CN103018451B - The enzyme linked immunological kit of chlorine detection mycin and application thereof - Google Patents
The enzyme linked immunological kit of chlorine detection mycin and application thereof Download PDFInfo
- Publication number
- CN103018451B CN103018451B CN201110279098.5A CN201110279098A CN103018451B CN 103018451 B CN103018451 B CN 103018451B CN 201110279098 A CN201110279098 A CN 201110279098A CN 103018451 B CN103018451 B CN 103018451B
- Authority
- CN
- China
- Prior art keywords
- chloromycetin
- sample
- enzyme
- liquid
- kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention provides a kind of enzyme linked immunological kit of chlorine detection mycin, it contains: the ELISA Plate being coated with coating antigen, enzyme marker, chloromycetin specific antibody working fluid (when envelope antigen in ELISA Plate and enzyme marker be enzyme labeling antiantibody or ELISA Plate are wrapped by antiantibody and enzyme marker is enzyme-labelled antigen time contain), chloromycetin standard solution, substrate nitrite ion, stop buffer, concentrated cleaning solution, the concentrated liquid that redissolves.The invention also discloses a kind of method applying above-mentioned enzyme linked immunological kit chlorine detection mycin, it comprises: first carry out sample pre-treatments, then detects with kit, ultimate analysis testing result.Enzyme linked immunological kit provided by the invention can be used for the residual quantity detecting chloromycetin in the sample such as animal tissue's (muscle, liver etc.), prepared food, aquatic products, royal jelly, urine, feed and milk, it is easy and simple to handle, low cost, highly sensitive, can on-site supervision and be applicable to the examination of great amount of samples.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, be specifically related to a kind of enzyme linked immunological kit for chlorine detection mycin, it is particularly suitable for the detection of residual chloromycetin in animal tissue's (muscle, liver etc.), prepared food, aquatic products, royal jelly, urine, feed and milk equal samples.
Technical background
Chloromycetin (Chloramphenicol, CAP) be a kind of broad-spectrum antibiotic that 20 middle of century find, good inhibiting effect is all had to Gram-negative bacteria and positive bacteria, due to its excellent antibiotic property, the stable property of medicine and cheap price, once within one period, be applied to humans and animals as the medicine of bacteriosis clinical, and be widely used in Animal husbandry production as feed addictive.But in use it is found that chloromycetin has toxicity, the residual meeting in animal food produces toxic action by animal foods such as meat, egg, milk to the health of people.Therefore, the Ministry of Agriculture of China announces No. 235 file regulation, and chloromycetin and salt thereof, ester (comprising Chloramphenicol Succinate) must not detect in all Edible tissues of all food animals.But due to the good antimicrobial effect of chloromycetin, price is low, people is still had to use it in poultry, domestic animals, aquatic products.In order to ensure the healthy of the people and expand the trade contacts of food, it is necessary for setting up highly sensitive, high specificity, quick, economic residual chloromycetin detection method.
At present, be used for most popular method mainly gas chromatography, liquid chromatography and the chromatography mass spectrometry of chlorine detection mycin, although these methods have the advantages such as highly sensitive, result is accurate, reproducible, false positive is few, but still there is certain shortcoming, as sample pretreatment process is complicated, instrumentation degree is high and expensive, and analysis speed is slow, can not meet developed country and food processing enterprises gradually to the requirement of detection method detectability.By comparison, immunological assay method sensitivity is higher, high specificity, sample pretreatment are simple, analysis time is short, is applicable to the examination of on-site supervision and great amount of samples.
Summary of the invention
The object of the invention is to provide a kind of enzyme linked immunological kit detected for chloromycetin for above-mentioned deficiency, it is simple to operate, is applicable to the screening of on-the-spot batch samples.
Kit of the present invention, it contains:
(1) ELISA Plate of coating antigen is coated with;
(2) enzyme marker;
(3) chloromycetin standard solution;
(4) substrate nitrite ion;
(5) stop buffer;
(6) concentrated cleaning solution;
(7) the concentrated liquid that redissolves.
The enzyme linked immunological kit of chlorine detection mycin provided by the present invention, comprises ELISA Plate and the enzyme marker working fluid of pre-coated coating antigen; Described coating antigen is chloromycetin antigen, antibody or antiantibody, described enzyme marker is enzyme labeling chloromycetin haptens, enzyme labeling chloramphenicol antibody or enzyme labeling antiantibody, when envelope antigen in ELISA Plate and enzyme marker be enzyme labeling antiantibody or ELISA Plate are wrapped by antiantibody and enzyme marker is enzyme-labelled antigen time also containing chloromycetin specific antibody working fluid.
Described chloromycetin haptens is obtained by catalytic hydrogenation reaction by the nitro in chloromycetin molecular structure.
Described chloromycetin specific antibody is chloromycetin monoclonal antibody; Described chloromycetin monoclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, is preferably chloromycetin mouse monoclonal antibody; Described antiantibody is sheep anti mouse antiantibody.
Above antibody all can be prepared as immunogene with the conjugate of chloromycetin haptens and carrier protein.Described carrier protein can be the common carrier albumen such as mouse haemocyanin, thyroprotein (BCG), bovine serum albumin, rabbit serum proteins, human albumin, ovalbumin, hemocyanin and fibrinogen; The conjugate of described chloromycetin haptens and carrier protein obtains by chloromycetin haptens and carrier protein glutaraldehyde method are carried out coupling.
The marker enzyme of described enzyme marker is horseradish peroxidase or alkaline phosphatase, wherein preferred horseradish peroxidase; The sheep anti mouse antiantibody of enzyme labeling adopts glutaraldehyde method or Over-voltage protection that marker enzyme and antiantibody are carried out coupling and obtains; Enzyme labeling chloromycetin haptens adopts diazotising method or glutaraldehyde method marker enzyme and chloromycetin hapten conjugation to be obtained; Enzyme labeling specific antibody adopts glutaraldehyde method or Over-voltage protection marker enzyme and specific antibody coupling to be obtained, horseradish peroxidase can adopt multiple method of the prior art that itself and antiantibody are carried out coupling, as glutaraldehyde method, Over-voltage protection etc., Over-voltage protection is improved through long-term labor and creation by the present invention, make it save time, the concentration rate of reduce horseradish peroxidase (HRP) and antiantibody, save starting material.
In order to more convenient on-site supervision and great amount of samples examination, described kit also comprises chloromycetin standard solution, substrate nitrite ion, stop buffer, concentrated cleaning solution, the concentrated liquid that redissolves.
Described chloromycetin standard solution: 6 bottles, concentration is respectively 0 μ g/L, 0.025 μ g/L, 0.075 μ g/L, 0.3 μ g/L, 1.2 μ g/L, 4.8 μ g/L.
When marker enzyme is horseradish peroxidase, described substrate nitrite ion is made up of nitrite ion A liquid and nitrite ion B liquid, A liquid is hydrogen peroxide or urea peroxide, and B liquid is o-phenylenediamine or tetramethyl benzidine, and described stop buffer is sulfuric acid or the hydrochloride buffer of 1 ~ 2mol/L; When marker enzyme is alkaline phosphatase, described substrate nitrite ion is to nitro phosphate buffer, and described stop buffer is 1 ~ 2mol/L sodium hydroxide solution.
It is 7.1 ~ 7.5 that described concentrated cleaning solution is preferably pH value, and the phosphate buffer containing 0.8% ~ 1.2% Tween-20,0.01 ‰ ~ 0.03 ‰ thiomersal preservative, 0.01 ~ 0.03mol/L, described number percent is percent weight in volume.
It is 7.2 ~ 7.7 that described concentrated redissolution liquid is preferably pH value, the phosphate buffer containing 8% ~ 12% ovalbumin, 0.1 ~ 0.4mol/L, and described number percent is percent weight in volume.
It is pH value is 9.6 that bag wherein used in ELISA Plate preparation process is buffered liquid, the carbonate buffer solution of 0.05mol/L, confining liquid used is pH value is 9.1 ~ 9.5, carbonate buffer solution containing 3% ~ 10% calf serum, 0.2% Tween-20,0.1 ~ 0.3mol/L, described number percent is percent weight in volume.
In the present invention, the preparation process of ELISA Plate is: be buffered liquid with bag and coating antigen is diluted to 0.1 ~ 0.2 μ g/ml, every hole adds 100 μ l, 37 DEG C of incubation 2h or 4 DEG C spend the night, and incline coating buffer, wash 2 times with cleansing solution, each 30s, pat dry, in every hole, then add 150 ~ 200 μ l confining liquids, 37 DEG C of incubation 1 ~ 2h, the liquid in hole that inclines pats dry, and preserves after dry with the vacuum seal of aluminium film.
In the present invention, immunogenic building-up process is:
1. chloromycetin hapten synthesis (synthetic route is as Fig. 2)
Chloromycetin haptens is obtained by catalytic hydrogenation reaction by the nitro in chloromycetin (structural formula is as Fig. 1) molecular structure, and concrete reactions steps is as follows:
Be dissolved in by chloromycetin in methyl alcohol, add the palladium carbon catalyst (Pd/C) of 5%, pass into hydrogen, keep certain pressure, room temperature reaction 2h, cross and filter Pd/C, solvent evaporated obtains faint yellow thick liquid, is chloromycetin haptens; The mol ratio of wherein said Pd/C and chloromycetin is 1: 10.
2. immunogenic synthesis
Adopt glutaraldehyde method to carry out coupling chloromycetin haptens and carrier protein and obtain immunogene, concrete preparation method is as follows:
Extracting chloromycetin haptens 30mg, by the water-soluble solution of 1.5ml, obtains (I) liquid; Glutaraldehyde (GA) the 10 μ l getting 50% adds in (I), and stirred at ambient temperature reaction 18h, obtains (II) liquid; Add in (II) liquid after getting the dilution of bovine serum albumin(BSA) (BSA) 100mg 1.5ml water, reaction adds 24mgNaBH after spending the night
4reaction 3h; Namely immunogene is obtained with tri-distilled water dialysis 48h.
In the present invention, the preparation process of chloromycetin monoclonal antibody is:
(1) animal immune program: adopt Balb/c mouse as immune animal, with chloromycetin haptens with carrier protein couplet thing for immunogene, after obtaining good polyvalent antibody, taking-up spleen carries out Fusion of Cells.
(2) Fusion of Cells and cloning: get immune Balb/c mouse boosting cell and SP2/0 myeloma cell fusion, screening cell line, until obtain the hybridoma cell strain of stably excreting monoclonal antibody.
In the present invention, the preparation process of sheep anti mouse antiantibody is: using sheep as immune animal, carries out immunity, obtain sheep anti mouse antiantibody with mouse source antibody for immunogene to pathogen-free domestic sheep.
Cleaning Principle of the present invention is:
When on capillary strip during pre-coated chloromycetin coupled antigen, after adding sample solution or standard solution, add enzyme labeling chloromycetin specific antibody solution again, chloromycetin coupled antigen chloromycetin in sample and ELISA Plate being wrapped quilt competes chloromycetin specific antibody, develop the color with nitrite ion, the content of sample absorbance and chloromycetin is negative correlation, compares the residual quantity that can draw chloromycetin in sample with typical curve; Simultaneously according to the depth of color in ELISA Plate, can the concentration range of chloramphenicol residue in judgement sample roughly with comparing of the chloromycetin standard solution color of series concentration.
When on capillary strip during pre-coated chloromycetin coupled antigen, after adding sample solution or standard solution, add chloromycetin specific antibody solution again, chloromycetin coupled antigen chloromycetin in sample and ELISA Plate being wrapped quilt competes chloromycetin specific antibody, add enzyme labeling antiantibody and carry out amplification, with nitrite ion colour developing, the content of sample absorbance and chloromycetin is negative correlation, compares the residual quantity that can obtain chloromycetin in sample with typical curve; Simultaneously according to the depth of color in ELISA Plate, can the concentration range of chloramphenicol residue in judgement sample roughly with comparing of the chloromycetin standard solution color of series concentration.
When on capillary strip during pre-coated chloromycetin specific antibody, after adding sample solution or standard solution, add enzyme labeling chloromycetin haptens solution again, chloromycetin in sample and ENR-HRP compete the chloromycetin specific antibody be coated in ELISA Plate, develop the color with nitrite ion, the content of sample absorbance and chloromycetin is negative correlation, compares the residual quantity that can draw chloromycetin in sample with typical curve; Simultaneously according to the depth of color in ELISA Plate, can the concentration range of chloramphenicol residue in judgement sample roughly with comparing of the chloromycetin standard solution color of series concentration.
When on capillary strip during pre-coated antiantibody, add after chloramphenicol antibody hatches, add sample solution or standard solution, add enzyme labeling chloromycetin haptens solution again, chloromycetin in sample and enzyme labeling chloromycetin haptens compete chloramphenicol resistance specific antibody, with nitrite ion colour developing, the content of sample absorbance and chloromycetin is negative correlation, compares the residual quantity that can draw chloromycetin in sample with typical curve; Simultaneously according to the depth of color in ELISA Plate, can the concentration range of chloramphenicol residue in judgement sample roughly with comparing of the chloromycetin standard solution color of series concentration.
Present invention also offers a kind of method applying above-mentioned enzyme linked immunological kit chlorine detection mycin, it comprises step:
(1) sample pre-treatments;
(2) detect with kit;
(3) testing result is analyzed.
The pre-treatment of sample mainly in order to obtain chloromycetin solution from sample, thus for follow-up detection.Here is the pre-treating method of common several samples:
1. organize (chicken/liver, pork/liver, shrimp, fish etc.) pre-treating method
After homogenizer homogeneous structure sample, take the equal pledge of 3.0 ± 0.05g in 50ml polystyrene centrifuge tube, add 6ml ethyl acetate, use oscillator vibrates 5min, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Pipette 4ml upper organic phase (being about equivalent to the sample of 2g) in the clean glass test tube of 10ml, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up; Add 1ml normal hexane, with vortex instrument whirling motion 1min, then add 1ml redissolution working fluid, with vortex instrument whirling motion 15s, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Removing upper organic phase, takes off layer 50 μ l for analyzing.
2. prepared food pre-treating method
After homogenizer homogeneous sample, take the equal pledge of 3.0 ± 0.05g in 50ml polystyrene centrifuge tube, add 3ml deionized water, add 6ml ethyl acetate again, use oscillator vibrates 10min, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 10min; Pipette 4ml upper organic phase (being about equivalent to the sample of 2g) in the clean glass test tube of 10ml, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up; Add 1ml normal hexane, with vortex instrument whirling motion 1min, then add 1ml redissolution working fluid, with vortex instrument whirling motion 15s, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Removing upper organic phase, takes off layer 50 μ l for analyzing.
3. urine pre-treating method
Getting the limpid urine specimen of 100 μ l, add 900 μ l wash operating solutions, mixing, getting 50 μ l for analyzing (if urine specimen is not limpid, need by urine 4000rpm, the centrifugal 5min of room temperature).
4. royal jelly pre-treating method
Take 1.0 ± 0.05g sample in 50ml polystyrene centrifuge tube, add 1ml0.1mol/LCB (take 4.66g natrium carbonicum calcinatum, 0.5g sodium bicarbonate add 500ml deionized water dissolving mixing), 8ml ethyl acetate, add 2g natrium carbonicum calcinatum again, use oscillator vibrates 5min, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Pipette 2ml upper organic phase (being about equivalent to the sample of 0.25g) in the clean glass test tube of 10ml, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up; Add 1ml normal hexane, whirling motion 1min, then add 1ml redissolution working fluid, with vortex instrument whirling motion 15s, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Removing upper organic phase, gets 50 μ l liquid for analyzing.
5. milk pre-treating method
Pipetting 500 μ l milk, add 500 μ l wash operating solutions, fully shake mixing, getting 50 μ l for analyzing.
6. feed pre-treating method
Take 1.0 ± 0.05g feed in 50ml polystyrene centrifuge tube, add the vibration of 10ml ethyl acetate 5min, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Pipette 2ml upper organic phase in the clean glass tube of 10ml, flow down in 50 ~ 60 DEG C of nitrogen and dry up; Add 1ml normal hexane, with vortex instrument whirling motion 1min, then add 1ml redissolution working fluid, with vortex instrument whirling motion 15s, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Removing upper organic phase, gets 50 μ l for analyzing.
7. egg pre-treating method
After homogenizer homogeneous sample, take 1 ± 0.05g egg in 50ml polystyrene centrifuge tube, add 8ml ethyl acetate, 1ml0.1mol/LCB (take 4.66g natrium carbonicum calcinatum, 0.5g sodium bicarbonate add 500ml deionized water dissolving mixing), use oscillator vibrates 5min, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Pipette 4ml upper organic phase (being about equivalent to the sample of 0.5g) in the clean glass test tube of 10ml, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up; Add 1ml normal hexane, with vortex instrument whirling motion 1min, then add 1ml redissolution working fluid, with vortex instrument whirling motion 15s, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Removing upper organic phase, takes off layer 50 μ l for analyzing.
When detecting with kit in the present invention: when coating antigen is chloromycetin coupled antigen, add standard solution or sample solution adds enzymic-labelled antibody again in ELISA Plate micropore, after incubation, washing pats dry, and colour developing, stops, and measures absorbance by microplate reader; When coating antigen is chloromycetin coupled antigen, add standard solution or sample solution adds antibody again in ELISA Plate micropore, after incubation, washing pats dry, then adds enzyme mark antiantibody, and after incubation, washing pats dry, and colour developing, stops, and measures absorbance by microplate reader; When coating antigen is chloromycetin specific antibody, add standard solution or sample solution adds enzyme labeling chloromycetin haptens again in ELISA Plate micropore, after incubation, washing pats dry, and colour developing, stops, and measures absorbance by microplate reader; When coating antigen is antiantibody, in ELISA Plate micropore, add chloramphenicol antibody, after incubation, washing pats dry, enzyme mark chloromycetin haptens is added after adding standard solution or sample solution again, after incubation, washing pats dry, and colour developing, termination, measure absorbance by microplate reader.
In the present invention Analysis of test results process for: with the absorbance values (B) of the standard solution of each concentration the obtained absorbance (B divided by first standard solution (0 standard)
0) be multiplied by 100% again, i.e. percentage absorbance.Computing formula is:
Percentage absorbance (%)=(B/B
0) × 100%
With the logarithm value of the concentration of chloromycetin standard solution (μ g/L) for X-axis, percentage absorbance is Y-axis, drawing standard curve map.With the percentage absorbance of same way calculation sample solution, the chloromycetin content of each sample corresponding then can read from typical curve.
In the present invention, the analysis of testing result also can adopt regression equation method, calculates sample solution concentration.
In the present invention, the analysis of testing result can also utilize computer professional software, and this method is more convenient for the express-analysis of a large amount of sample, and whole testing process at most only needs can complete for 75 minutes.
The enzyme linked immunological kit of chlorine detection mycin of the present invention mainly adopts the content of chloromycetin in the qualitative or quantitative detection sample of competitive ELISA method; Require low to the pre-treatment of sample, sample pretreatment process is simple, can detect batch samples fast simultaneously; Adopt the chloromycetin monoclonal antibody of high specific, main agents provides with the form of working fluid, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, degree of accuracy is high, accuracy high.Enzyme linked immunological kit of the present invention, structure is simple, easy to use, low price, carrying convenience, detection method are efficient, accurate, easy, be suitable for the qualitative, quantitative of batch samples screening.Kit of the present invention plays a significant role in the detection of chloromycetin.
Accompanying drawing explanation
Fig. 1: chloromycetin structural drawing
Fig. 2: chloromycetin hapten synthesis route map
Fig. 3: with the conjugate of chloromycetin haptens and carrier protein be coating antigen, the canonical plotting of the enzymic-labelled antibody kit that is enzyme marker
Embodiment
The present invention is set forth further below in conjunction with specific embodiment.Should be understood that these embodiments are only for illustration of the present invention, and be not used for limiting the scope of the invention.
The preparation of embodiment 1 reagent constituents
1. the synthesis of antigen
A. chloromycetin hapten synthesis
Be dissolved in by chloromycetin in methyl alcohol, add the palladium carbon catalyst (Pd/C) of 5%, pass into hydrogen, keep certain pressure, room temperature reaction 2h, cross and filter Pd/C, solvent evaporated obtains faint yellow thick liquid, is chloromycetin haptens; The mol ratio of wherein said Pd/C and chloromycetin is 1: 10.
B. immunogene synthesis
Adopt glutaraldehyde method to carry out coupling chloromycetin haptens and bovine serum albumin(BSA) and obtain immunogene.
Immunogenic preparation process:
Extracting chloromycetin haptens 30mg, by the water-soluble solution of 1.5ml, obtains (I) liquid; Glutaraldehyde (GA) the 10 μ l getting 50% adds in (I), and stirred at ambient temperature reaction 18h, obtains (II) liquid; Add in (II) liquid after getting the dilution of bovine serum albumin(BSA) (BSA) 100mg 1.5ml water, reaction adds 24mgNaBH after spending the night
4reaction 3h; Namely immunogene is obtained with tri-distilled water dialysis 48h.
C. coating antigen synthesis
Chloromycetin haptens and ovalbumin coupling are obtained coating antigen.
The preparation process of coating antigen:
Extracting chloromycetin haptens 30mg, by the water-soluble solution of 1.5ml, obtains (I) liquid; Glutaraldehyde (GA) the 10 μ l getting 50% adds in (I), and stirred at ambient temperature reaction 18h, obtains (II) liquid; Add in (II) liquid after getting the dilution of ovalbumin (OVA) 30mg 1.5ml water, reaction adds 24mgNaBH after spending the night
4reaction 3h; Namely coating antigen is obtained with tri-distilled water dialysis 48h.
2. the synthesis of monoclonal antibody
Animal immune: immunogene be injected in Balb/c Mice Body, immunizing dose is 100 μ g/, makes it produce polyclonal antibody.
Fusion of Cells and cloning: after mice serum measurement result is higher, get its splenocyte, in 8: 1 ratios and SP2/0 myeloma cell fusion, adopts indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Limiting dilution assay is utilized to carry out cloning to positive hole, until obtain the hybridoma cell strain of secrete monoclonal antibody.
Obtain the monoclonal hybridoma strain of chloromycetin through screening, generation chloromycetin specific antibody that can be endless, and this antibody specificity is for chloromycetin, sensitivity can reach 0.025 μ g/L.
Cell cryopreservation and recovery: the monoclonal hybridoma strain cryopreserving liquid of chloromycetin is made 1 × 10
6the cell suspension of individual/ml, preserves for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, after centrifugal segregation cryopreserving liquid, move into and cultivate culture in glassware.
The production of monoclonal antibody and purifying: only Balb/c mouse peritoneal is injected sterilizing paraffin oil 0.5ml/, within 7 days, pneumoretroperitoneum injects stable chloromycetin monoclonal hybridoma strain 1 × 10
6individual/only, gather ascites after 7 days.Carry out ascites by sad-saturated ammonium sulfate method to purify ,-20 DEG C of preservations.
3. the preparation of enzyme labeling chloromycetin monoclonal antibody
The Over-voltage protection after improveing is adopted to carry out coupling chloromycetin monoclonal antibody and horseradish peroxidase (HRP).Traditional Over-voltage protection requires that the molar concentration rate of enzyme and antibody in reaction system is 4: 1, because horseradish peroxidase produces many sites be combined with antibody under strong oxidation, the horseradish peroxidase molecule of such activation act as the bridge connecting each molecule, reduce the enzymatic activity of enzyme marker, make to be mixed with many condensates in the conjugate of preparation.In order to address this problem, traditional method improves by we, that is:
(1) amino closed process is eliminated, because it is seldom actual to produce self the amino amino connected;
(2) reduce horseradish peroxidase: the volumetric molar concentration ratio to 2 of antibody: 1, the method after improvement is easier than traditional method, reduces the loss of enzymatic activity.
4. the preparation of ELISA Plate
Be buffered liquid with bag and chloromycetin coupled antigen is diluted to 0.2 μ g/ml, every hole adds 100 μ l, 37 DEG C of incubation 2h, and incline coating buffer, 2 times are washed with the concentrated cleaning solution of dilution 20 times, each 30s, pats dry, and then adds 200 μ l confining liquids in every hole, 37 DEG C of incubation 2h, incline liquid in hole, preserves after dry with the vacuum seal of aluminium film
The establishment of the enzyme linked immunological kit of embodiment 2 chlorine detection mycin
Set up the enzyme linked immunological kit of chlorine detection mycin, make it comprise following component:
(1) bag is by the ELISA Plate of chloromycetin coupled antigen;
(2) chloromycetin standard solution 6 bottles, concentration is respectively 0 μ g/L, 0.025 μ g/L, 0.075 μ g/L, 0.3 μ g/L, 1.2 μ g/L, 4.8 μ g/L;
(3) chloromycetin high standard product solution 1 bottle, concentration is 100 μ g/L;
(4) with the chloromycetin monoclonal antibody working fluid of horseradish peroxidase-labeled;
(5) substrate nitrite ion is made up of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine;
(6) stop buffer is 2mol/L sulfuric acid;
(7) concentrated cleaning solution is pH value is 7.1 ~ 7.5, the phosphate buffer containing 0.8% ~ 1.2% Tween-20,0.01 ‰ ~ 0.03 ‰ thiomersal preservative, 0.01 ~ 0.03mol/L, and described number percent is percent weight in volume;
(8) concentrated redissolution liquid is pH value is 7.2 ~ 7.7, and the phosphate buffer containing 8% ~ 12% ovalbumin, 0.1 ~ 0.4mol/L, described number percent is percent weight in volume.
The detection of chloromycetin in embodiment 3 sample
1. sample pre-treatments
(1) tissue (chicken/liver, pork/liver, shrimp, fish etc.) pre-treating method
After homogenizer homogeneous structure sample, take the equal pledge of 3.0 ± 0.05g in 50ml polystyrene centrifuge tube, add 6ml ethyl acetate, use oscillator vibrates 5min, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Pipette 4ml upper organic phase (being about equivalent to the sample of 2g) in the clean glass test tube of 10ml, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up; Add 1ml normal hexane, with vortex instrument whirling motion 1min, then add 1ml redissolution working fluid, with vortex instrument whirling motion 15s, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Removing upper organic phase, takes off layer 50 μ l for analyzing.
(2) prepared food pre-treating method
After homogenizer homogeneous sample, take the equal pledge of 3.0 ± 0.05g in 50ml polystyrene centrifuge tube, add 3ml deionized water, add 6ml ethyl acetate again, use oscillator vibrates 10min, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 10min; Pipette 4ml upper organic phase (being about equivalent to the sample of 2g) in the clean glass test tube of 10ml, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up; Add 1ml normal hexane, with vortex instrument whirling motion 1min, then add 1ml redissolution working fluid, with vortex instrument whirling motion 15s, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Removing upper organic phase, takes off layer 50 μ l for analyzing.
(3) urine pre-treating method
Getting the limpid urine specimen of 100 μ l, add 900 μ l wash operating solutions, mixing, getting 50 μ l for analyzing (if urine specimen is not limpid, need by urine 4000rpm, the centrifugal 5min of room temperature).
(4) royal jelly pre-treating method
Take 1.0 ± 0.05g sample in 50ml polystyrene centrifuge tube, add 1ml0.1mol/LCB (take 4.66g natrium carbonicum calcinatum, 0.5g sodium bicarbonate add 500ml deionized water dissolving mixing), 8ml ethyl acetate, add 2g natrium carbonicum calcinatum again, use oscillator vibrates 5min, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Pipette 2ml upper organic phase (being about equivalent to the sample of 0.25g) in the clean glass test tube of 10ml, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up; Add 1ml normal hexane, whirling motion 1min, then add 1ml redissolution working fluid, with vortex instrument whirling motion 15s, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Removing upper organic phase, gets 50 μ l liquid for analyzing.
(5) milk pre-treating method
Pipetting 500 μ l milk, add 500 μ l wash operating solutions, fully shake mixing, getting 50 μ l for analyzing.
(6) feed pre-treating method
Take 1.0 ± 0.05g feed in 50ml polystyrene centrifuge tube, add the vibration of 10ml ethyl acetate 5min, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Pipette 2ml upper organic phase in the clean glass tube of 10ml, flow down in 50 ~ 60 DEG C of nitrogen and dry up; Add 1ml normal hexane, with vortex instrument whirling motion 1min, then add 1ml redissolution working fluid, with vortex instrument whirling motion 15s, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Removing upper organic phase, gets 50 μ l for analyzing.
(7) egg pre-treating method
After homogenizer homogeneous sample, take 1 ± 0.05g egg in 50ml polystyrene centrifuge tube, add 8ml ethyl acetate, 1ml0.1mol/LCB (take 4.66g natrium carbonicum calcinatum, 0.5g sodium bicarbonate add 500ml deionized water dissolving mixing), use oscillator vibrates 5min, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Pipette 4ml upper organic phase (being about equivalent to the sample of 0.5g) in the clean glass test tube of 10ml, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up; Add 1ml normal hexane, with vortex instrument whirling motion 1min, then add 1ml redissolution working fluid, with vortex instrument whirling motion 15s, 4000rpm, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Removing upper organic phase, takes off layer 50 μ l for analyzing.
2. detect with kit
Chloromycetin standard solution or sample solution 50 μ l is added in the ELISA Plate micropore being coated with chloromycetin coupled antigen, add the chloromycetin monoclonal antibody working fluid 50 μ l/ hole of horseradish peroxidase-labeled again, after cover plate membrane cover plate, put lucifuge reaction 30min in 25 DEG C of constant temperature ovens, pour out liquid in hole, every hole adds 250 μ l cleansing solutions, pours out liquid in hole after 30s, repeat operation and wash plate 5 times, pat dry with thieving paper; Every hole adds substrate nitrite ion A liquid urea peroxide 50 μ l, substrate nitrite ion B liquid tetramethyl benzidine (TMB) 50 μ l, to vibrate gently mixing, with lucifuge colour developing 20min in the rearmounted 25 DEG C of constant temperature ovens of cover plate membrane cover plate, every hole adds stop buffer 2mol/L sulfuric acid 50 μ l, to vibrate gently mixing, be set in 450nm place with microplate reader wavelength, measure every hole absorbance (OD value).
3. Analysis of test results
With the absorbance values (B) of the standard solution of the obtained each concentration absorbance (B divided by first standard solution (0 standard)
0) be multiplied by 100% again, obtain percentage absorbance.With the logarithm value of chloromycetin standard concentration (μ g/L) for X-axis, percentage absorbance is Y-axis, drawing standard curve map, as shown in Figure 3.With the percentage absorbance of same way calculation sample solution, the chloromycetin content of each sample corresponding then can read from typical curve.
The determination test of embodiment 4 kit technical parameter
1. standard items precision test
Respectively the ELISA Plate prepared from three different time periods respectively extract a collection of ELISA Plate out, often criticize each extraction 10 kits, 20 micropores extracted out by every plate, measure the absorbance of 0.3 μ g/L standard solution, calculate the coefficient of variation.
Table 1 standard repeatability test (CV%)
Can be drawn by above-mentioned test findings, often criticize each 10 the standard items coefficient of variation of kit between 3.8% ~ 10.2%, meet the regulation that precision is less than or equal to 25%.
2. sample preci-sion and accuracy test
(1) sample precision test
With the chloromycetin of 0.2 μ g/kg concentration, chicken, pork, pork liver, shrimp sample are carried out interpolation and measured, with the chloromycetin of 1 μ g/kg (L) concentration, egg, milk sample are carried out interpolation and measured, with the chloromycetin of 2 μ g/kg concentration, royal jelly, feed sample are carried out interpolation and measured, carry out interpolation with the chloromycetin of 5 μ g/L concentration to pig urine sample to measure, get each three of the kit of three different batches respectively, each concentration repeats 5 times, calculates the coefficient of variation, the results are shown in Table 2 ~ 10.
The test of table 2 chicken sample repeatability
The test of table 3 pork sample repeatability
The test of table 4 pork liver sample repeatability
The test of table 5 shrimp sample repeatability
The test of table 6 egg sample repeatability
The test of table 7 milk sample repeatability
The test of table 8 royal jelly sample repeatability
The test of table 9 feed sample repeatability
The test of table 10 pig urine sample repeatability
Result shows, the coefficient of variation of chicken, pork, pork liver, shrimp, egg, milk, royal jelly, feed, pig urine sample, all between 4.7% ~ 12.1%, has met " Ministry of Agriculture's file " agriculture doctor's method [2005] No. 17 annex 2 kits and has put on record with reference to the 4th precision standard in judgment criteria.
(2) sample accuracy test
With the chloromycetin of 0.2,0.4 μ g/kg concentration, chicken, pork, pork liver, shrimp sample are carried out interpolation and measured, with the chloromycetin of 1,2 μ g/kg (L) concentration, egg, milk sample are carried out interpolation and measured, with the chloromycetin of 2,4 μ g/kg concentration, royal jelly, feed sample are carried out interpolation and measured, carry out interpolation with the chloromycetin of 5,10 μ g/L concentration to pig urine sample to measure, each concentration do 4 parallel, accuracy in computation, the results are shown in Table 11 respectively.
The accuracy of table 11 kit
Result shows, chicken, pork, pork liver, shrimp sample TIANZHU XINGNAO Capsul are between 76.5% ~ 98.3%, egg sample TIANZHU XINGNAO Capsul is between 83.9% ~ 102.4%, milk sample TIANZHU XINGNAO Capsul is between 81.5% ~ 97.4%, royal jelly sample TIANZHU XINGNAO Capsul is between 86.2% ~ 103.0%, feed sample TIANZHU XINGNAO Capsul is between 84.2% ~ 98.7%, and pig urine sample TIANZHU XINGNAO Capsul is between 80.7% ~ 95.6%.
3. cross reacting rate test
Select the drug monitoring cross reacting rate with chloromycetin with similar structures and similar functions, obtain its 50% inhibition concentration respectively by the typical curve of various medicine, calculate kit to the cross reacting rate of other medicines with following formula.Cross reacting rate is larger, and so this kit is better to the specificity of the detection of chloromycetin.
Cross reacting rate (%)=(causing the analog concentration of the chloramphenicol concentration of 50% suppression/cause 50% suppression) × 100%
The specificity of table 12 kit
4. stabilization of kit test
Kit preservation condition is 2 ~ 8 DEG C, and through the mensuration of 12 months, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, chloromycetin added practical measurement value all within normal range.Consider in transport and use procedure, have improper preservation condition and occur, placed 15 days under 37 DEG C of preservation conditions by kit, carry out accelerated aging tests, result shows that this kit indices meets the requirements completely.Consider that the freezing situation of kit occurs, kit is put into-20 DEG C of refrigerator freezings 15 days, measurement result also shows that kit indices is completely normal.Can show that kit at least can be preserved more than 12 months at 2 ~ 8 DEG C from above result.
Claims (3)
1. a preparation method for chlorine detection mycin enzyme linked immunological kit, it comprises:
(1) preparation bag is by the ELISA Plate of chloromycetin antigen;
(2) by horseradish peroxidase-labeled chloromycetin monoclonal antibody;
(3) the chloromycetin standard solution that concentration is respectively 0 μ g/L, 0.025 μ g/L, 0.075 μ g/L, 0.3 μ g/L, 1.2 μ g/L, 4.8 μ g/L is prepared;
(4) prepare urea peroxide solution as substrate nitrite ion A liquid, tetramethyl biphenyl amine aqueous solution is as substrate nitrite ion B liquid;
(5) hydrochloride buffer of 2mol/L is prepared as stop buffer;
(6) preparing pH value is 7.1 ~ 7.5, and the phosphate buffer containing 0.8% ~ 1.2% (w/v) Tween-20,0.01 ‰ ~ 0.03 ‰ (w/v) thimerosal, 0.01 ~ 0.03mol/L is as concentrated cleaning solution;
(7) preparing pH value is 7.2 ~ 7.7, and the phosphate buffer containing 8% ~ 12% (w/v) ovalbumin, 0.1 ~ 0.4mol/L is as concentrated redissolution liquid;
It is characterized in that, described chloromycetin antigen is obtained by glutaraldehyde method coupling by chloromycetin haptens and carrier protein, and described chloromycetin monoclonal antibody is prepared by described coupled antigen; Described carrier protein is mouse haemocyanin, thyroprotein, bovine serum albumin, rabbit serum proteins, human albumin, ovalbumin, hemocyanin or fibrinogen, described chloromycetin haptens is dissolved in methyl alcohol by chloromycetin, add the Pd/C of 5%, pass into hydrogen, keep certain pressure, room temperature reaction 2h, cross and filter Pd/C, solvent evaporated obtains; The mol ratio of described Pd/C and chloromycetin is 1: 10.
2. the preparation method of chlorine detection mycin enzyme linked immunological kit as claimed in claim 1, it is characterized in that bag used to be buffered liquid be pH value is the carbonate buffer solution of 9.6,0.05mol/L; Confining liquid used is pH value is 9.1 ~ 9.5, the carbonate buffer solution containing 3% ~ 10% (w/v) calf serum, 0.2% (w/v) Tween-20,0.1 ~ 0.3mol/L.
3. detect a method for residual chloromycetin in sample, comprise step:
(1) sample pre-treatments;
(2) detect with kit prepared by the preparation method of the chlorine detection mycin enzyme linked immunological kit described in any one of claim 1 ~ 2;
(3) testing result is analyzed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110279098.5A CN103018451B (en) | 2011-09-20 | 2011-09-20 | The enzyme linked immunological kit of chlorine detection mycin and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110279098.5A CN103018451B (en) | 2011-09-20 | 2011-09-20 | The enzyme linked immunological kit of chlorine detection mycin and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103018451A CN103018451A (en) | 2013-04-03 |
| CN103018451B true CN103018451B (en) | 2016-04-20 |
Family
ID=47967290
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110279098.5A Active CN103018451B (en) | 2011-09-20 | 2011-09-20 | The enzyme linked immunological kit of chlorine detection mycin and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103018451B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105319369A (en) * | 2014-07-24 | 2016-02-10 | 江苏维赛科技生物发展有限公司 | Enzyme linked immunosorbent assay kit used for detecting chloramphenicol, and detection method thereof |
| CN106370870A (en) * | 2016-09-21 | 2017-02-01 | 中国农业大学 | Kit for detecting clenbuterol |
| CN108196046A (en) * | 2016-12-08 | 2018-06-22 | 丹阳亿太生物科技发展有限公司 | A kind of remaining enzyme-linked immune detection method of gibberellin |
| CN106932566A (en) * | 2017-04-17 | 2017-07-07 | 四川精卫食品检测科技有限公司 | A kind of carbendazim and the two-in-one detection enzyme linked immunological kit of thiophanate-methyl |
| CN109724930B (en) * | 2018-12-27 | 2021-10-26 | 北京普赞生物技术有限公司 | Detection kit and detection method for dibutyl phthalate in edible oil and grease-containing food |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4013004A1 (en) * | 1990-04-24 | 1991-10-31 | Elvira Schecklies | ELISA determn. of low mol. wt. pesticides, antibiotics or toxins - using enzyme labelled antibody reactant, providing lower detection limit |
| CN101078723A (en) * | 2007-06-25 | 2007-11-28 | 四川大学 | ELISA method for determining sudan 1 in food |
| CN101349696A (en) * | 2008-08-25 | 2009-01-21 | 深圳市绿诗源生物技术有限公司 | Enzyme-linked immunologic reagent and method for detecting alficetin |
| CN101526537A (en) * | 2009-01-12 | 2009-09-09 | 深圳市绿诗源生物技术有限公司 | Elisa reagent for detecting chloramphenicol and method thereof |
| CN101571540A (en) * | 2009-06-01 | 2009-11-04 | 北京望尔康泰生物技术有限公司 | Enzyme-linked immunosorbent kit for inspecting porcine immunoglobulin G and application thereof |
| CN101571539A (en) * | 2009-06-01 | 2009-11-04 | 北京望尔康泰生物技术有限公司 | Elisa kit for detecting cephalo-type medicine and application thereof |
| CN101788560A (en) * | 2010-03-17 | 2010-07-28 | 北京阿匹斯生物技术有限公司 | Enzyme-linked immunoassay kit for detecting residual chloramphenicol and detection method thereof |
| CN101915840A (en) * | 2010-07-20 | 2010-12-15 | 浙江大学 | Enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibody |
| CN101987836A (en) * | 2010-08-31 | 2011-03-23 | 华南农业大学 | Chlorpromazine hapten, artificial antigen, antibody as well as preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308686C (en) * | 2004-04-30 | 2007-04-04 | 中国农业大学 | Kit for detecting chloromycetin |
-
2011
- 2011-09-20 CN CN201110279098.5A patent/CN103018451B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE4013004A1 (en) * | 1990-04-24 | 1991-10-31 | Elvira Schecklies | ELISA determn. of low mol. wt. pesticides, antibiotics or toxins - using enzyme labelled antibody reactant, providing lower detection limit |
| CN101078723A (en) * | 2007-06-25 | 2007-11-28 | 四川大学 | ELISA method for determining sudan 1 in food |
| CN101349696A (en) * | 2008-08-25 | 2009-01-21 | 深圳市绿诗源生物技术有限公司 | Enzyme-linked immunologic reagent and method for detecting alficetin |
| CN101526537A (en) * | 2009-01-12 | 2009-09-09 | 深圳市绿诗源生物技术有限公司 | Elisa reagent for detecting chloramphenicol and method thereof |
| CN101571540A (en) * | 2009-06-01 | 2009-11-04 | 北京望尔康泰生物技术有限公司 | Enzyme-linked immunosorbent kit for inspecting porcine immunoglobulin G and application thereof |
| CN101571539A (en) * | 2009-06-01 | 2009-11-04 | 北京望尔康泰生物技术有限公司 | Elisa kit for detecting cephalo-type medicine and application thereof |
| CN101788560A (en) * | 2010-03-17 | 2010-07-28 | 北京阿匹斯生物技术有限公司 | Enzyme-linked immunoassay kit for detecting residual chloramphenicol and detection method thereof |
| CN101915840A (en) * | 2010-07-20 | 2010-12-15 | 浙江大学 | Enzyme-linked immunosorbent assay kit for analyzing chloramphenicol residues based on rabbit monoclonal antibody |
| CN101987836A (en) * | 2010-08-31 | 2011-03-23 | 华南农业大学 | Chlorpromazine hapten, artificial antigen, antibody as well as preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103018451A (en) | 2013-04-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100403030C (en) | ELISA kit for detecting Sudan red medicines and detection method thereof | |
| CN101013129B (en) | Enzyme linked immunosorbent reagent casing for detecting furacilin metabolite and uses thereof | |
| CN101571541B (en) | Enzyme-linked immunosorbent inspect kit for inspecting sulfa drugs and method thereof | |
| CN100501409C (en) | ELISA kit for detecting chloramphenicols in animal derived food | |
| CN101571539B (en) | Elisa kit for detecting cephalo-type medicine and application thereof | |
| CN100501410C (en) | ELISA kit for detecting ractopamine in animal derived food | |
| CN101256188A (en) | ELISA kit for detecting lincomycin medicine as well as usage thereof | |
| CN101788560B (en) | Enzyme-linked immunoassay kit for detecting residual chloramphenicol and detection method thereof | |
| CN102967709B (en) | Detect enzyme linked immunological kit and the application thereof of zearalenone medicine | |
| CN103018451B (en) | The enzyme linked immunological kit of chlorine detection mycin and application thereof | |
| CN1811436B (en) | Method for detecting Ennoxacin and its special enzyme-linked immune reagent kit | |
| CN101021536B (en) | Enzyme-linked immunological kit for detecting neomycin drug and method | |
| CN101013130A (en) | Enzyme linked immunosorbent reagent casing for detecting furantoin metabolite and uses thereof | |
| CN101776685B (en) | Enzyme linked immunosorbent assay kit for detecting trimethoprim medicament and application thereof | |
| CN104655847A (en) | Enzyme linked immunosorbent assay kit (ELISA kit) for detecting adprin and detection method thereof | |
| CN101526537A (en) | Elisa reagent for detecting chloramphenicol and method thereof | |
| CN101839918B (en) | Method for detecting spiramycin and special enzyme-linked immunoassay kit thereof | |
| CN101021535B (en) | Enzyme-linked immunalogical kit for detecting gentamicin medicine and method | |
| CN105277708A (en) | Enzyme linked immunosorbent assay kit for detecting aflatoxin B1 in chili | |
| CN104897864B (en) | The enzyme linked immunological kit of detection amantadine and application thereof | |
| CN101013131B (en) | Furantoin metabolite enzyme linked immunosorbent analytical reagent casing and uses thereof | |
| CN101349693A (en) | Fluorobenzene niekau series medicament fast detecting reagent kit and uses thereof | |
| CN104977406A (en) | Enzyme-linked immunoassay kit for detecting fluoroquinolone medicine and application of kit | |
| CN101349696A (en) | Enzyme-linked immunologic reagent and method for detecting alficetin | |
| CN105021815A (en) | Enzyme linked immunosorbent assay kit detecting sodium pentachlorophenate and application of enzyme linked immunosorbent assay kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |