CN103193873A - Immunogen for preparing antibodies used for identification of denatured Bt proteins, antibody prepared by same and application thereof - Google Patents
Immunogen for preparing antibodies used for identification of denatured Bt proteins, antibody prepared by same and application thereof Download PDFInfo
- Publication number
- CN103193873A CN103193873A CN201310070514XA CN201310070514A CN103193873A CN 103193873 A CN103193873 A CN 103193873A CN 201310070514X A CN201310070514X A CN 201310070514XA CN 201310070514 A CN201310070514 A CN 201310070514A CN 103193873 A CN103193873 A CN 103193873A
- Authority
- CN
- China
- Prior art keywords
- solution
- albumen
- immunogen
- sex change
- antibody
- 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
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an immunogen for preparing antibodies used for identification of denatured Bt proteins, an antibody prepared by same and an application thereof. The invention provides the immunogen for preparing the antibodies used for identification of the denatured Bt proteins, and the immunogen is a product obtained by mixing and emulsifying a solution containing the denatured Bt proteins and adjuvants. The polyclonal antibodies obtained by immunizing animals with the immunogen are also within the protective scope of the invention. The polyclonal antibodies can be used as primary antibodies for coating antigens and/or detecting, and thus can be used for detecting whether to-be-detected samples contain the denatured Bt proteins. The to-be-detected samples can be cooked transgenic crops, foodstuff or forage obtained by processing the transgenic crops and the like. The immunogen and the antibody have practical value for biological safety evaluation of genetically modified crops, and are great in potential social benefits.
Description
Technical field
The present invention relates to a kind of immunogen for the preparation of the antibody of identifying sex change Bt albumen and antibody and the application of preparation thereof.
Background technology
The insecticidal crystal protein of bacillus thuringiensis (Bacillus thuringiensis) (claiming Bt insecticidal crystal protein or Bt albumen again) has pest-resistant effect, so its encoding gene (Bt gene) is widely used crops such as transgene cottons.According to the homology of coding gene sequence and the insecticidal spectrum of proteins encoded, Bt albumen is divided into cry family and cryt family, is divided into the subclass that quantity does not wait under each family again.The Bt gene of expressing in plant at present has genes such as Cry1Aa gene, Cry1Ab gene, Cry1Ac gene, Cry2Aa gene, Cry3Bb gene and Cry9c.Mainly changed in the business-like trans Bt gene crops on the Chinese market at the virose Cry1Ac gene of lepidopterous insects larva.
After transgene cotton seed production Oleum Gossypii semen, can obtain the by product cottonseed cake, cottonseed cake is typically used as the feed of poultry or livestock.In the cottonseed cake, usually residual have sex change Bt albumen, poultry or livestock had potential harm, thereby may cause potential harm to the mankind of food poultry or domestic animal.Use domestic and international business-like immunity detection reagent or test strip at present, detect less than the sex change Bt albumen in the cottonseed cake substantially, press for antibody and the method that to identify sex change Bt albumen.
Summary of the invention
The purpose of this invention is to provide a kind of immunogen for the preparation of the antibody of identifying sex change Bt albumen and antibody and the application of preparation thereof.
The invention provides the immunogen for the preparation of the antibody of identification sex change Bt albumen, is to contain the solution of sex change Bt albumen and the product that adjuvant mixes and emulsification obtains.
The described preparation method who contains the solution of sex change Bt albumen can be as follows: handle the solution that contains Bt albumen with protein denaturant, obtain containing the solution of sex change Bt albumen.Described denaturing agent can be beta-mercaptoethanol and/or SDS(sodium laurylsulfonate).
The described preparation method who contains the solution of sex change Bt albumen can be as follows: will contain and carry out denaturing treatment after the solution of Bt albumen and solution third mix, and obtain containing the solution of sex change Bt albumen; The volume proportion of the solution of the described Bt of containing albumen and described solution third is 1:(1-10); Described solution third is by solute and solvent composition, and described solvent is water, and described solute and concentration thereof are as follows: volume ratio is the beta-mercaptoethanol of 0.1-2% and the SDS of 0.1-10.0mg/100mL.Described denaturing treatment can be water-bath in 40-100 ℃ of water.Described denaturing treatment can be in 100 ℃ of boiling water water-bath 3-5 minute.Described denaturing treatment can be at-20 ℃ of refrigerator multigelations more than 3 times.Described denaturing treatment can be below the accent pH to 3 or transfers more than the pH to 10.Specifically be to realize by the form that adds aqueous sulfuric acid below the described accent pH to 3.Described aqueous sulfuric acid specifically can be the 1M aqueous sulfuric acid.Specifically be to realize by the form that adds aqueous sodium hydroxide solution more than the described accent pH to 10.Described aqueous sodium hydroxide solution specifically can be the 1M aqueous sodium hydroxide solution.
The described preparation method who contains the solution of sex change Bt albumen can be as follows: the solution that will contain Bt albumen carries out denaturing treatment, obtains containing the solution of sex change Bt albumen.Described denaturing treatment can be water-bath in 40-100 ℃ of boiling water.Described denaturing treatment can be in 100 ℃ of boiling water water-bath 3-5 minute.Described denaturing treatment can be at-20 ℃ of refrigerator multigelations more than 3 times.Described denaturing treatment can be below the accent pH to 3 or transfers more than the pH to 10.Specifically be to realize by the form that adds aqueous sulfuric acid below the described accent pH to 3.Described aqueous sulfuric acid specifically can be the 1M aqueous sulfuric acid.Specifically be to realize by the form that adds aqueous sodium hydroxide solution more than the described accent pH to 10.Described aqueous sodium hydroxide solution specifically can be the 1M aqueous sodium hydroxide solution.
Described Bt albumen can be for extracting the Bt albumen of purifying or the Bt albumen that is purchased in the Bt albumen of the parasporal crystal of bacillus thuringiensis, prokaryotic expression, the genetically modified crops.
More than the solution of the arbitrary described Bt of containing albumen specifically can be the Bt protein solution that protein concentration is 0.1-1mg/mL.The PB damping fluid of available pH7.5,0.1mol/L is adjusted protein concentration.
Described Bt albumen specifically can be prepared as follows:
(1) carries out the extraction of solubility total protein after the Insect Resistant Cotton seed is shelled, obtain the solution first;
(2) described solution first is carried out the ammonium sulfate precipitation of 60% saturation ratio, centrifugal collecting precipitation;
(3) precipitation that obtains of dissolving step (2) and dialysing obtains solution second;
(4) described solution second is carried out immunoaffinity chromatography, the protein ingredient of the monoclonal antibody specific combination of collection and anti-Bt Cry1Ab/Ac albumen is the Bt toxalbumin; The monoclonal antibody of described anti-Bt Cry1Ab/Ac albumen is to be the monoclonal antibody that is produced by hybridoma Bt2F9CGMCC No.5162 secretion.
Hybridoma Bt2F9 is the monoclonal hybridoma strain of Bt Cry1Ab/Ac protein antibodies in the anti-transgenic plant of stably excreting, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on August 19th, 2011 and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.5162.
In the described step (1), can adopt the described solubility total protein of CAPS buffer extraction of pH10.5,0.01M.
In the described step (1), the preparation method of described solution first is specific as follows: described Insect Resistant Cotton seed is shelled and pulverizes, and (extract temperature and specifically can be 4 ℃, extraction time specifically can be 4 hours to use the CAPS buffer extraction of pH10.5,0.01M then; Available magnetic stirrer in the leaching process is to increase extraction efficiency), avoid the top layer grease after centrifugal (centrifugal condition specifically can be: centrifugal 20 minutes of 4 ℃, 10000rpm) and get upper liquid, be described solution first.
In the described step (2), the implementation method of described " carrying out the ammonium sulfate precipitation of 60% saturation ratio " is as follows: add ammonium sulfate and make it reach 60% saturation ratio in described solution first, ice bath is placed then.The time that described ice bath is placed specifically can be 40 minutes.Available magnetic stirrer in the described ice bath put procedure.Described centrifugal parameter specifically can be: centrifugal 20 minutes of 4 ℃, 7000rpm.
In the described step (3), the described precipitation of available deionized water dissolving is also transferred pH to 10.5 with the Tris aqueous solution.The described Tris aqueous solution specifically can be the 1M Tris aqueous solution.Described dialysis is specifically carried out in the PBS damping fluid.The condition of described dialysis specifically can be: with 6 hours (the PBS damping fluid that more renewed in per two hours) of 4 ℃ of dialysis of PBS damping fluid.
In the described step (4), the step of described immunoaffinity chromatography can be as follows: the immunochromatography post that 1. described solution second is splined on the monoclonal antibody that is connected with described anti-Bt Cry1Ab/Ac albumen; 2. clean pillar with scavenging solution, remove foreign protein; 3. carry out wash-out with elutriant, collected elutriant behind the post, be the solution that contains target protein.
The stopping composition of described immunoaffinity chromatography can be CNBr-activated Sepharose4B.
Described scavenging solution specifically can be the Gly-HCl damping fluid of pH2.5,0.01M.
Described elutriant specifically can be and contains the Gly-HCl damping fluid that volume ratio is pH2.5, the 0.01M of 50% ethylene glycol.
In the described immunoaffinity chromatography process, specifically can adopt 4 rev/mins last sample flow velocity, clean flow velocity and elution flow rate.
Described step 2. in concrete available 2 times of scavenging solutions of going up the sample volumes clean pillars.
Described step 3. in the elutriant of concrete available 3 times of volumes carry out wash-out.
Described step specifically can be collected elutriant behind the post by the every pipe of 1ml in 3..
Also can comprise elutriant behind the described post is excessively transferred pH to 7.5 with the Tris aqueous solution step in the described method.The described Tris aqueous solution specifically can be the 1M Tris aqueous solution.
Also can comprise the step of transferring pH to 7.5 to dialyse then with the Tris aqueous solution elutriant behind the described post excessively in the described method.The described Tris aqueous solution specifically can be the 1M Tris aqueous solution.Described dialysis is specifically carried out in the PBS damping fluid.The condition of described dialysis specifically can be: with 6 hours (the PBS damping fluid that more renewed in per two hours) of 4 ℃ of dialysis of PBS damping fluid.
Also can comprise in the described method with described cross post after elutriant transfer pH to 7.5 with the Tris aqueous solution, detect wherein whether contain the Bt toxalbumin then, will contain the step that the solution of Bt toxalbumin is dialysed at last.The described Tris aqueous solution specifically can be the 1M Tris aqueous solution.Described dialysis is specifically carried out in the PBS damping fluid.The condition of described dialysis specifically can be: with 6 hours (the PBS damping fluid that more renewed in per two hours) of 4 ℃ of dialysis of PBS damping fluid.
The concrete grammar of described " detect and wherein whether contain the Bt toxalbumin " is as follows:
(ⅰ) with the rabbit polyclonal coated elisa plate of anti-Bt Cry1Ac albumen;
(ⅱ) in the enzyme plate of step (ⅰ), add to transfer and cross elutriant behind the post, 37 ℃ of incubation 30min behind the pH to 7.5;
(ⅲ) the anti-Bt Cry1Ac protein monoclonal antibody of adding horseradish peroxidase-labeled in the enzyme plate of step (ⅱ), 37 ℃ of incubation 30min;
(ⅳ) in the enzyme plate of step (ⅲ), add substrate buffer solution, termination reaction behind the room temperature reaction 15min;
(ⅴ) under 492nm, measure the OD value;
(ⅵ) blade that will contrast cotton variety grinds in mortar, and in 4 ℃ of extractions 12 hours, 8000r/min4 ℃ of centrifuging and taking supernatant carried out supernatant above-mentioned steps (ⅰ) then to (ⅴ) with the PBS damping fluid;
If (ⅶ) the OD value that obtains of step (ⅴ) is more than three times of OD value that step (ⅵ) obtains, the result is positive.
The concrete grammar of described " detect and wherein whether contain the Bt toxalbumin " is as follows:
(ⅰ) rabbit polyclonal antibody of the anti-Bt Cry1Ac of 1mg/mL albumen is cushioned with the bag of 500 times of volumes joins in the enzyme plate after liquid dilutes, every hole 100 μ L, 37 ℃ of incubations 3 hours;
(ⅱ) in the enzyme plate of step (ⅰ), add to transfer and cross elutriant behind the post, every hole 100 μ L, 37 ℃ of incubation 30min behind the pH to 7.5;
(ⅲ) the anti-Bt Cry1Ac protein monoclonal antibody of 1mg/mL horseradish peroxidase-labeled is diluted in the enzyme plate that the back adds step (ⅱ) every hole 100 μ L, 37 ℃ of incubation 30min with the PBS damping fluid of 1000 times of volumes;
(ⅳ) add substrate buffer solution in the enzyme plate of step (ⅲ), every hole 100 μ L add 50 μ L2.0M aqueous sulfuric acid termination reactions in every hole behind the room temperature reaction 15min;
(ⅴ) under 492nm, measure the OD value;
(ⅵ) blade that will contrast cotton variety grinds in mortar, and in 4 ℃ of extractions 12 hours, 8000r/min4 ℃ of centrifuging and taking supernatant carried out supernatant above-mentioned steps (ⅰ) then to (ⅴ) with the PBS damping fluid;
If (ⅶ) the OD value that obtains of step (ⅴ) is more than three times of OD value that step (ⅵ) obtains, the result is positive.
Described contrast cotton variety is the cotton variety that does not contain the encoding gene of Bt Cry1Ac albumen, and it is far away by 321 specifically to can be cotton variety stone.
Described Insect Resistant Cotton can be the cotton of the encoding gene that changes Bt Cry1Ac albumen, specifically can be state glad cotton No. 6.
More than arbitrary described Bt albumen specifically can be Bt Cry1Ac albumen.
More than arbitrary described Bt Cry1Ac albumen can be following (a) or (b):
(a) protein of being formed by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and with the protein of being derived by sequence 1 that the insecticidal crystal protein activity is just arranged.
More than the encoding gene of arbitrary described Bt Cry1Ac albumen can be following 1) or 2) or 3) dna molecular:
1) dna molecular of coding region shown in sequence in the sequence table 2;
2) under stringent condition with 1) the dna sequence dna hybridization that limits and the dna molecular of coded insect-killing crystallin;
3) with 1) dna sequence dna that limits has homology more than 90% and the dna molecular of coded insect-killing crystallin.
Described adjuvant can be freund's adjuvant, specifically can be Freund's complete adjuvant or formula Freund not.Described adjuvant can be the water-based adjuvant.
But described solution and the concrete equal-volume of described adjuvant that contains sex change Bt albumen mixes.
More than the polyclonal antibody that obtains of arbitrary described immunogen immune animal also belong to protection scope of the present invention.Described animal is Mammals, as rabbit or mouse.
Described polyclonal antibody can be used as envelope antigen and/or detection primary antibodie, thereby for detection of whether containing sex change Bt albumen in the sample to be tested.Described sample to be tested can be the genetically modified crops of boiling, the food that genetically modified crops processing obtains or feed etc.The present invention has practical value to the biological safety evaluation of transgenosis food crop, and potential social benefit is huge.
Description of drawings
Fig. 1 is the polyacrylamide gel electrophoresis figure in the purge process of embodiment 1.
Fig. 2 is the photo of the step 2 of embodiment 5.
Fig. 3 is the photo of step 3 and the step 4 of embodiment 5.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all arranges repeated experiments three times, results averaged.Goat anti-mouse igg-HRP: available from Jackson company, cat. no is 79556).Freund's complete adjuvant: available from Sigma company, cat. no is F5881.Freund's incomplete adjuvant: available from Sigma company, cat. no is F5506.Beta-mercaptoethanol: available from Sigma company, cat. no is M6250.Sodium laurylsulfonate (SDS) and O-Phenylene Diamine (OPD) wait all the other conventional reagent all available from Beijing chemical reagents corporation.
Hybridoma Bt2F9 is the monoclonal hybridoma strain of Bt Cry1Ab/Ac protein antibodies in the anti-transgenic plant of stably excreting, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on August 19th, 2011 and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.5162.
The MONOCLONAL ANTIBODIES SPECIFIC FOR method of anti-Bt Cry1Ab/Ac albumen: hybridoma Bt2F9CGMCC No.5162 is placed cell culture medium, cultivated 2 days for 37 ℃, with sad-saturated ammonium sulphate method the nutrient solution that obtains is carried out purifying, obtain the monoclonal anti liquid solution of anti-Bt Cry1Ab/Ac albumen, freeze-drying-20 ℃ preservation.
The preparation method of cell culture medium: in the DMEM substratum, add calf serum (available from GIBCOBRL, catalog number is 26170-043) and sodium bicarbonate, the final concentration of calf serum is 20%(quality percentage composition), the final concentration of sodium bicarbonate is 0.2%(quality percentage composition), pH is 7.4.
The Bt Cry1Ac albumen that is purchased: grand bio tech ltd is helped in Shanghai, and products catalogue is numbered C010301.
Cotton variety stone is far away by 321: Hebei legendary god of farming's high-tech share company limited.
The seed of state glad cotton No. 6 (changeing the pest-resistant cultivar of the encoding gene of Bt Cry1Ac albumen) grinds meeting available from the glad farming of Hejian City, Hebei province state.
Used PBS cushions all as follows in the present embodiment if no special instructions: the prescription of PBS damping fluid (pH7.5): solvent is water, contains 0.02M Na
2HPO
4, 0.0015M KH
2PO
4With 0.14M NaCl.
The preparation of embodiment 1, Bt Cry1Ac albumen
1, the extraction of solubility total protein
Glad cotton No. 6 seed of state is shelled and grind to form fine powder, get the 10g fine powder, add 100ml pH10.5,0.01MCAPS damping fluid, using magnetic stirring apparatus to stir for 4 ℃ extracted 4 hours, centrifugal 20 minutes of 4 ℃ then, 10000rpm, avoid the top layer grease and get upper liquid, this liquid is the solubility total protein extracting solution.
2, ammonium sulfate precipitation
Add the ammonium sulfate powder in the solubility total protein extracting solution that obtains to step 1, make its saturation ratio in extracting solution reach 60%, in ice bath, used magnetic stirrer 40 minutes.
3, Chen Dian redissolution and dialysis
With centrifugal 20 minutes of 4 ℃ of extracting solutions, the 7000rpm of completing steps 2, collecting precipitation; Add the 2ml deionized water in precipitation, transfer pH to 10.5 with the 1M Tris aqueous solution, dissolution precipitation is transferred in the dialysis tubing then carefully, with 6 hours (the PBS damping fluid that more renewed in per two hours) of 4 ℃ of dialysis of 2000ml PBS damping fluid.
4, immunoaffinity chromatography
Filler: CNBr-activated Sepharose4B(is available from GE company, and marque is 17-0430-01).
The antibody that is used for immunoaffinity chromatography: the monoclonal antibody of anti-Bt Cry1Ab/Ac albumen.
Level pad: PBS damping fluid.
The Gly-HCl damping fluid of scavenging solution: pH2.5,0.01M.
Elutriant: the Gly-HCl damping fluid that contains volume ratio and be pH2.5, the 0.01M of 50% ethylene glycol.
Flow velocity: equilibrium velocity is 20 rev/mins, and last sample flow velocity, cleaning flow velocity and elution flow rate are 4 rev/mins.
The immunoaffinity chromatography process: (1) prepares immune affinity chromatographic column with filler and the antibody that is used for immunoaffinity chromatography according to the subsidiary working instructions of filler, and column volume is 3.5ml; (2) with the level pad balance pillar of 5 times of column volumes; (3) go up the solution that sample step 3 obtains; (4) with 2 times of scavenging solution washing pillars of going up the sample volume, to remove foreign protein; (5) carry out wash-out with 3 times of elutriants of going up the sample volumes, collected elutriant behind the post by the every pipe of 1ml.
5, every pipe is crossed behind the post elutriant and transferred pH to 7.5 with the 1M Tris aqueous solution immediately.
6, adopt application number to detect the solution that every pipe step 5 obtains for the test kit of the step 1 preparation of the embodiment 2 of the patent application of " 201210012498.4 ", method is as follows:
(1) the bag quilt of polyclonal antibody: the rabbit polyclonal antibody of the anti-Bt Cry1Ac of 1mg/mL albumen is cushioned liquid with bag dilutes, the volume ratio that is cushioned liquid according to the rabbit polyclonal antibody of anti-Bt Cry1Ac albumen and bag is to join in the enzyme plate after the dilution proportion of 1:500, every hole 100 μ L, 37 ℃ of incubations 3 hours; Remove the solution in the enzyme plate, wash plate 4 times with the PBS damping fluid, dry.
(2) in the enzyme plate of step (1), add the solution that step 5 obtains, every hole 100 μ L, control wells adds 100 μ L PBS damping fluids; 37 ℃ of incubation 30min; Outwell the solution in the enzyme plate, wash plate 4 times with the PBS damping fluid, dry.
(3) the anti-Bt Cry1Ac protein monoclonal antibody (being enzymic-labelled antibody) with the 1mg/mL horseradish peroxidase-labeled dilutes with the PBS damping fluid, after being the 1:1000 dilution according to the volume ratio of enzymic-labelled antibody and PBS damping fluid, add the enzymic-labelled antibody after the 100 μ L dilution respectively in the enzyme plate of above-mentioned steps (2); 37 ℃ of incubation 30min; Outwell the solution in the enzyme plate, wash plate 4 times with the PBS damping fluid, dry.
(4) in the enzyme plate of step (3), add 100 μ L substrate buffer solutions respectively, behind the room temperature reaction 15min, add the aqueous sulfuric acid termination reaction of 50 μ L2.0M again in every hole.
(5) under 492nm, measure the OD value.
(6) blade with cotton variety stone far away 321 grinds in mortar, and in 4 ℃ of extractions 12 hours, 8000r/min4 ℃ of centrifuging and taking supernatant carried out supernatant above-mentioned steps (1) then to (5) with the PBS damping fluid.
(7) if more than three times of OD value that the OD value that step (5) obtains is step (6) to be obtained, the result is positive.
7, the solution in the pipe of test positive in the step 6 is merged, be transferred in the dialysis tubing, with 6 hours (the PBS damping fluid that more renewed in per two hours) of 4 ℃ of dialysis of 2000ml PBS damping fluid.
Polyacrylamide gel electrophoresis figure in the purge process sees Fig. 1.Among Fig. 1, swimming lane 1 corresponding sample is the solution first, and swimming lane 2 corresponding sample are the solution that dissolution precipitation obtains in the step 3, and swimming lane 3 corresponding sample are that step 7 obtains solution, swimming lane 4 corresponding sample are protein marker, swimming lane 5 correspondences be the Bt Cry1Ac albumen of prokaryotic expression.The Bt Cry1Ac albumen that is purchased is consistent with the result of swimming lane 3.
Reclaim the purpose band in the swimming lane 3 and carry out the order-checking of 15 amino-acid residues of N end, the result shows that this purpose band is Bt Cry1Ab/Ac albumen really.
One, immunogenic preparation
1, the PB damping fluid with pH7.5,0.1mol/L is mixed with the solution A that protein concentration is 1mg/mL with the Bt Cry1Ac albumen that embodiment 1 prepares.
The preparation method of the PB damping fluid of pH7.5,0.1mol/L: with 0.2g KH
2PO
4With 2.96g Na
2HPO
412H
2O is dissolved in the distilled water, uses the distilled water constant volume to 1L.
2, solution A and solution B equal-volume are mixed, obtain solution C.
Solution B by solute and solvent composition, solvent is water, solute and concentration thereof are: the 2%(volume ratio) beta-mercaptoethanol and 10g/100mLSDS.
3, with solution C water-bath 3-5 minute (making the abundant sex change of albumen) in 100 ℃ of boiling water, be cooled to after the room temperature and the Freund's complete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the fundamental immunity of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former I of fundamental immunity.
4, with solution C water-bath 3-5 minute (making the abundant sex change of albumen) in 100 ℃ of boiling water, be cooled to after the room temperature and the Freund's incomplete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the booster immunization of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former I of booster immunization.
Two, immunogenic preparation
1, the PB damping fluid with pH7.5,0.1mol/L is mixed with the solution D that protein concentration is 0.1mg/mL with the Bt Cry1Ac albumen that embodiment 1 prepares.
2, solution D and solution B equal-volume are mixed, obtain solution E.
3, with solution E water-bath 3-5 minute (making the abundant sex change of albumen) in 100 ℃ of boiling water, be cooled to after the room temperature and the Freund's complete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the fundamental immunity of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former II of fundamental immunity.
4, with solution E water-bath 3-5 minute (making the abundant sex change of albumen) in 100 ℃ of boiling water, be cooled to after the room temperature and the Freund's incomplete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the booster immunization of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former II of booster immunization.
Three, immunogenic preparation
1,1 solution A for preparing and the 10 parts by volume solution F with 1 parts by volume step 1 mix, and obtain solution G.
Solution F by solute and solvent composition, solvent is water, solute and concentration thereof are: the 0.1%(volume ratio) beta-mercaptoethanol and 0.1g/100mL SDS.
2, with solution G water-bath 3-5 minute (making the abundant sex change of albumen) in 100 ℃ of boiling water, be cooled to after the room temperature and the Freund's complete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the fundamental immunity of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former III of fundamental immunity.
3, with solution G water-bath 3-5 minute (making the abundant sex change of albumen) in 100 ℃ of boiling water, be cooled to after the room temperature and the Freund's incomplete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the booster immunization of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former III of booster immunization.
Four, immunogenic preparation
1,1 solution D for preparing and the 10 parts by volume solution F with 1 parts by volume step 2 mix, and obtain Solution H.
2, with Solution H water-bath 3-5 minute (making the abundant sex change of albumen) in 100 ℃ of boiling water, be cooled to after the room temperature and the Freund's complete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the fundamental immunity of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former IV of fundamental immunity.
3, with Solution H water-bath 3-5 minute (making the abundant sex change of albumen) in 100 ℃ of boiling water, be cooled to after the room temperature and the Freund's incomplete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the booster immunization of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former IV of booster immunization.
Five, immunogenic preparation
1, the PB damping fluid with pH7.5,0.1mol/L is mixed with the solution that protein concentration is 1mg/mL with the Bt Cry1Ac albumen that is purchased.
2, the solution that step 1 is obtained water-bath 3-5 minute (making the abundant sex change of albumen) in 100 ℃ of boiling water, be cooled to after the room temperature and the Freund's complete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the fundamental immunity of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former V of fundamental immunity.
3, the solution that step 1 is obtained water-bath 3-5 minute (making the abundant sex change of albumen) in 100 ℃ of boiling water, be cooled to after the room temperature and the Freund's incomplete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the booster immunization of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former V of booster immunization.
Six, immunogenic preparation
1, the PB damping fluid with pH7.5,0.1mol/L is mixed with the solution that protein concentration is 0.1mg/mL with the Bt Cry1Ac albumen that is purchased.
2, the solution that step 1 is obtained water-bath 3-5 minute (making the abundant sex change of albumen) in 100 ℃ of boiling water, be cooled to after the room temperature and the Freund's complete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the fundamental immunity of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former VI of fundamental immunity.
3, the solution that step 1 is obtained water-bath 3-5 minute (making the abundant sex change of albumen) in 100 ℃ of boiling water, be cooled to after the room temperature and the Freund's incomplete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the booster immunization of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former VI of booster immunization.
Seven, immunogenic preparation
1, the solution that 1 of step 5 is obtained multigelation 3 times (making the abundant sex change of albumen) in-20 ℃ of refrigerators, be cooled to after the room temperature and the Freund's complete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the fundamental immunity of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former VII of fundamental immunity.
2, the solution that 1 of step 5 is obtained multigelation 3 times (making the abundant sex change of albumen) in-20 ℃ of refrigerators, be cooled to after the room temperature and the Freund's incomplete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the booster immunization of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former VII of booster immunization.
Eight, immunogenic preparation
1, the solution that 1 of step 6 is obtained multigelation 3 times (making the abundant sex change of albumen) in-20 ℃ of refrigerators, be cooled to after the room temperature and the Freund's complete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the fundamental immunity of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former VIII of fundamental immunity.
2, the solution that 1 of step 6 is obtained multigelation 3 times (making the abundant sex change of albumen) in-20 ℃ of refrigerators, be cooled to after the room temperature and the Freund's incomplete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the booster immunization of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former VIII of booster immunization.
Nine, immunogenic preparation
1, the solution that 1 of step 5 is obtained makes the abundant sex change of albumen with 1M aqueous sulfuric acid adjust pH to 3(), be cooled to after the room temperature and the Freund's complete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the fundamental immunity of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former IX of fundamental immunity.
2, the solution that 1 of step 5 is obtained makes the abundant sex change of albumen with 1M aqueous sulfuric acid adjust pH to 3(), be cooled to after the room temperature and the Freund's incomplete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the booster immunization of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former IX of booster immunization.
Ten, immunogenic preparation
1, the solution that 1 of step 6 is obtained makes the abundant sex change of albumen with 1M aqueous sulfuric acid adjust pH to 3(), be cooled to after the room temperature and the Freund's complete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the fundamental immunity of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former X of fundamental immunity.
2, the solution that 1 of step 6 is obtained makes the abundant sex change of albumen with 1M aqueous sulfuric acid adjust pH to 3(), be cooled to after the room temperature and the Freund's incomplete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the booster immunization of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former X of booster immunization.
11, immunogenic preparation
1, the solution that 1 of step 5 is obtained makes the abundant sex change of albumen with 1M aqueous sodium hydroxide solution adjust pH to 10(), be cooled to after the room temperature and the Freund's complete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the fundamental immunity of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former XI of fundamental immunity.
2, the solution that 1 of step 5 is obtained makes the abundant sex change of albumen with 1M aqueous sodium hydroxide solution adjust pH to 10(), be cooled to after the room temperature and the Freund's incomplete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the booster immunization of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former XI of booster immunization.
12, immunogenic preparation
1, the solution that 1 of step 6 is obtained makes the abundant sex change of albumen with 1M aqueous sodium hydroxide solution adjust pH to 10(), be cooled to after the room temperature and the Freund's complete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the fundamental immunity of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former XII of fundamental immunity.
2, the solution that 1 of step 6 is obtained makes the abundant sex change of albumen with 1M aqueous sodium hydroxide solution adjust pH to 10(), be cooled to after the room temperature and the Freund's incomplete adjuvant equal-volume mixes and with the abundant stirring and emulsifying of magnetic stirring apparatus (indiffusion in splashing into water), obtain for the preparation of the booster immunization of the antibody of sex change Bt Cry1Ac albumen formerly, be called for short the former XII of booster immunization.
The preparation of the antibody of embodiment 3, sex change Bt Cry1Ac albumen
One, the preparation of rabbit polyclonal antibody
Laboratory animal is New Zealand's large ear rabbit (available from Military Medical Science Institute's Experimental Animal Center) in rabbit age in March, more than the body weight 2kg.
1, fundamental immunity
The former I of fundamental immunity is adopted rear foot interdigit injection laboratory animal, and with Bt Cry1Ac albumimeter, injected dose is 1mg/.
2, booster immunization
Fundamental immunity adopts back leg femoral lymph node, back, the subcutaneous multi-point injection laboratory animal of neck with the former I of booster immunization after 4 weeks, and with Bt Cry1Ac albumimeter, injected dose is 1mg/; Carried out booster immunization once every 20 days according to identical method afterwards; Behind the 5th booster immunization the 7th day, carotid artery was adopted whole blood, and separation of serum adopts sad-saturated ammonium sulphate method to carry out purifying, obtains rabbit polyclonal antibody ,-20 ℃ of preservations.
Two, mouse Polyclonal Antibody Preparation
Laboratory animal is the 8-10 Bal b/C small white mouse (available from Military Medical Science Institute's Experimental Animal Center) in age in week.
1, fundamental immunity
The former II of fundamental immunity is adopted abdominal cavity, the subcutaneous multi-point injection laboratory animal in back, and with Bt Cry1Ac albumimeter, injected dose is 0.1mg/.
2, booster immunization
Fundamental immunity adopts abdominal cavity, the subcutaneous multi-point injection laboratory animal in back with the former II of booster immunization after 2 weeks, and with the BtCry1Ac albumimeter, injected dose is 0.1mg/; Carried out booster immunization once every 10 days according to identical method afterwards; From booster immunization for the third time, antibody titer is measured in the eye socket blood sampling in the 7th day after the immunity, and tiring is defined as OD
450nmValue is 1 o'clock serum diluting multiple, waits to tire greater than behind the 1:8000, wins eyeball and gets blood, and separation of serum adopts sad-saturated ammonium sulphate method to carry out purifying, obtains the mouse polyclonal antibody ,-20 ℃ of preservations.
One, the preparation of albumen sample
1, the cottonseed cake of the glad No. 6 cotton varieties preparation of weighing 1g state grinds with mortar, adds protein extract 2mL, and 4 ℃, the vibration of 300rpm room temperature extracted 1 hour, and 4 ℃ left standstill 1 hour then, got supernatant liquor, were sample-I.
Protein extract is by NaCl, KH
2PO
4, Na
2HPO
412H
2O, Tween-20 and water are formed, and the concentration of NaCl is 8.0g/L, KH
2PO
4Concentration be 0.2g/L, Na
2HPO
412H
2The concentration of O is 2.96g/L, and the concentration of Tween-20 is the 0.1%(volume ratio).
2, be mixed with the solution that protein concentration is 1mg/mL with the PB damping fluid of pH7.5,0.1mol/L, the Bt Cry1Ac albumen that embodiment 1 is prepared, be sample-II (positive control).
3, with sample-II in 100 ℃ of boiling water water-bath 3-5 minute, be sample-III (positive control).
4, the PBS damping fluid with pH7.5,0.1mol/L is mixed with the solution that protein concentration is 1mg/mL with bovine serum albumin (purchasing in Merck-Calbiochem article No. 12659), is sample-IV (negative control).
5, the PBS damping fluid of pH7.5,0.1mol/L is sample-V (blank).
Two, the prescription of various damping fluids
Bag is cushioned the carbonate buffer solution of liquid: pH9.6,0.05M.
Confining liquid: the commercially available skim-milk of 2mg is dissolved in the PBS damping fluid of 100mL pH7.5,0.1M, obtains confining liquid.
Sample diluting liquid: the PBS damping fluid of 0.5ml polysorbas20,0.5g gelatin and 500ml pH9.6,0.1M is mixed, obtain sample diluting liquid.
Citrate trianion-phosphate buffered saline buffer (pH5.5): by trisodium citrate, Na
2HPO
4Form with water, the concentration of trisodium citrate is 0.01M, Na
2HPO
4Concentration be 0.03M.
Substrate colour developing liquid: be purchased Sigma TMB colour developing liquid, article No. T0440-1L.
The aqueous hydrochloric acid of stop buffer: 1.0M.
Washings: by NaCl, KH
2PO
4, Na
2HPO
412H
2O, Tween-20 and water are formed, and the concentration of NaCl is 8.0g/L, KH
2PO
4Concentration be 0.2g/L, Na
2HPO
412H
2The concentration of O is 2.96g/L, and the concentration of Tween-20 is the 0.1%(volume ratio).
Three, use polyclonal antibody and detect sex change Bt albumen
1, bag quilt
Every hole adds 100 μ L rabbit polyclonal antibodies (get the rabbit polyclonal antibody of embodiment 2 preparations, being cushioned liquid adjustment protein concentration with bag is 100ng/mL) in 96 hole enzyme plates, hatches 3 hours for 37 ℃, then with washings washing 4 times.
2, sealing
Every hole adds 150 μ L confining liquids, seals 1h in 37 ℃ of wet boxes, abandons confining liquid, then with washings washing 4 times.
3, application of sample
Every hole adds 100 μ L testing samples (sample-V, sample-IV, sample-III, sample-II or sample-I), puts in the wet box incubation 30min under 37 ℃ of conditions, then with washings washing 4 times.
4, add mouse resists more
Every hole adds 100 μ L mouse polyclonal antibodies (get the mouse polyclonal antibody of embodiment 3 preparation, adjusting protein concentration with sample diluting liquid is 1000ng/mL), puts in the wet box incubation 30min under 37 ℃ of conditions, then with washings washing 4 times.
5, add ELIAS secondary antibody
Every hole adds 100 μ L ELIAS secondary antibody (getting goat anti-mouse igg-HRP, with 1000 times of sample diluting liquid dilutions), puts in the wet box incubation 30min under 37 ℃ of conditions, then with washings washing 4 times.
6, colour developing
Every hole adds 100 μ L substrates colour developing liquid, and lucifuge is placed 15min.
7, stop
Every hole adds 100 μ L stop buffers, measures the OD value with microplate reader 450nm place.
Each sample to be tested arranges three multiple holes, is averaged the OD value.
The average OD value of sample-V (blank) is 0.084.The average OD value of sample-IV (negative control) is 0.169.The average OD value of sample-III (positive control) is 2.178.The average OD value of sample-II (positive control) is 3.036.The average OD value of sample-I is 1.876.
If the average OD value of sample to be tested is higher than the average OD value of negative control more than 0.2, the Bt Cry1Ac albumen that contains sex change in the testing sample is described then.
Four, using the available reagent box detects
Adopt QualiPlate Kit for Cry1Ac/Cry1Ac test kit (catalog number (Cat.No.): AP003CRBS, Kit Lot:043230) and the by specification of ENVIROLOGIX company that testing sample (sample-V, sample-IV, sample-III, sample-II or sample-I) is detected.Each sample to be tested arranges three multiple holes, is averaged the OD value.
The average OD value of sample-V (blank) is 0.059.The average OD value of sample-IV (negative control) is 0.060.The average OD value of sample-III is 0.070.The average OD value of sample-II (positive control) is 3.274.The average OD value of sample-I is 0.178.
1, with the PB damping fluid of pH7.5,0.1mol/L, the Bt Cry1Ac albumen that is purchased is mixed with the solution that protein concentration is 1mg/mL, in 100 ℃ of boiling water water-bath 3-5 minute, is sample liquid VI.
2, with the PB damping fluid gradient dilution of sample liquid with pH7.5,0.1mol/L, obtain each sample diluent.The method of the step 3 of employing embodiment 4 detects the sex change Bt albumen in each sample diluent, and photo is seen Fig. 2.
3, with the sample-IV among the embodiment 4 (negative control), sample-V (blank), sample liquid VI (sex change antibody), adopt the method for the step 3 of embodiment 4 to detect sex change Bt albumen respectively, photo is seen Fig. 3.
4, replace mouse polyclonal antibody in the method for step 3 of embodiment 4 with external commercially available antibody (external antibody), sample liquid VI is detected, photo is seen Fig. 3.
Claims (10)
1. for the preparation of the immunogen of the antibody of identifying sex change Bt albumen, be to contain the solution of sex change Bt albumen and the product that adjuvant mixes and emulsification obtains.
2. immunogen as claimed in claim 1 is characterized in that: the preparation method of the described solution that contains sex change Bt albumen is as follows: will contain and carry out denaturing treatment after the solution of Bt albumen and solution third mix, and obtain containing the solution of sex change Bt albumen; The volume proportion of the solution of the described Bt of containing albumen and described solution third is 1:(1-10); Described solution third is by solute and solvent composition, and described solvent is water, and described solute and concentration thereof are as follows: volume ratio is the beta-mercaptoethanol of 0.1-2% and the SDS of 0.1-10.0mg/100mL.
3. immunogen as claimed in claim 2 is characterized in that: described denaturing treatment is in 100 ℃ of boiling water water-bath 3-5 minute.
4. immunogen as claimed in claim 2 is characterized in that: described denaturing treatment is at-20 ℃ of refrigerator multigelations more than 3 times.
5. immunogen as claimed in claim 2 is characterized in that: described denaturing treatment is for transferring pH to 3 following or transfer more than the pH to 10.
6. immunogen as claimed in claim 1 is characterized in that: the preparation method of the described solution that contains sex change Bt albumen is as follows: the solution that will contain Bt albumen carries out denaturing treatment, obtains containing the solution of sex change Bt albumen.
7. immunogen as claimed in claim 6 is characterized in that: described denaturing treatment is in 100 ℃ of boiling water water-bath 3-5 minute.
8. immunogen as claimed in claim 6 is characterized in that: described denaturing treatment is at-20 ℃ of refrigerator multigelations more than 3 times.
9. as arbitrary described method in the claim 1 to 8, it is characterized in that: described adjuvant is freund's adjuvant, specifically can be Freund's complete adjuvant or formula Freund not.
10. the polyclonal antibody that arbitrary described immunogen immune animal in the claim 1 to 8 is obtained.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310070514.XA CN103193873B (en) | 2013-03-06 | 2013-03-06 | For the preparation of identifying the immunogen of antibody of sex change Bt albumen and the antibody of preparation thereof and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310070514.XA CN103193873B (en) | 2013-03-06 | 2013-03-06 | For the preparation of identifying the immunogen of antibody of sex change Bt albumen and the antibody of preparation thereof and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103193873A true CN103193873A (en) | 2013-07-10 |
| CN103193873B CN103193873B (en) | 2015-11-18 |
Family
ID=48716703
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310070514.XA Expired - Fee Related CN103193873B (en) | 2013-03-06 | 2013-03-06 | For the preparation of identifying the immunogen of antibody of sex change Bt albumen and the antibody of preparation thereof and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103193873B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105693856A (en) * | 2016-04-25 | 2016-06-22 | 江苏省农业科学院 | Monoclonal antibody, cell strain secreting monoclonal antibody, preparation method and application |
| CN113493512A (en) * | 2021-07-26 | 2021-10-12 | 中国农业科学院生物技术研究所 | Preparation method of Cry3Bb rabbit polyclonal antibody |
| CN114371285A (en) * | 2021-12-28 | 2022-04-19 | 中国农业科学院生物技术研究所 | Insect-resistant protein Cry3Bb colloidal gold immunochromatographic assay rapid test strip and use method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590527A (en) * | 2012-01-16 | 2012-07-18 | 中国农业大学 | Method for detecting insecticidal crystal proteins Bt Cry1Ab/Ac and special enzyme linked immunosorbent assay kit thereof |
-
2013
- 2013-03-06 CN CN201310070514.XA patent/CN103193873B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102590527A (en) * | 2012-01-16 | 2012-07-18 | 中国农业大学 | Method for detecting insecticidal crystal proteins Bt Cry1Ab/Ac and special enzyme linked immunosorbent assay kit thereof |
Non-Patent Citations (4)
| Title |
|---|
| 严吉明等: "转Bt基因植物中杀虫蛋白的酶联免疫检测技术研究Ⅱ.Bt杀虫晶体蛋白抗体的制备", 《四川农业大学学报》, vol. 23, no. 2, 31 January 2005 (2005-01-31), pages 163 - 167 * |
| 孙明等: "苏云金芽胞杆菌不同类型晶体蛋白抗原的制备", 《杀虫微生物》, vol. 4, 31 December 1993 (1993-12-31), pages 57 - 59 * |
| 孙硕等: "双抗夹心酶免疫法检测转Bt基因抗虫棉种子的研究", 《棉花学报》, vol. 25, no. 1, 15 January 2013 (2013-01-15), pages 45 - 50 * |
| 王保民等: "苏云金芽胞杆菌杀虫晶体蛋白CryIA单克隆抗体的制备及在转Bt基因棉毒蛋白检测上的应用", 《棉花学报》, vol. 12, no. 1, 31 December 2000 (2000-12-31), pages 34 - 39 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105693856A (en) * | 2016-04-25 | 2016-06-22 | 江苏省农业科学院 | Monoclonal antibody, cell strain secreting monoclonal antibody, preparation method and application |
| CN113493512A (en) * | 2021-07-26 | 2021-10-12 | 中国农业科学院生物技术研究所 | Preparation method of Cry3Bb rabbit polyclonal antibody |
| CN114371285A (en) * | 2021-12-28 | 2022-04-19 | 中国农业科学院生物技术研究所 | Insect-resistant protein Cry3Bb colloidal gold immunochromatographic assay rapid test strip and use method thereof |
| CN114371285B (en) * | 2021-12-28 | 2024-06-04 | 中国农业科学院生物技术研究所 | Insect-resistant protein Cry 3Bb colloidal gold immunochromatography rapid test strip and application method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103193873B (en) | 2015-11-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102747040B (en) | Anti-influenza A virus nucleoprotein monoclonal antibody, its preparation and application | |
| CN101818129B (en) | Hybridoma cell line for anti-African swine fever virus monoclonal antibody and monoclonal antibody secreted thereby | |
| CN101825633B (en) | Competitive ELISA kit for detecting african swine fever virus antibody and purposes thereof | |
| CN101831407A (en) | Hybridoma cell line of monoclonal antibody against African swine fever virus and secreted monoclonal antibody thereof | |
| CN103992988B (en) | A kind of monoclonal antibody of the anti-canine distemper disease viral N proteins of hybridoma cell strain and generation thereof | |
| CN106282125A (en) | Hybridoma cell strain NaN 1 and the application thereof of anti-sulfa antibiotics monoclonal antibody is secreted in one strain | |
| CN103995126A (en) | ELISA kit for detecting Salmonella pullorum antibody | |
| CN101445558B (en) | Monoclonal antibody of glycinin and application thereof | |
| CN107422112A (en) | A kind of immune reagent kit for detecting ethopabate, preparation method and application | |
| CN103193873A (en) | Immunogen for preparing antibodies used for identification of denatured Bt proteins, antibody prepared by same and application thereof | |
| CN101441222B (en) | Method for detecting Beta accompany glycinin and specific antibody and reagent kit thereof | |
| CN102911919A (en) | Bar/PAT protein monoclonal antibody, method for preparing same and application of bar/PAT protein monoclonal antibody | |
| CN105087498A (en) | TCMTB monoclonal antibody hybridoma cell strain and application thereof | |
| CN102690351B (en) | Preparation method of plasmodium vivax aldolase protein monoclonal antibody | |
| CN1945335A (en) | Reagent kit for detecting hepatitis B virus e antigen and use | |
| CN103145815A (en) | Method of purifying Bt toxalbumin from insect-resistant cotton seeds | |
| CN102417895B (en) | Monoclonal antibody for beta-conglycinin beta-subunit and application of monoclonal antibody | |
| CN105004860A (en) | Florfenicol rapid detection kit and preparation and use method thereof | |
| CN107083370A (en) | Anti-treeing Shrew interferon gammas monoclonal antibody and hybridoma cell strain and the application for secreting the antibody | |
| CN102998461B (en) | Quick qualitative detection reagent for wheat globulin in milk and protein drinks and preparation method thereof | |
| CN102174104A (en) | Duck plague virus cyst membrane gI protein polyclonal antibody as well as preparation method and application thereof | |
| CN107188961A (en) | A kind of aspergillus flavus resisting toxin M1 monoclonal antibodies and immunosorbent, immune affinity column and preparation method thereof | |
| CN106970216A (en) | A kind of ELISA detection kit and detection method for detecting pyrimethanil residual | |
| CN101633920A (en) | Preparation method of monoclonal antibody of resisting treeshrew IgG | |
| CN109336974A (en) | A kind of preparation method of anti-TMP rabbit polyclonal antibody |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151118 Termination date: 20170306 |