CN105137009B - The enzyme linked immunological kit of detection carbofuran and application thereof - Google Patents
The enzyme linked immunological kit of detection carbofuran and application thereof Download PDFInfo
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Abstract
The invention provides a kind of enzyme linked immunological kit detecting carbofuran, it includes: be coated with the ELISA Plate of carbofuran coupled antigen, carbofuran monoclonal antibody, enzyme labelling anti antibody, carbofuran standard solution, substrate nitrite ion, stop buffer, cleaning mixture, redissolution liquid.The invention also discloses a kind of method applying above-mentioned enzyme linked immunological kit detection carbofuran, it includes: first carries out sample pre-treatments, then detects with test kit, ultimate analysis testing result.The enzyme linked immunological kit that the present invention provides can be used for detecting the content of carbofuran in vegetable and Folium Camelliae sinensis, it is easy and simple to handle, low cost, highly sensitive, can on-site supervision and the examination of applicable great amount of samples.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, be specifically related to a kind of enzyme linked immunological kit for detecting carbofuran, it is special
Be not suitable to the detection of carbofuran content in vegetable and Folium Camelliae sinensis.
Background technology
Carbofuran (carbofuran), chemical name 2,3-dihydro-2,2-dimethyl-7-benzofuran-N-methyl carbamate, be
Absorption parasite killing, nematicide in the carbamates of a kind of high-efficiency broad spectrum, be widely used in Cotton Gossypii, Oryza sativa L., Caulis Sacchari sinensis, Semen arachidis hypogaeae,
The preventing and treating of the crop pest such as Semen sojae atricolor, Nicotiana tabacum L..Carbofuran is the most degradable in soil, easily causes earth's surface and ground by runoff and infiltration
Lower water pollutes.Although in China's trace standard of pesticide, regulation carbofuran must not be in vegetable and fruit and detected, but Misuse gram
The phenomenon of Budweiser is the most universal, causes the event of poisoning to happen occasionally because eating the foods such as the vegetable that polluted by carbofuran by mistake.
The residue detection of carbofuran mainly has gas chromatography (GC), high performance liquid chromatography (HPLC), compounds GC-MS at present
Method (GC-MS) and liquid-mass chromatography method (HPLC-MS).These analysis methods are used to need expensive instrument and special technology
Personnel, sample pretreatment process length complex and expensive, time-consuming, it is difficult to meet a large amount of sample and need that field sample quickly detects
Want.Enzyme linked immunosorbent assay analysis method (ELISA) have easy quick, special sensitive, sample capacity is big, analysis cost is low
Feature, can simplify and even save sample purification step, demonstrates uniqueness in great amount of samples and field samples rapid screening detect
Advantage, it is possible to preferably meet China's food enterprise, government function supervision department etc. and carry out detection work, great development potentiality.
Summary of the invention
It is an object of the invention to provide a kind of simple in construction, easy to use, low price, portable examine for carbofuran
The enzyme linked immunological kit surveyed, and provide a kind of efficiently, accurately, easy, be suitable to high-volume screening sample qualitative, quantitatively examine
Survey method.
Test kit of the present invention, it includes: be coated with the ELISA Plate of carbofuran coupled antigen, carbofuran monoclonal antibody, enzyme labelling
Anti antibody, carbofuran standard solution, substrate nitrite ion, stop buffer, cleaning mixture, redissolution liquid；Described carbofuran coupled antigen
Being to be obtained with carrier protein couplet by carbofuran hapten, described carrier protein can be Mus serum albumin, thyroprotein, Sanguis Bovis seu Bubali
Pure albumen, rabbit serum proteins, human albumin, ovalbumin, hemocyanin or Fibrinogen, described carbofuran half is anti-
Former is by nitrofuran phenol under the effect of phosgene, with methylamine by acylation reaction, obtains band nitro carbofuran product, then warp
Nitro-reduction reaction obtains, and molecular structural formula is:
Described carbofuran monoclonal antibody is to prepare using carbofuran coupled antigen as immunogen.
Described anti antibody is sheep anti mouse anti antibody.
The marker enzyme of described enzyme labelling anti antibody is horseradish peroxidase or antibacterial extraction alkaline phosphatase, wherein preferred Radix Cochleariae officinalis
Peroxidase；The anti antibody of enzyme labelling is to use glutaraldehyde method or Over-voltage protection marker enzyme and anti antibody to be carried out coupling and obtains
's.
For more convenient on-site supervision and great amount of samples examination, described test kit also includes that carbofuran standard solution, substrate develop the color
Liquid, stop buffer, cleaning mixture, redissolution liquid.
Described carbofuran standard solution 6 bottles, concentration be respectively 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L,
40.5μg/L。
When marker enzyme is horseradish peroxidase, described substrate nitrite ion is made up of substrate solution A liquid and substrate solution B liquid, A liquid
For hydrogen peroxide or urea peroxide, B liquid is o-phenylenediamine or tetramethyl benzidine, described stop buffer be 1～2mol/L sulphuric acid or
Hydrochloride buffer；When marker enzyme is antibacterial extraction alkaline phosphatase, described substrate nitrite ion is to nitro phosphate buffer,
Described stop buffer is 1～2mol/L sodium hydroxide solution.
Described cleaning mixture preferably pH value is 7.4, containing 0.5%～1.0% tween 20,0.01 ‰～0.03 ‰ sodium azide preservatives,
The phosphate buffer of 0.1～0.3mol/L, described percentage ratio is quality percent by volume.
Described redissolution liquid preferably pH value is 7.0, the phosphate buffer of 0.1mol/L.
Wherein in ELISA Plate preparation process used be coated buffer be pH value be 9.6, the carbonate buffer solution of 0.05mol/L,
Confining liquid be pH value be 7.1～7.5, containing 1%～3% casein, 0.1～the phosphate buffer of 0.3mol/L, described percentage ratio
For quality percent by volume.
In the present invention, the preparation process of ELISA Plate is: with being coated buffer, coating antigen being diluted to 20 μ g/mL, every hole adds 100 μ L,
37 DEG C of lucifuges hatch 2h or 4 DEG C overnight, and liquid in hole of inclining washs 2 times with cleaning mixture, each 30s, pats dry, then exist
Adding 150～200 μ L confining liquids in every hole, 37 DEG C of lucifuges hatch 1～2h, and in hole of inclining, liquid pats dry, dried by aluminum film vacuum
Seal and preserve.
The Cleaning Principle of the present invention is:
Pre-coated carbofuran coupled antigen on capillary strip, after adding sample solution or standard solution, adds carbofuran Dan Ke
Grand antibody-solutions, the carbofuran in sample and coated carbofuran coupled antigen competition carbofuran monoclonal antibody in ELISA Plate, add
Entering enzyme labelling anti antibody and be amplified effect, develop the color with nitrite ion, sample absorbance is negative correlation with the content of carbofuran, with
Standard curve more i.e. can get the residual quantity of carbofuran in sample；Simultaneously according to the depth of color in ELISA Plate, with series concentration
The comparison of standard solution color can the concentration range of Determination of carbofuran amount in judgment sample roughly.
Present invention also offers a kind of method applying above-mentioned enzyme linked immunological kit detection carbofuran, it includes step:
(1) Sample pretreatment；
(2) detect with test kit；
(3) testing result is analyzed.
The present invention detects the enzyme linked immunological kit of carbofuran and mainly uses indirect competitive ELISA method qualitatively or quantitatively to detect sample
The content of middle carbofuran；Pre-treatment to sample requires low, and Sample pretreatment process is simple, can the most quickly detection high-volume sample
This；Main agents provides with the form of working solution, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, degree of accuracy
High, accuracy high.The enzyme linked immunological kit of the present invention, simple in construction, easy to use, low price, carries just
Profit, detection method are efficient, accurate, easy, be suitable to the qualitative and quantitative analysis of high-volume screening sample.
Accompanying drawing explanation
Fig. 1: carbofuran hapten synthesis route map
Fig. 2: carbofuran hapten hydrogen nuclear magnetic resonance spectrogram
Fig. 3: kit standard curve chart
Detailed description of the invention
The present invention is expanded on further below in conjunction with specific embodiment.Should be understood that these embodiments are merely to illustrate the present invention,
And it is not limited to the scope of the present invention.
The preparation of embodiment 1 reagent constituents
1, the haptenic synthesis of carbofuran (synthetic route is shown in accompanying drawing 1) and qualification
A, take benzofuranol 1.0g, add 30mL dichloromethane and dissolve, add phosgene, 0～5 DEG C of reaction 4h, add 90mL stone
Oil ether, stands, and bottom has grease to separate out, proceeds to separatory funnel, divides and goes upper organic phase, grease to add oxolane dissolving,
It is subsequently adding the tetrahydrofuran solution that 2mL contains methylamine, 0～5 DEG C of reaction 2h.Stopped reaction, rotation is steamed, and removes oxolane,
Obtaining red oil, upper silicagel column, petrol ether/ethyl acetate (20/1, v/v) eluting separates, obtains product I；
B, take zinc powder 1.2g, add distilled water and 0.5mL glacial acetic acid, 80 DEG C of heat-activated 30min, add the second containing product I
Alcohol 30mL, 60 DEG C of reaction 4h, TCL monitorings, react completely, stopped reaction, filter, remove zinc powder.Ethanol water
Being evaporated, the ethyl acetate that adds water extract and separate, separate organic facies washing and drying, petrol ether/ethyl acetate (30/1, v/v) is heavily tied
Crystalline substance, i.e. obtains carbofuran hapten product.
Taking above-mentioned hapten to identify through proton nmr spectra, result is shown in accompanying drawing 2.1H-NMR(CDCl3, 300MHz) and δ: 8.0
(s, 1H ,-NH-), 6.27 (s, 2H ,-NH2), 6.19 (s, 1H, ArH), 6.31 (s, 1H, ArH), 2.89 (s, 2H ,-CH2-),
2.58(s,3H,-CH3), 1.46 (s, 6H ,-CH3).The existence of the characteristic absorption peak of aromatic amine on the phenyl ring of δ=6.27 in collection of illustrative plates,
Feature in conjunction with other resonance absorbing peaks, it was demonstrated that carbofuran hapten structure is correct.
2, the synthesis of carbofuran coupled antigen and qualification
Immunizing antigen is prepared carbofuran hapten and is obtained immunogen with bovine serum albumin (BSA) coupling.
Weigh BSA 50mg, be allowed to be substantially dissolved in 3.8mL 0.1mol/L PBS (pH7.2)；Take 30mg carbonization two sub-
Amine (EDC) and N-hydroxy-succinamide (NHS) add in protein solution after fully dissolving with 0.2mL water, stir under room temperature
Mix 30min；Take 12mg hapten, be dissolved in 1mL DMF (DMF), be then slowly added to egg
In white solution, reaction 24h is stirred at room temperature；With 0.01mol/L PBS 4 DEG C dialysis 3d, change 3 dialysis solution every day；Subpackage, in
-20 DEG C save backup.
Envelope antigen is prepared carbofuran hapten and is obtained immunogen with ovalbumin (OVA) coupling.
Weigh OVA 50mg, be allowed to be substantially dissolved in 3.8mL 0.1mol/L PBS (pH7.2)；Take 30mg EDC and NHS
Add after fully dissolving with 0.2mL water in protein solution, under room temperature, stir 30min；Take 10mg hapten, be dissolved in 1mL DMF
In, it is then slowly added in protein solution, reaction 24h is stirred at room temperature；With 0.01mol/L PBS 4 DEG C dialysis 3d, change every day
3 dialysis solution；Subpackage, saves backup in-20 DEG C.
In synthesis carbofuran coupled antigen reaction hapten used, carrier protein and the ratio of coupled product, carry out ultraviolet
(200nm～400nm) sweep measuring, calculates its combination ratio at the light absorption value of 260nm and 280nm respectively by comparing three.
The maximum absorption band of conjugate carbofuran hapten-carrier albumen and carbofuran hapten, carrier protein maximum absorption band compared with send out
Give birth to obvious change, shown that the synthesis of carbofuran hapten-carrier albumen is successful.Being computed, hapten is with BSA's
In conjunction with than being 15:1, the ratio of the combination with OVA is for 11:1.
3, the preparation of carbofuran monoclonal antibody
(1) acquisition of hybridoma
1) first immunisation: by the most newborn with the Freund's complete adjuvant of equivalent for carbofuran hapten-BSA conjugate (immunizing antigen)
Change, the Balb/c mice of subcutaneous injection 6 week old, every 0.2mL；
2) booster immunization twice: from the beginning of first immunisation, booster immunization is once every two weeks, replaces Freund with not formula Freund's incomplete adjuvant
The same first immunisation of Freund's complete adjuvant, method and dosage；
3) last booster immunization after one week eyeground vein blood sampling survey titer and suppression, have suppression and titer reach 1:10000 with
Carried out following final immunization time upper: lumbar injection is not added with the immunogen solution 0.1mL of any adjuvant, put to death mice after three days, take
Its spleen and myeloma cell fusion；
4) use indirect competitive enzyme-linked immunosorbent to analyze method and measure cell supernatant, the positive hole of screening.Utilize limiting dilution assay to sun
Property hole carries out cloning, obtains and sets up the hybridoma cell strain of stably excreting carbofuran monoclonal antibody, take and be in logarithmic growth
The hybridoma frozen stock solution of phase makes cell suspension, is sub-packed in cryopreservation tube, preserves for a long time in liquid nitrogen.
(2) preparation of monoclonal antibody
1) cell recovery: take out carbofuran monoclonal antibody hybridoma cell strain cryopreservation tube, be immediately placed in 37 DEG C of water-bath middling speeds and melt,
After centrifugal segregation frozen stock solution, move into and cultivate culture in glassware；
2) ascites and antibody purification are prepared: use and internal induce method, by Balb/c mice (8 week old) Intraperitoneal injection sterilizing paraffin
Only, within 7 days, pneumoretroperitoneum injects hybridoma 5 × 10 to oil 0.5mL/5Individual/only, gather ascites after 7 days.With octanoic acid-saturated ammonium sulfate
Method is purified, and obtains carbofuran monoclonal antibody solution (-20 DEG C of preservations).
(3) mensuration of antibody titer
The titer measuring antibody with indirect competitive ELISA method is 1:(100000～150000).
Indirect competitive ELISA method: with carbofuran hapten-OVA conjugate coated elisa plate, add carbofuran standard substance molten
The sheep anti mouse anti antibody solution of liquid, carbofuran monoclonal antibody solution and horseradish peroxidase-labeled, 25 DEG C of reaction 30min,
Pour out liquid in hole, wash 3～5 times with cleaning mixture, pat dry with absorbent paper；Add substrate nitrite ion, after 25 DEG C of reaction 15min,
Add stop buffer and terminate reaction；Set microplate reader at wavelength 450nm, measure every hole absorbance.
(4) mensuration of monoclonal antibody specificity
Antibody specificity refers to ability and the comparison with such antigen-analogues ability of its homospecificity antigen combination, conventional
Cross reacting rate is as evaluation criterion.Cross reaction is the least, and the specificity of antibody is the highest.
This experiment by carbofuran, the metabolite (benzofuranol) of carbofuran, carbamate chemicals for agriculture (carbosulfan, carbaryl,
Mobucin, meta-tolyl-N-methylcarbamate (MTMC), Methomyl, Bassa) do serial dilution with organophosphorus pesticide (Bayer 71628, Azodrin, dichlorvos),
Carry out indirect competitive ELISA respectively with monoclonal antibody, make standard curve, analyze and obtain IC50, then it is calculated as follows friendship
Fork response rate:
Result shows that the cross reacting rate of each analog is: carbofuran 100%, benzofuranol ＜ 1%, carbosulfan ＜ 1%, first
Naphthalene prestige ＜ 1%, Mobucin ＜ 1%, meta-tolyl-N-methylcarbamate (MTMC) ＜ 1%, Methomyl ＜ 1%, Bassa ＜ 1%, Bayer 71628 ＜ 1%, Azodrin
＜ 1%, dichlorvos ＜ 1%.Antibody of the present invention to benzofuranol, carbosulfan, carbaryl, Mobucin, meta-tolyl-N-methylcarbamate (MTMC), go out many
Other pesticide no cross reactions such as prestige, Bassa, Bayer 71628, Azodrin, dichlorvos, have specific binding just for carbofuran.
4, the preparation of sheep anti mouse anti antibody
With sheep as immune animal, with Mus source antibody for immunogen immune pathogen-free domestic sheep, obtain sheep anti mouse anti antibody.
5, the preparation of enzyme labelling anti antibody
Over-voltage protection after sheep anti mouse anti antibody is used improvement with horseradish peroxidase (HRP) carries out coupling.Traditional
Over-voltage protection requires that in reaction system, the molar concentration rate of enzyme and antibody is 4:1, owing to horseradish peroxidase is at strong oxidation
The lower many sites with antibodies, so the horseradish peroxidase molecule of activation of producing act as connecting the bridge of each molecule,
Reduce the enzymatic activity of enzyme marker, make the conjugate of preparation is mixed with many polymers.In order to solve this problem, we will
Traditional method is improved, it may be assumed that
(1) closed process of amino is eliminated, because the amino that can produce the connection of self amino is actual seldom；
(2) reduction horseradish peroxidase and the molar concentration ratio of antibody are to 2:1, and the method after improvement is than traditional method letter
Just, the loss to enzymatic activity reduces.
6, the preparation of ELISA Plate
With being coated buffer, envelope antigen (carbofuran hapten-OVA conjugate) being diluted to 20 μ g/mL, every hole adds 100 μ L,
37 DEG C of lucifuges hatch 2h, liquid in hole of inclining, and wash 2 times with cleaning mixture, each 30s, pat dry, and then add in every hole
200 μ L confining liquids, 37 DEG C of lucifuges hatch 2h, and in hole of inclining, liquid pats dry, and dried sealing by aluminum film vacuum preserves.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of carbofuran
Set up the enzyme linked immunological kit of detection carbofuran so that it is comprise following component:
(1) ELISA Plate of carbofuran coupled antigen it is coated；
(2) carbofuran standard solution 6 bottles, concentration be respectively 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L,
40.5μg/L；
(3) carbofuran monoclonal antibody working solution；
(4) with the sheep anti mouse anti antibody of horseradish peroxidase-labeled；
(5) substrate nitrite ion is made up of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine；
(6) stop buffer is 2mol/L sulphuric acid；
(7) cleaning mixture be pH value be 7.4, containing 0.5%～1.0% tween 20,0.01 ‰～0.03 ‰ sodium azide preservatives,
The phosphate buffer of 0.1～0.3mol/L, described percentage ratio is quality percent by volume；
(8) redissolve liquid be pH value be 7.0, the phosphate buffer of 0.1mol/L.
The detection of carbofuran in embodiment 3 vegetable and Folium Camelliae sinensis sample
1, Sample pretreatment
Vegetable sample:
Weigh the vegetable sample after 1.0g ± 0.05g homogenizing in 10mL polystyrene centrifuge tube, add 5mL sample extraction agent and (claim
Take 4.4g disodium hydrogen phosphate dodecahydrate and 13.68g bis-hypophosphite monohydrate sodium dihydrogen add the mixing of 500mL deionized water dissolving), use
Agitator vibration 5min, mixing, 3000g room temperature (20～25 DEG C) is centrifuged 5min；Pipette 200 μ L of supernatant liquid to 2mL polyphenyl
In ethylene centrifuge tube, add 600 μ L redissolution liquid, with vortex instrument whirling motion 2min, fully mix；Take 50 μ L for analyzing.
Folium Camelliae sinensis sample:
Weigh 1g Folium Camelliae sinensis sample in 50mL polystyrene centrifuge tube, add 10mL acetonitrile, with vortex instrument whirling motion 1min, mixed
Even, 3000g room temperature (20～25 DEG C) is centrifuged 5min；Pipette 1mL upper organic phase in 10mL clean dried glass tubing,
Flow down in 20～30 DEG C of water-bath nitrogen and dry up；Add 1mL redissolution liquid, with vortex instrument whirling motion 2min, fully mix；Take 50 μ L
For analyzing.
2, detect with test kit
Carbofuran standard solution or the sample solution through pre-treatment is added in the ELISA Plate micropore be coated with carbofuran coupled antigen
50 μ L/ holes, are subsequently adding carbofuran monoclonal antibody working solution 50 μ L/ hole, mixing of vibrating gently, rearmounted 25 DEG C with cover plate membrane cover plate
Lucifuge reaction 30min；Pouring out liquid in hole, every hole adds 250 μ L cleaning mixture and fully washs 4～5 times, every minor tick 10s, uses
Absorbent paper pats dry；Add the sheep anti mouse anti antibody 100 μ L/ hole of horseradish peroxidase-labeled, mixing of vibrating gently, use cover plate
Membrane cover plate rearmounted 25 DEG C of lucifuges reaction 30min, takes out and repeats to wash plate step；Every hole adds substrate nitrite ion A liquid urea peroxide
50 μ L, substrate nitrite ion B liquid tetramethyl benzidine (TMB) 50 μ L, mixing of vibrating gently, rearmounted 25 DEG C with cover plate membrane cover plate
Lucifuge colour developing 15min, every hole adds stop buffer 2mol/L sulphuric acid 50 μ L, mixing of vibrating gently, is set in microplate reader wavelength
At 450nm, measure every hole absorbance (OD value).
3, Analysis of test results
With the absorbance values (B) of the standard solution of each concentration obtained divided by first standard solution (0 standard)
Absorbance (B0) it is multiplied by 100% again, obtain percentage absorbance.Logarithm value with carbofuran standard concentration (μ g/L)
For X-axis, percentage absorbance is Y-axis, draws standard curve, as shown in Figure 3.Sample solution is calculated by same way
Percentage absorbance, the carbofuran content of each sample corresponding then can read from standard curve.
The determination test of embodiment 4 carbofuran enzyme linked immunological kit technical parameter
1, test kit sensitivity and detection limit
Conventionally measure test kit sensitivity, kit standard curve minimum point is 0.5 μ g/L, standard curve in the range of
0.5～40.5 μ g/L, IC50(50% inhibition concentration) domain of walker is 1.5～3.0 μ g/L；To blank Rhizoma Solani tuber osi, Fructus Cucumidis sativi, Plantula Brassicae chinensis,
Each 20 parts of Folium Camelliae sinensis sample detects, and finds the concentration corresponding to each percentage absorbance from standard curve, with 20 parts of samples
The meansigma methods of concentration represents detection limit plus 3 times of standard deviations, and result obtains the method and the detection of vegetable sample is limited to 10 μ g/kg, right
The detection of Folium Camelliae sinensis sample is limited to 5 μ g/kg.
2, sample preci-sion and accuracy test
Using the response rate as accuracy estimating index, with the testing result relative standard deviation of a certain concentration samples of replication
(RSD%) as precision evaluation index.Computing formula is: the response rate (%)=practical measurement value/theoretical value × 100%,
The wherein interpolation concentration of theoretical value sample；Relative standard deviation RSD%=SD/X × 100%, wherein SD is standard deviation,
X is the meansigma methods of determination data.
By 10 μ g/kg, 20 μ g/kg, tri-concentration carbofurans of 40 μ g/kg, blank Rhizoma Solani tuber osi, Fructus Cucumidis sativi, Plantula Brassicae chinensis sample are added back
Receiving and measure, blank Folium Camelliae sinensis sample is added reclaiming and measures by 5 μ g/kg, 10 μ g/kg, tri-concentration carbofurans of 20 μ g/kg, each
Sample do 4 parallel, be measured with three batches of different test kits, calculate the average recovery rate of sample and precision result see table.
Table 1 precision and accuracy test
With the carbofuran of 10,20,40 tri-concentration of μ g/kg, blank Rhizoma Solani tuber osi, Fructus Cucumidis sativi, Plantula Brassicae chinensis sample are added, 5,
10, blank Folium Camelliae sinensis sample is added by the carbofuran of 20 tri-concentration of μ g/kg, and average recovery rate is between 70%～110%；Batch
In, batch between relative standard deviation be respectively less than 15%.
3, stabilization of kit test
Test kit preservation condition is 2～8 DEG C, through the mensuration of 12 months, and the maximum absorbance value (zero standard) of test kit, 50%
Inhibition concentration, carbofuran add practical measurement value all within normal range.Consider, during transport and using, to have anon-normal
Often preservation condition occurs, is placed 7 days by test kit, be accelerated senile experiment under 37 DEG C of preservation conditions, and result shows this examination
Agent box indices complies fully with requirement.Occur in view of test kit freezing situation, test kit put into-20 DEG C of refrigerator freezings 7 days,
Measurement result also indicates that test kit indices is the most normal.Can show that test kit at least can preserve at 2～8 DEG C from result above
More than 12 months.
Claims (7)
1. the enzyme linked immunological kit detecting carbofuran, it is characterised in that including: be coated with the enzyme mark of carbofuran coupled antigen
Plate, carbofuran monoclonal antibody, enzyme labelling anti antibody, carbofuran standard solution, substrate nitrite ion, stop buffer, cleaning mixture,
Redissolution liquid；Described carbofuran coupled antigen is to be obtained with carrier protein couplet by carbofuran hapten, and described carrier protein is Mus blood
Albumin, thyroprotein, bovine serum albumin, rabbit serum proteins, human albumin, ovalbumin, hemocyanin or fibre
Fibrillarin is former, and described carbofuran hapten is by nitrofuran phenol under the effect of phosgene, with methylamine by acylation reaction,
To band nitro carbofuran product, then obtaining through nitro-reduction reaction, molecular structural formula is:
2. test kit as claimed in claim 1, it is characterised in that described carbofuran monoclonal antibody is with carbofuran coupled antigen
Prepare as immunogen.
3. test kit as claimed in claim 1, it is characterised in that described anti antibody is sheep anti mouse anti antibody.
4. test kit as claimed in claim 1, it is characterised in that the marker enzyme of described enzyme labelling anti antibody is horseradish peroxidase
Enzyme, described substrate nitrite ion is made up of substrate solution A liquid and substrate solution B liquid, and substrate nitrite ion A liquid is hydrogen peroxide or peroxidating
Urea, substrate nitrite ion B liquid is o-phenylenediamine or tetramethyl benzidine, and described stop buffer is the sulfuric acid solution of 1～2mol/L.
5. test kit as claimed in claim 1, it is characterised in that described cleaning mixture be pH value be 7.4, containing quality volume hundred
Proportion by subtraction is 0.5%～1.0% tween 20,0.01 ‰～0.03 ‰ Hydrazoic acid,sodium salt, 0.1～the phosphate buffer of 0.3mol/L；Described multiple
Solution be pH value be 7.0, the phosphate buffer of 0.1mol/L.
6. test kit as claimed in claim 1, it is characterised in that the concentration of described carbofuran standard solution be respectively 0 μ g/L,
0.5μg/L、1.5μg/L、4.5μg/L、13.5μg/L、40.5μg/L。
7. detect a method for carbofuran content in sample, including step:
(1) sample pre-treatments；
(2) detect with the test kit described in any one of claim 1～6；
(3) testing result is analyzed.
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| CN113049812B (en) * | 2021-03-26 | 2024-03-19 | 信达安检测技术(天津)有限公司 | ELISA method for detecting carbofuran and kit thereof |
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| US5283066A (en) * | 1992-02-19 | 1994-02-01 | Development Center For Biotechnology | Method of stimulating an immune response by using a hapten |
| CN1384103A (en) * | 2001-04-30 | 2002-12-11 | 浙江大学 | Carbofuran half-antigen, antigen and antibody and their prepn process |
| CN1385703A (en) * | 2002-06-07 | 2002-12-18 | 浙江大学 | Enzyme-linked immunosorbant assay kit applicable for carbofuran residue analysis |
| CN1624479A (en) * | 2004-10-20 | 2005-06-08 | 扬州大学 | Solid phase antibody direct competition carbofuran enzyme joint immune abzorption analysis technology and its kit |
| CN1987469A (en) * | 2006-09-30 | 2007-06-27 | 华南农业大学 | Enzyme-linked immune analytic method for detecting carbofuran |
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