CN102721808A - Spiramycin assay kit and method for making the same - Google Patents
Spiramycin assay kit and method for making the same Download PDFInfo
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- CN102721808A CN102721808A CN2011100764473A CN201110076447A CN102721808A CN 102721808 A CN102721808 A CN 102721808A CN 2011100764473 A CN2011100764473 A CN 2011100764473A CN 201110076447 A CN201110076447 A CN 201110076447A CN 102721808 A CN102721808 A CN 102721808A
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- spiramvcin
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Abstract
A spiramycin assay kit and a method for making the same are provided in the invention, and relate to a field of determination technologies for biology immunity methods. The assay kit is composed of a sample pad (1), a colloidal gold pad (2), a nitrocellulose membrane (3), a sample suction pad (4) and a PVC support plate (5), wherein the sample pad, the colloidal gold pad, the nitrocellulose membrane and the sample suction pad are consecutively adhered to the PVC support plate in turn. The colloidal gold pad is a spiramycin monoclonal antibody polyester film which is labelled by the colloidal gold, and the nitrocellulose membrane, in order, is coated with a spiramycin coupling carrier protein as a test line (T line) and a goat-anti-mouse IgG polyclonal antibody as a quality control line (C line). As spiramycin that may be contained in a detected sample and the spiramycin coupling carrier protein coated on the nitrocellulose membrane compete to combine with the colloidal gold-labelled spiramycin monoclonal antibody, a colloidal gold immunochromatography technique is used to detect whether or not the sample contains spiramycin.
Description
Technical field
The present invention relates to the determination techniques field of biology immunization method, particularly relate to a kind of a kind of spiramvcin detection kit of utilizing the colloidal gold immunochromatographimethod fabrication techniques and preparation method thereof.
Background technology
Spiramvcin, English name Spiramycin is a macrolide antibiotic common in the veterinary drug.Spiramvcin has antibacterial action to staphylococcus, micrococcus scarlatinae, streptococcus pneumonia, meningococcus, gonococcus etc., is mainly used in an anti-brutish bacterium and a choamydiae infection, also can be used as feed addictive and promotes weightening finish and improve feed conversion rate.After animal was taken for a long time, metabolin can be accumulated in the tissue of animal and apparatus or store, and reaches the infringement that finite concentration can cause animal vestibular and Russia's cochlea nerve, causes dizzy and dysacousis, and severe patient also will cause the infringement of liver kidney.Spiramvcin can get into human body through food chain, will cause serious harm to human health.The international food code council, European Union and Japan stipulate that all the highest residual volume value of limiting the quantity of of this medicine is 200 μ g/L, therefore, need set up a kind of quick, sensitive spiramvcin detection method.
At present; Be mainly high performance liquid chromatography (HPLC), liquid chromatograph mass spectrography method (LC-MS), thin layer chromatography, the microorganism method etc. of tiring about the check and analysis method of spiramycin residues; But these methods exist instrument costliness, testing process loaded down with trivial details, reviewer's technical ability is required high shortcoming; Be not suitable for the examination of on-site supervision and great amount of samples, in the application of reality, be restricted.And the inventive method has overcome the shortcoming of said method, can realize the fast detecting to spiramvcin.
The present invention adopts the colloidal gold immunochromatographimethod technology to prepare a kind of spiramvcin detection kit.That the present invention has is simple to operate, detect fast, sensitive, need not characteristics such as complex instrument.
Summary of the invention
The object of the present invention is to provide a kind of simple, quick, highly sensitive, spiramvcin detection kit that cost is low and preparation method thereof.
A kind of spiramvcin detection kit comprises sample pad, collaurum pad, nitrocellulose filter, suction appearance pad and PVC back up pad, and tight adhesion has sample pad, collaurum pad, nitrocellulose filter and suction appearance pad successively on the PVC back up pad.Said sample pad is a spun glass; The spiramvcin monoclonal antibody polyester film that said collaurum pad is a colloid gold label; Be coated with detection line (T line) and nature controlling line (C line) on the said nitrocellulose filter successively, wherein encapsulated spiramvcin coupling carrier albumen on the detection line (T line), encapsulated the sheep anti-mouse igg polyclonal antibody on the nature controlling line (C line); Said suction appearance pad is thieving paper.
The carrier protein of coupling spiramvcin is a bovine serum albumin(BSA), or oralbumin, or hemocyanin.
The present invention adopts nano-colloid technology for gold and antigen and antibody specific reaction; The principle of using immunity competition inhibitory reaction is prepared from; Be the spiramvcin antibody of the spiramvcin coupling carrier protein competition association colloid gold mark that detection line (T line) encapsulates on the spiramvcin that possibly contain in the sample to be checked and the nitrocellulose filter, judge whether contain spiramvcin in the sample to be checked through the colour developing of T line.
The invention provides a kind of preparation method of spiramvcin detection kit, may further comprise the steps:
(1) preparation spiramvcin coupling carrier albumen
With spiramvcin and carrier protein couplet, synthetic spiramvcin coupling carrier albumen is as immunogene and coating antigen.
(2) preparation spiramvcin monoclonal antibody
Adopting spiramvcin coupling carrier albumen is the immunogen immune BALB/C mice, preparation spiramvcin monoclonal antibody.
(3) preparation collaurum
Get 0.01% aqueous solution of chloraurate, heated and boiled.Add 1% citric acid, three sodium water solution 1ml as required rapidly, continue to boil about 5min, occur orange red.The size of the colloid gold particle of processing like this is 20-40nm.
(4) preparation collaurum pad
Get the colloidal gold solution that grain size is 20-40nm, use 0.2mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 7.0-9.0, and room temperature was placed 30 minutes; In above-mentioned solution, add the spiramvcin monoclonal antibody, the concentration that makes the spiramvcin monoclonal antibody is 20-100 μ g/ml collaurum, and after mixing, room temperature was placed 30 minutes; The collaurum of getting the amount of initial colloidal gold solution volume 5% redissolves liquid in above-mentioned solution, mixes, and room temperature was placed 10 minutes; Above-mentioned solution left the heart 30 minutes in 12000, carefully drew supernatant, discarded, and remaining deposition is redissolved the liquid dissolving with the collaurum of the initial collaurum volume of 30-70%, obtains spiramvcin monoclonal antibody-colloid gold label thing; Spiramvcin monoclonal antibody-colloid gold label thing is pressed 15-35 μ l/cm
2Ratio be layered on uniformly on the polyester film, put into 45 ℃ of drying boxes dry 2.5-3 hour, process the collaurum pad.
Above-mentioned collaurum redissolution liquid is to contain 0.30% Tris, 5% sucrose, 0.50%PVP, 0.30%Casein-Na, the solution of pH value 7.0-9.0.
(5) encapsulate spiramvcin coupling carrier albumen, sheep anti-mouse igg polyclonal antibody
The NC film is sticked on the PVC plate, and thieving paper is attached to and pushes down film 1-1.5mm on the PVC plate.Open and draw the film appearance, press cancel key and clean a stroke film appearance, being provided with and drawing a film instrument parameter is C line 1.0 μ l/cm, T line 1.0 μ l/cm.
The PVC plate that posts is placed on stroke film appearance, on the NC film, draws C, T line respectively with C, T line coating buffer.Draw the red marking pen of the underproof usefulness of film and mark, will encapsulate plate and put drying box and be provided with 45 ℃, dry 1-1.5 hour.
Wherein, T line coating buffer is the spiramvcin coupling carrier albumen of 0.8-3.5mg/ml; C line coating buffer is the sheep anti-mouse igg polyclonal antibody of 0.6-2.0mg/ml.
(6) processing of sample pad
Spun glass is soaked 20-40min in the PBS of 0.01M pH 7.0-8.0, wherein contain 0.5-1.5%BSA in the PBS, 0.5-1.0%Tweeen-20,38 ℃ of oven dry in drying baker are preserved, and are subsequent use.
(7) assembling kit
Get the above-mentioned collaurum pad for preparing, PVC plate and sample pad; The collaurum pad is cut into the stripe shape that width is 10mm, then the collaurum pad be affixed on NC film on the PVC plate near the T line end, press NC film 1-1.5mm; Sample pad is affixed on the top of collaurum pad, exposes gold pad 2-3mm (as shown in Figure 1).Open cutting cutter, cut width, the above-mentioned PVC plate that assembles is put on the cutting cutter, carry out slitting, obtain the said test strips that is used to detect spiramvcin by width is set by the product requirement setting; Test strips can be packed in the plastic clip, is assembled into test card.
The detection method of kit according to the invention is: with the test sample balance to room temperature; Take out the spiramvcin pick-up unit, horizontal positioned; In sample pad, add 2-3 and drip sample, observe and write down the colour developing situation of C, T line in the time of 5-10 minute, judge testing result; Or with after about 10 seconds in the terminal immersion of the spiramvcin test strip sample pad sample solution, horizontal positioned; Observe and write down the colour developing situation of C, T line in the time of 5-10 minute, judge testing result.
Kit of the present invention adopts colloidal gold immunochromatographimethod technical measurement spiramvcin, during detection, sample is added on the sample pad on the kit, can observe directly immunoreactive result, accomplishes sample detection.The present invention can be used for detecting the spiramvcin that possibly exist in the sample, have easy to use, simple to operate, be swift in response, characteristics such as economical and practical.
Description of drawings
Fig. 1 spiramvcin detection kit structural representation;
The reference numeral explanation:
1: sample pad;
2: collaurum pad (polyester film of colloid gold label spiramvcin monoclonal antibody)
3: nitrocellulose filter (T: the detection line that has encapsulated spiramvcin coupling carrier albumen; C: the nature controlling line that has encapsulated the sheep anti-mouse igg polyclonal antibody);
4: inhale the appearance pad;
The 5:PVC back up pad;
The testing result synoptic diagram of Fig. 2 kit of the present invention.
Be followed successively by line positive test symbol of C line from left to right; T, two line negative result of C; Invalid.
Embodiment 1: the preparation of spiramvcin detection kit
1. prepare spiramvcin coupling bovine serum albumin(BSA)
With spiramvcin and bovine serum albumin(BSA) coupling, synthetic spiramvcin coupling bovine serum albumin(BSA) is as immunogene and coating antigen.
2. prepare the spiramvcin monoclonal antibody
Adopting spiramvcin coupling bovine serum albumin(BSA) is the immunogen immune BALB/C mice, preparation spiramvcin monoclonal antibody.
3. preparation collaurum
Get 0.01% aqueous solution of chloraurate, heated and boiled.Add 1% citric acid, three sodium water solution 1ml as required rapidly, continue to boil about 5min, occur orange red.The size of the colloid gold particle of processing like this is 20-40nm.
4. prepare the collaurum pad
Getting grain size is the colloidal gold solution 5ml of 20-40nm, uses 0.2mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 8.0, and room temperature was placed 30 minutes; In above-mentioned solution, adding 70 μ l concentration is the spiramvcin monoclonal antibody of 3.5mg/ml, and after mixing, room temperature was placed 30 minutes; The collaurum of getting 250 μ l redissolves liquid in above-mentioned solution, mixes, and room temperature was placed 10 minutes; Above-mentioned solution left the heart 30 minutes in 12000, carefully drew supernatant, discarded, and remaining deposition is redissolved the liquid dissolving with the collaurum of 2.5ml, obtains spiramvcin monoclonal antibody-colloid gold label thing; Spiramvcin monoclonal antibody-colloid label is pressed 20 μ l/cm
2Ratio be layered on uniformly on the polyester film, put into 45 ℃ of drying boxes dry 3 hours, process the collaurum pad.
Above-mentioned collaurum redissolution liquid is to contain 0.30% Tris, 5% sucrose, 0.50%PVP, 0.30%Casein-Na, the solution of pH value 8.0.
5. encapsulate spiramvcin coupling bovine serum albumin(BSA), sheep anti-mouse igg polyclonal antibody
The NC film is sticked on the PVC plate, and thieving paper is attached to and pushes down film 1-1.5mm on the PVC plate.Open and draw the film appearance, press cancel key and clean a stroke film appearance, being provided with and drawing a film instrument parameter is C line 1.0 μ l/cm, T line 1.0 μ l/cm.
The PVC plate that posts is placed on stroke film appearance, on the NC film, draws C, T line respectively with C, T line coating buffer.Draw the red marking pen of the underproof usefulness of film and mark, will encapsulate plate and put drying box and be provided with 45 ℃, dry 1 hour.
Wherein, T line coating buffer is the spiramvcin coupling bovine serum albumin(BSA) of 3.0mg/ml; C line coating buffer is the sheep anti-mouse igg polyclonal antibody of 1.0mg/ml.
6. the processing of sample pad
Spun glass is soaked 20-40min in the PBS of 0.01M pH 8.0, wherein contain 1.0%BSA in the PBS, 0.5%Tweeen-20,38 ℃ of oven dry in drying baker are preserved, and are subsequent use
7. assembling kit
Get the above-mentioned collaurum pad for preparing, PVC plate and sample pad; The collaurum pad is cut into the stripe shape that width is 10mm, then the collaurum pad be affixed on NC film on the PVC plate near the T line end, press NC film 1-1.5mm; Sample pad is affixed on the top of collaurum pad, exposes gold pad 2-3mm (as shown in Figure 1).Open cutting cutter, being provided with and cutting width is 3.8mm, and the above-mentioned PVC plate that assembles is put on the cutting cutter, carries out slitting by width is set, and obtains the said test strips that is used to detect spiramvcin; Test strips can be packed in the plastic clip, is assembled into test card.
Embodiment 2: the detection of spiramvcin detection kit
1. the processing of sample
Serum: extract serum, centrifugal or leave standstill after get transparent supernatant and use;
Urine: directly test with urine, if urine is visible muddy shape, need centrifugal earlier, filter or treat that its post precipitation gets supernatant and detect;
Milk: 4 ℃, the centrifugal 15min of 3000r/min, detect behind the fat of removal upper strata.
Honey, egg: use 0.01mol/L, the PBS of pH 7.5 processes 1: 2 testing sample suspending liquid, concussion 10min, and the centrifugal 15min of 3000r/min gets supernatant and detects.
Tissue sample: after getting the 5-10g tissue sample and smashing to pieces, add 20-30ml (0.01mol/L, pH 7.5) PBS; The concussion 10min, in 37 ℃ of constant temperature ovens the reaction 30min after, water-bath 10min; In 4 ℃, the centrifugal 15min of 3000r/min, remove upper strata fat, get supernatant and detect.
Feed: get the feed to be measured that 0.5-2g grinds, add 8-20ml (0.01mol/L, pH 7.5) PBS, behind the reaction 30min, water-bath 10min in 4 ℃, the centrifugal 15min of 3000r/min, gets supernatant and detects in 37 ℃ of constant temperature ovens.
2. detection method:
Take out the spiramvcin detection kit, horizontal positioned; On sample pad, splash into 3 samples, observe and write down the colour developing situation of C, T line after 10 minutes, judge testing result.
3. the result judges
Positive: the T line does not develop the color, and only C line colour developing is judged to be positive findings;
Negative: T line, C line all develop the color, and are judged to be negative findings;
Invalid: the C line does not develop the color, and the rotten damage of maloperation or kit is described.
During test sample, sample fills up an end chromatography because of capillarity to inhaling appearance.If contain spiramvcin in the sample; They will and detection line (T line) on limited antibody combining site on the spiramvcin monoclonal antibody of the spiramvcin coupling bovine serum albumin(BSA) competition association colloid gold mark that encapsulates; When the spiramvcin in the sample reaches finite concentration; Also saturated fully with the spiramvcin monoclonal antibody generation immune response of colloid gold label; This moment, colloidal gold composite did not have the spiramvcin coupling bovine serum albumin(BSA) combination that encapsulates on vacant site and the detection line, and this moment, the T line did not develop the color this positive result; If do not contain spiramvcin in the sample; Mark the colloid gold particle of spiramvcin monoclonal antibody will be to the T line position in company with the sample chromatography; Immune association reaction takes place with the spiramvcin coupling bovine serum albumin(BSA) that encapsulates on the T line; Colloid gold particle is piled up at the T line position and is made the T line demonstrate a macroscopic red stripes, this negative result.No matter whether contain spiramvcin in the sample; The colloid gold label thing all can form a macroscopic red stripes with the sheep anti-mouse igg reaction that the C line encapsulates; Whether the C line develops the color; Be to judge whether enough samples are arranged, whether normal chromatography process standard simultaneously also as the inner quality standard of reagent.
Claims (6)
1. spiramvcin detection kit and preparation method thereof; It is characterized in that being made up of sample pad, collaurum pad, nitrocellulose filter, suction appearance pad and PVC back up pad, tight adhesion has sample pad, collaurum pad, nitrocellulose filter and suction appearance pad successively on the PVC back up pad.Said sample pad is a spun glass; The spiramvcin monoclonal antibody polyester film that said collaurum pad is a colloid gold label; Be coated with detection line (T line) and nature controlling line (C line) on the said nitrocellulose filter successively, wherein encapsulated spiramvcin coupling carrier albumen on the detection line (T line), encapsulated the sheep anti-mouse igg polyclonal antibody on the nature controlling line (C line); Said suction appearance pad is thieving paper.
2. described spiramvcin detection kit of claim 1 and preparation method thereof is characterized in that described spiramvcin coupling carrier albumen is spiramvcin coupling bovine serum albumin(BSA), or oralbumin, or hemocyanin; Described spiramvcin monoclonal antibody is obtained as the immunogen immune BALB/C mice by spiramvcin coupling carrier albumen.
3. described spiramvcin detection kit of claim 1 and preparation method thereof is characterized in that described collaurum is by gold chloride (HAuCl
4) under the effect of reductive agent citric acid trisodium, process size and be the colloid gold particle of 20-40nm.
4. described spiramvcin detection kit of claim 1 and preparation method thereof is characterized in that the preparation method of described collaurum pad is: get the colloidal gold solution that grain size is 20-40nm, use 0.2mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 7.0-9.0, and room temperature was placed 30 minutes; In above-mentioned solution, add the spiramvcin monoclonal antibody, the concentration that makes the spiramvcin monoclonal antibody is 20-100 μ g/ml collaurum, and after mixing, room temperature was placed 30 minutes; The collaurum of getting the amount of initial colloidal gold solution volume 5% redissolves liquid in above-mentioned solution, mixes, and room temperature was placed 10 minutes; Above-mentioned solution left the heart 30 minutes in 12000, carefully drew supernatant, discarded, and remaining deposition is redissolved the liquid dissolving with the collaurum of the initial collaurum volume of 30-70%, obtains spiramvcin monoclonal antibody-colloid gold label thing; Spiramvcin monoclonal antibody-colloid gold label thing is pressed 15-35 μ l/cm
2Ratio be layered on uniformly on the polyester film, put into 45 ℃ of drying boxes dry 2.5-3 hour, process the collaurum pad.
5. described spiramvcin detection kit of claim 1 and preparation method thereof is characterized in that described collaurum redissolution liquid is to contain 0.30% Tris, 5% sucrose, 0.50%PVP, 0.30%Casein-Na, the solution of pH value 7.0-9.0.
6. described spiramvcin detection kit of claim 1 and preparation method thereof is characterized in that the method for coating of T on the described nitrocellulose filter, C line does; The NC film is sticked on the PVC plate, and thieving paper is attached to and pushes down film 1-1.5mm on the PVC plate.Open and draw the film appearance, press cancel key and clean a stroke film appearance, the PVC plate that posts is placed on draws on the film appearance, being provided with and drawing the film instrument parameter is C line 1.0 μ l/cm, T line 1.0 μ l/cm; On the NC film, draw C, T line respectively with C, T line coating buffer.Draw the red marking pen of the underproof usefulness of film and mark, will encapsulate plate and put drying box and be provided with 45 ℃, dry 1-1.5 hour.Wherein, T line coating buffer is the spiramvcin coupling carrier albumen of 0.8-3.5mg/ml; C line coating buffer is the sheep anti-mouse igg polyclonal antibody of 0.6-2.0mg/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100764473A CN102721808A (en) | 2011-03-29 | 2011-03-29 | Spiramycin assay kit and method for making the same |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011100764473A CN102721808A (en) | 2011-03-29 | 2011-03-29 | Spiramycin assay kit and method for making the same |
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| CN102721808A true CN102721808A (en) | 2012-10-10 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2515678Y (en) * | 2002-01-10 | 2002-10-09 | 河南省农业科学院生物技术研究所 | Test paper strip for fast detecting drug residue of animal body and its product |
| CN101576563A (en) * | 2009-06-19 | 2009-11-11 | 暨南大学 | Test paper for rapidly detecting immunochromatography of cadmium ion colloidal gold and preparation method and application thereof |
| CN101839918A (en) * | 2009-11-11 | 2010-09-22 | 北京望尔康泰生物技术有限公司 | Method for detecting spiramycin and special enzyme-linked immunoassay kit thereof |
| KR20100138619A (en) * | 2009-06-25 | 2010-12-31 | 경북대학교 산학협력단 | Strip for the detection of epn and immunochromatographic assay using thereof |
-
2011
- 2011-03-29 CN CN2011100764473A patent/CN102721808A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2515678Y (en) * | 2002-01-10 | 2002-10-09 | 河南省农业科学院生物技术研究所 | Test paper strip for fast detecting drug residue of animal body and its product |
| CN101576563A (en) * | 2009-06-19 | 2009-11-11 | 暨南大学 | Test paper for rapidly detecting immunochromatography of cadmium ion colloidal gold and preparation method and application thereof |
| KR20100138619A (en) * | 2009-06-25 | 2010-12-31 | 경북대학교 산학협력단 | Strip for the detection of epn and immunochromatographic assay using thereof |
| CN101839918A (en) * | 2009-11-11 | 2010-09-22 | 北京望尔康泰生物技术有限公司 | Method for detecting spiramycin and special enzyme-linked immunoassay kit thereof |
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Application publication date: 20121010 |