CN106324240A - Enzyme-linked immunoassay kit for detecting chlorpyrifos and application of kit - Google Patents
Enzyme-linked immunoassay kit for detecting chlorpyrifos and application of kit Download PDFInfo
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- CN106324240A CN106324240A CN201610631416.2A CN201610631416A CN106324240A CN 106324240 A CN106324240 A CN 106324240A CN 201610631416 A CN201610631416 A CN 201610631416A CN 106324240 A CN106324240 A CN 106324240A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/10—Insecticides
Abstract
The invention provides an enzyme-linked immunoassay kit for detecting chlorpyrifos. The kit comprises an enzyme label board coated with chlorpyrifos coupling antigen, a chlorpyrifos monoclonal antibody, an enzyme labeling antiantibody, a chlorpyrifos standard product solution, a substrate developing solution, TMAH, a cleaning solution and a combination solution. The invention further discloses a method for detecting chlorpyrifos by means of the enzyme-linked immunoassay kit. The method includes the steps that firstly, sample pretreatment is conducted; secondly, detection is conducted by means of the kit; finally, a detection result is analyzed. The enzyme-linked immunoassay kit can be used for detecting the content of chlorpyrifos in vegetables, fruits and tea leaves, is easy to operate, low in cost, high in sensitivity, capable of achieving in-situ monitoring and suitable for screening a lot of samples.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, be specifically related to a kind of ELISA reagent for detecting chlopyrifos
Box, it is particularly suitable for veterinary antibiotics and the detection of Folium Camelliae sinensis Chlorpyrifos content.
Background technology
Chlopyrifos (Chlorpyrifos), chemical name O, O-diethyl-O-(3,5,6-trichloro-2-pyridyl) sulfur is for phosphorus
Acid esters, trade name chlopyrifos, Le Siben, Coptotermes formosanus Shtrari. are clear, be the highly effective pesticide such as Bayer 71628 and parathion-methyl new and effective,
Low toxicity substitute species, belongs to broad spectrum type organophosphorus ester insecticides, can be used for the evil of grain, veterinary antibiotics and industrial crops
Worm prevents and treats.Chlopyrifos is one of important goal thing of agricultural product and food Pesticide Residue Monitoring, not only residual in crops
Benefit of studying in Japan is serious, and can be shifted in environment by approach such as metabolism, and human health is constituted potential threat, and it can affect
Brain development, to thyroid system generation effect, causes blood Triiodothyronine to reduce, and excess ingestion may cause spasm
And dizziness, especially bigger to children's health harm.
In view of this, developed country has carried out strict regulation to the use of chlopyrifos, and improves constantly chlopyrifos in agriculture
Residue limits in product.European Union is 0.05mg/kg to the highest limitation of Herba Spinaciae Chlorpyrifos；Japan's country's mark to chlopyrifos
Accurate most stringent, in the agriculture residual limit standard table that its in April, 2002 announces, the chlopyrifos limitation of its Herba Spinaciae is 0.01mg/kg；State
Border Codex Committee on Food (CAC) is 0.05mg/kg to Herba Spinaciae Chlorpyrifos Residue limitation.MRL country of China marks
Standard is: grain 0.1mg/kg；Melonidum, leaf vegetables 1mg/kg；Oleum Gossypii semen 0.05mg/kg.
Therefore, in order to prevent and tackle environment and the food pollution problem that chlopyrifos residue causes, it is necessary to set up one
Chlopyrifos detection method sensitive, easy, quick.The residue detection of chlopyrifos mainly has high performance liquid chromatography at present
(HPLC), gas chromatographymass spectrum (GC-MS).These analysis methods are used to need expensive instrument and special technical staff, sample
Pretreatment process length complex and expensive, time-consuming, it is difficult to meet a large amount of sample and needs that field sample quickly detects.Enzyme connection is exempted from
Epidemic disease adsorption analysis method (ELISA) has easy sensitive, sample capacity is big, analysis cost is low feature quick, special, can simplify
Even save sample purification step, in great amount of samples and field samples rapid screening detect, demonstrate unique advantage, it is possible to more
Meet well 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 for poisoning with poison
The enzyme linked immunological kit of Ticks detection, and provide a kind of efficiently, accurately, easy, be suitable to the qualitative, quantitative of high-volume screening sample
Detection method.
Test kit of the present invention, it includes: be coated with the ELISA Plate of chlopyrifos coupled antigen, Dursban monoclonal antibody, enzyme
Labelling anti antibody, chlopyrifos standard solution, substrate nitrite ion, stop buffer, cleaning mixture, redissolution liquid；Described chlopyrifos coupling resists
Former is to be obtained with carrier protein couplet by chlopyrifos hapten, and described carrier protein is Mus serum albumin, thyroprotein, Sanguis Bovis seu Bubali
Pure albumen, rabbit serum proteins, human albumin, ovalbumin, hemocyanin or Fibrinogen, described chlopyrifos hapten
It is that to be carried out nucleophilic displacement of fluorine with the chlorine of neighbouring nitrogen-atoms with aminobutyric acid on pyridine ring in the basic conditions by the former medicine of chlopyrifos anti-
Should obtain, molecular structural formula is:
Described Dursban monoclonal antibody is to prepare using chlopyrifos 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；The anti antibody of enzyme labelling is to use glutaraldehyde method
Or marker enzyme and anti antibody are carried out what coupling obtained by Over-voltage protection.
For more convenient on-site supervision and great amount of samples examination, described test kit also includes chlopyrifos standard solution, the end
Thing nitrite ion, stop buffer, cleaning mixture, redissolution liquid.
Described chlopyrifos standard solution 6 bottles, concentration is respectively 0 μ g/L, 0.6 μ g/L, 1.8 μ g/L, 5.4 μ g/L, 16.2 μ
G/L and 48.6 μ g/L.
Described substrate nitrite ion is made up of substrate solution A liquid and substrate solution B liquid, and A liquid is hydrogen peroxide or urea peroxide, B liquid
For o-phenylenediamine or tetramethyl benzidine, described stop buffer is the sulfuric acid solution of 1～2mol/L.
Described cleaning mixture preferably pH value is 7.4, containing 0.5%～1.0% tween 20,0.01 ‰～0.03 ‰ nitrine
Change 0.1～0.3mol/L phosphate buffer of sodium.
Described redissolution liquid preferably pH value is the 0.1mol/L phosphate buffer of 7.0.
Wherein in ELISA Plate preparation process used be coated buffer be pH value be the 0.05mol/L carbonate of 9.6
Buffer, confining liquid be pH value be 7.1～7.5, containing 1%～3% caseic 0.1～0.3mol/L phosphate buffer.
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, liquid in hole of inclining, and wash 2 times with cleaning mixture, each 30s, pat dry, then
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, and uses aluminum film after drying
Vacuum seals and preserves.
The Cleaning Principle of the present invention is:
Pre-coated chlopyrifos coupled antigen on capillary strip, after adding sample solution or standard solution, adds and poisons with poison
Ticks monoclonal antibody solution, the chlopyrifos in sample and coated chlopyrifos coupled antigen competition Dursban monoclonal in ELISA Plate
Antibody, adds enzyme labelling anti antibody and is amplified effect, develop the color with nitrite ion, and sample absorbance and the content of chlopyrifos are in negative
Relevant, the residual quantity that i.e. can get sample Chlorpyrifos is compared with standard curve；Simultaneously according to the depth of color in ELISA Plate, with
The comparison of the standard solution color of series concentration can the concentration range of judgment sample Chlorpyrifos Residue amount roughly.
Present invention also offers a kind of method applying above-mentioned enzyme linked immunological kit detection chlopyrifos, 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 chlopyrifos and mainly uses indirect competitive ELISA method qualitatively or quantitatively to examine
The content of this Chlorpyrifos of test sample；Pre-treatment to sample requires low, and Sample pretreatment process is simple, can the most quickly detect big
Sample in batches；Main agents provides with the form of working solution, and the method for inspection is convenient and easy, has specificity high, highly sensitive, smart
Exactness is high, accuracy high.The enzyme linked immunological kit of the present invention, simple in construction, easy to use, low price, carries
Facility, detection method are efficient, accurate, easy, be suitable to the qualitative and quantitative analysis of high-volume screening sample.
Accompanying drawing explanation
Fig. 1: chlopyrifos hapten synthesis route map
Fig. 2: chlopyrifos 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 this
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 chlopyrifos (synthetic route is shown in accompanying drawing 1) and qualification
The former medicinal 30mL ethanol of 1.0g chlopyrifos dissolves, and hydro-oxidation sodium 0.41g adds aminobutyric acid 0.35g, back flow reaction
3h, detection, raw material total overall reaction completes.Be evaporated ethanol, add water-ethyl acetate extraction, aqueous phase, regulation pH value to 3, ethyl acetate
Extraction, organic facies is evaporated, and with dichloromethane and normal hexane recrystallization, i.e. obtains chlopyrifos hapten product.
Taking above-mentioned hapten to identify through proton nmr spectra, result is shown in accompanying drawing 2.In collection of illustrative plates, chemical shift is at 11.0ppm
Carboxyl signal peak, the methylene signals peak at 2.30ppm, 1.89ppm and 3.35ppm, it was demonstrated that chlopyrifos hapten structure is just
Really.
2, the synthesis of chlopyrifos coupled antigen and qualification
Immunogen is prepared chlopyrifos hapten and is obtained immunogen with bovine serum albumin (BSA) coupling.
Take 9mg hapten, be dissolved in 1mL DMF (DMF)；Take 30mg carbodiimides (EDC) and
N-hydroxy-succinamide (NHS) adds in hapten solution after fully dissolving with 0.2mL water, stirs 24h under room temperature,
To reactant liquor A；Weigh BSA 50mg, be allowed to be substantially dissolved in 3.8mL CB (pH9.0), reactant liquor A is dropwise slowly added to
In protein solution, and stir 24h at room temperature；With 0.01mol/L PBS 4 DEG C dialysis 3d, change 3 dialysis solution every day；Point
Dress, saves backup in-20 DEG C.
Coating antigen is prepared chlopyrifos hapten and is obtained immunogen with ovalbumin (OVA) coupling.
Take 9mg hapten, be dissolved in 1mL DMF；Take 30mg EDC and NHS 0.2mL water fully dissolve after add half
In antigenic solution, stir 24h under room temperature, i.e. can get reactant liquor A；Weigh OVA 50mg, be allowed to be substantially dissolved in 3.8mL CB
(pH9.0) in, reactant liquor A is dropwise slowly added in protein solution, and stirs 24h at room temperature；Use 0.01mol/L PBS
4 DEG C of dialysis 3d, change 3 dialysis solution every day；Subpackage, saves backup in-20 DEG C.
In synthesis chlopyrifos 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.Even
Maximum absorption band and the chlopyrifos hapten of connection thing chlopyrifos hapten-carrier albumen, carrier protein maximum absorption band compared with
There occurs significantly change, show that the synthesis of chlopyrifos hapten-carrier albumen is successful.
3, the preparation of Dursban monoclonal antibody
(1) acquisition of hybridoma
1) first immunisation: by the most newborn with the Freund's complete adjuvant of equivalent for chlopyrifos hapten-BSA conjugate (immunogen)
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 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 to reach 1:
Carry out following final immunization when more than 10000: lumbar injection is not added with the immunogen solution 0.1mL of any adjuvant, put to death after three days
Mice, takes 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
Method carries out cloning to positive hole, obtains and sets up the hybridoma cell strain of stably excreting Dursban monoclonal antibody, take and be in
The hybridoma frozen stock solution of exponential 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 Dursban monoclonal antibody hybridoma cell strain cryopreservation tube, be immediately placed in 37 DEG C of water-baths
Speed is melted, and after centrifugal segregation frozen stock solution, moves into and cultivates 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 stone
Only, within 7 days, pneumoretroperitoneum injects hybridoma 5 × 10 to wax oil 0.5mL/5Individual/only, gather ascites after 7 days.With octanoic acid-saturated sulphuric acid
Ammonium method is purified, and obtains Dursban 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 chlopyrifos hapten-OVA conjugate coated elisa plate, add chlopyrifos standard substance
The sheep anti mouse anti antibody solution of solution, Dursban monoclonal antibody solution and horseradish peroxidase-labeled, 25 DEG C of reactions
30min, pours out liquid in hole, washs 3～5 times with cleaning mixture, pats dry with absorbent paper；Add substrate nitrite ion, 25 DEG C of reactions
After 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 its ability of homospecificity antigen combination and the ratio with such antigen-analogues ability
Relatively, conventional cross reacting rate is as evaluation criterion.Cross reaction is the least, and the specificity of antibody is the highest.
This experiment is by chlopyrifos, chlorpyrifos-methyl, basudin, parathion-methyl, fenifrothion, Malathion, parathion
Do serial dilution, carry out indirect competitive ELISA respectively with monoclonal antibody, make standard curve, analyze and obtain IC50, then press
Following formula calculating cross reacting rate:
Result shows that the cross reacting rate of each analog is: chlopyrifos 100%, chlorpyrifos-methyl 7.08%, basudin ＜
1%, parathion-methyl ＜ 1%, fenifrothion ＜ 1%, Malathion ＜ 1%, parathion ＜ 1%.Antibody of the present invention is to methyl
Other pesticide no cross reactions such as chlopyrifos, basudin, parathion-methyl, fenifrothion, Malathion, parathion, just for
Chlopyrifos has specific binding.
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.Pass
The Over-voltage protection of system requires that in reaction system, the molar concentration rate of enzyme and antibody is 4:1, owing to horseradish peroxidase is by force
Produce many sites with antibodies, so the horseradish peroxidase molecule of activation under Oxidation to act as connecting each point
The bridge of son, reduces the enzymatic activity of enzyme marker, makes to be mixed with many polymers in the conjugate of preparation.Ask to solve this
Topic, traditional method is improved by we, 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 side
Method is easy, and the loss to enzymatic activity reduces.
6, the preparation of ELISA Plate
With being coated buffer, coating antigen (chlopyrifos 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 chlopyrifos
Set up the enzyme linked immunological kit of detection chlopyrifos so that it is comprise following component:
(1) ELISA Plate of chlopyrifos coupled antigen it is coated；
(2) chlopyrifos standard solution 6 bottles, concentration is respectively 0 μ g/L, 0.6 μ g/L, 1.8 μ g/L, 5.4 μ g/L, 16.2 μ
G/L and 48.6 μ g/L；
(3) Dursban 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 ‰ Hydrazoic acid,sodium salt
0.1～0.3mol/L phosphate buffer；
(8) redissolve liquid be pH value be the 0.1mol/L phosphate buffer of 7.0.
Embodiment 3 veterinary antibiotics and the detection of Folium Camelliae sinensis sample Chlorpyrifos
1, Sample pretreatment
Weigh 1.0g ± 0.05g sample in 10mL polystyrene centrifuge tube, add 5mL methanol, with vortex mixed instrument whirlpool
Dynamic 2min, mixing；3000g room temperature (20～25 DEG C) is centrifuged 5min, takes the 50 μ L supernatantes in 2mL polystyrene centrifuge tube,
Add 950 μ L redissolution liquid, whirling motion 20s；Take 50 μ L for analyzing.
2, detect with test kit
Chlopyrifos standard solution is added or through pre-treatment in the ELISA Plate micropore be coated with chlopyrifos coupled antigen
Sample solution 50 μ L/ hole, is subsequently adding Dursban monoclonal antibody working solution 50 μ L/ hole, mixing of vibrating gently, uses cover plate membrane cover
Plate rearmounted 25 DEG C of lucifuges reaction 30min；Pouring out liquid in hole, every hole adds 250 μ L cleaning mixture and fully washs 4～5 times, every time between
Every 10s, pat dry with absorbent paper；Adding the sheep anti mouse anti antibody 100 μ L/ hole of horseradish peroxidase-labeled, vibration is mixed gently
Even, with cover plate membrane cover plate rearmounted 25 DEG C of lucifuges reaction 30min, take out and repeat to wash plate step；Every hole adds substrate nitrite ion A liquid mistake
Oxidation urea 50 μ L, substrate nitrite ion B liquid tetramethyl benzidine (TMB) 50 μ L, mixing of vibrating gently, with cover plate membrane cover plate rearmounted 25
DEG C 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.Right with chlopyrifos standard concentration (μ g/L)
Numerical value is X-axis, and percentage absorbance is Y-axis, draws standard curve, as shown in Figure 3.Sample solution is calculated by same way
Percentage absorbance, the chlopyrifos content of each sample corresponding then can read from standard curve.
The determination test of embodiment 4 chlopyrifos enzyme linked immunological kit technical parameter
1, test kit sensitivity and detection limit
Conventionally measuring test kit sensitivity, kit standard curve minimum point is 0.6 μ g/L, standard curve
Scope is 0.6～48.6 μ g/L, IC50(50% inhibition concentration) domain of walker is 1.0～2.0 μ g/L；To blank Chinese cabbage, Fructus Mali pumilae,
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 limits the detection of veterinary antibiotics, Folium Camelliae sinensis sample
It is 100 μ g/kg.2, sample preci-sion and accuracy test
Using the response rate as accuracy estimating index, the testing result relative standard with a certain concentration samples of replication is inclined
Difference (RSD%) is 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
Meansigma methods for determination data.
By 100 μ g/kg, 200 μ g/kg, tri-concentration chlopyrifos of 400 μ g/kg, blank Chinese cabbage, Fructus Mali pumilae, Folium Camelliae sinensis sample are entered
Row adds to reclaim and measures, each sample do 4 parallel, be measured with three batches of different test kits, calculate averagely reclaiming of sample
Rate and precision result see table.
Table 1 precision and accuracy test
With the chlopyrifos of 100,200,400 tri-concentration of μ g/kg, blank Chinese cabbage, Fructus Mali pumilae, Folium Camelliae sinensis sample are added, flat
All response rate are 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, the maximum absorbance value (zero standard) of test kit,
50% inhibition concentration, chlopyrifos add practical measurement value all within normal range.Consider, during transport and using, to have
Improper preservation condition occurs, being placed 7 days under 37 DEG C of preservation conditions by test kit, be accelerated senile experiment, result shows
This test kit indices complies fully with requirement.Occur in view of test kit freezing situation, test kit is put into-20 DEG C of refrigerators cold
Freezing 7 days, measurement result also indicates that test kit indices is the most normal.Can show that test kit can be at 2～8 DEG C from result above
At least preserve more than 12 months.
Claims (7)
1. the enzyme linked immunological kit detecting chlopyrifos, it is characterised in that including: be coated with the enzyme of chlopyrifos coupled antigen
Target, Dursban monoclonal antibody, enzyme labelling anti antibody, chlopyrifos standard solution, substrate nitrite ion, stop buffer, cleaning mixture,
Redissolution liquid；Described chlopyrifos coupled antigen is to be obtained with carrier protein couplet by chlopyrifos hapten, and described carrier protein is Mus
Serum albumin, thyroprotein, bovine serum albumin, rabbit serum proteins, human albumin, ovalbumin, hemocyanin or fibre
Fibrillarin is former, described chlopyrifos hapten be by the former medicine of chlopyrifos in the basic conditions with aminobutyric acid on pyridine ring with neighbouring
The chlorine of nitrogen-atoms carries out nucleophilic substitution and obtains, and molecular structural formula is:
2. test kit as claimed in claim 1, it is characterised in that described Dursban monoclonal antibody is to resist with chlopyrifos coupling
Original work are that immunogen prepares.
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 Radix Cochleariae officinalis peroxidating
Thing 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 0.5%～
1.0% tween 20,0.01 ‰～0.03 ‰ 0.1～0.3mol/L phosphate buffers of Hydrazoic acid,sodium salt；Described redissolution liquid is pH
Value is the 0.1mol/L phosphate buffer of 7.0.
6. test kit as claimed in claim 1, it is characterised in that the concentration of described chlopyrifos standard solution is respectively 0 μ g/
L, 0.6 μ g/L, 1.8 μ g/L, 5.4 μ g/L, 16.2 μ g/L and 48.6 μ g/L.
7. the method detecting sample Chlorpyrifos content, 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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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107064494A (en) * | 2017-04-17 | 2017-08-18 | 四川精卫食品检测科技有限公司 | A kind of chlopyrifos detects enzyme linked immunological kit |
| CN109061168A (en) * | 2018-08-27 | 2018-12-21 | 北京勤邦生物技术有限公司 | Detect enzyme linked immunological kit and its application of diclazuril |
| CN111961002A (en) * | 2020-09-01 | 2020-11-20 | 北京望尔生物技术有限公司 | Pyraclostrobin hapten, artificial antigen and antibody as well as preparation method and application thereof |
| CN112114147A (en) * | 2020-09-01 | 2020-12-22 | 北京望尔生物技术有限公司 | Test strip and method for detecting pyraclostrobin |
| CN113156127A (en) * | 2021-04-01 | 2021-07-23 | 北京勤邦生物技术有限公司 | Test strip and method for detecting chlorpyrifos |
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| CN109061168B (en) * | 2018-08-27 | 2022-10-21 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting diclazuril and application thereof |
| CN111961002A (en) * | 2020-09-01 | 2020-11-20 | 北京望尔生物技术有限公司 | Pyraclostrobin hapten, artificial antigen and antibody as well as preparation method and application thereof |
| CN112114147A (en) * | 2020-09-01 | 2020-12-22 | 北京望尔生物技术有限公司 | Test strip and method for detecting pyraclostrobin |
| CN113156127A (en) * | 2021-04-01 | 2021-07-23 | 北京勤邦生物技术有限公司 | Test strip and method for detecting chlorpyrifos |
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