centrifuged 7 min immediately, abandons supernatant, 10 mL HAT culture solutions is added, gently pressure-vaccum makes sedimentation cell suspend；
(3015) cell suspension is transferred in sterile petri dish, adding into HAT culture solution to total volume is 80mL；
(3016) cell suspension is separately added into 8 pieces to be covered in 96 porocyte culture plates of feeder cells, the hole 100nL/ is placed in
37°C、5 %CO2Incubator in cultivate；
(3017) cell growth status is observed and recorded daily, partly changes liquid, 5- after fusion with HAT culture solution within the 3rd day after fusion
Liquid is changed entirely with HT culture solution within 6 days, supernatant detection is sucked out when length to hole floor space 1/5 ~ 1/4.
In step (4), the method for cloning positive hybridoma cell specifically:
(401) clone prepares word the previous day and supports cell, preparation method such as step (3)；
(402) positive hybridoma cell wait clone is blown and beaten after hooking repeatedly, carries out cell count；
(403) it according to count results, is carried out suitably being diluted to 100/mL with HT culture solution, each clone cell does 3 dilutions
Degree, respectively 20/mL, 10/mL, 5/mL, point kind is in 96 porocyte culture plates, and every hole l00 μ L, i.e. number of cells points
Not Wei 2/hole, 1/hole, 0.5/hole, each dilution at least 24 holes；
(404) culture plate is placed in 37 °C, incubator culture 7-10 days of 5% CO2, when clone cell is long to the 1/ of hole floor space
When 4 ~ 1/3, ELISA detection is carried out to its supernatant；
(405) it takes positive colony to expand culture, freeze, while continuing to be cloned into positive rate up to 100% by upper method；
(406) by finally positive rate up to 100% cell expansion culture and freezes a part twice;It is remaining to be used to produce ascites, it makes
Standby monoclonal antibody；
Hybridoma is frozen and recovered, specifically includes the following steps:
(407) freezing preceding 12 ~ 36h and changing liquid makes hybridoma be in logarithmic growth phase, cell viability preferably when freeze and can mention
High viability.The cell that preparation freezes is blown down from bottle wall, is moved into centrifuge tube, 1000 rpm are centrifuged 5 min, abandon supernatant.
Cell is suspended (IxlO6/mL) with frozen stock solution, is transferred in cryopreservation tube, is put into 4 °C of refrigerators after Ih after mixing, move into-
80 °C of refrigerator overnights are then moved into liquid nitrogen credit and are saved；
(408) cell cryopreservation tube for needing to take out preparation recovery from liquid nitrogen container when recovery, puts into 37 DEG C of water-baths rapidly, freezes
After content all melts in pipe, 1000 rpm are centrifuged 5 min, abandon supernatant, and complete culture solution is added, is transferred to Tissue Culture Flask
In, 37 °C are placed in, is cultivated in the constant incubator of 5%C02, liquid is changed after staying overnight, continues to cultivate spare；
(409) it recovers after Cryopreservation of Hybridoma Cells 3 months, after continuous passage 10 times, detects cell supernatant, identify miscellaneous
Hand over the stability of oncocyte secretion monoclonal antibody specific.
As a further improvement of the present invention, in step (5), a large amount of methods for preparing monoclonal antibodies, specifically include with
Lower step:
(1) health Balb/c female mice is selected, 1 ~ 2 week before being inoculated with hybridoma, first to injection 0.5mL in mouse peritoneal
Sterile stone pays for oil.It can be used in pretreated mouse 2 ~ 3 months；
(2) well-grown positive hybridoma cell is collected, 1000 rpm are centrifuged 5 mim and abandon supernatant, and cell is resuspended in not exclusively
In culture solution, cell number is adjusted to 1 ~ 2*106/mL, every mouse peritoneal injects 0.5 mL cell suspension；
(3) after inoculating cell 7-12 days, it is seen that mouse web portion obviously expands, and sterilizes mouse part skin, connects 8 with 5niL syringe
Number syringe needle is pierced into abdominal cavity and extracts ascites, and generally collectable 2-5 mL can be extracted after ascites regeneration aggregation, again every 1-3 days pumpings
It takes 1 time, can generally extract 3 ~ 5 times；
(4) the ascites 3000rpm collected is centrifuged 10 min, collects faint yellow supernatant, dispenses, -80 DEG C save backup.
Claims (5)
1. the preparation method of monoclonal antibody, which comprises the following steps:
(1) 8 week old Balb/c female mice Shu 0 is taken, observation one week before being immunized, and eye socket blood sampling obtains negative serum, uses OTA-
BSA is immunized mouse as immunogene, and immunizing dose is 100 μ g/, when head exempts from, takes 2 mg of immunogene, it is dense to prepare antigen
Degree is the Freund's complete adjuvant antigen 1 ml of 1 mgtnri, and fully emulsified Water-In-Oil (W/0) emulsion processed carries out Balb/c mouse
It is immune；Two are carried out after being spaced 2 weeks to exempt from, and mouse is immunized with incomplete Freund's adjuvant antigen 1 ml；Reinforced exempting from interval of 2 weeks
Epidemic disease 1 time, altogether plus exempt from 4 times, the last time booster immunization diluted immune stoste of PBS；
(2) it prepares immune serum and screens immune serum, myeloma cell line is selected, to the myeloma cell of selection
Recovery and culture；
(3) feeder cells are prepared and prepare immune mouse spleen cell suspension, carry out the fusion of cell；
(4) hybridoma is screened, positive hybridoma cell is cloned, hybridoma is frozen and recovered；
(5) monoclonal antibody is largely prepared.
2. the preparation method of monoclonal antibody according to claim 1, which is characterized in that in the step (2), preparation is exempted from
The method of epidemic disease mouse resisting anteserum, specifically are as follows: three exempt from 10 days afterwards, carry out to mouse thin in eye socket;Take a blood sample 0.2 ml, is stored at room temperature
Ih, 4 °C of refrigerator overnights after 3000 rpm are centrifuged lOmin, are collected upper serum and are dispensed, saved backup in 4 °C；Screening
Antiserum, specifically are as follows: when carrying out serum detection, the best work of coating antigen, antibody is first determined with indirect ELISA square matrix titration
Make concentration, then the specificity with indirect competitive ELISA method detection antibody；Antiserum titre height is selected according to result, inhibits effect
The good mouse extracting spleen cell of fruit prepares hybridoma, selects SP2/0 myeloma cell line, to myeloma cell carry out recovery and
Culture, specifically includes the following steps:
(201) the SP2/0 myeloma cell frozen is taken out from liquid nitrogen container, is put into rapidly in 37 °C of water-baths, to melt completely
Afterwards, it is slowly added to the endless full nutrient solution cell of 8 mL to it in super-clean bench and gently vibrates to mix hook-shaped at suspension and move to 15 mL
In centrifuge tube, subsequent lOOOrpm is centrifuged 5min；(202) it discards supernatant, DEME is slowly added into cell precipitation and is cultivated completely
Liquid, side edged mix gently, and then move into cell bottle sedimentation cell and DEME complete culture solution is added, be placed in CO2Culture
37 DEG C of cultures in case；
(203) myeloma cell after recovery is cultivated using the complete culture solution containing 20 % calf serums, selects logarithm raw
Long-term cell (viable count should reach 90%) is passed on, and is gently blown down cell with pipette before fusion, is collected in 50
In mL centrifuge tube, lOOOrpm is centrifuged 5min, cannots be used up full culture medium (serum-free) and cleans cell 2 times,
Then it cannots be used up full culture medium to be resuspended, Trypan Blue is spare after counting.
3. the preparation method of monoclonal antibody according to claim 1, which is characterized in that in the step (3), preparation is raised
Support cell specifically includes the following steps:
(301) Balb/c mouse cervical vertebra de- 0 is put to death, is soaked in 75 % ethyl alcohol after about 5 min, move into super-clean bench；
(302) mouse web portion is fixed on dissecting pan upwards, wipes mouse web portion with cotton ball soaked in alcohol, lifts abdomen skin with tweezers
Skin cuts off an osculum (attention is sure not to break peritonaeum) with eye scissors, then tears skin, sufficiently exposes peritonaeum, uses alcohol swab
Ball sterilizes peritonaeum；
(303) the endless full nutrient solution of 3 ~ 5mL is drawn with syringe and inject mouse peritoneal, gently rub 2 min of abdominal cavity with cotton balls, then with infusing
Intraperitoneal liquid is sucked out in emitter, injects in centrifuge tube, is repeated several times to 10 mL of suction；
(304) gained liquid 1000rpm is centrifuged 5 min, abandons supernatant, with being counted after a small amount of culture solution suspension cell, is added
Appropriate HAT culture solution to cell concentration divides after being 2X105/mL to be laid in 96 porocyte culture plates, 100 holes nL/；
Prepare immune mouse spleen cell suspension the following steps are included:
(305) mouse cervical vertebra is taken off into white execution, be soaked in 75% ethyl alcohol after about 5min, move into super-clean bench；
(306) mouse web portion is fixed on dissecting pan upwards, spleen can be seen after peeling off left abdomen skin, use eye scissors
Peritonaeum is cut off, sterile taking-up spleen is put into the plate for having filled the endless full nutrient solution of 5-10 mL and cleans, and dials and remove spleen
The connective tissue of surrounding；
(307) spleen is moved into the plate that another fills the endless full nutrient solution of 10mL, with syringe needle the one of spleen
Several apertures are pricked at end, and call action emitter draws other end inserting needle of the endless system-wide nutrient solution from spleen, until rinsing spleen to whiting, are allowed
Splenocyte is completely into culture solution Cao；
(308) spleen cell suspension being transferred in 50 mL centrifuge tubes, adds endless full nutrient solution to 30 mL, bubble hooks, and 1000
Rpm is centrifuged 5 min, abandons supernatant, then cannots be used up full nutrient solution and clean cell 1 ~ 2 time；
(309) cell is resuspended with the endless full nutrient solution of 10mL, blows and beats mixed sentence, it is spare after cell count；
Cell fusion specifically includes the following steps:
(3010) draw respectively containing 1X108 splenocyte and containing 2X107 myeloma cell cell suspension (two kinds of cell numbers it
Than being mixed in 50 mL centrifuge tubes for 5:1), endless full nutrient solution is added to 30 mL, 1000 rpm and is centrifuged 5min, abandons supernatant,
Centrifuge tube is inverted, is blotted residual liquid with blotting paper；
(3011) centrifuge tube tube bottom is flicked with hand, keeps precipitating loose uniformly in the pasty state；
(3012) right hand holds 50% PEG that pipette draws 37 °C of 0.7 mL preheatings, left hand uniform rotation centrifuge tube, along tube wall
It is slowly added into, and controls and added in Imin；Then cell suspension is slowly sucked in pipette (time control 30s with
It is interior), after standing 30 s, then cell suspension is slowly blown back into centrifuge tube to (time controls within 30 s)；
(3013) the endless full nutrient solution of 25 mL is added in 5min immediately, terminates reaction, the specific speed that is added is, in the 1st min
In power Jie 1 mL, the 2nd min plus 4 mL, remaining 20 mL are added in 3 min, and when liquid feeding should gently rotate centrifuge tube；
(3014) 800rpm is centrifuged 7 min immediately, abandons supernatant, and 10 mL HAT culture solutions are added, and gently pressure-vaccum keeps sedimentation cell outstanding
It is floating；
(3015) cell suspension is transferred in sterile petri dish, adding into HAT culture solution to total volume is 80mL；
(3016) cell suspension is separately added into 8 pieces to be covered in 96 porocyte culture plates of feeder cells, the hole 100nL/ is placed in
37°C、5 %CO2Incubator in cultivate；
(3017) cell growth status is observed and recorded daily, partly changes liquid, 5- after fusion with HAT culture solution within the 3rd day after fusion
Liquid is changed entirely with HT culture solution within 6 days, supernatant detection is sucked out when length to hole floor space 1/5 ~ 1/4.
4. the preparation method of monoclonal antibody according to claim 1, which is characterized in that in the step (4), Ke Longyang
The method of property hybridoma specifically:
(401) clone prepares word the previous day and supports cell, preparation method such as step (3)；
(402) positive hybridoma cell wait clone is blown and beaten after hooking repeatedly, carries out cell count；
(403) it according to count results, is carried out suitably being diluted to 100/mL with HT culture solution, each clone cell does 3 dilutions
Degree, respectively 20/mL, 10/mL, 5/mL, point kind is in 96 porocyte culture plates, and every hole lOO μ L, i.e. number of cells points
Not Wei 2/hole, 1/hole, 0.5/hole, each dilution at least 24 holes；
(404) culture plate is placed in 37 °C, incubator culture 7-10 days of 5% CO2, when clone cell is long to the 1/ of hole floor space
When 4 ~ 1/3, ELISA detection is carried out to its supernatant；
(405) it takes positive colony to expand culture, freeze, while continuing to be cloned into positive rate up to 100% by upper method；
(406) by finally positive rate up to 100% cell expansion culture and freezes a part twice;It is remaining to be used to produce ascites, it makes
Standby monoclonal antibody；
Hybridoma is frozen and recovered, specifically includes the following steps:
(407) freezing preceding 12 ~ 36h and changing liquid makes hybridoma be in logarithmic growth phase, cell viability preferably when freeze and can mention
High viability blows down the cell that preparation freezes from bottle wall, moves into centrifuge tube, and 1000 rpm are centrifuged 5 min, abandons supernatant,
Cell is suspended (IxlO6/mL) with frozen stock solution, is transferred in cryopreservation tube, is put into 4 °C of refrigerators after Ih after mixing, move into-
80 °C of refrigerator overnights are then moved into liquid nitrogen credit and are saved；
(408) cell cryopreservation tube for needing to take out preparation recovery from liquid nitrogen container when recovery, puts into 37 DEG C of water-baths rapidly, freezes
After content all melts in pipe, 1000 rpm are centrifuged 5 min, abandon supernatant, and complete culture solution is added, is transferred to Tissue Culture Flask
In, 37 °C are placed in, is cultivated in the constant incubator of 5%C02, liquid is changed after staying overnight, continues to cultivate spare；
(409) it recovers after Cryopreservation of Hybridoma Cells 3 months, after continuous passage 10 times, detects cell supernatant, identify miscellaneous
Hand over the stability of oncocyte secretion monoclonal antibody specific.
5. the preparation method of monoclonal antibody according to claim 1, which is characterized in that a large amount of to make in the step (5)
The method of standby monoclonal antibody, specifically includes the following steps:
(1) health Balb/c female mice is selected, 1 ~ 2 week before being inoculated with hybridoma, first to injection 0.5mL in mouse peritoneal
Sterile stone pays for oil, can be used in pretreated mouse 2 ~ 3 months；
(2) well-grown positive hybridoma cell is collected, 1000rpm is centrifuged 5 mim and abandons supernatant, and cell is resuspended in incomplete training
In nutrient solution, cell number is adjusted to 1 ~ 2*106/mL, every mouse peritoneal injects 0.5 mL cell suspension；
(3) after inoculating cell 7-12 days, it is seen that mouse web portion obviously expands, and sterilizes mouse part skin, connects 8 with 5niL syringe
Number syringe needle is pierced into abdominal cavity and extracts ascites, and generally collectable 2-5 mL can be extracted after ascites regeneration aggregation, again every 1-3 days pumpings
It takes 1 time, can generally extract 3 ~ 5 times；
(4) the ascites 3000rpm collected is centrifuged 10 min, collects faint yellow supernatant, dispenses, -80 DEG C save backup.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110314414A (en) * | 2019-07-23 | 2019-10-11 | 宝锐生物科技泰州有限公司 | Immune affinity chromatographic column filler, immune affinity chromatographic column, the isolation and purification method of factor XIII and its application |
| CN114437224A (en) * | 2021-12-31 | 2022-05-06 | 武汉雁达生物技术有限公司 | Preparation method and application of mouse anti-chicken IgY monoclonal antibody |
| CN114573693A (en) * | 2022-04-25 | 2022-06-03 | 中国人民解放军陆军军医大学 | Preparation method of immunoregulatory molecule MIF antibody in auditory development |
| CN115299405A (en) * | 2022-08-09 | 2022-11-08 | 新疆医科大学第一附属医院 | Method for making nude mouse transplantation tumor model |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110314414A (en) * | 2019-07-23 | 2019-10-11 | 宝锐生物科技泰州有限公司 | Immune affinity chromatographic column filler, immune affinity chromatographic column, the isolation and purification method of factor XIII and its application |
| CN114437224A (en) * | 2021-12-31 | 2022-05-06 | 武汉雁达生物技术有限公司 | Preparation method and application of mouse anti-chicken IgY monoclonal antibody |
| CN114573693A (en) * | 2022-04-25 | 2022-06-03 | 中国人民解放军陆军军医大学 | Preparation method of immunoregulatory molecule MIF antibody in auditory development |
| CN115299405A (en) * | 2022-08-09 | 2022-11-08 | 新疆医科大学第一附属医院 | Method for making nude mouse transplantation tumor model |
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