CN107807235A - Clenbuterol ELISA kit, its preparation method and its application - Google Patents
Clenbuterol ELISA kit, its preparation method and its application Download PDFInfo
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- CN107807235A CN107807235A CN201711055603.1A CN201711055603A CN107807235A CN 107807235 A CN107807235 A CN 107807235A CN 201711055603 A CN201711055603 A CN 201711055603A CN 107807235 A CN107807235 A CN 107807235A
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- clenbuterol
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- kit
- potassium nitrite
- elisa plate
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses a kind of clenbuterol ELISA kit, its preparation method and its application, it is intended to solves the problems, such as that clenbuterol does not possess immunogenicity and traditional ELISA Plate method for coating and needs to deposit overnight.The kit includes the antigen protein of coated coupling clenbuterol, and its preparation method is：Potassium nitrite is taken to be dissolved in the potassium nitrite solution being configured in distilled water；With H2SO4Dissolve clenbuterol；Potassium nitrite solution is slowly added to after cooling and is stirred continuously, is stood；Carrier protein is dissolved in phosphate buffer；Diazotising clenbuterol is added, keeps acid-base value stable, overnight；With product of the PBS after coupled until can not detect any small-molecule substance in dialyzate；Product filtering after will be coupled, packing preserve；ELISA Plate is taken, clenbuterol comlete antigen is added per hole, dries rear enclosed；It is fixed after washing repeatedly, produced after drying.Present invention detection high specificity, reagent dosage is few, shortens kit fabrication cycle.
Description
Technical field
The present invention relates to technical field of food safety detection, and in particular to a kind of clenbuterol ELISA kit, its
Preparation method and applications.
Background technology
Clenbuterol（Clenbuterol）It is a kind of potent β2- adrenoceptor agonists, are added to feed
In, growth of animal can be promoted, increase the synthesis of protein, improve poultry lean meat ratio, but Clenbuterol can be in the edibility of animal
Accumulation causes eater to produce toxic side effect in tissue, and the Ministry of Agriculture of China makes regulation, strictly forbids using in aquaculture gram
Lun Teluo, and require to strengthen supervising residue detection box of the material in animal derived food.Measure clenbuterol is residual at present
The method stayed is more, there is GC methods, HPLC methods, GC/MS methods, LC-MS methods, although these method detection sensitivities are high, accuracy is good,
As a result stablize, and possess can be qualitative and the features such as can quantify for Part Methods, but for scene and the detection work of grass-roots unit
For work, also there are many weak points, such as instrument and equipment costliness, be not suitable for extensive use；Pre-treatment is cumbersome, operates skill
Art requires higher, and detection cycle is long, and once can not in high volume carry out the detection of sample.
Methods of the ELISA as the most frequently used screening clenbuterol, the detection method of chemistry, biology with routine
Compare, with detection time it is short, it is easy to operate, with higher sensitivity and higher specific advantage, and available for one
It is secondary large batch of to be detected.But the reagent that current domestic production uses is numerous and diverse there is coating technique, and cost is of a relatively high to be ordered
The shortcomings that purchase cycle is longer.
Therefore develop that a kind of coating technique is succinct, wrapper sheet cost is low, the product of kit short preparation period is used to detect
Clenobuterol hydrochloride residue in animal-derived food, by with boundless application prospect.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of clenbuterol ELISA kit, its preparation method and
It is applied.
In order to solve the above technical problems, the present invention uses following technology path：
Because clenbuterol is micromolecular compound, belonging to haptens, itself only has reactionogenicity, does not possess immunogenicity,
It could be coated on so comlete antigen need to be coupled into macromolecular carrier albumen with ELISA on ELISA Plate, traditional enzyme
Target method for coating is coated with using wet method, it is necessary to deposit overnight, and change coating environmental selection on this basis is coated with using dry method,
It is substantially shorter the coating time.
Concrete technical scheme is as follows：
A kind of clenbuterol ELISA kit is designed, includes the antigen protein of coated coupling clenbuterol.
The preparation method of described kit comprises the following steps：
（1）Potassium nitrite is taken to be dissolved in the potassium nitrite solution being configured in distilled water, temperature regulating is to 0~5 DEG C；
（2）With H2SO4Clenbuterol is dissolved, water-bath acts on 20 min；
（3）Treat that supernatant cools down, be slowly added to potassium nitrite solution and be stirred continuously, stand reaction；
（4）Carrier protein is dissolved in phosphate buffer, cooled down；
（5）Diazotising clenbuterol is slowly added to and is stirred continuously, keeps acid-base value stable, reaction overnight；
（6）With product of the PBS after coupled until can not detect any small-molecule substance in dialyzate；
（7）Product after will be coupled is preserved with membrane filtration, -20 DEG C of packing；
（8）Clean ELISA Plate is taken, clenbuterol comlete antigen is added per hole, is dried；
（9）BSA closings are added per hole；
（10）Washed repeatedly with PBST；
（11）Add fixer to fix, produced after being completely dried.
Preferably, the step（8）In the drying temperature be 37 DEG C.
Preferably, the step（11）In the drying temperature be 37 DEG C.
Application of the described kit in food safety detection.
Compared with prior art, advantageous effects of the invention are：
1. the present invention makes clenbuterol be coupled into comlete antigen with macromolecular carrier albumen, with competitive enzyme-linked immune method bag
By on ELISA Plate, detection sensitivity is high, and high specificity is detected for clenbuterol material.
2. reagent dosage of the present invention is few, make kit preparation process significantly simple and easy to do, it is cost-effective, it is easy to use, easily
In promotion and application.
It is coated with 3. the present invention changes coating environmental selection using dry method, is substantially shorter the coating time, ELISA Plate prepares consumption
When it is short, shorten kit fabrication cycle, save the plenty of time.
Brief description of the drawings
Fig. 1 is the comparison figure of dry method coating and wet method coating detection OD values；
Fig. 2 is coefficient of variation figure in the plate of each standard liquid.
Embodiment
Illustrate the embodiment of the present invention with reference to the accompanying drawings and examples, but following examples are used only in detail
Describe the bright present invention in detail, the scope not limiting the invention in any way.Involved instrument and equipment is such as in the examples below
It is routine instrument device without special instruction；Involved reagent is commercially available conventional reagent unless otherwise instructed；It is involved
Detection method, be conventional method unless otherwise instructed.
Embodiment one：A kind of preparation of clenbuterol ELISA kit
Detailed operating procedures are as follows：
1. potassium nitrite is taken to be dissolved in the potassium nitrite solution for being configured to 0.2 M in distilled water, with ice-water bath temperature regulating to 0~5
℃；
2. with the M of 4 mL 0.5 H2SO412 mg clenbuterols are dissolved, 37 DEG C of water-baths act on 20 min；
3. treating that supernatant is cooled to 4 DEG C, it is slowly added to 0.2 M potassium nitrites solution and is stirred continuously, stands 30 min of reaction；
4. by 100 mg ovalbumins（OVA）It is dissolved in as carrier protein in 4 mL phosphate buffers, is cooled to 4 DEG C or so；
5. diazotising clenbuterol to be slowly added to and be stirred continuously, to keep the stabilization of acid-base value in adition process, add simultaneously
Enter 0.2 M NaOH, regulation pH value to 7.4,4 DEG C of reaction overnights；
6. liquid is changed once with product 3 d, every 6 h of the PBS after coupled under the conditions of 4 DEG C, until being detected not in dialyzate
Go out any small-molecule substance；
7. the product after will be coupled is preserved with 0.45 μm of membrane filtration, -20 DEG C of packing；
8. take clean ELISA Plate, 100 μ L are added per hole, and with PBS to be diluted to the clenbuterol that concentration is 1.25 μ g/mL complete
Antigen, being placed in 37 DEG C of insulating boxs is completely dried coating buffer；
9. the BSA that 200 μ L mass volume ratios are 1% is added per hole closes 1h in 37 DEG C；
10. washed repeatedly 4~5 times with the PBST that mass volume ratio is 0.05%；
11. it is that 10% sucrose, the mixed solution of trehalose fix 0.5 h as fixer to add 200 μ L mass volume ratios；
12. being placed in 37 DEG C of insulating boxs makes to obtain being coated with complete ELISA Plate after being completely dried.
Embodiment two：The comparison of clenbuterol ELISA kit and conventional reagents box
（One）Testing result compares
1. ELISA reagent and ELISA Plate are taken out from cold storage environment, 30 min are balanced at room temperature, and every kind of liquid uses
Before must shake up, wherein titer need to be cooked 2 parallel tests.
2. add standard items：Clenbuterol standard items or the μ L of sample 20 are added per hole（Concentration is 0 ppb, 0.1 ppb, 0.3
Ppb, 0.9 ppb, 2.7 ppb, 8.1 ppb）, then add the μ L of sheep anti-Mouse enzyme marker 50（1：1500 dilution proportion liquid）, then add
Enter the μ L of clenbuterol monoclonal antibody working solution 80（Concentration is defined as 0.125 μ g/mL）, gently vibration mixes, with cover plate film
Cover, lucifuge reacts 30 min at 25 DEG C of room temperature；
3. board-washing：Carefully open cover plate film, liquid in hole dried, adds 0.05% PBST to wash repeatedly, every time soak 15~
30s, fully wash 4~5 times, patted dry with blotting paper；
4. colour developing：It is first each per hole to add TMB nitrite ions A（Main component is H2O2）50 μ L, then each addition TMB nitrite ions B（It is main
It is 3,3,5,5- tetramethyl benzidines to want composition）50 μ L, gently vibration mix, and good with cover plate membrane cover, lucifuge is anti-at 25 DEG C of room temperature
Answer 15 min；
5. measure：Add 50 μ L sulfuric acid terminate liquids per hole, set the nm dual wavelengths of ELIASA 450/630, determine OD values, 5 min
Inside run through data.
Compared with clenbuterol ELISA kit kit detection data coated with conventional wet, as a result such as
Shown in table 1 and Fig. 1：
The wet method of table 1 is coated with and dry method coated elisa plate testing result
。
（Two）Precision compares
Precision represents with the coefficient of variation, the coefficient of variation=SD average value/average Percentage bound
As a result it is as shown in Figure 2：Dry method wrapper sheet compares with wet method wrapper sheet, and the coefficient of variation is below 0.035 in plate, dry method wrapper sheet
The wrapper sheet time is substantially shorter, reduces the kit production cycle.
The present invention is described in detail above in conjunction with drawings and examples, still, those of skill in the art
Member can also be carried out it is understood that on the premise of present inventive concept is not departed to each design parameter in above-described embodiment
Change, forms multiple specific embodiments, is the common excursion of the present invention, is no longer described in detail one by one herein.
Claims (5)
1. a kind of clenbuterol ELISA kit, it is characterised in that the antigen egg of coupling clenbuterol is coated with containing dry method
White ELISA Plate.
2. the preparation method of the kit described in claim 1, it is characterised in that comprise the following steps：
（1）Potassium nitrite solution is configured, temperature adjustment is to 0~5 DEG C；
（2）With H2SO4Dissolve clenbuterol；
（3）Cool down and be slowly added to potassium nitrite solution, stand；
（4）Carrier protein is dissolved in phosphate buffer, cooled down；
（5）Diazotising clenbuterol is slowly added to, reaction overnight；
（6）With PBS step（5）Resulting solution；
（7）By step（6）Resulting solution filters, and -20 DEG C of packing preserve；
（8）ELISA Plate is taken, adds clenbuterol comlete antigen, is dried；
（9）Add BSA closings；
（10）Washed repeatedly with PBST；
（11）Fixer is added, is produced after drying.
3. the preparation method of kit according to claim 2, it is characterised in that the step（8）In the drying
Temperature is 37 DEG C.
4. the preparation method of kit according to claim 2, it is characterised in that the step（11）In the drying
Temperature is 37 DEG C.
5. the application in food safety detection of the kit described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711055603.1A CN107807235A (en) | 2017-11-01 | 2017-11-01 | Clenbuterol ELISA kit, its preparation method and its application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711055603.1A CN107807235A (en) | 2017-11-01 | 2017-11-01 | Clenbuterol ELISA kit, its preparation method and its application |
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| CN107807235A true CN107807235A (en) | 2018-03-16 |
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| CN201711055603.1A Pending CN107807235A (en) | 2017-11-01 | 2017-11-01 | Clenbuterol ELISA kit, its preparation method and its application |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111528219A (en) * | 2020-05-13 | 2020-08-14 | 上海市计量测试技术研究院 | Freeze-drying protective agent for T lymphocyte subpopulation counting standard substance and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1779462A (en) * | 2004-11-24 | 2006-05-31 | 国家海洋环境监测中心 | Hyperimmune serum against Vibrio parahaemolyticus, its preparation and inspecotion for astacin pathogenic bacterium |
| CN1885038A (en) * | 2006-07-11 | 2006-12-27 | 华南农业大学 | ELISA kit for detecting clenbuterol and detection method thereof, and animal tissue sample preparing method before detection |
| CN101368952A (en) * | 2008-09-24 | 2009-02-18 | 四川大学 | ELISA adsorption analysis method for measuring clenobuterol hydrochloride content in milk, pork liver, chicken liver and animal feed |
| CN101692087A (en) * | 2009-09-30 | 2010-04-07 | 暨南大学 | Method for preparing streptavidin pre-coated elisa plate and application |
| CN103063830A (en) * | 2012-12-21 | 2013-04-24 | 杭州茂天赛科技有限公司 | Preparation method for pre-coated enzyme-linked immunosorbent assay (ELISA) plate |
-
2017
- 2017-11-01 CN CN201711055603.1A patent/CN107807235A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1779462A (en) * | 2004-11-24 | 2006-05-31 | 国家海洋环境监测中心 | Hyperimmune serum against Vibrio parahaemolyticus, its preparation and inspecotion for astacin pathogenic bacterium |
| CN1885038A (en) * | 2006-07-11 | 2006-12-27 | 华南农业大学 | ELISA kit for detecting clenbuterol and detection method thereof, and animal tissue sample preparing method before detection |
| CN101368952A (en) * | 2008-09-24 | 2009-02-18 | 四川大学 | ELISA adsorption analysis method for measuring clenobuterol hydrochloride content in milk, pork liver, chicken liver and animal feed |
| CN101692087A (en) * | 2009-09-30 | 2010-04-07 | 暨南大学 | Method for preparing streptavidin pre-coated elisa plate and application |
| CN103063830A (en) * | 2012-12-21 | 2013-04-24 | 杭州茂天赛科技有限公司 | Preparation method for pre-coated enzyme-linked immunosorbent assay (ELISA) plate |
Non-Patent Citations (2)
| Title |
|---|
| 周群标 等: "动物性食品中盐酸克伦特罗ELISA检测方法的建立及应用", 《中国食品学报》 * |
| 夏宗范: "《化工百科全书》", 31 December 1991 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111528219A (en) * | 2020-05-13 | 2020-08-14 | 上海市计量测试技术研究院 | Freeze-drying protective agent for T lymphocyte subpopulation counting standard substance and application thereof |
| CN111528219B (en) * | 2020-05-13 | 2022-03-15 | 上海市计量测试技术研究院 | Freeze-drying protective agent for T lymphocyte subpopulation counting standard substance and application thereof |
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Application publication date: 20180316 |