CN202735343U - Kit for testing chloramphenicol - Google Patents
Kit for testing chloramphenicol Download PDFInfo
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- CN202735343U CN202735343U CN 201220227859 CN201220227859U CN202735343U CN 202735343 U CN202735343 U CN 202735343U CN 201220227859 CN201220227859 CN 201220227859 CN 201220227859 U CN201220227859 U CN 201220227859U CN 202735343 U CN202735343 U CN 202735343U
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- chloromycetin
- chloramphenicol
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Abstract
The utility model provides a kit for testing chloramphenicol, and the kit comprises a kit body, a fixed appliance with 12 holes and 12 reagent barrels with corks, which are arranged in the fixed appliance; microporous reagent strips and test paper strips are arranged in the reagent barrels; each reagent strip comprises a bottom plate, and a sample absorption pad, a reaction film, a water absorption pad and a protective film which are attached on the bottom plate and are sequentially connected tightly; and chloramphenicol monoclonal antibodies and colloidal gold markers are frozen out in microporous reagent. A test line imprint '|' coated with chloramphenicol hapten and carrier protein coupling and a quality control line imprint '|' coated with goat antibodies and mouse antibodies are arranged on each reaction film. The kit has the characteristics of good portability, high sensitivity, short testing time and the like, and can play an important role in the testing of residual chloramphenicol.
Description
Technical field
The utility model relates to a kind of kit of chlorine detection mycin, particularly detects the kit of chloromycetin in the animal derived food.
Background technology
Chloromycetin (Chloramphenicol, CAP) is a kind of high-efficiency broad spectrum microbiotic, can suppress gram-positive bacteria and Gram-negative bacteria, is widely used in the communicable disease control in all kinds of poultry, domestic animal, the aquatic products production run.But the residual meeting of chloromycetin in food consists of potential hazard to health, can cause that people's marrow hemopoiesis is in disorder, causes the diseases such as alpastic anemia, granular white blood cells deficiency disease and neonate, premature's Synthetic Grey disease.Residual chloromycetin consists of potential harm to human health and more and more is familiar with by people in the animal derived food, and the U.S. only allows chloromycetin to be used for non-edible animal, is defined in the animal derived food and must not detects chloromycetin; European Union does not allow chloromycetin to be used for producing cow and laying hen, and is restricted in the use of other animals yet, and the chloramphenicol residue in the strict regulations meat must not surpass 10 μ g/kg; China Ministry of Agriculture announces No. 235 file and also stipulates, chloromycetin and salt thereof, ester (comprising Chloramphenicol Succinate) must not detect in all Edible tissues of all food animals.But because good antimicrobial effect, the price of chloromycetin are low, still have the people to use it in poultry, domestic animals, the aquatic products.For the trade contacts that ensure the healthy of the people and enlarge food, set up highly sensitive, high specificity, quick, economic residual chloromycetin detection method is necessary.
At present, being used for the most popular method of chlorine detection mycin mainly contains microbial method, physico-chemical analysis method and immunoassay three major types.Microbial method is easy and simple to handle, amount of samples is few, testing cost is low, but is subject to other antibiotic impact in the tissue, and specificity is low, and sensitivity is not high yet; The physico-chemical analysis method comprises liquid phase chromatography, Chromatography/Mass Spectrometry coupling technique, vapor-phase chromatography etc., it is the main method of present chlorine detection mycin, have highly sensitive, result accurately, the advantage such as good reproducibility, false positive be few, but it exists that testing cost is high, equipment needed thereby is complicated, the testing staff had relatively high expectations, detects the shortcoming such as length consuming time; Immunoassay commonly used is mainly enzyme linked immunosorbent detection method and radio immunoassay at present, but the testing cost that needs is still higher, is not suitable for enterprise and detects cheaply needs.Therefore, be necessary in practice to set up that a Species sensitivity is high, simple to operate, cost is low, the detection of drugs kind is many, be fit to the detection method of large-scale promotion.
The utility model content
The purpose of this utility model provides a kind of kit of highly sensitive, cost is low, simple to operate, detection time is short chlorine detection mycin.
Kit of the present utility model comprises the kit box body, has holding appliance and placement 12 tool plug reagent barrels wherein in 12 holes, reagent barrel comprises micropore reagent strip and 8 test strips, the micropore reagent strip has 8 reacting holes, have the micropore plug on the reacting hole, wherein freeze-drying has chloromycetin monoclonal antibody-colloid gold label thing.Test strips by base plate, be attached on the base plate absorption of sample pad, reaction film, adsorptive pads and the diaphragm that closely link to each other successively and form, have the detection line trace " | " that is coated with chloromycetin hapten-carrier protein conjugate and the nature controlling line trace " | " that is coated with the sheep anti mouse antiantibody on the described reaction film.Detection line is parallel with nature controlling line, and detection line is positioned at apart from absorption of sample pad one side 5-8mm place, and nature controlling line is positioned at the place apart from detection line 4-7mm, and coated with protective film on the test strips, diaphragm cover on the absorption of sample pad, are the test side, and the above is printed on the MAX mark line.
Test strips base plate in the utility model kit can be the material that PVC base plate or other hard do not absorb water; The absorption of sample pad can be suction strainer paper or filter paper for oil; Adsorptive pads is thieving paper; Reaction film can be nitrocellulose filter or cellulose acetate membrane; Diaphragm is PE material diaphragm; The kit box body is carton box; Reagent barrel is the plastics reagent barrel; Holding appliance is the rigid support material.
The utility model kit has following beneficial effect:
1, highly sensitive.Chloramphenicol detection reagent box is prepared from as the basis take the monoclonal antibody of colloid gold label high-affinity, priceless strong formation between gold grain and the antibody molecule in the gold labeling antibody, the two combines by the Van der Waals force between the charges of different polarity, collaurum is very little on monoclonal antibody specificity and affinity impact, and has higher mark rate.Chloromycetin monoclonal antibody wherein-colloid gold label thing freeze-drying in testing process, can make golden labeling antibody fully contact with sample liquid to be checked in the micropore reagent strip, fully reaction, thus reduce error, increase the reaction sensitivity of whole system.Therefore, the utility model kit has higher specificity and sensitivity.This kit is 0.1 μ g/L to the detection sensitivity of chloromycetin.
2, easy and simple to handle quick.Need not any other reagent when using kit to detect, as long as sample solution to be checked is dripped in micropore reagent, behind the mixing, room temperature (20-25 ℃) is hatched 5min, an end that test paper is indicated the MAX mark line is downward, timing behind the micropore reagent after insertion is hatched, observations behind the 10min.
3, show testing result image, directly perceived, accurate.Test strip is to show that red line " | " and " ‖ " trace are as the positive and negative marker, namely show that at nitrocellulose filter a red line " | " trace represents that the content of chloromycetin is higher than in the detected sample liquid and equals kit to the lowest detectable limit of chloromycetin, article two, red line " ‖ " trace represents that the content of chloromycetin in the detected sample is lower than kit to the lowest detectable limit of chloromycetin, the result judges image, directly perceived, accurate, simple and clear, is not easy to occur result's erroneous judgement.
4, cost is low, small investment.Use the utility model kit, do not need to join in addition instrument and equipment and other reagent, Site Detection is settled at one go, with low cost, small investment, instant effect.
5, be easy to apply on a large scale.Kit is simple to operate, can better satisfy different levels personnel's needs, such as specialty chemical examination, customs quarantine control, health and epidemic prevention, quality monitoring, food enterprise etc., has wide market outlook and larger economical, societal benefits.
Description of drawings
Fig. 1 is the utility model test strips structural representation;
Fig. 2 is the utility model test strips vertical view;
Fig. 3 is the utility model micropore reagent figure;
Fig. 4 is as a result process decision chart of the utility model ELISA test strip;
Fig. 5 is the utility model reagent barrel structural representation;
Fig. 6 is the vertical view of the utility model holding appliance;
Fig. 7 is the side view of the utility model box body and holding appliance.
Embodiment
One, the assembling of Chloramphenicol detection reagent box preparation
(1) test strips
Absorption of sample pad 1, reaction film 2, adsorptive pads 3 and diaphragm 7 are pasted successively in order on described base plate 6; the end of absorption of sample pad links to each other with the top of reaction film; the end of reaction film links to each other with adsorptive pads; the top of absorption of sample pad aligns with the top of base plate; the end of adsorptive pads aligns with the end of base plate; test paper is pasted with diaphragm; diaphragm 7 covers on the absorption of sample pad; be the test side; the above is printed on the MAX mark line; detection line 4 is coated with chloromycetin hapten-carrier protein conjugate, and nature controlling line 5 is coated with the sheep anti mouse antiantibody.
Base plate is the PVC base plate, and the absorption of sample pad is suction strainer paper, and reaction film is nitrocellulose filter, and adsorptive pads is thieving paper, and diaphragm is PE material diaphragm.
(2) micropore reagent strip
Micropore reagent strip 8 has plug 9, and freeze-drying has chloromycetin monoclonal antibody-colloid gold label thing in the plate hole.
The plastics reagent barrel 10 of (3) (1) test strips and (2) micropore reagent strip being packed into and having gland bonnet is positioned over the plastics reagent barrel in the holding appliance 11 in the kit box body 12.
Two, the use of kit
(1) sample pre-treatments
Animal tissue (pork, chicken, fish, shrimp) sample: take by weighing tissue samples that 5g ± the 0.05g homogeneous is crossed to 50ml polystyrene centrifuge tube, add 8ml ethyl acetate, with the oscillator 5min that vibrates, the centrifugal 5min of the above room temperature of 3000g (20-25 ℃); Pipette whole upper organic phase to the clean glass test tube of 10ml, flow down in 50 ~ 60 ℃ of water-bath nitrogen and dry up; Add the 1ml normal hexane, with vortex instrument whirling motion 1min, add again 1ml 0.02mol/L PBS damping fluid, with vortex instrument whirling motion 30s, the centrifugal 5min of the above room temperature of 3000g (20-25 ℃); Remove upper organic phase, take off layer inorganic phase and be used for analyzing.
Milk sample: take by weighing 4.0ml ± 0.05ml milk to the 50ml centrifuge tube, add 6ml ethyl acetate, 2ml acetonitrile, the 5min that vibrates up and down, the centrifugal 5min of the above room temperature of 3000g (20-25 ℃); Pipette the 6ml upper organic phase to 10ml dry glass test tube, flow down in 50 ~ 60 ℃ of water-bath nitrogen and dry up, add 0.5ml 0.02mol/L PBS damping fluid, the dry residue of whirling motion 1min dissolving, to be checked.
Honey sample: take by weighing 4.0g ± 0.05g honey to the 50ml centrifuge tube, add the 4ml deionized water, vibrate to honey with oscillator and all dissolve; Add 4ml ethyl acetate, 4ml acetonitrile, the 5min that vibrates up and down, the centrifugal 5min of the above room temperature of 3000g (20-25 ℃); Pipette the 6ml upper organic phase to 10ml dry glass test tube, flow down in 50 ~ 60 ℃ of water-bath nitrogen and dry up, add 0.5ml 0.02mol/L PBS damping fluid, the dry residue of whirling motion 1min dissolving, to be checked.
(2) detect with kit
Drip sample solution 200 μ l to be checked in micropore reagent, behind the mixing, room temperature (20-25 ℃) is hatched 5min, and an end that test paper is indicated the MAX mark line is downward, timing behind the micropore reagent after insertion is hatched, observations behind the 10min.
Three, Analysis of test results
The content of chloromycetin in sample is greater than or equal to kit its lowest detection is prescribed a time limit, chloromycetin monoclonal antibody-colloid gold label thing is combined with chloromycetin, antigen binding site on the gold labeling antibody is closed, thereby in detection zone, because competitive reaction, can not be combined with chloromycetin hapten-carrier protein conjugate and red stripes do not occur.Negative sample owing to lack the antigen-antibody competitive reaction, will red stripes occur at detection line and nature controlling line in testing process.As shown in Figure 4.
Positive: when red stripes of nature controlling line (C) demonstration, and detection line (T) does not develop the color, and test strips is judged to the positive, shown in Fig. 4 .a figure to show red line " | ".
Negative: when red stripes of nature controlling line (C) demonstration, detection line (T) also demonstrates a red stripes simultaneously, and test strips is judged to feminine gender, shown in Fig. 4 .b to show red line " ‖ ".
Invalid: (C) do not demonstrate red stripes when nature controlling line, and then no matter whether detection line (T) demonstrates red stripes, and it is invalid that this test strips all is judged to, shown in Fig. 4 .c, 4.d.
Four, the material preparation method of using in the detection kit
1, the synthetic and evaluation of chloromycetin hapten-carrier protein conjugate
Chloromycetin is small-molecule substance, only has immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.
(1) the chloromycetin haptens is synthetic
A. get 1.0g chloromycetin and add 30ml N, dinethylformamide (DMF) dissolving, add imidazoles 0.44g, tert-butyl chloro-silicane 0.37g, stirring at room 3h adds the water ethyl acetate extraction and separates, the organic phase washing, evaporate to dryness, upper silicagel column, sherwood oil: ethyl acetate=5:1 wash-out obtains product 1.20g;
B. the 1.2g eluted product adds succinic anhydride 0.65g with pyridine 30ml dissolving, and 70 ℃ add thermal agitation 12h.After reaction stops, solvent evaporated, sherwood oil: ethyl acetate=3:1 wash-out obtains product 1.33g;
C. the 1.3g eluted product adds the methyl alcohol dissolving, adds p-toluenesulfonic acid 0.2g, and stirring at room 3h adds the water ethyl acetate extraction and separates evaporate to dryness, upper silicagel column.Sherwood oil: ethyl acetate=1:1 wash-out obtains chloromycetin haptens product 0.46g.
(2) immunogenic preparation---chloromycetin haptens and bovine serum albumin(BSA) conjugate are synthetic
Get the 20mg haptens, be dissolved among the 1.5ml DMF, get the solution I; In adding I, stir 24h under the room temperature after getting 50mg carbodiimides (EDC) and fully dissolving with 0.2ml water, get the solution II; Take by weighing bovine serum albumin(BSA) (BSA) 60mg, make it fully to be dissolved in 4.3ml PBS(pH 7.2) in, dropwise slowly be added drop-wise in the protein solution solution II, and under room temperature, stir 24 h, with 4 ℃ of dialysis of 0.01mol/L PBS 3d, change dislysate every day 3 times, to remove unreacted small-molecule substance; Packing saves backup in-20 ℃.
(3) preparation of coating antigen---chloromycetin haptens and ovalbumin conjugate are synthetic
Get the 15mg haptens, be dissolved among the 1.5ml DMF, get the solution I; In adding I, stir 24 h under the room temperature after getting 36mg dicyclohexylcarbodiimide (DCC) and 18mg N-hydroxy-succinamide (NHS) and fully dissolving with 0.2ml DMF, get the solution II; Take by weighing ovalbumin (OVA) 45mg, make it fully to be dissolved in 4.3ml PBS(pH 7.2) in, dropwise slowly be added drop-wise in the protein solution solution II, and under room temperature, stir 24h, with 4 ℃ of dialysis of 0.01mol/L PBS 3d, change dislysate every day 3 times, to remove unreacted small-molecule substance; Packing saves backup in-20 ℃.
(4) evaluation of chloromycetin hapten-carrier protein conjugate
Carrier protein, chloromycetin haptens, chloromycetin hapten-carrier protein conjugate are made into the solution of 0.5mg/ml with the PBS of pH7.4, return to zero with 0.01mol/L pH7.4 PBS, in the interscan of wavelength 200-800nm scope, obtain the absorption curve of carrier protein, chloromycetin haptens, chloromycetin hapten-carrier protein conjugate with ultraviolet spectrophotometer.Different absorption curves appears in the three, shows the success of chloromycetin haptens and carrier protein couplet.
2, the preparation of chloromycetin monoclonal antibody
(1) animal immune
The immunogene that step 1 obtains is injected in the Balb/c Mice Body, and immunizing dose is 150 μ g/, makes it produce antiserum.
(2) Fusion of Cells and cloning
Get immune Balb/c mouse boosting cell, in the 8:1(quantitative proportion) ratio and SP2/0 myeloma cell's fusion, screening obtains the chloromycetin monoclonal hybridoma strain of stably excreting chloromycetin monoclonal antibody.
(3) cell cryopreservation and recovery
Hybridoma is made 1 * 10 with cryopreserving liquid
6The cell suspension of individual/ml is in the medium-term and long-term preservation of liquid nitrogen.Take out cryopreservation tube during recovery, put into immediately 37 ℃ of water-bath middling speeds and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
(4) preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed cell culture medium, under 37 ℃ of conditions, cultivate, with sad-saturated ammonium sulfate method the nutrient solution that obtains is carried out purifying, obtain monoclonal antibody ,-20 ℃ of preservations.
Described cell culture medium is to add calf serum and sodium bicarbonate in the RPMI1640 nutrient culture media, making the final concentration of calf serum in cell culture medium is 20%(quality percentage composition), the final concentration of sodium bicarbonate in cell culture medium is 0.2%(quality percentage composition); The pH of described cell culture medium is 7.4.
3, the preparation of sheep anti mouse antiantibody
As immune animal, as immunogene the pathogen-free domestic sheep is carried out immunity take mouse source antibody with sheep, obtain the sheep anti mouse antiantibody.
4, the preparation of chloromycetin monoclonal antibody-colloid gold label thing
(1) preparation of collaurum
With two ionized waters that boil off 1% gold chloride (is purchased from sigma company, catalog number T09041) is diluted to 0.01%(quality percentage composition), get 100ml and place conical flask, be heated to boiling with the thermostatic electromagnetic stirrer, under continuous high temperature, the lasting stirring, add 2.5ml 1% trisodium citrate and (be purchased from Guangzhou Chemical Reagent Factory, catalog number BG11-AR-01KG), continuing at the uniform velocity to be heated with stirring to solution is bright and stops when red, return to original volume with deionized water after being cooled to room temperature, 4 ℃ of preservations.The collaurum outward appearance for preparing is pure, bright, nothing precipitates and floating thing.
(2) preparation of chloromycetin monoclonal antibody-colloid gold label thing
Under magnetic agitation, transfer the pH to 7.2 of collaurum with 0.2mol/L sal tartari, in colloidal gold solution, add above-mentioned chloromycetin monoclonal antibody by the standard that adds 50 ~ 100 μ g in every milliliter of colloidal gold solution, stir and evenly mix, after room temperature leaves standstill 10min, adding 10% bovine serum albumin(BSA) (BSA), to make its final concentration in colloidal gold solution be the 1%(volumn concentration), leave standstill 10min.12000r/min, 4 ℃ of centrifugal 40min abandon supernatant, precipitation is with redissolving the damping fluid washed twice, it is resuspended to be with volume that the redissolution damping fluid of initial collaurum volume 1/10 will precipitate, put 4 ℃ for subsequent use.
Redissolution damping fluid: contain bovine serum albumin(BSA) 0.1% ~ 0.3%(volumn concentration), Tween-80 0.05% ~ 0.2%(quality percentage composition), the 0.02mol/L phosphate buffer of pH7.2.
5, with chloromycetin monoclonal antibody-colloid gold label thing freeze-drying to micropore reagent
In micropore reagent microwell plate, add 100 μ l chloromycetin monoclonal antibody-colloid gold label things, put into freeze drier, under condenser temperature-50 ℃ condition, behind the pre-freeze 3h, vacuum drying 15h again, can take out, obtain the micropore reagent that freeze-drying has chloromycetin monoclonal antibody-colloid gold label thing, sealing is preserved.
6, the preparation of absorption of sample pad
The absorption of sample pad placed contain 0.5% bovine serum albumin(BSA) (volumn concentration), pH7.2,0.1mol/L phosphate buffer and soak 2h, 37 ℃ of baking 2h are for subsequent use.
7, the preparation of reaction film
Coated process: with phosphate buffer chloromycetin haptens-ovalbumin conjugate is diluted to 10mg/ml, with Isoflow point film instrument it is coated in detection line (T) on the nitrocellulose filter, package amount is 1.0 μ g/cm
2Phosphate buffer with 0.01mol/L, pH7.4 is diluted to 200 μ g/ml with sheep anti-mouse igg antibody, with Isoflow point film instrument it is coated in nature controlling line (C) on the nitrocellulose filter, and package amount is 1.0 μ g/cm
2Coated good reaction film is placed dry 2h under 37 ℃ of conditions, for subsequent use.
Claims (9)
1. the kit of a chlorine detection mycin; it is characterized in that kit comprises the kit box body, has the holding appliance in 12 holes and places wherein 12 tool plug reagent barrels; comprise micropore reagent strip and 8 test strips in the reagent barrel, test strips by base plate, be attached on the base plate absorption of sample pad, reaction film, adsorptive pads and the diaphragm that closely link to each other successively and form.
2. kit as claimed in claim 1 is characterized in that having on the described reaction film detection line trace " | " that is coated with chloromycetin hapten-carrier protein conjugate and the nature controlling line trace " | " that is coated with the sheep anti mouse antiantibody.
3. kit as claimed in claim 2 is characterized in that described detection line is parallel with nature controlling line, all is the ribbon vertical with the appearance of test strips.
4. kit as claimed in claim 2 or claim 3 is characterized in that described detection line is positioned at apart from absorption of sample pad one side 5-8mm place, and described nature controlling line is positioned at the place apart from detection line 4-7mm.
5. kit as claimed in claim 1 is characterized in that coated with protective film on the described test strips, and diaphragm covers on the absorption of sample pad, is the test side, and the above is printed on the MAX mark line.
6. kit as claimed in claim 1 is characterized in that described base plate can be the PVC base plate, and the absorption of sample pad can be suction strainer paper or filter paper for oil, and adsorptive pads is thieving paper, and reaction film can be nitrocellulose filter or cellulose acetate membrane, and diaphragm is PE material diaphragm.
7. kit as claimed in claim 1 is characterized in that described micropore reagent strip has 8 reacting holes, has the micropore plug on the reacting hole.
8. kit as claimed in claim 1 is characterized in that freeze-drying has chloromycetin monoclonal antibody-colloid gold label thing in the described micropore reagent.
9. kit as claimed in claim 1 is characterized in that described kit box body is carton box, and reagent barrel is the plastics reagent barrel, and holding appliance is the rigid support material.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220227859 CN202735343U (en) | 2012-05-18 | 2012-05-18 | Kit for testing chloramphenicol |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220227859 CN202735343U (en) | 2012-05-18 | 2012-05-18 | Kit for testing chloramphenicol |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN202735343U true CN202735343U (en) | 2013-02-13 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201220227859 Expired - Fee Related CN202735343U (en) | 2012-05-18 | 2012-05-18 | Kit for testing chloramphenicol |
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| Country | Link |
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| CN (1) | CN202735343U (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105759040A (en) * | 2015-12-31 | 2016-07-13 | 贵州勤邦食品安全科学技术有限公司 | Enzyme-linked immunosorbent reagent kit for detecting chloramphenicol residues |
-
2012
- 2012-05-18 CN CN 201220227859 patent/CN202735343U/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105759040A (en) * | 2015-12-31 | 2016-07-13 | 贵州勤邦食品安全科学技术有限公司 | Enzyme-linked immunosorbent reagent kit for detecting chloramphenicol residues |
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| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130213 Termination date: 20210518 |
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| CF01 | Termination of patent right due to non-payment of annual fee |