CN101387644A - Melamine immune chromatography test paper detecting method - Google Patents
Melamine immune chromatography test paper detecting method Download PDFInfo
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- CN101387644A CN101387644A CNA2008101970940A CN200810197094A CN101387644A CN 101387644 A CN101387644 A CN 101387644A CN A2008101970940 A CNA2008101970940 A CN A2008101970940A CN 200810197094 A CN200810197094 A CN 200810197094A CN 101387644 A CN101387644 A CN 101387644A
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- melamine
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Abstract
The invention relates to a melamine immunity-chromatography test strip test method, comprising: the liner of the test strip is attached with a sample solution absorption part, an adhesive gold mark part, a test reaction part and a water adsorption part in turn, wherein the test reaction part is enveloped by one melamine multi-clone antibody or one detection antigen as the detection line and is enveloped by one IgG resisting the second animal protein as the reference line. The fast detection test strop has strong specificity, can realize half-quantitative detection, can be used in the environment temperature of 4 to 40 DEG C, is suitable for the fast test on the food and feeds of melamine pollution for milk product enterprises, individual cultivators, food health quality monitor departments, customs and supermarkets and the like. The melamine immunity-chromatography test strip test method has the advantages of strong specificity, high sensitivity, half-quantitative test and simple operation and the like.
Description
Technical field
Belong to the food safety detection field, be specifically related to the detection method of hazard residue thing in the food, particularly a kind of melamine test paper detecting method.
Background technology
Melamine (English name Melamine) is called for short triamine, cries 2 again, 4,6-triamido-1,3,5-triazine, 1,3,5-triazines-2,4,6-triamine, 2,4,6-triamido urea, melamine, melamine, cyanogen urea triamide, being a kind of triazines nitrogen heterocyclic ring organic compound, is important azacyclo-Organic Chemicals.Melamine molecule formula C
3N
6H
6, C
3N
3(NH
2)
3, molecular weight 126.12.
In the production and selling process of food, need to measure the protein content of food, and directly measure the technical more complicated of protein, so adopt Kjeldahl to measure protein content usually, promptly calculate Protein content indirectly by the content of measuring nitrogen in the food.Because melamine is compared with protein and is contained more nitrogen-atoms, so the melamine that is added in the food can cause the higher illusion of content in food, thereby is utilized by illegal retailer.Melamine is ingested behind the human body, enters blood circulation and finally enters kidney by little intestinal absorption, forms kidney stone, especially for the baby of lactation, and the easier calculus that causes.
The main at present method of instrument detecting that adopts detects melamine.Main method has: GC-MS method, the online detection of Spectra-Quad, Ultra Performance Liquid Chromatography-esi-msn method, reversed-phased high performace liquid chromatographic, high performance liquid chromatography-diode array, high performance liquid chromatography (HPLC), high performance liquid chromatography-quadrupole rod mass spectrometry, Solid-Phase Extraction and high performance liquid chromatography coupling, liquid chromatography tandem mass spectrometry (LC-MSMS) etc.These instrumental methods cost an arm and a leg, and the method complicated operation, could operate by the personnel of specialized training, and exist problems such as narrow application range, apparatus expensive, cost height.At present the present technique field also do not have a kind of high specificity, highly sensitive and simple to operation, suitable on-the-spot fast detect and the dairy products of the requirement of extensive generaI investigation in the melamine detection method.
Summary of the invention
The objective of the invention is provides a kind of high specificity, highly sensitive and simple to operation in order to overcome the deficiencies in the prior art, and sample is passed through the quick test paper semi-quantitative detection method of melamine that simple process can detect.
Purpose of the present invention can be achieved through the following technical solutions.
Its technical essential of melamine immune chromatography test paper detecting method is: posted sample liquid absorption portion and colloid gold label part and detection reaction part and the part that absorbs water on the quilt lining of test paper successively, the material that colloid gold label partly is labeled is that the second kind animal protein and melamine detect with the potpourri of antigen or the potpourri of the second kind animal protein and melamine antibody; Be coated with 1 of melamine antibody above the detection reaction part as detection line, 1 of the IgG that also is coated with the anti-second kind animal protein simultaneously is as reference line or be coated with melamine and detect with the antigen 1 bar as detection line, and 1 of the IgG that also is coated with the anti-second kind animal protein simultaneously is as reference line.By regulating the concentration of detection line and reference line encrusting substance, the colour developing depth of detection line and reference line during control detection, and its colour developing depth and standard substance concentration be mapped reaches half-quantitative detection when test paper prepares.
Described detection is meant the conjugates that melamine and carrier mass gelatin form with antigen, and immunity is meant the conjugates that melamine and carrier mass keyhole limpet hemocyanin (KLH) form with antigen.
Described carrier mass also can be selected other macromolecular substances for use, as: lipoprotein, polyamino acid, glucosan, oralbumin etc., do not have cross-immune reaction but require to detect with antigen and immune carrier with antigen.
The described second kind animal protein refers to that the non-antibody source belongs to the albumen of animal, and for example, antibody is rabbit source property, and then the second kind animal protein can be chicken, pig or other non-rabbit source property animal proteins such as oralbumin, porcine hemoglobin.
The each several part of test paper described in the invention is handled with function as follows:
Served as a contrast:,, play fixing other ingredients of test paper of supporting as the PVC plate for simultaneously scribbling the toughness material that does not absorb water of adhesive sticker.
The preparation of sample liquid absorption portion: with 2min among the PBS of filter paper or all-glass paper immersion pH7.0-8.4, take out, 80 ℃ of oven dry or other mode dryings promptly as sample liquid absorption portion, play a part during detection to absorb sample solution, are convenient to sample solution and move up.
The preparation of colloid gold label part: this part plays the fixedly antigen or the antibody of colloid gold label.Preparation process comprises preparation, colloid gold label melamine antigen or melamine antibody, the colloid gold label section processes of colloidal gold solution.
(1) preparation of colloidal gold solution: with gold chloride (HauCl
4) be mixed with 1% mother liquor with ultrapure water, get the mother liquor of 1mL, be settled to 100mL with ultrapure water, be made into 0.01% solution, be heated to boiling, add the trisodium citrate aqueous solution of 1-5mL1%, continue to be heated to occur transparent orange red till, be colloidal gold solution.
(2) colloid gold label melamine antigen: the melamine detection is used PBS (0.01mol/L respectively with the antigen and the second kind animal protein, pH7.0-7.5) dissolved dilution is to 2-4mg/mL, every 100mL colloidal gold solution adds the detection of 1-3mL 2-4mg/mL with antigen and 1-1.5mL 2-4mg/mL second kind animal protein concussion 2min, with the K of 0.2mol/L
2CO
3Regulate pH to 8.4, concussion 5min adds 11% PEG-10000 2mL, concussion 5min, the centrifugal 15min of 8000-15000r/min removes supernatant, will precipitate (the 0.01mol/L with PBS, pH7.0-7.5) redissolve, with the centrifugal 15min of 6000-13000r/min, remove supernatant, will precipitate (0.01mol/L with PBS, pH7.0-7.5) dilution is 500-2000 times, and product is as golden mark melamine antigen (GAg).
(3) colloid gold label melamine antibody: melamine monoclonal antibody or polyclonal antibody and the second kind albumen are used PBS (0.01mol/L respectively, pH7.0-7.5) dissolved dilution is to 2-4mg/mL, every 100mL colloidal gold solution adds melamine antibody and the 1-1.5mL2-4mg/mL second kind animal protein of 1-3mL 2-4mg/mL, concussion 2min is with the K of 0.2mol/L
2CO
3Regulate pH to 8.4, concussion 5min adds 11% PEG-10000 2mL, concussion 5min, the centrifugal 15min of 8000-15000r/min removes supernatant, will precipitate (the 0.01mol/L with PBS, pH7.0-7.5) redissolve, with the centrifugal 15min of 6000-13000r/min, remove supernatant, will precipitate (0.01mol/L with PBS, pH7.0-7.5) dilution is 500-2000 times, and product is marked melamine antibody (GAb) as gold.
(4) colloid gold label section processes: GAg or GAb are poured in the groove, glass fibre or filter paper are immersed 1min, take out, after the drying at room temperature, promptly as the colloid gold label part.
Detection reaction partly prepares: glutaraldehyde solution with 0.8% or 0.2% carbodiimide solution soak nitrocellulose membrane or cellulose acetate film or nylon membrane 30min, take out, 37 ℃ of oven dry, the top bag is detected by the melamine of 1 variable concentrations uses antigen line or melamine antibody line as detection line, wraps simultaneously by the IgG line of 1 anti-second kind animal protein as the reference line.When colloid gold label part tagged object was the melamine antibody and the second kind animal protein, detection line then wrapped by melamine detection antigen; When colloid gold label part tagged object was the melamine detection usefulness antigen and the second kind albumen, detection line then wrapped by melamine antibody.This is the detection reaction part, and it is that reaction result is come out with macroscopic characterization that this part mainly acts on.
Suction part preparation: after all-glass paper or filter paper or thieving paper drying at room temperature, promptly as the suction part.This part mainly acts on the unnecessary sample solution that is moving up and absorbs.
Test paper assembling: on backing, paste sample liquid absorption portion, colloid gold label part, detection reaction part and suction part successively, melamine detect test paper.
Detect principle: detect the object difference of principle because of colloid gold label, and variant slightly.
When colloid gold label part tagged object is the melamine detection usefulness antigen and the second kind animal protein, if contain melamine in the sample, sample solution is by the absorption of the sample liquid absorption portion of test paper and by moving on the capillary action, the little translational speed of free melamine molecule amount in the sample is fast, arrive detection line earlier, earlier with detection line on wrap quilt melamine antibody combine, because of the melamine antibody that wraps quilt on the detection line has only specific binding site, and the melamine binding ability of dissociating in the sample is stronger with antigen than the melamine detection of parataxic, so the detection of colloid gold label is caught with the melamine antibody of antigen on can not tested again survey line, so detection line is colourless, this is promptly positive; If there is not melamine in the sample, then the melamine of colloid gold label detects with catching with regard to the melamine antibody of bag quilt on the tested survey line behind the antigen arrival detection line, forms macroscopic redness, and this is promptly negative.No matter whether contain melamine in the sample, move on on the second kind animal protein of colloid gold label all can be caught by the IgG of the anti-second kind animal protein of bag quilt on the reference line when reaching reference line and form macroscopic redness, this is reference line.
When colloid gold label part tagged object is the melamine antibody and the second kind animal protein, if contain melamine in the sample, sample solution is absorbed by the sample liquid absorption portion of test paper and reaches the colloid gold label part by moving on on the capillary action, melamine in the sample solution and the reaction of the melamine antibody of colloid gold label form bond, bond moves on to detection line on continuing, because of the melamine antibody of colloid gold label has only specific binding site, melamine in the sample solution with it in conjunction with after, detection on the detection line just can not combine with the melamine antibody of colloid gold label with antigen again, so detection line is colourless, this is promptly positive; When not having melamine in the sample, the detected antigen capture of the using when melamine antibody of colloid gold label arrives detection line then forms macroscopic redness.No matter whether contain melamine in the sample, move on on the second kind albumen of colloid gold label all can be caught by the IgG of the anti-second kind animal protein of bag quilt on the reference line when reaching reference line and form macroscopic redness, this is reference line.
The test paper of above dual mode, when preparing, passes through by test paper to regulate detection line and reference line encrusting substance concentration, the colour developing depth of detection line and reference line during control detection, and its colour developing depth and standard substance concentration is mapped, can reach the half-quantitative detection purpose.
The beneficial effect that the present invention had
1. high specificity.Melamine in the energy specific recognition sample.
2. highly sensitive.Be limited to 5 μ g/kg under the lowest detection of this test strips.Although China as yet not in the regulation food the residual of melamine limit the quantity of, the lowest detection lower limit of this test strips has been significantly less than the significant amount of mingling of melamine in dairy products or other food.
3. can realize half-quantitative detection.This test paper can reach the half-quantitative detection purpose according to detection line color and reference line color contrast.
4. simple to operation.The liquid towards dairy products can directly detect, and can detect after dissolving or simple process solid-state or Powdered foodstuff samples.This test paper does not need professional training, and without any need for complex instrument equipment, common layman can detect.
Description of drawings
Accompanying drawing detects the structural representation of test paper for the present invention.1 is that sample liquid absorption portion, 2 is that colloid gold label part, 3 is that detection reaction part, 4 is that detection line, 5 is that reference line, 6 is the part that absorbs water among the figure.
Embodiment
Below in conjunction with accompanying drawing the present invention is described in further detail:
Article 1, the test paper of detection line+1 a reference line form
Detection line is coated with melamine polyclonal antibody or melamine detection the antigen 1 bar, wide 1mm that concentration is 0.1-30 μ g/mL; Reference line is coated with 1 of the IgG that concentration is the anti-second kind animal protein of 0.1-30 μ g/mL, wide 1mm; Article two, 2-6mm at interval between the line.When the detection line color was identical with the reference line color, then sample was negative; When detection line was more of light color than reference line, then sample was positive, and concentration is greater than A μ g/kg, and the detection line of A for setting can be a certain concentration more than or equal to 5 μ g/kg.Reference line develops the color all the time, if reference line does not develop the color, shows that test paper lost efficacy.
Further specify below:
Melamine coupling antigen is synthetic
Melamine coupling antigen is divided into immunity and uses antigen with antigen and detection, and the former is used for immune animal, and the latter is used for colloid gold label or the detection line bag quilt in Antibody Preparation detection of antibodies and the test paper preparation.
Immunity antigen preparation: adopt glutaraldehyde to make crosslinking chemical, make the amino of keyhole limpet hemocyanin (KLH) carrier and micromolecule haptens melamine be connected synthetic immune holoantigen with covalent bond.The 200mg melamine is dissolved in the pyridine of 30mL, get among the PBS (pH7.2) that 400mg KLH is dissolved in 25mL 0.01mol/L, under agitation slowly add melamine solution in the KLH solution, slowly splash into 0.40mL 25% glutaraldehyde solution again, behind the stirring at room reaction 4h, under 4 ℃, synthetic product was dialysed 3 days with 0.01molL-1PBS (pH7.2), change dislysate every day 4 times, to remove unreacted small-molecule substance such as glutaraldehyde, melamine etc.Can obtain melamine-KLH holoantigen after the freeze-drying.
Detect and use antigen: replace KLH with gelatin, adopt synthetic identical step with melamine-KLH,
Melamine and synthetic detection of gelatin coupling are used antigen melamine-gelatin.
The melamine Polyclonal Antibody Preparation
4 of male new zealand rabbits about selection body weight 2kg are used immunizing antigen melamine-KLH, adopt routine immunization program immunity new zealand white rabbit, the preparation polyclonal antibody.Concrete steps are: the immunizing antigen of preparation is dissolved with 1% salt solution, and fully emulsified with isopyknic complete Freund's adjuvant, making the immunizing antigen final concentration is 2mg/mL.In the hypodermic injection of rabbit back, every injection 1mL.After two weeks, the full Freund replacement complete Freund's adjuvant that toos many or too much for use with quadrat method emulsification immunizing antigen, in rabbit vola hypodermic injection, every is injected 1mL after the emulsification by above-mentioned.Carried out the primary reinforcement immunity later in per 7 days, each reinforced immunological adopts the immunizing antigen that does not add any adjuvant, and the concentration of used immunizing antigen is 2mg/mL, and consumption is 1mL, in the hypodermic injection of rabbit vola.Adopt immunoprecipitation experiment to detect antibody titer before each reinforced immunological, when antibody titer no longer continues to raise, stop reinforced immunological, carry out 5-7 time reinforced immunological usually altogether.Can adopt the arteria carotis blood taking method to collect antiserum on the 3rd day behind the last reinforced immunological, sad-ammonium sulfate method antibody purification, 0.01mol/L PBS (pH7.2) dialysis is till extracellular fluid dialysis and barium chloride reaction nothing precipitation, add final concentration and be 0.01% Sodium azide, after the packing in-70 ℃ of cryopreservation.
Sample liquid absorption portion is handled
Filter paper or all-glass paper etc. are immersed 2min among the PBS of 0.1mol/L pH7.4, take out 80 ℃ and dry or other mode dryings, sample liquid absorption portion.
The preparation and the processing of colloid gold label part
The preparation of colloid gold label part and processing comprise colloidal gold solution preparation, collaurum mark melamine antibody, colloid gold label section processes.
Colloidal gold solution preparation: with gold chloride (HAuCl
4) be mixed with 1% mother liquor with ultrapure water, get the mother liquor of 1mL, be settled to 100mL with ultrapure water, be made into 0.01% solution, be heated to boiling, add a certain amount of 1% trisodium citrate aqueous solution, continue to be heated to occur transparent orange red till, be colloidal gold solution.
Colloid gold label melamine polyclonal antibody preparation: get the 10mL colloidal gold solution, add 10% K
2CO
3Solution is to pH7.4, the melamine polyclonal antibody that adds 120 μ L 3mg/mL then, the room temperature lower magnetic force stirs 15min, the adding final concentration is 0.2% Macrogol 2000 0, continue to stir until dissolving, put 4 ℃ of refrigerator 2h then, in 4 ℃, the centrifugal 15min of 15000rpm can see mobile golden labeling antibody precipitation at the pipe end, abandon supernatant, use dilution (containing 8% sucrose, 0.05% Tween20, the 0.01mol/LPBS of the pH7.4 of 0.5% bovine serum albumin(BSA)) that precipitation is returned to original volume then, in 4 ℃, the centrifugal once more 15min of 15000rpm abandons supernatant, precipitates with above-mentioned dilution 1mL mixing, adding Sodium azide, to make final concentration be 0.01%, and it is standby to be placed on 4 ℃ of refrigerators.
The colloid gold label section processes: gold is marked the melamine polyclonal antibody pour in the groove, all-glass paper or filter paper are immersed 1min, take out, drying at room temperature promptly becomes the colloid gold label part.
The processing of detection paper reactive moieties
The processing of detection paper reactive moieties
Glutaraldehyde solution with 0.8% or 0.2% carbodiimide solution soak nitrocellulose membrane 30min, takes out, and 37 ℃ of oven dry, the top is detected with the antigen line as detection line by 1 melamine with flush coater spraying bag, and concentration is 2 μ g/mL.As the reference line, concentration is 2 μ g/mL to bag by the IgG line of 1 goat anti-rabbit immunoglobulin.Be the detection reaction part.
Suction section processes and test paper assembling
After the thieving paper drying at room temperature, promptly as the suction part.
On by lining, paste sample liquid absorption portion, colloid gold label part, detection reaction part and suction part successively, array configuration be the quick detection test paper of the melamine of 1 detection line+1 reference line.
Detect liquid diary product: concentration and viscosity are done suitable dilution (1-10 doubly) with PBS solution per sample, and gained solution is as detecting liquid.Take out after directly detection paper partly being immersed detection liquid 2min then, observe color.If detection line presents red color, show that sample is negative; If detection line does not develop the color, show that content of melamine is more than the 5 μ g/kg in the sample.Regardless of content of melamine in the sample, reference line should develop the color, if reference line does not develop the color, shows that test paper lost efficacy.
Solid-state dairy products: with PBS solution with sample dissolution and do suitable dilution (by solid-to-liquid ratio 1: 5-10), press the detection of liquid diary product same procedure then.
Other foodstuff samples: fatty sample is removed fat earlier, then sample is pulverized or is smashed to pieces powdered or be dissolved in the PBS solution, detects according to above-mentioned dairy products disposal route again; Not fatty sample after can directly sample being pulverized or smash to pieces, be dissolved in the PBS solution, detects according to above-mentioned dairy products disposal route again.
Claims (4)
1. melamine immune chromatography test paper detecting method, it is characterized in that: post sample liquid absorption portion and colloid gold label part and detection reaction part and the part that absorbs water on the quilt lining of test paper successively, the material that colloid gold label partly is labeled is that the second kind animal protein and melamine detect with the potpourri of antigen or the potpourri of the second kind animal protein and melamine antibody; Be coated with 1 of melamine antibody above the detection reaction part as detection line, 1 of the IgG that also is coated with the anti-second kind animal protein simultaneously is as reference line or be coated with melamine and detect with the antigen 1 bar as detection line, and 1 of the IgG that also is coated with the anti-second kind animal protein simultaneously is as reference line.
2. melamine immune chromatography test paper detecting method according to claim 1 is characterized in that: it is the conjugates that melamine and carrier mass form that melamine detects with antigen.
3. melamine immune chromatography test paper detecting method according to claim 2 is characterized in that: described carrier mass is protein or protein fragments or synthetic polypeptide or semi-synthetic polypeptide or polysaccharide.
4. melamine immune chromatography test paper detecting method according to claim 1 is characterized in that: the described second kind animal protein is the protein of non-antibody source animal.
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|---|---|---|---|
| CNA2008101970940A CN101387644A (en) | 2008-09-27 | 2008-09-27 | Melamine immune chromatography test paper detecting method |
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|---|---|---|---|
| CNA2008101970940A CN101387644A (en) | 2008-09-27 | 2008-09-27 | Melamine immune chromatography test paper detecting method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101650314B (en) * | 2009-09-15 | 2011-07-20 | 中国人民解放军第三军医大学 | Chroma detection method of tripolycyanamide contained in raw milk and milk product and kit |
| CN102297964A (en) * | 2011-05-20 | 2011-12-28 | 广州万孚生物技术有限公司 | Immunity chromatography detection test strip of melamine |
| CN102297970A (en) * | 2011-05-27 | 2011-12-28 | 重庆市科学技术研究院 | Two-joint detection test strip for rapidly detecting residue of cyromazine (CA) and melamine (MA) and preparation method thereof |
| CN103293312A (en) * | 2013-06-14 | 2013-09-11 | 张峰 | Ultrasensitive lateral chromatography rapid detection method based on luminescent quantum dots, and preparation method of test strip |
| CN104950089A (en) * | 2014-03-25 | 2015-09-30 | 兰州红虫生物工程有限责任公司 | Rapid melamine detection test paper strip |
-
2008
- 2008-09-27 CN CNA2008101970940A patent/CN101387644A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101650314B (en) * | 2009-09-15 | 2011-07-20 | 中国人民解放军第三军医大学 | Chroma detection method of tripolycyanamide contained in raw milk and milk product and kit |
| CN102297964A (en) * | 2011-05-20 | 2011-12-28 | 广州万孚生物技术有限公司 | Immunity chromatography detection test strip of melamine |
| CN102297970A (en) * | 2011-05-27 | 2011-12-28 | 重庆市科学技术研究院 | Two-joint detection test strip for rapidly detecting residue of cyromazine (CA) and melamine (MA) and preparation method thereof |
| CN103293312A (en) * | 2013-06-14 | 2013-09-11 | 张峰 | Ultrasensitive lateral chromatography rapid detection method based on luminescent quantum dots, and preparation method of test strip |
| CN104950089A (en) * | 2014-03-25 | 2015-09-30 | 兰州红虫生物工程有限责任公司 | Rapid melamine detection test paper strip |
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Application publication date: 20090318 |