CN105277703A - Kit for rapidly detecting content of milneb in crops - Google Patents
Kit for rapidly detecting content of milneb in crops Download PDFInfo
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- CN105277703A CN105277703A CN201410350671.0A CN201410350671A CN105277703A CN 105277703 A CN105277703 A CN 105277703A CN 201410350671 A CN201410350671 A CN 201410350671A CN 105277703 A CN105277703 A CN 105277703A
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- milneb
- kit
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- antibody
- standard
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- JZFICWYCTCCINF-UHFFFAOYSA-N Thiadiazin Chemical compound S=C1SC(C)NC(C)N1CCN1C(=S)SC(C)NC1C JZFICWYCTCCINF-UHFFFAOYSA-N 0.000 title claims abstract description 83
- 239000007788 liquid Substances 0.000 claims abstract description 26
- 239000000758 substrate Substances 0.000 claims abstract description 12
- 238000002965 ELISA Methods 0.000 claims abstract description 10
- 239000000427 antigen Substances 0.000 claims description 25
- 102000036639 antigens Human genes 0.000 claims description 25
- 108091007433 antigens Proteins 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 17
- 238000002360 preparation method Methods 0.000 claims description 17
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- 238000010790 dilution Methods 0.000 claims description 13
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- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims description 12
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 11
- 229940005654 nitrite ion Drugs 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 10
- 241000699670 Mus sp. Species 0.000 claims description 9
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- 210000004408 hybridoma Anatomy 0.000 claims description 9
- 230000001900 immune effect Effects 0.000 claims description 8
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 7
- 230000003053 immunization Effects 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 241001494479 Pecora Species 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 239000002671 adjuvant Substances 0.000 claims description 6
- 238000010367 cloning Methods 0.000 claims description 6
- 230000004927 fusion Effects 0.000 claims description 6
- 238000002649 immunization Methods 0.000 claims description 6
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- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims description 5
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- 239000007924 injection Substances 0.000 claims description 5
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- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 claims description 3
- 206010003445 Ascites Diseases 0.000 claims description 3
- 239000005662 Paraffin oil Substances 0.000 claims description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 3
- 206010068676 Pneumoretroperitoneum Diseases 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 208000005727 Retropneumoperitoneum Diseases 0.000 claims description 3
- 238000012870 ammonium sulfate precipitation Methods 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
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- 239000004615 ingredient Substances 0.000 claims description 3
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- 239000012452 mother liquor Substances 0.000 claims description 3
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 3
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- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
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- 210000004989 spleen cell Anatomy 0.000 claims description 3
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 2
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 claims description 2
- 108010058846 Ovalbumin Proteins 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 claims description 2
- RLRHPCKWSXWKBG-UHFFFAOYSA-N n-(2-azaniumylethyl)carbamate Chemical compound NCCNC(O)=O RLRHPCKWSXWKBG-UHFFFAOYSA-N 0.000 claims description 2
- NFJCQBGAUBIGKV-UHFFFAOYSA-N nitro dihydrogen phosphate Chemical compound OP(O)(=O)O[N+]([O-])=O NFJCQBGAUBIGKV-UHFFFAOYSA-N 0.000 claims description 2
- 229940092253 ovalbumin Drugs 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 11
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- 239000004411 aluminium Substances 0.000 description 2
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- 230000000890 antigenic effect Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000012113 quantitative test Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 1
- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 description 1
- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 244000241257 Cucumis melo Species 0.000 description 1
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- PDQAZBWRQCGBEV-UHFFFAOYSA-N Ethylenethiourea Chemical compound S=C1NCCN1 PDQAZBWRQCGBEV-UHFFFAOYSA-N 0.000 description 1
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- 208000031320 Teratogenesis Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
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- 230000007670 carcinogenicity Effects 0.000 description 1
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- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
The invention provides an enzyme-linked immunosorbent assay kit capable of detecting milneb in fruits and vegetables, and relates to the immunoassay detection technology. The kit includes an ELISA plate coated with milneb, a milneb antibody, a milneb standard, a standard diluent, an enzyme-labelled secondary antibody, a substrate developing liquid, a washing buffer liquid and a stopping liquid. The key technology comprises that a monoclonal antibody which can identify milneb is prepared. The kit adopts the high-specificity milneb monoclonal antibody, a main reagent is provided in a form of a working liquid, the operation steps can be reduced, and the time is saved.
Description
Technical field
The invention belongs to pesticide residue analysis and immunoassay detection technique field, be specifically related to a kind of monoclonal antibody preparation and the enzyme-linked immunologic detecting kit thereof that can identify milneb.The invention discloses monoclonal antibody specific, coating antigen, immunogenic preparation method and enzyme-linked immune detection method.Compared with prior art, monoclonal antibody prepared by the present invention can identify milneb, and kit of the present invention and method have the advantages such as easy, quick, sensitive, accurate.
Background technology
Milneb is mainly used in the control of multiple diseases on vegetables, fruit tree, also more remarkable to the downy mildew and wheat rust effect of preventing and treating melon and Chinese cabbage.Milneb catabolite ethylene thiourea in the environment causes animal used as test thyroid adenoma, and have carcinogenicity, mutagenesis type and teratogenesis, therefore safety problem is paid much attention to.
At present, HPLC is the Main Means measuring milneb, there is the deficiencies such as the pre-treatment step that analysis time is long, the required sample size of detection is large and loaded down with trivial details, be unwell to and measure a large amount of sample due to these class methods.In contrast to this, immunological method has low cost, fast, without the need to features such as complex sample pre-treatments, can be used as a kind of detection method efficiently.
Summary of the invention
(1) the technical problem to be solved in the present invention
The object of the invention is to provide that a kind of structure is simple, easy to use, low price, be easy to carry, the highly sensitive enzyme linked immunological kit simultaneously detected fast for milneb, and provide a kind of efficient, accurate, sensitive, quantitative detecting method of being applicable to a large amount of screening.
(2) technical scheme of the present invention
For solving described problem, the present invention comprehensively adopt protein molecule and biological chemistry to prepare etc. technology has prepared the monoclonal antibody that can identify milneb.Milneb and carrier protein couplet are prepared into artificial immunity antigen and envelope antigen; The monoclonal antibody of milneb is prepared with this artificial immunizing antigen immune animal; Milneb envelope antigen bag is adsorbed on solid phase carrier; The reagent detected is configured to the reagent that can directly use.During use, milneb standard items or testing sample are added milneb antibody working fluid, milneb residual in testing sample and solid phase carrier wrap the milneb antigenic competition milneb monoclonal antibody of quilt, add that enzyme labeling two is anti-carries out enzymatic activity amplification again, stop after colour developing, the absorbance of working sample, in this value and sample, milneb is residual in negative correlation, compares the content that can draw milneb in sample with typical curve.
This enzyme-linked immunologic detecting kit is made up of following ingredients:
(1) bag is by the ELISA Plate of milneb antigen
(2) milneb antibody
(3) milneb standard items
(4) standard dilutions
(5) ELIAS secondary antibody
(6) substrate nitrite ion
(7) lavation buffer solution
(8) stop buffer
Wherein, the preparation method of milneb antigen and milneb monoclonal antibody is:
(1) synthesis of milneb antigen
The carboxyl ethylenediamine of carrier protein (BSA) is activated, then by water-soluble carbodiimide method (EDC), milneb and the amino phase coupling of activated carrier albumen (BSA) is prepared milneb antigen.It is 98.6% that the immunogene of the present invention's synthesis adopts immunoelectrophoresis to measure its purity.
(2) milneb monoclonal antibody preparation
Animal immune program: adopt milneb antigen to be immunogene, immune balb/c mice, immunizing dose is 50 μ g, first immunisation is the immunogene containing Freund's complete adjuvant, later every surrounding, with the immunogene booster immunization containing incomplete Freund's adjuvant, totally four times, front and back, the mouse containing milneb antigen-specific antibodies in blood can be obtained; Latter 10 days of last immunity, blood sampling, extracting spleen cell.
Fusion of Cells and cloning: get immune balb/c mice splenocyte and murine myeloma cell hybrid fusion, prepare hybridoma, adopt indirect competitive enzyme-linked immunosorbent method to measure cell conditioned medium liquid, screen positive hole.Limiting dilution assay is utilized to carry out cloning to positive hole, the hybridoma cell strain of screening energy stably excreting milneb monoclonal antibody.
The preparation and purification of monoclonal antibody: adopt in body and induce method, by BALB/C mice Intraperitoneal injection sterilizing paraffin oil, 7-14 days pneumoretroperitoneum injection hybridomas, gather ascites after 7-10 days, through ammonium sulfate precipitation purifying, packing is stand-by ,-20 DEG C of preservations.
Wherein, the compound method of kit agents useful for same of the present invention is as follows:
(1) milneb standard solution preparation: accurately take standard milneb 1mg, be accurate to 0.00001g, be made into 1 μ g/mL standard solution mother liquor; During use, be diluted to required concentration (10-1000ng/mL) with standard dilutions;
(2) standard dilutions is the damping fluid of 0.05mol/LTris-HCl, pH8.0,0.9%NaCl;
(3) bag is buffered liquid is 0.1mol/L carbonic acid buffer, and pH7.8, containing 0.05%NaN
3, 0.9%NaCl;
(4) confining liquid is 0.05mol/LTris-HCl solution, and pH8.0, containing 0.5%BSA, 0.9%NaCl, 0.04%NaN
3;
(5) lavation buffer solution is 0.05mol/LTris-HCl, pH8.0,0.9%NaCl, 0.04%Tween20;
(6) milneb monoclonal antibody working fluid: by antibody pH7.4,0.02mol/L, it is that 0.1-1 μ g/L uses that the phosphate buffer containing 1% ovalbumin is diluted to protein concentration;
(7) ELIAS secondary antibody working fluid: the sheep anti mouse two of horseradish peroxidase-labeled resists;
(8) substrate nitrite ion A liquid: hydrogen peroxide or urea peroxide;
(9) substrate nitrite ion B liquid: o-phenylenediamine (OPD) or tetramethyl benzidine (TMB);
(10) substrate nitrite ion is to nitro phosphate buffer: pH8.1, containing MgCl
20.01%100mmol/LTris-HCl;
(11) stop buffer: 2mol/L sulfuric acid.
Wherein, the method for coating of ELISA Plate is: envelope antigen is buffered liquid with suitable concn and bag and mixes, and to make an addition in ELISA Plate and to react 14h under being placed in room temperature.After washing 3 times with lavation buffer solution, carry out closing (about 1h) at 37 DEG C with Block buffer, remove liquid in hole, preserve with the vacuum seal of aluminium film after dry.
The Cleaning Principle of kit of the present invention and detection method:
Milneb antigen conjugates bag is adsorbed on solid phase carrier, add milneb standard items or testing sample, and add milneb antibody working fluid, milneb residual in testing sample and solid phase carrier wrap the milneb antigenic competition milneb monoclonal antibody of quilt, free antigen antibody complex is removed in washing, add that enzyme labeling two is anti-carries out enzymatic activity amplification again, stop after colour developing, the absorbance of working sample, in this value and sample, milneb is residual in negative correlation, compares the content that can draw milneb in sample with typical curve.
(3) beneficial effect of the present invention
The enzyme linked immunological kit that can identify milneb prepared by the present invention, has the features such as easy, quick, sensitive, accurate, can be used for the residual quantity of milneb in the samples such as qualitative or quantitative detection fruits and vegetables; Its lowest detection is limited to 0.01ng/g, and detection time only needs 3 hours.This method average recovery rate is 97-121%, and in batch, error is less than 8%, and between batch, error is less than 13%.
Kit of the present invention adopts the milneb monoclonal antibody of high specific, and main agents provides with working fluid form, can reduce operation steps, save time.
Accompanying drawing explanation
Fig. 1 is the typical curve of milneb.
Embodiment
the preparation of embodiment 1 reagent constituents
1, the synthesis of antigen
A, get carrier bovine serum albumin(BSA) (BSA) 10g and be dissolved in 35mLpH9.6 carbonate buffer solution, add 10g ethylenediamine and activate;
B, get milneb 1g and be dissolved in the sodium hydroxide solution of 10mL0.5M;
C, get 1g carbodiimides and be dissolved in 5mL pure water, be then added to stirring at room temperature in milneb solution and react 3 hours;
D, the carrier protein BSA of activation is added drop-wise to 4 DEG C of stirring reactions in milneb solution spends the night;
E, to dialyse having reacted the obtained PBS damping fluid of artificial antigen to 0.1M 5 days, exchange buffering every day liquid 4 times; The artificial antigen of the purifying obtained is preserved through ultrafiltration concentration or freeze-drying.
2, milneb monoclonal antibody preparation
A, animal immune program: adopt milneb artificial antigen to be immunogene, immune balb/c mice, immunizing dose is 50 μ g, first immunisation is the immunogene containing Freund's complete adjuvant, neck dorsal sc multi-point injection, later every surrounding, with the immunogene booster immunization containing incomplete Freund's adjuvant, totally four times, front and back, can obtain the mouse containing milneb specific antibody in blood; Latter 10 days of last immunity, blood sampling, extracting spleen cell.
B, Fusion of Cells and cloning: get immune balb/c mice splenocyte, according to ratio and the mouse SP2/0 myeloma cell hybrid fusion of 5:1, prepare hybridoma, adopts indirect competitive enzyme-linked immunosorbent method to measure cell conditioned medium liquid, screen positive hole.Limiting dilution assay is utilized to carry out cloning to positive hole, the hybridoma cell strain of screening energy stably excreting milneb monoclonal antibody.
The preparation and purification of c, monoclonal antibody: adopt in body and induce method, by BALB/C mice Intraperitoneal injection 0.5mL sterilizing paraffin oil, 7-14 days pneumoretroperitoneum injection hybridomas, gather ascites after 7-10 days, through ammonium sulfate precipitation purifying, packing is stand-by ,-20 DEG C of preservations.
3, the preparation of ELIAS secondary antibody
Horseradish peroxidase (HRP) is carried out coupling with sheep anti mouse two is anti-, and method is as follows:
A, 8mg horseradish peroxidase is dissolved in 2mL distilled water;
B, add the 100mmol/LNaIO of existing preparation
4solution 0.4mL, stirring at room temperature reacts 20 minutes;
C, under 4 DEG C of conditions, by 1mmol/L acetate buffer dialysed overnight;
D, add pH8.6,0.5mol/L phosphate buffer 40 μ L and the pH8.6 containing the anti-protein I gG16mg of sheep anti mouse two, 5mol/L phosphate buffer 2mL, stirring at room temperature reacts 5 hours;
E, add the NaBH of existing preparation
4aqueous solution (1mol/L) 0.1mL4 DEG C reaction 5 hours;
F, purification storage.
4, the preparation of ELISA Plate
A, be buffered liquid with bag and envelope antigen be diluted to 0.05 μ g/mL;
B, in ELISA Plate, every hole adds 150 μ L, and reacts 14h under being placed in room temperature;
C, removal coating buffer, after washing 3 times, carry out closing (about 1h) at 37 DEG C with Block buffer 150 μ L, remove liquid in hole with lavation buffer solution, with aluminium film vacuum seal preservation after dry.
the establishment of the enzyme linked immunological kit that embodiment 2 detects fast for milneb
This enzyme-linked immunologic detecting kit is made up of following ingredients:
(1) bag is by the ELISA Plate of milneb antigen;
(2) protein concentration is the milneb monoclonal antibody working fluid of 0.5 μ g/L;
(3) concentration is 1 μ g/mL milneb standard items;
(4) standard dilutions: the damping fluid of 0.05mol/LTris-HCl, pH8.0,0.9%NaCl;
(5) with the sheep anti mouse ELIAS secondary antibody working fluid of horseradish peroxidase-labeled;
(6) substrate nitrite ion A liquid level hydrogen peroxide, substrate nitrite ion B liquid level o-phenylenediamine;
(7) lavation buffer solution is 0.05mol/LTris-HCl, pH8.0,0.9%NaCl, 0.04%Tween20;
(8) stop buffer is 2mol/L sulfuric acid.
the detection that in embodiment 3 sample, milneb is residual
1, sample pre-treatments
A, get 1g and pulverize and representative sample, add 5mL acetonitrile;
B, fully vibration mixing 3-5 minute, get the centrifugal 5min of 5mL supernatant 4000r/min or use quantitative test Filter paper filtering after stratification;
C, get centrifugal after supernatant or filter after filtrate 1mL, 50 ~ 60 DEG C of water-bath nitrogen dry up;
D, add standard dilutions 1mL and fully mixing, the centrifugal 5min of 4000r/min or use quantitative test Filter paper filtering;
E, get centrifugal after supernatant or filter after filtrate analyze.
2, detect with kit
Compound concentration is 1000ng/mL milneb mother liquor, carry out doubling dilution, dilution is 0 respectively, 0.3,0.9,2.7,8.1, the standard items of 24.3ng/mL6 concentration, according to 100 μ L standard items or testing sample and 50 μ L milneb monoclonal antibody working fluids totally 150 μ L make an addition to and be coated with in the ELISA Plate micropore of milneb antigen, in 25 DEG C of reaction 1h that are at war with.After competitive reaction terminates, pour out liquid in hole, every hole adds 250 μ L cleansing solutions, pours out liquid in hole after 30 seconds, repeats operation and washes plate altogether 5 times, blot with thieving paper.Add the sheep anti mouse two anti-working fluid 100 μ L of horseradish peroxidase-labeled, with cover plate film shrouding, in 25 DEG C of isothermal reaction 30min.Take out ELISA Plate, wash plate as aforementioned 5 times.Every hole adds substrate nitrite ion A liquid 50 μ L, then adds B liquid 50 μ L, shakes mixing gently, in 25 DEG C of isothermal reaction 15min.Add stop buffer 50 μ L/ hole, mixing of vibrating gently, setting microplate reader detects in 450nm place or dual wavelength 450/630nm, measures every hole absorbance (please running through data in 5min).
Interpretation of result:
Measure absorbance (OD value) and drawing standard curve, survey standard items absorbance (OD value) corresponding milneb concentration make canonical plotting, in each sample corresponding, the concentration of milneb can read from typical curve, thus obtains the milneb content of institute's test sample product.
Claims (3)
1., for the enzyme linked immunological kit that milneb detects fast, it is characterized in that it comprises following ingredients:
(1) bag is by the ELISA Plate of milneb antigen
(2) milneb antibody
(3) milneb standard items
(4) standard dilutions
(5) ELIAS secondary antibody
(6) substrate nitrite ion
(7) lavation buffer solution
(8) stop buffer
Wherein, the compound method of kit agents useful for same of the present invention is as follows:
(1) milneb standard solution preparation: accurately take standard milneb 1mg, be accurate to 0.00001g, be made into 1 μ g/mL standard solution mother liquor; During use, be diluted to required concentration (10-1000ng/mL) with standard dilutions;
(2) standard dilutions is the damping fluid of 0.05mol/LTris-HCl, pH8.0,0.9%NaCl;
(3) bag is buffered liquid is 0.1mol/L carbonic acid buffer, and pH7.8, containing 0.05%NaN
3, 0.9%NaCl;
(4) confining liquid is 0.05mol/LTris-HCl solution, and pH8.0, containing 0.5%BSA, 0.9%NaCl, 0.04%NaN
3;
(5) lavation buffer solution is 0.05mol/LTris-HCl, pH8.0,0.9%NaCl, 0.04%Tween20;
(6) milneb monoclonal antibody working fluid: by antibody pH7.4,0.02mol/L, it is that 0.1-1 μ g/L uses that the phosphate buffer containing 1% ovalbumin is diluted to protein concentration;
(7) ELIAS secondary antibody working fluid: the sheep anti mouse two of horseradish peroxidase-labeled resists;
(8) substrate nitrite ion A liquid: hydrogen peroxide or urea peroxide;
(9) substrate nitrite ion B liquid: o-phenylenediamine (OPD) or tetramethyl benzidine (TMB);
(10) substrate nitrite ion is to nitro phosphate buffer: pH8.1, containing MgCl
20.01%100mmol/LTris-HCl;
(11) stop buffer: 2mol/L sulfuric acid.
2. kit as claimed in claim 1, it is characterized in that the preparation of envelope antigen: activated by the carboxyl ethylenediamine of carrier protein (BSA), then by water-soluble carbodiimide method (EDC), milneb and the amino phase coupling of activated carrier albumen (BSA) are prepared milneb antigen.
3. kit as claimed in claim 1, is characterized in that identifying milneb monoclonal antibody preparation:
A, animal immune program: adopt milneb artificial antigen to be immunogene, immune balb/c mice, immunizing dose is 50 μ g, first immunisation is the immunogene containing Freund's complete adjuvant, neck dorsal sc multi-point injection, later every surrounding, with the immunogene booster immunization containing incomplete Freund's adjuvant, totally four times, front and back, can obtain the mouse containing milneb specific antibody in blood; Latter 10 days of last immunity, blood sampling, extracting spleen cell;
B, Fusion of Cells and cloning: get immune balb/c mice splenocyte, according to ratio and the mouse SP2/0 myeloma cell hybrid fusion of 5:1, prepare hybridoma, adopts indirect competitive enzyme-linked immunosorbent method to measure cell conditioned medium liquid, screen positive hole; Limiting dilution assay is utilized to carry out cloning to positive hole, the hybridoma cell strain of screening energy stably excreting milneb monoclonal antibody;
The preparation and purification of c, monoclonal antibody: adopt in body and induce method, by BALB/C mice Intraperitoneal injection 0.5mL sterilizing paraffin oil, 7-14 days pneumoretroperitoneum injection hybridomas, gather ascites after 7-10 days, through ammonium sulfate precipitation purifying, packing is stand-by ,-20 DEG C of preservations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410350671.0A CN105277703A (en) | 2014-07-23 | 2014-07-23 | Kit for rapidly detecting content of milneb in crops |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410350671.0A CN105277703A (en) | 2014-07-23 | 2014-07-23 | Kit for rapidly detecting content of milneb in crops |
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| CN105277703A true CN105277703A (en) | 2016-01-27 |
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| CN201410350671.0A Pending CN105277703A (en) | 2014-07-23 | 2014-07-23 | Kit for rapidly detecting content of milneb in crops |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001090153A2 (en) * | 2000-05-23 | 2001-11-29 | Nexell Therapeutics, Inc. | Reagents for cell selection and methods of use |
| CN103619171A (en) * | 2010-11-03 | 2014-03-05 | 陶氏益农公司 | Pesticidal compositions and methods related thereto |
| CN103808931A (en) * | 2012-11-06 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Enzyme linked immunosorbent assay kit for detecting sulfadiazine and detection method thereof |
-
2014
- 2014-07-23 CN CN201410350671.0A patent/CN105277703A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001090153A2 (en) * | 2000-05-23 | 2001-11-29 | Nexell Therapeutics, Inc. | Reagents for cell selection and methods of use |
| CN103619171A (en) * | 2010-11-03 | 2014-03-05 | 陶氏益农公司 | Pesticidal compositions and methods related thereto |
| CN103808931A (en) * | 2012-11-06 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Enzyme linked immunosorbent assay kit for detecting sulfadiazine and detection method thereof |
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