CN103728450B - A kind of detection clenobuterol hydrochloride chemiluminescence detection kit - Google Patents
A kind of detection clenobuterol hydrochloride chemiluminescence detection kit Download PDFInfo
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- CN103728450B CN103728450B CN201310551775.3A CN201310551775A CN103728450B CN 103728450 B CN103728450 B CN 103728450B CN 201310551775 A CN201310551775 A CN 201310551775A CN 103728450 B CN103728450 B CN 103728450B
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- G—PHYSICS
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- 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
- G01N33/545—Synthetic resin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
- G01N21/763—Bioluminescence
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Abstract
The present invention relates to a kind of detection clenobuterol hydrochloride chemiluminescence detection kit, comprise the antibody of the goat-anti rabbit of luminous plaque, clenobuterol hydrochloride polyclonal antibody, ELIAS secondary antibody and horseradish peroxidase mark, clenobuterol hydrochloride series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, luminous substrate A, luminous substrate B, negative control and positive control; Clenobuterol hydrochloride chemiluminescence detection kit of the present invention, highly sensitive, its sensitivity can reach 0.05ng/ml, chemiluminescence immune assay can detect the material that the method such as radiommunoassay and diagnostic analysis cannot detect, sensing range is wide, easy and simple to handle fast and nontoxic pollution-free, is of great significance early diagnosis tool clinically.
Description
Technical field
The present invention relates to field of immunodetection, specifically relate to a kind of detection clenobuterol hydrochloride chemiluminescence detection kit.
Background technology
Be applied to the quantitative measurement technology detecting clenobuterol hydrochloride in edible animal urine, tissue, serum at present and mainly contain following several method:
1, gas chromatography and mass spectromentry coupling method (Gc-Ms)
GC-MS can be used for the β-anti-depressant qualitative and quantitative analysis such as Clenbuterol in animal hair, urine and tissue.In the method for gas phase-mass spectroscopy Clenbuterol, detect the residual of CL in ox hair, lowest detection is limited to 5ng/g.Utilize feeding big white mouse Clenbuterol to obtain positive organisms material, extract Clenbuterol through O.Olmol/L hydrochloric acid solution, ether defatting purifies, and ethyl acetate is evaporated to dry after extracting, and is loaded onto alumina column on after dissolving with ethanol.With 0.Olmol/L hydrochloric acid solution wash-out.Be evaporated to after doing, derive with BSTFA (two TMS trifluoroacetamide)+1% (ψ) TMCS (trimethyl chlorosilane), finally adopt GC-MS to measure.The Clenbuterol recovery is 75.5%, and O.5ug/kg detect and track is.
GC-MS method advantage to be carried out qualitative to certain specific residue in the simultaneous situation of multiple residue, quantitative test, and have higher sensitivity.Shortcoming is complicated operation, time-consuming, and cost is high.
2, high pressure lipuid chromatography (HPLC) (HPLC)
Detect CL time, HPLC analyze do not need CL derivatization, and accurately, quick, reproducible, false positive rate is low, is applicable to quantitative detection.Beta-adrenergic agonist molecule can with the interaction of molecules of 18C or 8C Stationary liquid, so reversed-phase column is often applied in HPLC technology detect beta-2-agonists class material.HPLC is applicable to the beta-2-agonists and the metabolic product thereof that measure thermally labile and strong polarity, and HPLC can with post before extract, the system coupling such as fluorescence derivation reaction and mass spectrum (MS) after purifying and post, the robotization of easy Realization analysis process.HPLC method can adopt different detecting devices, has electrochemical detector, UV-detector, photodiode array detector and mass detector.At present, China oneself using HPLC method as detecting half residual authenticity method of CL, lowest detectable limit scope is 1-15ng/g, but its shortcoming to be testing process loaded down with trivial details, detection time is long, needs expensive instrument, is difficult to operate, expensive.
3, enzyme linked immunological (ELISA)
The general treatment step somewhat expensive of HPLC or GC-MS. and usually utilize competition elisa immunoassay Rapid detection test strip (card) to carry out detecting more convenient economy, it measures basis is competitive antigen-antibody reaction.Micropore is coated with the goat-anti body for rabbit igg (CLENbuerol antibody).Clenbuterol (CLENbuterol) antibody is added into, through hatching and after washing step, adding CL and CL enzyme marker competition CL antibody, do not have the CL enzyme marker connected to be removed by washing, use corresponding chromogenic enzyme substrate.The color comparator result of determination of hole and gauge orifice per sample.
ELISA has the superiority of its uniqueness compared with GC-MS and HPLC: sample processes fairly simple early stage, and the data detected are more stable, high specificity, and detection speed is fast; Equipment cost is not high, is easy to promote, and highly sensitive, detectability is lower than 0.5ug/kg.CL antibody for EIA have polyclonal antibody and monoclonal antibody point, apply and how anti-ly there is different cross reactivities, apply monoclonal antibody and then greatly improve its atopic.At present, detect more mostly by resist of application for import Rapid detection test strip (card).
4, radiating immuning analysis technology (RlA)
Radio immunoassay detects clenobuterol hydrochloride residue and has good specificity and high sensitivity.Within 1976, Ropitar and Zimmer reported first radio immunoassay detects β-adrenal gland rope activator.The people such as DelAhaut establish more special radioimmunoassay, and O.5ng/g lowest detectable limit can reach.The external Rapid detection test strip (card) having produced utilization RIA mensuration CL, also establishes the radioreceptor assay of homologous radioimmunoassay method, and this method replaces antibody in conjunction with CL with acceptor, and detectability can reach 2.4ng/g.Domestic research in this regard still belongs to blank.But RIA method also exists instrument price costliness, requirement for experiment condition is harsh, and radioactive isotope is to environment and all unsafe shortcoming of staff.
5, fluoroimmunoassay (FIA)
BacigalupoMA etc. (1995) utilize monoclonal technigue and fluoroimmunoassay technology for detection Clenbuterol residual in the urine of ox, its detectability 10ng/ml.Compare radio immunoassay, this method is radiationless, only need compare simple process to sample, simultaneously can the multiple sample of one-time detection, but depends on excitation wavelength due to its fluorescence intensity.And to temperature strong depend-ence, make it apply and be very restricted.
6, By Capillary Zone Electrophoresis (CE)
CE method is highly suitable for those separation and analysis being difficult to the ionization of sample be separated by traditional liquid phase chromatography.Its separation efficiency can reach millions of theoretical cam curve.Gausepohl etc. apply the residual of CL in CE technology for detection people urine, and lowest detectable limit reaches 0.5ng/mL, and degree of accuracy, accuracy have met the examination criteria of residue in biological sample all.The advantage of this method is easy and simple to handle, amount of samples few (general needs a few ng), and due to its many separation parameter, as the composition of damping fluid and pH value, kapillary type and the waveform etc. of electric field that uses can regulate and seem very flexible.Its shortcoming is that detection required time is long, and instrument is complicated, lacks suitable, supporting detecting instrument at present.Therefore the advantage of CE method just can must give full play of when developing the higher detection system of sensitivity.
Summary of the invention
The object of the invention is the deficiency for solving the problems of the technologies described above, providing a kind of and detecting clenobuterol hydrochloride chemiluminescence detection kit, highly sensitive, sensing range is wide, easy and simple to handle fast and nontoxic pollution-free.
The present invention is the deficiency solved the problems of the technologies described above, the technical scheme adopted is: a kind of detection clenobuterol hydrochloride chemiluminescence detection kit, comprises the antibody of the goat-anti rabbit of luminous plaque, clenobuterol hydrochloride polyclonal antibody, ELIAS secondary antibody and horseradish peroxidase mark, clenobuterol hydrochloride series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, luminous substrate A, luminous substrate B, negative control and positive control;
Described luminous plaque is milky opaque polystyrene 96 hole Chemiluminescent plate, and the plate hole surface bottom portion of luminous plaque is coated with envelope antigen;
Described envelope antigen concentration is 3ug/mL;
The working concentration of the antibody of the goat-anti rabbit of described horseradish peroxidase mark is 1:5000;
Described clenobuterol hydrochloride series standard solution concentration is respectively 0.1ng/ml, 0.2ng/ml, 0.5ng/ml, 1ng/ml, 2ng/ml and 5ng/ml;
Described concentrated cleaning solution be containing volume fraction 0.05% polysorbas20 mmol/l, pH be the phosphate buffer of 7.8;
Described envelope antigen is made up by the coupling of diazotising method of clenobuterol hydrochloride haptens and bovine serum albumin(BSA) (BSA), is prepared as follows:
A, getting 40mg bovine serum albumin(BSA) (BSA), to be dissolved in concentration be in the phosphate buffer of 0.1mol/L, and adjustment PH to 8.6, obtains bovine serum albumin(BSA) (BSA) solution for standby;
B, getting 10mg clenobuterol hydrochloride haptens, to be dissolved in concentration be in the HCl solution of 0.01mol/L, is chilled to 0 ~-5 DEG C in advance, then adds the NaNO of 10mg
2, at 4 DEG C, stir 6h, then add bovine serum albumin(BSA) (BSA) solution of step a.Continue to stir 6h at 4 DEG C, finally obtain clenobuterol hydrochloride-bovine serum albumin(BSA) (BSA) conjugate with sephadex SephadexG-25 is gel-filtration purified;
The preparation method of described luminous substrate liquid A: the phosphate buffer of configuration 0.2MpH7.4, adds luminol and tetraphenylboron sodium, mix in this damping fluid;
The preparation method of described luminous substrate liquid B: the phosphate buffer of configuration 0.2MpH7.4, adds 0.1%H in this damping fluid
2o
2;
The preparation method of described negative controls: select more than 5 parts clenobuterol hydrochlorides to be detected as negative pig urine mixing, aseptic filtration is stored in 2 ~ 8 DEG C;
The preparation method of described positive control solution: the pig urine mixing of selecting more than 5 parts clenobuterol hydrochloride test positive, aseptic filtration is stored in 2 ~ 8 DEG C.
beneficial effect
(1) highly sensitive, its sensitivity can reach 0.05ng/ml, and chemiluminescence immune assay can detect the material that the method such as radiommunoassay and diagnostic analysis cannot detect, and is of great significance early diagnosis tool clinically.
(2) there is wide linear dynamics scope, linear detection range can widen 0.01 ~ 10ng/ml, luminous intensity is between 4 ~ 6 magnitudes and measure between material concentration linear, compared with this is the scope of 2.0 with the EIA enzyme immunoassay absorbance (O value) of colour developing, with the obvious advantage. although RIA also has wider linear dynamics scope, and radioactivity limits its application.
(3), this product luminous substrate stablizes, and the light signal duration is long.
(4), analytical approach is fast easy.
(5), stable, the error of result.
(6) security is good and the operating period is long, eliminates use radiomaterial.Up to the present, its harmfulness is not also found; Stable reagent, storage life can reach six months to more than 1 year.
Accompanying drawing explanation
Fig. 1 is the preparation flow figure of clenobuterol hydrochloride chemiluminescence detection kit of the present invention.
Fig. 2 is the canonical plotting that clenobuterol hydrochloride chemiluminescence detection kit of the present invention carries out when result judges.
Embodiment
Below specific embodiments of the invention:
As wrapping by with antigen after described clenobuterol hydrochloride specific antigen and bovine serum albumin(BSA) (BSA) coupling, detectable antigens solution appearance is clarified, without naked eyes visible foreign matters, without the precipitation of cannot not shaking loosely, purity higher (equal > 99%), and there is higher tiring and good activity, specificity and sensitivity better, can be used for producing clenbuterol hydrochloride assay kit, dilute by the carbonic acid buffer of the suitableeest working concentration by clenobuterol hydrochloride specific antigen 0.05mol/LpH9.6, wrap by Chemiluminescent plate by every hole 100 μ l, put 2-8 DEG C and hatch more than 20 hours, wash plate 1 time to add 150 μ l confining liquids and hatch more than 20 hours in 2-8 DEG C in every hole afterwards, blot the environmental drying 5 hours of rearmounted relative humidity≤30% of confining liquid, aluminium foil bag packs, 2-8 DEG C of preservation.
Prepare enzyme labeling antibody of clenbuteral hydrochloride with Over-voltage protection, reaction system is: the 0.15mlNaIO first using HRP solution 1ml and the 0.2ml of 5mg/ml
4solution mixes 4 DEG C of lucifuges 0.5 hour, the ethylene glycol adding 0.25ml shakes up room temperature lucifuge 1 hour, 4 DEG C of dialysed overnight, be 9.2-9.5 with the CB of 0.2ml0.2MpH9.6 by the hydroformylation HRP solution adjust pH after dialysis, add immediately after a certain amount of antigen being dissolved in the CB of 1.0ml0.01MpH9.6 in above-mentioned hydroformylation HRP and react certain hours in 37 DEG C, add 0.1mlNaBH
4solution (3.5 mg/ml), mixes and places 2 hours in 4 DEG C, carry out purifying with gel permeation chromatography.
The detection method of clenobuterol hydrochloride in sample is detected for detecting clenbuterol hydrochloride assay kit
(1) from refrigerator, kit is taken out, equilibrium at room temperature more than 30 minutes.
(2) get concentrated washing lotion one bottle (25ml), be diluted to 500ml with distilled water or deionized water, make into working fluid, mix for subsequent use.
(3) establish blank 1 hole, it does not add sample and CLEN enzyme conjugates, and all the other steps are identical with other holes; If negative control 2 hole, positive control 2 hole; Except blank well, every hole adds 100 μ lCLEN enzyme conjugates, in respective aperture, then add negative control, positive control, each 20 μ l of sample to be tested respectively, with the sealing of shrouding film, mixes rearmounted 37 DEG C of incubator incubations 60 minutes gently.
(4) wash plate with washing lotion working fluid, fill washing lotion (every hole at least 400 μ l) by after the liquid exhaustion in every hole, after soaking 10-60 second, washing lotion in every hole that exhausts, 5 times so repeatedly, finally pats dry on thieving paper.
(5) every hole successively adds chemical luminous substrate A liquid, each 50 μ l of B liquid, reacts 5 minutes gently after mixing in room temperature (18-30 DEG C) lucifuge.
(6) relative light units (RLU) in each hole is measured in lucifuge reaction immediately on chemiluminescent analyzer after terminating, and Measuring Time is set as 0.1 second/hole.
(7) result judges: get standard items log concentration and do horizontal ordinate, and standard items detect luminous value logarithm and do ordinate, and do typical curve, the concentration of each sample can be calculated from typical curve.
The result of appraisal of kit of the present invention: test through three pig cultivation centers, testing result sees the following form:
Interpretation of result:
(1) first hand test result display: this kit negative match-rate is 100%, and positive coincidence rate is 98.7%.
(2) second test result displays: this kit negative match-rate is 99.8%, and positive coincidence rate is 100%.
(3) the 3rd test displays: this kit negative match-rate is 100%, and positive coincidence rate is 100%.
The total sensitivity of this diagnostic kit is 98.8%, and specificity is 100%, and diagnostic kit sensitivity is 98.8%, and specificity is 100%, is suitable for the examination of clenobuterol hydrochloride.
Claims (1)
1. detect a clenobuterol hydrochloride chemiluminescence detection kit, it is characterized in that: comprise luminous plaque, clenobuterol hydrochloride monoclonal antibody, the antibody of goat-anti rabbit of horseradish peroxidase mark, clenobuterol hydrochloride series standard solution, concentrated phosphoric acid salt buffer, concentrated cleaning solution, luminescent solution, negative control and positive control;
Described luminous plaque is milky opaque polystyrene 96 hole chemiluminescence luminous plaque, and the plate hole surface bottom portion of luminous plaque is coated with envelope antigen;
Described envelope antigen concentration is 3 μ g/mL;
The working concentration of the antibody of the goat-anti rabbit of described horseradish peroxidase mark is 1:5000;
Described clenobuterol hydrochloride series standard solution concentration is respectively 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL and 5ng/mL;
Described concentrated cleaning solution is for containing volume fraction 0.5% tween, and 20mmol/L, pH are the phosphate buffer of 7.8;
Described envelope antigen is made up by the coupling of diazotising method of clenobuterol hydrochloride haptens and ovalbumin, is prepared as follows:
A, getting 40mg ovalbumin, to be dissolved in concentration be in the phosphate buffer of 0.1mol/L, and adjustment pH to 8.6, obtains egg albumin solution for subsequent use;
B, getting 10mg clenobuterol hydrochloride haptens, to be dissolved in concentration be in the HCl solution of 0.01mol/L, is chilled to 0 ~-5 DEG C in advance, then adds the NaNO of 10mg
2, at 4 DEG C, stir 6h, then add the egg albumin solution of step a;
Continue to stir 6h at 4 DEG C, finally obtain clenobuterol hydrochloride-ovalbumin conjugate with sephadex SephadexG-25 is gel-filtration purified;
The preparation method of luminous substrate liquid A: the phosphate buffer of configuration 0.2MpH7.4, adds luminol and tetraphenylboron sodium, mix in this damping fluid;
The preparation method of luminous substrate liquid B: the phosphate buffer of configuration 0.2MpH7.4, adds 0.1%H in this damping fluid
2o
2;
The preparation method of described negative control: select more than 5 parts clenobuterol hydrochlorides to be detected as negative pig urine mixing, aseptic filtration is stored in 2 ~ 8 DEG C;
The preparation method of described positive control: the pig urine mixing of selecting more than 5 parts clenobuterol hydrochloride test positive, aseptic filtration is stored in 2 ~ 8 DEG C.
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| CN107356745A (en) * | 2017-08-23 | 2017-11-17 | 太原瑞盛生物科技有限公司 | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of Clenbuterol |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307303A (en) * | 2007-05-16 | 2008-11-19 | 哈尔滨仁皇药业股份有限公司 | Kit for detecting clenobuterol hydrochloride residue and method for preparing same |
| CN101936984A (en) * | 2010-08-03 | 2011-01-05 | 中国农业大学 | Method and special chemical luminous immunoassay kit for detecting clenbuterol |
| CN103018450A (en) * | 2011-09-20 | 2013-04-03 | 北京勤邦生物技术有限公司 | Chloramphenicol chemiluminescence enzyme-linked immunodetection kit |
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| GB0514678D0 (en) * | 2005-07-18 | 2005-08-24 | Europ Cardiovascular Genetics | Method for high-throughput screening |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307303A (en) * | 2007-05-16 | 2008-11-19 | 哈尔滨仁皇药业股份有限公司 | Kit for detecting clenobuterol hydrochloride residue and method for preparing same |
| CN101936984A (en) * | 2010-08-03 | 2011-01-05 | 中国农业大学 | Method and special chemical luminous immunoassay kit for detecting clenbuterol |
| CN103018450A (en) * | 2011-09-20 | 2013-04-03 | 北京勤邦生物技术有限公司 | Chloramphenicol chemiluminescence enzyme-linked immunodetection kit |
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