CN105486872A - Test strip for detecting triadimenol and preparation method and application thereof - Google Patents
Test strip for detecting triadimenol and preparation method and application thereof Download PDFInfo
- Publication number
- CN105486872A CN105486872A CN201510973179.3A CN201510973179A CN105486872A CN 105486872 A CN105486872 A CN 105486872A CN 201510973179 A CN201510973179 A CN 201510973179A CN 105486872 A CN105486872 A CN 105486872A
- Authority
- CN
- China
- Prior art keywords
- triadimenol
- test strips
- sample
- pad
- coated
- 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
- NASSELZHMMRSRS-YFKPBYRVSA-N CC[C@@H](NC)OC Chemical compound CC[C@@H](NC)OC NASSELZHMMRSRS-YFKPBYRVSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a test strip for detecting triadimenol, a preparation method of the test strip and application of the test strip. The test strip comprises a sample absorbing pad, a conjugate releasing pad, a reaction film, a water absorbing pad and a bottom plate, and is characterized in that the reaction film is provided with a detection line which is coated by a triadimenol hapten-carrier protein conjugate, and a quality control line which is coated by a goat anti-mouse antibody; the conjugate releasing pad is sprayed with a triadimenol monoclonal antibody-colloidal gold marker. The invention further provides a method for detecting the triadimenol in a sample by utilization of the test strip. The test strip and the detection method provided by the invention have the advantages of simplicity in operation, high sensitivity, quick detection speed, low cost, freeness from restriction of detection equipment, and capability of performing quick detection and field monitoring on large batches of triadimenol samples.
Description
Technical field
The present invention relates to the detection of Triadimenol, specifically a kind of colloidal gold strip for detecting Triadimenol, it is specially adapted to the detection that in tobacco juice, Triadimenol is residual.
Background technology
Triadimenol (triadimenol, C
14h
18cN
3o
2) have another name called hundred loads, chemical name is 1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-2-butanols, be a kind of low toxicity, wide spectrum, efficiently systemic fungicide, its Main Function mechanism suppresses the biosynthesizing of gibberellin and ergosterol and then affects cell division speed, has good prevention effect to fungal diseases such as black root of tobacco, powdery mildew, frog eyes.。Therefore, the application in China's agricultural production widely, is applicable to the crops such as wheat class, corn, Chinese sorghum, fruits and vegetables, tobacco.
In tobacco, the residual international tobacco scientific research Cooperation Centre (CORESTA) of Triadimenol is paid close attention to, and recall rate in recent years in some place of production tobacco of China is also higher.Along with widely using and the attention of people to food and Crop securify of Triadimenol, residual also receive of Triadimenol in food and crops is shown great attention to.In January, 2007, European Union disclosed in import the early warning circular detecting in domestic honey that Triadimenol residual quantity exceeds standard.In May in the same year, Japanese MHLW finds Triadimenol residual quantity in 1 batch, China carrot of defeated day and exceeds standard, and Japanese side requires the Triadimenol project implementation 50% monitor check in the carrot of China's export Japan thus.At present, a lot of country clearly limits the maximum residue limit(MRL) of this agricultural chemicals in food, as Japan specifies that the residue limits of Triadimenol in food (MRL) is 0.2 ~ 0.5mg/kg; European Union specifies that the MRL of Triadimenol in beans is 0.1mg/kg; In Codex Alimentary Commission (CAC) regulation tomato, the MRL of Triadimenol is 0.5mg/kg.Remain for avoiding Triadimenol and work the mischief and breaks through foreign trade barrier to human body, the detection method setting up triazole residual quantity of alcohol in simple, quick, accurate, reliable tobacco is necessary.
Triadimenol detection method conventional at present mainly contains vapor-phase chromatography (GC), liquid phase chromatography (LC), gas chromatographymass spectrum (GC-MS) and liquid-mass chromatography method (LC-MS).Adopt these analytical approachs to need expensive instrument and special technician, sample pretreatment process is complicated and spend high, time-consuming length, is difficult to the needs that satisfied a large amount of sample and field sample detect fast.Therefore, develop a kind of not examined device-restrictive and can realize carrying out to batch samples the product that detects fast and method becomes problem in the urgent need to address.
Summary of the invention
It is high to device dependence that the method that the object of the invention is to overcome existing detection Triadimenol exists, and the feature of the quick detection to batch samples can not be realized, there is provided a kind of simple to operate, highly sensitive, detection speed is fast, cost is low, the test strips of not examined device-restrictive and its preparation method and application, to realize detecting fast and on-site supervision large batch of Triadimenol sample.
In order to realize object of the present invention, the invention provides a kind of test strips detecting Triadimenol, this test strips comprises absorption of sample pad, bond release pad, reaction film, adsorptive pads and base plate; Wherein, described reaction film has the detection line being coated with Triadimenol hapten-carrier protein conjugate and the nature controlling line being coated with sheep anti mouse antiantibody, described bond release pad is coated with Triadimenol monoclonal antibody-colloid gold label thing.
Described Triadimenol hapten-carrier protein conjugate is obtained by Triadimenol haptens and carrier protein couplet, described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins, described Triadimenol haptens is at organic base 1 by triazolone and aminocaproic acid, the condensation reaction carrying out ketoamine under the catalysis of 8-diazabicylo 11 carbon-7-alkene obtains, and its molecular structural formula is:
。
Described Triadimenol monoclonal antibody prepares using Triadimenol hapten-carrier protein conjugate as immunogene, and described sheep anti mouse antiantibody carries out immunity for immunogene to goat with mouse source antibody to prepare.
Described absorption of sample pad, bond release pad, reaction film, adsorptive pads are pasted onto on base plate successively, under described bond release pad 1/3 ~ 1/2 is capped on absorption of sample pad.
Described base plate can be the material that PVC base plate or other hard do not absorb water; Described absorption of sample pad can be suction strainer paper or filter paper for oil; Described bond release pad can be glass wool or polyester material; Described adsorptive pads is thieving paper; Described reaction film can be nitrocellulose filter or cellulose acetate membrane.
Prepare a method for above-mentioned test strips, comprise the following steps:
1) preparation is coated with the bond release pad of Triadimenol monoclonal antibody-colloid gold label thing;
2) preparation has the reaction film of the detection line being coated with Triadimenol hapten-carrier protein conjugate and the nature controlling line being coated with sheep anti mouse antiantibody;
3) by 1) and 2) bond for preparing release pad, reaction film and absorption of sample pad, adsorptive pads and base plate be assembled into test strips.
Specifically, step comprises:
1) triazolone and aminocaproic acid are carried out under the catalysis of organic base 1,8-diazabicylo 11 carbon-7-alkene the condensation reaction of ketoamine, prepare Triadimenol haptens;
2) by Triadimenol haptens and carrier protein couplet, Triadimenol hapten-carrier protein conjugate is prepared;
3) with Triadimenol hapten-carrier protein conjugate immune mouse, mouse boosting cell and myeloma cell are passed through to merge, screen, obtains the hybridoma cell strain secreting Triadimenol monoclonal antibody;
4) extract mouse IgG immune health goat, obtain sheep anti mouse antiantibody;
5) on the detection line (T) respectively Triadimenol hapten-carrier protein conjugate and sheep anti mouse antiantibody being coated in reaction film and nature controlling line (C);
6) react with trisodium citrate and gold chloride and prepare collaurum;
7) the Triadimenol monoclonal antibody of preparation is joined in the collaurum of preparation, obtain Triadimenol monoclonal antibody-colloid gold label thing;
8) Triadimenol monoclonal antibody-colloid gold label thing is sprayed on bond release pad, takes out after 37 DEG C of baking 1h, be placed in dry environment and save backup;
9) the 0.1mol/L phosphate buffer of absorption of sample pad containing 0.5% bovine serum albumin(BSA) (massfraction), pH7.2 is soaked 2h, at 37 DEG C, dry 2h;
10) on base plate, paste absorption of sample pad, bond release pad, reaction film, adsorptive pads in order, bond release pad has 1/3 region to be covered by absorption of sample pad from initiating terminal.Finally be cut into the little bar that 3mm is wide, add plastic casing, vacuum packaging, under 4 ~ 30 DEG C of conditions, can 12 months be preserved.1/3 of bond release pad to be covered by absorption of sample pad and can extend testing result observing time, absorption of sample pad can be made fully to be absorbed by tracer liquid and fully react with golden labeling antibody, thus reduce error.
Apply the method for Triadimenol in above-mentioned ELISA test strip sample, comprise the steps:
(1) pre-treatment is carried out to sample;
(2) detect by test strips;
(3) testing result is analyzed.
The test strips of detection Triadimenol of the present invention adopts antibody antigen reaction and the immunochromatographiassays assays technology of high degree of specificity, Triadimenol monoclonal antibody-colloid gold label thing is fixed on bond release pad, Triadimenol in sample is in flow process, discharge the monoclonal antibody-colloid gold label thing of the Triadimenol on padding with bond to be combined, form Triadimenol-antibody-colloidal gold label.Triadimenol hapten-carrier protein conjugate competition binding Triadimenol monoclonal antibody-colloid gold label thing on Triadimenol in sample and reaction film detection line, judges whether remain containing Triadimenol in analyte sample fluid according to the detection line red stripes depth.
During detection, sample instills absorption of sample pad after treatment, when Triadimenol concentration in the sample to which lower than detectability or be zero time, monoclonal antibody-colloid gold label thing in chromatography process can be fixed on the Triadimenol hapten-carrier protein conjugate on reaction film and be combined, respectively occur a red stripes at detection line (T) and nature controlling line (C) place, and the colour developing of T line is darker than the colour developing of C line or develop the color consistent with C line; If Triadimenol concentration is in the sample to which equal to or higher than detectability, monoclonal antibody-colloid gold label thing all can be combined with Triadimenol, thus does not occur red stripes at T line place because competitive reaction can not be combined with Triadimenol hapten-carrier protein conjugate or develop the color shallow than C line.As shown in Figure 2.
Negative: when nature controlling line (C) demonstrates red stripes, detection line (T) also demonstrates red stripes simultaneously, and (T) line color is close or when being deeper than (C) line, be judged to feminine gender.
Positive: when nature controlling line (C) demonstrates red stripes, and when detection line (T) does not develop the color or (T) line color is shallower than (C) line, to be judged to the positive.
Invalid: when nature controlling line (C) does not demonstrate red stripes, then no matter whether detection line (T) demonstrates red stripes, and it is invalid that this test strips is all judged to.
Test strips of the present invention have highly sensitive, high specificity, cost are low, simple to operate, detection time is short, not examined device-restrictive, be applicable to various units and use, store advantage that is simple, long shelf-life.By the method for ELISA test strip Triadimenol of the present invention, easy, quick, directly perceived, accurate, applied widely, cost is low, easily promote the use of.
Accompanying drawing explanation
Fig. 1 is test strips cross-sectional view, in figure: 1, absorption of sample pad; 2, bond release pad; 3, reaction film; 4, adsorptive pads; 5, detection line; 6, nature controlling line; 7, base plate;
Fig. 2 is ELISA test strip result process decision chart;
Fig. 3 is Triadimenol hapten synthesis figure.
Embodiment
The present invention is set forth further below in conjunction with specific embodiment.Should be understood that these embodiments are only for illustration of the present invention, and be not used for limiting the scope of the invention.In addition, those skilled in the art may carry out various change or modification to the present invention in the scope that appended claims limits, and these are changed or modify the protection domain that should fall into invention equally.
Embodiment 1 detects the preparation of the test strips of Triadimenol
The preparation method of this test strips mainly comprises the following steps:
1) preparation is coated with the bond release pad of Triadimenol monoclonal antibody-colloid gold label thing;
2) preparation has the reaction film of the detection line being coated with Triadimenol hapten-carrier protein conjugate and the nature controlling line being coated with sheep anti mouse antiantibody;
3) by 1) and 2) bond for preparing release pad, reaction film and absorption of sample pad, adsorptive pads and PVC base plate be assembled into test strips.
Substep describes in detail below:
1, the haptenic synthesis of Triadimenol (synthetic route is shown in accompanying drawing 3) and qualification
Get 1.0g triazolone and add ethanol dissolving, add 0.35g1,8-diazabicylo 11 carbon-7-alkene (DBU), stir, then add 0.87g aminocaproic acid aqueous solution, heating reflux reaction 12h; Stop reaction, revolve steaming, removing ethanol, add water and 1.3g potassium hydroxide, concussion, adds extraction into ethyl acetate, divide and go organic phase, aqueous phase adjust ph, to 4, adds extraction into ethyl acetate, organic phase is washed, and anhydrous sodium sulfate drying evaporate to dryness, obtains light yellow oil, methylene chloride/sherwood oil (1:10, V/V) recrystallization, obtains haptens product 0.76g, yield 69%.
Get above-mentioned haptens to identify through proton nmr spectra,
1h-NMR(CDCl
3, 300MHz) δ: 11.00(1H, s), 7.51(1H, ddd, J=8.132, J=5.483, J=1.670), 7.46(1H, ddd, J=8.132, J=5.485, J=1.670), 6.94(1H, dd, J=8.132, J=5.485), 6.94(1H, dd, J=8.132, J=5.483), 6.52(1H), 8.270(1H, d, J=1.903), 8.1(1H, d, J=1.903), 3.87(2H, t, J=7.434), 1.25(3H, s), 1.25(3H, s), 1.25(3H, s), 1.84(2H, tt, J=7.434, J=7.022), 1.41(2H, tt, J=7.434, J=7.022), 1.55(2H, tt, J=7.434, J=7.367), 2.30(2H, t, J=7.367).In collection of illustrative plates, chemical shift=11.0 is carboxyl hydrogen resonance absorbing peak on spacerarm, δ=1.85,1.41,2.30 be the resonance absorbing peak of methylene hydrogen on spacerarm, the existence at these peaks, prove spacerarm coupling success, Triadimenol haptens structure is correct.
2, the synthesis of Triadimenol coupled antigen and qualification
Prepared by immunizing antigen---and Triadimenol haptens and bovine serum albumin(BSA) (BSA) coupling obtain immunizing antigen.
Get 14mg haptens, be dissolved in 1mLN, in dinethylformamide (DMF); Take BSA50mg, make it fully to be dissolved in 3.8mL2-(N-morpholine) ethanesulfonic acid buffer (MES) (pH5.0), haptens is slowly added drop-wise in protein solution; Get 30mg carbodiimides (EDC) 0.2mLMES(pH5.0) fully dissolve after add in haptens mixed liquid of protein, stirred at ambient temperature 24h; With 0.01mol/L phosphate buffer (PBS) 4 DEG C dialysis 3d, change 3 dislysates every day; Packing, saves backup in-20 DEG C.
Triadimenol haptens and hemocyanin coupling obtain immunogene.
Take hemocyanin 50mg, make it the phosphate buffer PBS(PH7.2 being fully dissolved in 3.8mL0.1M) in, obtain solution A; Get 30mg carbodiimides (EDC) and N-hydroxy-succinamide (NHS) 0.2mL water fully dissolve after in adding in solution A, stirred at ambient temperature 30min.Get 6mg haptens, be dissolved in 1mLN, in dinethylformamide (DMF), then slowly join in protein dissolution, stirring at room temperature reaction 24h.3 dislysates are changed every day with 0.01mol/LPBS4 DEG C of dialysis 3d.Packing, saves backup in-20 DEG C.
Triadimenol haptens and thyroprotein coupling obtain immunogene.
Take thyroprotein 50mg, make it the phosphate buffer PBS(PH7.2 being fully dissolved in 3.8mL0.1M) in, obtain solution A; Get 30mg carbodiimides (EDC) and N-hydroxy-succinamide (NHS) 0.2mL water fully dissolve after in adding in solution A, stirred at ambient temperature 30min.Get 8mg haptens, be dissolved in 1mLN, in dinethylformamide (DMF), then slowly join in protein dissolution, stirring at room temperature reaction 24h.3 dislysates are changed every day with 0.01mol/LPBS4 DEG C of dialysis 3d.Packing, saves backup in-20 DEG C.
Prepared by envelope antigen---and Triadimenol haptens and ovalbumin (OVA) coupling obtain envelope antigen.
Get 14mg haptens, be dissolved in 1mLDMF; Take OVA50mg, make it fully to be dissolved in 3.8mLMES(pH5.0) in, haptens is slowly added drop-wise in protein solution; Get 30mgEDC 0.2mLMES(pH5.0) fully dissolve after add in haptens mixed liquid of protein, stirred at ambient temperature 24h; With 0.01mol/LPBS4 DEG C of dialysis 3d, change 3 dislysates every day; Packing, saves backup in-20 DEG C.
Triadimenol haptens and human serum albumins coupling obtain coating antigen
Take human serum albumins 50mg, make it fully to be dissolved in 3.8mL0.1MPBS(PH7.2) in, obtain solution B; Get 30mgEDC and NHS 0.2mL water fully dissolve after in adding in solution B, stirred at ambient temperature 30min.Get 14mg haptens, be dissolved in 1mLDMF, then slowly join in protein dissolution, stirring at room temperature reaction 24h.3 dislysates are changed every day with 0.01mol/LPBS4 DEG C of dialysis 3d.Packing, saves backup in-20 DEG C.
Triadimenol haptens and albumin rabbit serum coupling obtain coating antigen
Take albumin rabbit serum 50mg, make it fully to be dissolved in 3.8mL0.1MPBS(PH7.2) in, obtain solution B; Get 30mgEDC and NHS 0.2mL water fully dissolve after in adding in solution B, stirred at ambient temperature 30min.Get 14mg haptens, be dissolved in 1mLDMF, then slowly join in protein dissolution, stirring at room temperature reaction 24h.3 dislysates are changed every day with 0.01mol/LPBS4 DEG C of dialysis 3d.Packing, saves backup in-20 DEG C.
Reacting the ratio of haptens used, carrier protein and coupled product in synthesis Triadimenol coupled antigen, carry out ultraviolet (200 ~ 400nm) sweep measuring, calculating its combination ratio at the light absorption value of 260nm and 280nm respectively by comparing three.The maximum absorption band of conjugate Triadimenol hapten-carrier albumen there occurs obvious change compared with the maximum absorption band of Triadimenol haptens, carrier protein, shows that the synthesis of Triadimenol hapten-carrier albumen is successful.As calculated, haptens is 13:1 with the combination ratio of BSA, is 8:1 with the combination ratio of OVA.
3, the preparation of Triadimenol monoclonal antibody
(1) acquisition of hybridoma
1) first immunisation: by fully emulsified for the Freund's complete adjuvant of Triadimenol haptens-BSA conjugate (immunizing antigen) and equivalent, the Balb/c mouse in 6 week age of hypodermic injection, every 0.2mL;
2) booster immunization twice: from first immunisation, every two weeks booster immunizations once, use not formula Freund's incomplete adjuvant to replace Freund's complete adjuvant, method and the same first immunisation of dosage;
3) the rear eyeground vein blood sampling survey in a week of last booster immunization is tired and suppresses, have and suppress and tire when reaching more than 1:10000 to carry out following final immunization: lumbar injection does not add the immunogen solution 0.1mL of any adjuvant, put to death mouse after three days, get its spleen and myeloma cell fusion;
4) adopt indirect competitive enzyme-linked immunosorbent analytical approach to measure cell conditioned medium liquid, screen positive hole.Limiting dilution assay is utilized to carry out cloning to positive hole, obtain and set up the hybridoma cell strain of stably excreting Triadimenol monoclonal antibody, get the hybridoma cryopreserving liquid being in exponential phase and make cell suspension, be sub-packed in cryopreservation tube, preserve for a long time in liquid nitrogen.
(2) preparation of monoclonal antibody
1) cell recovery: take out Triadimenol monoclonal antibody hybridoma cell strain cryopreservation tube, put into 37 DEG C of water-bath middling speeds immediately and melt, after centrifugal segregation cryopreserving liquid, move into and cultivate culture in glassware;
2) ascites and antibody purification is prepared: adopt in body and induce method, by Balb/c mouse (8 week age) Intraperitoneal injection sterilizing paraffin oil 0.5mL/ only, 7 days pneumoretroperitoneum injection hybridomas 5 × 10
5individual/only, gather ascites after 7 days.Carry out purifying by sad-saturated ammonium sulfate method, obtain Triadimenol monoclonal antibody solution (-20 DEG C of preservations).
(3) mensuration of antibody titer
Tiring as 1:(200000 ~ 500000 of antibody is measured) with indirect competitive ELISA method.
Indirect competitive ELISA method: with Triadimenol haptens-OVA conjugate coated elisa plate, add the sheep anti mouse antiantibody solution of Triadimenol standard solution, Triadimenol monoclonal antibody solution and horseradish peroxidase-labeled, 25 DEG C of reaction 30min, pour out liquid in hole, with cleansing solution washing 3 ~ 5 times, pat dry with thieving paper; Add substrate nitrite ion, after 25 DEG C of reaction 15min, add stop buffer cessation reaction; Setting microplate reader measures every hole absorbance in wavelength 450nm place.
(4) mensuration of monoclonal antibody specificity
Antibody specificity refers to the ability that its homospecificity antigen combines and comparing with such antigen-analogues ability, and conventional cross reacting rate is as evaluation criterion.Cross reaction is less, and the specificity of antibody is then higher.
Triadimenol series bactericidal agent (Triadimenol, cyproconazole, Flutriafol, Tebuconazole, own azoles alcohol, olefin conversion, bitertanol) is done serial dilution by this experiment, carries out indirect competitive ELISA respectively, production standard curve with monoclonal antibody, analyzes and obtains IC
50, be then calculated as follows cross reacting rate:
The cross reacting rate that result shows each analog is: Triadimenol 100%, cyproconazole < 1%, Flutriafol < 1%, Tebuconazole < 1%, own azoles alcohol < 1%, olefin conversion < 1%, bitertanol < 1%.Antibody of the present invention, to other Triadimenol insecticides no cross reactions such as cyproconazole, Flutriafol, Tebuconazole, own azoles alcohol, olefin conversion, bitertanols, only has specific binding for Triadimenol.
4, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, for immunogene, immunity is carried out to pathogen-free domestic sheep with mouse source antibody, obtain sheep anti mouse antiantibody.
5, the preparation of Triadimenol monoclonal antibody-colloid gold label thing
(1) preparation of collaurum
With two boil off ionized water by massfraction be 1% chlorauric acid solution be diluted to 0.01%, get 100mL and be placed in conical flask, boiling is heated to thermostatic electromagnetic stirrer, the citric acid three sodium solution that 1.5mL massfraction is 1% is added under continuous high temperature, Keep agitation, continue at the uniform velocity to be heated with stirring to when solution is bright claret to stop, original volume is returned to deionized water, 4 DEG C of preservations after being cooled to room temperature.It is limpid transparent for preparing that good collaurum detects by an unaided eye, and do not have muddiness, liquid surface is without floating thing, and the color of observing colloid gold is claret in the sunlight.
(2) preparation of Triadimenol monoclonal antibody-colloid gold label thing
Under magnetic stirring, adjust the pH label range of pH to the 7.2(different antibodies of collaurum between 7 ~ 8 with 0.2mol/L solution of potassium carbonate, can change), in colloidal gold solution, above-mentioned Triadimenol monoclonal antibody is added by the standard adding 20 ~ 50 μ g antibody in every milliliter of colloidal gold solution, stir and evenly mix, room temperature leaves standstill 10min, and adding the whole massfraction that 10%BSA makes it in colloidal gold solution is 1%, leaves standstill 10min.12000r/min, 4 DEG C of centrifugal 40min, abandon supernatant, precipitation with redissolve buffer solution twice, with volume be initial colloid gold volume 1/10 redissolution damping fluid will precipitate resuspended, put 4 DEG C for subsequent use.
Redissolve damping fluid: the massfraction containing BSA is 0.1% ~ 0.3%, the massfraction of Tween-80 is 0.05% ~ 0.2%, the 0.02mol/L phosphate buffer of pH7.2.
6, the preparation of bond release pad
Bond is discharged pad to be soaked in containing 0.5%BSA(massfraction), in the 0.5mol/L phosphate buffer of pH7.2, evenly soak 1h, 37 DEG C to dry 3h for subsequent use.Spray film instrument with Isoflow the Triadimenol monoclonal antibody-colloid gold label thing even application prepared is padded in bond release, after every 1cm bond release pad spraying 0.01mL Triadimenol monoclonal antibody-colloid gold label thing, take out after being placed in 37 DEG C of environment (humidity < 20%) 60min, be placed in dry environment (humidity < 20%) and save backup.
7, the preparation of reaction film
Triadimenol haptens-ovalbumin conjugate bag is formed detection line by reaction film, sheep anti mouse antiantibody is coated on reaction film and forms nature controlling line.
Bag is by process: with phosphate buffer, Triadimenol haptens-ovalbumin conjugate is diluted to 1mg/mL, be coated in the detection line (T) on nitrocellulose filter with Isoflow point film instrument, package amount is 1.0 μ L/cm; With the phosphate buffer of 0.01mol/L, pH7.4, sheep anti mouse antiantibody is diluted to 200 μ g/mL, be coated in the nature controlling line (C) on nitrocellulose filter with Isoflow point film instrument, package amount is 1.0 μ L/cm.Bag is placed in dry 2h under 37 DEG C of conditions by good reaction film, for subsequent use.
8, the preparation of absorption of sample pad
The 0.1mol/L phosphate buffer of absorption of sample pad containing 0.5% bovine serum albumin(BSA) (massfraction), pH7.2 is soaked 2h, and it is for subsequent use to dry 2h at 37 DEG C.
9, the assembling of test strips
Absorption of sample pad, bond release pad, reaction film, adsorptive pads are pasted onto on PVC base plate successively in order; Bond release pad has 1/3 region to be covered by absorption of sample pad from initiating terminal, the end of bond release pad is connected with the top of reaction film, the end of reaction film is connected with the top of adsorptive pads, the top of absorption of sample pad aligns with the top of PVC base plate, and the end of adsorptive pads aligns with the end of PVC base plate; Described reaction film has detection line and nature controlling line, detection line (T) and nature controlling line (C) are all in the strip tape vertical with the appearance of described test strips; Detection line is positioned at the side of the end near bond release pad; Nature controlling line is positioned at the side of the end away from bond release pad; Test strips machine is cut into the little bar that 3mm is wide, is contained in special plastics fabrication, under 4 ~ 30 DEG C of conditions, can 12 months be preserved.
The detection of Triadimenol in embodiment 2 sample
1, the pre-treatment of sample
Take the tobacco leaf sample of 1.0 ± 0.05g pulverizing in polystyrene centrifuge tube; Add 10mL50% methanol solution, with refiner, it is fully smashed, obtain sample liquid; Pipetting 50 μ L sample liquid and 575 μ L deionized waters (dry tobacco) or 100 μ L sample liquid and 650 μ L deionized waters (wet tobacco) mix to be checked afterwards.
2, detect by test strips
Get sample solution 100 μ L to be checked with pipettor vertically to drip in well, during liquid flow, start timing, reaction 10min, result of determination.
3, testing result is analyzed
Negative (-): the colour developing of T line is darker than the colour developing of C line or develop the color consistent with C line, represents that in sample, Triadimenol concentration is lower than detectability, as Fig. 2 a, 2b.
Positive (+): T line colour developing or T line more shallow than the colour developing of C line does not develop the color, and represents that in sample, Triadimenol concentration is equal to or higher than detectability, as Fig. 2 c, 2d.
Invalid: not occur C line, show the deterioration failure of incorrect operating process or test strips, as Fig. 2 e, 2f.In the case, again should read over instructions, and retest by new test strips.
Embodiment 3 sample detection example
1, detectability test
Get blank tobacco sample, add respectively wherein Triadimenol to final concentration be dry tobacco 2.5,5,10mg/kg, wet tobacco 1.5,3,6mg/kg, get test strips and detect, each sample replication three times.
During with ELISA test strip tobacco sample, when wherein Triadimenol interpolation concentration is dry tobacco 2.5mg/kg, wet tobacco 1.5mg/kg, test strips demonstrates the colour developing of T line and develop the color dark than C line or develop the color consistent with C line, be negative; When wherein Triadimenol interpolation concentration is dry tobacco 5,10mg/kg, when wet tobacco 3,6mg/kg, test strips demonstrates the colour developing of T line not develop the color than the shallow or T line of C line colour developing, be positive, show that this test strips is limited to dry tobacco 5mg/kg, wet tobacco 3mg/kg to the detection of Triadimenol in tobacco leaf.
2, false positive rate, false negative rate test
Get known Triadimenol content and be greater than each 20 parts of the wet tobacco positive that the dry tobacco positive of 5mg/kg and content is greater than 3mg/kg, known Triadimenol content is less than each 20 parts of the wet tobacco negative sample that the dry tobacco negative sample of 5mg/kg and content are less than 3mg/kg, detect by three batches of test strips, calculate its yin and yang attribute rate.
Result shows: during the ELISA test strip positive of producing with 3 batches, and result is positive entirely, and known positive coincidence rate is 100%, and false negative rate is 0; When detecting negative sample, result is negative entirely, and known negative sample coincidence rate is 100%, and false positive rate is 0.Illustrate that the test strips of detection Triadimenol of the present invention can detect fast to the Triadimenol in tobacco leaf.
3, specific test
Cyproconazole, Flutriafol, Tebuconazole, own azoles alcohol, olefin conversion, bitertanol etc. are diluted to 5mg/L with the phosphate buffer of pH7.2,0.2mol/L, detect by Triadimenol test strips.Result shows, and during with this ELISA test strip 5mg/L cyproconazole, Flutriafol, Tebuconazole, own azoles alcohol, olefin conversion, bitertanol, the colour developing of test strips T line is darker than the colour developing of C line or develop the color consistent with C line, is negative.Illustrate that this test strips is to cyproconazole, Flutriafol, Tebuconazole, own azoles alcohol, olefin conversion, bitertanol no cross reaction.
Claims (8)
1. detect a test strips for Triadimenol, comprise absorption of sample pad, bond release pad, reaction film, adsorptive pads and base plate; It is characterized in that: described reaction film has the detection line being coated with Triadimenol hapten-carrier protein conjugate and the nature controlling line being coated with sheep anti mouse antiantibody, described bond release pad is coated with Triadimenol monoclonal antibody-colloid gold label thing.
2. test strips according to claim 1, is characterized in that, described absorption of sample pad, bond release pad, reaction film, adsorptive pads are pasted onto on base plate successively, and bond discharges under pad 1/3 ~ 1/2 is capped on absorption of sample pad.
3. test strips according to claim 1, it is characterized in that, described Triadimenol hapten-carrier protein conjugate is obtained by Triadimenol haptens and carrier protein couplet, and described carrier protein can be bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein, human serum albumins, albumin rabbit serum.
4. the test strips according to claim 1 or 3, is characterized in that, described Triadimenol haptens is that the condensation reaction carrying out ketoamine by triazolone and aminocaproic acid under the catalysis of organic base 1,8-diazabicylo 11 carbon-7-alkene obtains, and its molecular structural formula is:
。
5. test strips according to claim 1, is characterized in that, described Triadimenol monoclonal antibody prepares using Triadimenol hapten-carrier protein conjugate as immunogene.
6. test strips according to claim 1, is characterized in that, described sheep anti mouse antiantibody carries out immunity for immunogene to goat with mouse source antibody to prepare.
7. prepare a method for test strips described in any one of claim 1-6, it is characterized in that, comprise the following steps:
1) preparation is coated with the bond release pad of Triadimenol monoclonal antibody-colloid gold label thing;
2) preparation has the reaction film of the detection line being coated with Triadimenol hapten-carrier protein conjugate and the nature controlling line being coated with sheep anti mouse antiantibody;
3) by 1) and 2) bond for preparing release pad, reaction film and absorption of sample pad, adsorptive pads and base plate be assembled into test strips.
8. application rights requires a method for Triadimenol in the arbitrary described ELISA test strip tobacco sample of 1-6, and it is characterized in that, the method comprises the following steps:
1) pre-treatment is carried out to sample;
2) detect by the test strips described in any one of claim 1-6;
3) testing result is analyzed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510973179.3A CN105486872B (en) | 2015-12-23 | 2015-12-23 | A kind of test strips for detecting Triadimenol and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510973179.3A CN105486872B (en) | 2015-12-23 | 2015-12-23 | A kind of test strips for detecting Triadimenol and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105486872A true CN105486872A (en) | 2016-04-13 |
| CN105486872B CN105486872B (en) | 2017-07-07 |
Family
ID=55673989
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510973179.3A Active CN105486872B (en) | 2015-12-23 | 2015-12-23 | A kind of test strips for detecting Triadimenol and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105486872B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109298191A (en) * | 2018-09-30 | 2019-02-01 | 云南省烟草农业科学研究院 | A kind of nitrile bacterium azoles colloidal gold colloidal gold detection test paper strip and the preparation method and application thereof |
| CN110044894A (en) * | 2019-03-14 | 2019-07-23 | 昆明理工大学 | A kind of colorimetric detection method of Triadimenol |
| CN110441518A (en) * | 2019-07-17 | 2019-11-12 | 北京勤邦生物技术有限公司 | A kind of test strips and method detecting gibberellin |
| CN111044728A (en) * | 2019-09-27 | 2020-04-21 | 广州医科大学附属第一医院 | IgM antibody colloidal gold test strip for rapidly detecting adenovirus and preparation method thereof |
| CN114685387A (en) * | 2022-05-31 | 2022-07-01 | 深圳市易瑞生物技术股份有限公司 | Flutriafol hapten, antigen, antibody, detection device, preparation and application thereof |
| CN114957141A (en) * | 2022-06-20 | 2022-08-30 | 云南省烟草质量监督检测站 | Hapten for detecting content of triadimenol and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1680407A (en) * | 2005-01-21 | 2005-10-12 | 浙江大学 | Preparation of triazoline artificial semi-antigen, antigen and antibody |
| CN101518257A (en) * | 2009-04-10 | 2009-09-02 | 利尔化学股份有限公司 | Bactericidal pesticide composition |
| CN103940999A (en) * | 2013-01-19 | 2014-07-23 | 北京勤邦生物技术有限公司 | A dipstick used for testing betamethasone and application thereof |
-
2015
- 2015-12-23 CN CN201510973179.3A patent/CN105486872B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1680407A (en) * | 2005-01-21 | 2005-10-12 | 浙江大学 | Preparation of triazoline artificial semi-antigen, antigen and antibody |
| CN101518257A (en) * | 2009-04-10 | 2009-09-02 | 利尔化学股份有限公司 | Bactericidal pesticide composition |
| CN103940999A (en) * | 2013-01-19 | 2014-07-23 | 北京勤邦生物技术有限公司 | A dipstick used for testing betamethasone and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| WILLIAM H. NEWSOME: "Development of an Enzyme-Linked Immunosorbent Assay for Triadimefon in Foods", 《BULLETIN OF ENVIRONMENTAL CONTAMINATION AND TOXICOLOGY》 * |
| 胡静 等: "三唑醇分子印迹聚合物的制备及在食品检测中的应用", 《药物分析杂志》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109298191A (en) * | 2018-09-30 | 2019-02-01 | 云南省烟草农业科学研究院 | A kind of nitrile bacterium azoles colloidal gold colloidal gold detection test paper strip and the preparation method and application thereof |
| CN110044894A (en) * | 2019-03-14 | 2019-07-23 | 昆明理工大学 | A kind of colorimetric detection method of Triadimenol |
| CN110044894B (en) * | 2019-03-14 | 2020-04-21 | 昆明理工大学 | Colorimetric detection method of triadimenol |
| CN110441518A (en) * | 2019-07-17 | 2019-11-12 | 北京勤邦生物技术有限公司 | A kind of test strips and method detecting gibberellin |
| CN110441518B (en) * | 2019-07-17 | 2022-10-18 | 北京勤邦生物技术有限公司 | Test strip and method for detecting gibberellin |
| CN111044728A (en) * | 2019-09-27 | 2020-04-21 | 广州医科大学附属第一医院 | IgM antibody colloidal gold test strip for rapidly detecting adenovirus and preparation method thereof |
| CN114685387A (en) * | 2022-05-31 | 2022-07-01 | 深圳市易瑞生物技术股份有限公司 | Flutriafol hapten, antigen, antibody, detection device, preparation and application thereof |
| CN114957141A (en) * | 2022-06-20 | 2022-08-30 | 云南省烟草质量监督检测站 | Hapten for detecting content of triadimenol and application thereof |
| CN114957141B (en) * | 2022-06-20 | 2023-08-29 | 云南省烟草质量监督检测站 | Hapten for detecting triadimenol content and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105486872B (en) | 2017-07-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105486872A (en) | Test strip for detecting triadimenol and preparation method and application thereof | |
| CN105807055B (en) | A kind of test strips for detecting dichloro quinolinic acid and its preparation method and application | |
| CN105675874B (en) | A kind of colloidal gold strip for detecting imidacloprid and its application | |
| CN104569399B (en) | A kind of test strips detecting ochratoxin A and application thereof | |
| CN104849467A (en) | Fluorescent microsphere immunochromatography test paper strip for detecting clenbuterol hydrochloride residue, preparation method and applications thereof | |
| CN105424939B (en) | A kind of test strips for detecting carbendazim and its preparation method and application | |
| CN102087284A (en) | Preparation method of reagent board for rapidly detecting aflatoxin B1 | |
| CN105259351A (en) | Test paper for detecting metalaxyl residue and application thereof | |
| CN105759043A (en) | Test paper for simultaneous detection of carbendazim and thiophanate-methyl residues in crops, and application and preparation method thereof | |
| CN105572348B (en) | Detect enzyme linked immunological kit and its application of Triadimenol | |
| CN104007255B (en) | A kind of aflatoxin B1 Rapid detection test strip and preparation method thereof and application | |
| CN110850090A (en) | Test strip for detecting bifenthrin and application thereof | |
| CN105785011B (en) | A kind of test strips for detecting maleic hydrazide and its preparation method and application | |
| CN109061146A (en) | A kind of test strips and its preparation method and application detecting Acetamiprid | |
| CN103575887B (en) | A kind of test card and application thereof detecting aflatoxin B1 | |
| CN109917126A (en) | A method of test strips, the preparation method of imidacloprid haptens and the detection Determination of Imidacloprid Residue of detection imidacloprid | |
| CN108196054A (en) | A kind of test strips for detecting glycyrrhizic acid and its preparation method and application | |
| CN105044365A (en) | Preparation method of time-resolved fluoroimmunoassay test paper for detection of Enrofloxacin residue | |
| CN105388286A (en) | Test strip for detecting iprodione and preparation method and application of test strip | |
| CN106918705A (en) | Detect test paper and its application of Fenpropathrin | |
| CN108931640A (en) | A kind of test strips and its application detecting organophosphorus pesticide | |
| CN103940999B (en) | A kind of test strip detecting Betamethasone Valerate and its preparation method and application | |
| CN109682961A (en) | A kind of test strips and its application detecting cyphenothrin | |
| CN105785010A (en) | Test paper for detecting residual triadimefon in crops and application and preparation method of test paper | |
| CN104215763B (en) | A kind of test strips detecting T-2 toxin and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |