CN105349495A - Hybridomas cell strain of anti-human coagulation factor IX monoclonal antibody - Google Patents
Hybridomas cell strain of anti-human coagulation factor IX monoclonal antibody Download PDFInfo
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Abstract
The invention relates to a hybridomas cell strain secreting anti-human coagulation factor IX monoclonal antibody and a preparation method thereof. The preparation method comprises using a conjugate obtaining by performing coupling on bovine serum albumin (BSA) and human coagulation factor IX as an antigen for immunizing Balb/c mouse, getting spleen cell of the mouse and performing cell fusion with SP2/0 myeloma cell, performing selective culture by using HAT medium, performing multiple cloning by using a limiting dilution process, and finally screening and obtaining the hybridomas cell strain stably secreting anti-human coagulation factor IX monoclonal antibody. The prepared hybridomas cell strain is preserved in China Center for Type Culture Collection with the preservation number of CCTCC C2015197.
Description
Technical field
The present invention relates to hybridoma cell strain and preparation method thereof, particularly, relate to hybridoma cell strain of anti-human coagulation factor IX monoclonal antibody and preparation method thereof.
Background technology
Thrombin is a kind of key protein composition participating in coagulation process.Its physiological action is, is activated when angiorrbagia, mends the leak on plug blood vessel together with PA.This process is called as blood coagulation.In haemophiliac, some patient is hemophilia B, i.e. hemophilia B, and hemophilia B (hemophiliaB) has another name called plasma thromboplastin component (PTC) deficiency disease or IX factor deficiency.This kind of clinical manifestation exactly likes A type, and mode of inheritance is also x linked recessive heredity, and because heterozygote IX factor active is only normal 1/3, some heterozygote can occur symptom, therefore female patients is common compared with A type.Such patient mainly lacks the plasma thromboplastin component that normal people has, the method for still not effecting a radical cure at present, can only be treated by infusion Human factor IX substitute products.
Human factor IX molecular structure is complicated, and molecular weight is 60-80kDa.Its content in blood plasma is only 0.1-0.2 μ g/mL.Because its extractive technique is very difficult, and China is technically relatively backward in extensive affinity chromatography, and the high-purity thrombin series products still not having affinity chromatography to prepare at present comes out.Therefore, cause plasma thromboplastin component product wretched insufficiency on domestic market, can not fully in supplier hemophilia B patient use, often there will be the situation of disconnected medicine, endanger the health and lives safety of domestic patient.
The difficult point of the chromatographic separation technology of plasma proteins is the high viscosity of blood plasma and the enormousness of primary treatment.For the separation of blood clotting factors trace plasma proteins, affinity chromatography is best way of purification.At present, most enterprise in the world, comprises maximum blood products enterprise CSL and also adopts the method for cold ethanol associating chromatography to carry out separated plasma albumen.In the nineties in last century, hundred spies have adopted antibody affinity chromatography method to prepare the ultrapure VIII Summing Factor VII factor, and specific activity can reach 3000IU/mg, and the general activity rate of recovery is more than 30%.China prepares plasma thromboplastin component at present and then still adopts the rough acquisition of cryoprecipitate, and specific activity is many lower than 100IU/mg, and activity recovery is about about 6%.
Therefore, in order to the separation efficiency utilizing affinity chromatography to increase substantially thrombin, its gordian technique bottleneck is the effective affinity chromatography antibody of preparation.Thus, there is the demand to specific coagulation factors IX monoclonal antibody in this area.
Summary of the invention
In order to solve the problem, the invention provides and a kind ofly secrete hybridoma cell strain of anti-human coagulation factor IX monoclonal antibody and preparation method thereof.
In first, the invention provides a kind of method (in this article sometimes also referred to as " method of the present invention ") preparing the hybridoma cell strain of secretion anti-human coagulation factor IX monoclonal antibody, said method comprising the steps of:
1) prepare immunizing antigen: by homotype bifunctional protein linking agent by Human factor IX and bovine serum albumin coupling, obtain Human factor IX-bovine serum albumin conjugate;
2) immune mouse: adopt low dose of long period immunization protocol, utilize step 1) in Human factor IX-bovine serum albumin conjugate acting immune antigen of obtaining, immunity is carried out to Balb/c mouse;
3) cytogamy: by SP2/0 murine myeloma cell and step 2) spleen cell of immune Balb/c mouse that obtains merges, in the RPMI-1640 substratum containing HAT, carry out selectivity cultivation;
4) selectivity of hybridoma is cultivated: in step 3) in merge after cell after selectivity is cultivated in HAT substratum, select the fused cell of Mouse spleen cells and mouse hybridoma cell SP2/0; And
5) mono-clonal: by step 4) in obtain after testing for positive hybridoma carries out repeatedly through limiting dilution assay the hybridoma cell strain that mono-clonal obtains stably excreting anti-human coagulation factor IX monoclonal antibody.
In second, the invention provides the hybridoma cell strain (being hereinafter sometimes referred to as " hybridoma cell strain of the present invention ") of secretion anti-human coagulation factor IX monoclonal antibody, described hybridoma cell strain is prepared by method of the present invention.
In a preferred embodiment, the strain of hybridoma strain prepared by method of the present invention is preserved in China typical culture collection center on November 6th, 2015, address is: Wuhan University's preservation center, Wuchang District, Wuhan City, Hubei Province, deposit number is CCTCCC2015197, and culture name is called: hybridoma cell strain F9-M-01.
Embodiment
Definition:
" protein-crosslinking agent " is small molecule compound, and its molecule two ends respectively have one or more for specific groups (-NH
2,-COOH ,-HS etc.) reactive terminal, can with the respectively coupling of the active group in biomolecules, thus these molecular juction are combined.Protein-crosslinking agent can be divided into homotype bi-functional cross-linking agent (also referred to as " homology linking agent "), Heterobifunctional Reagent (also referred to as " allos linking agent ") and photoactivated cross-linking agent (also referred to as " photoreactivity linking agent ") three classes.
The molecule two ends of homotype bifunctional protein linking agent have identical reactive terminal or priming reaction group.Conventional homotype bi-functional cross-linking agent is known in the art, its unrestricted example includes but not limited to, NHS ester class linking agent such as N-hydroxy-succinamide (NHS), dithio two (succinyl phosphorons amino propyl acid ester) (DSP) and 3, 3'-dithio two (sulfonic acid Succinimidyl Propionate) (DTSSP), succsinic acid suberic acid diimine (DSS) and butadiene-styrene copolymer (BS), tartrate two succinimide ester (DST) and Sulfo-DST, two (2-[succinimidyloxycarbonyl oxygen) ethyl) sulfone (BSOCOES) and sulfo-BSOCOES, ethylene glycol bis [succinimido succinic acid] (EGS) and sulfo-EGS, succsinic acid suberic acid glutarate (DSG), N, N'-bis-succinimidyl carbonate (DSC), carbodiimide class is 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) such as, polyurethane class linking agent is dimethyl diiminoester (DMA), dimethylimino ester (DMP), dimethyl benzene two acid imide ester (DMS), ditertiary butyl peroxide (DTBP) such as, sulfydryl response class linking agent is Isosorbide-5-Nitrae-two (3`-[2`-disulfide group pyridine] propionic acid amido) butane (DPDPB) such as, 1,5-bis-fluoro-2,4-dinitrobenzenes (DFDNB) of difluoro bezene derivative, two (the fluoro-3-nitrophenyl of 4-) sulfoxide (DFDNPS) etc., photoreactivity linking agent class linking agent such as two-[β-(azido-salicyloyl is amino) ethyl] disulphide (BASED) etc., aldehyde crosslinking agent is formaldehyde, glutaraldehyde etc. such as, the BDO glycidaldehyde etc. of di-epoxide class, hydrazides class linking agent such as adipic dihydrazide, carbohydrazide etc., double derivative thing class linking agent is such as by the o-tolidine, bis-diazotized benzidine etc. of diazonium, and two alkyl halides etc.
Photoreactivity linking agent is the linking agent with photoreactive group.The priming reaction group of homotype bi-functional cross-linking agent also can be photoreactive group.
The character such as type, reactive group, protein binding group, photoactivity of part homotype bifunctional protein linking agent is listed in table 1.
Table 1
" antibody " (antibody) refers to that the immunity system of body is under antigenic stimulation, that the plasmocyte be divided into by bone-marrow-derived lymphocyte or memory cell prolifera produces, can with the immunoglobulin (Ig) of corresponding antigens generation specific binding.
The invention provides a kind of preparation method secreting the hybridoma cell strain of anti-human coagulation factor IX monoclonal antibody.
In one embodiment, method of the present invention comprises the following steps:
1) immunizing antigen is prepared: by homotype bifunctional protein linking agent by Human factor IX and bovine serum albumin (BSA) coupling, obtain Human factor IX-BSA conjugate;
2) immune mouse: adopt low dose of long period immunization protocol, utilize step 1) in Human factor IX-BSA conjugate acting immune antigen of obtaining, immunity is carried out to Balb/c mouse;
3) cytogamy: by SP2/0 murine myeloma cell and step 2) spleen cell of immune Balb/c mouse that obtains merges, in containing 1640 substratum of HAT, carry out selectivity cultivation;
4) selectivity of hybridoma is cultivated: in step 3) in merge after cell after selectivity is cultivated in HAT substratum, select the fused cell of Mouse spleen cells and mouse hybridoma cell SP2/0; And
5) mono-clonal: by step 4) in obtain after testing for positive hybridoma carries out repeatedly through limiting dilution assay the hybridoma cell strain that mono-clonalization obtains stably excreting anti-human coagulation factor IX monoclonal antibody.
In the method for the invention, any homotype bifunctional protein linking agent as known in the art can be adopted, preferred carbodiimide proteinoid linking agent, more preferably EDC.
In the present invention, animal immune adopts low dose repeatedly immunization protocol, for mouse, conventional with 100 ~ 200 μ g/ dose immunization only, immunity 3 ~ 4 times.A concrete immunization protocol example is: use the female Balb/c mouse of immunizing antigen to 7 week age to carry out immunity, by 100 μ g/ immunizing dose only, with Freund's complete adjuvant by volume 1:1 mix, emulsification reaches water-in-oil shape, by the complete antigen of emulsification, nape portion multiple spot subcutaneous injection immune mouse, after 2 weeks, with same antigen and isopyknic Freund's incomplete adjuvant emulsification, 100 μ g/ only carry out booster immunization, row third time immunity after 2 weeks, dosage and method are with second time immunity, before cytogamy, for the last time immunity is impacted to mouse, 100 μ g/ immunizing dose abdominal injection only.
In the method for the invention, the method for cytogamy is unrestricted, can adopt method as known in the art and flow process.
In the method for the invention, the method for antibody test is unrestricted, can adopt detection method as known in the art, such as, can adopt ELISA method.
By method of the present invention, the hybridoma cell strain of secretion anti-human coagulation factor IX monoclonal antibody can be filtered out.
As an example, provided below is an exemplary fabrication flow of the hybridoma cell strain of secretion anti-human coagulation factor IX monoclonal antibody of the present invention:
1) preparation of immunizing antigen:
Get 1mg Human factor IX to be dissolved in 0.25mLDMSO solution, add EDC, under room temperature, lucifuge acutely shakes 2 hours, and 4 DEG C of reactions are spent the night, and slowly dripped by above-mentioned solution in BSA solution, under room temperature, lucifuge shakes 2 hours, and reaction product is in 0.05molL
-1in PBS damping fluid, lucifuge is dialysed 72 hours, obtains the conjugate of Human factor IX and carrier proteins BSA.
2) mouse immune:
End user's plasma thromboplastin component-BSA conjugate is as immunizing antigen, immunity is carried out to the female Balb/c mouse in 7 week age, by 100 μ g/ immunizing dose only, with Freund's complete adjuvant by volume 1:1 mix, emulsification reaches water-in-oil shape, by the complete antigen of emulsification, nape portion multiple spot subcutaneous injection immunity Balb/c mouse, after 2 weeks, with same antigen and isopyknic Freund's incomplete adjuvant emulsification, 100 μ g/ only carry out booster immunization, row third time immunity after 2 weeks, method is with second time immunity, before cytogamy, for the last time immunity is impacted to mouse, 100 μ g/ immunizing dose abdominal injection only.
3) selectivity of cytogamy and hybridoma is cultivated:
Get SP2/0 murine myeloma cell to mix in the ratio of 1:5-1:10 with the Balb/c Mouse spleen cells of immunity, centrifugal 7 minutes of 1300rpm, put 40 DEG C of metal bath preheatings, in 45s, the 1mL50%PEG4000 being preheated to 40 DEG C is added with 1mL suction pipe, limit edged vibrates gently, then in 90s, add the 1640 incomplete substratum that 30mL is preheated to 37 DEG C, room temperature leaves standstill 10 minutes, centrifugal 5 minutes of 1000rpm, abandon supernatant, add 20mL resuspended containing 1640 substratum of 20% foetal calf serum and HAT, be dispensed on the culture plate of existing feeder cell, cultivate in 5% CO2gas incubator, after 7 days, the upgrowth situation of observation of cell, to swap out 1/2 substratum with 1640 substratum containing 20% foetal calf serum and HT, after 14 days, use 20% foetal calf serum and HT1640 culture medium culturing instead.
4) hybridoma of screening secretion anti-human coagulation factor IX antibody:
Being buffered liquid by antibody dilution to protein content with the carbonate bag of 0.05MpH9.6 is 2ug/ml, adds 50 μ l in the reacting hole of each polystyrene board, and 4 DEG C are spent the night.Next day, discard solution in hole, wash 3 times with lavation buffer solution, each 3 minutes (being called for short washing, lower same).In the reacting hole of each polystyrene board, add skim-milk (BD company) the PBS300 μ l containing 5%, put 37 DEG C and hatch 2 hours, then wash.The measuring samples 50 μ l adding certain dilution in above-mentioned wrapped by reacting hole in, put 37 DEG C and hatch 1 hour, then wash (doing blank well, negative control hole and Positive control wells) simultaneously.In each reacting hole, add with diluted enzyme labelled antibody (by specification dilutes) 50 μ l, hatch 1 hour, then wash for 37 DEG C.The tmb substrate solution 50 μ l of Extemporaneous is added, 37 DEG C of colour developings 15 minutes in each reacting hole.2M sulfuric acid 50 μ l termination reaction is added in each reacting hole.In white background, directly detect by an unaided eye result: in reacting hole, color is darker, and positive degree is stronger, and negative reaction is colourless or extremely shallow, according to be the depth of color, represent with "+", "-" number.As a kind of alternative decision procedure, also can survey OD value on ELISA detector, detect in 450nm and 630 dual wavelengths, to survey each hole OD value after blank control wells zeroing, if be greater than 2.1 times of the negative control OD value of regulation, be the positive.
5) mono-clonal:
The hybridoma of the positive is carried out repeatedly through limiting dilution assay the hybridoma cell strain that mono-clonalization obtains stably excreting anti-human coagulation factor IX monoclonal antibody.
In a preferred embodiment, the strain of hybridoma strain called after F9-M-01 prepared by method of the present invention, and be deposited in China typical culture collection center on November 6th, 2015, address is: Wuhan University's preservation center, Wuchang District, Wuhan City, Hubei Province, deposit number is CCTCCC2015197.
The preparation method of hybridoma cell strains F9-M-01 comprises the following steps:
Preparation Human factor IX (DMSO) solution, adds 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) and activates.Above-mentioned solution is slowly dripped the coupling carrying out Human factor IX and BSA in bovine serum albumin (BSA) solution, reaction product phosphate buffered saline buffer (PBS) dialysis, obtains the conjugate of Human factor IX and carrier proteins BSA.Detect by immune mouse tail vein by enzyme linked immunosorbent assay after four immunity, get serum titer and be greater than the splenocyte of the mouse of 1:10000 and myeloma cell SP2/0 merges; With containing xanthoglobulin (H), aminopterin-induced syndrome (A) and thymidine (T) selectivity nutrient solution (HAT, be purchased from SIGMA company) and 20% foetal calf serum RPMI-1640 (being purchased from HYCLONE company) nutrient solution cultivate, merge latter seven days cells and start to form colony; Carry out bag quilt with Human factor IX antigen, screened by enzyme-linked immunosorbent assay (ELISA); By four time clonings, finally obtain the hybridoma cell strain of a strain stably excreting anti-human coagulation factor IX monoclonal antibody, called after F9-M-01.Apply this strain of hybridoma with 1 × 10
6individual/amount abdominal injection only uses the Balb/c female mice in the 8-10 of whiteruss sensitization all ages in advance, gathers ascites after 10-14 days, and it is positive for detecting ascites by enzyme linked immunosorbent assay, and proves that it can identify Human factor IX by Western blotting.The monoclonal antibody hypotype measuring hybridoma cell strain F9-M-01 secretion through mouse monoclonal antibody subclass parting kit is IgM.
Embodiment
1, material and instrument
1.1 cell strains, tissue sample and animal
A. myeloma cell (SP2/0): derive from ATCC (American Type Culture collection warehousing).
B.Balb/c mouse: purchased from Tsing-Hua University's animal platform.
1.2 main agents
A. Freund's complete adjuvant, freund 's incomplete adjuvant, HAT, HT, TMB, PEG (molecular weight 4000), EDC: available from Sigma;
B.HRP marks sheep anti-mouse igg: purchased from Gibco company;
C.RPMI1640 substratum: purchased from HyClone company;
D. cellulose acetate film (NC): purchased from Hybond company;
E. foetal calf serum: purchased from PAN company;
F. mouse monoclonal antibody subclass parting kit: divide department purchased from Thermo;
G. bag is buffered liquid: add the sodium carbonate of 0.795g and the sodium bicarbonate composition of 1.465g in every 500ml deionized water, pH value is 9.6.
H. substrate buffer solution: add the disodium hydrogen phosphate of 9.2g and the citric acid composition of 2.55g in every 500ml deionized water, pH value is 5.0.
I.TMB (tetramethyl benzidine) uses liquid: TMB (10mg/5ml dehydrated alcohol) 0.5ml substrate buffer solution (pH5.5) 10ml30%H
2o
20.01 μ l;
J. incomplete RPMI-1640: the sodium bicarbonate 15ml adding 7.5% in the RPMI-1640 stoste of every 500ml forms.
K. complete RPMI-1640: the foetal calf serum composition adding 20ml in every incomplete RPMI-1640 of 80ml.
L. antibody diluent: the PBS of every 100ml adds the BSA composition of 0.1g.
M. lavation buffer solution: the PBS of every 100ml adds the Tween-20 composition of 0.5ml.
N.PEG prepares liquid: the 7.5% sodium bicarbonate composition adding DMSO and 0.1ml of 1ml in every incomplete RPMI-1640 of 8.9ml.
1.3 key instrument
A. CO2gas incubator: Heraeus company;
B. Bechtop: Suzhou treating plant instrument plant;
C. inverted microscope: Leica company;
D.37 a DEG C thermostat(t)ed water educates case: Yuyao City east electric instrument factory;
E. Polystyrene plastic plate (enzyme plate) 96 hole, Tissue Culture Plate (96 holes and 24 holes): NEST company;
F. whizzer: Eppendorf company
G. pure water instrument: Milipore company
H. miniature vertical disk electrophoresis groove: Bio-Rad company
I. microplate reader: TECAN company
2, method
2.1 cell cultures: myeloma cell SP2/0 is incubated in complete RPMI-1640, are placed in 37 DEG C, the carbon dioxide cell incubator of 5%, change liquid every other day 1 time, within 3-4 days, go down to posterity 1 time.
2.2 antigen preparations: get 1mg Human factor IX and be dissolved in 0.25mLDMSO solution, measure 2mmolEDC and add in above-mentioned solution, under room temperature, lucifuge acutely shakes 2h, and 4 DEG C of reactions are afterwards spent the night.The coupling of Human factor IX: above-mentioned solution is slowly dripped in BSA solution that (2mg albumen is dissolved in 1mL0.1molL
-1in sodium hydrogen carbonate solution), add magnetic rotor, be placed on magnetic force vibrator, lucifuge vibration 2h under room temperature; Reaction product is in 0.05molL
-1lucifuge dialysis 72h at 4 DEG C in PBS damping fluid, every 5h changes dialyzate once, obtains the conjugate of Human factor IX and carrier proteins BSA.
2.3.1 mouse immune
Antigen immune Balb/c mouse after coupling (4-8 age in week, female) 3, concrete steps are as follows:
A. first time immunity: Human factor IX-BSA100 μ g/ only, add equal-volume Freund's complete adjuvant fully mix after with 1ml/ only subcutaneous multi-point injection Balb/c mouse, 0.2ml/ point; 2 weeks, interval.
B. second time immunity: dosage and approach the same, this adds equal-volume freund 's incomplete adjuvant, 2 weeks, interval.
C. third time immunity: dosage is the same, this adds equal-volume freund 's incomplete adjuvant, get after 10 days by the tail vein of immune mouse (namely put 4 DEG C of refrigerator overnight after collection, next day stays after centrifugal 10 minutes supernatant for subsequent use with 2000 revs/min), measure serum titer by ELISA.
D. the 4th immunity: dosage is the same, does not add adjuvant, direct abdominal injection immunity.
2.3.2ELISA method detects and to be tired flow process by immune serum:
A. detect the day before yesterday, be buffered liquid dilution antigen Human factor IX to 15 μ g/ml with bag, add in Enzyme-linked Immunosorbent Assay plate, every hole 100 μ l, puts 4 DEG C of refrigerator overnight;
B. pour out the liquid wrapped in the enzyme linked plate holes of quilt next day, with lavation buffer solution washing totally 3 times, pat dry at every turn;
C. the serum to be checked (serum: PBS is respectively 1:100 with the capable gradient dilution of PBS is added respectively; 1:1000; 1:2000; 1:4000; 1:8000; 1:16000) 100 μ l/ holes, with the serum of non-immune Balb/c mouse for negative control, put 37 DEG C of constant water bath box 1 hour;
D. pour out the liquid in enzyme linked plate holes, with lavation buffer solution washing totally 3 times, pat dry at every turn;
E. every hole adds sheep anti-mouse igg antibody (dilution with the capable 1:5000 of antibody diluent) the 100 μ l of HRP mark, puts 37 DEG C of constant water bath box 1 hour;
F. pour out the liquid in enzyme linked plate holes, with lavation buffer solution washing totally 3 times, pat dry at every turn;
G. develop the color: the tmb substrate solution 50 μ l adding Extemporaneous in each reacting hole, 37 DEG C 15 minutes.
H. result judges: microplate reader detects.
Judge that three immune mouses all produce the antibody for Human factor IX by ELISA detected result, the mouse of getting wherein OD value the highest (tire and be greater than 10000) carries out subsequent step.
2.3.3 the preparation (merge and get Turnover of Mouse Peritoneal Macrophages the day before yesterday) of feeder cell:
Adopt the Balb/c female mice in 7 week age; Neck is drawn to put to death, be soaked in 75% alcohol and sterilize 5 minutes, with sterile scissors abdominal cut skin to expose peritonaeum, with the incomplete RPMI-1640 of asepsis injector per injection 8ml, repeatedly rinse, sucking-off washing fluid puts into 50ml centrifuge tube (sharing about 40ml), centrifugal 5 minutes with 1300 revs/min; Abandon with the resuspended precipitation of complete RPMI-1640 after supernatant, adjustment cell count is 4 × 10
5individual/ml, adds 24 porocyte culture plates, 500 μ l/ holes, puts into 37 DEG C, the carbon dioxide cell incubator of 5% cultivates.
2.3.4 cytogamy
A. observe myeloma cell SP2/0 state (requiring at logarithmic phase best), incomplete for 50%PEG and 20ml prepared RPMI-1640 is put into 37 DEG C of cell culture incubator preheatings; (preparation of 50%PEG: to put 4 DEG C of refrigerators for subsequent use in merging the PEG preparing 50% the day before yesterday, the PEG of 1g to put into after the sterilizing of penicillin bottle rearmounted pressure kettle inner high voltage about liquid 1ml, the PEG adding 1ml in the inner prepares the PEG that namely liquid obtain 50%);
B. SP2/0 is collected in 50ml sterile centrifugation tube and counts and (require that total cellular score is 1 × 10
6individual), centrifugal 1300 revs/min, totally 7 minutes;
C. get out sterilized petri dishes, screen cloth, penicillin bottle, draw neck to put to death immune by four times BLAB/c mouse, put in 75% alcohol and soak 5 minutes;
D. get SP2/0 murine myeloma cell to mix in the ratio of 1:5-1:10 with the Balb/c Mouse spleen cells of immunity, centrifugal 7 minutes of 1300rpm, abandons supernatant, touches at the bottom of pipe with palm, make cell evenly loose;
E. put 40 DEG C of metal bath preheatings, add the 1mL50%PEG4000 being preheated to 40 DEG C with 1mL suction pipe in 45s, limit edged vibrates gently;
F. in 90s, then add the 1640 incomplete substratum that 30mL is preheated to 37 DEG C, room temperature leaves standstill 10 minutes;
G.1000rpm centrifugal 5 minutes, abandon supernatant, it is resuspended containing 1640 substratum of 20% foetal calf serum and HAT to add 20mL, is dispensed on the culture plate of existing feeder cell, cultivates in 5% CO2gas incubator.
2.3.5 the selectivity of hybridoma is cultivated
Merge and start the nutrient solution that brings Selection In after 24 hours, detailed process is as follows:
A. merge after 24 hours, add in the complete RPMI-1640 of 20ml with HAT1.6ml and HT0.4ml and mix, add in 24 orifice plates, 500 μ l/ holes;
B. after 5 days, change liquid, after hole sucking-off 1500 μ l every in 24 orifice plates, add the complete RPMI-1640 (adding the HT composition of HAT and 0.6ml of 0.6ml in the complete RPMI-1640 of every 60ml) intending changing;
C. after 7 days, change liquid again, after hole sucking-off 1500 μ l every in 24 orifice plates, add the complete RPMI-1640 intending changing and (in the complete RPMI-1640 of every 60ml, add the HT composition of 1.2ml.)。
Carrying out step c after 3 to 7 days, collecting in 24 orifice plates the cell grown in the culture hole of cell colony, carry out subsequent step.
2.3.6 the screening of hybridoma
By the hybrid tumor cell monoclonal (mono-clonal method is with reference to 2.3.7 below) obtained in 2.3.5, get its culture supernatant and carry out following step:
A. quilt is wrapped: being buffered liquid by antibody dilution to protein content with the carbonate bag of 0.05MpH9.6 is 2 μ g/ml.In the reacting hole of each polystyrene board, add 50 μ l, 4 DEG C are spent the night.Next day, discard solution in hole, wash 3 times with lavation buffer solution, each 3 minutes (being called for short washing, lower same).
B. close: in the reacting hole of each polystyrene board, add skim-milk (BD company) the PBS300 μ l containing 5%, put 37 DEG C and hatch 2 hours, then wash.
C. application of sample: the measuring samples 50 μ l adding certain dilution in above-mentioned wrapped by reacting hole in, put 37 DEG C and hatch 1 hour, then wash (doing blank well, negative control hole and Positive control wells) simultaneously.
D. add enzyme labelled antibody: in each reacting hole, add with diluted enzyme labelled antibody (by specification dilutes) 50 μ l.Hatch 1 hour for 37 DEG C, washing.
E. add substrate solution colour developing: the tmb substrate solution 50 μ l adding Extemporaneous in each reacting hole, 37 DEG C 15 minutes.
F. termination reaction: add 2M sulfuric acid 50 μ l in each reacting hole.
G. result judges: can in white background, and directly detect by an unaided eye result: in reacting hole, color is darker, and positive degree is stronger, and negative reaction is colourless or extremely shallow, foundation be the depth of color, represent with "+", "-" number.Also in 450nm and 630 double UV check OD values on ELISA detector, to survey each hole OD value after blank control wells zeroing, if be greater than 2.1 times of the negative control OD value of regulation, the positive can be.
H. mark positive hole, select the cell in positive hole to carry out mono-clonal.
I. result: first time is detected supernatant by microplate reader and obtains 15 strain positive hybridoma cells altogether.Positive hybridoma is carried out mono-clonal (see 2.3.7) by limiting dilution assay.
2.3.7 the mono-clonal of hybridoma, enlarged culturing and frozen
The cloning of hybridoma adopts limiting dilution assay to carry out, and concrete grammar is as follows:
A. get and merge rear hybridoma in sterile centrifugation tube, the HT mixing of 3.2ml is added again after adding complete RPMI-1640 to 80ml, this suspension mixed is added in 96 porocyte wall panels respectively, 100 μ l/ holes, last three holes (H10, H11, H12) in the lower right corner of every block plate do not add feeder cell suspension in order to dilution use, the plate added are put into 37 DEG C, the carbon dioxide cell incubator of 5%;
B. will cell suspension (with 200 μ l rifle featheriness) in the hole of mono-clonal counting, this cell suspension is moved in the dilution holes H10 in the plate having feeder cell, from H10 hole, take out 20 μ l cell suspensions put into the complete RPMI-1640 in H11 hole and do 10 times of dilutions, then from H11 hole, take out 20 μ l put into H12 hole and do 10 times of dilutions;
C. it is 1 cell/100 μ l in theory that complete 1640 liquid of cell suspension got in H12 hole are diluted to, then this diluent is instilled respectively (it is 48 holes that rear point of every hole dilution is dripped) in 96 orifice plates containing feeder cell suspension, every hole 100 μ l; Put 37 DEG C, the carbon dioxide cell incubator of 5% cultivates;
D.1 mark mono-clonal hole after week, with ELISA method (the same 2.3.2), the supernatant in mono-clonal hole is detected, select the cloning that the cell in positive hole carries out next time, so repeatedly obtaining after three times can hybridoma 8 strain (all mono-clonal holes of carrying out a strain cell cloning next time of cloning are the positive) of stably excreting monoclonal antibody;
E. result: the cell in mono-clonal hole is proceeded in 24 orifice plates and cultivate (1 hole turns 1 hole), be divided into 2 holes after 3 days in 24 orifice plates, then be divided into 4 holes after 3 days, then after 2 days, the cell in this 4 hole is proceeded to 50ml Tissue Culture Flask enlarged culturing in the lump.Its strongest positives strain of hybridoma called after F9-M-01.
F. hybridoma is frozen: when 50ml culturing bottle inner cell is paved with bottle floorage about 70% → the cell suction pipe in culturing bottle is blown and beaten, it is made to suspend completely, cell suspension to be moved in 50ml sterile centrifugation tube and to count, centrifugal 1200 revs/min, 6 minutes → abandon supernatant, with cells frozen storing liquid (cells frozen storing liquid composition: 50% foetal calf serum; 40% incomplete RPMI-1640; 10%DMSO) resuspended precipitation, regulates cell density to be 1 × 10
7/ L → be sub-packed in cryopreservation tube, 1ml/ prop up → put and be concealed in liquid nitrogen by frozen cell-70 DEG C of refrigerator overnight → next day.
2.3.8 a large amount of productions of monoclonal antibody
For hybridoma cell strain F9-M-01, the Balb/c female mice getting 10 8-10 age in week first uses whiteruss 0.3ml/ abdominal injection pre-sensitization, and after 1 week, collection hybridoma cell strain F9-M-01 is with 1 × 10
6the mouse of the above-mentioned pre-sensitization of amount abdominal injection of individual/(normal saline is only diluted to 1ml/), observe after 10-14 days mouse abdominal distension obviously, be slow in action, anorexia and hair entanglement time collect ascites, neck is drawn to put to death mouse, with clean dropper, ascites is sucked in centrifuge tube, centrifugal 2000 revs/min, 5 minutes.
Result: detect ascites through ELISA method and be the positive.
The qualification of 2.4 monoclonal antibody subclass
Adopt mouse monoclonal antibody subclass parting kit (Thermo company), concrete steps are with reference to its specification sheets.
Result: through kit measurement, the monoclonal antibody hypotype that gained hybridoma cell strain F9-M-01 secretes is IgM.
2.5 Western blottings (Westernblotting)
2.5.1 SDS-PAGE (SDS-PAGE)
A. conventionally preparative separation glue and spacer gel;
B. get a certain amount of protein example to mix, in 100 DEG C of sex change 5 minutes with isopyknic 2 × SDS sample-loading buffer (100mmol/lTrisbase, 200mmol/l dithiothreitol (DTT), 4%SDS, 0.2 bromine Finland, 20%Glycerol);
C. load in discontinuous polyethylene acrylamide gel sample well according to every swimming lane 100 μ g;
D. gel base is arrived at constant voltage 50V (spacer gel)/100V (separation gel) electrophoresis to tetrabromophenol sulfonphthalein;
E. take out gel and be used for Westernblotting.
2.5.2Westernblotting
A.SDS-PAGE gel soaks 10-20 minute through transfering buffering liquid (25mmol/lTrisbase, 192mmpl/lDlycin, 20% formaldehyde);
B. according to the order assembled box type transfer tower of 3 filter paper, gel, NC film, 3 filter paper, and it is loaded transfer device with the direction of NC film anode;
C. under room temperature condition, current stabilization 0.8mA/cm
2electrotransfer, 90 minutes time;
D. with ponceau dye liquor, NC film is dyeed, with each band position of marker pen labelled protein molecular weight standard amount, then wash away ponceau with deionized water;
E.NC film closes 2 hours through 10% skim-milk in room temperature;
F. be diluted in 5% skim-milk antibody (freshly prepd monoclonal antibody 1:1000, anti-β-actin1:5000) 4 DEG C of overnight incubation, TBST (10mmol/lTrisbase, 150mmol/lNaCl, 0.05%Tween20, pH8.0) shakes and washes 4 times × 15 minutes;
G. the sheep anti-mouse igg marked with the HRP being diluted in 4% skim-milk was in incubated at room 4 hours;
H.TBST shakes and washes totally 4 times, 15 minutes/time;
I. A+B liquid in balanced mix ECL Color Appearance System, drops to NC film;
J. imaging.
K. result: can Human factor IX be identified by the monoclonal antibody that hybridoma F9-M-01 detects the generation of this hybridoma cell strain of proof through the ascites that mouse peritoneal injection produces through WesternBlot method.
Claims (6)
1. prepare a method for the hybridoma cell strain of secretion anti-human coagulation factor IX monoclonal antibody, it is characterized in that said method comprising the steps of:
1) prepare immunizing antigen: by homotype bifunctional protein linking agent by Human factor IX and bovine serum albumin coupling, obtain Human factor IX-bovine serum albumin conjugate;
2) immune mouse: adopt low dose repeatedly immunization protocol, utilize step 1) in Human factor IX-bovine serum albumin conjugate acting immune antigen of obtaining, immunity is carried out to Balb/c mouse;
3) cytogamy: by SP2/0 murine myeloma cell and step 2) spleen cell of immune Balb/c mouse that obtains merges, in the RPMI-1640 substratum containing HAT, carry out selectivity cultivation;
4) selectivity of hybridoma is cultivated: in step 3) in merge after cell after selectivity is cultivated in HAT substratum, select the fused cell of Mouse spleen cells and mouse hybridoma cell SP2/0; And
5) mono-clonal: by step 4) in the positive hybridoma cell that obtains carry out repeatedly through limiting dilution assay the hybridoma cell strain that mono-clonal obtains stably excreting anti-human coagulation factor IX monoclonal antibody.
2. method according to claim 1, it is characterized in that described homotype bifunctional protein linking agent is selected from: NHS ester class linking agent such as N-hydroxy-succinamide (NHS), dithio two (succinyl phosphorons amino propyl acid ester) (DSP) and 3, 3'-dithio two (sulfonic acid Succinimidyl Propionate) (DTSSP), succsinic acid suberic acid diimine (DSS) and butadiene-styrene copolymer (BS), tartrate two succinimide ester (DST) and Sulfo-DST, two (2-[succinimidyloxycarbonyl oxygen) ethyl) sulfone (BSOCOES) and sulfo-BSOCOES, ethylene glycol bis [succinimido succinic acid] (EGS) and sulfo-EGS, succsinic acid suberic acid glutarate (DSG), N, N'-bis-succinimidyl carbonate (DSC), carbodiimide class is 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) such as, polyurethane class linking agent is dimethyl diiminoester (DMA), dimethylimino ester (DMP), dimethyl benzene two acid imide ester (DMS), ditertiary butyl peroxide (DTBP) such as, sulfydryl response class linking agent is Isosorbide-5-Nitrae-two (3`-[2`-disulfide group pyridine] propionic acid amido) butane (DPDPB) such as, 1,5-bis-fluoro-2,4-dinitrobenzenes (DFDNB) of difluoro bezene derivative, two (the fluoro-3-nitrophenyl of 4-) sulfoxide (DFDNPS) etc., photoreactivity linking agent class linking agent such as two-[β-(azido-salicyloyl is amino) ethyl] disulphide (BASED) etc., aldehyde crosslinking agent is formaldehyde, glutaraldehyde etc. such as, the BDO glycidaldehyde etc. of di-epoxide class, hydrazides class linking agent such as adipic dihydrazide, carbohydrazide etc., double derivative thing class linking agent is such as by the o-tolidine, bis-diazotized benzidine etc. of diazonium, and two alkyl halide.
3. method according to claim 2, is characterized in that described homotype bifunctional protein linking agent is carbodiimide proteinoid linking agent.
4. method according to claim 3, is characterized in that described carbodiimide proteinoid linking agent is EDC.
5. the hybridoma cell strain of the secretion anti-human coagulation factor IX monoclonal antibody prepared by the method according to any one of claim 1-4.
6. hybridoma cell strain according to claim 5, it is characterized in that: described hybridoma cell strain is called after F9-M-01, be preserved in China typical culture collection center on November 6th, 2015, deposit number is the hybridoma cell strain of CCTCCC2015197.
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| CN112094346A (en) * | 2020-06-01 | 2020-12-18 | 普众发现医药科技(上海)有限公司 | Monoclonal antibody of mouse anti-cell single-chain transmembrane glycoprotein CD142 capable of being applied to tumor cell capture |
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| CN103237811A (en) * | 2010-05-06 | 2013-08-07 | 诺瓦提斯公司 | Compositions and methods of use for therapeutic low density lipoprotein-related protein 6 (LPR6) multivalent antibodies |
| CN103396494A (en) * | 2013-04-27 | 2013-11-20 | 江苏省疾病预防控制中心 | Monoclonal antibody of blood coagulation factor IX |
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| CN103237811A (en) * | 2010-05-06 | 2013-08-07 | 诺瓦提斯公司 | Compositions and methods of use for therapeutic low density lipoprotein-related protein 6 (LPR6) multivalent antibodies |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112094346A (en) * | 2020-06-01 | 2020-12-18 | 普众发现医药科技(上海)有限公司 | Monoclonal antibody of mouse anti-cell single-chain transmembrane glycoprotein CD142 capable of being applied to tumor cell capture |
| CN112094346B (en) * | 2020-06-01 | 2024-01-05 | 普众发现医药科技(上海)有限公司 | Monoclonal antibody of mouse anti-cell single-chain transmembrane glycoprotein CD142 capable of being applied to tumor cell capture |
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