CN105349495B - The hybridoma cell strain of anticoagulin IX monoclonal antibody - Google Patents

Abstract

The present invention relates to the hybridoma cell strains and preparation method thereof of secretion anti-human coagulation factor IX monoclonal antibody.Balb/c mouse is immunized as antigen by the conjugate after using bovine serum albumin(BSA) BSA and Human factor IX to be coupled in the present invention, its spleen cell and SP2/0 myeloma cell is taken to carry out cell fusion, selective culture is carried out with HAT culture medium, using limiting dilution assay by multiple cloning, finally screening obtains the hybridoma cell strain of stably excreting anti-human coagulation factor IX monoclonal antibody.Strain of hybridoma strain prepared in accordance with the present invention is preserved in China typical culture collection center, and deposit number is CCTCC C2015197.

Description

The hybridoma cell strain of anticoagulin IX monoclonal antibody

Technical field

The present invention relates to hybridoma cell strains and preparation method thereof, and in particular, to anti-human coagulation factor IX monoclonal is anti- Hybridoma cell strain of body and preparation method thereof.

Background technique

Coagulation factor is a kind of key protein ingredient for participating in coagulation process.Its physiological action is, in angiorrbagia When be activated together with platelet adhesion mend plug blood vessel on leak.This process is referred to as blood coagulation.In haemophiliac In, some patient is hemophilia B, i.e. hemophilia B, and hemophilia B (hemophilia B) also known as blood plasma blood coagulation are living Enzyme component (PTC) deficiency disease or Section IX factor deficiency.This type of clinical manifestation exactly likes A type, and mode of inheritance is also x linked recessive Heredity, since heterozygote IX factor active is only normal 1/3, certain heterozygotes may occur in which symptom, therefore female patients are more compared with A type See.Such patient mainly lacks the plasma thromboplastin component that normal person is possessed, and at present still without the method for radical cure, can only pass through infusion Human factor IX substitute products are treated.

Human factor IX molecular structure is complicated, molecular weight 60-80kDa.Its content in blood plasma is only 0.1-0.2 μg/mL.Since its extractive technique is extremely difficult, and China is relatively backward in extensive affinity chromatography technology, does not have still at present High-purity blood clotting factors product of affinity chromatography preparation comes out.Therefore, plasma thromboplastin component on domestic market is caused to produce Product wretched insufficiency cannot be sufficiently fed the case where domestic hemophilia B patient uses, often will appear off-drug, endanger state's troubles The health and lives safety of person.

The difficult point of the chromatography technology of plasma protein is the high viscosity of blood plasma and the enormousness of single treatment.For The separation of the micro plasma protein of blood clotting factors, affinity chromatography are optimal way of purification.Currently, most enterprises in the world, packet It includes the method that maximum blood product enterprise CSL also uses cold ethanol joint chromatography and carrys out separated plasma albumen.In last century 90 Age, hundred it is special prepared the ultrapure VIII factor and the VII factor using antibody affinity chromatography method, specific activity up to 3000IU/mg, The general activity rate of recovery is more than 30%.And China prepares plasma thromboplastin component then still using the crude acquisition of cryoprecipitate, specific activity at present It is lower than 100IU/mg, activity recovery is about 6% or so more.

Therefore, in order to increase substantially the separative efficiency of coagulation factor using affinity chromatography, key technology bottleneck is system Standby effective affinity chromatography antibody.Thus, there is the demands to specific coagulation factors IX monoclonal antibody in this field.

Summary of the invention

To solve the above-mentioned problems, the present invention provides a kind of hybridomas for secreting anti-human coagulation factor IX monoclonal antibody Cell strain and preparation method thereof.

In the first aspect, the present invention provides a kind of hybridomas for preparing secretion anti-human coagulation factor IX monoclonal antibody The method (being also referred to as " method of the invention " sometimes herein) of cell strain, the described method comprises the following steps:

1) immunizing antigen is prepared: by homotype bifunctional protein crosslinking agent that Human factor IX and bovine serum albumin(BSA) is even Connection, obtains Human factor IX-bovine serum albumin(BSA) conjugate；

2) mouse is immunized: using low dose of long period immunization protocol, utilizing Human factor IX-ox obtained in step 1) Seralbumin conjugate acting immune antigen, is immunized Balb/c mouse；

3) cell fusion: the spleen for the immune Balb/c mouse that SP2/0 murine myeloma cell and step 2) are obtained is thin Born of the same parents' fusion carries out selective culture in the RPMI-1640 culture medium containing HAT；

4) the selectivity culture of hybridoma: fused cell is selectively trained in HAT culture medium in step 3) After supporting, the fused cell of Mouse spleen cells and mouse hybridoma cell SP2/0 is selected；And

5) monoclonal: the hybridoma for being detected as the positive obtained in step 4) is more through limiting dilution assay progress Secondary monoclonal obtains the hybridoma cell strain of stably excreting anti-human coagulation factor IX monoclonal antibody.

In the second aspect, the present invention provides the hybridoma cell strains of secretion anti-human coagulation factor IX monoclonal antibody (hereinafter sometimes called " hybridoma cell strain of the invention "), the hybridoma cell strain is to prepare by means of the present invention 's.

In a preferred embodiment, strain of hybridoma strain prepared by the method for the present invention was in 2015 November 6 was preserved in China typical culture collection center, address are as follows: in the preservation of Wuhan City, Hubei Province Wuchang District Wuhan University The heart, deposit number are CCTCC C2015197, culture title are as follows: hybridoma cell strain F9-M-01.

Specific embodiment

Definition:

" protein-crosslinking agent " is small molecule compound, and molecule both ends respectively have one or more for special base Group (- NH₂,-COOH ,-HS etc.) reactive terminal, can be coupled respectively with the active group in biomolecule, to make this A little molecules are combined together.Protein-crosslinking agent can be divided into homotype bi-functional cross-linking agent (also referred to as " homologous crosslinking agent "), special-shaped pair Functional cross-link agent (also referred to as " heterologous crosslinking agent ") and photoactivated cross-linking agent (also referred to as " photoreactivity crosslinking agent ") three classes.

The molecule both ends reactive terminal having the same or priming reaction group of homotype bifunctional protein crosslinking agent.It is common Homotype bi-functional cross-linking agent be well known in the present art, non-limiting example includes but is not limited to that NHS esters crosslinking agent is all Such as bis- thiobis (sulfonic acid of n-hydroxysuccinimide (NHS), two thiobis (succinyl phosphorons amino propyl acid ester) (DSP) and 3,3'- Succinimidyl Propionate) (DTSSP), succinic acid suberic acid diimine (DSS) and butadiene-styrene copolymer (BS), Two succinimide ester of tartaric acid (DST) and Sulfo-DST, bis- (2- [succinimidyloxycarbonyl oxygen) ethyls) sulfone (BSOCOES) and sulfo-BSOCOES, ethylene glycol bis- [succinimido succinic acid] (EGS) and sulfo-EGS, succinic acid Suberic acid glutarate (DSG), N, bis- succinimidyl carbonate of N'- (DSC)；Carbodiimide class such as 1- ethyl -3- [3- Dimethylaminopropyl] carbodiimide hydrochloride (EDC)；Imido esters crosslinking agent such as dimethyl diiminoester (DMA), diformazan Base imines ester (DMP), two acid imide ester (DMS) of dimethyl benzene, di-tert-butyl peroxide (DTBP)；Sulfydryl reacts class crosslinking agent Such as 1,4- bis- (3`- [2`- disulfide group pyridine] propionic acid acylamino-) butane (DPDPB)；The 1,5- bis- fluoro- 2 of difluoro bezene derivative, 4- dinitrobenzene (DFDNB), bis- (the fluoro- 3- nitrobenzophenone of 4-) sulfoxides (DFDNPS) etc.；Photoreactivity crosslinking agent class crosslinking agent is all Such as double-[β-(azido salicyl amino) ethyl] disulphide (BASED)；Aldehyde crosslinking agent such as formaldehyde, glutaraldehyde Deng；1,4- butanediol glycidaldehyde of di-epoxide class etc.；Hydrazides crosslinking agent such as adipic dihydrazide, carbohydrazide Deng；Double derivative species crosslinking agent is by the o- tolidine of diazonium, bis-diazotized benzidine etc.；And double alkyl halides Deng.

Photoreactivity crosslinking agent is the crosslinking agent with photoreactive group.The priming reaction base of homotype bi-functional cross-linking agent Group is also possible to photoreactive group.

Type, the reactive group, protein binding group, light work of part homotype bifunctional protein crosslinking agent are listed in table 1 The properties such as the property changed.

Table 1

" antibody " (antibody) refers to that the immune system of body under antigenic stimulus, is increased by bone-marrow-derived lymphocyte or memory cell Grow the immunoglobulin that the thick liquid cell being divided into is generated, can specifically bind with corresponding antigens.

The present invention provides a kind of preparation methods of hybridoma cell strain for secreting anti-human coagulation factor IX monoclonal antibody.

In one embodiment, the method for the present invention includes the following steps:

1) immunizing antigen is prepared: by homotype bifunctional protein crosslinking agent by Human factor IX and bovine serum albumin(BSA) (BSA) it is coupled, obtains Human factor IX-BSA conjugate；

2) mouse is immunized: using low dose of long period immunization protocol, utilizing Human factor IX-obtained in step 1) BSA conjugate acting immune antigen, is immunized Balb/c mouse；

3) cell fusion: the spleen for the immune Balb/c mouse that SP2/0 murine myeloma cell and step 2) are obtained is thin Born of the same parents' fusion carries out selective culture in 1640 culture mediums containing HAT；

4) the selectivity culture of hybridoma: fused cell is selectively trained in HAT culture medium in step 3) After supporting, the fused cell of Mouse spleen cells and mouse hybridoma cell SP2/0 is selected；And

5) monoclonal: the hybridoma for being detected as the positive obtained in step 4) is more through limiting dilution assay progress Secondary monoclonalization obtains the hybridoma cell strain of stably excreting anti-human coagulation factor IX monoclonal antibody.

In the method for the invention, any homotype bifunctional protein crosslinking agent as known in the art can be used, preferably Carbodiimide albuminoid crosslinking agent, more preferable EDC.

In the present invention, animal immune is using low dose of multiple immunization protocol, conventional with 100~200 μ by taking mouse as an example The dose immunization of g/ only, is immunized 3~4 times.One specific immunization protocol example is: the female using immunizing antigen to 7 week old Balb/c mouse is immunized, and by the immunizing dose of 100 μ g/ only, 1:1 is mixed by volume with Freund's complete adjuvant, and emulsification reaches To Water-In-Oil shape, by the comlete antigen of emulsification, immune mouse is subcutaneously injected in the nape of the neck multiple spot, after 2 weeks, with same antigen and waits bodies Long-pending incomplete Freund's adjuvant emulsification, 100 μ g/ only carry out booster immunization, and row third time is immune after 2 weeks, and dosage and method are the same as the Secondary immunity carries out last time to mouse before cell fusion and impacts immune, the immunizing dose intraperitoneal injection of 100 μ g/ only.

In the method for the invention, the method for cell fusion is unrestricted, can using method as known in the art and Process.

In the method for the invention, the method for antibody test is unrestricted, can use detection side as known in the art Method, for example, ELISA method can be used.

By means of the invention it is also possible to filter out the hybridoma of secretion anti-human coagulation factor IX monoclonal antibody Strain.

As example, the hybridoma cell strain of secretion anti-human coagulation factor IX monoclonal antibody of the invention is provided below An exemplary fabrication flow:

1) preparation of immunizing antigen:

It takes 1mg Human factor IX to be dissolved in 0.25mLDMSO solution, EDC is added, be protected from light that be aggressively shaken 2 small at room temperature When, 4 DEG C reaction overnight, above-mentioned solution is slowly added dropwise in BSA solution, be protected from light at room temperature concussion 2 hours, reaction product in 0.05molL^-1It is protected from light dialysis 72 hours in PBS buffer solution, obtains the conjugate of Human factor IX Yu carrier protein BSA.

2) mouse immune:

Use Human factor IX-BSA conjugate as immunizing antigen, the female Balb/c mouse of 7 week old is exempted from Epidemic disease, by the immunizing dose of 100 μ g/ only, 1:1 is mixed by volume with Freund's complete adjuvant, and emulsification reaches Water-In-Oil shape, will be emulsified Comlete antigen, immune Balb/c mouse is subcutaneously injected in the nape of the neck multiple spot, after 2 weeks, not with same antigen and isometric Freund Freund's complete adjuvant emulsification, 100 μ g/ only carry out booster immunization, and row third time is immune after 2 weeks, and method is immune with second, melt in cell Last time is carried out to mouse before closing and impacts immune, the immunizing dose intraperitoneal injection of 100 μ g/ only.

3) the selectivity culture of cell fusion and hybridoma:

SP2/0 murine myeloma cell is taken to mix with immune Balb/c Mouse spleen cells in the ratio of 1:5-1:10 Even, 1300rpm is centrifuged 7 minutes, sets the preheating of 40 DEG C of metal baths, is added in 45s with 1mL suction pipe and is preheated to 40 DEG C of 1mL50% PEG4000, side edged gently vibrate, and 1640 incomplete culture mediums that 30mL is preheated to 37 DEG C are then added in 90s, and room temperature is quiet It setting 10 minutes, 1000rpm is centrifuged 5 minutes, abandons supernatant, and 1640 culture mediums of the 20mL containing 20% fetal calf serum and HAT are added and are resuspended, It is dispensed on the culture plate of existing feeder cells, in 5% carbon dioxide incubator culture, after 7 days, observes the growth shape of cell Condition is swapped out 1/2 culture medium with 1640 culture mediums containing 20% fetal calf serum and HT, after 14 days, use instead 20% fetal calf serum and HT1640 culture medium culture.

4) hybridoma of screening secretion anti-human coagulation factor IX antibody:

It is 2ug/ml that antibody, which is diluted to protein content, with the carbonate of 0.05M pH9.6 coating buffer, each Add 50 μ l in the reacting hole of polystyrene board, 4 DEG C overnight.Next day discards solution in hole, washes 3 times with washing buffer, and every time 3 Minute (referred to as washs, similarly hereinafter).Add skimmed milk power (BD company) PBS containing 5% in the reacting hole of each polystyrene board 300 μ l set 37 DEG C and are incubated for 2 hours, are washed out.Add certain diluted 50 μ l of measuring samples in the above-mentioned reacting hole being coated with In, it sets 37 DEG C and is incubated for 1 hour, be washed out (while doing blank well, negative control hole and Positive control wells).In each reacting hole In, the 50 μ l of enzyme labelled antibody (by specification is diluted) of diluted is added, 37 DEG C are incubated for 1 hour, are washed out. The 50 μ l of tmb substrate solution of Extemporaneous is added in each reacting hole, develops the color 15 minutes at 37 DEG C.2M is added in each reacting hole 50 μ l of sulfuric acid terminates reaction.In the result that in white background, directly detects by an unaided eye: color is deeper in reacting hole, and positive degree is got over By force, negative reaction is colourless or extremely shallow, according to the depth of be in color, is indicated with "+", "-" number.As a kind of alternative judgement Mode can also be surveyed OD value on ELISA detector, be detected in 450nm and 630 dual wavelengths, after being returned to zero with blank control wells Each hole OD value is surveyed, it is as positive if more than 2.1 times of defined negative control OD value.

5) monoclonal:

By positive hybridoma through limiting dilution assay carry out multiple monoclonalization obtain the anti-human blood coagulation of stably excreting because The hybridoma cell strain of sub- IX monoclonal antibody.

In a preferred embodiment, strain of hybridoma strain prepared by the method for the present invention is named as F9-M-01, and China typical culture collection center, address are deposited on November 6th, 2015 are as follows: Wuhan City, Hubei Province Wuchang Wuhan University of area collection, deposit number are CCTCC C2015197.

The preparation method of hybridoma cell strains F9-M-01 the following steps are included:

Human factor IX (DMSO) solution is prepared, 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide (EDC) is added It is activated.Above-mentioned solution is slowly added dropwise and carries out the idol of Human factor IX and BSA in bovine serum albumin(BSA) (BSA) solution Connection, reaction product are dialysed with phosphate buffer (PBS), obtain the conjugate of Human factor IX Yu carrier protein BSA.Through four It is secondary it is immune after with enzyme-linked immunosorbent assay detection by immune mouse tail vein blood, take mouse of the serum titer greater than 1:10000 Splenocyte merged with myeloma cell SP2/0；With containing hypoxanthine (H), aminopterin-induced syndrome (A) and thymidine (T) choosing The RPMI-1640 (being purchased from HYCLONE company) of selecting property culture solution (HAT is purchased from SIGMA company) and 20% fetal calf serum culture Liquid culture, seven days cells initially form colony after fusion；It is coated with Human factor IX antigen, passes through Enzyme-linked Immunosorbent Assay Test (ELISA) screening；By four time clonings, one plant of stably excreting anti-human coagulation factor IX monoclonal antibody is finally obtained Hybridoma cell strain is named as F9-M-01.Using this strain of hybridoma with 1 × 10⁶It is a/only amount intraperitoneal injection in advance use The Balb/c female mice of the 8-10 week old of atoleine sensitization, acquires ascites, passes through enzyme-linked immunosorbent assay after 10-14 days Detecting ascites is the positive, and proves that it can identify Human factor IX by immunoblotting.Through mouse monoclonal antibody subclass The monoclonal antibody hypotype that parting kit measures hybridoma cell strain F9-M-01 secretion is IgM.

Embodiment

1, material and instrument

1.1 cell strains, tissue specimen and animal

A. it myeloma cell (SP2/0): derives from ATCC (American Type Culture collection warehousing).

B.Balb/c mouse: it is purchased from Tsinghua University animal platform.

1.2 main agents

A. Freund's complete adjuvant, freund 's incomplete adjuvant, HAT, HT, TMB, PEG (molecular weight 4000), EDC: are purchased from Sigma company；

B.HRP marks sheep anti-mouse igg: being purchased from Gibco company；

C.RPMI1640 culture medium: it is purchased from HyClone company；

D. cellulose acetate film (NC): it is purchased from Hybond company；

E. fetal calf serum: it is purchased from PAN company；

F. mouse monoclonal antibody subclass parting kit: purchased from Thermo points of departments；

G. it is coated with buffer: the sodium carbonate of 0.795g and the sodium bicarbonate group of 1.465g being added in every 500ml deionized water At pH value 9.6.

H. the disodium hydrogen phosphate of 9.2g and the lemon of 2.55g substrate buffer solution: are added in every 500ml deionized water Acid composition, pH value 5.0.

I.TMB (tetramethyl benzidine) uses liquid: TMB (10mg/5ml dehydrated alcohol) 0.5ml substrate buffer solution (pH5.5) 10ml 30%H₂O₂0.01μl；

J. 7.5% sodium bicarbonate 15ml group incomplete 1640 culture medium: is added in the 1640 culture medium stoste of every 500ml At.

K. the fetal calf serum composition of 20ml complete 1640 culture medium: is added in every incomplete 1640 culture medium of 80ml.

L. antibody diluent: the BSA composition of 0.1g is added in the PBS of every 100ml.

M. washing buffer: the Tween-20 composition of 0.5ml is added in the PBS of every 100ml.

N.PEG prepares liquid: 7.5% carbonic acid of the DMSO and 0.1ml of 1ml being added in every incomplete 1640 culture medium of 8.9ml Hydrogen sodium composition.

1.3 key instrument

A. carbon dioxide incubator: Heraeus company；

B. superclean bench: Suzhou cleaning equipment instrument plant；

C. inverted microscope: Leica company；

D.37 a DEG C thermostatted water educates case: Yuyao City east electric instrument factory；

E. 96 hole of Polystyrene plastic plate (ELISA Plate), tissue culture plate (96 holes and 24 holes): NEST company；

F. centrifuge: Eppendorf company

G. pure water meter: Milipore company

H. miniature vertical disk electrophoresis slot: Bio-Rad company

I. microplate reader: TECAN company

2, method

2.1 cell culture: myeloma cell SP2/0 is incubated in complete 1640 culture medium, is placed in the dioxy of 37 DEG C, 5% Change carbon cell incubator, changes liquid 1 time, pass on 1 time every other day within 3-4 days.

The preparation of 2.2 antigens: taking 1mg Human factor IX to be dissolved in 0.25mL DMSO solution, measures 2mmol EDC and adds Enter in above-mentioned solution, be protected from light be aggressively shaken 2h at room temperature, 4 DEG C of reactions later are overnight.The coupling of Human factor IX: will be above-mentioned molten Liquid is slowly added dropwise that (2mg albumen is dissolved in 1mL 0.1mol L in BSA solution^-1In sodium bicarbonate solution), magnetic rotor is added, is placed in On magnetic force oscillator, it is protected from light oscillation 2h at room temperature；Reaction product is in 0.05mol L^-1Dialysis is protected from light in PBS buffer solution at 4 DEG C 72h, every 5h replacement dialyzate is primary, obtains the conjugate of Human factor IX Yu carrier protein BSA.

2.3.1 mouse immune

Balb/c mouse (4-8 week old, female) 3 is immunized in antigen after coupling, the specific steps are as follows:

A. immune for the first time: Human factor IX-BSA100 μ g/, after adding isometric Freund's complete adjuvant to mix well With 1ml/ subcutaneous multi-point injection Balb/c mouse, 0.2ml/ point；Interval 2 weeks.

B. be immunized for second: dosage and approach are same as above, and this time add isometric freund 's incomplete adjuvant, are spaced 2 weeks.

C. third time is immune: dosage is same as above, and is this time added isometric freund 's incomplete adjuvant, is taken after 10 days and mouse is immunized Tail vein (sets 4 DEG C of refrigerator overnights after acquisition, next day stays supernatant spare after ten minutes with 2000 revs/min of centrifugations), passes through ELISA measures serum titer.

D. the 4th time it is immune: dosage is same as above, and adjuvant is not added, and directly intraperitoneal injection is immunized.

2.3.2ELISA method detection is by immune serum potency process:

A. detection the previous day dilutes antigen Human factor IX to 15 μ g/ml with coating buffer, enzyme linked immunological is added and inhales In attached plate, every 100 μ l of hole sets 4 DEG C of refrigerator overnights；

B. next day pours out the liquid in coated enzyme linked plate holes, with washing buffer washing totally 3 times, pats dry every time；

C. it is separately added into that (serum: PBS is respectively 1:100 with the serum to be checked of PBS row gradient dilution；1:1000；1: 2000；1:4000；1:8000；37 are set using the serum of non-immune Balb/c mouse as negative control in 1:16000) 100 hole μ l/ DEG C constant water bath box 1 hour；

D. the liquid in enzyme linked plate holes is poured out, with washing buffer washing totally 3 times, is patted dry every time；

E. sheep anti-mouse igg antibody (with the dilution of antibody diluent row 1:5000) 100 μ l of HRP label are added in every hole, set 37 DEG C constant water bath box 1 hour；

F. the liquid in enzyme linked plate holes is poured out, with washing buffer washing totally 3 times, is patted dry every time；

G. develop the color: being added the tmb substrate solution 50 μ l of Extemporaneous in each reacting hole, 37 DEG C 15 minutes.

H. result judgement: microplate reader detection.

Determine that three immune mouse generate the antibody for Human factor IX by ELISA testing result, takes wherein The mouse of OD value highest (potency is greater than 10000) carries out subsequent step.

2.3.3 the preparation (fusion the previous day takes Turnover of Mouse Peritoneal Macrophages) of feeder cells:

Using the Balb/c female mice of 7 week old；It draws neck to put to death, is soaked in 75% alcohol and sterilizes 5 minutes, cut with sterile Knife abdominal cut skin is to expose peritonaeum, and with the incomplete 1640 culture medium of asepsis injector per injection 8ml, repeated flushing is inhaled Flushing liquor is put into 50ml centrifuge tube and (shares about 40ml) out, is centrifuged 5 minutes with 1300 revs/min；It abandons after supernatant with complete 1640 Precipitating is resuspended in culture solution, and adjustment cell number is 4 × 10⁵A/ml, be added 24 porocyte culture plates, 500 holes μ l/, be put into 37 DEG C, 5% carbon dioxide cell incubator culture.

2.3.4 cell fusion

A. observe myeloma cell SP2/0 state (it is required that best in logarithmic growth phase), by prepared 50%PEG and The incomplete 1640 culture medium of 20ml is put into 37 DEG C of cell incubator preheatings；It (preparation of 50%PEG: is prepared in fusion the previous day It is spare that 50% PEG sets 4 DEG C of refrigerators, the PEG of 1g be put into penicillin bottle postposition pressure cooker after high pressure sterilization about liquid 1ml, the PEG that 1ml is added in the inner prepare liquid up to 50% PEG)；

B. SP2/0 is collected in 50ml sterile centrifugation tube and counted (it is required that total number of cells are 1 × 10⁶It is a), centrifugation 1300 Rev/min, totally 7 minutes；

C. sterilized petri dishes, sieve, penicillin bottle are got out, draws neck to put to death by four immune BLAB/c mouse, sets It is impregnated 5 minutes in 75% alcohol；

D. SP2/0 murine myeloma cell is taken to mix with immune Balb/c Mouse spleen cells in the ratio of 1:5-1:10 Uniformly, 1300rpm is centrifuged 7 minutes, abandons supernatant, is tapped tube bottom with palm, is kept cell loosely uniform；

E. the preheating of 40 DEG C of metal baths is set, is added in 45s with 1mL suction pipe and is preheated to 40 DEG C of 1mL50%PEG4000, side Edged gently vibrates；

F. 1640 incomplete culture mediums that 30mL is preheated to 37 DEG C are then added in 90s, are stored at room temperature 10 minutes；

G.1000rpm it is centrifuged 5 minutes, abandons supernatant, 1640 culture base weights of the 20mL containing 20% fetal calf serum and HAT are added It is outstanding, it is dispensed on the culture plate of existing feeder cells, in 5% carbon dioxide incubator culture.

2.3.5 the selectivity culture of hybridoma

Fusion was initially added into selection culture solution after 24 hours, and detailed process is as follows:

A. it after merging 24 hours, is added in the complete 1640 culture medium of 20ml and is mixed with HAT 1.6ml and HT 0.4ml, be added In 24 orifice plates, 500 holes μ l/；

B. liquid is changed after 5 days, after 1500 μ l are sucked out in hole every in 24 orifice plates, it is (every that the quasi- complete 1640 culture medium changed is added The HT composition of the HAT and 0.6ml of 0.6ml is added in the complete 1640 culture medium of 60ml)；

C. liquid is changed after 7 days again, after 1500 μ l are sucked out in hole every in 24 orifice plates, the quasi- complete 1640 culture medium changed is added (the HT composition of 1.2ml is added in the complete 1640 culture medium of every 60ml.).

After carrying out step c 3 to 7 days, the cell grown in the culture hole of cell colony in 24 orifice plates is collected, is carried out subsequent Step.

2.3.6 the screening of hybridoma

By the hybrid tumor cell monoclonal obtained in 2.3.5 (monoclonal method refers to following 2.3.7), its training is taken It supports supernatant and carries out following step:

A. be coated with: it is 2 μ g/ml that antibody, which is diluted to protein content, with the carbonate of 0.05M pH9.6 coating buffer. Add 50 μ l in the reacting hole of each polystyrene board, 4 DEG C overnight.Next day discards solution in hole, is washed 3 times with washing buffer, 3 minutes every time (referred to as washing, similarly hereinafter).

B. it closes: adding skimmed milk power (BD company) PBS300 μ l containing 5% in the reacting hole of each polystyrene board, set 37 DEG C are incubated for 2 hours, are washed out.

C. it is loaded: adding certain diluted 50 μ l of measuring samples in the above-mentioned reacting hole being coated with, it is small to set 37 DEG C of incubations 1 When, it is washed out (while doing blank well, negative control hole and Positive control wells).

D. enzyme labeling antibody: in each reacting hole, the enzyme labelled antibody of diluted is added, and (by specification carries out dilute Release) 50 μ l.37 DEG C are incubated for 1 hour, washing.

Plus substrate solution colour developing e.: be added the tmb substrate solution 50 μ l of Extemporaneous in each reacting hole, 37 DEG C 15 minutes.

F. it terminates reaction: 50 μ l of 2M sulfuric acid being added in each reacting hole.

G. result judgement: can be in the result that in white background, directly detects by an unaided eye: color be deeper in reacting hole, positive journey Degree is stronger, and to be colourless or extremely shallow, the depth of the be in color of foundation is indicated negative reaction with "+", "-" number.It can also be examined in ELISA It surveys on instrument in 450nm and 630 double UV check OD values, each hole OD value is surveyed after returning to zero with blank control wells, if more than defined yin Property control 2.1 times of OD value, it is as positive.

H. positive hole is marked, the cell in positive hole is selected to carry out monoclonal.

I. result: supernatant is detected by microplate reader for the first time and obtains 15 plants of positive hybridoma cells altogether.By positive hybridoma Carry out monoclonal by limiting dilution assay (see 2.3.7).

2.3.7 the monoclonal of hybridoma, expansion are cultivated and are frozen

The cloning of hybridoma is carried out using limiting dilution assay, and the specific method is as follows:

A. hybridoma adds after complete 1640 culture medium to 80ml is added in sterile centrifugation tube after taking fusion The HT of 3.2ml is mixed, this suspension mixed is separately added into 96 hole cell wall panels, 100 holes μ l/, the lower right corner of every block of plate Feeder cells suspension is not added for last three hole (H10, H11, H12) in case the plate added, is put into the dioxy of 37 DEG C, 5% by dilution use Change carbon cell incubator；

B. will the cell in the hole of monoclonal suspend (with 200 μ l rifle featheriness) and count, by this cell suspension It moves in the dilution holes H10 in the plate for having feeder cells, is put into the hole H11 from 20 μ l cell suspensions are taken out in the hole H10 with complete 1640 culture medium does 10 times of dilutions, then does 10 times of dilutions from taking out 20 μ l in the hole H11 and being put into the hole H12；

C. the cell suspension in the hole H12 is taken to be diluted to theoretically 1 cell/100 μ l with complete 1640 liquid, then this is dilute It releases liquid and is respectively dropped into 96 orifice plates containing feeder cells suspension (a point drop is 48 holes after every hole dilution), every 100 μ l of hole；Set 37 DEG C, 5% carbon dioxide cell incubator culture；

D.1 monoclonal hole is marked after week, and the supernatant in monoclonal hole is detected with ELISA method (the same 2.3.2), choosing sun Property hole in cell carry out cloning next time, obtain afterwards three times repeatedly can stably excreting monoclonal antibody 8 plants of hybridoma (into All monoclonal holes of one plant of cell cloning next time of row cloning are the positive)；

E. result: being transferred to culture in 24 orifice plates (1 hole turns 1 hole) for the cell in monoclonal hole, after 3 days in 24 orifice plates point At being divided into 4 holes behind 2 holes, then 3 days, then after 2 days the cell in this 4 hole is transferred to 50ml Tissue Culture Flask together and expands culture.Its The middle strongest strain of hybridoma of the positive is named as F9-M-01.

F. hybridoma freezes: when 50ml culture bottle inner cell is paved with bottom of bottle area about 70% → will be in culture bottle Cell blown and beaten with suction pipe, so that it is suspended completely, cell suspension moved into the centrifugation 1200 in 50ml sterile centrifugation tube and counting Rev/min, 6 minutes → abandon supernatant, with cells frozen storing liquid (cells frozen storing liquid composition: 50% fetal calf serum；40% incomplete 1640 training Nutrient solution；Precipitating 10%DMSO) is resuspended, adjusting cell density is 1 × 10⁷/ L → is sub-packed in cryopreservation tube, 1ml/ branch → set -70 DEG C of ice Case overnight → cell frozen is concealed in liquid nitrogen by next day.

2.3.8 the mass production of monoclonal antibody

By taking hybridoma cell strain F9-M-01 as an example, the Balb/c female mice of 10 8-10 week old is taken first to use atoleine Pre-sensitization is only injected intraperitoneally in 0.3ml/, and hybridoma cell strain F9-M-01 is collected after 1 week with 1 × 10⁶A/(same amount of normal saline Be diluted to 1ml/ only) amount the mouse of above-mentioned pre-sensitization is injected intraperitoneally, observed after 10-14 days mouse abdominal distension obviously, be slow in action, Ascites is collected when do not feel like eating and hair entanglement, neck is drawn to put to death mouse, is sucked ascites in centrifuge tube with clean dropper, centrifugation 2000 revs/min, 5 minutes.

As a result: being the positive through ELISA method detection ascites.

The identification of 2.4 monoclonal antibody subclass

Using mouse monoclonal antibody subclass parting kit (Thermo company), specific steps are referring to its specification.

As a result: through kit measurement, the monoclonal antibody hypotype of gained hybridoma cell strain F9-M-01 secretion is IgM.

2.5 immunoblottings (Western blotting)

2.5.1 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

A. conventionally preparative separation glue and spacer gel；

B. take a certain amount of protein example and isometric 2 × SDS sample-loading buffer (100mmol/l Trisbase, 200mmol/l dithiothreitol (DTT), 4%SDS, 0.2 bromine Finland, 20%Glycerol) mixing, it is denaturalized 5 minutes in 100 DEG C；

C. it is loaded in discontinuous polyethylene acrylamide gel sample well according to every 100 μ g of swimming lane；

D. gel bottom edge is arrived at constant pressure 50V (spacer gel)/100V (separation gel) electrophoresis to bromophenol blue；

E. it takes out gel and is used for Western blotting.

2.5.2Western blotting

The transferred buffer of a.SDS-PAGE gel (25mmol/l Tris base, 192mmpl/l Dlycin, 20% first Aldehyde) it impregnates 10-20 minutes；

B. tower is shifted according to the sequence assembled box type of 3 filter paper, gel, NC film, 3 filter paper, and it is faced south with NC film The direction of pole is packed into transfer device；

C. under room temperature, current stabilization 0.8mA/cm²Electrotransfer, the time 90 minutes；

D. NC film is dyed with Ponceaux dye liquor, with marker pen labelled protein molecular weight standard amount, each band institute is in place It sets, then Ponceaux is washed with deionized water；

E.NC film is closed 2 hours through 10% skimmed milk power in room temperature；

F. be diluted in 5% skimmed milk power antibody (freshly prepd monoclonal antibody 1:1000, anti-β-actin 1:5000) 4 DEG C be incubated overnight, TBST (10mmol/l Tris base, 150mmol/l NaCl, 0.05%Tween20, pH8.0), which shakes, to be washed 4 times × 15 minutes；

G. the sheep anti-mouse igg marked with the HRP for being diluted in 4% skimmed milk power was in incubation at room temperature 4 hours；

H.TBST, which shakes, washes totally 4 times, 15 minutes/time；

I. A+B liquid in mixed in equal amounts ECL Color Appearance System, is added dropwise to NC film；

J. it is imaged.

K. result: being detected with the ascites that hybridoma F9-M-01 is generated through mouse peritoneal injection through WesternBlot method proves The monoclonal antibody that the hybridoma cell strain generates can identify Human factor IX.

Claims (1)

1. a kind of hybridoma cell strain for secreting anti-human coagulation factor IX monoclonal antibody, it is characterised in that: the hybridoma is thin Born of the same parents' strain is to be named as F9-M-01, is preserved in China typical culture collection center on November 6th, 2015, deposit number is The hybridoma cell strain of CCTCC C2015197, wherein the monoclonal antibody hypotype of hybridoma cell strain F9-M-01 secretion For IgM.