CN200989901Y - Tylosion residual fast detection test paper strip - Google Patents
Tylosion residual fast detection test paper strip Download PDFInfo
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- CN200989901Y CN200989901Y CN 200620135124 CN200620135124U CN200989901Y CN 200989901 Y CN200989901 Y CN 200989901Y CN 200620135124 CN200620135124 CN 200620135124 CN 200620135124 U CN200620135124 U CN 200620135124U CN 200989901 Y CN200989901 Y CN 200989901Y
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- tylosin
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- test
- fibrage
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Abstract
The utility model discloses a tylosin residue fast detecting test-paper strip. A bottom layer of the test-paper strip is a support layer; a middle layer of the test-paper strip is an adsorbing layer and is fixed on the support layer; an external layer of the test-paper strip is a protecting membrane which is fixed on the adsorbing layer which is sequentially provided, from a test end, with a fiber layer, an adsorption colloidal-gold antibody fiber layer, a cellulose film and a water adsorption material layer of a handle end; wherein the cellulose film is provided with a beeline-type detecting print which is printed by a carrier albumen liquor of coupled tylosin, and a beeline-type comparing print which is printed by a goat-anti or rabbit-anti mouse IgG liquor; the detecting print and the comparing print are arranged as a upright parallel shape in a combined and parallel way. The test-paper strip has the advantages of that the specificity is strong, the sensitivity is high, minim tylosin residue of 0.1 nanogram can be detected, the operation is simple, convenient and fast, the detecting result can be determined within two minutes, the result display is visual, intuitionistic and exact, the mistaken determination such as false negative and false positive can not occur, the cost is low, the applicable range is wide, and the utility model is convenient to be popularized and generalized.
Description
One, technical field:
The utility model relates to a kind of utensil that detects veterinary drug, particularly relates to the residual quick test strip of a kind of tylosin (Tylosin is called for short TL).
Two, technical background:
Tylosin is a Tri-Biocin, is used for gramnegative bacterium and mycoplasma infection.From a similar strain cultured solution of streptomyces fradiae, obtain, be white crystals, be slightly soluble in water, the tool alkalescent, soluble in water behind the general salify, tylosin has antibacterial action to gram-positive bacteria and some negative bacterium, and is all responsive to staphylococcus aureus, streptococcus pyogenes, streptococcus pneumonia, corynebacterium pyogenes.In honeybee breed, its feeding process, the veterinary drug tylosin not only is used to prevent and control the disease of livestock and poultry, honeybee, and has growth promoting function, it is the medicine that extensively makes an addition at present in the feed, its drug resistance problem becomes increasingly conspicuous, and this is one of products such as honeybee, milk cow, live pig, poultry key factor of becoming non-safe food.European Union has forbidden tylosin as far back as 1999 and has used as feed addictive, and maximum residue limit(MRL) in the products such as livestock and poultry meat and milk, honey has been stipulated in Hong Kong.China's forbidding compound tylosin, simultaneously in The Ministry of Agriculture of the People's Republic of China, MOA's bulletin (No. 278) strict regulations the withdrawal time of tylosin in the breed of pig ox, " feed and feed addictive criterion " clearly provides against and uses tylosin as medicine forage adjuvant in the first batch of green livestock products authentication criterion of China, nuisanceless bee product has been stipulated the examination criteria and the method for tylosin residue detection, and the tylosin conventional detection mainly contains high pressure lipuid chromatography (HPLC), vapor-phase chromatography, makings logotype method and ELISA etc.Chromatographic technique is very effective, accurate, responsive method, but sample to be checked needs through a series of pre-service, and is loaded down with trivial details time-consuming, needs 2～3 day time from sample pretreatment at least to drawing assay, and testing cost is very high.This on the other hand detection method also must have expensive instrument and equipment.Have only specific professional to use, and each sample that detects is limited, the examination of incompatibility gross sample has seriously hindered applying of this detection method, does not more fit into field test.ELISA also is a kind of detection technique commonly used, and it is to serve as to detect principle with competitiveness enzyme-linked reaction, detects overall process and needs 2-4 hour approximately.Because ELISA is the very strong detection method of a kind of susceptibility, different operators tends to occur different results, so this method usually causes artificial flase drop and omission.Therefore in actual production, be badly in need of a kind of detection method of fast detecting tylosin.Present Food Inspection at different levels department, especially foreign export unit have strengthened the inspecting force of tylosin, to guarantee the people's animal food safety.Therefore development is sensitive, fast, and tylosin detection method accurately detects product such as honey, milk and domestic animals and fowls and organizes and whether contains tylosin in the feed crucial meaning is arranged.
Three, utility model content:
The purpose of this utility model: develop special, responsive, quick, easy quick testing strip for residual pesticide.
The technical solution of the utility model:
A kind of quick testing strip for residual pesticide; bottom is a supporting layer; the middle layer adsorbed layer is fixed on the supporting layer; outer protective film is fixed on the adsorbed layer; adsorbed layer is followed successively by fibrage from test lead; ADSORPTION OF GOLD labeling antibody fibrage; the absorbent material layer of cellulose rete and handle end; wherein the orthoscopic that the carrier protein solution of useful coupling tylosin is printed on the cellulose rete detects trace; with the orthoscopic contrast trace that goat-anti or the anti-mouse IgG of rabbit solution are printed, detecting trace is " ‖ " with the parallel assembled arrangement of contrast trace.
Supporting layer is to make with the hard plastic slip or the cardboard bar that do not absorb water.
The test lead fibrage is made with glass wool or nylon membrane or polyvinylidene fluoride (PVDF) film or polyester film, the preferred glass cotton, and absorbent material layer is made with thieving paper.
Cellulose rete nitrocellulose filter or pure cellulose film, or the carboxylation cellulose membrane is made.
The gold labeling antibody fibrage golden labeling antibody glass wool of absorption tylosin, tylosin gold labeling antibody is the monoclonal antibody or the polyclonal antibody of colloid gold label tylosin, and the carrier protein solution of coupling tylosin is the composite solution of tylosin and carrier protein couplet.
On test lead adsorbing fiber layer, golden labeling antibody fibrage and absorbent material layer, be coated with diaphragm; on the test lead fibrage diaphragm corresponding, be printed with the sample mark line, the about 0.5cm of this mark line deflection test lead fibrage one side place with golden labeling antibody fibrage intersection.
The carrier protein of coupling tylosin is bovine serum albumin(BSA) (BSA), or is the pure albumen of ovum gallinaceum (OVA), or is ferritin, or is hemocyanin.
Quick testing strip for residual pesticide of the present utility model has the following advantages:
(1) high specificity, the susceptibility height.This test strips is that the basis is prepared from the monoclonal antibody (W1) of colloid gold label high-affinity, no covalent bond 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, the collaurum mark is very little to monoclonal antibody (W1) specificity and affinity influence, and has higher mark rate.Therefore, the quick detection test paper bar has higher specificity and susceptibility, and the micro-TL that can detect 0.1 nanogram is residual.
(2) easy and simple to handle, quick.Need not any other reagent when using the utility model test strips, as long as be inserted into sample to be checked about 10 seconds, was the decidable testing result in 2 minutes.
(3) result shows image, directly perceived, accurate.This test paper slip is to show that rufous " | " and " ‖ " trace are as the positive and the negative marker that detect, showing on cellulose membrane promptly that a brownish red " | " trace is illustrated in the detected sample liquid contains TL, article two, brownish red " ‖ " trace is illustrated in and does not contain TL in the test sample, the result judges image, directly perceived, accurate, simple and clear, be not prone to the erroneous judgement of false negative and false positive.
(4) cost is low, small investment.Use the quick detection test paper bar, do not need to join in addition instrument and equipment and other reagent, detect whenever and wherever possible, expense is cheap, can save a large amount of expensive instruments and cost of equipment.
(5) applied range, easy to utilize.The utility model quick detection test paper is simple to operate, can satisfy different levels personnel's needs, comprise that professional chemical examination, customs quarantine control, health and epidemic prevention, quality monitoring, livestock products processing, intensive culture arrive individual breed etc., have vast market prospect and better economic, social benefit.
Four, description of drawings:
Fig. 1 is the quick testing strip for residual pesticide structural representation.
Fig. 2 is a quick testing strip for residual pesticide plan structure synoptic diagram.
Among the figure, 1: supporting layer, 2: test lead fibrage, 3: golden labeling antibody fibrage; 4: cellulose rete, 5: absorbent material layer, 6: the stealthy trace band that detects, 7: stealthy contrast trace band; 8-1: test lead diaphragm, 8-2: handle end diaphragm, 9: mark line.
Five, embodiment:
The preparation quick testing strip for residual pesticide at first need prepare carrier protein and the golden labeling antibody of coupling TL, detects trace and golden labeling antibody fiber thereby prepare; Secondly need preparation goat-anti or the anti-mouse IgG antibody of rabbit, be used for preparation contrast trace.
1. the coupling of tylosin TL and carrier protein Xi
Adopt ethyloic hydroxylamine assay or carbodiimides (EDC) method
Carbodiimides (EDC) method: TL and carrier protein are carried out coupling prepare immunizing antigen.The 5mg tylosin is dissolved in the phosphate buffer in (pH7.0), adds 10mgEDC, the room temperature lucifuge stirred 6 hours.Carrier bovine serum albumin(BSA) (or pure albumen of ovum gallinaceum or ferritin, or hemocyanin) is dissolved in the PBS damping fluid, added in 25: 1 in molar ratio and to go up in the reaction, the greenhouse stirs and spends the night.Reactant liquor is got final product with PBS (phosphate buffer) dialysis.
2. the preparation of anti-tylosin monoclonal antibody (W1)
Carrier protein immunity BALB/c with 50～100 μ g/ coupling TL only is mouse three times, each 15～30 days at interval; Behind the booster immunization 3～4 days for the third time, with the bloodletting of immune mouse eyeball, draw neck to cause death, with 75% alcohol-pickled 5～10min, aseptic its splenocyte of getting; Shred and through 100 order nylon net filters, the centrifugal 10min of 1000r/min collects splenocyte; With 1 * 10
8Splenocyte and 2～5 * 10
7NSO myeloma cell, 1000r/min is centrifugal, and 10min abandons supernatant, cell precipitation slowly adds 40～50%PEG4000 (pH8.5～9.0) effect 1min of 0.7～1ml in 37 ℃ water-soluble, slowly add serum-free 1640 nutrient culture media 15ml then, to stop the effect of PEG, 37 ℃ of water-bath 5～10min, 1000r/min is centrifugal, and .10mm abandons supernatant, cell precipitation is resuspended in HAT selects in the nutrient culture media, and add 96 well culture plates (100～200 μ l/ hole), place 37 ℃ of 5%CO
2Cultivate in the incubator.Cultivate after 7～10 days,, detect the culture supernatant of hybridoma, picking epistasis cell clone (OD with enzyme linked immunosorbent assay (ELISA) with the carrier protein coated elisa plate (96 holes/piece) of the coupling TL of 5～10 μ g/ml
450More than=0.5), carry out continuous three times limiting dilution assay cloning, the hybridoma chromosome number of being produced is 92～98, the monoclonal antibody of its secretion (W1) specifically with TL reaction, and not with other albumen generation cross reaction, affinity constant reaches 10
9～10, light chain subtype is κ or λ, the heavy chain hypotype is IgG
1, IgG
2a, IgG
2b, IgG
3, the monoclonal antibody (W1) at TL specific antigen determinant is used to prepare golden labeling antibody.
3. the preparation of tylosin gold labeling antibody (W1) and golden labeling antibody glass wool
Prepare aurosol with the sodium citrate reducing process, promptly in 0.01～0.05% aqueous solution of chloraurate of 50～100ml boiling, add 0.5～2% citric acid three sodium solution of 2～4ml, obtain the collaurum about diameter 15nm.K with 0.1mol/L
2CO
3Transfer collaurum pH to 8.5～9.5, place the aurosol that TL monoclonal antibody (M1) to be marked is added pH8.5～9.5 with 1: 1000～1300 mark ratio, behind the mark 10min, add 20%PEG10000 to final concentration 0.05%, 4 ℃, the centrifugal 20min of 1500～3000rpm, remove unconjugated colloid gold particle, 4 ℃, the centrifugal 1h of 15000rpm, abandon supernatant, after obtaining preliminary purification gold labeling antibody protein mixture, with propylene glucosan S-400 column chromatography, separation and purification gold mark albumen obtains TL colloid gold label antibody.With 1: the glue gold labelled antibody of (100～500) dilution is adsorbed in the processed glass cotton, 4 ℃ of low-temperature vacuum dryings, preparation TL gold labeling antibody glass wool.
4. the preparation of the anti-mouse IgG of goat-anti or rabbit
Extract mice serum IgG with saturated ammonium sulfate, get 1 part of mice serum and add 2 parts of PBS (pH72) mixing, add equal-volume saturated ammonium sulfate mixing, put 4 ℃ of refrigerator 2h, 4 ℃, the centrifugal 15min of 1200r/min abandon supernatant; With an amount of PBS (pH7.2) dissolution precipitation, add saturated ammonium sulfate to final concentration 33%, put 4 ℃ of refrigerator 2h, 4 ℃, the centrifugal 15min of 1200r/min, abandon supernatant, with a small amount of PBS (pH7.2) dissolution precipitation, put in 4 ℃ of refrigerators with PBS (pH7.2) dialyzed overnight, change liquid 2～3 times, 4 ℃, the centrifugal 15min of 12000r/min, collect supernatant, measure its protein concentration with ultraviolet spectrophotometer, with 50～100 μ g/kg body weight (mice serum IgG) through subcutaneous and intramuscular injection immune health sheep or rabbit 3～4 times, the last immunity is after 10 days, venous blood collection is measured its serum antibody titer more than 1: 2000 with ELISA, heart blood sampling or arteria carotis bloodletting, collect its hyper-immune serum, extract goat-anti or the anti-mouse IgG of rabbit (method is identical with extraction mice serum IgG, does not repeat) with saturated ammonium sulfate, be used for the preparation that the contrast of TL quick detection test paper bar shows trace.
5. tylosin TL quick detection test paper bar is implemented the detection reaction principle
After tylosin TL quick detection test paper bar sample end inserts detected sample solution, solution to be checked spreads to nitrocellulose filter together by the golden labeling antibody (W1) that siphon drives in TL to be checked and the golden labeling antibody cotton, and finally infiltrate in the handle end filter paper, TL to be checked can combine with golden labeling antibody (W1) in the diffusion process, and then seal the antigen-combining site that golden labeling antibody (W1) is gone up TL, stop the detection trace of the carrier protein (Xi) of coupling TL on golden labeling antibody (W1) and the cellulose membrane to combine, can not show the detection trace, the anti-mouse IgG of goat-anti or rabbit (Y) then can combine with golden labeling antibody (W1), form rufous contrast trace " | ", the i.e. positive mark of a rufous " | " trace, otherwise no TL then can not stop the carrier protein (Xi) of coupling TL on golden labeling antibody (W1) and the cellulose membrane to detect trace to combine in the sample solution, show that rufous detects trace " | ", same goat-anti or the anti-mouse IgG of rabbit (Y) also combine with golden labeling antibody (W1), show rufous contrast trace " | ", form the negative mark of two rufous " ‖ ", if do not have the rufous mark to show on the cellulose membrane, show that then test strips lost efficacy.
Embodiment one: the structure of quick testing strip for residual pesticide, and referring to Fig. 1, Fig. 2.1 is supporting layer among the figure, make with plastic strip, 2 is the fibrage of specimen end, make with glass wool, 3 for being adsorbed with golden labeling antibody (W1) fibrage of tylosin TL, according to the preparation method described in the above-mentioned embodiment 3, preparation is adsorbed with golden labeling antibody (W1) glass wool of tylosin monoclonal antibody, 4 is the cellulose rete, adopt nitrocellulose filter, 5 is absorbent material layer, makes with absorbent filter, to number 2,3,4,5 each layers and be pasted and fixed on successively from left to right on the plastic strip 1, the infiltration that crosses one another of each layer intersection fiber.On cellulose nitrate rete 4,6 for bovine serum albumin(BSA) (BSA) solution with the coupling tylosin stamps detection trace " | ", and 7 for stamping contrast trace " | " with goat anti-mouse igg (Y) solution, and two traces are arranged in parallel and form combination trace band " ‖ ".8-1 covers fibrage 2 and the white of the specimen end above the golden labeling antibody fibrage 3 diaphragm; on the corresponding diaphragm 8-1 of 2 and 3 intersections position, be partial to fibrage 2 one side 0.5cm places and be printed on mark line 9; 9 right-hand member is printed on arrow and max printed words, is coated with other color (as yellow) diaphragm 8-2 on the handle end water accepting layer 5.
The preparation of test sample liquid:
If detect the urine sample of animal, can directly get the urine of animal as test sample; If detect meat, meat products or other tissue sample, tissue sample can be shredded, grinds, meat sample and physiological saline are made sample suspension with 1: 1～100 ratio; If detected object is egg, milk or honey, can directly egg, milk or honey be made 1: 1～100 detected sample suspensions with physiological saline; If detect to as if the blood of animal, then extract serum, with physiological saline serum is made 1: 1～100 as testing sample.
The detecting operation method: TL quick detection test paper bar sample end is inserted in the detected sample liquid, and insertion depth is no more than mark line, takes out test strip after about 10 seconds, about 2 minutes of horizontal positioned, observations.
Testing result is judged: if there is a rufous mark " | " to show that testing result is positive on cellulose membrane, be illustrated in the detected sample liquid to contain TL, the product that content surpasses national residue criterion can not circulate on market; If occur two rufous marks " ‖ " on the cellulose membrane on the TL quick detection test paper bar, testing result is negative, and represents not contain in the sample to be checked the tylosin composition.
Embodiment two: quick testing strip for residual pesticide structure and embodiment one are basic identical, difference is: the cardboard bar that supporting layer 1 usefulness does not absorb water is made, test lead fibrage 2 is used nylon membrane, gold labeling antibody fibrage 3 is adsorbed with the tylosin TL polyclonal antibody of glue gold mark, cellulose rete 4 adopts the pure cellulose film, coupling TL carrier protein solution is ferritin in the stealthy detection trace band 6, and the anti-mouse IgG of stealthy contrast trace band 7 usefulness rabbits solution is prepared from cellulose membrane.
Test sample preparation: detect the meat sample,, meat sample and physiological saline are made the detected sample suspension with 1: 30 ratio with sample chopping, grinding.
Other method of operating and result judge all with embodiment one, do not repeat.
Embodiment three: quick testing strip for residual pesticide structure and embodiment one are basic identical, difference is: test lead adsorbing fiber layer 2 usefulness polyvinylidene fluoride (PVDF) film, cellulose rete 4 adopts the carboxylation cellulose membrane, coupling TL carrier protein solution is the pure albumen of ovum gallinaceum (OVA) in the stealthy detection trace band 6, and stealthy contrast trace band 7 usefulness goat anti-mouse igg solution are prepared from cellulose membrane.
Other comprises that test sample preparation, method of operating and judgement as a result etc. all with embodiment one, do not repeat.
Embodiment four: quick testing strip for residual pesticide structure and embodiment one are basic identical, difference is: test lead adsorbing fiber layer 2 is used polyester film, gold labeling antibody fibrage 3 is adsorbed with the TL polyclonal antibody of glue gold mark, and coupling TL carrier protein solution is hemocyanin in the stealthy detection trace band 6.
Other comprises that test sample preparation, method of operating and result judge all with embodiment one, do not repeat.
Claims (8)
1. quick testing strip for residual pesticide; bottom is a supporting layer; the middle layer adsorbed layer is fixed on the supporting layer; outer protective film is fixed on the adsorbed layer; it is characterized in that: adsorbed layer is followed successively by the absorbent material layer of fibrage, golden labeling antibody fibrage, cellulose rete and handle end from test lead; wherein the orthoscopic that the carrier protein solution of useful coupling tylosin is printed on the cellulose rete detects trace; the orthoscopic contrast trace that the anti-mouse IgG of useful goat-anti or rabbit solution is printed, detecting trace is " ‖ " with the parallel assembled arrangement of contrast trace.
2. test strips according to claim 1 is characterized in that: supporting layer is to make with the hard plastic bar or the cardboard bar that do not absorb water.
3. test strips according to claim 1 is characterized in that: the test lead fibrage is made with glass wool or nylon membrane or PVDF membrane or polyester film.
4. test strips according to claim 1, it is characterized in that: absorbent material layer is made with thieving paper.
5. test strips according to claim 1 is characterized in that: the cellulose rete is made with nitrocellulose filter or pure cellulose film or carboxylation cellulose membrane.
6. test strips according to claim 1, it is characterized in that: the golden labeling antibody fibrage golden labeling antibody glass wool of absorption tylosin, tylosin gold labeling antibody is the tylosin monoclonal antibody or the polyclonal antibody of colloid gold label, and the carrier protein solution of coupling tylosin is the composite solution of tylosin and carrier protein couplet.
7. test strips according to claim 1; it is characterized in that: on test lead adsorbing fiber layer, golden labeling antibody fibrage and absorbent material layer, be coated with diaphragm; on the test lead fibrage diaphragm corresponding, be printed with the sample mark line, the about 0.5cm of this mark line deflection test lead fibrage one side place with golden labeling antibody fibrage intersection.
8. test strips according to claim 1 is characterized in that: the carrier protein of coupling tylosin is a bovine serum albumin(BSA), or is the pure albumen of ovum gallinaceum, or is ferritin, or is hemocyanin.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200620135124 CN200989901Y (en) | 2006-12-19 | 2006-12-19 | Tylosion residual fast detection test paper strip |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200620135124 CN200989901Y (en) | 2006-12-19 | 2006-12-19 | Tylosion residual fast detection test paper strip |
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| CN200989901Y true CN200989901Y (en) | 2007-12-12 |
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| CN 200620135124 Expired - Fee Related CN200989901Y (en) | 2006-12-19 | 2006-12-19 | Tylosion residual fast detection test paper strip |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102411053A (en) * | 2011-08-11 | 2012-04-11 | 浙江省农业科学院 | Co-detection test strip of colibacillus 0157 and listeria monocytogenes |
| CN102590516A (en) * | 2012-01-17 | 2012-07-18 | 重庆师范大学 | Immune colloidal gold test strip for simultaneously carrying out residue detection and analysis on Tylosin and Tilmicosin and preparation method thereof |
| CN103353525A (en) * | 2012-12-12 | 2013-10-16 | 河南省农业科学院 | Test strip for detecting aureomycin residue, and test card |
| CN103389379A (en) * | 2012-05-08 | 2013-11-13 | 北京勤邦生物技术有限公司 | Test strip and method for detecting tylosin and tilmicosin |
| CN104678102A (en) * | 2013-11-27 | 2015-06-03 | 北京维德维康生物技术有限公司 | Neomycin and tylosin two-in-one gold-labeled microporous test paper strip |
-
2006
- 2006-12-19 CN CN 200620135124 patent/CN200989901Y/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102411053A (en) * | 2011-08-11 | 2012-04-11 | 浙江省农业科学院 | Co-detection test strip of colibacillus 0157 and listeria monocytogenes |
| CN102590516A (en) * | 2012-01-17 | 2012-07-18 | 重庆师范大学 | Immune colloidal gold test strip for simultaneously carrying out residue detection and analysis on Tylosin and Tilmicosin and preparation method thereof |
| CN103389379A (en) * | 2012-05-08 | 2013-11-13 | 北京勤邦生物技术有限公司 | Test strip and method for detecting tylosin and tilmicosin |
| CN103353525A (en) * | 2012-12-12 | 2013-10-16 | 河南省农业科学院 | Test strip for detecting aureomycin residue, and test card |
| CN104678102A (en) * | 2013-11-27 | 2015-06-03 | 北京维德维康生物技术有限公司 | Neomycin and tylosin two-in-one gold-labeled microporous test paper strip |
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| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Assignee: Henan Baiao Biotechnology Ltd. Assignor: Biological Technology Institute, Henan Agricultural Academy Contract fulfillment period: 2009.2.20 to 2014.2.20 Contract record no.: 2009410000128 Denomination of utility model: Quick testing strip for residual pesticide Granted publication date: 20071212 License type: Exclusive license Record date: 20090513 |
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| LIC | Patent licence contract for exploitation submitted for record |
Free format text: EXCLUSIVE LICENSE; TIME LIMIT OF IMPLEMENTING CONTACT: 2009.2.20 TO 2014.2.20; CHANGE OF CONTRACT Name of requester: HENAN BAO AO BIOLOGY ENGINEERING CO., LTD. Effective date: 20090513 |
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| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20071212 Termination date: 20111219 |