CN105572391A - Chemiluminescence enzyme-linked immunoassay (ELISA) method for detecting chlopyrifos in wheat - Google Patents
Chemiluminescence enzyme-linked immunoassay (ELISA) method for detecting chlopyrifos in wheat Download PDFInfo
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- CN105572391A CN105572391A CN201410542833.0A CN201410542833A CN105572391A CN 105572391 A CN105572391 A CN 105572391A CN 201410542833 A CN201410542833 A CN 201410542833A CN 105572391 A CN105572391 A CN 105572391A
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Abstract
The invention discloses a chemiluminescence enzyme-linked immunoassay (ELISA) kit for detecting chlopyrifos. The kit comprises a kit body and an enzyme label plate and reagents in the kit. Pores of the enzyme label plate are coated with a coating antigen which is a chlopyrifos nuclear parent-carrier protein conjugate. The reagents comprise a chlopyrifos monoclonal antibody, a horseradish peroxidase-labeled goat anti-mouse antibody, a series of chlopyrifos standard solutions, a condensed phosphate buffer solution, a condensed washing liquid and a chemiluminescence liquid. The chemiluminescence enzyme-linked immunoassay kit has the characteristics of high sensitivity, fastness, simpleness, high accuracy and multiple detection agents. Compared with the traditional colorimetric ELISA method, the method using the kit greatly reduces operation time.
Description
Technical field
The present invention relates to a kind of enzyme-linked immunologic detecting kit, particularly relate to a kind of chemical luminescence ELISA detection kit detecting wheat 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.
At present, the method detecting chlopyrifos mainly contains: radioimmunology, high performance liquid chromatography (HPLC), look/matter combination analysis method (LC-MS), liquid/matter combination analysis method (LC-MS/MS).The defect of thin-layered chromatography is: operating process is complicated, and the time is long; Operating personnel need through professional training; The disturbing factor of impact analysis is more, result poor repeatability.Radioimmunology, high performance liquid chromatography, look/matter are used in conjunction analytic approach, the defect of liquid/these physico-chemical methods of matter combination analysis method is that instrument and equipment is expensive, sample pre-treatments is complicated, time-consuming, effort, not easily popularize, testing cost is high, particularly radioimmunology also needs to be equipped with radioactive source, has certain risk.Given this, a kind of method setting up effective, quick, simple, sensitive detection wheat Chlorpyrifos is significant.
Summary of the invention
The object of this invention is to provide the chemical luminescence ELISA detection kit of a kind of chlopyrifos.This kit has that detection sensitivity is high, applying flexible, easily feature.
The chemical luminescence ELISA detection kit of chlopyrifos of the present invention, comprise box body, be located at ELISA Plate in box body and be located at chlopyrifos series standard solution in box body, enzyme mark goat anti-rabbit antibody, chlopyrifos antibody, luminescent solution, wash solution, bag by solution and lock solution; It is characterized in that:
Each hole of described ELISA Plate is coated with the envelope antigen made with chlopyrifos and ovalbumin coupling, wherein envelope antigen concentration preferably 10 μ g/mL.
Preferred 6.7KDa ~ the 6.8KDa of molecular weight ranges of described ovalbumin.
Described chlopyrifos series standard solution is that described in 0mg/kg, 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg and 8.1mg/kg, enzyme mark goat anti-rabbit antibody is horseradish peroxidase-goat anti-rabbit igg stoste respectively, and its working concentration is preferably 1:1000.
Described chlopyrifos antibody is the obtained polyclonal antibody of the bovine serum albumin coupling being 6.7KDa ~ 6.8KDa by chlopyrifos and the molecular weight ranges artificial immunogen immune animal of making, and its working concentration is preferably 1:1000.
Described luminescent solution is three (methylol) aminomethane solution (pH=8.8) and 3/10000 (volume ratio) H of 0.01M luminol and 0.001M p-cresol
2o
2mixed liquor.Described luminol is luminous substrate, and p-cresol is luminescence enhancer.
Described wash solution is pH7.5,0.1mol/L phosphate buffer containing volume fraction 0.05% Tween-20.
Described bag is the solution containing 1.59g sodium carbonate and 2.53g sodium bicarbonate in every premium on currency by solution, and pH is 9.5.
Described lock solution contains 10g ovalbumin (OVA, ovalbumin, also claim chicken ovalbumin or chicken ovalbumin, be made up of 386aa, molecular weight is about 43Kd) in often liter of wash solution and adds weight fraction 0.5%NaN
3solution.
Kit maximum detection range of the present invention is 0.1mg/kg ~ 8.1mg/kg.
The sensitivity impact that the chlopyrifos standard solution related in kit of the present invention, enzyme mark goat anti-rabbit antibody solution, chlopyrifos antibody-solutions, luminescent solution and wash solution and formula thereof detect kit of the present invention is very large; Wherein the principal ingredient of each solution and compound method thereof are:
1, chlopyrifos standard solution: compound concentration is respectively 0mg/kg in conventional manner, the chlopyrifos standard solution of 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg and 8.1mg/kg;
2, enzyme mark goat anti-rabbit antibody solution: enzyme mark goat anti-rabbit antibody is horseradish peroxidase-goat anti-rabbit igg stoste, is mixed with the working concentration of 1:1000 during use with wash solution;
3, chlopyrifos antibody-solutions: chlopyrifos antibody is with the obtained polyclonal antibody of artificial immunizing antigen immune animal, gained chlopyrifos antibody wash solution is diluted to the working concentration of 1:1000;
4, luminescent solution: three (methylol) aminomethane solution (pH=8.8) and 3/10000 (volume ratio) H of being 0.01M luminol and 0.001M p-cresol
2o
2mixed liquor;
5, wash solution: refer to pH7.5,0.1mol/L phosphate buffer containing volume fraction 0.05% Tween-20;
6, wrap by solution: 1.59g sodium carbonate and 2.53g sodium bicarbonate are dissolved in 1L water, regulate pH to be 9.5;
7, lock solution preparation: 10gOVA is dissolved in 1L wash solution, then adds the NaN that weight ratio is 0.05%
3.
The preparation of ELISA Plate of the present invention:
The method for coating of ELISA Plate of the present invention adopts chlopyrifos-OVA at the bag of setting by solution, with the concentration set, and reaction overnight bag quilt in 4 DEG C.
The present invention adopt to be pH be 9.5 sodium carbonate-bicarbonate buffer solution.In ELISA Plate of the present invention, the chlopyrifos-ovalbumin (OVA) of institute's bag quilt can well be combined on microwell plate frosting under alkaline environment, can stand repeatedly to wash plate, and the envelope antigen concentration of employing is 10 μ g/mL.
Bag can be closed by lock solution by good ELISA Plate, and in confining liquid, the preferred ovalbumin of inert protein (OVA), need add NaN
3prevent from going bad.
The preparation of chlopyrifos antibody-solutions and enzyme mark goat anti-rabbit antibody solution:
Chlorpyrifos antibody-solutions of the present invention, enzyme mark goat anti-rabbit antibody solution concentration are the key factors determining Ractopamine enzyme linked immunological test kit measurement range and sensitivity in the present invention.
It is 0.1 ~ 8.1mg/kg solution that the chlopyrifos antibody-solutions related in the present invention can be mixed with concentration with wash solution; Or the working concentration of 1:1000 is diluted to wash solution.
The enzyme mark goat anti-rabbit antibody solution related in the present invention preferably with wash solution preparation concentration be 1:1000.
The sensitivity (0.1mg/kg) that the kit prepared according to above-mentioned chlopyrifos antibody-solutions concentration and enzyme mark goat anti-rabbit antibody solution concentration can reach the good range of linearity (standard lines scope can reach 0.1mg/kg ~ 8.1mg/kg) and become reconciled.
The preparation of luminescent solution:
The present invention adopts horseradish peroxidase labeled substrate luminescent system, mainly luminol-hydrogen peroxide system.
Described luminescent solution is three (methylol) aminomethane solution (pH=8.8) and 3/10000 (volume ratio) H of 0.01M luminol and 0.001M p-cresol
2o
2mixed liquor.Described luminol is luminous substrate, and p-cresol is luminescence enhancer.
Principle of the present invention is combined with enzymatic high sensitivity by the high degree of specificity that antibody-antigene reacts, and utilizes the chemiluminescence reaction of substrate for enzymatic activity to detect production concentration.
Chemical luminescence ELISA detection kit of the present invention has highly sensitive, easy point fast and accurately, compares with traditional colorimetric ELISA method, and sensitivity can improve an order of magnitude.Play a significant role during the chlopyrifos residue be expected in crop products detects.
Accompanying drawing explanation
Fig. 1 is the canonical plotting of chlopyrifos.
Embodiment
Embodiment 1, immunogene, envelope antigen and the preparation of antibody
(1) immunogenic synthesis
Adopt p-aminobenzoic acid method to carry out coupling chlopyrifos and bovine serum albumin (BSA) and obtain immunogene.Specifically comprise the following steps:
A, take 14mg (100 μm of ol) p-aminobenzoic acid (ABA) and dissolve in 1.5mL0.2M hydrochloric acid, then take the sodium nitrite (NaNO of 8.3mg (120 μm of ol)
2) be dissolved in the distilled water of 0.24mL, 0-4 DEG C of stirrings, by sodium nitrite (NaNO
2) dropwise joins in p-aminobenzoic acid solution, lucifuge reacts 1 hour, obtains solution A;
B, take 34mg (100 μm of ol) chlopyrifos be dissolved in 5mL ice-cold borax buffering (0.05M) (pH8.5, NaCl containing 0.15M) in, 0-4 DEG C of stirring, above-mentioned solution A 2mL is dropwise joined in this solution, lucifuge reacts 2 hours, obtains orange solution;
C, solution is added a small amount of boric acid crystal adjust pH to 7.4, then 136mg (2 μm of ol) cBSA (bovine serum albumin(BSA)) is added, add 160mg (834 μm of ol) water-soluble carbodiimide (EDC) simultaneously, 48mg (417 μm of ol) N-hydroxy-succinamide (NHS), stirring at room temperature 3 hours, obtains orange solution;
D, reactant liquor is transferred in semi-permeable diaphragm, dialyse 3 days by phosphate buffered solution (PBS) (0.01M, pH7.4) at 0-4 DEG C, often within 4-6 hour, change a dislysate therebetween; Dialyse 3 days with high purity water subsequently, often within 4-6 hour, change a dislysate therebetween; Dialyse complete, use freeze drier freeze-drying, obtain yellow orange solid powder and be immunogene (conjugate of chlopyrifos and bovine serum albumin) ,-20 DEG C of preservations, for subsequent use.
(2) synthesis of envelope antigen
Adopt Isosorbide-5-Nitrae-Ding diether method to carry out coupling chlopyrifos and ovalbumin (OVA) and obtain envelope antigen.Specifically comprise the following steps:
A. taking 66mg ovalbumin (OVA) is dissolved in 5mL50mM carbonate (pH10.7) damping fluid, then in solution, add 1 of 28 μ L (147.9 μm of ol), 4-fourth diether (BDE), room temperature reaction 24 hours, obtains solution A;
B. taking 76mg (277.1 μm of ol) chlopyrifos is dissolved in 1mlDMF (anhydrous N-N dimethyl formamide), add in 1mL50mM carbonate (pH10.7) damping fluid again, solution A is passed into nitrogen, subsequently chlopyrifos dropwise is added in solution A, room temperature reaction 24 hours, obtains yellow solution;
C. reactant liquor is transferred in semi-permeable diaphragm, dialyse 3 days by phosphate buffered solution (PBS) (0.01M, pH7.4) at 0-4 DEG C, often within 4-6 hour, change a dislysate therebetween; Dialyse 3 days with high purity water subsequently, often within 4-6 hour, change a dislysate therebetween; Dialyse complete, use freeze drier freeze-drying, obtain white solid powder and be envelope antigen (conjugate of chlopyrifos and ovalbumin) ,-20 DEG C of preservations, for subsequent use.
(3) preparation of chlopyrifos polyclonal antibody
Adopt new zealand white rabbit as immune animal, be the conjugate of the bovine serum albumin of 6.7KDa ~ 6.8KDa with chlopyrifos and molecular weight ranges be immunogene, first immunisation dosage is 500 μ g/mL, when head exempts from, immunogene is dissolved in and makes emulsifying agent into isopyknic physiological saline and Freund's complete adjuvant, nape portion multi-point injection, later immunity is got the immunogene that dosage reduces by half and is dissolved in isopyknic physiological saline and incomplete Freund's adjuvant mixing and emulsifying, head exempts from and two exempts from interval 20 days, once be total to immunity five times every immunity in two weeks later, do not add adjuvant for the last time.Culling heart blood after last immune 7 days, centrifugal antiserum, obtains chlopyrifos polyclonal antibody.
The foundation of embodiment 2, CL-ELISA detection method
(1) preferred (square formation) of antibody and envelope antigen concentration
Longitudinally with the dilution series coated elisa plate of often kind of envelope antigen by 40.0 μ g/mL, 20.0 μ g/mL, 10.0 μ g/mL, 5.0 μ g/mL, 2.5 μ g/mL, 1.25 μ g/mL, 0.625 μ g/mL, 0.3125 μ g/mL, 100 μ L/ holes, 0-4 DEG C of placement is spent the night, wash plate three times with cleansing solution, pat dry at every turn; 250 μ L/ hole lock solution are closed, and room temperature places 2 hours, washes plate three times, pats dry at every turn; Add the antibody (1:100 to 1:1024000) of the 100 a series of dilutions in μ L/ hole, room temperature places 2.5 hours, washes plate three times, pats dry at every turn; Add the horseradish peroxidase-goat anti-rabbit igg antibody of the 1:1000 in 100 μ L/ holes, room temperature places 1 hour, washes plate three times, pats dry at every turn; Add the luminescent solution in 100 μ L/ holes, measure luminous value.The envelope antigen concentration of obvious graded and antibody dilution is had to carry out specific assay for optium concentration with luminous value with the concentration of envelope antigen.
(2) mensuration of antibody sensitivity
According to the optimization experiment of above-mentioned antagonist and envelope antigen concentration, applicant selects and determines that antibody concentration is 1:1000, and envelope antigen concentration is the mensuration that 10 μ g/mL carry out the sensitivity of antibody:
A, bag quilt: the solution by solution, the envelope antigen of chlopyrifos being made into 10 μ g/mL with the carbonate bag of 0.05MpH9.6, add 100 μ L in the reacting hole of each polystyrene board, 4 DEG C are spent the night;
Next day, discard solution in hole, wash 3 times with lavation buffer solution, 300 μ L/ holes, each 5 minutes, pat dry; (this step is called for short washing, lower same);
B, close: close above-mentioned ELISA Plate of having wrapped quilt by lock solution, 250 μ L/ holes, room temperature incubates 2-4 hour, then washs;
C, application of sample: add the chlopyrifos solution 50 μ L/ hole of dilution chlopyrifos antibody (1:1000) 50 μ L/ hole and variable concentrations in the above-mentioned reacting hole closed, room temperature 2-4 hour, then washs;
D, add enzyme labelled antibody: in each reacting hole, add antibody (1:1000) the 100 μ L/ hole of diluted fresh horseradish peroxidase-goat anti-rabbit igg, 1.5 hours, washing;
E, luminescence: the luminescent solution 100 μ L/ hole adding Extemporaneous in each reacting hole, detect with chemical illumination immunity analysis instrument immediately;
F, testing result calculate with inhibiting rate:
Inhibiting rate (%)=B/Bo%, B is the luminous value of medicine as rival of variable concentrations, and Bo is the luminous value of not dosing
The concentration calculating medicine during 50% inhibiting rate is the sensitivity of this antibody.
The chemiluminescence enzyme linked immunoassay reagent kit of embodiment 3, detection chlopyrifos
(1) composition of the chemiluminescence enzyme linked immunoassay reagent kit of chlopyrifos is detected
A, be coated with the solid phase carrier (ELISA Plate) of envelope antigen (conjugate of chlopyrifos and carrier protein);
B, chlopyrifos standard solution: 0mg/kg, 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg and 8.1mg/kg;
C, enzyme mark goat anti-rabbit antibody solution: enzyme mark goat anti-rabbit antibody is horseradish peroxidase-goat anti-rabbit igg stoste, load, be mixed with the working concentration of 1:1000 during use with wash solution;
D, chlopyrifos antibody-solutions: the polyclonal antibody preparing gained with artificial immunizing antigen immune animal, be diluted to 1:1000 working concentration by gained chlopyrifos antibody wash solution;
E, luminescent solution: use three (methylol) aminomethane solution preparation of 0.0001M p-cresol of pH8.8 to become the luminol solution of 0.01M, then with H
2o
2mix according to the volume ratio of 3:10000;
F, wash solution: pH7.5,0.1mol/L phosphate buffer containing volume fraction 0.05% Tween-20;
G, wrap by solution: 1.59g sodium carbonate and 2.53g sodium bicarbonate are dissolved in 1L water, regulate pH9.5;
H, lock solution are prepared: 10g ovalbumin (OVA) is dissolved in 1L wash solution, then adds the NaN that weight ratio is 0.05%
3.
(2) preparation of ELISA Plate
With coating buffer, envelope antigen is diluted to 10 μ g/mL, every hole adds 100 μ L, and 4 DEG C are spent the night, and incline coating buffer, every hole adds 250 μ L cleansing solutions and washs 3 times, pat dry, then every hole adds confining liquid 250 μ L, hatches 1h for 37 DEG C, incline liquid in hole, cleansing solution washs 3 times, pats dry, and preserves with masking foil vacuum seal.
Embodiment
4, the application of the chemiluminescence enzyme linked immunoassay reagent kit of chlopyrifos is detected
(1) preparation of reagent
A. sample diluting liquid: use after the concentrated phosphoric acid salt buffer solution distilled water diluting 10 times provided in kit;
B. wash solution: use after the concentrated cleaning solution distilled water diluting 10 times provided in kit;
C. luminescent solution: three (methylol) aminomethane solution (pH8.8)+3/10000 (volume ratio) H of 0.01M luminol+0.001M p-cresol
2o
2.
(2) sample pre-treatments
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.
(3) detecting step
A. application of sample: add chlopyrifos series standard strength solution or the molten 50 μ L of sample in ELISA Plate micropore, then add chlopyrifos antibody-solutions 50 μ L, room temperature (25 DEG C) constant-temperature incubation 2.5h;
B. wash: incline the middle liquid that portals, and every hole adds wash solution 250 μ L, washs 3 times, pat dry;
C. enzyme-added mark goat anti-rabbit antibody solution: every hole adds enzyme mark goat anti-rabbit antibody solution 100 μ L, room temperature constant-temperature incubation 1h;
D. wash: incline the middle liquid that portals, and every hole adds wash solution 250 μ L, washs 3 times, pat dry;
E. luminescent solution is added: every hole adds luminescent solution 100 μ L;
F. detect: the luminous intensity measuring every hole with chemical illumination immunity analysis instrument.
(4) result judges
The mean value of the standard items obtained and sample luminous value is multiplied by 100 again divided by the luminous value of first standard (0 standard), take inhibiting rate as ordinate, the logarithm of chlopyrifos concentration is that horizontal ordinate makes typical curve, and the concentration of each sample can read from typical curve.
Inhibiting rate (%)=standard items luminous value (or sample) × 100%/0 standard items luminous value.
Embodiment 5 kit preci-sion and accuracy is tested
Get 0mg/kg, the chlopyrifos standard specimen of 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg and 8.1mg/kg, adds in wheat samples, detects the chlopyrifos recovery.The interassay coefficient of variation of each concentration calculates with 5 repeating datas of different 5 days, and variation within batch coefficient calculates with time repeating data of 5 on the same day.
The quantitative calculating of the recovery is carried out according to the linear equation of the typical curve formulated.
From said determination result, the coefficient of variation is lower than 24.7%, and the recovery is between 78-120%.Show that this kit has well repeatability and accuracy.
Claims (9)
1. a chemical luminescence ELISA detection kit for chlopyrifos, comprises box body, the reagent being located at the ELISA Plate in box body and being located in box body; It is characterized in that, each hole of described ELISA Plate is coated with the envelope antigen made with chlopyrifos parent nucleus and ovalbumin coupling; Described reagent comprises: the sheep anti-mouse antibody of chlopyrifos class monoclonal antibody, horseradish peroxidase-labeled, chlopyrifos class series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, chemical luminescence for liquid.
2. the chemical luminescence ELISA detection kit of chlopyrifos class according to claim 1, is characterized in that: described ELISA Plate is milky opaque polystyrene 96 hole chemiluminescence ELISA Plate.
3. the chemical luminescence ELISA detection kit of chlopyrifos class according to claim 1, is characterized in that: described envelope antigen concentration is 10 μ g/mL.
4. the chemical luminescence ELISA detection kit of chlopyrifos class according to claim 1, is characterized in that: the working concentration of described Dursban monoclonal antibody is 1: 64000.
5. the chemical luminescence ELISA detection kit of chlopyrifos class according to claim 1, is characterized in that: the monoclonal antibody of described chlopyrifos is that the conjugate be made up of chlopyrifos parent nucleus and bovine serum albumin coupling prepares as immunogen immune Balb/c mouse.
6. the chemical luminescence ELISA detection kit of chlopyrifos according to claim 1, is characterized in that: described chlopyrifos series standard solution concentration is respectively: 0mg/kg, 0.1mg/kg, 0.3mg/kg, 0.9mg/kg, 2.7mg/kg and 8.1mg/kg.
7. the chemical luminescence ELISA detection kit of chlopyrifos according to claim 1, is characterized in that: described concentrated phosphoric acid salt buffer be often liter containing NaH
2pO
42H
2o5.74g, Na
2hPO
412H
2the aqueous solution of O32.6g.
8. the chemical luminescence ELISA detection kit of chlopyrifos according to claim 1, is characterized in that: described thickening and washing solution is the pH=7.4 containing volume fraction 0.05% Tween-20,0.1mol/L phosphate buffer.
9. the chemical luminescence ELISA detection kit of chlopyrifos class according to claim 1, is characterized in that: three (methylol) aminomethane solution that described chemical luminescence for liquid A liquid is luminol content is 0.01M, p-cresol content is 0.001MpH8.8; B liquid is that 100mL solution contains citric acid 2.1g, anhydrous Na
2hPO
4the solution of 2.82g, the carbamide peroxide 0.64mL of 0.75%, described number percent is mass percent.
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Cited By (1)
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| CN1438485A (en) * | 2003-01-13 | 2003-08-27 | 浙江大学 | Enzyme-linked immunosorbentassay reagent box suitable to chlorpyrifos residual analysis |
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| CN103018450A (en) * | 2011-09-20 | 2013-04-03 | 北京勤邦生物技术有限公司 | Chloramphenicol chemiluminescence enzyme-linked immunodetection kit |
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