CN101993486A - Clenbuterol immunogen, coatingen and application thereof in colloidal gold test paper - Google Patents
Clenbuterol immunogen, coatingen and application thereof in colloidal gold test paper Download PDFInfo
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- CN101993486A CN101993486A CN2009101897615A CN200910189761A CN101993486A CN 101993486 A CN101993486 A CN 101993486A CN 2009101897615 A CN2009101897615 A CN 2009101897615A CN 200910189761 A CN200910189761 A CN 200910189761A CN 101993486 A CN101993486 A CN 101993486A
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Abstract
The invention relates to clenbuterol immunogen, coatingen and application thereof in colloidal gold test paper, and belongs to the technical field of immunology. The immunogen and the coatingen are products of hapten coupling carrier proteins obtained by reaction of clenbuterol and 4-bromobutyric acid. The hapten is similar to the clenbuterol corresponding body on molecular structure, stereo chemistry and electronic distribution. The hapten molecules have active groups convenient for coupling with protein carriers, and the presence of the active groups has no influence on the electronic distribution of molecules of an object to be detected. After the hapten is coupled with hemocyanin, the body can produce clenbuterol-orientated antigens with high titer and good specificity, wherein the antigens are adsorbed on a glass cellulose membrane; and the coatingen constructed after coupling the human albumin can be adsorbed on a nitrocellulose membrane to construct the clenbuterol colloidal gold test paper.
Description
Technical field
The present invention relates to a kind of clenbuterol immunogen, coating antigen, belong to the immunological technique field.
Background technology
Along with global economic integration and food trade internationalization, food safety has become the important public health problem in the whole world.Chemical residue in agricultural-food and the animal product has tangible acute toxicity and chronic toxicity to human body, comprises specific toxicities such as cancer and chromosome mutation, directly national healthy the and life of harm.A large amount of uses of these medicines also constitute direct threat to ecological and livestock birds health.Food-safety problem not only endangers people health, influences international trade, causes heavy losses economically, the serious instability that also will cause society.Along with China joined WTO, the international trade of our animal products and fishery products will welcome keen competition environment more.With regard to aquaculture, the overall quality level of our animal product still has one section gap compared with developed countries, and basic food safety can't be guaranteed fully; Now veterinary drug not only is used for preventing and treating Animal diseases, and increasing medicine is used for promoting non-therepic use such as growth, and the residual potential hazard to public health and environment of food animal tissue of supervening therefrom and product Chinese traditional medicine also is on the rise.For ensureing human beings'health, the task of top priority that the residue problem of veterinary drug in animal food become China is controlled in protection environmental health and the normal exit trade that ensures China.
Clenbuterol (clenbuterol hydrochloride) is as a kind of residue of veterinary drug, be widely applied in the Production of Livestock and Poultry eighties in 20th century, be used for improving lean ratio, the residual harm that human health is caused of clenbuterol and the destruction that ecologic food chain caused has been caused the attention of height in the livestock product in recent years.If the edible excessive meat of clenbuterol that contains of people, palpitating speed, elevation of blood pressure, palpitaition, headache will occur, feel sick, symptoms such as vomiting, hand tremor, severe patient has a convulsion, faints, and is bigger to patient's harm of suffering from hypertension, heart trouble, hyperthyroidism, prostatomegaly.Particularly cause people's special concern recent years especially, the dispatch of departments such as the Ministry of Agriculture, State General Administration for Quality Supervision is forbidded strictly to use clenbuterol in animal produces.But clenbuterol is also used illegal in domestic some area, and a lot of poisonings take place.China's " clenbuterol hydrochloride " incident in the last few years occurs repeatedly especially, and as on October 6th, 1999, Jiaxing City, Zhejiang Province 57 people poisoned; On November 7 calendar year 2001, extensive poisoning takes place in Heyuan, has 747 people to poison; Just mid-September in 2006, the poisoning of tens of people's clenbuterol hydrochloride took place in Shanghai, and these incidents are stimulating the original just very responsive food safety nerve of people invariably.
For this reason, easy, reliable, quick, sensitive residue analysis method is set up in research, the method and the reagent of exploitation rapid screening and detection by quantitative residual of kelengtelu, to detecting and control the residue of veterinary drug in the animal food, guarantee the safety of animal food, improve the quality of China's animal-derived food product, strengthen the competitive power of China's animal-derived food product on the world, domestic market, promote the consumption safety that China's aquaculture is stable, develop in a healthy way and ensure China people, promote international trade, have crucial strategic importance.
Clenbuterol is a micromolecular compound, and detection method commonly used has high efficiency liquid phase chromatographic analysis method (HPLC), gas chromatography analytical method (GC/MS) or liquid chromatography/mass spectrometry coupling analytical method (LC/MS).These method ubiquities the instrument costliness, the method complexity, operate loaded down with trivial details, the limitation that reagent consumption is big.The colloidal gold immunochromatographimethod that rises the eighties in 20th century detects, and based on the immunology detection principle, complicated experimental skill and equipment are judged, be need not to clear being easy to of simple and quick, result, is particularly suitable for on-the-spot the detection.Can detect residual of kelengtelu in the fishery products in the very first time, significant to the illegal use of containment clenbuterol.But clenbuterol Radioactive colloidal gold speed is at present on the market surveyed product and is existed detectability can't satisfy the present situation that country limits the quantity of and requires.Head it off is following two approach: (one) improves antibody quality and competition thing.Also be to optimize the immunogen structure and improve the antibody quality; (2) improve the coating antigen structure, thereby optimize antigen-antibody identification from the angle of competition thing.
Summary of the invention
At the appeal problem, the invention provides a kind of clenbuterol immunogen, coating antigen, this immunogen, coating antigen can greatly improve the sensitivity that clenbuterol Radioactive colloidal gold speed is surveyed.
Another object of the present invention provides this immunogen, the application of coating antigen in colloid gold test paper is produced.
The immunogenic preparation of above-mentioned clenbuterol may further comprise the steps:
(1) clenbuterol is haptenic synthetic;
(2) the active ester of clenbuterol haptens is synthetic;
(3) the active ester of clenbuterol haptens carries out coupling in hemocyanin;
(4) immunogenic purifying;
(5) immunogen protein concentration determines packing.
Described haptens has structure shown in the formula (I):
Described immunogen has structure shown in the formula (II):
The preparation of above-mentioned clenbuterol coating antigen may further comprise the steps:
(1) clenbuterol is haptenic synthetic;
(2) the active ester of clenbuterol haptens is synthetic;
(3) the active ester of clenbuterol haptens carries out coupling in the human serum protein;
(4) purifying of coating antigen;
(5) the coating antigen protein concentration determines packing.
Described envelope antigen has structure shown in the formula (III):
Invention is applied to the production of clenbuterol colloid gold test paper by following approach:
(1) clenbuterol immunogen immune experimental animal is produced antibody of clenbuteral;
(2) preparation of colloid gold particle;
(3) colloid gold label antibody of clenbuteral;
(4) preparation of sample pad, this pad is installed with the film of one section colloid gold label antibody of clenbuteral;
(5) preparation of clenbuterol solid phase nitrocellulose filter, this film bag by a detection band that contains the clenbuterol coating antigen and a bag by the quality control band of sheep anti-mouse igg;
(6) processing of sample pad: select suitable closed reagent, tensio-active agent and (or) non-ionic detergent separately or evenly to be dipped in the plain film of glass fibre after the suitable proportion combination, drying at room temperature is standby;
(7) assembling clenbuterol Radioactive colloidal gold test card, as backing, at the stacked nitrocellulose filter in the middle part of PVC liner plate, two ends are stacked absorbent pad and sample pad respectively with PVB for this test card, and nitrocellulose filter connects mutually with absorbent pad, sample pad.
Compared with prior art, the present invention has following beneficial effect:
(1) remains with the skeleton structure of clenbuterol, thereby improve specificity;
(2) used the arm of certain-length, reduced high molecular weight protein, be easy to body and produce antibody at the clenbuterol feature structure to the influence of small molecules haptens on sterically hindered and electron distributions;
(3) adopt high molecular weight protein as the immunogen conjugate, improved immunogenic immunogenicity;
(4) adopt the coupling protein of human serum protein, reduced the non-specific identification of antibody the coating antigen carrier proteins as coating antigen.
Embodiment
Embodiment 1 clenbuterol is haptenic synthetic
Take by weighing clenbuterol 1mmol, 4-bromo-butyric acid 5mmol, sodium hydroxide 10mmol, back flow reaction 48h in 20mL methyl alcohol.Cooling reaction system is to room temperature, regulates pH to neutral, rotation evaporate to dryness component, use silicagel column separate target clenbuterol haptens.
The haptens structure of preparation is:
Embodiment 2 clenbuterols are immunogenic synthetic
Get clenbuterol haptens 0.1mmol and be dissolved among the 2mLDMF, stir adding 27.5mg DCC and 14.4mg NHS.4 ℃ of lower magnetic force stirring reactions spend the night, and centrifugal back supernatant is an A liquid, takes by weighing hemocyanin (KLH) 140mg and is dissolved among the PBS that 10mL concentration is 0.1mol/L (pH8.0).Add DMF1mL, stirring and dissolving prepares B liquid, and under the magnetic agitation, A liquid splashes in the B liquid gradually, and 4 ℃ are reacted 12h down.After centrifugal, get supernatant, use normal saline dialysis 3d, every day to change dialyzate 3 times down for 4 ℃.The holoantigen that obtains is sub-packed in the 0.5mL centrifuge tube with the concentration of 1mg/mL.Frozen in-20 ℃ of refrigerators.
The immunizing antigen structure of preparation is:
(II)
Embodiment 3 laboratory animal immunity
With the respectively immune 6 all female balb/c mouse of haptens clenbuterol immunizing antigen, 3 every group.During the first immunisation injection, the immunizing antigen 100 μ L of 100 μ g/mL are fully emulsified with the equivalent Freund's complete adjuvant respectively, the abdominal cavity direct injection.After two weeks, take the antigen of sample at interval,, inject with quadrat method with 100 μ L Freund emulsifications.
Embodiment 4 cytogamy
The 1d or the same day draw neck to put to death Kunming mouse before cytogamy, are immersed in 70% alcohol body surface sterilization.With pin fixedly Kunming mouse on stencil plate, cut off belly on the Bechtop, provoke peritonaeum with pincet, inject 5mL RPMI-1640 complete culture solution, gently rub the abdominal cavity with have gentle hands, liquid in its body is moved in the 75mL HAT complete culture solution (adding 0.75mL100 * HAT liquid in the 75mL RPMI-1640 complete culture solution) 2～3 times repeatedly with aseptic straw.Use the suction pipe mixing, spread 24 orifice plates, every hole adds 0.5mL, places 37 ℃ of CO
2In the incubator.
Serum is collected in the mouse orbit bloodletting, draws neck to put to death, 70% alcohol-pickled sterilization body surface, aseptic taking-up spleen is put into the RPMI-1640 basic culture solution, and carefully rejects manadesma and fat, shred, place in the stainless steel sift (100 order), aseptic grinding discharges single splenocyte, the liquid that absorption contains splenocyte places the aseptic centrifuge tube of 50mL, and is centrifugal.
Myeloma cell and the above-mentioned splenocyte for preparing are added in the centrifuge tube of same 50mL with 5: 1 ratio of number, add the incomplete nutrient solution 20mL of RPMI-1640 that 37 ℃ of temperature are bathed, mix, centrifugal (1500r/min, 6min).Remove supernatant, touch the centrifuge tube bottom, make precipitation mixing such as pasty state with finger.Get the PEG 1mL of 37 ℃ of preheatings with transfer pipet, splash into centrifuge tube, leave standstill 1min after, in 37 ℃ of water-baths, in 2min, drip RPMI-1640 complete culture solution 10mL.The centrifugal 6min of 1000r/min, supernatant discarded adds 75mL HAT nutrient solution, and mixing is sub-packed in the mixing suspension in 24 orifice plates of feeder cell gently, and every hole 0.5mL is in 37 ℃, 5%CO
2Hatch in the incubator of saturated humidity.
The cultivation and the screening of embodiment 5 hybridomas
Merge back 6～9d, change liquid 1 time, behind 12～14d, use complete culture solution instead according to the propagation situation with HT nutrient solution half amount.Treat cell attachment when accounting for plate hole 1/3, the hole count and the total cellular score of the growth of meter hybridoma.Get supernatant liquor, indirect ELISA is selected to tire high and indirect competitive ELISA selects medicine to suppress strong positive hybridoma cell.
Adopt indirect ELISA and indirect competitive ELISA method to carry out the positive hybridoma cell screening, show the positive and the hole of the hole of competition inhibited reaction occur, and can be used for further subclone for the product antibody of clenbuteral.
Under the aseptic condition, the cell in the positive hole of wash-out, with the elbow suction pipe with cell transfer to 96 well culture plates with the feeder cell bed board in advance, each archioporus is cloned into 8 holes, treat that cell attachment covers with at the bottom of 1/2～1/3 hole after, get supernatant liquor, the indirect ELISA detection.Get the strong subclone that is positive, 2～5 times so repeatedly, antibody positive rate is 100% o'clock in 8 hole supernatant liquors of waiting to be cloned, the picking single cell clone, detect and to be transferred to 24 porocyte culture plates or 25mL Tissue Culture Flask enlarged culturing, build strain and with packing, frozen for complete positive person.Carry and inject the 0.5mL pristane the last week to the Balb/c mouse peritoneal.Get the freeze-stored cell strain, after the recovery, breed through a large amount of the cultivation, collecting cell toos many or too much for use behind the full substratum washing secondary, suspends counting again with the incomplete substratum of 10mL.(every mouse 1mL contains 3.1 * 10 with cell
7Individual cell) intraperitoneal injection of mice belly, behind 10～15d, when treating that mouse web portion obviously expands with No. 16 syringe aseptic collection ascites.The centrifugal 10min of 2000r/min removes upper strata fat and lower floor's scleroproein and cell, and the collection middle level is got part ELISA method and measured it and tire, and all the other packing-70 are ℃ frozen standby.
Embodiment 6 clenbuterol MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
Get the about 3mL of ascites, add 0.06mol/L, pH 4.5 sodium-acetate buffers of 2 times of volumes.N-caprylic acid (33 μ g/mL ascites) is dropwise slowly added in the sample, and the limit edged stirs, and adds the back and continues to stir 30min, and 4 ℃ of centrifugal 30min of following 10000r/min go precipitation (albumin and other non-IgG albumen).Get supernatant through 0.45 μ m micro-pore-film filtration, mix (10 * PBS:80gNaCl, 2g KCl, 11.5g Na with 1/10 volume, 10 * PBS
2HPO
4, 2g KH
2PO
4, 0.5845g EDTA, 1000mL distilled water, pH 7.4), with 1mol/LNaOH solution adjust pH to 7.4.Supernatant is cooled to 4 ℃, adds ammonium sulfate (0.277g/mL, making final saturation ratio is 45%).Stir 30min, 4 ℃ of centrifugal 30min of following 10000r/min abandon supernatant.With a small amount of PBS solution dissolution precipitation,, change liquid 3 times with the PBS dialysed overnight of 50～100 times of volumes.Solution after the dialysis suitably concentrates with PEG-6000, and 4 ℃ storage is standby down.
The preparation of embodiment 7 clenbuterol coating antigens
Get clenbuterol haptens 0.1mmol and be dissolved among the 2mLDMF, stir adding 27.5mg DCC and 14.4mg NHS.4 ℃ of lower magnetic force stirring reactions spend the night, and centrifugal back supernatant is an A liquid, takes by weighing human serum protein (HSA) 140mg and is dissolved among the PBS that 10mL concentration is 0.1mol/L (pH8.0).Add DMF1mL, stirring and dissolving prepares B liquid, and under the magnetic agitation, A liquid splashes in the B liquid gradually, and 4 ℃ are reacted 12h down.After centrifugal, get supernatant, use normal saline dialysis 3d, every day to change dialyzate 3 times down for 4 ℃.The holoantigen that obtains is sub-packed in the 0.5mL centrifuge tube with the concentration of 1mg/mL.Frozen in-20 ℃ of refrigerators.
The envelope antigen structure of preparation is:
The preparation of embodiment 8 colloid gold particles
Get 1% chlorauric acid solution 1ml, add the chlorauric acid solution that the 99ml ultrapure water becomes final concentration 0.01%, behind the ebuillition of heated, get in the chlorauric acid solution that the disposable rapid adding of 1% trisodium citrate 1.6ml boils, continue to be heated to solution and transfer the black-and-blue shiny red that finally becomes to by faint yellow, continue heating 5min behind the colour stable, the room temperature cooling replenishes dehydration to original volume.
The preparation of embodiment 9 colloid gold label clenbuterol monoclonal anti nanocrystal composition
After being determined, the optimal ph of the suitableeest proteinic stable quantity and mark just can carry out mark.Concrete steps are as follows: calculate required proteinic total amount to be marked by minimum steady quantitative 120%.Under magnetic agitation, protein soln is added (pH is modulated to 5.9～6.2) in the colloidal gold solution, should dropwise add when adding protein, the about 5min of the protein of 1mg adds.Get 1ml Radioactive colloidal gold-staphylococcal protein A,SPA binding substances liquid (experimental group) and 1ml Radioactive colloidal gold stoste (control group) respectively and in test tube, add 10% sodium chloride solution 0.1ml, room temperature leaves standstill 1h, observations: if control group test tube solution transfers blueness to by redness, even can see polymer precipitation, and experimental group solution still keeps red, do not have precipitation, can continue next step experiment, otherwise need add mark albumen staphylococcal protein A,SPA.Add final concentration at last and be 0.2% polyoxyethylene glycol (PEG MW20000), continue to stir 30min.
The preparation of embodiment 10 sample pad
Preserve liquid dilution colloid gold label mono-clonal antibody of clenbuteral mixture stoste to working concentration, evenly be dipped in the plain film of glass fibre in proportion ,-20 ℃ frozen, and after the vacuum freezedrying, sealing is preserved.
The preparation of embodiment 11 clenbuterol envelope antigen solid phase nitrocellulose filters:
Getting clenbuterol envelope antigen 2mg/ml uses the PB damping fluid (pH 7.2) of 0.01mol/L with 250 μ g/ml interval, through the linear bag of BIO-DOT type XYZ3000 point sample instrument dispenser by in the test reaction district of the observations of nitrocellulose filter, be defined as the detection band, distance detects band 5mm quality control band far away and uses the linear bag of dispenser by normal mice IgG (2mg/ml).37 ℃ of dry 2h, 4 ℃ of sealings are preserved.
The assembling of embodiment 12 test strip
As backing, at the stacked nitrocellulose filter in the middle part of PVC liner plate, two ends are stacked absorbent pad and sample pad respectively with PVB, and nitrocellulose filter connects mutually with absorbent pad, sample pad.The test strip that assembles places portrayal to have and detects in the plastic casing of band and quality control band sign.
Claims (6)
1. clenbuterol immunogen, coating antigen is characterized in that its preparation method is that clenbuterol haptens and albumen carry out crosslinked.
2. haptens according to claim 1 is characterized in that described haptens has structure shown in the formula (I).
3. immunogen according to claim 1 is characterized in that described immunogen is clenbuterol haptens and hemocyanin link coupled product, has structure shown in the formula (II).
4. to it is characterized in that it is used for the antibody that the immunization experiment animal produces the specific recognition clenbuterol with regard to 1 described immunogen according to right, behind the antibody labeling Radioactive colloidal gold, on the absorption glass fibre membrane, place near the sample pad specific recognition clenbuterol molecule.
5. coating antigen according to claim 1 is characterized in that described coating antigen is the product after clenbuterol haptens and the human serum protein's coupling, has structure shown in the formula (III).
6. coating antigen according to claim 1 is characterized in that it is used for bag by near the detection band, discerns excessive colloid gold label antibody of clenbuteral.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104849467A (en) * | 2014-02-15 | 2015-08-19 | 北京勤邦生物技术有限公司 | Fluorescent microsphere immunochromatography test paper strip for detecting clenbuterol hydrochloride residue, preparation method and applications thereof |
| CN104892442A (en) * | 2014-03-07 | 2015-09-09 | 北京维德维康生物技术有限公司 | Clenbuterol hapten and antigen and dedicated chemiluminescence immune kit |
| 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 |
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