CN105349524A - Hybridomas cell strain of anti-human coagulation factor VII polyclonal antibody - Google Patents
Hybridomas cell strain of anti-human coagulation factor VII polyclonal antibody Download PDFInfo
- Publication number
- CN105349524A CN105349524A CN201510792378.4A CN201510792378A CN105349524A CN 105349524 A CN105349524 A CN 105349524A CN 201510792378 A CN201510792378 A CN 201510792378A CN 105349524 A CN105349524 A CN 105349524A
- Authority
- CN
- China
- Prior art keywords
- factor vii
- coagulation factor
- linking agent
- mouse
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The invention relates to a hybridomas cell strain secreting anti-human coagulation factor VII polyclonal 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 VII 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, selecting fusion cell of the mouse spleen cell and the mouse myeloma cell SP2/0, and finally identifying the hybridomas cell strain secreting anti-human coagulation factor VII polyclonal antibody through ELISA. The prepared hybridomas cell strain is preserved in China Center for Type Culture Collection with the preservation number of CCTCC C2015195.
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 VII polyclonal antibody and preparation method thereof.
Background technology
Human blood coagulation factor VII is a kind of single chain glycoprotein of the vitamin K dependent synthesized by liver cell, is the initiation factor of body external source coagulation pathway.The concentration of human blood coagulation factor VII in blood plasma is only (0.5 ~ 2) mg/L, and the transformation period is 6 ~ 8 hours, no matter is that primary or insecondary proconvertin lack, all likely causes hemorrhage, even threat to life in human body.Research confirm, human blood coagulation factor VII not only has important effect in physiological hemostasis, and the height gene pleiomorphism of its blood plasma level also with some diseases there is dependency.Human blood coagulation factor VII polyclonal antibody, except for except fundamental research, also can be used for preparing high sterling human blood coagulation factor VII and preparing human blood coagulation factor VII detection reagent.In addition, human blood coagulation factor VII coagulation activity and antigenic content detect and can utilize human blood coagulation factor VII antibody.The domestic report having not yet to see development human plasma proconvertin polyclonal antibody, also without high purity human blood coagulation factor VII product, human blood coagulation factor VII detection reagent used substantially purchased from abroad, and will first must prepare the how grand antibody of human blood coagulation factor VII by the high sterling human blood coagulation factor VII of preparation and human blood coagulation factor VII detection reagent.
Thus, there is the demand to human blood coagulation factor VII polyclonal 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 VII polyclonal 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 VII polyclonal antibody, said method comprising the steps of:
1) prepare immunizing antigen: by homotype bifunctional protein linking agent by human blood coagulation factor VII and bovine serum albumin coupling, obtain human blood coagulation factor VII-bovine serum albumin conjugate;
2) immune mouse: adopt low dose repeatedly immunization protocol, utilize step 1) in human blood coagulation factor VII-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) identify: detecting step 4) in the culture supernatant of hybridoma of fusion that obtains, the hybridoma cell strain of qualification secretion anti-human coagulation factor VII polyclonal 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 VII polyclonal 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 CCTCCC2015195, and culture name is called: hybridoma cell strain F7-P-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 and a kind ofly secrete hybridoma cell strain of anti-human coagulation factor VII polyclonal antibody and preparation method thereof.
In one embodiment, method of the present invention comprises the following steps:
1) prepare immunizing antigen: by homotype bifunctional protein linking agent by human blood coagulation factor VII and bovine serum albumin coupling, obtain human blood coagulation factor VII-bovine serum albumin conjugate;
2) immune mouse: adopt low dose repeatedly immunization protocol, utilize step 1) in human blood coagulation factor VII-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) identify: detecting step 4) in the culture supernatant of hybridoma of fusion that obtains, the hybridoma cell strain of qualification secretion anti-human coagulation factor VII polyclonal 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 VII polyclonal 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 VII polyclonal antibody of the present invention:
1) preparation of immunizing antigen:
Get 1mg human blood coagulation factor VII 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 blood coagulation factor VII and carrier proteins BSA.
2) mouse immune:
End user's proconvertin-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 qualification secretion anti-human coagulation factor VII antibody:
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).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 enzyme labelled antibody (by specification dilutes) the 50 μ l by diluted, 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 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.
In a preferred embodiment, the hybridoma cell strain called after F7-P-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 CCTCCC2015195.
The preparation method of hybridoma cell strains F7-P-01 comprises the following steps:
Preparation human blood coagulation factor VII (DMSO) solution, adds 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) and activates.Above-mentioned solution is slowly dripped the coupling carrying out human blood coagulation factor VII and BSA in bovine serum albumin (BSA) solution, reaction product phosphate buffered saline buffer (PBS) dialysis, obtains the conjugate of human blood coagulation factor VII 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 coagulation factor VII antigen, by enzyme-linked immunosorbent assay (ELISA) qualification, obtain the hybridoma cell strain of stably excreting anti-human coagulation factor VII polyclonal antibody, called after F7-P-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, ascites is gathered after 10-14 days, it is positive for detecting ascites by enzyme linked immunosorbent assay, and proves that it can identify human blood coagulation factor VII by Western blotting.The polyclonal antibody hypotype measuring hybridoma cell strain F7-P-01 secretion through mouse polyclonal 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 polyclonal 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 blood coagulation factor VII 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 blood coagulation factor VII: 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 blood coagulation factor VII 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 blood coagulation factor VII-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 blood coagulation factor VII 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 blood coagulation factor VII 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 qualification of the hybridoma of anti-human coagulation factor VII polyclonal antibody is secreted
By the Hybridoma Cell Culture 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. result: detect culture supernatant by microplate reader is the positive.
2.3.7 the enlarged culturing of hybridoma and frozen
A. enlarged culturing: the polyclonal cells getting the supernatant positive proceeds in 24 orifice plates cultivates (1 hole turns 1 hole), in 24 orifice plates, 2 holes are divided into after 3 days, be divided into 4 holes after 3 days again, then after 2 days, the cell in this 4 hole proceeded to 50mL Tissue Culture Flask enlarged culturing in the lump.A wherein strain of hybridoma called after F7-P-01.
B. hybridoma is frozen: when 50mL culturing bottle inner cell is paved with bottle floorage about 70% → and the cell suction pipe in culturing bottle cut and beats, 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 polyclonal antibody
For hybridoma cell strain F7-P-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 F7-P-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 polyclonal antibody subclass
Adopt mouse polyclonal antibody subclass parting kit (Thermo company), concrete steps are with reference to its specification sheets.
Result: through kit measurement, the polyclonal antibody hypotype that gained hybridoma cell strain F7-P-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 100ug;
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 polyclonal 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 blood coagulation factor VII be identified with the polyclonal antibody that hybridoma F7-P-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 VII polyclonal antibody, it is characterized in that said method comprising the steps of:
1) prepare immunizing antigen: by homotype bifunctional protein linking agent by human blood coagulation factor VII and bovine serum albumin coupling, obtain human blood coagulation factor VII-bovine serum albumin conjugate;
2) immune mouse: adopt low dose repeatedly immunization protocol, utilize step 1) in human blood coagulation factor VII-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) identify: detecting step 4) in the culture supernatant of hybridoma of fusion that obtains, the hybridoma cell strain of qualification secretion anti-human coagulation factor VII polyclonal 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 VII polyclonal 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 F7-P-01, be preserved in China typical culture collection center on November 6th, 2015, deposit number is the hybridoma cell strain of CCTCCC2015195.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510792378.4A CN105349524B (en) | 2015-11-17 | 2015-11-17 | The hybridoma cell strain of anticoagulin VII polyclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510792378.4A CN105349524B (en) | 2015-11-17 | 2015-11-17 | The hybridoma cell strain of anticoagulin VII polyclonal antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105349524A true CN105349524A (en) | 2016-02-24 |
| CN105349524B CN105349524B (en) | 2019-02-22 |
Family
ID=55325586
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510792378.4A Active CN105349524B (en) | 2015-11-17 | 2015-11-17 | The hybridoma cell strain of anticoagulin VII polyclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105349524B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906159A (en) * | 2010-06-21 | 2010-12-08 | 南京农业大学 | Hybridoma cell line D6D |
-
2015
- 2015-11-17 CN CN201510792378.4A patent/CN105349524B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906159A (en) * | 2010-06-21 | 2010-12-08 | 南京农业大学 | Hybridoma cell line D6D |
Non-Patent Citations (3)
| Title |
|---|
| 吕茂民等: "人凝血因子VII单克隆抗体的制备与鉴定", 《生物技术通讯》 * |
| 吕鑫钰等: "人肝脏凝血因子XI的原核表达及其兔多克隆抗体的制备", 《哈尔滨医科大学学报》 * |
| 徐世洲: "人凝血因子VII单克隆抗体的制备与应用初探", 《中国优秀硕士学位论文全文数据库（硕士） 基础科学辑》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105349524B (en) | 2019-02-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110642793A (en) | Azoxystrobin hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN105906711B (en) | The preparation method of a kind of resisting O-type and A type foot and mouth disease virus Yolk antibody, product and application thereof | |
| CN101339195A (en) | Elisa kit for checking soluble PD-1 protein and checking method | |
| CN106589124B (en) | CD146 monoclonal antibody detects and separates the application in identification in glioma perivascular cell | |
| CN101906159B (en) | Hybridoma cell line D6D | |
| CN101793897A (en) | Method for improving screening efficiency of hybridoma | |
| CN105349496B (en) | The hybridoma cell strain of anti-macroglobulin monoclonal antibody | |
| CN105349497A (en) | Hybridomas cell strain of anti-human alpha-antitrysin polyclonal antibody | |
| CN102180950B (en) | Human-derived voltage-gated potassium channel 1.3 immunogenic peptide fragment and application thereof | |
| CN104164407B (en) | Anti(pig erythrocyte CR1-like) monoclonal antibody and preparation thereof | |
| CN105400769B (en) | The hybridoma cell strain of anticoagulin VIII monoclonal antibody | |
| CN105349495A (en) | Hybridomas cell strain of anti-human coagulation factor IX monoclonal antibody | |
| CN102702357B (en) | Recombination cyclophilin A antibody, preparation method thereof, enzyme-linked immuno sorbent assay (ELISA) kit and cell strains | |
| CN108103002A (en) | Preparation of mdck cell host's residual protein and application thereof | |
| CN101985476A (en) | Preparation, identification and application of antihuman NKp30 monoclonal antibody | |
| CN107892713A (en) | The monoclonal antibody of the anti-main albumen 3 of royal jelly and the enzyme linked immunological kit for detecting the main albumen 3 of royal jelly | |
| CN105349524A (en) | Hybridomas cell strain of anti-human coagulation factor VII polyclonal antibody | |
| CN104789535B (en) | Anti- methadone metabolin EDDP hybridoma cell strains and its preparation method and application | |
| CN105039260B (en) | Diphtheria toxoid monoclonal antibody and application thereof | |
| CN101921336B (en) | Monoclonal antibody against human alpha1 acid glycoprotein and preparation method thereof | |
| CN107188961A (en) | A kind of aspergillus flavus resisting toxin M1 monoclonal antibodies and immunosorbent, immune affinity column and preparation method thereof | |
| CN101240021B (en) | Monoclonal antibody for anti-human CDK5RAP2 protein and application thereof | |
| CN111961002A (en) | Pyraclostrobin hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN104031887A (en) | Monoclonal antibody and application thereof in diphacinone detection | |
| CN105368786B (en) | Secrete the hybridoma cell strain of 1 type hepatitis A virus hypotype monoclonal antibody of duck |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |