CN105572343A - ELISA kit and detection method for detecting chlopyrifos - Google Patents
ELISA kit and detection method for detecting chlopyrifos Download PDFInfo
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- CN105572343A CN105572343A CN201410537499.XA CN201410537499A CN105572343A CN 105572343 A CN105572343 A CN 105572343A CN 201410537499 A CN201410537499 A CN 201410537499A CN 105572343 A CN105572343 A CN 105572343A
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Abstract
The invention discloses an ELISA kit and detection method for detecting chlopyrifos. The ELISA kit and detection method are sensitive, accurate and rapid in detection, simple to operate, high in specificity and applicable to detection of large-scale samples. The kit comprises an enzyme-labeled plate coated with chlopyrifos antigen, a chlopyrifos standard substance, a chlopyrifos antibody working solution, a chlopyrifos enzyme-labeled secondary antibody working solution, a substrate solution A, a substrate solution B, a stopping solution, a concentrated dilution solution and a concentrated washing solution. The principle of the ELISA kit for detecting chlopyrifos is a solid-phase indirect competitive ELISA reaction. An extracted sample, the enzyme-labeled secondary antibody working solution and the antibody working solution are added into corresponding micropores in the enzyme-labeled plate; after incubation for a certain period of time, the substrate solution A and the substrate solution B are added during plate washing; and a color developing agent is blue under the action of an enzyme and turns yellow from blue when the stopping solution is added. The degree of color development is inversely proportional to the content of chlopyrifos in the standard substance or sample. The method can be directly used for detecting the residual quantity of chlopyrifos in rice.
Description
Technical field
The present invention relates to detection of veterinary drugs in food technical field, particularly detect the enzyme linked immunological kit of rice Chlorpyrifos.
Background technology
In the last few years, scientist constantly found that some chemical substances derived from environment can be simulated endocrine hormone function thus cause biosome endocrine system disorder, was called environmental hormone or Environmental Hormone.Environmental hormone has become the third-largest environmental problem in the whole world after ozonosphere, terrestrial climate warm, becomes the heat subject in Research of Environmental Sciences field.The definition of the environmental hormone that USEPA provides, refer in a class interfering bodies to the normal behaviour of biosome and with reproduction, the synthesis of growing relevant normal hormonal, storage, secretion, body in transport, combine and the exogenous compounds of the process such as removing.
Environmental hormone is the hormone analogs in environment, and it is by being combined with hormone receptor, and the normal physiological metabolism of interference biosome, endocrine and Reproductive Performance, cause all negative biological effects.Environmental hormone is entered in human body or animal body by surrounding medium and food chain, disturbs its internal system and reproductive function system, affects existence and the procreation of offspring.Therefore safety problem is paid much attention to.In GB, in regulation wheat, maximum residue limit is 0.5mg/kg.
Enzyme linked immunosorbent assay is a kind of accurate, reliable, quick, special detection method, is suitable for the rapid screening of gross sample, has been widely used in food safety detection industry in recent years.The present invention is intended to set up a kind of enzyme linked immunological kit and the detection method thereof that detect chlopyrifos.
Summary of the invention
In order to overcome chromatographic deficiency, the invention provides a kind of enzyme linked immunological kit and the detection method thereof that detect chlopyrifos.The method is sensitive, accurate, quick, easy and simple to handle, high specificity, is applicable to the quick detection of gross sample.
The present invention detects the enzyme linked immunological kit of chlopyrifos, comprises ELISA Plate, chlopyrifos standard items, chlopyrifos antibody working fluid, chlopyrifos ELIAS secondary antibody working fluid, substrate solution A, substrate solution B, stop buffer, concentration and dilution liquid and concentrated cleaning solution.
The present invention detects the preparation of the enzyme linked immunological kit of chlopyrifos, comprises the following steps: the preparation of the preparation of the preparation of the preparation of ELISA Plate, the preparation of chlopyrifos standard items, chlopyrifos antibody working fluid, the preparation of chlopyrifos ELIAS secondary antibody working fluid, substrate solution A, the preparation of substrate solution B, stop buffer, the preparation of concentration and dilution liquid and the preparation of concentrated cleaning solution.
It is further characterized in that: described ELISA Plate is via the antigen coated preparation of chlopyrifos, concrete steps are that chlopyrifos haptens and the pure albumen of carrier proteins Bovine (BSA) coupling are obtained envelope antigen, with carbonate (CBS) damping fluid of 0.05mol/LpH9.6 as coating buffer, chlopyrifos envelope antigen is diluted to 1:40000 ratio, 100 μ L/ holes, 37 DEG C of lucifuges hatch 2h, take out ELISA Plate and get rid of liquid in plate, add diluted concentrated cleaning solution 300 μ L/ hole, wash plate 2 times, 30s/ time, then 0.5% bovine serum albumin(BSA) (BSA) confining liquid is added, 150 μ L/ holes, place 1.5h for 37 DEG C, get rid of confining liquid directly to pat dry, ELISA Plate after patting dry is placed (25 DEG C) between constant temperature and is dried, inspect by random samples qualified after ELISA Plate is vacuum-sealed in 4 DEG C of conditions under preserve.
Chlopyrifos standard concentration is respectively 0mg/kg, 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg, 8.1mg/kg.
Described chlopyrifos antibody working fluid adopts chlorpyrifos artificial mice immunized with antigen to obtain monoclonal antibody, is diluted to the preparation of 1:60000 ratio with antibody diluent.
Described chlopyrifos ELIAS secondary antibody working fluid adds diluted by ELIAS secondary antibody and becomes 1:2000 ratio, described substrate solution A is the citrate-phosphate disodium hydrogen buffer solution of the carbamide peroxide containing 0.5mmol/L, described substrate solution B is the ethanolic solution of tetramethyl biphenyl diamines, described stop buffer is the sulfuric acid of 2mol/L, described concentration and dilution liquid is 10 times of concentration and dilution liquid, for the PBS of 0.1mol/L, between pH value range 7.0-7.5, described concentrated cleaning solution is 10 times of concentrated cleaning solutions, for containing 0.5% Tween-20, the PBST of 0.1mol/L, between pH value range 7.0-7.5.
Detect enzyme linked immunological kit and the detection method thereof of chlopyrifos, based on the indirect competitive enzyme-linked immunosorbent reaction principle of antigen-antibody, the method comprises the following steps:
(1) pre-service testing sample is fluid sample by sample preparation to be tested, or uses Solvent Extract methods testing sample, and is redissolved in sample diluting liquid;
(2) required reagent is taken out from cold storage environment, be placed in room temperature (20 ~ 25 DEG C) balance more than 30min, notice that often kind of liquid reagent must shake up before using;
(3) ELISA Plate being coated with chlopyrifos antigen is got, add standard items/sample 50 μ L/ hole in the micropore of correspondence, add chlopyrifos ELIAS secondary antibody working fluid, 50 μ L/ holes, then chlopyrifos antibody working fluid is added, 50 μ L/ holes, mixing of vibrating gently, reacts 30min with in the rearmounted room temperature of cover plate membrane cover plate 25 DEG C of light protected environment;
(4) carefully open cover plate film, liquid in hole is dried, with wash operating solution 260 μ L/ hole, fully washs 4 ~ 5 times, every minor tick 10s, pat dry (bubble be not eliminated after patting dry can be poked with original rifle head) with thieving paper;
(5) add substrate solution A50 μ L/ hole, substrate solution B50 μ L/ hole, mixing of vibrating gently, react 15 ~ 20min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate;
(6) add stop buffer 50 μ L/ hole, mixing of vibrating gently, setting microplate reader detects in 450nm place or dual wavelength 450/630nm, measures every hole absorbance (please running through data in 5min);
(7) with the logarithm of standard concentration (ppb) for horizontal ordinate, standard items percentage absorbance is ordinate, drawing standard curve, the content of reference standard curve calculation sample Chlorpyrifos.
Wherein, described testing sample following methods carries out pre-service:
A. representative sample (make the sample of 50% can by 20 object filter screens) is ground;
B. the sample weighed after 5g grinding filtration adds in 50mL centrifuge tube, adds 3g sodium chloride, 10mL water and 20mL normal hexane-acetone soln (volume ratio is 2:1);
C. mix in the container of sealing, concussion vortex 1min;
D. room temperature centrifugal more than 4000r/min, 10min; Get centrifugal after supernatant or filter after filtrate 1mL, 50 ~ 60 DEG C of water-bath nitrogen dry up;
E. add standard dilutions 1mL and fully mixing, the centrifugal 5min of 4000r/min or use quantitative test Filter paper filtering, get centrifugal after supernatant or filtrate after filtration analyze.
The present invention detects the enzyme linked immunological kit of chlopyrifos and the measuring principle of detection method thereof: antigen-specific sexual competition antibody fixing in the chlopyrifos in sample and ELISA Plate, add ELIAS secondary antibody, with antibody response, by enzymatic chromogenic reagent, carry out the content of judgement sample Chlorpyrifos according to the depth of colour developing.If the chlopyrifos content in sample is few, colour developing is dark; Otherwise, then develop the color shallow.Kit test method of the present invention is easy and simple to handle, detects sensitive, accurate, quick, is applicable to the quick detection of batch samples.
Embodiment
The preparation of chlopyrifos protein conjugate:
Succinic anhydride method is adopted to obtain being with the chlopyrifos hapten derivant of carboxyl, get 0.05mmol and the carrier protein BSA combination by 10:1 afterwards than being blended in the carbonate buffer solution (CBS) of 0.05mol/LpH9.6, then 0.15mmol carbodiimide is added, room temperature reaction 24h is put in stirring, finally dialyse two days in the PBS damping fluid of 0.2mol/LpH7.6, remove unreacted haptens, the protein conjugate solution obtained is saved backup in-20 DEG C.
The preparation of chlopyrifos antibody:
Select the purebred BALA/C mouse of healthy adult, get the immunizing antigen 50 μ g prepared with protein molecule to mix with equivalent complete Freund's adjuvant and adopt lumbar injection to carry out initial immunity, adding equivalent incomplete Freund's adjuvant every 3 weeks with same dose immunizing antigen afterwards adopts lumbar injection to carry out secondary, three immunity, after after each immune 6 days, tail vein blood measures antiserum titre to certain titre, do not add adjuvant with same dose and carry out final immunization, get spleen after 3 days and prepare Spleen cell suspensions and myeloma cell carries out Fusion of Cells, filter out required hybridoma cell line and carry out cloning, the hybridoma being in exponential phase is selected to carry out frozen, prepare for ascites, first lumbar injection 0.5ml whiteruss is in BALB/C mouse sensitization, pneumoretroperitoneum injection 1 × 10 in 2 weeks
6individual hybridoma, ascites can be produced after inoculating cell 7-10 days, ascites is extracted with syringe when ascites is many as far as possible, repeatedly collect for several times, the centrifugal 15min of 4000rpm, collect supernatant, adopt caprylic acid-ammonium purifying ascites to carry out purifying to monoclonal antibody, freeze drying saves backup in-20 DEG C after obtaining freeze-dried powder.
Preparation is coated with the ELISA Plate of chlopyrifos envelope antigen:
Chlopyrifos haptens and the pure albumen of carrier proteins Bovine (BSA) coupling obtain by envelope antigen, with carbonate (CBS) damping fluid of 0.05mol/LpH9.6 as coating buffer, chlopyrifos antigen diluent is become 1:40000 ratio, 100 μ L/ holes, place 2h for 37 DEG C, take out ELISA Plate and get rid of liquid in plate, with the concentrated cleaning solution 300 μ L/ hole after dilution, wash plate 2 times, 30s/ time, then add 0.5% bovine serum albumin(BSA) (BSA) to close, 150 μ L/ holes, place 1.5h for 37 DEG C, discard confining liquid, ELISA Plate after patting dry is placed (25 DEG C) between constant temperature and is dried, inspect by random samples qualified after by rearmounted for ELISA Plate vacuum seal 4 DEG C preserve.
Chlopyrifos standard items compound concentration is respectively 0mg/kg, 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg, 8.1mg/kg.
The preparation of chlopyrifos antibody working fluid: adopt chlorpyrifos artificial mice immunized with antigen to obtain monoclonal antibody, is diluted to the preparation of 1:60000 ratio with antibody diluent.
Chlopyrifos ELIAS secondary antibody working fluid adds diluted by ELIAS secondary antibody and becomes 1:2000 ratio, substrate solution A is the citrate-phosphate disodium hydrogen buffer solution of the carbamide peroxide containing 0.5mmol/L, substrate solution B is the ethanolic solution of tetramethyl biphenyl diamines, stop buffer is the sulfuric acid of 2mol/L, concentration and dilution liquid is 10 times of concentration and dilution liquid, for the PBS of 0.1mol/L, between pH value range 7.0-7.5, concentrated cleaning solution is 10 times of concentrated cleaning solutions, for containing 0.5% Tween-20, the PBST of 0.01mol/L, between pH value range 7.0-7.5.
Based on the reagent of above-mentioned preparation, the present invention comprises following material for the enzyme linked immunological kit detecting chlopyrifos:
(1) 96 ELISA Plate × 1 piece, hole;
(2) titer × 6 bottle: (1mL/ bottle) 0mg/kg, 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg, 8.1mg/kg;
(3) antibody working fluid 7mL;
(4) ELIAS secondary antibody working fluid 7mL;
(5) substrate solution A7mL;
(6) substrate solution B7mL;
(7) stop buffer 7mL;
(8) 10 × concentration and dilution liquid 40mL;
(9) 10 × concentrated cleaning solution 40mL;
When kit of the present invention is for detecting rice sample Chlorpyrifos Residue amount, implemented by following steps: sample pretreatment, with kit of the present invention carry out detecting, analysis result.
(1) sample pretreatment
A. 2.0 ± 0.02g homogenised sample is accurately taken in 50mL centrifuge tube;
B. first add 5mL ethyl acetate, vortex instrument fully vibrates and mixes 10min;
C.5000r/min centrifugal 10min;
D. get supernatant 1mL to flow down in 50 ~ 60 DEG C of nitrogen and dry up;
E. add 1mL normal hexane (sample that fat content is high can add 2mL normal hexane), abundant whirling motion 10s, then add 1mL sample diluting liquid, low speed whirling motion 10s; 4000r/min, centrifugal 5min, discard upper strata normal hexane and middle layer impurity completely;
F. layer 50 μ L is taken off for analyzing.
(2) testing sample Chlorpyrifos Residue amount is detected with kit of the present invention
Get the ELISA Plate being coated with chlopyrifos antigen, add standard items/sample 50 μ L/ hole in the micropore of correspondence; Add ELIAS secondary antibody working fluid, 50 μ L/ holes, then add chlopyrifos antibody working fluid, 50 μ L/ holes, mixing of vibrating gently, react 30min with in the rearmounted room temperature of cover plate membrane cover plate 25 DEG C of light protected environment; Carefully open cover plate film, dried by liquid in hole, with wash operating solution 300 μ L/ hole, fully washing 4 times, soaks 15-30s, pats dry with thieving paper; Add substrate solution A50 μ L/ hole, substrate solution B50 μ L/ hole, mixing of vibrating gently, react 15min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate; Add stop buffer 50 μ L/ hole, mixing of vibrating gently, setting microplate reader detects in 450nm place or dual wavelength 450/630nm, measures every hole absorbance (please running through data in 5min); The absorbance size of contrast testing sample and standard items, quantitative test testing sample Chlorpyrifos Residue amount.
(3) analysis result
The calculating of percentage absorbance, the percentage absorbance of standard items or sample equals the absorbance of mean value (diplopore) divided by first standard (0 standard) of the absorbance of standard items or sample, then is multiplied by 100%, namely
Percentage absorbance (%)=B/B
0× 100%
The wherein mean absorbance values of B-standard solution or sample solution, B
0the mean absorbance values of-0ppb standard solution.
With the logarithm of the standard concentration of chlopyrifos (ppb) for horizontal ordinate, standard items percentage absorbance is ordinate, and drawing standard curve, obtains straight-line equation.Substituted in typical curve by the percentage absorbance of sample, read the concentration corresponding to sample from typical curve, the extension rate being multiplied by its correspondence is the actual concentrations of sample Chlorpyrifos.
Claims (8)
1. detect enzyme linked immunological kit and the detection method thereof of chlopyrifos, comprise ELISA Plate, chlopyrifos standard items, chlopyrifos antibody working fluid, chlopyrifos ELIAS secondary antibody working fluid, substrate solution A, substrate solution B, stop buffer, concentration and dilution liquid and concentrated cleaning solution.
2. detect the enzyme linked immunological kit of chlopyrifos and detection method thereof, comprise the following steps: the preparation of the preparation of the preparation of the preparation of ELISA Plate, the preparation of chlopyrifos standard items, chlopyrifos antibody working fluid, the preparation of chlopyrifos ELIAS secondary antibody working fluid, substrate solution A, the preparation of substrate solution B, stop buffer, the preparation of concentration and dilution liquid and the preparation of concentrated cleaning solution.
3. enzyme linked immunological kit and the detection method thereof detecting chlopyrifos according to claim 2, it is characterized in that: described ELISA Plate preparation method is for obtain chlopyrifos envelope antigen by chlopyrifos haptens and the pure albumen of carrier proteins Bovine (BSA) coupling, with carbonate (CBS) damping fluid of 0.05mol/LpH9.6 as coating buffer, envelope antigen is diluted to 1:40000 ratio, 100 μ L/ holes, hatch 2h for 37 DEG C, take out ELISA Plate and get rid of liquid in plate, add the concentrated cleaning solution after dilution 300 μ L/ hole, wash plate 2 times, 30s/ time, then add 0.5% bovine serum albumin(BSA) (BSA) to close, 150 μ L/ holes, place 1.5h for 37 DEG C, discard confining liquid directly to pat dry, ELISA Plate after patting dry is placed (25 DEG C) between constant temperature and is dried, inspect by random samples qualified after ELISA Plate is vacuum-sealed in 4 DEG C of conditions under preserve.
4. enzyme linked immunological kit and the detection method thereof detecting chlopyrifos according to claim 2, is characterized in that: the concentration of chlopyrifos standard items is respectively 0mg/kg, 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg, 8.1mg/kg.
5. enzyme linked immunological kit and the detection method thereof detecting chlopyrifos according to claim 2, it is characterized in that: described chlopyrifos antibody working fluid adopts chlorpyrifos artificial mice immunized with antigen to obtain monoclonal antibody, is diluted to the preparation of 1:60000 ratio with antibody diluent.
6. a kind of enzyme linked immunological kit and detection method thereof detecting chlopyrifos according to claim 2, it is characterized in that: described chlopyrifos ELIAS secondary antibody working fluid adds diluted by ELIAS secondary antibody and becomes 1:2000 ratio, described substrate solution A is the citrate-phosphate disodium hydrogen buffer solution of the carbamide peroxide containing 0.5mmol/L, described substrate solution B is the ethanolic solution of tetramethyl biphenyl diamines, described stop buffer is the sulfuric acid of 2mol/L, described concentration and dilution liquid is 10 times of concentration and dilution liquid, it is the PBS of 0.1mol/L, between pH value range 7.0-7.5, described concentrated cleaning solution is 10 times of concentrated cleaning solutions, it is for containing 0.5% Tween-20, the PBST of 0.01mol/L, between pH value range 7.0-7.5.
7. enzyme linked immunological kit and the detection method thereof detecting chlopyrifos according to claim 2, based on the indirect competitive enzyme-linked immunosorbent reaction principle of antigen-antibody, the method is characterized in that: pre-service testing sample, get the ELISA Plate being coated with chlopyrifos antigen, add standard items/sample respectively according to the order of sequence, chlopyrifos ELIAS secondary antibody working fluid, the each 50 μ L/ holes of chlopyrifos antibody working fluid are in the micropore of correspondence, to vibrate gently mixing, 30min is reacted with in the rearmounted room temperature of cover plate membrane cover plate 25 DEG C of light protected environment, liquid in hole is dried, 4 ~ 5 times are fully washed with wash operating solution, every minor tick 10s, substrate solution A50 μ L/ hole is added after patting dry, substrate solution B50 μ L/ hole, to vibrate gently mixing, 15 ~ 20min is reacted with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate, add stop buffer 50 μ L/ hole, to vibrate gently mixing, setting microplate reader detects in 450nm place or dual wavelength 450/630nm, measure every hole absorbance (please running through data in 5min), with the logarithm of standard concentration (ppb) for horizontal ordinate, standard items percentage absorbance is ordinate, drawing standard curve, the content of reference standard curve calculation sample Chlorpyrifos.
8. method according to claim 7, wherein, described testing sample following methods carries out pre-service:
A. 2.0 ± 0.02g homogenised sample is accurately taken in 50mL centrifuge tube;
B. first add 5mL ethyl acetate, vortex instrument fully vibrates and mixes 10min;
C.5000r/min centrifugal 10min;
D. get supernatant 1mL to flow down in 50 ~ 60 DEG C of nitrogen and dry up;
E. add 1mL normal hexane (sample that fat content is high can add 2mL normal hexane), abundant whirling motion 10s, then add 1mL sample diluting liquid, low speed whirling motion 10s; 4000r/min, centrifugal 5min, discard upper strata normal hexane and middle layer impurity completely;
F. layer 50 μ L is taken off for analyzing.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106324240A (en) * | 2016-08-04 | 2017-01-11 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for detecting chlorpyrifos and application of kit |
| CN108614109A (en) * | 2016-12-12 | 2018-10-02 | 丹阳亿太生物科技发展有限公司 | A kind of enzyme linked immunological kit and its detection method of detection sodium sulfocyanate |
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| CN1431213A (en) * | 2003-01-13 | 2003-07-23 | 浙江大学 | Method for preparing artificial hapten, artificial antigen and specific antibody of chlorpyrifos and its usage |
| CN1438485A (en) * | 2003-01-13 | 2003-08-27 | 浙江大学 | Enzyme-linked immunosorbentassay reagent box suitable to chlorpyrifos residual analysis |
| CN103901137A (en) * | 2012-12-28 | 2014-07-02 | 中粮营养健康研究院有限公司 | Tea pretreatment method for detecting organophosphorus pesticides and application of as well as method for detecting organophosphorus pesticides in tea |
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Patent Citations (3)
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|---|---|---|---|---|
| CN1431213A (en) * | 2003-01-13 | 2003-07-23 | 浙江大学 | Method for preparing artificial hapten, artificial antigen and specific antibody of chlorpyrifos and its usage |
| CN1438485A (en) * | 2003-01-13 | 2003-08-27 | 浙江大学 | Enzyme-linked immunosorbentassay reagent box suitable to chlorpyrifos residual analysis |
| CN103901137A (en) * | 2012-12-28 | 2014-07-02 | 中粮营养健康研究院有限公司 | Tea pretreatment method for detecting organophosphorus pesticides and application of as well as method for detecting organophosphorus pesticides in tea |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106324240A (en) * | 2016-08-04 | 2017-01-11 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for detecting chlorpyrifos and application of kit |
| CN108614109A (en) * | 2016-12-12 | 2018-10-02 | 丹阳亿太生物科技发展有限公司 | A kind of enzyme linked immunological kit and its detection method of detection sodium sulfocyanate |
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Application publication date: 20160511 |