CN100370256C - Enzyme-linked immunologic kit for detecting clenbuterol - Google Patents
Enzyme-linked immunologic kit for detecting clenbuterol Download PDFInfo
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- CN100370256C CN100370256C CNB2004100465679A CN200410046567A CN100370256C CN 100370256 C CN100370256 C CN 100370256C CN B2004100465679 A CNB2004100465679 A CN B2004100465679A CN 200410046567 A CN200410046567 A CN 200410046567A CN 100370256 C CN100370256 C CN 100370256C
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Abstract
The present invention discloses an enzyme-linked immunologic kit for detecting clenbuterol. The enzyme-linked immunologic kit for detecting clenbuterol provided by the present invention comprises custodite antibodies, a clenbuterol specific antibody and enzyme labeling clenbuterol. The enzyme-linked immunologic kit for detecting clenbuterol of the present invention has a low requirement to the sample pre-processing, and a sample pretreatment process is simple; a urine sample can be directly detected after centrifugation, the detection is fast, and the time is about 1.5 hours. A standard curve contains a concentration point of 1 mu g/L, and qualitative and quantitative integration is realized. The lowest detection limitation is 0.1 mu g/L, and the kit has a very low crossing-over rate with other beta excitants, and only has 3.6 % of cross reaction with salbutamol. The present invention has the characteristics of high specificity, high sensitivity, high precision, high accuracy, etc. The present invention can exert important functions in the clenbuterol residue detection for animal foods.
Description
Technical field
The present invention relates to a kind of enzyme linked immunological kit that detects clenbuterol in enzyme linked immunological and the detection of veterinary drugs in food analysis technical field.
Background technology
Clenbuterol claims Clenbuterol again, is a kind of beta-stimulants.Can change the metabolic pathway of nutrient after Clenbuterol enters in the body, protein is synthetic in the promotion animal muscle, particularly skeletal muscle, suppress the synthetic and accumulation of fat, thereby improve carcass quality, the speed of growth is accelerated, lean meat increases relatively, so, claim that it is " clenbuterol hydrochloride ".The phenomenon that in the raising of edible animal, all has this medicine of illegal use to increase economic efficiency both at home and abroad, yet long-term use can make this drug accumulation in the tissue of edible animal, behind edible this tissue of people, can produce that skeletal muscle trembles, a series of bad reactions such as heartbeat is overrun, headache, serious even threat to life.Therefore European and American countries all forbids using clenbuterol on animal husbandry is produced, and China also forbids clenbuterol is used for all edible animals and classifies it emphasis of residue of veterinary drug monitoring as.The regulation clenbuterol must not be and detected medicine in China's " animal food herbal medicine maximum residue limit(MRL) ".
The Clenbuterol residual quantity analytical approach of now having reported mainly contains GC-MS(gas chromatography-mass spectrography), high performance liquid chromatogram-tandem mass spectrometry and test strips detection method, and wherein, GC-MS(gas chromatography-mass spectrography) has been classified industry or national standard as.Though GC-MS(gas chromatography-mass spectrography), high performance liquid chromatogram-tandem mass spectrometry can carry out quantitative test, need through loaded down with trivial details sample pretreatment process, the instrument and equipment costliness is difficult to the detection of a large amount of samples; And the sensitivity of test strips detection method does not reach the detection requirement.
The innovation and creation content
The purpose of this invention is to provide a kind of enzyme linked immunological kit that detects clenbuterol.
The enzyme linked immunological kit of detection clenbuterol provided by the present invention comprises that two of quilt are anti-, clenbuterol specific antibody and enzyme mark clenbuterol.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises A, B, C, D, E, F reagent solution, G reagent solution, H reagent solution, I reagent solution, J reagent solution, K reagent solution, L reagent solution.
Described A, B, C, D, E, F reagent solution are clenbuterol series concentration standard solution.
Described G reagent solution is an enzyme mark clenbuterol concentrate.
Described H reagent solution is the clenbuterol antibody concentrated solution.
Described I reagent solution is a phosphate buffer.
Described J reagent solution is a tetramethyl benzidine.
Described K reagent solution is a citrate buffer solution.
Described L reagent solution is a stop buffer.
Wherein, described clenbuterol specific antibody can be clenbuterol monoclonal antibody or clenbuterol polyclonal antibody; Described clenbuterol monoclonal antibody or polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, pig source, rabbit source and cavy source antibody, described clenbuterol monoclonal antibody is preferably the clenbuterol mouse monoclonal antibody, and described clenbuterol polyclonal antibody is preferably the clenbuterol rabbit polyclonal antibody.
Above antibody all can prepare as immunogene according to a conventional method with the conjugate of clenbuterol and carrier protein.
Described carrier protein can be bovine serum albumin(BSA) (BSA), human serum albumins (HSA), ovalbumin (OVA), albumin rabbit serum (RSA), thyroglobulin (TG) or hemocyanin common carrier albumen such as (KLH); The conjugate of described clenbuterol and carrier protein can obtain by clenbuterol and carrier protein are carried out coupling with glutaraldehyde method, sodium periodate method or diazonium method.
Described marker enzyme can be horseradish peroxidase or alkaline phosphatase, is preferably horseradish peroxidase.Horseradish peroxidase can be marked on the clenbuterol by glutaraldehyde method, sodium periodate method or diazonium method.Described two anti-can be anti-mouse or anti-rabbit antiantibody, are preferably goat anti-rabbit igg or sheep anti-mouse igg.
The material that can be used as fixing described two carriers that resist is a lot, as polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber, Ago-Gel etc.The form of this carrier can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc.
Detection principle of the present invention is adsorbed on the solid phase carrier for resisting two, add sample and enzyme mark clenbuterol, add the clenbuterol specific antibody again, clenbuterol and enzyme mark clenbuterol residual in the testing sample are competed specific antibody, the colour developing back stops, the working sample light absorption value, Clenbuterol residual quantity is negative correlation in this value and the sample, relatively can draw the content of clenbuterol with typical curve.Simultaneously according to the depth of the color sample on the ELISA Plate, but with the concentration range of the comparison judgement sample of the clenbuterol standard solution color of series concentration.
The enzyme linked immunological kit of detection clenbuterol of the present invention mainly adopts the residual quantity of clenbuterol in the qualitative or samples such as detection by quantitative animal tissue (musculature of pig, chicken, ox, sheep and liver, kidney urine etc.), urine sample of ELISA competing method; Pre-treatment requirement to sample is low, and sample pretreatment process is simple, can directly detect after urine sample is centrifugal, detects fast, and the time is about 1.5 hours; 1 this concentration point of μ g/L is arranged in the typical curve, realized that qualitative and quantitative is integrated; Lowest detectable limit reaches 0.1 μ g/L, this kit and other beta-agonist crossing-over rate are very low, 3.6% cross reaction is only arranged with salbutamol, have characteristics such as high specific, high sensitivity, pinpoint accuracy, pin-point accuracy, can in animal food clenbuterol residue detection, play a significant role.
Embodiment
The preparation of embodiment 1, clenbuterol antigen and antibody
(1) clenbuterol antigen is synthetic
With the human serum albumins is carrier, and clenbuterol is a haptens, with diazotising method, glutaraldehyde method, multi-anhydride method or EDC method clenbuterol is coupled on the human serum albumins, obtains clenbuterol antigen.
(2) preparation of clenbuterol rabbit polyclonal antibody
Adopting the big ear rabbit of Japan as immune animal, is immunogene with antigen synthetic in the step (1), and immunizing dose be the 2mg/mL immunizing antigen at every turn, and first immunisation is with the complete freund adjuvant emulsification of 2mL, all around after booster immunization with the incomplete freund adjuvant emulsification of 2mL.Each booster immunization is 2 weeks at interval, and immune 5-10 time altogether, for the last time not with the direct intramuscular injection of immunologic adjuvant, blood sampling detects after 7 days, measure serum antibody titer after, the arteria carotis bloodletting, extraction serum obtains the polyclonal antibody of purifying through ammonium sulfate precipitation.
(3) clenbuterol Monoclonal Antibody
Adopting BALB/C mice as immune animal, is immunogene with antigen synthetic in the step (1), and dosage is that 50 μ g (0.1mL) add the complete freund adjuvant emulsification of equal-volume, carries out subcutaneous first multi-point injection immunity.After January, get same amount immunizing antigen and add incomplete freund adjuvant, booster immunization is carried out in emulsification, and immunizing antigen does not add the adjuvant lumbar injection and carries out booster immunization after January, and antibody titer is measured in blood sampling in 5 days afterwards.Extracting spleen cell carries out Fusion of Cells in 4: 1 ratios and myeloma cell SP2/0.Adopt limiting dilution or soft agar flat band method screening hybridoma, obtain the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain.
Cell cryopreservation and recovery are got the hybridoma that is in exponential phase and are made 1 * 10 with cryopreserving liquid
6-5 * 10
6The cell suspension of individual/mL is sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.Make regular check on the stability of cell activity and secretory antibody.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
Adopt in the body and induce method, BALB/c mouse (8 age in week) abdominal cavity is only injected sterilization paraffin oil 0.5ml/, 7-14 days pneumoretroperitoneum injection hybridomas 5 * 10
5-10
6Individual/as only, to gather ascites after 7-10 days.Carry out the ascites purifying through sad-ammonium sulfate precipitation method, bottle packing ,-20 ℃ of preservations.
(4) preparation of clenbuterol-horseradish peroxidase bond
With clenbuterol being marked on the horseradish peroxidase with diazotising method, glutaraldehyde method, multi-anhydride method or EDC method, obtain clenbuterol-horseradish peroxidase bond, clenbuterol residue detection sensitivity (i.e. 50% inhibition concentration) is less than or equal to 1 μ g/kg.
(5) two anti-preparations
As immune animal, is that immunogene carry out immunity with mouse or rabbit igg with sheep, obtains sheep anti mouse or goat-anti rabbit antiantibody.Concrete grammar is as follows: will be behind the rabbit or the emulsification of rat immune globulin usefulness equivalent Fu Shi Freund's complete adjuvant of ammonium sulfate precipitation method purifying, muscle, subcutaneous, the adult sheep of intracutaneous multiple spot immunity, immunizing dose is a 10mg albumen/only, after January, rabbit or rat immune globulin with half amount add freund 's incomplete adjuvant muscle, subcutaneous or intracutaneous injection sheep, afterwards, per two all booster immunizations once.Fine jade expands to tire and reaches 1: 32 when above, blood sampling, and separation of serum after slightly carrying with ammonium sulfate precipitation method, with immune affinity column chromatography extraction goat anti-rabbit igg or sheep anti-mouse igg, obtains the goat anti-rabbit igg or the sheep anti-mouse igg of purifying.
The enzyme linked immunological kit of embodiment 2, detection clenbuterol
1, detects the structure of the enzyme linked immunological kit of clenbuterol
This kit is mainly by box body, ELISA Plate, A, B, C, D, E, F reagent solution (clenbuterol series concentration standard solution), G reagent solution (clenbuterol-horseradish peroxidase bond concentrate), H reagent solution (clenbuterol antibody concentrated solution), I reagent solution (phosphate buffer); J reagent solution (tetramethyl benzidine); K reagent solution (citrate buffer solution); L reagent solution (stop buffer); Cover plate film and carriage are formed.Mentioned reagent liquid leaves in the reagent bottle.Above-mentioned A is housed, B, C, D, E, F, G, H, J reagent bottle in the carriage.Carriage, ELISA Plate, cover plate film, I reagent bottle, K reagent bottle and L reagent bottle are installed in the box body.ELISA Plate is made up of plastic stent and the plastic strip with holes that separates separately.
2, the preparation of agents useful for same
A, B, C, D, E, F reagent solution are clenbuterol series concentration standard solution, its concentration is respectively 0,0.1,0.3,1,3,9 μ g/L.
The G reagent solution is clenbuterol-horseradish peroxidase bond concentrate, contains clenbuterol-horseradish peroxidase bond of 5-500mg/L.
The H reagent solution is the clenbuterol antibody concentrated solution, and containing protein concentration is clenbuterol mouse monoclonal antibody or the rabbit polyclonal antibody of 5-500mg/L.
The I reagent solution is a 0.01M pH7.2 phosphate buffer.
The J reagent solution is a 0.1-10mg/mL tetramethyl biphenyl amine aqueous solution.
The K reagent solution is a 0.1M pH5.0 citrate buffer solution.
The L reagent solution is a stop buffer, 1-2mol/L sulfuric acid or hydrochloric acid.
3, the preparation of ELISA Plate
Be cushioned liquid (0.05M pH9.6 carbonate buffer solution) with bag goat anti-rabbit igg or sheep anti-mouse igg are diluted to 0.1-5 μ g/ml, every hole adds 100 μ l, 4 ℃ are spent the night, and the bag that inclines is cushioned liquid, wash 3 times with the 0.05M pH7.2 phosphate buffer of 0.05% tween, each 30 seconds, pat dry, in every hole, add 5% skim milk 0.05M phosphate buffer, 250 μ l then, room temperature incubation 1-2h, the liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
The pre-treatment of embodiment 3, sample and detection
1, pig urine: centrifuging and taking supernatant after the freeze thawing, directly detect.
2, pork liver
(1) extracts
Take by weighing minced tissues 1.0g, place centrifuge tube, add 50mmol/L hydrochloric acid 5mL, the vibration 1.5h, in 10-15 ℃ with 5000r/min or the higher centrifugal 15min of speed, supernatant goes in another centrifuge tube.Add 1mol/L sodium hydroxide solution 300 μ L, mixing.Jolting 15min adds 500mmol/L potassium phosphate buffer (pH3.0) 4mL, and mixing is placed 1.5h or spent the night for 4 ℃.With 5000r/min or speed 10-15 ℃ of higher centrifugal 15min, it is standby to get limpid supernatant again.
(2) purify
C
18Solid-phase extraction column is used absolute methanol 3mL, 50mmol/L potassium phosphate buffer (pH3.0) 2mL prewashing successively.Get whole reserve liquids and cross post,, extract with 50mmol/L potassium phosphate buffer 2mL drip washing.With absolute methanol 2mL wash-out, collect eluent, in 50-60 ℃ with nitrogen or air blow drying.Residue water 1.0mL dissolving (centrifugal with the speed more than the 5000r/min if any precipitation), supernatant is as supplying to have a try material.
3, detect
Before detecting kit is recovered room temperature (18 ℃-30 ℃), be made into antibody-solutions, in the elisa plate micropore, add 20 μ l A, B respectively with reagent solution H and I, C, D, E, F reagent solution and sample solution add l00 μ l antibody-solutions in every then micropore, hatch 30min for 18 ℃-30 ℃.Wash ELISA Plate 3 times with deionized water, pat dry.Be made into enzyme conjugates solution with reagent solution G and I, add 100 μ l enzyme conjugates solution in every then micropore, cover the cover plate film, hatch 30min for 18 ℃-30 ℃.Wash ELISA Plate 3 times with deionized water again, pat dry, with J reagent solution and K reagent solution preparation substrate mixed liquor (citrate buffer solution of 0.1% tetramethyl benzidine), get 100 μ l and add mixing behind the ELISA Plate aperture, the about 15min of lucifuge colour developing, add M stop buffer (2mol/L sulfuric acid) at last, measure the 450nm A of place with microplate reader
450Value.Press
Formula is calculated the percentage absorbance:
(B-is the mean light absorbency value of standard solution or sample; B
0-be the standard solution mean light absorbency value of 0 concentration.)
Logarithm with clenbuterol concentration in the standard solution is an X-axis, and the percentage absorbance is a Y-axis, and the drawing standard curve calculates clenbuterol concentration the sample solution from typical curve.
Embodiment 4, kit sensitivity, specificity, precision and accuracy
It is goat anti-rabbit igg antibody that two of this kit resists, and the clenbuterol specific antibody is the clenbuterol rabbit polyclonal antibody, and enzyme mark clenbuterol is clenbuterol-horseradish peroxidase bond.
1, sensitivity
Carried out the kit sensitivity test according to a conventional method, the result shows that standard curve range is 0.1 μ g/L-9 μ g/L, wherein comprises this critical concentration point of 1 μ g/L; The lowest detection of kit is limited to 0.1 μ g/L, detectability≤1.0 μ g/L (kg) in samples such as pig urine and pork liver.
2, specific assay
Select and the similar eight kinds of medicines of clenbuterol 26S Proteasome Structure and Function, measure cross reacting rate respectively.The result is as shown in table 1, shows that this kit and other beta-agonist crossing-over rate are very low, and 3.6% cross reaction is only arranged with salbutamol, and the measurement result of sample can be represented Clenbuterol residual quantity.
Table 1. cross reaction
| Medicine name | Cross reacting rate % |
| Clenbuterol salbutamol bambuterol Formoterol Procaterol Hydrochloride theophylline ephedrine hydrochloride adrenalin hydrochloride 3.5-(N.N-dimethylamino formyloxy) acetophenone | 100 3.6 2.2 ＜0.1 ＜0.1 ＜0.1 ＜0.1 ＜0.1 ＜0.1 |
3, accuracy, precision are measured
Adding clenbuterol titer to final concentration respectively in blank pig urine, pork liver is 1.0 μ g/L and 2.0 μ g/L.Each concentration prepares 5 parts in sample respectively, measures its content then respectively, repeats 3 times.The result shows that in pork liver the recovery that 1 μ g/kg adds concentration is 40%～110%; In pig urine, the recovery that 1 μ g/L adds concentration is 60%～120%; Add in pork liver and the pig urine samples variation within batch coefficient CV≤20%, interassay coefficient of variation CV≤25% in blank.
Claims (3)
1. enzyme linked immunological kit that detects clenbuterol, by wrapping by two solid phase carriers that resist, protein concentration is clenbuterol mouse monoclonal antibody or the rabbit polyclonal antibody of 5-500mg/L, concentration is respectively 0,0.1,0.3,1,3, the clenbuterol series concentration standard solution of 9 μ g/L, the enzyme mark clenbuterol concentrate that contains clenbuterol-horseradish peroxidase bond of 5-500mg/L, 0.01M the phosphate buffer of pH7.2,0.1-10mg/mL the tetramethyl biphenyl amine aqueous solution, the citrate buffer solution of 0.1M pH5.0 and the sulfuric acid of 1-2mol/L or hydrochloric acid stop buffer are formed; Described bag by two anti-solid phase carriers prepare with following method: be cushioned liquid with 0.05M pH9.6 carbonate bag goat anti-rabbit igg or sheep anti-mouse igg be diluted to 0.1-5 μ g/ml, every hole adds 100 μ l, 4 ℃ are spent the night, the bag that inclines is cushioned liquid, 0.05M pH7.2 phosphate buffer with 0.05% tween washs 3 times, each 30 seconds, pat dry, in every hole, add then and contain 5% skim milk 0.05M phosphate buffer, 250 μ l, room temperature incubation 1-2h, the liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
2. enzyme linked immunological kit according to claim 1 is characterized in that: described clenbuterol monoclonal antibody or clenbuterol polyclonal antibody are that the conjugate with clenbuterol and carrier protein obtains as immunogen preparing.
3. enzyme linked immunological kit according to claim 2 is characterized in that: described carrier protein is bovine serum albumin(BSA), human serum albumins, albumin rabbit serum, thyroglobulin, ovalbumin or hemocyanin.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101398424A (en) * | 2007-09-26 | 2009-04-01 | 上海乾隽生物技术有限公司 | Rapid detection kit for brown meat essence and method for manufacturing same |
| CN102539747A (en) * | 2011-12-28 | 2012-07-04 | 于洪侠 | Enzyme-linked immunoassay kit for detecting phenylethanolamine A by direct competition method |
| CN102735834B (en) * | 2012-05-11 | 2014-09-03 | 中国兽医药品监察所 | Enzyme-linked immunoassay kit for detecting phenylethanolamine A |
| CN103630689B (en) * | 2013-12-03 | 2015-08-05 | 河北省科学院生物研究所 | A kind ofly detect enzyme linked immunological kit of Cimaterol medicament residue and preparation method thereof and application |
| CN103852581A (en) * | 2014-03-11 | 2014-06-11 | 河南工业大学 | 3,4-benzopyrene enzyme-linked immune detection kit |
| CN104359900B (en) * | 2014-11-07 | 2017-02-15 | 福建农林大学 | Kit for quickly detecting content of proline in honey |
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| US5660995A (en) * | 1992-03-02 | 1997-08-26 | Enfer Technology Ltd. | Veterinary drug residue surveillance method |
| CN1403814A (en) * | 2001-12-15 | 2003-03-19 | 河南省农业科学院生物技术研究所 | Fast clenbuterol hydrochloride detecting test paper strip |
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