CN110205300A - Bar monoclonal antibody hybridoma cell strain, antibody of generation and preparation method thereof - Google Patents
Bar monoclonal antibody hybridoma cell strain, antibody of generation and preparation method thereof Download PDFInfo
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- CN110205300A CN110205300A CN201910483419.XA CN201910483419A CN110205300A CN 110205300 A CN110205300 A CN 110205300A CN 201910483419 A CN201910483419 A CN 201910483419A CN 110205300 A CN110205300 A CN 110205300A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1292—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Actinomyces; from Streptomyces (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention discloses Bar monoclonal antibody hybridoma cell strain, antibody of generation for belonging to bioengineering field and preparation method thereof.The high-purity herbicide resistant protein Bar that utilizing works bacterial strain of the present invention is expressed, purifying obtains is as antigen, it is prepared for secreting the Bar monoclonal antibody hybridoma cell strain (PA1-18030030/1B8 3D6) of special, the sensitive monoclonal antibody of anti-Bar by hybridoma technology and Bar monoclonal antibody hybridoma cell strain (PA1-18030030/6F5 1D2), deposit number is followed successively by CGMCC No.17083 and CGMCC No.17084.The antibody indirect ELISA potency that cell strain secretion odd contradictive hydroperitoneum obtains after purification is respectively 1:1024000 and 1:640000, antibody subtype is IgG1, the monoclonal antibody can provide substance and technical support with the endogenous Bar albumen in the recombination Bar albumen and transgenic paddy rice of specific recognition prokaryotic expression, the detection for being configured to the albumen in transgenic paddy rice for secreting the mouse monoclonal antibody hybridoma cell strain of anti-Bar herbicide resistant protein.
Description
Technical field
The present invention relates to bioengineering fields, and in particular to Bar monoclonal antibody hybridoma cell strain, the antibody of generation and its preparation
Method.
Background technique
Turn the plantation for having anti-herbicide gene crop, make to become effective management of weeds mode in field sprinkling herbicide,
Herbicide resistance is thus set to become one of the character of most advantage in agricultural production.Turn currently, Resistant Herbicide Crops account for the whole world
80% or more of gene crops.From development trend, the crop ratio of transgenosis is still being continuously increased.
The albumen of Bar gene expression can be catalyzed in the presence of acetyl coenzyme A in streptomycete (streptomyces)
Acetyl coenzyme A makes L-glufosinate-ammonium become acetyl-L-glufosinate-ammonium in conjunction with the free amine group of herbicide glufosinate-ammonium, to make weeding
Agent inactivation, assigns genetically modified crops Tolerance To Herbicides.Bar gene is widely used in base as the marker gene in genetic transformation
Because in Engineering Breeding.
While bringing tremendous economic interests with the rapid development of genetically modified crops, people also increasingly pay close attention to transgenosis
The possible not expected edible safety of crop and environmental safety problem.Therefore suitable method is established to eat transgenosis
Product transgenic ingredient is identified and is detected, and can promote the safety management of agriculture genetically modified organism, ensure people, animal and
The safety of microorganism can preserve the ecological environment.It is quick in order to be carried out to Bar albumen in transgenic paddy rice or its derivative
Quantitative or qualitative analysis, is studied and the monoclonal antibody for obtaining antiweed Bar albumen has very big meaning.
Summary of the invention
The purpose of the present invention is to provide Bar monoclonal antibody hybridoma cell strain (PA1-18030030/1B8 3D6) and Bar are mono-
Anti- hybridoma cell strain (PA1-18030030/6F5 1D2), two kinds of antibody generated and preparation method thereof.
To achieve the above object, the present invention provides the preparation methods of Bar monoclonal antibody hybridoma cell strain, including following step
It is rapid:
A) Bar recombinant protein is obtained by prokaryotic expression；
B) animal is immunized: BALB/c mouse is immunized using Bar recombinant protein as antigen；
C) it cell fusion: collects the splenocyte of immune BALB/c mouse and is merged with SP2/0 cell；
D) cell strain built: being subcloned by limiting dilution assay, is subcloned progress ELISA detection in 5-7 days, until screening
The hybridoma cell strain of stably excreting positive antibody expand and is further cultured for and saves out.
In another embodiment, the splenocyte of mouse and the integration percentage of SP2/0 cell are 1 to above-mentioned preparation method:
(5-10)。
The present invention also provides the Bar monoclonal antibody hybridoma cell strains that above-mentioned preparation method is prepared, including biological deposits to compile
Number for CGMCC No.17083 Bar monoclonal antibody hybridoma cell strain (PA1-18030030/1B8 3D6) and biological deposits compile
Number be CGMCC No.17084 Bar monoclonal antibody hybridoma cell strain (PA1-18030030/6F5 1D2).
The present invention also provides monoclonal antibody caused by above-mentioned Bar monoclonal antibody hybridoma cell strain, the Bar monoclonal antibody
Hybridoma cell strain is Bar monoclonal antibody hybridoma cell strain (PA1-18030030/1B8 3D6) and Bar monoclonal antibody hybridoma cell strain
(PA1-18030030/6F5 1D2)。
Said monoclonal antibody in another embodiment, Bar monoclonal antibody hybridoma cell strain (PA1-18030030/
1B8 3D6) and Bar monoclonal antibody hybridoma cell strain (PA1-18030030/6F5 1D2) caused by monoclonal antibody class
Type is IgG1.
Said monoclonal antibody in another embodiment, Bar monoclonal antibody hybridoma cell strain (PA1-18030030/
1B8 3D6) and Bar monoclonal antibody hybridoma cell strain (PA1-18030030/6F5 1D2) caused by monoclonal antibody pass through
Indirect ELISA detects obtained potency and is followed successively by 1:1024000 and 1:640000.
The present invention also provides the preparation methods of said monoclonal antibody, Bar monoclonal antibody hybridoma cell strain are inoculated into small
Ascites is prepared in mouse abdominal cavity, is then carried out Protein A- agarose affinity chromatography column purification, is obtained monoclonal antibody.
The present invention also provides application of the said monoclonal antibody in detection Bar herbicide resistant protein.
The present invention also provides said monoclonal antibodies in the reagent of preparation ELISA method detection Bar herbicide resistant protein
Application.
The present invention also provides said monoclonal antibodies to detect Bar antiweed egg in preparation ELISA double antibody sandwich method
Application in white reagent.
Bar monoclonal antibody hybridoma cell strain (PA1-18030030/1B8 3D6) provided by the invention and Bar monoclonal antibody hybridoma
Cell strain (PA1-18030030/6F5 1D2) is successively preserved in Chinese microorganism strain preservation pipe on December 21st, 2018
Reason committee common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.Postcode: 100101, preservation
Number is followed successively by CGMCC No.17083 and CGMCC No.17084.
Compared with prior art, the invention has the following beneficial effects: utilizing works bacterial strain of the present invention expression, purifying to obtain
High-purity herbicide resistant protein Bar as antigen, special, the sensitive lists of 2 plants of anti-Bar of secretion are prepared for by hybridoma technology
Anti- Bar monoclonal antibody hybridoma cell strain (PA1-18030030/1B8 3D6) and Bar monoclonal antibody hybridoma cell strain (PA1-
18030030/6F5 1D2), the antibody indirect ELISA potency that the cell strain secretion odd contradictive hydroperitoneum obtains after purification is respectively 1:
1024000 and 1:640000, antibody subtype are IgG1, and the monoclonal antibody can be with the recombination Bar albumen of specific recognition prokaryotic expression
And the endogenous Bar albumen in transgenic paddy rice, secrete being configured to for the mouse monoclonal antibody hybridoma cell strain of anti-Bar herbicide resistant protein
The detection of the albumen provides substance and technical support in transgenic paddy rice.
Detailed description of the invention
Fig. 1 is Bar recombinant protein SDS-PAGE electrophoresis result figure.
Fig. 2 is that Bar recombinant protein monoclonal antibody screens western result figure.
Fig. 3 is Bar monoclonal antibody hybridoma cell strain (PA1-18030030/1B8 3D6) and Bar monoclonal antibody hybridoma cell strain
The SDS-PAGE electrophoresis result figure of the monoclonal antibody of (PA1-18030030/6F5 1D2) purifying.
Fig. 4 is monoclonal antibody titre caused by Bar monoclonal antibody hybridoma cell strain (PA1-18030030/1B8 3D6)
Figure.
Fig. 5 is monoclonal antibody titre caused by Bar monoclonal antibody hybridoma cell strain (PA1-18030030/6F5 1D2)
Figure.
Specific embodiment
The present invention is described in detail with reference to the accompanying drawings and examples, but implementation of the invention is not limited only to this.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, it is unless otherwise specified, commercially commercially available.
Embodiment 1Bar monoclonal antibody hybridoma cell strain obtains and its preparation of monoclonal antibody
1. the preparation of immunizing antigen
1) recombinant protein Bar expresses strain construction, thallus culture and cracking
Bar encoding gene amplification: using transgenic paddy rice T1C-19 genomic DNA as template, with high-fidelity Fastpfu into
Row PCR amplification, the PCR product expanded, by purpose band gel extraction, are used after the identification of (1.0%) agarose gel electrophoresis
Universal DNA purification and recovery kit (Tiangeng) recycling, recovery product through restriction enzyme Nde I and Xho I digestion,
It is connect overnight with the pET28a plasmid fragments for carrying out same digestion, glue recovery processing in 4 DEG C through glue recycling, conversion Trans10 sense
By state cell (Beijing Quan Shijin), picked clones carry out bacterium colony PCR identification, and positive colony is by Beijing six directions Huada gene company
It is sequenced.
It will identify correct recombinant expression carrier pET28a-Bar conversion e. coli bl21 (DE3) competent cell, bacterium
Liquid is coated on the LB plate containing kanamycins (50 μ g/mL), and 37 DEG C are incubated overnight.Next day picking monoclonal on plate is inoculated with
In the LB liquid medium of the kanamycins containing same concentrations, it is incubated overnight in 37 DEG C.Resulting expression bacterial strain bacterium solution and 50%
Glycerite mixes in equal volume, in -80 DEG C of cold storage.
By the recombinant protein Bar expression bacterial strain recovery culture of preservation, bacterium solution is coated on containing kanamycins (50 μ g/mL)
On LB plate, 37 DEG C are incubated overnight.Picking monoclonal is inoculated in liquid LB (the 50 μ g/mL containing kanamycins) culture medium and stays overnight for 37 DEG C
Culture.With the inoculation of 1% inoculum concentration, it is 0.8 or so in 37 DEG C of cultures to OD600,1mM IPTG is added and is stayed overnight in 16 DEG C within second day
Induction expression protein.Induction terminates bacterium solution ice bath, and in 4 DEG C, thalline were collected by centrifugation by 4000rpm, 10min, abandons supernatant, thallus warp
It is suspended after PBS washing with lysate (50mM Tris pH7.5,300mM NaCl, 5%Glycerol and 20mM imidazoles)；Ultrasound
Broken (2s on/4s off, Amp:45%, Time:3min)；4 DEG C of 15000rpm are centrifuged 1hr, collect supernatant.
2) recombinant protein purification
Cellular lysate liquid supernatant and the Ni Sepharose 6FF Beads (GE Healthcare) through lysate balance are mixed
It closes, 2-3hrs, 3000rpm is combined to be centrifuged 3min and abandon supernatant in 4 DEG C；Protein-bonded Ni Sepharose 6FF beads warp
Cleaning solution (50mM Tris pH7.5,300mM NaCL, 5%Glycerol and 50mM imidazoles) is washed 2-3 times；Eluent is used again
(50mM Tris pH7.5,300mM NaCL, 5%Glycerol and 200mM imidazoles) elution.Collecting eluent is that albumen is thick
Pure solution.
The protein sample that affinity purification is obtained is dialysed to protein storage solution (50mM Tris pH7.5,300mM
NaCL, 5%Glycerol), and with the 10/30 GL (GE of Superdex 200Increase balanced through same solution
Healthcare it) is further purified, collects protein peak component and the purity through SDS-PAGE detection protein sample, it can by Fig. 1
, electrophoretically pure Bar albumen is finally obtained by gel filtration, molecular weight is about 70kDa.
2. immune animal
Use the Bar recombinant protein of Quality Control qualification in step 2) that the SPF grade BALB/c female of 88 week old is immunized as antigen
Mouse (it purchases in Hubei Province Animal Experimental Study center, credit number:
SCXK (Hubei Province) 2015-0018), antigen (adds with isometric complete Freund's adjuvant (head exempts from) and incomplete Freund's adjuvant
It is strong immune) it mixes and is emulsified, it is fully mixed to the subcutaneous multiple spot of Water-In-Oil state progress and is immunized, 2-3 booster immunization, often
In 2 weeks secondary immunization interval periods, carry out bioactivity later, be higher than > 1:10000 after abdominal cavity impact is carried out in 1 week, will directly exempt from
The antigen of epidemic disease dosage is dissolved in the PBS of 250 μ L, and specific immune time and immunizing dose are as shown in table 1:
1 immune time of table and immunizing dose
Immune example: one exempts from, and the antigen of 50ug is dissolved in PBS, is then mixed with adjuvant by volume 1:1.
3. cell fusion
After last impacts 3 days, positive control blood is collected, spleen is taken, is prepared into single cell suspension；SP2/ in logarithmic phase
It after the processing of 0 cell, is mixed in a certain proportion (1:5-1:10) with splenocyte, 50%PEG1450 acts on 1min, with basis culture
Base DMEM dilution terminates, and after low-speed centrifugal, then with the HAT culture medium for containing 20% fetal calf serum is gently suspended and is mixed, according to 2 ×
107/ plate is spread to preprepared feeder cells plate, is placed in 5%CO2, cultivate at 37 DEG C.
4. cell strain built
1) fusion plate detection:
Plate to be fused changes liquid cell length to about 10,000 cells of median size or more and starts to detect, qualified in ELISA Quality Control
(i.e. negative control OD450< 0.2, positive control OD450> 1.0) positive hole (general OD is selected after450>=0.5) it is subcloned.
2) subcloning procedures and detection:
Choose the hole progress limiting dilution that positive value high (OD450 > 2.0) is detected in fusion plate, with the Dan Ke of every plate 60%
Grand hole number counting is subcloned, and the picking positive is worth higher monoclonal hole progress limiting dilution every time, is subcloned 5-7 every time
It can carry out ELISA detection, until the monoclonal cell strain that finishing screen selects energy stably excreting positive antibody carries out expansion training
It supports.
3) cell strain is established:
The cell strain for the stably excreting positive antibody that the subclone stage is filtered out, expansion is incubated at 24 orifice plates, after expansion
It collects supernatant and carries out antigen detection, its stability is verified using ELISA gradient dilution and western-blotting, passes through Fig. 2
It is found that Bar monoclonal antibody hybridoma cell strain (PA1-18030030/1B8 3D6) and Bar monoclonal antibody hybridoma cell strain (PA1-
18030030/6F5 1D2) secretion monoclonal antibody energy specific detection arrive endogenous sample and recombinant protein Bar, collection cell expansion in
In 10cm culture dish, collects supernatant again and detect the potency of wherein antibody, pick out 2 plants of cell strain cultures of OD450 > 2.0
It in cell bottle, is frozen, i.e. Bar monoclonal antibody hybridoma cell strain (PA1-18030030/1B8 3D6) and the hybridization of Bar monoclonal antibody
Tumor cell strain (PA1-18030030/6F5 1D2) is successively preserved in Chinese microorganism strain preservation on December 21st, 2018
Administration committee's common micro-organisms center (abbreviation CGMCC), deposit number are followed successively by CGMCC No.17083 and CGMCC
No.17084。
4) cell strain, which freezes, identifies that one in the same batch that must recover after cell strain freezes is identified, is reflected
Calibration is quasi-:
1. recovery viable count >=1,000,000 cell/；2. vibrant cell >=500,000/strain in living cells；3. recovery is thin
There cannot be other microorganisms (such as: bacterium fungi mycoplasma) in addition to cell strain cell to occur in born of the same parents；4. recovery cell
It grows into after certain amount to select the cell grown and make monoclonal and counts bed board, and the secretory antibody ability for detecting monoclonal is
No full sun has antibody-secreting；5. cells and supernatant also needs to make ELISA (OD450 > 2.0), to determine whether that secretion is positive anti-
The identification of western-blotting is done while body, as shown in Figure 3, Bar monoclonal antibody hybridoma cell strain (PA1-
18030030/1B8 3D6) and the monoclonal antibody of Bar monoclonal antibody hybridoma cell strain (PA1-18030030/6F5 1D2) secretion can be special
Detect endogenous sample and recombinant protein Bar.
5. preparing ascites
It is first injected with norphytane or atoleine row mouse peritoneal, after a week respectively by Bar monoclonal antibody hybridoma cell strain
(PA1-18030030/1B8 3D6) and Bar monoclonal antibody hybridoma cell strain (PA1-18030030/6F5 1D2) are inoculated into two small
Expand culture in mouse abdominal cavity, after cell singling and select 10% fetal calf serum culture medium, when cell density reaches with 1 × 106-2×
106When/mL, 800rpm centrifugation collects precipitating, and after being resuspended with PBS, Intraperitoneal injection to mouse (atoleine) in vivo, 7-10
After it, collects ascites and prepare purifying.
6. antibody purification
Ascites after collection selects Protein A- agarose affinity chromatography column purification after pretreatment, and specific steps are such as
Under:
1) buffer: start buffer pH7.0,20mM phosphate buffer；Elution buffer is the sweet ammonia of pH2.7 0.1mM
Hydrochlorate acid.
2) prepare collecting pipe: taking 1.5mL centrifuge tube, every centrifuge tube adds 70 μ L pH9.0 1M Tris-HCL.
3) preparation of samples: the sample precipitated through 50%SAS sets start buffer and carries out dialysed overnight, and through 0.22 μ
The filtration of m miillpore filter.
4) purification process: Protein A- agarose affinity chromatography column is balanced with enough start buffers (8-10mL)
(HiTrap Protein A 1mL, Pharmacia Biotech).Take sample to be purified (every milliliter of sample .2- containing protein 10
Then 21.1mg) 15-25mL upper prop, flow velocity 0.5mL/min successively use start buffer 7-8mL, elution with same flow velocity
Buffer 6-7mL, start buffer 5mL washing, every pipe 1mL collect eluent.
5) purity and the monoclonal antibody (McAb) of activity identification purifying identify its purity with SDS-PAGE, are detailed in Fig. 3, lead to
Fig. 4 is crossed it is found that Bar monoclonal antibody hybridoma cell strain (PA1-18030030/1B8 3D6) and Bar monoclonal antibody hybridoma cell strain
The purified nearly all foreign protein of removal of (PA1-18030030/6F5 1D2) monoclonal antibody, there is 2 main bands of specificity
(55kDa and 30kDa).
7. the subgroup identification and titration of monoclonal antibody
By standard anti-the BALB/c mouse IgG1, IgG2a, IgG2b, IgG3, IgM of purified monoclonal antibody and sigma company
Antibody carries out ELISA detection, and testing result shows: monoclonal antibody type uses indirect ELISA using recombinant protein Bar as antigen for IgG1
Method detection purified monoclonal antibody potency is measured through ELISA as shown in Figure 4, the Bar monoclonal antibody hybridoma cell strain purified
(PA1-18030030/1B8 3D6) monoclonal antibody titre is 1:1024000；As shown in Figure 5, it measures, purifies through ELISA
Bar monoclonal antibody hybridoma cell strain (PA1-18030030/6F5 1D2) monoclonal antibody titre is 1:640000.
The concentration of monoclonal antibody caused by 2 Bar monoclonal antibody hybridoma cell strain (PA1-18030030/1B8 3D6) of table
And hypotype
The concentration of monoclonal antibody caused by 3 Bar monoclonal antibody hybridoma cell strain (PA1-18030030/6F5 1D2) of table
And hypotype
Transgenic paddy rice protein extracting method:
Liquid nitrogen flash freezer is organized, is ground, 1mL (by sample size, general 0.5g adds 1-2mL) protein extract is added, 4 DEG C mix 30
Minute, (12,000rpm 4 DEG C of centrifugation 15min, take supernatant), protein extraction formula of liquid is shown in Table 4:
4 protein extraction formula of liquid of table
| Ingredient | Dosage |
| 1M Tris,pH 7.5 | 500uL |
| 1M NaCL | 1.5mL |
| 0.5M EDTA | 20uL |
| 50%glycerol | 2mL |
| 10%SDS | 1mL |
| Distilled water (DDH2O) | It is made into 10mL |
| Protease inhibitors (Roche) | Used time addition is a piece of |
| 1mM PMSF (phenylmethylsulfonyl fluoride, Sigma) | Used time adds 50uL |
Disclosed above is only specific embodiments of the present invention, and still, the present invention is not limited to this, any this field
What technical staff can think variation should all fall into protection scope of the present invention.
Claims (9)
1. a kind of preparation method of Bar monoclonal antibody hybridoma cell strain, which comprises the following steps:
A) Bar recombinant protein is obtained by prokaryotic expression；
B) animal is immunized: BALB/c mouse is immunized using Bar recombinant protein as antigen；
C) it cell fusion: collects the splenocyte of immune BALB/c mouse and is merged with SP2/0 cell；
D) cell strain built: being subcloned by limiting dilution assay, is subcloned progress ELISA detection in 5-7 days, until filtering out steady
Surely the hybridoma cell strain for secreting positive antibody expand and is further cultured for and saves.
2. the preparation method of Bar monoclonal antibody hybridoma cell strain according to claim 1, which is characterized in that the spleen of the mouse
The integration percentage of cell and SP2/0 cell is 1:(5-10).
3. the Bar monoclonal antibody hybridoma cell strain that preparation method described in claim 1 is prepared, which is characterized in that the Bar is mono-
Anti- hybridoma cell strain is the Bar monoclonal antibody hybridoma cell strain PA1- that biological deposit number is CGMCC No.17083
The Bar monoclonal antibody hybridoma cell strain PA1- that 18030030/1B8 3D6 and biological deposits number are CGMCC No.17084
18030030/6F5 1D2。
4. monoclonal antibody caused by Bar monoclonal antibody hybridoma cell strain described in claim 3, which is characterized in that the Bar is mono-
Anti- hybridoma cell strain is Bar monoclonal antibody hybridoma cell strain PA1-18030030/1B8 3D6 and Bar monoclonal antibody hybridoma cell strain
PA1-18030030/6F5 1D2。
5. monoclonal antibody according to claim 4, which is characterized in that the Bar monoclonal antibody hybridoma cell strain PA1-
Monoclonal antibody caused by 18030030/1B8 3D6 and Bar monoclonal antibody hybridoma cell strain PA1-18030030/6F5 1D2
Type be IgG1.
6. monoclonal antibody according to claim 4, which is characterized in that the Bar monoclonal antibody hybridoma cell strain PA1-
Monoclonal antibody caused by 18030030/1B8 3D6 and Bar monoclonal antibody hybridoma cell strain PA1-18030030/6F5 1D2
Obtained potency, which is detected, by indirect ELISA is followed successively by 1:1024000 and 1:640000.
7. the preparation method of monoclonal antibody described in claim 4, which is characterized in that Bar monoclonal antibody hybridoma cell strain to be inoculated with
Ascites is prepared into mouse peritoneal, is then carried out Protein A- agarose affinity chromatography column purification, is obtained monoclonal antibody.
8. application of the monoclonal antibody described in claim 4 in detection Bar herbicide resistant protein.
9. application of the monoclonal antibody in detection Bar herbicide resistant protein according to claim 8, which is characterized in that institute
State application of the monoclonal antibody in the reagent of preparation ELISA method detection Bar herbicide resistant protein.
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