CN1811445A - Enzyme-linked immunosorbent analytical technique with specificity to clenbuterol and its detecting reagent kit - Google Patents
Enzyme-linked immunosorbent analytical technique with specificity to clenbuterol and its detecting reagent kit Download PDFInfo
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- CN1811445A CN1811445A CN 200610038011 CN200610038011A CN1811445A CN 1811445 A CN1811445 A CN 1811445A CN 200610038011 CN200610038011 CN 200610038011 CN 200610038011 A CN200610038011 A CN 200610038011A CN 1811445 A CN1811445 A CN 1811445A
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- clenbuterol
- enzyme
- antibody
- tool
- linked immunosorbent
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Abstract
The present invention discloses an enzyme-linked immcenosorbent analysis method for clenbuterol with specificity and its detection kit, belonging to the field of bio-chemical analysis technique. Said invention includes the following steps: using clenbuterol and halogenated carboxylic acid ester as initial raw material to synthesize semiantigen, making the semiantigen be covalently coupled with different proteins to respectively synthesize artificial antigen and coating source, using artificial antigen to immunize animal to prepare antibody with specific affinity for clenbuterol, using horseradish peroxidase to label antibody or semiantigen, using described antibody or coating source to coat microwell plate so as to prepare the invented quick detection kit for detecting residual clenbuterol in the samples of feed, urine and live-stock products.
Description
Technical field
The present invention relates to specific competitiveness enzyme immune absorption conalysis technology of Clenbuterol tool and detection kit thereof are mainly used in the fast detecting of residual Clenbuterol in feed, urine, the livestock products equal samples.
Background technology
Clenbuterol (Clenbuterol) has another name called Celenbuterol, clenbuterol hydrochloride, and clenbuterol is a kind of beta-2-adrenoreceptor agonists of synthetic, is used to prevent and treat respiratory diseases such as bronchial astehma, asthma chronic bronchitis and pulmonary emphysema.On veterinary clinic,, can reduce animal trunk fat deposition, increase the synthetic of muscle when its application dose reaches 5～10 times of therapeutic dose and application time when longer.The title that " clenbuterol hydrochloride " therefore arranged again.Clenbuterol absorbs soon in animal body, distribute wide, long half time, easily accumulation, symptom such as the people may cause palpitaition, trembles after having eaten the residual meat that Clenbuterol arranged, dizziness, headache even cause death.Therefore, must strengthen the monitoring of residual Clenbuterol in feed, urine, the livestock products equal samples.
At present, the method for detection residual of kelengtelu mainly contains high performance liquid chromatography, GC-MS(gas chromatography-mass spectrography), capillary electrophoresis and enzyme linked immunosorbent assay analysis method.Adopt instrumental method to need expensive instrument and equipment, specialized laboratory and well-trained specialized personnel, slow, the poor selectivity of requirement height, process complexity, speed to sample pre-treatments is difficult to adapt to the requirement of great amount of samples and field quick detection.That enzyme-linked immunosorbent analytical technique has is highly sensitive, analysis cost is low, simple and efficient, advantage such as instrument and equipment is simple, is suitable for the fast detecting of batch samples.But the development of existing Clenbuterol enzyme-linked immuno sorbent assay kit, because the synthetic of artificial antigen is earlier with the amino diazotising on the Clenbuterol phenyl ring, then under weak basic condition with protein on the tyrosine covalent coupling, so just caused the specificity part of Clenbuterol to be covered by protein macromolecule, the antibody that obtains and Clenbuterol analogue more serious cross reaction is arranged, false positive in the actual detected process, often occurs, have some positive not to be identified simultaneously.
Summary of the invention
The purpose of this invention is to provide and a kind of Clenbuterol is had high selectivity, high sensitivity, be fit to the competitiveness enzyme immune absorption conalysis technology of residual Clenbuterol fast detecting in feed, urine, the livestock products equal samples.
Another purpose of the present invention provides a kind of high specificity, highly sensitive, convenient and swift, Cheap highly effective, is fit to the competitiveness enzyme linked immuno sorbent assay kit of residual Clenbuterol field quick detection in feed, urine, the livestock products equal samples.
Technical scheme one of the present invention:, it is characterized in that with Clenbuterol and halogenated carboxylic ester being the haptens of the synthetic outstanding Clenbuterol architectural feature of initiation material to specific enzyme-linked immunosorbent analytical technique of Clenbuterol tool and detection kit thereof
(n=1-5), with haptens and synthetic respectively artificial antigen of different proteins covalent coupling and coating antigen, with the antibody of artificial antigen immune animal preparation to Clenbuterol tool specificity affinity, with horseradish peroxidase-labeled antibody or haptens, with described coating antigen or antibody sandwich polystyrene micropore plate, add the mixed liquor of testing sample (or Clenbuterol standard specimen) and enzyme labeling thing, the competitive combination takes place with insolubilized antibody or coating antigen in Clenbuterol, enzyme labeling thing; Educt is removed in washing, adds the substrate and the developer of enzyme, and the intensity of enzyme-catalytic chromogenic reaction is marked haptens with the enzyme on being combined in antibody or the enzyme labelled antibody amount that is combined on the coating antigen is directly proportional, and is inversely proportional to the content of Clenbuterol in sample or the standard specimen.
Described haptens is to be the derivant that initiation material reaction generates with Clenbuterol and halogenated carboxylic ester, and its molecular structural formula is
(n=1-5).
Described artificial antigen is to adopt active ester method or mixed anhydride method that described haptens and protein covalent coupling are formed.
Described antibody is to what Clenbuterol had the polyclonal antibody of specificity affinity or adopted the hybridoma technology preparation Clenbuterol to be had the monoclonal antibody of specificity affinity with animals preparations such as immunize rabbit, mouse behind described artificial antigen and an amount of Freund mixing and emulsifying.
Described enzyme mark haptens is to adopt mixed anhydride method or active ester method that described haptens and horseradish peroxidase covalent coupling are formed.
Described enzyme labelled antibody is to adopt the periodates method of improvement that described antibody and horseradish peroxidase covalent coupling are formed or adopt commercially available ELIAS secondary antibody.
With described antibody or coating antigen bag by polystyrene micropore plate, the site that is not adsorbed with 0.5% gelatin closed porosity surface.
The substrate of described enzyme is urea peroxide or hydrogen peroxide.
Described developer is 3 ', 3 ', 5 ', 5 '-tetramethyl benzidine (TMB) or o-phenylenediamine (OPD).
Technical scheme two of the present invention:, it is characterized in that by box body and place detachable polystyrene micropore plate in the box body, the antibody of Clenbuterol tool specificity affinity or coating antigen, damping fluid, confining liquid (also can be in advance be coated on described antibody or coating antigen on the microwell plate and seal in the rearmounted box body), Clenbuterol standard specimen, enzyme labeling thing, substrate solution, developer, stop buffer and operation instructions be formed to the specific Enzyme Linked Immunoadsorbent Assay detection kit of Clenbuterol tool.
Use above-mentioned the specific competitiveness enzyme immune absorption conalysis technology of Clenbuterol tool to be provided with related reagent and material in box, the preparation immunity detection reagent is used for the fast detecting of feed, urine, the residual Clenbuterol of livestock products sample.The linear concentration range that kit detects Clenbuterol is 10
-3~ 10
0μ g/mL, detectability＜0.1ng/mL.
Science of the present invention is strong, advanced technology, and cost is low, and application process is easy.Basic foundation is the micromolecular compound immunoassay principles of chemistry and technology.Molecular weight does not generally possess immunogenicity less than 5000 daltonian compounds, the haptens and the protein covalent coupling of outstanding Clenbuterol molecular characterization are prepared artificial antigen and coating antigen, with the antibody of artificial antigen immune animal generation to Clenbuterol tool specificity affinity.Do not have reactionogenicity though micromolecular compound does not possess immunogenicity, can immunity take place with corresponding antibodies under isolated condition and combine and meet the mass action law.With horseradish peroxidase-labeled haptens or antibody, the mixed liquor that in the hole of the microwell plate that is coated with antibody or coating antigen, adds testing sample (or Clenbuterol standard specimen) and enzyme labeling thing, Clenbuterol, competitive association reaction takes place with the antibody or the coating antigen that are coated on micropore surface in the enzyme labeling thing, the substrate and the developer that add enzyme behind the washing removal educt, the intensity of enzyme-catalytic chromogenic reaction is directly proportional with the amount of enzyme labeling thing on being combined in antibody or coating antigen, be inversely proportional to the content of Clenbuterol in the sample (or standard specimen), thereby set up the Clenbuterol enzyme-linked immunosorbent analytical technique.Use this technology, related reagent and material are set in box body, prepare immunity detection reagent, be used for the field quick detection of feed, urine, the residual Clenbuterol of livestock products equal samples.By referring to instructions, the general staff also can carry out on-the-spot check and analysis, is convenient to promote the use of.
Embodiment
One, to the specific competitiveness enzyme immune absorption conalysis technology of Clenbuterol tool embodiment
1, Clenbuterol and γ-bromo butyric ester reaction are generated derivant
(n=3) make haptens with this, adopt synthetic artificial antigen of covalent couplings such as active ester method or mixed anhydride method and bovine serum albumin(BSA), ovalbumin and coating antigen.
2, with animal preparations such as immunize rabbit, sheep, mouse behind described artificial antigen and an amount of Freund mixing and emulsifying Clenbuterol is had the polyclonal antibody of specificity affinity or adopts the hybridoma technology preparation Clenbuterol to be had the monoclonal antibody of specificity affinity.
3, adopt mixed anhydride method or active ester method that described haptens and horseradish peroxidase covalent coupling are prepared enzyme mark haptens.
4, with described antibody sandwich polystyrene micropore plate, the site of not adsorbing antibody with 0.5% gelatin closed porosity surface.
5, be the substrate of enzyme with urea peroxide or hydrogen peroxide, with 3 ', 3 ', 5 ', 5 '-tetramethyl benzidine (TMB) or o-phenylenediamine (OPD) they are developer.
6, in wrapping, add testing sample (or Clenbuterol standard specimen) and mark haptenic mixed liquor with enzyme by the hole of the microwell plate of good antibody, Clenbuterol, the competitive combination takes place with the antibody that is coated on micropore surface in enzyme mark haptens, educt is removed in washing, the substrate and the developer that add enzyme, the intensity of enzyme-catalytic chromogenic reaction is marked the haptens amount with the enzyme on being combined in antibody and is directly proportional, be inversely proportional to the content of Clenbuterol in the sample (or standard specimen), set up in view of the above the specific solid phase direct competitive of Clenbuterol tool enzyme-linked immunosorbent analytical technique, the residual Clenbuterol in the testing sample is carried out the qualitative, quantitative fast detecting.
Two, the specific competitiveness enzyme linked immunosorbent assay kit of Clenbuterol tool is prepared embodiment
1, the bag of microwell plate is dissolved into debita spissitudo with antibody with the damping fluid of 20 times of dilutions, add in the hole of microwell plate, and 100 μ L/ holes, 4 ℃ of absorption are spent the night.Remove the solution in the hole, pat dry, add confining liquid 150 μ L/ holes, 4 ℃ of sealings are spent the night or 37 ℃ of sealings 2 hours.Remove unnecessary confining liquid, pat dry, wash 3 times with the damping fluid of 20 times of dilutions, pat dry, air dry under 4 ℃ of conditions adds drying agent and packs, and preserves standby below 4 ℃.Also antibody, damping fluid, confining liquid can be respectively charged into specified containers, put in the kit, wrap quilt voluntarily by operation instruction before use by the user.
2, the preparation of standard specimen solution accurately takes by weighing Clenbuterol standard specimen 0.0100g, is dissolved in 100mL methyl alcohol, 4 ℃ of preservations.
3, enzyme is marked haptenic preparation and is adopted mixed anhydride method or active ester method with haptens and horseradish peroxidase covalent coupling, dialysis is removed and to be stored in behind the free micromolecular compound in 50% the glycerine, and the direct combined techniques of coated antibody is measured enzyme mark haptens and tired.Preparation is marked enzyme during kit haptens and is diluted to 100 times of working concentration, preservation below 4 ℃ with 50% glycerine.
4, the preparation 0.2mol/L NaH of damping fluid
2PO
4280mL adds 0.2mol/L Na
2HPO
4720mL, NaN
30.5g the dissolving mixing, 4 ℃ of preservations.
5, preparation 5.0g gelatin, the 0.5g NaN of confining liquid
3Be dissolved in the phosphate buffer of 0.01mol/L, pH7.2 and be settled to 1L, 4 ℃ of preservations.
6, the preparation 0.6g urea peroxide of substrate solution is dissolved in 1L citric acid-phosphate buffer (5.2g citric acid, 18.4g sodium hydrogen phosphate are dissolved in distilled water and are settled to 1L), 4 ℃ of preservations.
7, the preparation 0.44g TMB of developer is dissolved in the 3.2mL absolute ethyl alcohol, is settled to 1L with citric acid-phosphate buffer, fills N
2Or the decompression degassing, 4 ℃ of preservations.
8, the preparation 100mL concentrated sulphuric acid of stop buffer under agitation slowly adds in the 800mL distilled water, cooling.
9, reagent packing all ingredients is prepared on request, and it is aseptic subpackaged to measure qualified back.To the antibody of Clenbuterol tool specificity affinity an amount of/bottle, Clenbuterol standard specimen 0.1mL/ bottle, enzyme mark haptens 0.1mL/ bottle, damping fluid 10mL/ bottle, confining liquid 20mL/ bottle, substrate solution 6mL/ bottle, developer 6mL/ bottle, stop buffer 6mL/ bottle.Label after the packing, indicate the lot number and the term of validity, 4 ℃ of preservations.
10, the kit assembling is respectively with 1 of detachable microwell plate, antibody, Clenbuterol standard specimen solution, the enzyme of Clenbuterol tool specificity affinity are marked respectively 1 bottle of haptens, damping fluid, confining liquid, substrate solution, developer, stop buffer, and operation instructions are put assigned address in the kit for 1 part.Also can put assigned address in the kit in advance with antibody sandwich also sealing on microwell plate.Kit encapsulates after the assay was approved, 4 ℃ of preservations.
Claims (10)
1, a kind of to the specific enzyme-linked immunosorbent analytical technique of Clenbuterol tool, it is characterized in that with Clenbuterol and halogenated carboxylic ester being the haptens of the synthetic outstanding Clenbuterol architectural feature of initiation material
With haptens and synthetic respectively artificial antigen of different proteins covalent coupling and coating antigen, with the antibody of artificial antigen immune animal preparation to Clenbuterol tool specificity affinity, with horseradish peroxidase-labeled antibody or haptens, with described coating antigen or antibody sandwich polystyrene micropore plate, add the mixed liquor of testing sample and enzyme labeling thing, the competitive combination takes place with insolubilized antibody or coating antigen in Clenbuterol, enzyme labeling thing; Educt is removed in washing, adds the substrate and the developer of enzyme, and the intensity of enzyme-catalytic chromogenic reaction is marked haptens with the enzyme on being combined in antibody or the enzyme labelled antibody amount that is combined on the coating antigen is directly proportional, and is inversely proportional to the content of Clenbuterol in sample or the standard specimen.
2, described according to claim 1 to the specific enzyme-linked immunosorbent analytical technique of Clenbuterol tool, it is characterized in that described haptens is is the derivant that initiation material reaction generates with Clenbuterol and halogenated carboxylic ester, its molecular structural formula is
3, described according to claim 1 to the specific enzyme-linked immunosorbent analytical technique of Clenbuterol tool, it is characterized in that described artificial antigen is to adopt active ester method or mixed anhydride method that described haptens and protein covalent coupling are formed.
4, described according to claim 1 to the specific enzyme-linked immunosorbent analytical technique of Clenbuterol tool, it is characterized in that described antibody is to what Clenbuterol had the polyclonal antibody of specificity affinity or adopted the hybridoma technology preparation Clenbuterol to be had the monoclonal antibody of specificity affinity with animals preparations such as immunize rabbit, mouse behind described artificial antigen and an amount of Freund mixing and emulsifying.
5, described according to claim 1 to the specific enzyme-linked immunosorbent analytical technique of Clenbuterol tool, it is characterized in that described enzyme mark haptens is to adopt mixed anhydride method or active ester method that described haptens and horseradish peroxidase covalent coupling are formed.
6, described according to claim 1 to the specific enzyme-linked immunosorbent analytical technique of Clenbuterol tool, it is characterized in that described enzyme labelled antibody is to adopt the periodates method of improvement that described antibody and horseradish peroxidase covalent coupling are formed or ELIAS secondary antibody.
7, described according to claim 1 to the specific enzyme-linked immunosorbent analytical technique of Clenbuterol tool, it is characterized in that with described antibody or coating antigen bag by polystyrene micropore plate the site that is not adsorbed with 0.5% gelatin closed porosity surface.
8, described to the specific enzyme-linked immunosorbent analytical technique of Clenbuterol tool according to claim 1, the substrate that it is characterized in that described enzyme is urea peroxide or hydrogen peroxide.
9, described according to claim 1 to the specific enzyme-linked immunosorbent analytical technique of Clenbuterol tool, it is characterized in that described developer is 3 ', 3 ', 5 ', 5 '-tetramethyl benzidine or o-phenylenediamine.
10, a kind of to the specific Enzyme Linked Immunoadsorbent Assay detection kit of Clenbuterol tool, it is characterized in that by box body and place detachable polystyrene micropore plate in the box body, antibody or coating antigen, damping fluid, confining liquid, Clenbuterol standard specimen, enzyme labeling thing, substrate solution, developer, stop buffer and the operation instructions of Clenbuterol tool specificity affinity are formed.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610038011 CN1811445A (en) | 2006-01-25 | 2006-01-25 | Enzyme-linked immunosorbent analytical technique with specificity to clenbuterol and its detecting reagent kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610038011 CN1811445A (en) | 2006-01-25 | 2006-01-25 | Enzyme-linked immunosorbent analytical technique with specificity to clenbuterol and its detecting reagent kit |
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|---|---|---|---|
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1885038B (en) * | 2006-07-11 | 2010-08-18 | 华南农业大学 | ELISA kit for detecting clenbuterol and detection method thereof, and animal tissue sample preparing method before detection |
| CN104193637A (en) * | 2014-05-06 | 2014-12-10 | 湖州海创生物科技有限公司 | Specific clenbuterol semiantigen derivative, clenbuterol artificial antigen, preparing methods of the semiantigen derivative and the clenbuterol artificial antigen and applications of the clenbuterol artificial antigen |
| CN104892764A (en) * | 2014-03-07 | 2015-09-09 | 北京维德维康生物技术有限公司 | Quantum dot immunofluorescence kit for detecting clenbuterol or derivatives thereof |
| CN113960305A (en) * | 2021-10-15 | 2022-01-21 | 北京勤邦生物技术有限公司 | Immunomagnetic bead for clenbuterol enrichment and purification and preparation method and application thereof |
-
2006
- 2006-01-25 CN CN 200610038011 patent/CN1811445A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1885038B (en) * | 2006-07-11 | 2010-08-18 | 华南农业大学 | ELISA kit for detecting clenbuterol and detection method thereof, and animal tissue sample preparing method before detection |
| CN104892764A (en) * | 2014-03-07 | 2015-09-09 | 北京维德维康生物技术有限公司 | Quantum dot immunofluorescence kit for detecting clenbuterol or derivatives thereof |
| CN104193637A (en) * | 2014-05-06 | 2014-12-10 | 湖州海创生物科技有限公司 | Specific clenbuterol semiantigen derivative, clenbuterol artificial antigen, preparing methods of the semiantigen derivative and the clenbuterol artificial antigen and applications of the clenbuterol artificial antigen |
| CN113960305A (en) * | 2021-10-15 | 2022-01-21 | 北京勤邦生物技术有限公司 | Immunomagnetic bead for clenbuterol enrichment and purification and preparation method and application thereof |
| CN113960305B (en) * | 2021-10-15 | 2023-10-17 | 北京勤邦科技股份有限公司 | Immunomagnetic bead for clenbuterol Luo Fuji purification and preparation method and application thereof |
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Open date: 20060802 |