CN103197075A - Method for detecting Bt protein in transgenic rice by quantum dot - Google Patents
Method for detecting Bt protein in transgenic rice by quantum dot Download PDFInfo
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Abstract
The invention belongs to the technical field of transgenic ingredient detection and relates to a method for detecting a Bt protein in transgenic rice by a quantum dot. The method is fast and simple. The method comprises the following steps of preparing a CdSe/ZnS quantum dot-labeled CrylAb protein-resistant double-antibody sandwich by a CrylAb-resistant monoclonal antibody as a primary antibody and quantum dot (QDs)-labeled goat anti-rabbit IgG as a second antibody, and building a double-antibody sandwich fluorescent immunosorbent assay method for detecting a rice Bt protein. The method allows the minimum CrylAb normalized protein detection concentration value of 9pg/mL, and has a linear detection range of 6 to 200pg/mL, a recovery rate of 91.6 to 105.9 and a variation coefficient of 3.0 to 12.6. The quantum dot double-antibody sandwich fluorescent immunosorbent assay method is suitable for quantitative detection of transgenic rice and has the characteristics of high detection rate, simple operation and high sensitivity.
Description
Technical field
The invention belongs to the detection technique field of crop transgene component, be specifically related to utilize the quantum spot check to survey the method for Bt albumen in the transgenic paddy rice, the present invention is relevant with the ELISA method.
Background technology
The safety evaluation of genetically modified organism is genetically modified organism and products thereof marketization and commercial prerequisite, the detection of transgene component is an important content in safety evaluation, sets up that genetically modified organism is quick, easy, accurate test method is safety evaluation and management lays the foundation.
China is in world lead level to the research of transgenic paddy rice, wherein, some of research and development are disease-resistant, the pest-resistant transgenic rice strain has applied for producing plantation bio-safety certificate, but owing to their some reasons such as genetically modified organism safety problem, there is not the strain approval to commercially produce application so far as yet.In view of the maturation of transgenic paddy rice technology with and the trend of product marketization, therefore set up a kind of method of quick, easy detection Bt albumen, have necessity and urgency.
Can carry out from nucleic acid and two levels of protein of foreign gene for the detection of transgene component at present.The detection of nucleic acid level, be primarily aimed at the nucleotide sequence of the foreign gene that changes over to and according to the homology of foreign gene and endogenous gene design detection method, the main method that adopts is pcr amplification, namely according to changing exogenous gene promoter, genes of interest, terminator and expression (reorganization) carrier information over to, the design Auele Specific Primer, carry out the PCR, having or not or how much judge whether to be transgenic product according to product.The PCR reaction has the characteristics of high sensitivity, high specific, high efficiency, is the main detection method in transgenic product primary dcreening operation stage.
The detection of albumen inspection level mainly is based on the interactional immunological method of antigen and antibody, carries out qualitative and quantitative detection at the specific proteins of external source destination gene expression in genetically modified plants and products thereof.The people such as Engvall of Sweden in 1971 respectively with cellulose and the poly-third ethene test tube as solid phase carrier adsorption antigen/antibody, set up enzyme linked immunosorbent assay (Enzyme Linked Immunosrbent Assay is called for short the ELISA method).People such as Voller used the polystyrene micro-reaction plate instead as the solid-phase immunity absorption carrier in 1974, the ELISA method is applied, make the enzyme-labelled antibody technique that is used for the antigen location develop into the assay method of liquid sample micro substance, and become a kind of method the most commonly used in the antigen-antibody detection gradually.It combines the immune response of enzyme labeling thing synantigen antibody complex with the catalysis amplification of enzyme, both kept the susceptibility of enzymic catalytic reaction, has kept the specificity of antigen-antibody reaction again, thereby has improved sensitivity greatly.It is again a kind of heterogeneous immune analysis method simultaneously, namely in each step of reaction washing process is arranged, thereby has removed unreacted reactant and interfering material.Since the ELISA method have highly sensitive, high specificity, easy and simple to handle, detect rapidly and on-radiation and plurality of advantages such as can measure in batches, make the ELISA method obtain application more and more widely.
But along with the requirement of detection sensitivity is more and more higher, the quantum dot with unique fluorescent characteristic more and more is subjected to people's attention.
Quantum dot (quantumdots) be a kind of that formed by II family～VI family or III family～V group element, stable, water-soluble, between semiconductor nano crystal grain 1nm～100nm, that can accept excitation light generation fluorescence.When its physical size during less than the Bohr radius of exciton, because quantum limitation effect makes it have unique optics and electronics character.Its optical property depends primarily on the size of its radius, and irrelevant with its composition, just can obtain from the ultraviolet to the near infrared range by the size that changes quantum dot in the spectrum of arbitrfary point.
Because have unique fluorescent characteristic, quantum dot is compared with traditional organic fluorescent dye and is had the following advantages:
(1) has wide excitation wavelength range and narrow emission wavelength ranges, namely can use the exciting light less than any wavelength of its emission wavelength 10nm to excite, so just can use with a kind of exciting light and excite multiple quantum dot simultaneously, launch the fluorescence of different wave length, thereby detect when can be used for multiple label.
(2) emission peak of quantum dot is narrow and symmetrical, and continuous distribution, and is overlapping little, like this, can use a plurality of probes simultaneously in the detected spectral range at one, and that emission spectrum does not occur is overlapping, the multicomponent analysis of biomolecule detected become easy.And the emission peak of traditional organic fluorescent dye is not only wide, and asymmetric, and hangover is serious, and it is serious to overlap each other, and interferes with each other easily, brings an insoluble difficult problem to analyzing and testing.
(3) emission wavelength of quantum dot can be next tuning by size and the composition of controlling it, can synthesize the quantum dot of required wavelength arbitrarily, and quantum point spectrum of uniform size peak is symmetrical Gaussian-like distribution, and the peak shape of traditional organic fluorescent dye then is lognormal distribution.
(4) the most frequently used organic fluorescent dye rhodamine 6G dyestuff of the fluorescence intensity ratio of quantum dot is high 20 times, and its stability is more than 100 times of rhodamine 6G dyestuff especially, and the anti-photobleaching ability of quantum dot is strong.
(5) good biocompatibility especially passes through after the various chemical modifications, can carry out specificity and connect, cytotoxicity is low, and is little to biosome harm, can carry out biological living mark and detection, and traditional organic fluorescent dye general toxicity is bigger, and biocompatibility is poor.
(6) fluorescence lifetime is long, and the fluorescence lifetime of typical organic fluorescent dye only is several nanoseconds (ns), and this time with the autofluorescence decay of a lot of biological specimens is suitable.And the fluorescence lifetime of quantum dot sustainable reach tens of nanoseconds (20ns～50ns), this makes counts after nanosecond when optical excitation, most autofluorescence background has decayed, and quantum dot fluorescence still exists, and can obtain not have the fluorescence signal of background interference this moment.
The optical characteristics that these are unique makes quantum dot become a kind of desirable fluorescence probe.Therefore, use quantum dot to replace organic fluorescent dye, will be in molecular labeling, signal conduction, cell play an important role in the researchs such as the motion of molecule and migration.Utilize the success of quantum dot labelled antibody be used for carried out the fluorescence immunoassay detection to the sulfadimidine of chicken is residual.Therefore, utilize quantum dot as biological fluorescent labeling, in researchs such as immuno-biology and food inspection, have broad application prospects.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, a kind of high sensitivity method for quick that is applicable to Bt in the transgenic paddy rice is provided.Method of the present invention is based on quantum dot double antibodies sandwich fluorescence immunoassay adsorption measurement principle, set up the detection method that quantitatively detects Bt protein content in the transgenic paddy rice, the present invention accurately quick, simple, high-sensitivity detection goes out Bt protein content in the transgenic paddy rice, do not need specific installation, for the testing staff of basic unit provides a kind of fast and convenient method, method of the present invention also is that genetically modified organism and products thereof safety evaluation and management are offered reference simultaneously.
Technical scheme of the present invention is as follows:
A kind of fast and convenient method of utilizing the quantum spot check to survey Bt albumen in the transgenic paddy rice, its step is as follows:
(1) anti-CrylAb MONOCLONAL ANTIBODIES SPECIFIC FOR
Be that antigen obtains rabbit anteserum with CrylAb albumen, obtain anti-CrylAb monoclonal antibody as primary antibodie by purifying, secrete the anti-CrylAb monoclonal antibody hybridoma cell of this monoclonal antibody
HHX-001 is deposited in Chinese typical culture collection center (CCTCC), and its preserving number is CTCC NO:C201214.
(2) the quantum dot-labeled goat-anti of CdSe/ZnS is exempted from i.e. two preparations that resist of IgG antibody
1) 500 μ L goat-antis are exempted from the 50 μ L small tubes that the IgG antibody-solutions is distributed into 10 equal portions, be stored in-20 ℃ of refrigerators, treat that the time spent takes out a tubule, place 4 ℃ of refrigerator places, standby;
2) get 25 μ L CdSe/ZnS quantum dot mother liquors, 20 μ L 1-ethyl-(3-dimethylaminopropyl) end diimmonium salt hydrochlorate (EDC) solution and 10 μ LN-N-Hydroxysuccinimide (NHS) solution, pH7.0, hybrid reaction 5min, add the 0.2mg goat-anti again and exempt from the IgG antibody-solutions, add the label solution that sterilized water is made into 100 μ L, react 2-4h at normal temperatures; Above-mentioned label solution is put mixing in the magnetic stirring apparatus, and reaction finishes to be placed in 4 ℃ of refrigerators to spend the night, and obtains the quantum dot-labeled goat-anti of CdSe/ZnS and exempts from IgG antibody crude product;
3) goat-anti that CdSe/ZnS is quantum dot-labeled is exempted from IgG antibody crude product 100 μ L and is put into the sample memotron that contains ultra filtration membrane with the application of sample rifle, in another pipe, put into the water of equivalent as balance, be 10 at rotating speed, 000rpm, centrifugation time is 20min, and centrifuging temperature is centrifugal in 4 ℃ the hydro-extractor;
4) after the centrifugal end of step 3), add the PBS damping fluid, set by step 3) the condition repeated centrifugation once;
5) after the centrifugal end of step 4), add 100 μ L PBS damping fluids in the sample memotron, then it is tipped upside down on the centrifuge tube, be 500rpm at rotating speed, the time is 3min, temperature is centrifugal and recovery sample in 4 ℃ the hydro-extractor, is two anti-;
(3) preparation of the quantum dot-labeled anti-CrylAb albumen double-antibody sandwich of CdSe/ZnS
Be primary antibodie with anti-CrylAb monoclonal antibody, this primary antibodie and bag are cushioned liquid dilution in 1: 1000 by volume, join then in the ELISA Plate hole, 20 ℃ of overnight incubation, inferior daily PBS damping fluid is washed plate 3 times, each 5min, add the sealing damping fluid after patting dry, every hole 200 μ L leave standstill 1h under 37 ℃, afterwards with PBS damping fluid washing 3 times, 5min at every turn; Be added on bag by in the hole after the CrylAb protein solution is diluted to 6.0pg/ml, 12.5pg/ml, 25.0pg/ml, 50.0pg/ml, 100.0pg/ml, 200.0pg/ml gradient concentration with the PBS damping fluid, every hole 100 μ L, under 37 ℃, leave standstill 1h, afterwards with PBS damping fluid washing 3 times, 5min at every turn; Behind the purifying two anti-ly is diluted to 10.00ug/mL with the PBS damping fluid, add the bag of ELISA Plate by in the hole. every hole 100 μ L, under 37 ℃, leave standstill 1h, wash fast with the PBS damping fluid afterwards, obtain the quantum dot-labeled anti-CrylAb albumen double-antibody sandwich of CdSe/ZnS;
(4) typical curve of drafting CrylAb albumen
The CrylAb protein standard solution dilution of 0.4 μ g/ml is 200pg/mL, 100, pg/mL, 50, pg/mL, 25pg/mL, 12.5pg/mL and 6pg/mL gradient concentration, detect the content of paddy rice CrylAb albumen with the quantum dot-labeled anti-CrylAb albumen double-antibody sandwich of CdSe/ZnS, CrylAb protein solution concentration with this gradient concentration dilution is independent variable, deducting blank relative intensity of fluorescence value with the CrylAb albumen relative intensity of fluorescence value of correspondence is dependent variable, the drawing standard curve, formula is as follows:
Y＝0.0137X+0.02 R2＝0.994，
X is that the CrylAb protein solution concentration of serial gradient dilution is independent variable,
Y is corresponding CrylAb albumen relative intensity of fluorescence;
(5) with the value of microplate reader detection OD450, judge the content of CrylAb albumen in the paddy rice to be detected;
Wherein:
Component and the preparation of CdSe/ZnS quantum dot mother liquor:
Take by weighing 0.0127g (0.1mmol) CdO and 0.1140g stearic acid and put into the three-necked bottle of 50mL, under argon shield, be heated to 130 ℃, make CdO fully be dissolved in the stearic acid; After cooling the temperature to room temperature, take by weighing trioctylphosphine oxide (TOPO) and each 1.5g of hexadecylamine (HDA) puts into three-necked bottle, be stirred and heated to 230 ℃, other takes by weighing 0.032g (1mmol) sulphur powder, under argon shield, make it to be dissolved in the 2mL trioctylphosphine phosphorus (TOP), in the needle tubing of suction 5mL as storing solution; When Cd precursor temperature reaches 230 ℃, Se powder storing solution injected three-necked bottle fast after, make temperature be down to 180 ℃, keep 10min, collect the CdSe sample and be dissolved in the chloroform standby;
The sulphur powder that takes by weighing the zinc stearate of 0.632g and 0.032g adds respectively in the vaccenic acid (ODE) of 2mL and is heated to 120 ℃ of fully dissolvings, obtain zinc stearate solution and sulphur powder solution respectively, be cooled to 70 ℃, down preserve standby in 70 ℃ zinc stearate solution and sulphur powder solution mixing back; The CdSe sample that will be scattered in the chloroform adds in the bottle, is heated to 120 ℃ and keeps 30m in, and the evaporate to dryness chloroform is warming up to 200 ℃; Extract the vaccenic acid solution of zinc stearate and sulphur powder, inject the ZnS shell with 0.2mL/m in, inject finish after, be warming up to 230 ℃ and keep 1h, be cooled to 60 ℃, reaction product is with heavyization of methyl alcohol, eccentric cleaning three times obtains the CdSe/ZnS chloroformic solution; Getting each 1mL of CdSe/ZnS chloroformic solution, mercaptopropionic acid and deionized water puts into bottle and mixes, strong agitation 3h, (quantum dot is namely moved up in the aqueous solution by initial chloroformic solution transfer, this is a kind of chemical phenomenon, cannot write in claims, can only write in the instructions, have reasoning character, claim is conservation of nature phenomenon phenomenon and rule, mechanism scheduling theory achievement not), extract upper water solution, add the methyl alcohol eccentric cleaning three times, after be scattered in and namely obtain CdSe/ZnS quantum dot mother liquor in the 1mL deionized water;
PBS damping fluid component and preparation:
KH2PO4 0.2g, NaCl 8.0g, KCl 0.2g, Na2HPO4 2.9g, Tween-20 0.5mL, adding distil water is settled to 1L, transfers pH to 7.4;
Bag is cushioned component and the preparation of liquid:
Na2CO3 1.59g, NaHCO3 2.93g, adding distil water is settled to 1000mL;
Sealing damping fluid component and preparation:
Bovine serum albumin(BSA) 0.1g is dissolved in the 100mL PBS damping fluid.
Anti-Cry1Ab monoclonal antibody hybridoma cell HHX-001 with the monoclonal antibody of above-mentioned secretion Cry1Ab albumen, deliver China on January 6th, 2012. Wuhan. Chinese typical culture collection center (CCTCC) preservation in the Wuhan University, its preserving number is CCTCC NO:C201214.
The present invention has set up the new method that the double antibodies sandwich fluorescence immunoassay determining adsorption standard measure that uses quantum dot-labeled antibody detects the CrylAb albumen in the transgenic paddy rice.This method is minimum to the concentration of standard protein can the inspection value to be 9pg/mL, and linear detection range is 6～200pg/mL; The recovery is 91.6～105.9; The coefficient of variation is 3.0～12.6.And with this method Bt albumen in transgenic paddy rice and the non-transgenic paddy rice is quantitatively detected, the result shows that the double antibodies sandwich fluorescence immunoassay adsorption measurement of quantum dot-labeled antibody is fit to the quantitative detection of transgenic paddy rice.
The simple and practical detection method of Bt albumen in the designed transgenic paddy rice of the present invention.Compare this invention with other traditional detection method following advantage arranged:
1. the present invention is economical and practical: reaction is to carry out under the condition of constant temperature, thus do not need expensive PCR instrument, and also sense cycle is shorter.
2. of the present invention highly sensitive: this method is 9pg/mL to the minimal detectable concentration of standard Bt albumen, than the high 800-1000 of detection sensitivity of conventional ELISA method doubly.
3. the present invention detects simply: this method detects objective, the easy judgement of Bt albumen result in the transgenic paddy rice, can carry out quantitative test to the content of transgenic product.
More detailed technical scheme sees that " embodiment " is described.
Description of drawings
Fig. 1: be quantum dot double antibodies sandwich fluorescence immunoassay adsorption measurement mechanism synoptic diagram of the present invention.
Fig. 2: be techniqueflow chart of the present invention.
Fig. 3: the TEM sem image that is the CdTe quantum dot.
Fig. 4: be the emission spectrum image that the quantum dot-labeled goat-anti of CdTe is exempted from IgG.
The quantum dot-labeled goat-anti of Fig. 5: CdTe is exempted from the electrophoretogram of IgG
Fig. 6: quantum dot double antibodies sandwich fluorescence immunoassay adsorption measurement detects the concentration standard curve figure of CrylAb albumen.
Embodiment
The invention will be further described below by embodiment, but be not restriction the present invention.
The preparation of CrylAb albumen entrusts the beautiful minister bio tech ltd in Shanghai to finish, concrete steps are as follows: will be stored in-70 ℃ of Bacillus thuringiensis bacterial strain (kurstaki HD-1 in the refrigerator, a kind of bacterial strain of open report, referring to document: Linda Thorne, Fermin Garduno, Ted Thompson, Debra Decker, Maryann Zounes, Martha Wild, Alan m.Walfield, And Thomas J.Pollock*, Structural Similarity between the Lepidoptera-and Diptera-Specific Insecticidal Endotoxin Genes of Bacillus thuringiensis subsp " kurstaki " and " israelensis ", Journal of Bacteriology, June 1986, p.801-811), be scoring to the dull and stereotyped last 30 ℃ of grow overnight of LB, choose single bacterium colony and spend the night based on 30 ℃ of shaken cultivation to the LB cultivation of 10mL.Again the bacterium liquid of incubated overnight is added in the PGSM nutrient culture media of 200mL after 30 ℃ of shaken cultivation 3-4 days, examine under a microscope the cell that cell can be seen cracking.4 ℃ then, the centrifugal 15min of 4000rpm collects culture, with ice-cold 0.1mol/L NaCl washing precipitation once, 4 ℃, the centrifugal 15min of 4000rpm collects culture, uses ice-cold distilled water washing precipitation more once, at 4 ℃, the centrifugal 15min of 4000rpm collects culture, and precipitation is suspended in the ice-cold pure water of 15mL.Again with solution in 4 ℃, the centrifugal 15min collecting precipitation of 4000rpm, this precipitation is CrylAb albumen.Precipitation is suspended in 15min dissolves CrylAb albumen fully among the 0.1mol/LNaOH, with the pH to 7.0 of 1mol/LTris buffer solution regulator solution, behind the centrifugal 10min of 12000rpm, gets supernatant and dialyse three times with the PBS solution of 100 times of volumes at once.Adopt the Bradford method to measure its concentration;
Wherein:
Described reagent component and proportioning thereof are as follows:
The LB nutrient culture media: tryptone (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L, sodium chloride (NaCl) 10g/L, NaOH regulates the pH of this nutrient culture media, makes it reach 7.4;
The PGSM nutrient culture media: bacto peptone 7.5g, glucose 1g, KH2PO4 3.4g, K2HPO4 4.35g, adding distil water is settled to 1L, transfers pH to 7.2, and 30min sterilizes under 121 ℃ of high-temperature steams;
The PBS damping fluid: KH2PO4 0.2g, NaCl 8.0g, KCl 0.2g, Na2HPO4 2.9g, Tween-20 0.5mL, adding distil water is settled to 1L, transfers pH to 7.4;
Tris buffer solution: 50ml 0.1mol/L trishydroxymethylaminomethane (Tris) solution is regulated pH to 7.0 with 0.1mol/L hydrochloric acid, and thin up is to 100ml.
The preparation of embodiment 2 anti-CrylAb monoclonal antibodies (primary antibodie)
(1) anti-CrylAb MONOCLONAL ANTIBODIES SPECIFIC FOR
Present embodiment carries out with reference to reported method in Xue Qingshan " philosophy and technique of in vitro culture " the Science Press calendar year 2001 version.Concrete grammar is as follows:
Adopt the method immunizing rabbit of back multi-point injection, select the hypodermic injection of 4-6 point in rabbit backbone both sides with the CrylAb albumen that obtains among the embodiment 1, every some injection 0.1mL, two Zhou Houzai select the difference injection in above-mentioned position at interval.The about 100 μ g of CrylAb protein content of each immunity.Immune time gets final product at four or five times, and the antigen consumption can reduce number of times greatly.
Rabbit is bought back and raises about a week, carries out the immunity first time, and immune 2-4 carries out the immunity second time after week for the first time, afterwards every immunity in 7-10 days once.After one week of immunity, can get blood in arteria auricularis and both must resist CrylAb monoclonal antibody crude product.
(2) anti-CrylAb Purification of Monoclonal Antibodies
The antigen affinity purification specificity antagonism CrylAb monoclonal antibody that utilization is fixed on the NC film is carried out purifying.Concrete grammar is as follows:
1) massfraction that adds 0.1mL in the 1mL rabbit anteserum is 10% lauryl sodium sulfate aqueous solution (being SDS), and the beta-mercaptoethanol of 0.01mL is in 100 ℃ of following water-bath 5min;
2) add 100uL 10 * SDS-PAGE damping fluid (purchasing in the graceful bio tech ltd of last Hypon), mixing;
3) encapsulating, upper strata massfraction are 5%, and lower floor's massfraction is 8% SDS-PAGE glue;
4) determine albumen applied sample amount (general albumen applied sample amount be 20 or 40uL) according to the glue hole size, and point dyes albumen marker in advance;
5) electrophoresis concentrates glue (voltage of electrophoresis apparatus is 80V), separation gel (voltage of electrophoresis apparatus is 120V);
6) before the commentaries on classics film, cut the redundance of SDS-PAGE glue earlier, glue is placed transfering buffering liquid (purchasing in the graceful bio tech ltd of last Hypon) 15min.Cut and 8 of big filter paper such as glue, 1 of NC film and sponge are dipped in 15min in the transfering buffering liquid.
7) open film and shift folder;
1. put into (getting rid of bubble for every layer) successively: two-layer sponge, four metafiltration paper, the NC film is put four metafiltration paper, two-layer sponge again.Cut off unnecessary NC film and filter paper;
2. will shift in the clamping and also fix, glue places electrophoresis tank facing to negative pole;
3. add that transfering buffering liquid (voltage of electrophoresis apparatus is 5V) changes film and spends the night;
8) with the dyeing of Ponceaux dye liquor;
1. take out film and place double dish, add Ponceaux dye liquor dyeing 5-10min;
2. remove the Ponceaux dyestuff, wash twice with deionized water, develop the color until band.
9) antibody combination
1. scissors is cut the purpose band, places double dish, adds 1 * PBS damping fluid (prescription as described later), and about 10min decolours;
2. discard the PBS damping fluid, adding massfraction is 3% bovine serum albumin(BSA) (BSA now joins with 1 * PBS damping fluid), places jog on the shaking table, sealing 1h;
3. discard BSA, add fresh massfraction and be 3% BSA (now joining with 1 * PBS damping fluid), add the anti-CrylAb monoclonal antibody that obtains among the 0.5ml1 again.Shake 16h or 16 ℃ in 4 ℃ and shake 5h;
4. discard bovine serum albumin(BSA), wash 5 times with 1 * PBS damping fluid, each 5min;
10) antibody reclaims
The NC film is cut into 0.5cm
2The fragment of size is put into the EP pipe, adds 500uL elution buffer (0.2mol/L glycocoll), leaves standstill 20min; Be transferred in the new EP pipe, add 50 μ L 1mol/L trishydroxymethylaminomethanes and 60uL10 * PBS damping fluid mixing, 4 ℃ of preservations.Getting 200uL detects anti-CrylAb monoclonal antibody and tires.
(3) preservation of anti-CrylAb monoclonal antibody
Anti-CrylAb monoclonal antibody and the hybridoma of above-mentioned preparation success are carefully merged (concrete operation method reference: Xue Qingshan, " philosophy and technique of in vitro culture " Science Press
Reported method in the calendar year 2001 version), the anti-CrylAb monoclonal antibody hybridoma cell of screening secretion
HHX-001 (this hybridoma is deposited in Chinese typical culture collection center (CCTCC), and its preserving number is CTCC NO:C201214).
(4) using indirect elisa method to measure anti-CrylAb monoclonal antibody tires
1) the antigen coated carbonate buffer solution (prescription as described later) that prepared CrylAb albumen is used 0.05mol/L pH9.6 is with volume ratio dilution in 1: 500, and bag is by 96 hole polystyrene reaction plates, and the 100uL/ hole is put 4 ℃ and spent the night; Inferior daily PBS massfraction is washing lotion washing 3 times, each 10min at interval, and patting dry the back, to add massfraction be that the PBS damping fluid of 10%BSA seals, the 200uL/ hole, behind 37 ℃ of wet boxes sealing 1h, with PBS damping fluid washing 3 times, each 10min pats dry.Putting 4 ℃ of refrigerators preserves standby.
2) add antibody to be checked and be made into PBS damping fluid dilution bovine serum albumin(BSA) that to contain the bovine serum albumin massfraction be 0.1% bovine serum albumin solution (bovine serum albumin(BSA) is taken from the rabbit ear artery serum after the immunity), add bag by in the good reaction plate, set up negative control and blank simultaneously, the 100uL/ hole, act on 1h down at 37 ℃, with PBS damping fluid washing 3 times, each 10min pats dry.
3) add goat anti-rabbit igg (purchasing the beautiful minister bio tech ltd in Shanghai) that ELIAS secondary antibody will be purchased alkaline phosphatase (AP) mark by recommended density (volume ratio) doubly dilution in 1: 1000, the 100uL/ hole, reaction 1h under 37 ℃, with PBS damping fluid washing 3 times, each 10min pats dry.
4) develop the color in each reacting hole and to add the NPP solution of interim preparation, 75uL/ hole, lucifuge colour developing 30-60min under 37 ℃ or room temperature.
5) cessation reaction adds 25uL 0.5mol/L NaOH cessation reaction in each reacting hole.
6) result judges in microplate reader in OD
450Place's reading after the zeroing of blank hole, is surveyed each hole value.
Wherein:
Described reagent component and proportioning thereof are as follows:
Carbonate buffer solution: Na2CO3 1.59g, NaHCO3 2.93g, adding distil water is settled to 1000mL, transfers pH 9.6;
PBS damping fluid: KH
2PO
40.2g, NaCl 8.0g, KCl 0.2g, Na
2HDO
42.9g, Tween-20 0.5mL, adding distil water is settled to 1L, transfers pH to 7.4;
NPP solution: 5ml 1mmol/L MgCl
2, 2.5ml 1mmol/L ZnCl
2, 1.5ml 0.1mol/L glycocoll is regulated pH to 10.4 with 0.1mol/L NaOH.
The preparation of embodiment 3 water-soluble CdSes/ZnS quantum dot
Preparation reference literature reported method (the Xie HY of water-soluble CdSe/ZnS quantum dot, Liang JG, Liu Y, Zhang ZL, Pang DW, He ZK, et al.Preparation and characterization of overcoated II-VI quantum dots.J Nanosci Nanotechnol 2005; 5:880e6), concrete steps are: take by weighing the three-necked bottle that 0.0127g (0.1mmol) CdO and 0.1140g stearic acid are put into 50mL, under argon shield, be heated to 130 ℃, CdO fully is dissolved in the stearic acid.After cooling the temperature to room temperature; take by weighing trioctylphosphine oxide (TOPO) and each 1.5g of hexadecylamine (HDA) puts into three-necked bottle; be stirred and heated to 230 ℃; other takes by weighing 0.032g (1mmol) S powder; under argon shield; make it to be dissolved in the 2mL trioctylphosphine phosphorus (TOP), in the needle tubing of suction 5mL as storing solution.When Cd precursor temperature reaches 230 ℃, Se powder storing solution injected three-necked bottle fast after, temperature is down to 180 ℃, keeps 10min, collects the CdSe sample and is dissolved in the chloroform standby.
The sulphur powder that takes by weighing the zinc stearate of 0.632g and 0.032g adds respectively in the vaccenic acid (ODE) of 2mL and is heated to 120 ℃ of fully dissolvings, cool to 70 ℃, obtain zinc stearate solution and sulphur powder solution, above-mentioned two kinds of solution are mixed preserve standby down in 70 ℃.The CdSe sample that will be scattered in the chloroform adds in the bottle, is heated to 120 ℃, keeps 30min, and the evaporate to dryness chloroform is warming up to 200 ℃.Extract the vaccenic acid solution of zinc stearate and sulphur powder, inject the ZnS shell with the flow velocity of 0.2mL/min, after injection is finished, be warming up to 230 ℃, keep 1h, be cooled to 60 ℃ again, reaction product is with heavyization of methyl alcohol, and eccentric cleaning three times obtains the CdSe/ZnS chloroformic solution.Getting each 1mL of CdSe/ZnS chloroformic solution, mercaptopropionic acid and deionized water puts into bottle and mixes, strong agitation 3h, quantum dot is namely moved up in the aqueous solution by initial chloroformic solution transfer, extract upper water solution, add the methyl alcohol eccentric cleaning three times, after both be scattered in the 1mL deionized water CdSe/ZnS quantum dot mother liquor.Then, right its carries out transmission electron microscope, fluorescence spectrum characterizes (seeing Fig. 3, shown in Figure 4).
The quantum dot-labeled goat-anti of embodiment 4CdSe/ZnS is exempted from i.e. two preparations that resist of IgG antibody
1) 500 μ L goat-antis are exempted from the 50 μ L small tubes that IgG antibody-solutions (purchasing the beautiful minister bio tech ltd in Shanghai) is distributed into 10 equal portions, be stored in-20 ℃ of refrigerator places, treat that the time spent takes out a tubule, place 4 ℃ of refrigerator places, standby;
2) get the CdSe/ZnS quantum dot mother liquor that 25 μ L embodiment 3 obtain, 20 μ L 1-ethyl-(3-dimethylaminopropyl) end diimmonium salt acid salt solution (EDC solution) and 10 μ LN-N-Hydroxysuccinimide solution (NHS solution, pH7.0) hybrid reaction 5min, add the 0.2mg goat-anti again and exempt from the IgG antibody-solutions, add the label solution that sterilized water is made into 100 μ L, react 2-4h at normal temperatures; Described label solution is put mixing in the magnetic stirring apparatus, make its reaction evenly, reaction finishes to be placed in 4 ℃ of refrigerators to spend the night, and obtains the quantum dot-labeled goat-anti of CdSe/ZnS and exempts from IgG antibody crude product;
3) the quantum dot-labeled goat-anti of step 1 preparation CdSe/ZnS is exempted from IgG antibody crude product 100 μ L and put into the sample memotron that contains ultra filtration membrane with the application of sample rifle, in another pipe, put into the water of equivalent as balance, be 10 at rotating speed, 000rpm, centrifugation time is 20min, and centrifuging temperature is centrifugal in 4 ℃ the hydro-extractor;
4) after the centrifugal end of step 3), add the PBS damping fluid, set by step 3) the condition repeated centrifugation once;
5) after the centrifugal end of step 4), add 100 μ L PBS damping fluids in the sample memotron, then it is tipped upside down on the centrifuge tube, be 500rpm at rotating speed, the time is 3min, temperature is centrifugal and recovery sample in 4 ℃ the hydro-extractor, is two anti-;
The preparation of the anti-CrylAb albumen double-antibody sandwich that embodiment 5CdSe/ZnS is quantum dot-labeled
Be primary antibodie with the CrylAb monoclonal antibody, in the ELISA Plate hole, add the CrylAb monoclonal antibody that is cushioned liquid dilution (CrylAb monoclonal antibody and bag are cushioned liquid volume ratio 1: 1000) by bag, in 20 ℃ of overnight incubation, inferior daily PBS damping fluid is washed plate 3 times, and each 5min adds the sealing damping fluid after patting dry, every hole 200 μ L, under 37 ℃, leave standstill 1h, afterwards with PBS damping fluid washing 3 times, each 5min; Be added on bag by in the hole after the CrylAb protein solution is diluted to 6.0pg/ml, 12.5pg/ml, 25.0pg/ml, 50.0pg/ml, 100.0pg/ml, 200.0pg/ml gradient concentration with the PBS damping fluid, every hole 100 μ L, under 37 ℃, leave standstill 1h, afterwards with PBS damping fluid washing 3 times, 5min at every turn; Behind the purifying two anti-ly is diluted to 1.25ug/mL, 2.50ug/mL, 5.00ug/mL, 10.00ug/mL, 20.00ug/mL respectively as two anti-working concentrations through the PBS damping fluid, be added on bag by in the hole. every hole 100 μ L, under 37 ℃, leave standstill 1h, wash fast with the PBS damping fluid afterwards, obtain the quantum dot-labeled anti-CrylAb albumen double-antibody sandwich of CdSe/ZnS.
Wherein:
Component and proportioning that described bag is cushioned liquid are as follows:
Na
2CO
31.59g, NaHCO
32.93g adding distil water is settled to 1000mL;
Described sealing damping fluid component and proportioning are as follows:
Bovine serum albumin(BSA) 0.1g is dissolved in the 100mLPBS damping fluid.
The concentration standard curve of embodiment 6CrylAb albumen is set up
CrylAb protein standard solution (is purchased the beautiful minister bio tech ltd in Shanghai, 0.4 μ g/ml) accurately dilution is 6 gradient concentrations, be respectively 200,100,50,25,12.5,6pg/mL detects paddy rice Bt albumen with quantum dot double antibodies sandwich method, is independent variable with the CrylAb protein standard solution concentration of above-mentioned gradient dilution, deducting blank relative intensity of fluorescence value with the CrylAb albumen relative intensity of fluorescence value of correspondence is dependent variable, the drawing standard curve obtains the concentration standard curve computing formula of CrylAb albumen as shown in Figure 6, and this formula is as follows:
Y＝0.0137X+0.02 R
2＝0.994。
X is that the CrylAb protein solution concentration of serial gradient dilution is independent variable;
Y is corresponding CrylAb albumen relative intensity of fluorescence.
With the concentration standard curve of above-mentioned CrylAb albumen as the standard of judging CrylAb protein content in the paddy rice to be detected.
The sensitivity checking of the anti-CrylAb albumen double-antibody sandwich elisa detection method that embodiment 7CdSe/ZnS is quantum dot-labeled
Determine antigen coated concentration and antibody best effort concentration.Concrete steps are: the CrylAb protein of purifying is cushioned liquid with the bag of pH9.6 (was equivalent to that the CrylAb protein content is respectively 2.0 μ g, 1.0 μ g, 0.5 μ g, 0.25 μ g, 0.125 μ g, 0.0625 μ g among per 100 μ L by 1: 100,1: 200,1: 400,1: 800,1: 1600,1: 3200, add successively in each hole of ELISA Plate (100 μ L/ hole), each dilutability is two row, spends the night in 4 ℃.Inferior daily PBS damping fluid washing 3 times, each 10min at interval, with 10%BSA sealing back (200 μ L/ hole), behind 37 ℃ of preservation 1h, again with PBS damping fluid washing 3 times, each 10min pats dry, and respectively the cleer and peaceful non-immune rabbit ear artery serum of the rabbit ear arterial blood after the immunity is made 1: 100,1: 200,1: 400,1: 800,1: 1600,1: 3200,1: 6400 doubling dilution again.Measure with the square formation experiment.All stay 4 holes in every block of plate, wherein two holes add non-immune rabbit ear artery serum as negative control, and other two holes add 100 μ L PBST as blank.
(1) sets up indirect ELISA method criterion
Detect OD with microplate reader
450Value.With blank zeroing, P is the value in each detection hole, the mean value of the negative contrast of N, i.e. and the dilutability of the rabbit ear artery serum of P/N 〉=2.0, and OD450 value after close to 1 immunity is as best effort concentration.
(2) indirect ELISA experiment square formation result
Experiment is determined through square formation, as can be seen from Table 1, rabbit ear artery serum after the immunity is at the antigen amount coated elisa plate with 0.5 μ g/ hole, when the rabbit ear artery serum optimum dilution degree after the immunity is dilution in 1: 1600, P=1.015, N=0.186, P/N=5.46, far above 2.0, therefore with the CrylAb protein at this numerical value place, rabbit ear artery serum dilution after the immunity as the best effort concentration of indirect ELISA screening technique.
Table 1 quantum dot ELISA of the present invention method detects precision and the accuracy of CrylAb protein and compares
(3) the quantum dot-labeled anti-CrylAb albumen double-antibody sandwich elisa detection method precision of CdSe/ZnS and the checking of accuracy
In quantitative scope, to choose the CrylAb protein standard solution of 7 variable concentrations and measure, measured value is learned processing by statistics, and value for coefficient of variation is all less than 15.The detected value of each concentration albumen and the theoretical recovery are 90～110, and average is 100 (seeing Table 2).The core reagent that the present invention's preparation is described has good repeatability and accuracy, meet " agriculture genetically modified organism safety evaluation management method " that The Ministry of Agriculture of the People's Republic of China, MOA announces, July 1 2004 date issued, implementation date: on November 8th, 2007, referring to: requirement http://wenku.baidu.com/view/52d14bd428ea81c758f57852.html).
The result that table 2 quantum dot ELISA of the present invention method detects sample compares
(4) the quantum dot-labeled anti-CrylAb albumen double-antibody sandwich elisa detection method of CdSe/ZnS quantitatively detects sample
The extraction of sample albumen to be measured: after rice grain is milled to powdery with comminutor, therefrom take by weighing 0.5g in the 5mL test tube, add the 2mL sample extracting solution, at room temperature slightly shake 4h, centrifugal 5min under 5000r/min draws supernatant then.Concentration results (pg/mL) in the actual measurement sample is converted by the concentration standard curve (see figure 6) and obtains.Analyzing quantum dot double antibodies sandwich method testing result (seeing Table 2) shows, the transgenic paddy rice sample of obtaining from the field 1 and the CrylAb protein concentration the transgenic paddy rice sample 2 reach 151.2pg/mL and 178.2pg/mL respectively, but far above minimum detected value, thereby can judge that this testing result is positive.The CrylAb protein concentration of non-transgenic paddy rice is 1-7pg/mL (may cause for the pollination of field trace-pollen), but is lower than minimum detected value, thereby can be judged to be the testing result feminine gender.The control experiment result shows that testing result of the present invention is reliably, is suitable for the quantitative detection of transgenic paddy rice trans Bt gene albumen.
Wherein:
Component and the proportioning of described sample extracting solution are as follows:
Mtris-HCl (pH=8) 45ml, glycerine 75ml, polyvinylpyrrolidone 6g replenishes distilled water to 1L.
Reagent source described in the present embodiment and component thereof and its proportioning are as follows:
Reagent source
The special reagent of the main chemical reagent that the present invention is used is from following channel (omitting the channel of common agents): the CdO (Chemical Reagent Co., Ltd., Sinopharm Group) that is purchased; Argon gas (Chemical Reagent Co., Ltd., Sinopharm Group); Cd (Chemical Reagent Co., Ltd., Sinopharm Group); Carbodiimide (EDC) (Chemical Reagent Co., Ltd., Sinopharm Group); N-hydroxy-succinamide (NHS) (Shanghai traditional Chinese medicines group); CrylAb protein standard substance (the beautiful minister bio tech ltd in Shanghai); Bovine serum albumin (BSA) (Roche Holding Ag's production); Trishydroxymethylaminomethane Tris (Chemical Reagent Co., Ltd., Sinopharm Group); Cadmium oxide (CdO, content 99.99%), hexadecylamine (HDA, content 90%), trioctylphosphine oxide (TOPO, content 90%), trioctylphosphine phosphorus (TOP, content 90%), sulphur powder (S, 99%), mercaptopropionic acid (MPA, 98%).
The instrument source
The flat instrument and meter of current chart company limited on the FA1004 type electronic balance
The Ying Yu of Gongyi City of DC-2 type universal mixer Henan Province gives magnificent instrument plant
Evolution 300 type ultraviolet-visible spectrophotometer Thermo Electron Corporation
LS-55 fluorospectrophotometer type fluorospectrophotometer U.S. Perkin Elmer company
TEM-H-70000FA transmission electron microscope HIT
Milli-Q ultrapure water machine U.S. Millipore company.
Claims (3)
1. fast and convenient method that detects CrylAb albumen in the transgenic paddy rice, its spy is following steps:
(1) anti-CrylAb MONOCLONAL ANTIBODIES SPECIFIC FOR
Be that antigen obtains rabbit anteserum with CrylAb albumen, obtain anti-CrylAb monoclonal antibody as primary antibodie by purifying, the anti-CrylAb monoclonal antibody hybridoma cell HHX-001 that secretes this monoclonal antibody is deposited in Chinese typical culture collection center (CCTCC), and its preserving number is CTCC NO:C201214.
(2) the quantum dot-labeled goat-anti of CdSe/ZnS is exempted from the preparation of IgG antibody (namely two is anti-)
1) 500 μ L goat-antis are exempted from the 50 μ L small tubes that the IgG antibody-solutions is distributed into 10 equal portions, be stored in-20 ℃ of refrigerators, treat that the time spent takes out a tubule, place 4 ℃ of refrigerator places, standby;
2) get 25 μ L CdSe/ZnS quantum dot mother liquors, 20 μ L 1-ethyl-(3-dimethylaminopropyl) end diimmonium salt acid salt solution and 10 μ LN-N-Hydroxysuccinimide solution, pH7.0, hybrid reaction 5min, add the 0.2mg goat-anti again and exempt from the IgG antibody-solutions, add the label solution that sterilized water is made into 100 μ L, react 2-4h at normal temperatures; Above-mentioned label solution is put mixing in the magnetic stirring apparatus, and reaction finishes to be placed in 4 ℃ of refrigerators to spend the night, and obtains the quantum dot-labeled goat-anti of CdSe/ZnS and exempts from IgG antibody crude product;
3) goat-anti that CdSe/ZnS is quantum dot-labeled is exempted from IgG antibody crude product 100 μ L and is put into the sample memotron that contains ultra filtration membrane with the application of sample rifle, in another pipe, put into the water of equivalent as balance, be 10 at rotating speed, 000rpm, centrifugation time is 20min, and centrifuging temperature is centrifugal in 4 ℃ the hydro-extractor;
4) after the centrifugal end of step 3), add the PBS damping fluid, set by step 3) the condition repeated centrifugation once;
5) after the centrifugal end of step 4), add 100 μ L PBS damping fluids in the sample memotron, then it is tipped upside down on the centrifuge tube, be 500rpm at rotating speed, the time is 3min, temperature is centrifugal and recovery sample in 4 ℃ the hydro-extractor, is two anti-;
(3) preparation of the quantum dot-labeled anti-CrylAb albumen double-antibody sandwich of CdSe/ZnS
Be primary antibodie with anti-CrylAb monoclonal antibody, this primary antibodie and bag are cushioned liquid dilution in 1: 1000 by volume, join then in the ELISA Plate hole, 20 ℃ of overnight incubation, inferior daily PBS damping fluid is washed plate 3 times, each 5min, add the sealing damping fluid after patting dry, every hole 200 μ L leave standstill 1h under 37 ℃, afterwards with PBS damping fluid washing 3 times, 5min at every turn; Be added on bag by in the hole after the CrylAb protein solution is diluted to 6.0pg/ml, 12.5pg/ml, 25.0pg/ml, 50.0pg/ml, 100.0pg/ml, 200.0pg/ml gradient concentration with the PBS damping fluid, every hole 100 μ L, under 37 ℃, leave standstill 1h, afterwards with PBS damping fluid washing 3 times, 5min at every turn; Behind the purifying two anti-ly is diluted to 10.00ug/mL with the PBS damping fluid, add the bag of ELISA Plate by in the hole. every hole 100 μ L, under 37 ℃, leave standstill 1h, wash fast with the PBS damping fluid afterwards, obtain the quantum dot-labeled anti-CrylAb albumen double-antibody sandwich of CdSe/ZnS;
(4) typical curve of drafting CrylAb albumen
The CrylAb protein standard solution dilution of 0.4 μ g/ml is 200pg/mL, 100, pg/mL, 50, pg/mL, 25pg/mL, 12.5pg/mL and 6pg/mL gradient concentration, detect the content of paddy rice CrylAb albumen with the quantum dot-labeled anti-CrylAb albumen double-antibody sandwich of CdSe/ZnS, CrylAb protein solution concentration with this gradient concentration dilution is independent variable, deducting blank relative intensity of fluorescence value with the CrylAb albumen relative intensity of fluorescence value of correspondence is dependent variable, the drawing standard curve, formula is as follows:
Y＝0.0137X+0.02 R
2＝0.994，
X is that the CrylAb protein solution concentration of serial gradient dilution is independent variable,
Y is corresponding CrylAb albumen relative intensity of fluorescence;
(5) detect OD with microplate reader
450Value, judge the content of CrylAb albumen in the paddy rice to be detected;
Wherein:
Component and the preparation of described CdSe/ZnS quantum dot mother liquor:
Take by weighing 0.0127g, the CdO of 0.1mmol and the stearic acid of 0.11g are put into the three-necked bottle of 50mL, under argon shield, are heated to 130 ℃, make CdO fully be dissolved in the stearic acid; After cooling the temperature to room temperature, take by weighing each 1.5g of trioctylphosphine oxide and hexadecylamine and put into three-necked bottle, be stirred and heated to 230 ℃, other takes by weighing 0.032g, and the sulphur powder of 1mmol is under argon shield, make it to be dissolved in the 2mL trioctylphosphine phosphorus, in the needle tubing of suction 5mL as storing solution; When Cd precursor temperature reaches 230 ℃, Se powder storing solution injected three-necked bottle fast after, make temperature be down to 180 ℃, keep 10min, collect the CdSe sample and be dissolved in the chloroform standby;
The sulphur powder that takes by weighing the zinc stearate of 0.632g and 0.032g adds respectively in the vaccenic acid of 2mL and is heated to 120 ℃ of fully dissolvings, obtain zinc stearate solution and sulphur powder solution respectively, be cooled to 70 ℃, down preserve standby in 70 ℃ zinc stearate solution and sulphur powder solution mixing back; The CdSe sample that will be scattered in the chloroform adds in the bottle, is heated to 120 ℃ and keeps 30min, and the evaporate to dryness chloroform is warming up to 200 ℃; Extract the vaccenic acid solution of zinc stearate and sulphur powder, inject the ZnS shell with 0.2mL/min, inject finish after, be warming up to 230 ℃ and keep 1h, be cooled to 60 ℃, reaction product is with heavyization of methyl alcohol, eccentric cleaning three times obtains the CdSe/ZnS chloroformic solution; Get each 1mL of CdSe/ZnS chloroformic solution, mercaptopropionic acid and deionized water and put into bottle and mix, strong agitation 3h extracts upper water solution, adds the methyl alcohol eccentric cleaning three times, after be scattered in and namely obtain CdSe/ZnS quantum dot mother liquor in the 1mL deionized water;
Wherein:
PBS damping fluid component and preparation:
KH
2PO
40.2g, NaCl 8.0g, KCl 0.2g, Na
2HPO
42.9g, Tween-20 0.5mL, adding distil water is settled to 1L, transfers pH to 7.4;
Bag is cushioned fluid component and preparation:
Na
2CO
31.59g, NaHCO
32.93g adding distil water is settled to 1L;
Sealing damping fluid component and preparation:
Bovine serum albumin(BSA) 0.1g is dissolved in the 100mL PBS damping fluid.
2. an anti-CrylAb monoclonal antibody hybridoma cell HHX-001 who secretes the monoclonal antibody of CrylAb albumen is deposited in Chinese typical culture collection center, and its preserving number is CCTCC NO:C201214.
3. the application of monoclonal antibody in detecting the trans Bt gene paddy rice of the described hybridoma HHX-001 secretion of claim 2.
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