CN105486872B - A kind of test strips for detecting Triadimenol and its preparation method and application - Google Patents
A kind of test strips for detecting Triadimenol and its preparation method and application Download PDFInfo
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- CN105486872B CN105486872B CN201510973179.3A CN201510973179A CN105486872B CN 105486872 B CN105486872 B CN 105486872B CN 201510973179 A CN201510973179 A CN 201510973179A CN 105486872 B CN105486872 B CN 105486872B
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Abstract
The present invention provides a kind of test strips for detecting Triadimenol and its preparation method and application, and the test strips include sample absorption pad, conjugate release pad, reaction film, adsorptive pads and base plate；It is characterized in that：Have on the reaction film and be coated with the detection line of Triadimenol hapten-carrier protein conjugate and be coated with the nature controlling line of sheep anti mouse antiantibody, Triadimenol monoclonal antibody colloid gold label thing is coated with the conjugate release pad.The present invention also provides a kind of method for applying Triadimenol in above-mentioned ELISA test strip sample.The test strips and detection method that the present invention is provided have the advantages that simple to operate, sensitivity is high, detection speed is fast, low cost, not examined equipment limit, can realize being used for quickly detecting and on-site supervision large batch of Triadimenol sample.
Description
Technical field
The present invention relates to the detection of Triadimenol, specifically a kind of colloidal gold strip for detecting Triadimenol, it is special
The detection of Triadimenol residual suitable for tobacco juice.
Background technology
Triadimenol（Triadimenol, C14H18CN3O2）Also known as hundred load, chemical name be 1- (4- chlorophenoxies) -3,3- bis-
Methyl isophthalic acid-(1H-1,2,4- triazol-1-yls) -2- butanol, is a kind of low toxicity, wide spectrum, efficient systemic fungicide, and its is main
The mechanism of action is to suppress the biosynthesis of gibberellin and ergosterol and then influence cell division speed, to black root of tobacco,
The fungal diseases such as powdery mildew, frog eye have good prevention effect..Therefore, the application in China's agricultural production is very wide
It is general, it is adaptable to the crops such as wheat class, corn, sorghum, fruits and vegetables, tobacco.
Triadimenol residual is international tobacco scientific research Cooperation Centre in tobacco（CORESTA）Pay close attention to, and in recent years
The recall rate come in some place of production tobaccos of China is also higher.With widely using with people to food and crops for Triadimenol
The attention of safety, residual of the Triadimenol in food and crops also receives highest attention.In January, 2007 European Union disclose into
The exceeded early warning circular of Triadimenol residual quantity is detected in mouthful in domestic honey.May in the same year, Japanese MHLW finds me
Triadimenol residual quantity is exceeded in the defeated day carrot of 1 batch, state, and thus Japanese side requires Triadimenol in the carrot to China's export Japan
Mesh implements 50% monitor check.At present, many countries have clearly limited MRL of the agricultural chemicals in food, such as Japan
Residue limits of the regulation Triadimenol in food（MRL）It is 0.2 ~ 0.5 mg/kg；European Union specifies MRL of the Triadimenol in beans
It is 0.1 mg/kg；Codex Alimentary Commission（CAC）The MRL of Triadimenol is 0.5 mg/kg in regulation tomato.To avoid
Triadimenol remains do harm to huamn body and breaks through foreign trade barrier, sets up in simple, quick, accurate, reliable tobacco
The detection method of triazole residual quantity of alcohol is necessary.
Conventional Triadimenol detection method mainly has gas chromatography at present（GC）, liquid chromatography（LC）, compounds GC-MS
Method（GC-MS）With liquid-mass chromatography method（LC-MS）.Expensive instrument and special technical staff is needed using these analysis methods,
Sample pretreatment process is complex and expensive, time-consuming length, it is difficult to the need for meeting a large amount of samples and field sample quick detection.Cause
This, develop product that a kind of not examined equipment limit and can realizing is used for quickly detecting to batch samples and method into
It is problem in the urgent need to address.
The content of the invention
It is high to device dependence it is an object of the invention to overcome the existing method for detecting Triadimenol to exist, and not
The characteristics of quick detection to batch samples can be realized, there is provided a kind of simple to operate, sensitivity is high, detection speed is fast, into
Test strips of this low, not examined equipment limit and its preparation method and application, to realize entering large batch of Triadimenol sample
Row quick detection and on-site supervision.
In order to realize the purpose of the present invention, the invention provides a kind of test strips for detecting Triadimenol, the test strips include
Sample absorption pad, conjugate release pad, reaction film, adsorptive pads and base plate；Wherein, have on the reaction film and be coated with Triadimenol
The detection line of hapten-carrier protein conjugate and the nature controlling line for being coated with sheep anti mouse antiantibody, in the conjugate release pad
It is coated with Triadimenol monoclonal antibody-colloid gold label thing.
The 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, the Triadimenol
Haptens is to carry out ketoamine under the catalysis of the carbon -7- alkene of organic base 1,8- diazabicylos 11 by triazolone and aminocaproic acid
Condensation reaction is obtained, and its molecular structural formula is：
。
The Triadimenol monoclonal antibody is to prepare to obtain as immunogene using Triadimenol hapten-carrier protein conjugate
, the sheep anti mouse antiantibody is that immune preparing is carried out to goat by immunogene of mouse source antibody.
The sample absorption pad, conjugate release pad, reaction film, adsorptive pads are pasted onto on base plate successively, the conjugate
Release pad 1/3 ~ 1/2 is capped under sample absorption pad.
The base plate can be the material that PVC base plates or other hard do not absorb water；The sample absorption pad can for suction strainer paper or
Filter paper for oil；The conjugate release pad can be mineral wool or polyester material；The adsorptive pads are blotting paper；The reaction film can be
Nitrocellulose filter or CAM.
A kind of method for preparing above-mentioned test strips, comprises the following steps：
1）Preparation is coated with the conjugate release pad of Triadimenol monoclonal antibody-colloid gold label thing；
2）Preparing, there is the detection line for being coated with Triadimenol hapten-carrier protein conjugate to resist with sheep anti mouse is coated with
The reaction film of the nature controlling line of body；
3）By 1）With 2）Conjugate release pad, reaction film and sample absorption pad, adsorptive pads and the base plate for preparing are assembled into
Test strips.
Specifically, step includes：
1）Triazolone and aminocaproic acid are carried out into ketoamine under the catalysis of the carbon -7- alkene of organic base 1,8- diazabicylos 11
Condensation reaction, 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
Fusion, screening, obtain secreting the hybridoma cell strain of Triadimenol monoclonal antibody；
4）Mouse IgG immune health goat is extracted, sheep anti mouse antiantibody is obtained；
5）Triadimenol hapten-carrier protein conjugate and sheep anti mouse antiantibody are coated in the detection line of reaction film respectively
（T）And nature controlling line（C）On；
6）Collaurum is prepared with trisodium citrate and gold chloride reaction；
7）The Triadimenol monoclonal antibody of preparation is added in the collaurum of preparation, obtain Triadimenol monoclonal antibody-
Colloid gold label thing；
8）Triadimenol monoclonal antibody-colloid gold label thing is sprayed in conjugate release pad, is taken after 37 DEG C of 1 h of baking
Go out, be placed in dry environment and save backup；
9）By sample absorption pad with containing 0.5% bovine serum albumin(BSA)（Mass fraction）, pH 7.2 0.1 mol/L phosphate
Buffer solution soaks 2 h, and 2 h are dried at 37 DEG C；
10）Paste sample absorption pad, conjugate release pad, reaction film, adsorptive pads in order on base plate, conjugate is released
Put pad has 1/3 region to be absorbed by the sample pad covering from initiating terminal.3 mm small bars wide, plus plastic casing are finally cut into, are vacuum-packed,
Can be preserved 12 months under the conditions of 4 ~ 30 DEG C.The 1/3 of conjugate release pad is absorbed by the sample pad covering can extend testing result sight
The time is examined, sample absorption pad can be made to detect that liquid is fully absorbed and fully reacted with gold labeling antibody, so as to reduce error.
Using the method for Triadimenol in above-mentioned ELISA test strip sample, comprise the following steps：
（1）Pre-treatment is carried out to sample；
（2）Detected with test strips；
（3）Analysis testing result.
The test strips of detection Triadimenol of the invention are using the antibody antigen reaction of high degree of specificity and immunochromatographiassays assays
Technology, Triadimenol monoclonal antibody-colloid gold label thing is fixed in conjugate release pad, and the Triadimenol in sample is in flowing
During, combined with the Triadimenol monoclonal antibody-colloid gold label thing in conjugate release pad, form Triadimenol-antibody-glue
Body gold label.Triadimenol in sample is tied with the Triadimenol hapten-carrier protein conjugate competition in reaction film detection line
Close Triadimenol monoclonal antibody-colloid gold label thing, judged according to the detection line red stripes depth in analyte sample fluid whether
Contain Triadimenol residual.
During detection, sample instills sample absorption pad after treatment, when Triadimenol concentration in the sample less than test limit or
When being zero, monoclonal antibody-colloid gold label thing can be with the Triadimenol haptens-load being fixed on reaction film in chromatography process
Body protein conjugate is combined, in detection line（T）And nature controlling line（C）One red stripes of each appearance in place, and the colour developing of T lines is more aobvious than C line
Color depth is consistent with the colour developing of C lines；If Triadimenol concentration in the sample is equal to or higher than test limit, monoclonal antibody-colloid
Golden label can all be combined with Triadimenol, so that because competitive reaction will not be with Triadimenol hapten-carrier albumen at T lines
Conjugate combines and occurs without red stripes or developed the color than C line shallow.As shown in Figure 2.
It is negative：Work as nature controlling line（C）Show red stripes, detection line（T）Red stripes are also showed that simultaneously, and（T）Line
Color is close or is deeper than（C）During line, feminine gender is judged to.
It is positive：Work as nature controlling line（C）Show red stripes, and detection line（T）Do not develop the color or（T）Line color is shallower than（C）Line
When, it is judged to the positive.
It is invalid：Work as nature controlling line（C）Do not show red stripes, then no matter detection line（T）Whether show red stripes, should
It is invalid that test strips are judged to.
Test strips of the invention have sensitivity high, high specificity, low cost, simple to operate, detection time is short, do not examined
Measurement equipment limitation, suitable various units are used, storage is simple, the advantage of long shelf-life.With ELISA test strip Triadimenol of the present invention
Method, easy, quick, directly perceived, accurate, applied widely, low cost, easily promote the use of.
Brief description of the drawings
Fig. 1 is test strips cross-sectional view, in figure：1st, sample absorption pad；2nd, conjugate release pad；3rd, reaction film；
4th, adsorptive pads；5th, detection line；6th, nature controlling line；7th, base plate；
Fig. 2 is ELISA test strip result judgement figure；
Fig. 3 is Triadimenol hapten synthesis figure.
Specific embodiment
The present invention is expanded on further with reference to specific embodiment.It should be understood that these embodiments are merely to illustrate this
Invention, and it is not limited to the scope of the present invention.In addition, the scope that those skilled in the art limits in appended claims
Interior to carry out various changes or modification to the present invention, these are changed or modification should equally fall into the protection domain of invention.
The preparation of the test strips of the detection Triadimenol of embodiment 1
The preparation method of the test strips is mainly included the following steps that：
1）Preparation is coated with the conjugate release pad of Triadimenol monoclonal antibody-colloid gold label thing；
2）Preparing, there is the detection line for being coated with Triadimenol hapten-carrier protein conjugate to resist with sheep anti mouse is coated with
The reaction film of the nature controlling line of body；
3）By 1）With 2）Conjugate release pad, reaction film and the assembling of sample absorption pad, adsorptive pads and PVC base plates for preparing
Into test strips.
Substep narration in detail below：
1st, the synthesis of Triadimenol haptens（Synthetic route is shown in accompanying drawing 3）And identification
1.0 g triazolones plus ethanol dissolving are taken, 0.35 g 1, the carbon -7- alkene of 8- diazabicylos 11 is added（DBU）, stir
Mix, add the 0.87 g aminocaproic acid aqueous solution, the h of heating reflux reaction 12；Stop reaction, revolving, remove ethanol, add water and
1.3 g potassium hydroxide, concussion, plus ethyl acetate extraction, divide and go organic phase, and water mutually adjusts pH value to 4, plus ethyl acetate extraction,
Organic phase is washed, and anhydrous sodium sulfate drying is evaporated, and obtains light yellow oil, dichloromethane/petroleum ether（1:10, V/V）Tie again
Crystalline substance, obtains the g of haptens product 0.76, yield 69%.
Above-mentioned haptens is taken to be identified through proton nmr spectra,1H-NMR（CDCl3, 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）.Change in collection of illustrative plates
Displacement study=11.0 are carboxyl hydrogen resonance absorbing peak on spacerarm, and δ=1.85,1.41,2.30 are methylene hydrogen on spacerarm
Resonance absorbing peak, the presence at these peaks, it was demonstrated that spacerarm is coupled successfully, Triadimenol haptens structure is correct.
2nd, the synthesis and identification of Triadimenol coupled antigen
It is prepared by immunizing antigen --- Triadimenol haptens and bovine serum albumin(BSA)（BSA）Coupling obtains immunizing antigen.
14 mg haptens are taken, 1 mL DMFs are dissolved in（DMF）In；The mg of BSA 50 are weighed, is allowed to fill
Divide and be dissolved in 3.8 mL 2- (N- morpholines) ethanesulfonic acid buffer（MES）（pH 5.0）In, haptens is slowly dropped to albumen
In solution；Take 30 mg carbodiimides（EDC）With 0.2 mL MES（pH5.0）The mixing of haptens albumen is fully added after dissolving
In liquid, 24 h are stirred at room temperature；With 0.01 mol/L phosphate buffers（PBS）4 DEG C of 3 d of dialysis, change 3 dialyzates daily；
Packing, saves backup in -20 DEG C.
Triadimenol haptens obtains immunogene with hemocyanin coupling.
The mg of hemocyanin 50 is weighed, is allowed to be substantially dissolved in the phosphate buffer PBS of 3.8 mL 0.1M（PH 7.2）
In, obtain solution A；Take 30 mg carbodiimides（EDC）And N-hydroxy-succinamide（NHS）Fully dissolved with 0.2 mL water
In solution A is added, 30 min are stirred at room temperature.6 mg haptens are taken, 1 mL DMFs are dissolved in
（DMF）In, it is then slowly added in protein dissolution, 24 h of reaction are stirred at room temperature.With 0.01 mol/L PBS, 4 DEG C of 3 d of dialysis
Change 3 dialyzates daily.Packing, saves backup in -20 DEG C.
Triadimenol haptens obtains immunogene with thyroprotein coupling.
The mg of thyroprotein 50 is weighed, is allowed to be substantially dissolved in the phosphate buffer PBS of 3.8 mL 0.1M（PH
7.2）In, obtain solution A；Take 30 mg carbodiimides（EDC）And N-hydroxy-succinamide（NHS）It is abundant with 0.2 mL water
Dissolving stirs 30 min at room temperature in solution A is added.8mg haptens is taken, 1 mL DMFs are dissolved in
（DMF）In, it is then slowly added in protein dissolution, 24 h of reaction are stirred at room temperature.With 0.01 mol/L PBS, 4 DEG C of 3 d of dialysis
Change 3 dialyzates daily.Packing, saves backup in -20 DEG C.
It is prepared by envelope antigen --- Triadimenol haptens and ovalbumin（OVA）Coupling obtains envelope antigen.
14 mg haptens are taken, is dissolved in 1 mL DMF；The mg of OVA 50 are weighed, is allowed to be substantially dissolved in 3.8 mL MES
（pH 5.0）In, haptens is slowly dropped in protein solution；30 mg EDC are taken with 0.2 mL MES（pH5.0）It is fully molten
Added after solution in haptens mixed liquid of protein, 24 h are stirred at room temperature；With 0.01 mol/L PBS, 4 DEG C of 3 d of dialysis, 3 are changed daily
Secondary dialyzate；Packing, saves backup in -20 DEG C.
Triadimenol haptens obtains coating antigen with human serum albumins coupling
The mg of human serum albumins 50 is weighed, is allowed to be substantially dissolved in 3.8 mL 0.1M PBS（PH 7.2）In, obtain molten
Liquid B；Take 0.2 mL water of 30mg EDC and NHS fully to dissolve in solution B is added, 30 min are stirred at room temperature.Take 14mg
Haptens, is dissolved in 1mL DMF, is then slowly added in protein dissolution, and 24 h of reaction are stirred at room temperature.Use 0.01 mol/L
4 DEG C of 3 d of dialysis of PBS change 3 dialyzates daily.Packing, saves backup in -20 DEG C.
Triadimenol haptens obtains coating antigen with albumin rabbit serum coupling
The mg of albumin rabbit serum 50 is weighed, is allowed to be substantially dissolved in 3.8 mL 0.1M PBS（PH 7.2）In, obtain molten
Liquid B；Take 0.2 mL water of 30mg EDC and NHS fully to dissolve in solution B is added, 30 min are stirred at room temperature.Take 14mg
Haptens, is dissolved in 1mL DMF, is then slowly added in protein dissolution, and 24 h of reaction are stirred at room temperature.Use 0.01 mol/L
4 DEG C of 3 d of dialysis of PBS change 3 dialyzates daily.Packing, saves backup in -20 DEG C.
In the ratio of synthesis Triadimenol coupled antigen reaction haptens, carrier protein and coupled product used, carry out ultraviolet
（200 ~ 400 nm）Sweep measuring, by compare three respectively the light absorption value of 260 nm and 280 nm calculate its combine than.
The maximum absorption band of conjugate Triadimenol hapten-carrier albumen and Triadimenol haptens, the maximum absorption band phase of carrier protein
Than there occurs obvious change, show that the synthesis of Triadimenol hapten-carrier albumen is successful.It is computed, haptens and BSA
Combination ratio be 13:1, and the combination ratio of OVA is 8:1.
3rd, the preparation of Triadimenol monoclonal antibody
（1）The acquisition of hybridoma
1）First immunisation：By Triadimenol haptens-BSA conjugate（Immunizing antigen）Freund's complete adjuvant with equivalent is abundant
Emulsification, the Balb/c mouse of the week old of hypodermic injection 6, every 0.2 mL；
2）Booster immunization is twice：Since first immunisation, booster immunization once, is replaced with not formula Freund's incomplete adjuvant every two weeks
Freund's complete adjuvant, method and the same first immunisation of dosage；
3）Eyeground vein blood sampling survey potency and the suppression after one week of last time booster immunization, has suppression and potency reaches 1:
Following final immunization is carried out when more than 10000：Intraperitoneal injection is not added with the mL of immunogen solution 0.1 of any adjuvant, is put to death after three days
Mouse, takes its spleen and is merged with myeloma cell；
4）Cell supernatant, the positive hole of screening are determined using indirect competitive enzyme-linked immunosorbent analysis method.Using limiting dilution
Method carries out cloning to positive hole, obtains and set up the hybridoma cell strain of stably excreting Triadimenol monoclonal antibody, takes and is in
The hybridoma of exponential phase is made cell suspension with frozen stock solution, is sub-packed in cryopreservation tube, is preserved for a long time in liquid nitrogen.
（2）The preparation of monoclonal antibody
1）Cell recovery：Triadimenol monoclonal antibody hybridoma cell strain cryopreservation tube is taken out, is immediately placed in 37 DEG C of water-baths
Speed is melted, and after centrifugation removal frozen stock solution, moves into culture culture in glassware；
2）Prepare ascites and antibody purification：Using method is induced in vivo, by Balb/c mouse（8 week old）Intraperitoneal injection sterilizing stone
Only, pneumoretroperitoneum injects hybridoma 5 × 10 to the mL/ of wax oil 0.5 within 7 days5Individual/only, gather ascites after 7 days.With octanoic acid-saturation sulphur
Sour ammonium method is purified, and obtains Triadimenol monoclonal antibody solution（- 20 DEG C of preservations）.
（3）The measure of antibody titer
The potency for determining antibody with indirect competitive ELISA method is 1:(200000~500000).
Indirect competitive ELISA method：With Triadimenol haptens-OVA conjugate coated elisa plates, Triadimenol standard items are added
The sheep anti mouse antiantibody solution of solution, Triadimenol monoclonal antibody solution and horseradish peroxidase-labeled, 25 DEG C of reactions 30
Min, pours out liquid in hole, is washed with cleaning solution 3 ~ 5 times, is patted dry with blotting paper；Add substrate nitrite ion, 25 DEG C of 15 min of reaction
Afterwards, terminate liquid terminating reaction is added；Setting ELIASA is determined per hole absorbance at the nm of wavelength 450.
（4）The measure of monoclonal antibody specificity
Antibody specificity refers to the ability and the ratio with such antigen-analogues ability of its homospecificity antigen binding
Compared with conventional cross reacting rate is used as evaluation criterion.Cross reaction is smaller, and the specificity of antibody is then higher.
This experiment is by Triadimenol series bactericidal agent（Triadimenol, cyproconazole, Flutriafol, Tebuconazole, hexaconazole, olefin conversion, biphenyl
Triadimenol）It is serially diluted, carries out indirect competitive ELISA with monoclonal antibody respectively, make standard curve, analysis obtains IC50,
Then it is calculated as follows cross reacting rate：
Result shows that the cross reacting rate of each analog is：Triadimenol 100%, cyproconazole ＜ 1%, Flutriafol ＜ 1%, penta azoles
Alcohol ＜ 1%, hexaconazole ＜ 1%, olefin conversion ＜ 1%, bitertanol ＜ 1%.Antibody of the present invention to cyproconazole, Flutriafol, Tebuconazole,
Other Triadimenol insecticides no cross reactions such as hexaconazole, olefin conversion, bitertanol, have specific knot just for Triadimenol
Close.
4th, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, pathogen-free domestic sheep is immunized by immunogene of mouse source antibody, obtains sheep anti mouse and resist
Antibody.
5th, the preparation of Triadimenol monoclonal antibody-colloid gold label thing
（1）The preparation of collaurum
The chlorauric acid solution that mass fraction is 1% is diluted to 0.01% with double distilled deionized water, 100 mL is taken and is placed in taper
In bottle, boiling is heated to thermostatic electromagnetic agitator, it is 1% to add 1.5 mL mass fractions under continuous high temperature, lasting stirring
Citric acid three sodium solution, stops when continuation is at the uniform velocity heated with stirring to solution in bright claret, be cooled to after room temperature spend from
Sub- water returns to original volume, 4 DEG C of preservations.It is limpid transparent to prepare good collaurum and detect by an unaided eye, without muddiness, liquid
Surface is claret without floating object, the in the sunlight color of observing colloid gold.
（2）The preparation of Triadimenol monoclonal antibody-colloid gold label thing
Under magnetic stirring, the pH to 7.2 of collaurum is adjusted with 0.2 mol/L solution of potassium carbonate（The pH marks of different antibodies
Scope can change between 7 ~ 8）, by being added in every milliliter of colloidal gold solution, the standard of 20 ~ 50 μ g antibody is molten to collaurum
Above-mentioned Triadimenol monoclonal antibody is added in liquid, is stirred and evenly mixed, be stored at room temperature 10 min, add 10% BSA to make it in collaurum
Whole mass fraction in solution is 1%, stands 10 min.12000 r/min, 4 DEG C of 40 min of centrifugation, abandon supernatant, and precipitation is with again
Molten buffer solution is washed twice, with volume be initial colloid gold volume 1/10 redissolution buffer solution will precipitate resuspended, put 4 DEG C it is standby.
Redissolve buffer solution：Mass fraction containing BSA is 0.1% ~ 0.3%, the mass fraction of Tween-80 is 0.05% ~ 0.2%,
The 0.02 mol/L phosphate buffers of pH7.2.
6th, the preparation of conjugate release pad
Conjugate release pad is soaked in containing 0.5% BSA（Mass fraction）, pH 7.2 0.5 mol/L phosphate-buffereds
In liquid, 1 h is uniformly soaked, 37 DEG C of 3 h of baking are standby.Triadimenol monoclonal antibody-colloid that film instrument will be prepared is sprayed with Isoflow
In conjugate release pad, every 1 cm conjugates release pad sprays 0.01 mL Triadimenols monoclonal and resists golden label even application
After body-colloid gold label thing, it is placed in 37 DEG C of environment（Humidity ＜ 20%）Taken out after 60 min, be placed in dry environment（Humidity ＜
20%）In save backup.
7th, the preparation of reaction film
Detection line will be constituted in Triadimenol haptens-ovalbumin conjugate coating to reaction film, by sheep anti mouse antiantibody
It is coated on and nature controlling line is constituted on reaction film.
Coating process：Triadimenol haptens-ovalbumin conjugate is diluted to 1 mg/mL with phosphate buffer, is used
Isoflow point film instruments are coated in the detection line on nitrocellulose filter（T）, package amount is 1.0 μ L/cm；With 0.01
Sheep anti mouse antiantibody is diluted to 200 μ g/mL by the phosphate buffer of mol/L, pH7.4, is wrapped with Isoflow point film instruments
By in the nature controlling line on nitrocellulose filter（C）, package amount is 1.0 μ L/cm.The reaction film that will be coated with is placed in 37 DEG C of conditions
The lower h of drying 2, it is standby.
8th, the preparation of sample absorption pad
By sample absorption pad with containing 0.5% bovine serum albumin(BSA)（Mass fraction）, pH 7.2 0.1 mol/L phosphate delay
Fliud flushing soaks 2 h, 2h is dried at 37 DEG C standby.
9th, the assembling of test strips
Sample absorption pad, conjugate release pad, reaction film, adsorptive pads are pasted onto on PVC base plates in order successively；With reference to
Thing release pad has 1/3 region to be absorbed by the sample pad covering from initiating terminal, and the end of conjugate release pad connects with the top of reaction film
Connect, the end of reaction film is connected with the top of adsorptive pads, the top of sample absorption pad aligns with the top of PVC base plates, adsorptive pads
End alignd with the end of PVC base plates；There are detection line and nature controlling line, detection line on the reaction film（T）And nature controlling line（C）
In the strip tape perpendicular with the length of the test strips；Detection line is located at the side near the end of conjugate release pad；Quality Control
Line is located remotely from the side of the end of conjugate release pad；Test strips machine is cut into 3 mm small bars wide, mounted in special
In plastics fabrication, can be preserved 12 months under the conditions of 4 ~ 30 DEG C.
The detection of Triadimenol in the sample of embodiment 2
1st, the pre-treatment of sample
Weigh 1.0 ± 0.05 g crushing tobacco leaf sample to polystyrene centrifuge tube in；Add the methyl alcohol of 10 mL 50% molten
Liquid, is fully smashed it with refiner, obtains sample liquid；Pipette 50 μ L samples liquid and 575 μ L deionized waters（Dry tobacco）Or 100
μ L samples liquid and 650 μ L deionized waters（Wet tobacco）It is to be checked after mixing.
2nd, detected with test strips
The μ L of sample solution to be checked 100 are taken with pipettor vertically to drip in well, liquid starts timing when flowing, react
10 min, result of determination.
3rd, testing result is analyzed
It is negative（-）：The colour developing of T lines is deeper or consistent with the colour developing of C lines than the colour developing of C line, and triazole determining alcohol is less than inspection in representing sample
Survey limit, such as Fig. 2 a, 2b.
It is positive（+）：The colour developing of T lines does not develop the color than the C line shallow or T lines of colour developing, and triazole determining alcohol is equal to or higher than in representing sample
Test limit, such as Fig. 2 c, 2d.
It is invalid：There are not C lines, show the deterioration failure of incorrect operating process or test strips, such as Fig. 2 e, 2f.Herein
In the case of, specification should be again read over, and retested with new test strips.
The sample detection example of embodiment 3
1st, test limit experiment
Blank tobacco sample is taken, addition Triadimenol is wet to the mg/kg of final concentration of dry tobacco 2.5,5,10 respectively wherein
The mg/kg of tobacco 1.5,3,6, takes test strips and is detected, each sample is repeated three times.
During with ELISA test strip tobacco sample, when wherein Triadimenol addition concentration is the mg/kg of dry tobacco 2.5, wet tobacco
Show that the colour developing of T lines develops the color than C line during 1.5 mg/kg, in test strips deep or consistent with the colour developing of C lines, be negative；When wherein triazole
Alcohol addition concentration is dry tobacco 5,10 mg/kg, shows that the colour developing of T lines develops the color than C line during wet tobacco 3,6 mg/kg, in test strips
Shallow or T lines do not develop the color, and are positive, and show that detection of this test strips to Triadimenol in tobacco leaf is limited to the mg/kg of dry tobacco 5, wet tobacco
3 mg/kg。
2nd, false positive rate, false negative rate experiment
Take the wet tobacco of dry tobacco positive of the known triazole alcohol content more than 5 mg/kg and content more than 3 mg/kg
Each 20 parts of positive, it is known that dry tobacco negative sample of the triazole alcohol content less than 5 mg/kg and content are wet less than 3 mg/kg
Each 20 parts of tobacco negative sample, is detected with three batches of test strips, calculates its yin and yang attribute rate.
Result shows：During the ELISA test strip positive produced with 3 batches, as a result it is all positive, it is known that positive sample
Product coincidence rate is 100%, and false negative rate is 0；During detection negative sample, as a result it is all negative, it is known that negative sample coincidence rate is
100%, false positive rate is 0.Illustrate that the test strips of detection Triadimenol of the invention can quickly be examined to the Triadimenol in tobacco leaf
Survey.
3rd, specific test
By the pH7.2 such as cyproconazole, Flutriafol, Tebuconazole, hexaconazole, olefin conversion, bitertanol, 0.2 mol/L
Phosphate buffer is diluted to 5 mg/L, is detected with Triadimenol test strips.Result shows, with ELISA test strip 5mg/L
When cyproconazole, Flutriafol, Tebuconazole, hexaconazole, olefin conversion, bitertanol, test strips T line colour developing is than C line colour developing depth or and C
Line colour developing is consistent, is negative.Illustrate this test strips to cyproconazole, Flutriafol, Tebuconazole, hexaconazole, olefin conversion, bitertanol
No cross reaction.
Claims (7)
1. a kind of test strips for detecting Triadimenol, including sample absorption pad, conjugate release pad, reaction film, adsorptive pads and base plate；
Characterized in that, have on the reaction film and being coated with the detection line of Triadimenol hapten-carrier protein conjugate and being coated with
The nature controlling line of sheep anti mouse antiantibody, is coated with Triadimenol monoclonal antibody-colloid gold label thing, institute in the conjugate release pad
Stating Triadimenol haptens is entered under the catalysis of the carbon -7- alkene of organic base 1,8- diazabicylos 11 with aminocaproic acid by triazolone
The condensation reaction of row ketoamine is obtained, and specific synthetic method is：1.0 g triazolones plus ethanol dissolving are taken, 0.35 g 1,8- bis- is added
Carbon -7- the alkene of azabicyclic 11, stirring adds the 0.87 g aminocaproic acid aqueous solution, and the h of heating reflux reaction 12 stops anti-
Answer, revolving removes ethanol, adds water and 1.3 g potassium hydroxide, shakes, plus ethyl acetate extraction, dividing and go organic phase, water is mutually adjusted
PH value is to 4, plus ethyl acetate extraction, and organic phase washing, anhydrous sodium sulfate drying is evaporated, and obtains light yellow oil, dichloromethane
Alkane/petroleum ether v/v=1:10 recrystallizations, obtain haptens product, and the molecular structural formula of the Triadimenol haptens is：
。
2. test strips according to claim 1, it is characterised in that the sample absorption pad, conjugate release pad, reaction
Film, adsorptive pads are pasted onto on base plate successively, and conjugate release pad 1/3 ~ 1/2 is capped under sample absorption pad.
3. test strips according to claim 1, it is characterised in that the Triadimenol hapten-carrier protein conjugate by
Triadimenol haptens is obtained with carrier protein couplet, the carrier protein be bovine serum albumin(BSA), ovalbumin, hemocyanin,
Thyroprotein, human serum albumins or albumin rabbit serum.
4. test strips according to claim 1, it is characterised in that the Triadimenol monoclonal antibody is anti-with Triadimenol half
Original-carrier protein couplet thing is prepared as immunogene.
5. test strips according to claim 1, it is characterised in that it with mouse source antibody is immune that the sheep anti mouse antiantibody is
It is former that immune preparing is carried out to goat.
6. a kind of method for preparing test strips described in claim any one of 1-5, it is characterised in that comprise the following steps：
1）Preparation is coated with the conjugate release pad of Triadimenol monoclonal antibody-colloid gold label thing；
2）Prepare to have and be coated with the detection line of Triadimenol hapten-carrier protein conjugate and be coated with sheep anti mouse antiantibody
The reaction film of nature controlling line；
3）By 1）With 2）Conjugate release pad, reaction film and sample absorption pad, adsorptive pads and the base plate for preparing are assembled into test paper
Bar.
7. in a kind of any ELISA test strip tobacco samples of application claim 1-5 Triadimenol method, it is characterised in that
The method is comprised the following steps：
1）Pre-treatment is carried out to sample；
2）Detected with the test strips described in claim any one of 1-5；
3）Analysis testing result.
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| 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 |
| CN110044894B (en) * | 2019-03-14 | 2020-04-21 | 昆明理工大学 | Colorimetric detection method of triadimenol |
| 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 |
| CN111044728B (en) * | 2019-09-27 | 2023-05-30 | 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 |
| CN114685387B (en) * | 2022-05-31 | 2022-08-12 | 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 |
| 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 |
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