CN109265404A - A kind of preparation method and application of carbendazim haptens and antigen - Google Patents
A kind of preparation method and application of carbendazim haptens and antigen Download PDFInfo
- Publication number
- CN109265404A CN109265404A CN201811104645.4A CN201811104645A CN109265404A CN 109265404 A CN109265404 A CN 109265404A CN 201811104645 A CN201811104645 A CN 201811104645A CN 109265404 A CN109265404 A CN 109265404A
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- CN
- China
- Prior art keywords
- carbendazim
- haptens
- antigen
- preparation
- liquid
- 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.)
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- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 title claims abstract description 101
- 239000000427 antigen Substances 0.000 title claims abstract description 40
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Classifications
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
- C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
- C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
- C07D235/24—Benzimidazoles; Hydrogenated benzimidazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
- C07D235/30—Nitrogen atoms not forming part of a nitro radical
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- 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
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
A kind of preparation method and application of carbendazim haptens and antigen, it is characterised in that: the carbendazim haptens is to react to obtain with succinic anhydride by 2- aminobenzimidazole；The carbendazim antigen is to be obtained by carbendazim haptens with carrier protein couplet.Antigen prepared by the present invention shows the carbendazim antigenic determinant of specificity, makes it possible the carbendazim monoclonal antibody for filtering out high specific.The antibody specificity height of generation, high sensitivity, can be used for establishing enzyme-linked immunosorbent assay for measuring and colloid gold test paper rapid test method, to realize the quick detection of carbendazim in tobacco and food.
Description
Technical field
The present invention relates to the preparation method and applications of a kind of carbendazim haptens and antigen.Belong to pesticide immunochemical technique
Field.
Background technique
Carbendazim (Carbendazim) is benzimidazole germicide, is a kind of good wide spectrum, systemic fungicide,
It is all effective to most of pathogens in sac fungus, basidiomycetes and Fungi Imperfecti, it is widely used in crops, Chinese herbal medicine, cigarette
In the disease control work of grass etc..Carbendazim chemical property is stablized, and can be absorbed by the seed, root and leaf of plant, and the residual period is longer,
There is certain toxicity to people, animal.China is directed to Different Crop, has formulated the maximum residue limit standard of carbendazim, wherein more bacterium
Spirit is in the maximum residue limit in cereal between 0.05-2 mg/kg, and the maximum residue limit in vegetables is 0.02-5
Between mg/kg, in the maximum residue limit in fruit between 0.5-5 mg/kg, the maximum residue limit in nut is
0.1 mg/kg.The guiding residue limits of carbendazim are in international tobacco scientific research Cooperation Centre (CORESTA) regulation tobacco
2 mg/kg, in actual production, using 2 mg/kg as tobacco maximum residue limit criterion.
Currently, mainly having tablets by HPLC-MS, efficient liquid to the remaining detection method of carbendazim both at home and abroad
Phase chromatography.Instrumental method has the advantages such as detection sensitivity height, high specificity, but detection Sample pretreatment is cumbersome, time-consuming,
Sample also needs extraction and purified treatment, while instrument detection method needs expensive large-scale instrument and equipment, is equipped with the inspection of profession
Survey technology personnel operate and manage, and can not carry out live extensive detection, poor in timeliness, it is difficult to promote.It is anti-based on antigen
The immunoassay method of body specific recognition can be with the pesticide residue in qualitative and quantitative detection sample.This analysis method is to instrument
Equipment requirement is not high, fast and convenient, general without carrying out complicated pretreatment to sample, high sensitivity, high specificity, to using
The professional technique of personnel is of less demanding, is easy universal and promotes, can meet the needs of quick analysis detection, be particularly suitable for live sieve
The quick analysis of choosing and a large amount of samples.Immunoassay provides a new analysis detection approach for carbendazim residual research.Exempt from
Epidemic disease analysis has become a brand-new field of pesticide residue analysis research at present, and American Chemical Society is by immunoassay and gas phase color
Spectrum, liquid chromatogram are classified as three big mainstays of pesticide residue analysis jointly.China's pesticide immuno analytical method research starting phase
It to later, but quickly grows in recent years, about parathion, parathion-methyl, methyl paraoxon, carbendazim, chlopyrifos, triazole
The artificial antigen of the pesticides such as phosphorus, Fipronil, dichloro quinolinic acid, carbofuran, triazolone, acephatemet, Atrazine, 2 first, 4 chlorine and height
The preparation of the specific antibody of affinity and the report that the trace analysis of agricultural drugs in sample is carried out with enzyme-linked immunization.
The invention belongs to pesticide molecule compound immunochemistries and retention analysis technical field, are related to organic synthesis, exempt from
Epidemic disease chemistry and biochemistry etc., by immunology, immunochemistry basic principle and biotechnological method, design, synthesized micromolecule
Target analytes haptens, and be coupled with carrier protein, prepare effective artificial antigen.The antigen of preparation can be by immune dynamic
Object prepares the antibody to small molecule analyte specific recognition, is detected using the specificity immunology reaction of antigen-antibody with easy
The amplification of the marker of identification, ultramicron small molecule object in quantitative test sample.The MOLECULE DESIGN of haptens with
Synthesis is the committed step for generating specific antibody and establishing pesticide residue immunoassay method.The preparation of artificial antigen, including
Difference on binding site, combination, carrier and haptens and any structure of analyte substance of interest, such as molecule are big
Topological sex character including small, shape, ingredient, configuration, conformation, polarity, cloud density etc., all may strong influence it is corresponding
The property of antibody.It can design and synthesize the better haptens of performance, effect and antigen, emphasis of interest exactly of the invention。
Summary of the invention
The purpose of the present invention is based on above-mentioned prior art situation and provides the system of a kind of carbendazim haptens and antigen
Preparation Method and application.
The purpose of the present invention is achieved through the following technical solutions:
A kind of preparation method of carbendazim haptens is to react to obtain with succinic anhydride by 2- aminobenzimidazole, molecule knot
Structure formula are as follows:
。
Specific step is as follows:
Take 1.05 g(10.5 mmol) succinic anhydride is dissolved in 100 mL acetonitriles, be heated to reflux under (85-100 DEG C) by several times plus
Enter 1.33 g(10 mmol) 2- aminobenzimidazole, continues reflux 1 hour, is cooled to room temperature, the solid was filtered, through methanol, second
It is dry after nitrile washing, obtain 2.00 g of carbendazim haptens.
The carbendazim haptens can be used for making the antigen system raw material of animal immune.
A kind of preparation method of carbendazim antigen is to be obtained by carbendazim haptens with carrier protein couplet.The carrier
Albumen is thyroprotein, bovine serum albumin(BSA), rabbit serum proteins, human albumin, ovalbumin or hemocyanin.
Specific step is as follows:
The preparation of immunizing antigen: taking 15 mg carbendazim haptens, is dissolved in 1 mL n,N-Dimethylformamide (DMF), adds
0.22 mL of oxalyl chloride, is stirred overnight, and obtains haptens activating solution A liquid；50 mg of bovine serum albumin(BSA) (BSA) is taken, 0.05 M is added
3.6 mL of borate buffer solution dissolution, obtains B liquid, A drop is added in B liquid, and 4 DEG C of 5 h of stirring stop reaction, with 0.01
4 DEG C of 3 d of dialysis of mol/L phosphate buffer PBS, change 3 dialyzates daily.Packing, obtains carbendazim-BSA antigen, in-
20 DEG C save backup.
The preparation of envelope antigen: 13 mg carbendazim haptens are taken, are dissolved in 1 mL DMF, chlorination isopropyl formate
0.21 mL, 0.3 mL of triethylamine, is stirred at room temperature 7 h, obtains haptens activating solution A liquid；50 mg of ovalbumin (OVA) is taken, it is molten
In 4 mL 0.1M phosphate buffer PB, B liquid is obtained, A drop is added in B liquid, 8 h are stirred at room temperature.With 0.01 mol/L
4 DEG C of PBS 3 d of dialysis, change 3 dialyzates daily.Packing, obtains carbendazim-OVA coating antigen, saves backup in -20 DEG C.
The monoclonal antibody obtained using carbendazim antigen-immunized animal can be used for establishing enzyme-linked immunosorbent assay for measuring
With colloid gold test paper rapid test method, to realize the quick detection of carbendazim in tobacco and food.
The carbendazim haptens and antigen that are synthesized in the present invention with application No. is 201510973048.5 and
201510973216.0 patent in haptens and antigenic structure it is different.The carbendazim haptens synthesized in the present invention was both maximum
Degree remains the chemical structure of carbendazim, and has the linking arm of appropriate length, goes to exempt from immunogene prepared by the haptens
Epidemic disease animal, potency, specificity, the affinity of obtained antibody are all relatively good, low with the cross reacting rate of other pesticides, specificity
More preferably, sensitivity is higher.
Antigen prepared by the present invention shows the carbendazim antigenic determinant of specificity, so that filtering out the more of high specific
Bacterium spirit monoclonal antibody is possibly realized.The antibody specificity height of generation, high sensitivity, can be used for establishing enzyme linked immunosorbent assay (ELISA)
Method and colloid gold test paper rapid test method, to realize the quick detection of carbendazim in tobacco and food.
Detailed description of the invention
Fig. 1 is carbendazim hapten synthesis route map (figure is as Figure of abstract)；
Fig. 2 is test paper the schematic diagram of the section structure, in figure: 1, sample absorption pad；2, reaction film；3, water absorption pad；4, detection line；5, matter
Control line；6, bottom plate；7, protective film；
Fig. 3 is test paper top view；
Fig. 4 is micropore reagent figure, in figure: 8, micropore；9, micropore plug.
Specific embodiment
Below with reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this
Invention, and be not intended to limit the scope of the invention.
The preparation of 1 carbendazim haptens of embodiment
1, the synthesis of carbendazim haptens (synthetic route is shown in Fig. 1)
Taking 1.05 g(10.5 mmol) succinic anhydride is dissolved in 100 mL acetonitriles, and it is heated to reflux lower gradation and 1.33 g(10 is added
Mmol) 2- aminobenzimidazole continues reflux 1 hour, is cooled to room temperature, and the solid was filtered, does after acetonitrile washing through methanol
It is dry, obtain 2.00 g of carbendazim haptens, yield 86%.
2, the identification of carbendazim haptens
Nuclear-magnetism identify 1H NMR (DMSO-d6): 11.95 (br, 3H), 7.43 (d, d, J1=5.53 Hz, J2=
5.61 Hz), 7.07 (d,d, J1 = 5.53 Hz, J2 = 5.61 Hz), 2.70 (d, J = 6.61 Hz, 2H),
2.58 (d, J = 6.61 Hz, 2H)。
In map, chemical shift δ=2.70,2.58 for methylene hydrogen on spacerarm resonance absorbing peak, the presence at these peaks
Prove that spacerarm is coupled successfully, carbendazim haptens structure is correct.
The preparation of 2 carbendazim antigen of embodiment
1, the synthesis of carbendazim immunogene
Carbendazim haptens and bovine serum albumin(BSA) (BSA) coupling obtain immunogene.
15 mg carbendazim haptens are taken, is dissolved in 1 mL n,N-Dimethylformamide (DMF), adds oxalyl chloride 0.22
ML is stirred overnight, and obtains haptens activating solution A liquid；50 mg of bovine serum albumin(BSA) (BSA) is taken, 0.05 M borate buffer solution is added
3.6 mL dissolution, obtains B liquid, A drop is added in B liquid, and 4 DEG C of 5 h of stirring stop reaction, with 4 DEG C of 0.01 mol/L PBS
Dialyse 3 d, changes 3 dialyzates daily.Packing, obtains carbendazim-BSA antigen, saves backup in -20 DEG C.
2, the synthesis of carbendazim coating antigen
Carbendazim haptens and ovalbumin (OVA) coupling obtain coating antigen.
13 mg carbendazim haptens are taken, are dissolved in 1 mL DMF, 0.21 mL of chlorination isopropyl formate adds triethylamine
7 h are stirred at room temperature in 0.3 mL, obtain haptens activating solution A liquid；50 mg of ovalbumin (OVA) is taken, 4 mL 0.1M PB are dissolved in
In buffer, B liquid is obtained, A drop is added in B liquid, 8 h are stirred at room temperature.With 0.01 mol/L PBS, 4 DEG C of 3 d of dialysis, often
It changes 3 dialyzates.Packing, obtains carbendazim-OVA coating antigen, saves backup in -20 DEG C.
3, the identification of carbendazim immunogene and coating antigen
It determines whether coupling succeeds: generally identifying whether the coupling of haptens and carrier protein is effective idol by UV scanning method
Connection, because haptens has different characteristic absorptions from albumen under ultraviolet, when being coupled successfully, the UV absorption of conjugate is had
The superimposed effect of the two occurs, therefore certain offset can occur than individually protein specificity absorption, can be used for detecting coupling
Whether succeed.
The measurement of coupling ratio: with pure water dilution carbendazim haptens, bovine serum albumin(BSA), ovalbumin and two kinds of albumen with
The conjugate of carbendazim haptens is configured to certain density solution, then carries out full wavelength scanner with ultraviolet specrophotometer,
Obtain their uv absorption spectra.
The Molar Extinction of carbendazim haptens, bovine serum albumin(BSA), ovalbumin is calculated separately out according to formula K=A/CL
Coefficient.The absorbance value that conjugate is detected at the maximum wavelength of carrier protein and carbendazim haptens, is calculated by formula two kinds
Molar concentration rate of the substance in conjugate, i.e. coupling ratio:
Ca/Cb=(AEven 264×KBSA280-AEven 280×KBSA264)/(AEven 280×KCarbendazim 264-AEven 264×KCarbendazim 280)
The measurement of protein content: after conjugate is diluted to suitable multiple, the spectrophotometric value of 280 nm and 260 nm is measured, is pressed
Concentration, that is, conjugate concentration of formula calculating albumen:
Protein (mg/mL)=1.45 × OD280- 0.74 × OD260
The qualification result of immunogene and coating antigen: the coupling that haptens and carrier protein are identified by UV scanning method is effective idol
Connection estimates haptens and carrier egg in the molar absorption coefficient of specific wavelength according to carbendazim haptens, carrier protein, conjugate
Than being respectively 17:1 and 15:1, coupling effect is preferable for white combination, and the protein content of immunogene is 19.2 mg/mL, coating antigen
Protein content is 8.4 mg/mL.
The preparation of 3 carbendazim monoclonal antibody of embodiment
1, the acquisition of hybridoma
1) first immunisation: carbendazim haptens-BSA conjugate (immunogene) and 3 times of Freund's complete adjuvant is fully emulsified, skin
The Balb/c mouse of lower injection 8-10 week old, immunizing dose are 150 μ g/；
2) booster immunization is twice: since first immunisation, booster immunization is primary every two weeks, replaces Freund with not formula Freund's incomplete adjuvant
Freund's complete adjuvant, method and the same first immunisation of dosage；
3) potency and inhibition are surveyed in eyeground vein blood sampling to last time booster immunization after a week, have inhibition and potency reaches 1:10000
Following final immunization is carried out when above: 0.1 mL of immunogen solution of any adjuvant is not added in intraperitoneal injection, puts to death mouse after three days,
Its spleen is taken to merge with myeloma cell；
4) cell supernatant is measured using indirect competitive enzyme-linked immunosorbent analysis method, screens positive hole.Utilize limiting dilution assay pair
Positive hole carries out cloning, obtains and establishes the hybridoma cell strain of stably excreting carbendazim monoclonal antibody, take in logarithm
Cell suspension is made with frozen stock solution in the hybridoma in growth period, is sub-packed in cryopreservation tube, saves for a long time in liquid nitrogen.
2, the preparation of monoclonal antibody
1) cell recovery: taking out carbendazim monoclonal antibody hybridoma cell strain cryopreservation tube, be immediately placed in 37 DEG C of water-bath middling speeds and melt,
After centrifugation removal frozen stock solution, culture culture in glassware is moved into；
2) ascites and antibody purification are prepared: using method is induced in vivo, by Balb/c mouse (8 week old) Intraperitoneal injection sterilizing paraffin oil
Only, hybridoma 5 × 10 is injected intraperitoneally in 0.5 mL/ after 7 days5A/only, ascites is acquired after 7 days.With octanoic acid-saturated ammonium sulfate
Method is purified, and carbendazim monoclonal antibody solution (- 20 DEG C of preservations) is obtained.
3, the measurement of antibody titer
Potency with indirect competitive ELISA method measurement antibody is 1:(200000 ~ 400000).
Indirect competitive ELISA method: using carbendazim haptens-OVA conjugate coated elisa plate, and carbendazim standard product is added
The sheep anti mouse antiantibody solution of solution, carbendazim monoclonal antibody solution and horseradish peroxidase-labeled, 25 DEG C of reactions 30
Min pours out liquid in hole, is washed 3 ~ 5 times with cleaning solution, is patted dry with blotting paper；Substrate developing solution, 25 DEG C of 15 min of reaction are added
Afterwards, terminate liquid is added and terminates reaction；Setting microplate reader measures every hole absorbance value at 450 nm of wavelength.
4, the measurement of monoclonal antibody specificity
Antibody specificity refer to the ability of its homospecificity antigen binding compared with such antigen-analogues ability, often
Use cross reacting rate as evaluation criterion.Cross reaction is smaller, and the specificity of antibody is then higher.
Carbendazim and other benzimidazole germicides (thiophanate-methyl, benomyl, probenazole) are done series by this experiment
Dilution carries out indirect competitive ELISA with monoclonal antibody respectively, makes standard curve, and analysis obtains IC50, then count as the following formula
Calculate cross reacting rate:
The cross reacting rate of carbendazim and its analogue as the result is shown are as follows: carbendazim 100%, thiophanate-methyl < 1%, benzene bacterium
Clever < 1%, probenazole < 1%.Antibody of the present invention is to other benzimidazole germicides such as thiophanate-methyl, benomyl, probenazole
No cross reaction has specific binding just for carbendazim.
The preparation of 4 carbendazim colloidal gold strip of embodiment
1, carbendazim monoclonal antibody-colloid gold label object preparation
(1) preparation of colloidal gold
The chlorauric acid solution that mass fraction is 1% is diluted to 0.01% with double distilled deionized water, 100 mL is taken to be placed in conical flask,
It is heated to boiling with thermostatic electromagnetic blender, in continuous high temperature, is persistently added with stirring the lemon that 1.5 mL mass fractions are 1%
Sour three sodium solutions, continuation are at the uniform velocity heated with stirring to solution in stopping when bright claret, use deionized water after being cooled to room temperature
It is restored to original volume, 4 DEG C of preservations.Preparing good colloidal gold and detecting by an unaided eye is clear and transparent, no muddiness, liquid table
Face is without floating material, and the color of observing colloid gold is claret in the sunlight.
(2) carbendazim monoclonal antibody-colloid gold label object preparation
Under magnetic stirring, it is marked with the pH value of the pH value of 0.2 mol/L solution of potassium carbonate tune colloidal gold to 7.2(different antibodies
Range can change between 7 ~ 8), it is molten to colloidal gold by the standard that 20 ~ 50 μ g antibody are added in every milliliter of colloidal gold solution
Above-mentioned carbendazim monoclonal antibody is added in liquid, stirs and evenly mixs, is stored at room temperature 10 min, 10% BSA, which is added, makes it in colloidal gold
Whole mass fraction in solution is 1%, stands 10 min.12000 r/min, 4 DEG C of 40 min of centrifugation, abandon supernatant, and precipitating is used
Redissolve buffer wash twice, with the redissolution buffer that volume is initial colloid gold volume 1/10 will precipitating be resuspended, set 4 DEG C it is standby
With.
Redissolve buffer: the mass fraction containing BSA be the mass fraction of 0.1% ~ 0.3%, Tween-80 be 0.05% ~ 0.2%,
The 0.02 mol/L phosphate buffer that pH value is 7.2.
2, the preparation of micropore reagent
100 μ L carbendazim monoclonal antibodies-colloid gold label object is added into micropore reagent micropore, is put into freeze drier,
Under the conditions of condenser temperature is -50 DEG C, after 3 h of pre-freeze, then 15 h is dried in vacuo, that is, can be taken off, obtaining freeze-drying has carbendazim list
Clonal antibody-colloid gold label object micropore reagent, is sealed.
3, the preparation of sample absorption pad
It is 0.5% bovine serum albumin(BSA), the 0.1 mol/L phosphate-buffered that pH value is 7.2 that sample absorption pad, which is placed in volume fraction,
2h is impregnated in liquid, 37 DEG C of baking 2h are spare.
4, the preparation of reaction film
Coating process: carbendazim haptens-ovalbumin conjugate is diluted to 1 mg/mL with phosphate buffer, uses Isoflow
Point film instrument is coated in the detection line (T) on nitrocellulose filter, and package amount is 1.0 μ L/cm；With 0.01mol/L, pH value
Sheep anti mouse antiantibody is diluted to 200 μ g/mL for 7.4 phosphate buffer, is coated in nitre with Isoflow point film instrument
Nature controlling line (C) on acid cellulose film, package amount are 1.0 μ L/cm.It is dry under the conditions of the reaction film being coated with is placed in 37 DEG C
2h, it is spare.
5, the assembling of each component
(1) assembling of test paper
The sample absorption pad, reaction film, water absorption pad are successively pasted on the bottom plate in order；The end of sample absorption pad
It is connected with the beginning of reaction film, the end of reaction film is connected with the beginning of water absorption pad, the beginning of sample absorption pad and the beginning of bottom plate
End alignment, the end of water absorption pad is aligned with the end of bottom plate；The bonding protective film on the sample absorption pad of assembled test paper is protected
MAX mark line is printed on cuticula.
(2) assembling of test strips
Test paper and micropore reagent that above-mentioned steps 1 obtain are assembled into test strips, stored in 2 ~ 8 DEG C of environment, validity period 12
A month.
Embodiment 5 detects the application of the colloidal gold strip of carbendazim
1, the pre-treatment of sample
The smashed sample to be tested of 1.0 ± 0.05 g is weighed into 50 mL centrifuge tubes, 10 mL, 50% methanol aqueous solution, whirlpool is added
1 min is revolved, 3000 rpm or more are centrifuged 5 min；100 μ L supernatants are taken, 400 μ L samples are added and redissolve liquid, mix to be measured.
2, it is detected with test strips
With micropipettor draw 100 μ L sample to be tested solution in micropore reagent, slowly suction and sufficiently with reagent in micropore
It mixes, after (20 ~ 25 DEG C) 3 min of incubation of room temperature, test paper is indicated into MAX label line end and is downwardly into the micropore reagent after being incubated for
In, liquid starts timing when flowing, react 10 min, determines result according to schematic diagram.
3, analysis detection result
Negative (-): the colour developing of T line develops the color deep or develops the color unanimously with C line than C line, indicates that carbendazim concentration is limited lower than detection in sample.
Positive (+): the colour developing of T line is more shallow than the colour developing of C line or T line does not develop the color, and indicates that carbendazim concentration is equal to or higher than in sample
Detection limit.
It is invalid: not occur C line, show the deterioration failure of incorrect operating process or test strips.In the case, it answers
Specification is read over again, and is retested with new test strips.
Embodiment 6 detects the determination of the colloidal gold strip technical parameter of carbendazim
1, detection limit test
Take blank agricultural product and tobacco sample, wherein respectively addition carbendazim to final concentration of 0.1,0.2,0.4 mg/kg,
Test strips are taken to be detected, each sample is repeated three times.
When detecting agricultural product and tobacco sample with test strips, when wherein carbendazim addition concentration is 0.1 mg/kg, test paper
It shows that the colour developing of T line develops the color deep or develops the color unanimously with C line than C line on item, is negative；When wherein carbendazim addition concentration is 0.2,
It shows that the colour developing of T line is more shallow than the colour developing of C line or T line does not develop the color when 0.4 mg/kg, in test strips, is positive, shows this test strips pair
The detection of carbendazim is limited to 0.2 mg/kg in agricultural product.
2, false positive rate, false negative rate test
It takes known carbendazim content greater than 20 parts of positive sample of 0.2 mg/kg, 20 parts of negative sample, is carried out with three batches of test strips
Detection, calculates its yin and yang attribute rate.
The result shows that: when detecting positive sample with the test strips of 3 batch productions, as a result it is all positive, it is known that positive sample
Product coincidence rate is 100%, false negative rate 0；When detecting negative sample, as a result it is all negative, it is known that negative sample coincidence rate is
100%, false positive rate 0.Illustrate detection carbendazim of the invention test strips can to the carbendazim in agricultural product and tobacco into
Row quickly detection.
3, specific test
Thiophanate-methyl, benomyl, the probenazole of 1 mg/L are detected with carbendazim test strips.The results show that test strips T line develops the color
It develops the color deep than C line or develops the color unanimously with C line, be negative.Illustrate this test strips to thiophanate-methyl, benomyl, probenazole without friendship
Fork reaction.
Claims (9)
1. a kind of preparation method of carbendazim haptens, it is characterised in that: be to be reacted by 2- aminobenzimidazole with succinic anhydride
It obtains, molecular structural formula are as follows:
。
2. the preparation method of carbendazim haptens as described in claim 1, it is characterised in that: the specific steps of the preparation method
It is as follows:
It takes 1.05 g succinic anhydrides to be dissolved in 100 mL acetonitriles, is heated to reflux lower gradation and 1.33 g 2- amino benzo miaows are added
Azoles continues reflux 1 hour, is cooled to room temperature, and the solid was filtered, dry after acetonitrile washing through methanol, and it is anti-to obtain carbendazim half
2.00 g of original.
3. the preparation method of carbendazim haptens as claimed in claim 2, it is characterised in that: temperature when being heated to reflux is
85-100℃。
4. the application of the carbendazim haptens of method preparation as described in claim 1, it is characterised in that: the carbendazim haptens
It can be used for making the antigen system raw material of animal immune.
5. a kind of preparation method of carbendazim antigen, it is characterised in that: be the carbendazim haptens being prepared by claim 1
It is obtained with carrier protein couplet.
6. the preparation method of carbendazim antigen as claimed in claim 5, it is characterised in that: the carrier protein is thyroid gland egg
White, bovine serum albumin(BSA), rabbit serum proteins, human albumin, ovalbumin or hemocyanin.
7. such as the preparation method of carbendazim antigen described in claim 5 or 6, it is characterised in that: specific step is as follows: taking 15
Mg carbendazim haptens is dissolved in 1 mL n,N-Dimethylformamide, is added 0.22 mL of oxalyl chloride, is stirred overnight, and obtains half
Antigen-activated liquid A liquid；50 mg of bovine serum albumin(BSA) is taken, adds 0.05 M borate buffer solution, 3.6 mL to dissolve, B liquid is obtained, by A
Drop is added in B liquid, 4 DEG C of 5 h of stirring, stops reaction, with 0.01 mol/L phosphate buffer PBS, 4 DEG C of 3 d of dialysis, often
It changes 3 dialyzates, obtains carbendazim antigen；Packing, saves backup in -20 DEG C.
8. such as the preparation method of carbendazim antigen described in claim 5 or 6, it is characterised in that: specific step is as follows: taking 13
Mg carbendazim haptens is dissolved in 1 mL n,N-Dimethylformamide, 0.21 mL of chlorination isopropyl formate, triethylamine 0.3
7 h are stirred at room temperature in mL, obtain haptens activating solution A liquid；50 mg of ovalbumin is taken, 4 mL 0.1M phosphate buffer PB are dissolved in
In, B liquid is obtained, A drop is added in B liquid, 8 h are stirred at room temperature；With 0.01 mol/L PBS, 4 DEG C of 3 d of dialysis, change daily 3 times
Dialyzate obtains carbendazim antigen；Packing, saves backup in -20 DEG C.
9. the application of the carbendazim antigen of method preparation as claimed in claim 5, it is characterised in that: immune using carbendazim antigen
The monoclonal antibody that animal obtains can be used for establishing enzyme-linked immunosorbent assay for measuring and colloid gold test paper rapid test method, from
And realize the quick detection of carbendazim in tobacco and food.
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Cited By (4)
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| CN112986564A (en) * | 2021-03-16 | 2021-06-18 | 广州敏捷生物技术有限公司 | Carbendazim hapten, artificial antigen and immunofluorescence chromatography detection card thereof |
| CN113307776A (en) * | 2021-04-20 | 2021-08-27 | 华南农业大学 | Preparation and application of bispecific antibody for recognizing albendazole and carbendazim |
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| CN113307776A (en) * | 2021-04-20 | 2021-08-27 | 华南农业大学 | Preparation and application of bispecific antibody for recognizing albendazole and carbendazim |
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Application publication date: 20190125 |