CN103235117A - Beta 2-acceptor stimulant ELISA kit and usage method and application thereof - Google Patents

Beta 2-acceptor stimulant ELISA kit and usage method and application thereof Download PDF

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CN103235117A
CN103235117A CN2013101282706A CN201310128270A CN103235117A CN 103235117 A CN103235117 A CN 103235117A CN 2013101282706 A CN2013101282706 A CN 2013101282706A CN 201310128270 A CN201310128270 A CN 201310128270A CN 103235117 A CN103235117 A CN 103235117A
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sample
stimulating agent
solution
receptor stimulating
enzyme
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钟凯
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TIANJIN KANGLIYUAN BIOLOGY CO Ltd
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Abstract

The invention relates to a beta 2-acceptor stimulant ELISA kit, which comprises the following compositions in proportion: a coating plate, a calibrator, an enzyme labelling object operating fluid, a substrate liquid A, a substrate liquid B, a stopping solution, a condensed washing liquid and a condensed extract. The kit sensitivity is 0.1 ppb, and the precision is as follows: intra assay variation coefficient (CV%) is 5%, and inter assay variation coefficient (CV%) is 10%; the accuracy is shown by recovery rate, and the recovery rate is in the range of 80-110%. IC 50 range is between 0.3 and 0.5 ppb, and the linear correlation coefficient |r| is not less than 0.9900, and the product has the advantages of wide usage scope, convenient usage and accurate detection.

Description

A kind of β 2-receptor stimulating agent enzyme linked immunological kit and using method thereof and application
Technical field
The invention belongs to the detection kit field, relate to the clenbuterol hydrochloride detection kit, especially a kind of β 2-receptor stimulating agent enzyme linked immunological kit and using method thereof and application.
Background technology
In recent years, along with Developing of Animal Industry, the use of veterinary drug is also increasingly extensive, becomes one of indispensable material base gradually.Comprise β 2-receptor stimulating agent interior veterinary drug at growth promotion, improve food conversion ratio and utilization factor and improve aspects such as product quality and played very significant effect really.Yet, when promoting animal husbandry development, the use of veterinary drug can cause undoubtedly medicine itself and metabolic product thereof in animal body delay or accumulate, and enter human body and the ecosystem in residual mode, thereby work the mischief.The animal derived food safety problem, particularly the abuse or illegally use veterinary drug and illegal drug, the problem of fowl poultry kind product residue veterinary drug is become increasingly conspicuous, caused very big threat to people ' s health, and become a principal contradiction of animal husbandry development.Along with China's accession to the WTO, the animal derived food of developed country's low price, low-residual certainly will pour in China from now on.If actively do not adopt an effective measure, not only the animal derived food of China outlet is not smooth, and the status that is connected in the home market also can be subjected to serious impact, will be unfavorable for very much the sound development of aquaculture.
β 2-receptor stimulating agent is called β again 2-excitant is manually synthetic phenyl ethylamine class medicine of a class, and common have its woods of Clenbuterol, salbutamol, Tulobuterol, special cloth, fenoterol, Formoterol, Rabat sieve, Ractopamine, an alotec etc.β 2-receptor stimulating agent has the muscle growth of promotion, reduces fat deposition, improves the meat effect, therefore has some feed-grain makers and Livestock Production person to violate a ban and uses this medicine in livestock culturing.Because β 2-receptor stimulating agent has the long residence time in animal body and produces bigger residual quantity in a short time, particularly accumulate residual in the internal organ, enter human body by food chain, can make some Susceptible population, produce significantly toxicity symptom as old man, pregnant woman, heart body chump, the serious harm human health, the legislation of states such as America and Europe is forbidden using in poultry production, and identical regulation was also made by China Ministry of Agriculture in 1997.But under the ordering about of economic interests, the illegal abuse phenomenon in various countries is still more general.Surplus the poisoning that Italy, France and Spain take place because the food Clenbuterol pollutes between nineteen ninety to nineteen ninety-five surpasses 500, poisoning also took place in China.
In order to ensure the security of China's animal foodstuff, protect public health safety, it is very necessary to set up effective rapid quantitative conclusive evidence method.
1.1 β 2The structure of-receptor stimulating agent
β 2-receptor stimulating agent has phenolethanolamine structure parent nucleus, be connected with on the phenyl ring alkalescence β-azanol (secondary amine) side chain, the substituting group of side chain is generally the N-tert-butyl group, N-isopropyl or N-alkyl benzene, can with mineral acid or organic acid salify.Common have its woods of Clenbuterol, salbutamol, Tulobuterol, special cloth, fenoterol, Formoterol, Rabat sieve, Ractopamine, an alotec.Its basic structure is:
Figure BDA00003048399300021
According to substituent difference on the phenyl ring, can be with β 2-receptor stimulating agent is divided into aniline type and phenol type.Have the aryl primary amine base in the aniline type structure, middle polarity, the aryl primary amine base often is used to the derivatization analysis.Typical medicine has Clenbuterol, and its structure is:
Figure BDA00003048399300022
The phenol type generally have the neighbour () benzenediol or phenol structure, contain 1 or 2 phenolic hydroxyl group in C-3 ', C-5 ' position.The phenol type can be divided into catechol (the catechol type is as adrenaline) again, resorcinol (mine-mooring cable phenol type is as its woods of fenoterol, special cloth, alotec), and saligenin type (typical medicine has salbutamol), its structure is as follows:
The structure of fenoterol is:
Figure BDA00003048399300024
The structure of Terbutaline is:
The structure of alotec is:
Figure BDA00003048399300031
β 2-receptor stimulating agent belongs to the adrenomimetic drug medicine, it can with target tissue cell membrane β 2-receptors bind, activated adenyl cyclase, the catalysis atriphos is converted into cAMP, thereby brings out phosphorylation process and the physiological effect of a series of enzymes.β 2The physiological effect of-receptor stimulating agent: 1) lax bronchus, uterus and intestines wall smooth muscle increase heart rate.Be mainly used in expansion bronchus on medical science or the veterinary clinic and increase the lung qi amount, can treat diseases such as bronchial astehma, obstructive pneumonia, smooth muscle spasm and shock, also be used as ox, horse birth canal relaxant.2) redistribution effects.β 2-receptor stimulating agent can make multiple animal (ox, pig, sheep, poultry) endotrophic composition be shifted to musculature by adipose tissue, be called " redistribution effects ", consequently the catabolism of fat in the body strengthens, and protein is synthetic to be increased, and can significantly increase weight and improve food conversion ratio.3) to neural hormesis, thereby can be used as a kind of motion stimulant.In recent years, it is in rising trend that the sportsman uses the frequency of such medicine.The International Olympic Committee began first β in 1992 2-receptor stimulating agent Clenbuterol has been listed the forbidden drugs table in, has increased several such medicines in afterwards several years again successively newly.By in January, 2004, all β 2-receptor stimulating agent is all disabled.
1.2 β 2The abuse of-receptor stimulating agent reaches the spinoff to the people
1.2.1 the abuse in animal feeding
In animal husbandry, β 2-receptor stimulating agent can be used as a kind of lean meat growing agent, can promote the poultry growth and improve meat, has tempting economic interests.But, feed livestock with this class medicine, can in the livestock body, produce medicament residue.The people has eaten the health that the livestock (particularly internal organ) that has medicament residue just may endanger the people, and cardinal symptom is: dry, headache, dizziness, heartbeat, hand are trembled, twitched from head to foot, and be bigger for hypertension, cardiac's harm, even threat to life.This be because: 1) β 2-receptor stimulating agent can be accelerated lipolysis, makes more free fatty acid enter blood, has reduced vessel wall elasticity, and blood fat and blood pressure raises, and causes microcirculation to be expanded and the compressing peripheral termination of nerve, causes headache and dizzy.2) because it has reduced the Mg that suppresses the excitation of muscle 2+Concentration causes the bioelectricity disorder, and patient's the excitation of muscle strengthens, and causes muscular tremor, systremma, and vomiting and diarrhoea, myocardial contraction is accelerated simultaneously, and heart rate is accelerated, and causes that easily patient suffers a shock and hyperpietic's death.
Take place a lot of because using β both at home and abroad 2-receptor stimulating agent nutrition purposes, especially for feeding animals and the event of poisoning.For example, in Hispanic Catalonia 113 routine Clenbuterol poisonings took place in 1992, reason is the liver that has eaten the ox that derives from the Clenbuterol raising.For another example, in August, 1996,62 people poisoned because of utility beef at gondola Caserta.(Enzyme-linked immunosorbent assay ELISA), has detected Clenbuterol to the scientific research personnel in its edible beef with enzyme linked immunosorbent assay.Similarly event also occurs in France.In Holland, though Clenbuterol has been prohibited use, can the running check Clenbuterol in the ox urine of culturing.Clenbuterol and other β 2Its woods of-receptor stimulating agent such as salbutamol, special cloth etc. was banned use of by country of the European Economic Community in 1985.China Ministry of Agriculture forbidded strictly β on March 11st, 1997 2The application of-receptor stimulating agent in animal husbandry, single part provinces and cities just have the Clenbuterol intoxicating phenomenon to occur from the mid-90, after this are development trend, comparatively serious province municipalization Guangdong, Chongqing, Zhejiang, Beijing, Shanghai etc.Calendar year 2001,17 Clenbuterols food poisoning, poisoning number 106 people took place in Guangzhou altogether.
1.2.2 the abuse in sporting world
β 2After-receptor stimulating agent the oral administration, almost 100% is absorbed by body.Zooperal studies show that used β 2Clenbuterol in the-receptor stimulating agent can increase the muscle fat ratio and improve muscle power.This result is hankered after using in physical culture the personage of medicine to adopt very naturally, so just begun its abuse in physical culture, its degree is very serious.The sportsman uses β 2-receptor stimulating agent also has respiratory system and neural excitation except strengthening muscle strength.
From 1992, IOC Medical Comm. began clearly to forbid oral or injection Clenbuterol in sports tournament and training, as the spraying of therapeutic purposes with to suck also be unallowed.Several such medicines have been increased in afterwards several years again successively newly.By January calendar year 2001, all β 2-receptor stimulating agent is all disabled.
1.3 β 2The regulation of the high residue amount of-receptor stimulating agent
As from the foregoing, β 2-receptor stimulating agent serious harm human health, various countries are to some β in the animal derived food 2-receptor stimulating agent stipulated high residue amount (Maximum Residue Limits, MRLs).High residue amount refers to allow at food Chinese traditional medicine or the residual maximum amount of other chemical substance, also can be described as the permission residual quantity.High residue amount belongs to the compulsory standard that country announces, has determined the off-drug period of security and the production medication of public consumption, and its importance is apparent.
β 2Clenbuterol is to study maximum getting in-the receptor stimulating agent, and European and American countries all forbids using in animal husbandry is produced.The high residue amount of Food and Drug Administration and World Health Organization (WHO) suggestion Clenbuterol in animal tissue sees Table 1.Some countries as Holland, Britain, China carry out Clenbuterol maximum residue limit restriction regulation, concrete numerical value such as table 2.But so far without any the state approval Clenbuterol as feed addictive for animals.China also forecloses Clenbuterol in " the veterinary drug kind that allows to make the feed medicated premix is used regulation " of issue in 1997.
The high residue amount of table 1 Food and Drug Administration and World Health Organization (WHO) suggestion Clenbuterol in animal tissue
Tissue Maximum residue limit (ug/kg)
Meat 0.2
Liver 0.6
Kidney 0.6
Fat 0.2
Milk 0.05
Some countries of table 2 are about the regulation of the high residue amount of Clenbuterol
Country Tissue Maximum residue limit
Britain Edible tissue 0.5ug/kg
Holland Liver 1ug/kg
China Pig urine 5ng/mL
1.4 domestic and international β 2-receptor stimulating agent is analyzed present situation
1.4.1 β 2The pre-treatment of-receptor stimulating agent residual sample
For satisfying the needs of residual inspection card, β in recent years 2The development of-receptor stimulating agent residue analysis method is very fast.β 2The sample of the residual mensuration of-receptor stimulating agent is mainly based on edible tissues such as urine and liver, kidney, muscle.In the sample pre-treatments, soda acid liquid-liquid extraction, Solid-Phase Extraction and immune affinity chromatographic are basic extraction and cleaning method.
1.4.1.1 the hydrolysis of sample
Except Clenbuterol, other β 2-receptor stimulating agent mainly exists with forms such as sulfate conjugate compound or glucosiduronate conjugatess in animal tissue and biological sample.Therefore sample will carry out hydrolysis earlier, makes determinand free or discharge to carry out follow-up solvent extraction from tissue.Especially the conjugates ratio of phenol type medicament residue such as salbutamol, alotec is higher, must extract and purify after the hydrolysis.Method for hydrolysis mainly contains enzyme hydrolysis method, acid hydrolysis and alkali hydrolysis method.
(1) enzyme hydrolysis method
The normal hydrolytic enzyme that adopts of enzyme hydrolysis method mainly contains subtilopeptidase A, glucuronidase, sulfatase, aryl sulfatase.Wherein, glucuronidase, sulfatase or their mixed enzyme system are the most frequently used hydrolytic enzymes.Hydrolysising condition has appreciable impact to hydrolysis efficiency.Temperature and pH value directly influence the activity of enzyme, strict control.Usually in sample, add damping fluid and transfer pH value to 5.2, use glucuronidase/sulfatase hydrolysis 2h under 55 ℃ of conditions then.The general method of high-temperature boiling that adopts was destroyed enzyme after hydrolysis was finished.
(2) acid-hydrolysis method
Acid-hydrolysis method generally carries out in watery hydrochloric acid, dilute sulfuric acid or rare perchloric acid solution.Diluted acid also plays the effect of protein precipitation except the effect with hydrolysis conjugates.The general ultrasonic assisted extraction of water-bath, the hydrolysis 1h in the water-bath of uniform temperature then of adopting during with the dilute acid hydrolysis conjugates.Therefore, acid-hydrolyzed process also is leaching process.The sample interfering material that adopts acid-hydrolysis method to extract is few.
(3) alkali hydrolysis method
Alkali hydrolysis method is usually used in the hydrolysis of conjugates in the hair sample, and general operation is fairly simple.Fente etc. use the sodium hydroxide solution of 1mol/L at 80 ℃ of following 1h hydrolyzed bovine hairs, measure β wherein 2-receptor stimulating agent is residual.
Hydrolysis and extraction are carried out usually simultaneously, also can extract or extract earlier the back hydrolysis after the first hydrolysis.
1.4.1.2 the extraction of sample
⑴ solvent extraction is extracted
One of abstraction technique that fluid sample is the most frequently used is solvent extraction, is called liquid-liquid extraction usually.The liquid-liquid extraction technology utilizes the difference of solubleness or distribution ratio in two kinds of immiscible solution of different component in the sample to reach the purpose of separation, extraction or purifying.
Most β 2-receptor stimulating agent is the lyophobic dust of middle polarity, is alkalescent or faintly acid.Adopt polar organic solvent, diluted acid, damping fluid to extract, after centrifugal, do further to purify.Organic solvent and pH value directly influence extraction efficiency.Turberg etc. are with the ractopamine in methyl alcohol extraction pig and the turkey tissue, through liquid chromatogram measuring.
The ⑵ supercritical fluid extraction (Supercritical fluid extraction, SFE)
Supercritical fluid extraction is to utilize the solvent ability of supercritical fluid and the relation of its density, namely utilizes pressure and temperature that the influence of supercritical fluid dissolving power is carried out.Under supercriticality, supercritical fluid is contacted with material to be separated, it is extracted the composition of polarity size, boiling point height and molecular weight size selectively successively.At present the more supercritical fluid of research is carbon dioxide, it is nontoxic because having, do not burn, to most of material do not react, advantage such as inexpensive, the most commonly used.Mandy etc. adopt Clenbuterol and the salbutamol in the SFE extraction beef liver, and ELISA measures.
⑶ other sample extraction technology
Some new sample preparation technology also are used to β 2The extraction that-receptor stimulating agent is residual.Residual of kelengtelu during usefulness two-phase dialysis isolation technics such as Fente extraction liver and ox hair are sent out.This article adopts long 25cm, and the ion-exchange area is 196cm 2Dialysis tube, with in the moistening tissue sample that is equipped with after the dialysis tube that extracts solvent t-butyl methyl ether, methylene chloride is inserted homogenate in advance, under 37 ℃ with the 150rpm/min rotational speed, 4h is extracted in dialysis continuously, then the solution in the dialysis tube is changed in the separating funnel, aqueous phase discarded, organic phase are concentrated into dried behind anhydrous sodium sulfate dehydration with nitrogen, (Gas chromatography-mass spectrometry GC-MS) measures the laggard promoting the circulation of qi phase chromatography-mass spectroscopy of deriving.This extracting method integrates extraction and purifies, and is easy to be quick, the extraction efficiency height.
1.4.1.3 the purification of sample
The ⑴ liquid-liquid extraction (Liquid liquid extraction, LIE)
Liquid-liquid extraction (LIE) method is a kind of elementary purification techniques, and the liquid-liquid extraction technology has developed into complicated heterogeneous distribution system from a simple step solvent extraction and extracted and purify analyte, and liquid-liquid extraction separation method can reduce protein to be disturbed.Sometimes in order to improve the sensitivity of the method for inspection, in liquid-liquid extraction, need to concentrate the material of analyzing.
At β 2Liquid-liquid extraction is a kind of rough purification means commonly used in the retention analysis of-receptor stimulating agent.Sample is after preliminary the extraction, and general pH value of regulating sample earlier makes it be higher than β 2The pKa of-receptor stimulating agent, i.e. pH〉9, use the pleasantly surprised extracting and purifying of polarity hydrophobic solvent then.Use mixed extractant solvent can obtain higher selectivity.Extraction solvent commonly used has ethyl acetate-isopropyl alcohol, isooctane-methylene chloride, ether-two butanols, normal hexane-normal-butyl methyl esters etc.
The ⑵ Solid-Phase Extraction (Solid phase extraction, SPE)
Solid-Phase Extraction (SPE) is a kind of of many uses and more and more welcome sample pre-treatments technology, it is based upon on traditional liquid-liquid extraction (LIE) basis, high performance liquid chromatography (High performance liquid chromatography in conjunction with interactional similar mix mechanism and widespread use at present of material, HPLC), (Gas chromatography, GC) the fixedly phase ABC in grows up gas chromatography gradually.Many problems of using solid phase extraction can avoid liquid-liquid extraction to bring, such as: incomplete being separated, the lower quantitative test recovery, frangible glassware and a large amount of organic liquid wastes.Compare with liquid-liquid extraction, Solid-Phase Extraction is more effective, reaches quantified extract easily, and quick and robotization has also reduced solvent load and working time simultaneously.So SPE has characteristics such as consumption of organic solvent is few, convenient, safe, efficient.
Solid-Phase Extraction (SPE) is generally used for the pre-treatment of fluid sample, extracting and enriching half volatilization or involatile compound wherein, or remove the impurity that in the sample separation is caused interference, also can be used for handling the solid phase sample that can be dissolved in advance in the solvent in addition.Present domestic organic substance such as palycyclic aromatic and polychlorinated biphenyl analysis in the water, agricultural chemicals and herbicide residue analysis in fruit, vegetables and the food, microbiotic analysis, the aspects such as clinical medicine analysis of being mainly used in.Solid phase extraction techniques to the extraction of analyte, concentrate and purify all very good.Solid-Phase Extraction (SPE) can be divided into three kinds according to its similar mechanism that mixes: anti-phase SPE, positive SPE, ion-exchange SPE.
SPE is β 2Most widely used technology in the retention analysis of-receptor stimulating agent adopts different solid-phase extraction columns to realize purifying purpose in analytic process.At β 2The solid-phase extraction column that uses in the retention analysis of-receptor stimulating agent is of a great variety, as C18 Bond Elut post, Chem Elut CE 1020 posts, strong cation solid-phase extraction column (Strong cation exchange-solid phase extraction, SCX-SPE), the weak cation solid-phase extraction column (Weak cation exchange-solid phase extraction, WCX-SPE), neutral alumina post, molecular engram macromolecule solid phase extraction column, Extrelut TMSolid-phase extraction column etc.Wherein the weak cation solid-phase extraction column of C18 carboxylic acid bonding has the characteristic of C18 solid-phase extraction column and weak cation exchange concurrently, has become the important purification means of the residual method for supervising of European Union.The eluant, eluent of these SPE posts is generally 2%～3% ammonia-methyl alcohol or ammonia-ethanolic solution.Generally adopt combination S PE post to purify for many residual components or complex sample.Leyssens etc. earlier with ox urine and pork liver with Tris damping fluid (Ph=8) homogenate, with homogenate subtilopeptidase A enzymolysis, extract with normal butyl alcohol-ethyl acetate (3:7) after transferring enzymolysis liquid Ph=9.8, organic phase is carried out primary purifying with the neutral alumina post after concentrating and redissolving, transfer sample pH value=6.0 again, by Bond Elut Certif post, with methylene chloride-isopropyl alcohol (8:2) wash-out that contains 2% ammoniacal liquor, clean-up effect is good, adopts gas chromatography mass spectrometry method (GC-MS) to measure β behind the analyte derivative 2-receptor stimulating agent, the detectability of different residual components is between 0.5～5ng/g.Philippe etc. unite C18Bond Elut post and strong cation solid-phase extraction column (SCX-SPE) and use the Clenbuterol separate in beef and the pork liver, with LC-MS method (Liquid chromatography-mass spectrometry, LC-MS) measure, when the mark-on level was 0.4ng/g, the recovery was 63 ± 7%.
(3) immune affinity chromatographic (Immunoaffinity chromatography, IAC)
The immune affinity chromatographic principle is that antibody is fixed on the solid support material, makes immune affinity sorbent, and by adsorbent, the target compound in the sample is retained on the solid-phase adsorbent because with antibody immune affinity interaction taking place with sample solution.Then with damping fluid or organic solvent as the fixing phase of eluant, eluent wash-out, target compound is dissociated from antibody, thereby target compound is extracted and purifies.Because immune affinity interaction has very high specificity, so immune affinity column can separate the target compound that other extracting process are difficult to extract easily from complex sample matrix.Since characteristics such as highly sensitive, high selection that antigen-antibody reaction has, high specific, the component to be measured in the alternative absorption sample liquid, and remove all kinds of impurity such as lipid, protide, carbohydrate, thus improved clean-up effect and the detection sensitivity of sample greatly.IAC with its efficient selective reserve capability at β 2The crucial purifying step that-receptor stimulating agent is residual is only purification method sometimes.The IAC post of external existing many commodity is sold at present.This pillar is that the antibody that can be combined with target analytes is solidificated on the cylinder in advance, behind the treated sample upper prop, and single component β 2-receptor stimulating agent or polycomponent β 2-receptor stimulating agent is combined with antibody and is retained on the post, carries out wash-out through appropriate solvent again, reaches clean-up effect preferably.Hassnoot etc. use the Clenbuterol in IAC column purification urine sample or the tissue first.Lawrence etc. purify sample extracting solution with weak cation exchange SPE post and the coupling of IAC post, the ammonia with 2%-ethanolic solution wash-out Clenbuterol adopts HPLC directly to measure, and detectability reaches 0.3 μ g/kg.After reusing 10 times, the IAC post do not find that column capacity reduces.Compare with traditional SPE post, the immune affinity column that utilizes the antigen-antibody binding mechanism to purify has good selectivity, good purification, can repeat repeatedly to use, but the main limitation of IAC technology is to consume a large amount of antibody purifications, and the IAC post of general merchandiseization is expensive, therefore at β 2Analysis cost in the retention analysis of-receptor stimulating agent is than higher.
(4) the matrix solid phase extraction techniques (Matrix solid phase dispersion-extraction, MSPD)
Matrix solid phase dispersion technology (MSPD) is proposed by people such as Baeker and Long, based on the mechanical dispersion effect design of the hydrophobic lipotropism of C18 filler bonding phase and particle thereof, integrate the sample rapid treating technology that extracts and purify.Its basic operation is that (C18 C8) grinds the solid matrix that sample is direct and an amount of, is mixed into semisolid for silica gel, florisil, and the dress post is selected suitable eluent solvent.The matrix Solid-Phase Extraction is with the extraction of sample and purify a step and finish, avoided sample homogenizing, change molten, emulsification, concentrate the loss of the component to be measured that causes, be applicable to the extraction and cleaning of various molecular structures and polarity residues of pesticides.Horne etc. [33]Adopt this technology to extract the residual of kelengtelu of beef liver sample, the beef liver sample of 0.5g homogenate is mixed with the 2gC18 filler and grind evenly, the dress post, with 8mL normal hexane-ether (60:40) drip washing, with 8mL methanol-eluted fractions Clenbuterol, be concentrated into and do the back with acetate buffer solution redissolution, measured by radioimmunoassay.
(5) column switching technique
In recent years, many scholars have utilized column switching technique in research work, and biological sample is directly analyzed without solvent.At this moment the HPLC instrument is used for carrying out the preparation (pre-treatment) of sample and analyzes, and its chromatographic system is made up of a pre-column and an analytical column.Adopt column switching technique, enter before analytical column carries out compartment analysis at sample, sample is carried out the trace enrichment at pre-column.Sample enters pre-column, and pre-column keeps tested component, and impurity is poured useless pond, further washes pre-column to make sample cleaner, and the tested component pre-column that recoiled out enters analytical column, carries out quantitatively final with ultraviolet method, fluorescence method or electrochemical process etc.Whole process is that robotization is carried out, and is all controlled by microprocessor up to data, report from the beginning sample introduction, and this is a kind of application technology of joint pin.Be about to liquid-solid extraction micro-column and be connected with the HPLC instrument as pre-column, increase a pump, earlier with sample flow through pre-column, make and play purifying and inrichment, post by analysis again can improve the sensitivity of analysis.
The LC-MS of employings such as Schmeer band C18 pre-column has directly measured the salbutamol in the human plasma.Enter in the pre-column behind the mark in sample adds, switch to flow after the enrichment of component process pre-column and carry out wash-out and mensuration mutually, only need 8min the analysis time of each sample, and quantitative limit reaches 0.2ng/mL, and the coefficient of variation is less than 7%.Similarly method also is used for the analysis of special sieve of its woods and Fino of the special cloth of blood plasma.Oosterhuis B and Van C J [34]Adopt online cation exchange SPE post (SCX)-reversed-phase HPLC to measure salbutamol.
1.4.2 β 2-receptor stimulating agent analytical approach
After the extraction and purifying step of sample through necessity, β 2-receptor stimulating agent can use thin-layer chromatography (Thin layer chromatography, TLC), (Capillary electrophoresis CE), the screening of liquid chromatography, immunoassay separates, confirms with mass spectrum Capillary Electrophoresis.Gas chromatography-mass spectrum (GC-MS), being called gas chromatography mass spectrometry again is the most frequently used analytical approach, but because β 2-receptor stimulating agent polarity is stronger, need carry out suitable derivatization reaction usually and just be adapted to gas chromatographic technique, so liquid chromatography-mass spectrography method (LC-MS) in recent years, being called LC-MS has again become and analyze β 2The main qualitative and quantitative analysis method of-receptor stimulating agent.
1.4.2.1 thin-layered chromatography
Thin-layered chromatography is exactly that suitable solid phase is coated on glass plate, plastics or the aluminium substrate, becomes an even thin layer.After treating point sample, launching, with suitable tester by the same method the chromatogram of gained contrast, in order to the method for the discriminating, determination of foreign matter or the assay that carry out medicine.
High performance thin layer chromatography is mainly as β 2The sxemiquantitative of-receptor stimulating agent or prescreening method.Sangiorgi [36]Earlier the Clenbuterol in the sample is screened analysis with ELISA, to doubtful positive HPTLC plate development, inspect behind the Bratton-Marshall reaction solution, also spot can be scraped again and further analyze with HPLC or GC-MS.Same method is used to aniline type β in the samples such as urine 2The analysis of-receptor stimulating agent.Salbutamol can change into the indole aniline dyestuff after being launched by high performance thin layer chromatography, detects with micro-optical densitometric method under 650nm.
1.4.2.2 capillary electrophoresis
Capillary electrophoresis is the separate analytical technique that develops rapidly in recent years, its principle be according to different component under the effect of high-voltage electric field, the difference of migration rate and reach separation to component.Capillary electrophoresis technique has high separating efficiency, and little sample size, the fast and low reagent of analysis speed such as expend at advantage, also are applied in the mensuration of residual of kelengtelu.Gausepohl etc. have measured Clenbuterol pill taker's urine with capillary electrophoresis method.Employing constant speed capillary zone electrophoresis UV-detector such as Toussaint are measured Clenbuterol.Because it is not electro-chemical systems instrument and capillary electrophoresis apparatus all are not the standing detecting instruments of common laboratory, therefore very extensive in application aspect a large amount of sample determinations.
1.4.2.3 high performance liquid chromatography
Reversed-phase high-performance liquid chromatography is β 2The quantitative analysis method a kind of commonly used of the residual mensuration of-receptor stimulating agent.Usually adopt the C18 analytical column, buffer solution is mobile mutually with the acetonitrile preparation, degree of grade or gradient elution.Organic modifiers is the principal element that influence separates with the pH value.The detecting device that adopts comprises that uv absorption detects (Ultraviolet absorption detector, UVD), photodiode array detector (Photo-diode array detector, PDA), fluorescence detector (Fluorescence detector, FLD), electrochemical detector (Electrochemical detector, ECD), detectability generally can only reach 1～100 μ g/kg.Wherein bibliographical information is maximum is to adopt UV-detector (UV), adopts this method to measure Clenbuterol in beef liver and the chicken as Lawrence.Extract detects in 245nm earlier by WCX-SPE post and IAC column purification, and flowing is methyl alcohol+10mmol/L acetic acid-ammonium acetate (30:70) damping fluid (pH=4.6) mutually.The interpolation recovery of 2ng/g and 5ng/g level is between 53%～74% in beef liver and the chicken.Tsai etc. [43]Adopt Clenbuterol, salbutamol in HPLC/PDA mensuration blood plasma and the tissue, detect wavelength and be respectively 210nm 196nm, the recovery 62～786% quantitatively is limited to 100 μ g/kg.PDA can obtain complete ultraviolet absorpting spectrum, can differentiate the purity of component peaks, and positive findings is tentatively proved conclusively.
Some phenol type β 2-receptor stimulating agent has photoluminescent property, can adopt the higher FLD of sensitivity and selectivity to detect.Hashimoto etc. measure salbutamol in the muscle with HPLC/FLD, and excitation wavelength is 225nm, and emission wavelength is 310nm, quantitatively is limited to 55 μ g/kg, and the recovery reaches 75～81%.
The sensitivity of ECD is higher, but electrode polluted easily, thus the precision of having influence on, so require high to regeneration or the balance of electrode.Turberg etc. are with the ractopamine in the electrochemical detector determining porcine tissue, when voltage during for+600mV quantitative limit reach 0.5 μ g/kg, the recovery reaches 75～100%, the coefficient of variation 2～18%.
1.4.2.4 gas chromatography mass spectrometry analytic approach
Gas chromatography-mass spectrum (GC-MS) method not only can be per sample in the retention time of component to be measured on collection of illustrative plates, main is according to residual β in this retention time 2The characteristic ion fragment of-receptor stimulating agent cracking, by mass spectrometer by its molecular weight and molecular structure to β 2-receptor stimulating agent is accurately qualitative, and with this as quantitative foundation.GC-MS is β 2In the residual mensuration of-receptor stimulating agent the most frequently used quantitatively and the conclusive evidence method.Because β 2Have the hydroxyl and the amino that are difficult for gasification in the chemical constitution of-receptor stimulating agent, therefore the sample after extraction, the purified treatment need carry out derivatization, could measure in gas chromatography.Derivatization reagent commonly used have N-methyl-N-trimethyl silicon based-trifluoroacetamide and N, two (the trimethyl silicon based)-trifluoroacetamides of O-.
Whaites etc. adopt GC-MS to measure residual of kelengtelu in the ox urine, with N-methyl-N-trimethyl silicon based-trifluoroacetamide is derivatization reagent, adopt length than short capillary post (BP5,12.5m * 0.22mm) and selected ion monitoring pattern (Selective ion monitoring, SIM), detect and be limited to 0.07 μ g/L; When the mark-on level was 1ng/g and 0.2ng/g, the recovery was 100%.Leyssens uses the GC-MS method to 8 kinds of β in ox urine and the beef liver 2-receptor stimulating agent is residual to be measured, and detectability is between 0.5～5ng/g.If adopt negative ion chemistry ionization technique, cooperate deuterium for the stable isotope dilution technology, its sensitivity will get a greater increase.
1.4.2.5 LC-MS analytic approach
GC-MS is highly sensitive, but needs loaded down with trivial details derivatization step; Though HPLC can directly analyze, most detector sensitivities are on the low side, and can't prove conclusively.LC-MS becomes mensuration β just gradually in recent years 2The strong instrument of-receptor stimulating agent.
Progressively popularize β along with LC-MS and LC-MS/MS 2The reaction of-receptor stimulating agent retention analysis mensuration aspect is more and more.Doerge etc. use LC-MS with Atmosphere Pressure Chemical Ionization (APCI) (Atmospheric pressure chemical ionization, APCI) β in the mensuration bovine retina 2-receptor stimulating agent, sample directly detect and conclusive evidence after extract and separate.
1.4.2.6 immunoassay
Immunoassay is core reagent with antibody, utilize the specificity association reaction of antigen and antibody to be the analytical technology on basis, comprise fluoroimmunoassay (Fluoroimmunoassay, FIA), radio immunoassay (Radioimmunoassay, RIA), enzyme-linked immunosorbent assay.As screening technique, immunoassay has characteristics such as rapid sensitive is accurate.But for quantitatively also adopting other analytical approachs with conclusive evidence.
Euzymelinked immunosorbent assay (ELISA) is modal analytical approach in the immuno analytical method.Samples such as Chen Jiming enzyme-linked immunosorbent assay feed, urine sample and internal organs, this method sensing range is between 1.56 μ g/mL～1.9 μ g/mL.
In addition, radioimmunology, chemiluminescence enzyme linked immunosorbent assay etc. are also at β 2-receptor stimulating agent is applied in detecting.
In a word, in various detection methods, what be fit to be applied to large quantities of test sample is the ELISA detection method, it has fast, sensitive, simple to operate and characteristics that the one-time detection sample size is big, and can directly detect urine, blood sample, with HPLC, GC/MS the higher rate that conforms to is arranged, and be fit to very much the detection of living animal.And HPLC and GC/MS method all need expensive instrument, complicated sample handling procedure and special operative skill, and it is more time-consuming, therefore be not suitable for being used as the detection of gross sample, but its validity, accuracy and susceptibility make it still have the irreplaceable advantage of ELISA method.At present, when detecting, generally use the ELISA kit and carry out screening, with methods such as HPLC and GC/MS positive is confirmed again, thereby can guarantee the reliability of testing result.
Enzyme linked immunosorbent assay (ELISA) (ELISA) method is as a kind of screening technique, have the characteristics easy, quick, sensitive, that cost is low, therefore, during the extensive screening of monitoring and basic unit detects at the scene, immunological detection method has actual, has broad application prospects.
By retrieval, do not find the patent publication us relevant with patented claim of the present invention as yet.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art part, provide a kind of usable range wide, easy to use, detect β accurately 2-receptor stimulating agent linked immunoassay reagent kit and using method thereof.
The object of the present invention is achieved like this:
A kind of β 2-receptor stimulating agent enzyme linked immunological kit, its constituent and ratio are as follows:
Comprise that bag is by plate, calibration object, enzyme labeling thing working fluid, substrate solution A, substrate solution B, stop buffer, concentrated cleaning solution, concentrated extracting solution;
Described bag is coated with desertification butylamine alcohol polyclonal antibody at the bottom of for the hole by plate; Package amount is 5 μ g/mL, and bag is spent the night for 4 ℃ by temperature;
Described calibration object is the clenobuterol hydrochloride series calibration object of variable concentrations; Concentration is 0,0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb;
Described enzyme labeling thing working fluid is the salbutamol haptens that contains underlined horseradish peroxidase, for the sodium periodate method with horseradish peroxidase with the combination of salbutamol haptens; Enzyme labeling thing enzyme diluted;
Be weight percentage 0.07% hydrogen peroxide urea solution of described substrate solution A;
Described substrate solution B be weight percentage 0.04% 3,3', 5,5'-tetramethyl biphenyl amine aqueous solution;
Described stop buffer is 2M sulfuric acid;
Described concentrated extracting solution is 7.4 phosphate buffer for 0.2mol/L PH;
Described concentrated cleaning solution is that to contain mass concentration be 1% polysorbas20, and 0.2mol/lpH is 7.4 phosphate buffer.
And described enzyme labeling thing working fluid is: with the salbutamol haptens dilution with the mark horseradish peroxidase of enzyme dilution, to the salbutamol haptens: the volume ratio of enzyme dilution is 1:1000; The enzyme dilution is to contain the phosphate buffer that mass concentration is 1% bovine serum albumin(BSA).
And described substrate solution A uses acetic acid-citrate buffer solution dilution of 0.1M; Described substrate solution B uses acetic acid-citrate buffer solution of 0.1M with 3,3', and 5,5'-tetramethyl benzidine is diluted to 3,3', and the mass concentration of 5,5'-tetramethyl benzidine is 0.04%.
And described concentrated cleaning solution is the 20X concentrated cleaning solution, and use preceding according to 0.2M phosphate tween damping fluid: the volume ratio of distilled water is used behind the mixing as the dilution proportion of 1:19; Described concentrated extracting solution 20X concentrated extracting solution, use preceding according to 0.2M PBS phosphate buffer: the volume ratio of distilled water is used behind the mixing as the dilution proportion of 1:19.
And: described bag is as follows by step by the bag of plate:
⑴ antibody sandwich: with best the bag by the concentration coated elisa plate of desertification butylamine alcohol polyclonal antibody, 100uL/ hole, 4 ℃ are spent the night, and best package amount is 5 μ g/mL, and 0.05M pH9.6 carbonate buffer solution is used for bag by solid phase carrier as dilution;
⑵ wash plate: liquid in the hole is dried, and with cleansing solution 250 μ L/ holes washing 4～5 times, each 10s at interval pats dry with thieving paper;
⑶ sealing: will be that 1% bovine serum albumin(BSA) and massfraction are the confining liquid adding ELISA Plate that the phosphate buffer of 10% sucrose is formed by massfraction, 200uL/ hole, 37 ℃ of incubations 2 hours, liquid in the hole is dried, pat dry, vacuum drying adds the aluminium foil bag Vacuum Package.
Aforesaid β 2-receptor stimulating agent enzyme linked immunological kit detects β at fast quantification 2Application in the-receptor stimulating agent residual quantity.
And, described β 2-receptor stimulating agent derives from urine, serum, musculature, feed or the milk.
And, described detection β 2The lowest detection of-receptor stimulating agent is limited to 0.1ng/mL.
Aforesaid β 2The using method of-receptor stimulating agent enzyme linked immunological kit is characterized in that: step is as follows:
⑴ take out required reagent from cold storage environment, place more than the equilibrium at room temperature 30min, shakes up;
⑵ take out microwell plate, and no microwell plate is put into aluminide-coating bag, is stored in 2-8 ℃;
⑶ concentrated cleaning solution is returned to room temperature before use;
⑷ numbering: sample and the corresponding micropore of calibration object are numbered according to the order of sequence, and it is parallel that each sample and calibration object are done, and the position at record calibration hole and sample aperture place;
⑸ add calibration object/sample: add each 50 μ L of calibration object and sample in the micropore of correspondence, add enzyme labeling thing working fluid 50 μ L/ holes immediately, the vibration mixing is with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of shrouding film shrouding;
⑹ wash plate: open the shrouding film, liquid in the hole is dried, with concentrated cleaning solution 250 μ L/ holes washing 4～5 times, each 10s at interval pats dry;
⑺ colour developing: substrate A liquid and substrate B liquid is the 1:1 mixing by volume, and 100 μ L/ holes add in the microwell plate, and the vibration mixing behind shrouding film shrouding, is put in 25 ℃ of lucifuge environment and reacted 15min;
⑻ measure: add stop buffer 50 μ L/ holes, the mixing that vibrates is gently set microplate reader in the 450nm place, measures every hole OD value;
⑼ result: the mean value of the calibration object that obtains and sample absorbance multiply by 100 again divided by the absorbance of first calibration, can detect and draw β 2-receptor agonism agent dose.
Advantage of the present invention and good effect are:
1, the sensitivity of kit of the present invention is 0.1ppb, precision: variation within batch coefficient (CV%), CV%＜5%; Interassay coefficient of variation (CV%), CV%＜10%; Accuracy is represented accuracy with the recovery, and the recovery should be between 80%-110%.The IC50 scope: between 0.3-0.5ppb, linearly dependent coefficient | r|: be not less than 0.9900, have usable range wide, easy to use, detect advantage accurately.
2, the preparation method of kit of the present invention makes SAL and the synthetic immunizing antigen SAL-OVA of OVA coupling successfully with salbutamol antigen and carrier protein OVA by mixed anhydride method; Obtained highly purified polyclonal antibody by immune sheep and purification technique, and with 9 kinds of β 2-receptor stimulating agent all has cross reaction; Adopt the sodium periodate method with horseradish peroxidase and the crosslinked enzyme-labelled antigen that forms of salbutamol haptens, thereby set up a kind of kit of easy, quick, sensitive direct competition method, kit sensitivity 0.1ng/mL, and method is simple, easy operating, with low cost.
3, the exploitation of kit of the present invention and foundation, rely on ripe ELISA liquid system platform, through consulting a large amount of lists of references, and with reference to domestic and international similar kit, through a large amount of experiments and the optimization of system, finally establish production and the reaction conditions of kit, can be widely used in β in the samples such as urine sample or serum, muscle or tissue, feed, milk 2The fast quantification of-receptor stimulating agent residual quantity detects.
Description of drawings
Fig. 1 is that kit of the present invention is with β 2-receptor agonism agent concentration is horizontal ordinate, and variable concentrations calibration object OD value B/B0 value is ordinate, the typical curve graph of equation that draws;
Fig. 2 is that kit of the present invention is with β 2-receptor agonism agent concentration is horizontal ordinate, is that the variable concentrations calibration object OD value B/B0 value of matrix is ordinate with the urine, the typical curve graph of equation that draws.
Embodiment
Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Kit of the present invention by mixed anhydride method with salbutamol (SAL) and ovalbumin (OVA) synthetic immunogen, prepare polyclonal antibody with coupling immunogen immune sheep, and use the ammonium sulfate precipitation method preliminary purification, be further purified with the ELISA method with affinity chromatography and identify, enzyme-labelled antigen adopts sodium metaperiodate with horseradish peroxidase and the salbutamol haptens is crosslinked forms.Set up direct competitive enzyme linked immunosorbent assay analysis method (ELISA) with gained antibody and enzyme-labelled antigen and detect β 2-receptor stimulating agent, lowest detection is limited to 0.1ng/mL.
The measuring principle of kit of the present invention is: kit adopts the direct competitive ELISA method, pre-coated antibody on the ELISA Plate capillary strip, residual β in the sample 2-receptor stimulating agent and enzyme-labelled antigen competition desertification butylamine alcohol antibody are with tmb substrate colour developing, sample absorbance residue β contained with it 2The content of-receptor stimulating agent becomes negative correlation, multiply by its corresponding extension rate more again with typical curve and can draw β in the sample 2The content of-receptor stimulating agent.
One, a kind of β 2-receptor stimulating agent enzyme linked immunological kit, it is composed as follows:
This kit be by the bag by plate, calibration object, high concentration calibration object, enzyme labeling thing working fluid, substrate solution A, substrate solution B, stop buffer, 20X concentrated cleaning solution, the 20X concentrated extracting solution specifically sees Table 3.
Table 3 kit constituent of the present invention table
Figure BDA00003048399300151
Related main material preparation method is as follows for kit of the present invention:
1, enzyme conjugates
Enzyme in the enzyme-labelled antigen is horseradish peroxidase (HRP), and horseradish peroxidase-salbutamol label adopts the preparation of sodium periodate method, and step is as follows:
1) taking by weighing 5mgHRP is dissolved in the 0.5mL distilled water;
2) add the 0.1M NaIO4 solution that 0.5mL newly joins in last liquid, lucifuge stirred 20 minutes under the room temperature;
3) above-mentioned solution is packed in the bag filter (8000-12000Da), to sodium-acetate buffer (pH4.4) dialysis of 1mM, 4 ℃ are spent the night;
4) add the 0.2M carbonate buffer solution, make the pH of above hydroformylation HRP be elevated to 9.0～9.5, add 5mg salbutamol antibody then immediately, the room temperature lucifuge stirred 2 hours gently;
5) add the 4mg/mL NaBH4 liquid that 0.2mL newly joins, mixing is put 4 ℃, 2 hours again;
6) above-mentioned liquid is packed in the bag filter (8000-12000Da), to 0.15M pH7.4PBS dialysis, 4 ℃ are spent the night;
7) under agitation dropwise add the equal-volume saturated ammonium sulfate, put 4 ℃, 1 hour;
8) 3000rpm centrifugal half an hour, abandon supernatant, sediment is washed secondary with semi-saturation ammonium sulfate, and last sediment is dissolved among the PBS of a small amount of 0.15M pH7.4;
9) above-mentioned solution is packed in the bag filter, to the PBS buffer saline dialysis of 0.15M PH7.4, remove ammonium ion after, 10,000rpm removed precipitation in centrifugal 30 minutes, and supernatant is the enzyme conjugates of horseradish peroxidase-salbutamol label, after the packing, stored frozen.
2, immunogene
The summary of immunogenic preparation principle: salbutamol (SAL) be micromolecular compound, only has reactionogenicity and lacks immunogenicity, genus haptens.The conjugates that haptens and carrier protein couplet form not only has immunogenicity, and itself special reactionogenicity is arranged.With salbutamol antigen and carrier protein OVA, make SAL and the synthetic immunizing antigen SAL-OVA of OVA coupling by mixed anhydride method, carry out the scanning of ultraviolet-visible absorbing wavelength and infrared spectrum behind the purifying and identify whether coupling is successful, successfully namely can be used as immunogene.
Synthesizing of salbutamol succinic acid derivative:
According to the activity difference of three hydroxyls of Sal, under anhydrous condition, the methylol at Sal phenolic hydroxyl group ortho position can with succinic anhydride generation alcoholysis reaction, generate the succinic acid derivative (Sal-HS) of Sal, reaction equation is as follows:
Figure BDA00003048399300161
Experimental procedure is as follows:
⑴ take by weighing 100mgSa1, is dissolved in the 12.5mL methyl alcohol;
⑵ methyl alcohol is removed in decompression distillation (0.1mmHg, 60 ℃);
⑶ residue is dissolved in the 20mL absolute ethyl alcohol, and adds the 45mg succinic anhydride, and this moment, solution became turbid;
⑷ stirring at room reaction 3h;
With reactant liquor centrifugal (10000rpm, 30min), collecting precipitation;
⑹ clean centrifugal repeatedly with absolute ethyl alcohol, get precipitation;
⑺ suction filtration;
⑻ room temperature is dried in the shade, and namely gets the salbutamol succinic acid derivative.
The synthesis step of SaI-HS-OVA is as follows:
⑴ take by weighing 40mg Sal-HS and be dissolved in a certain amount of triethylamine one dioxane dimethyl formamide (0.15/3.75/3.75V/V/V) mixed solution, ice bath 30min;
⑵ add 4.875g chloro-carbonic acid isobutyl vinegar, stirring reaction 2h in the ice bath environment;
⑶ take by weighing 21mgOVA, with its fully be dissolved in 6mL sodium borate buffer liquid (0.lmol/L, pH8.5) in;
⑷ dropwise join the OVA solution that configures in the Sal-HS reaction mixture;
⑸ reaction mixture stirring reaction spends the night, during intermittently reactant liquor pH is monitored, make its pH remain on 8.5;
⑹ next day, reactant liquor is fully dialysed to the PBS solution of 0.01mol/L;
⑺ dialysis is after 4 days, and the product of will dialysing is sub-packed in the 1.5mL centrifuge tube, puts in-20 ℃ of refrigerators and preserves.
Reaction equation is as follows:
Figure BDA00003048399300171
3, desertification butylamine alcohol Polyclonal Antibody Preparation flow process
3.1 with above-mentioned synthetic SAL-OVA as immunogene, be diluted to 1mg/mL, with the freund 's incomplete adjuvant mixed in equal amounts, adopt intracutaneous injection inoculation sheep, whenever immunity was all around once taken a blood sample to test clearly at every turn and is tired in immune back 7 days, observation antiserum titre growth pattern, when reaching satisfied tiring, the jugular vein blood sampling separates antiserum.
3.2 purifying antibody
3.2.1 slightly carry
Antibody globulin with in the saturated ammonium sulfate salting out method purification serum is further purified with chromatographic column, and phosphate buffer is dialysed.Draw antiserum 10ml, balance adds the phosphate buffer of the 0.01mol/l PH7.4 of 10ml to room temperature, fully mixing, the saturated ammonium sulfate solution (transferring to PH7.4 with strong aqua) that adds 250ml, shake up, 4 ℃ of refrigerators left standstill 3 hours, with 5000rpm centrifugal 30 minutes, remove supernatant, add the phosphate buffer of 10ml0.01mol/l PH7.4, the dissolution precipitation thing repeats to saltout twice.The final phosphate buffer lytic immunity globulin IgG sediment that adds 10ml0.01mol/l PH7.4, the bag filter of packing into (8000-12000), to the phosphate buffer dialysis of 1000ml0.01mol/l PH7.4 24 hours, during change liquid 3 times.
3.2.2 cross the post purifying
1、DEAE-Sephadex
The A-50 pre-service:
Claim that DEAE-SephadexA-50(calls A-50 in the following text) 5g, be suspended from the 500ml distilled water, the upper strata particulate is removed in the 1h hypsokinesis.Add the ratio of 0.5mol/L NaOH15ml in every gram A-50, A-50 is soaked in the 0.5mol/L NaOH liquid, stir evenly, leave standstill 30min, suction filtration in the Buchner funnel of packing into (being lined with 2 metafiltration paper), and take out with distilled water repeatedly and be washed till pH and be neutral; Handle with the same operating process of 0.5mol/L HCl again, handle once again with 0.5mol/L NaOH at last.After handling, A-50 is soaked in the 0.1mol/L pH7.4PB damping fluid spends the night.
2, dress post
(1) with the chromatographic column vertical fixing on titration stand, post heelpiece one circular nylon yarn, water delivering orifice connects a latex or plastic tube and closes switch.
(2) with 0.1mol/L, pH7.4PB pours in the post to 1/4 height along glass bar, pours into through pre-service and with the A-50 of the same damping fluid furnishing scattered paste shape again.When high, open the water delivering orifice screw clamp Deng A-50 gel sedimentation 2～3cm, control flow velocity 1mL/min pours pasty state A-50 gel into continuously to desired height simultaneously.
(3) close water delivering orifice, treat the complete sedimentation of A-50 gel after, cylinder is put a circular filter paper sheet, and is suitable for reading with rubber stopper jam-pack post.Be connected with the eluent bottle by the syringe needle that inserts rubber stopper and the latex that connects or plastic tube.
3, balance
Breakdown water delivering orifice screw clamp, 12～14/min of control flow velocity.The eluent of about 2 times of bed volumes is flowed out.And measure eluent respectively and flow out pH value and the ionic strength of liquid with pH meter and conductivity meter, when reaching unanimity, both close water delivering orifice, stop balance.
4, application of sample and wash-out
Breakdown rubber suitable for reading is filled in the end opening screw clamp, makes that liquid slowly oozes in the post, when cylinder liquid and cylinder are tangent, closes water delivering orifice immediately, with capillary burette along post jamb add sample (volume should＜bed volume 2%, protein concentration is advisable with＜100mg).Unclamp the water delivering orifice screw clamp face sample is slowly entered in the post, when extremely tangent with cylinder, close end opening immediately, wash post jamb 2～3 times with a small amount of eluent; Decontrol water delivering orifice again, make washing lotion enter column, immediately add several milliliters of eluents on cylinder, tight stick harness is suitable for reading, makes whole elution process become a closed system.And 12～14/min of control flow velocity.
5, collect
Just collect with test tube in the time of the beginning wash-out.Every pipe is collected 3～5ml.Collect 10～15 pipes altogether.
6, survey albumen
Measure every pipe OD280nm respectively with 751 type ultraviolet spectrophotometers, with OD260nm, by formula calculate each tubulin content.And be ordinate with OD280nm, be numbered abscissa with test tube, draw elution curve.
7, merge, concentrate
Collect liquid and merge respectively washing each pipe of the upward slope section at sharp peak and lower slope section, with PEG(MW6000) be concentrated into volume requiredly, it is anticorrosion to add 0.02%NaN3, standby in 4 ℃ of preservations.Should store in-20 ℃ of refrigerators during long preservation.
4, the preparation of reagent
4.1 standard solution: accurately take by weighing clenobuterol hydrochloride 1mg, be mixed with the mother liquor of 1mg/mL with absolute methanol, be diluted to final required concentration (0.1ppb-8.1ppb), the can of 1ml/ bottle with PBS again.
4.2 enzyme-labelled antigen solution: enzyme-labelled antigen is diluted to the 1:1000(enzyme-labelled antigen with enzyme dilution (containing the phosphate buffer that mass concentration is 1%BSA): the volume ratio of enzyme dilution), the can of 7ml/ bottle.
4.3 substrate solution A: with acetic acid-citrate buffer solution of 0.1M urea peroxide being diluted to percentage by weight is the can of 0.07%, 7ml/ bottle.
4.4 substrate solution B: with acetic acid-citrate buffer solution of 0.1M TMB being diluted to percentage by weight is the can of 0.04%, 7ml/ bottle.
4.5 stop buffer: 2M sulfuric acid, the can of 7ml/ bottle.
4.6 cleansing solution: get the 50mL concentrated cleaning solution and add deionized water 950mL dilution, use behind the mixing, put 4 ℃ of preservations.
4.7 extract: get the 50mL concentrated extracting solution and add deionized water 950mL dilution, use behind the mixing, put 4 ℃ of preservations.
4.8 wrap as follows by step by the bag of plate:
A) bag quilt: antibody is wrapped by the concentration coated elisa plate with best, the 100ul/ hole, 4 ℃ are spent the night, best package amount is 5 μ g/mL, 0.05M pH9.6CBS(carbonate buffer solution) be used for bag by solid phase carrier as dilution.
B) wash plate: liquid in the hole is dried, fully wash 4～5 times with cleansing solution 250 μ L/ holes, each 10s at interval pats dry with thieving paper.
C) sealing: be the 1%BSA(bovine serum albumin(BSA) with massfraction) and massfraction be the confining liquid adding ELISA Plate that the phosphate buffer of 10% sucrose (sucrose) is formed, the 200uL/ hole, 37 ℃ of incubations 2 hours, liquid in the hole is dried, pat dry with thieving paper, vacuum drying adds the aluminium foil bag Vacuum Package.
Salbutamol ELISA diagnostic kit analytical performance of the present invention is as follows:
1, finished product calibrating
1.1 physical examination
Each component should meet outward appearance and sense organ requirement separately, and loading amount is accurate, and put correctly the position, and assembly is complete.Label is clear, and is complete, and print What is correct.
1.2 experiment calibrating
1.2.1 sensitivity: represent with lowest detectable limit.Measure 20 hole zero standards, the OD mean value that record records, the minimum medicine of sample when adding 3 times of standard deviations can reach 20 parts of blank samples mensuration averages is dense to be detectability.
1.2.2 accuracy: the recovery is measured the recovery with additive process, and two of selected 1ppb, 5ppb add concentration.Each concentration is made 5 parallel samples, and every sample is done 2 holes, is no less than revision test 3 times, and the recovery is 80～110%.
1.2.3 specificity:
Cross reaction
Clenbuterol ... ... ... ... ... ... 100%
Salbutamol ... ... ... ... ... ... 100%
The bromo Clenbuterol ... ... ... ... ... ... 150%
Bromine Boot sieve ... ... ... ... ... ... 110%
Mabuterol ... ... ... ... ... ... 75%
Ke Lunpanteluo ... ... ... ... ... ... 65%
Horse is sprayed special sieve ... ... ... ... ... ... 50%
Arubendol ... ... ... ... ... ... 35%
Carbuterol ... ... ... ... ... ... 30%
Gram human relations third sieve ... ... ... ... ... ... 12%
Cimaterol ... ... ... ... ... ... 10%
Dobutamine ... ... ... ... ... ... 0.5%
Sharp holder monarch ... ... ... ... ... ... 0.1%
Ractopamine ... ... ... ... ... ... 0.01%.
1.2.4 accuracy: selection standard curve 50% inhibition concentration (IC 50) near the standard solution replication of concentration.This reagent is selected 5ppb calibration object replication for use.
Variation within batch coefficient: CV＜5%;
Interassay coefficient of variation: CV＜10%.
1.2.5 linear: as serial calibration object to be measured, with the mapping of 4p simulation curve, calculated its R value, R〉0.9900.
1.2.6 stability:
Stability: 37 ℃ of stable experiments of acceleration are answered and were stablized in 5 days.The OD value of 0 titer, IC 50, represent the interpolation recovery, quality-control product measured value of concentration all in tolerance.
2, preservation and the term of validity: in 2-8 ℃ of drying, keep in Dark Place, examine and determine certainly qualified from the term of validity be 6 months.
Two, β as mentioned above 2-receptor stimulating agent enzyme linked immunological kit detects β 2The residual quantity test sample of-receptor stimulating agent is handled:
1, urine or serum (extension rate: 1)
Can directly detect analysis.If urine sample is muddy shape, 4000 rev/mins, centrifugal 5 minutes or filter after get supernatant and detect.For fear of high background value, available extract carries out 5 times of dilutions (1mL urine sample+4mL extract).
2, muscle or tissue
Method A:(extension rate: 0.5)
⑴ remove the fat in muscle or the tissue.Take by weighing the even quality sample of 3 ± 0.05g in the 50mL centrifuge tube, add 8mL acetonitrile and 1mL ethyl acetate, maximum (top) speed vortex vibration 10 minutes.4000 rev/mins of room temperatures, centrifugal 5 minutes;
⑵ get supernatant 6mL, 60-70 ℃ of decompression distillation, and perhaps 60-70 ° of C water-bath nitrogen dries up;
⑶ get the 1mL normal hexane and dissolve dry thing, and add the 1mL extract, maximum (top) speed vortex vibration 1 minute.(if emulsion, lower layer of water phase quantity not sufficient enough detects simultaneously, guarantees the pipe exhaust, 85 ℃ water-bath 3 minutes).Getting 50ul lower floor water detects.
Method B:(extension rate: 4)
⑴ remove the fat in muscle, liver or the kidney, takes by weighing sample behind 2 ± 0.05g homogeneous in the 10mL centrifuge tube, adds the 2mL3% trichloroacetic acid, vibrated 4000 rev/mins of room temperatures, centrifugal 10 minutes 10 minutes.
⑵ migrate out supernatant, with 0.05M NaOH1:1 mixing (1mL supernatant+1mL0.05M NaOH), gets 50 μ l and detect.
3, feed
Method A: direct dilution process (extension rate: 10)
Grind feed with mortar, the feed sample that claims 1g to grind adds the hydrochloric acid of 10mL0.01M, fully mixes 10 minutes.Check pH value whether between 6.5～8, otherwise with NaOH or hydrochloric acid adjusting.4000 rev/mins of room temperatures centrifugal 5 minutes, migrate out supernatant and (can improve rotating speed or filter with filter paper as supernatant still muddiness.Directly getting supernatant detects.
Method B: organic solvent extraction method (extension rate: 1.0)
Use suitable homogenizer to spare quality sample.Get the even quality sample of 1.5g, add 8mL acetonitrile and 1mL ethyl acetate, maximum (top) speed vortex vibration 10 minutes.4000 rev/mins of room temperatures, centrifugal 5 minutes, pipette the 6mL supernatant to another new test tube, 60-70 ℃ of decompression distillation, or 60-70 ° of C water-bath nitrogen dries up.Get the 1mL normal hexane and dissolve dry thing, and add the 1mL extract, maximum (top) speed vortex vibration 1 minute.4000 rev/mins of room temperatures, centrifugal 5 minutes, every hole was got 50 μ L lower floor waters and is detected.
4, milk (extension rate: 5)
Get 4000 rev/mins of 10mL milk room temperatures (20-25 ℃), centrifugal 10 minutes, absorb the upper strata fat deposit.Carry out 1:4 dilution (1mL milk+4mL extract) with extract, get 50ul liquid and detect.
Note the preservation of sample:
The freezing preservation of----untreated sample
----sample after the processing is preferably measured immediately, handles the back sample and can preserve 24 hours 2～8 ℃ of lucifuge conditions.
The using method of this kit is as follows: (operating under the room temperature 20-25 ℃ of condition)
1, required reagent is taken out from cold storage environment, place room temperature (20-25 ℃) more than the balance 30min, note to shake up before every kind of liquid reagent uses;
2, take out the microwell plate that needs quantity, no microwell plate is put into aluminide-coating bag, be stored in 2-8 ℃;
3, cleansing solution also need be returned to room temperature before use;
4, numbering: the corresponding micropore of sample and calibration object is numbered according to the order of sequence, and it is parallel that each sample and calibration object are done 2 holes, and the position at record calibration hole and sample aperture place;
5, add calibration object/sample: add each 50 μ l of calibration object and sample in the micropore of correspondence, add enzyme labeling thing 50 μ l/ holes immediately, the mixing that vibrates gently is with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of shrouding film shrouding;
6, wash plate: carefully open the shrouding film, liquid in the hole is dried, fully wash 4-5 time with cleansing solution 250 μ l/ holes, each 10s at interval pats dry (bubble that is not eliminated after patting dry can be poked with clean rifle head) with thieving paper;
7, colour developing: add colour developing liquid (substrate A liquid and substrate B liquid 1:1 mixing) 100 μ l/ holes, the mixing that vibrates gently behind shrouding film shrouding, is put in 25 ℃ of lucifuge environment and is reacted 15min;
8, measure: add stop buffer 50 μ l/ holes, the mixing that vibrates is gently set microplate reader in the 450nm place (suggestion detects with dual wavelength 450/630nm, runs through data in 5min), measures every hole OD value.(if no microplate reader does not then add stop buffer and can judge with ocular estimate);
9, result
The mean value of the calibration that obtains and sample absorbance multiply by 100 again divided by the absorbance of first calibration (0 calibration).Therefore 0 calibration equals 100% and provide absorbance with the form of number percent.
Formula is as follows:
The absorbance (or sample) of calibration
-------------------------x100=% absorbance
The absorbance of 0 calibration
It is a corresponding β that the calibration value that calculates plots 2The semilog coordinate system curve map of-receptor agonism agent concentration, the concentration of corresponding each sample can be read from calibration curve.
In order to obtain the β in the sample 2The actual concentrations of-receptor stimulating agent, the concentration value of reading from typical curve must multiply by corresponding dilution factor.
10) the directly inhibition ELISA method bioassay standard curve of the present invention's employing is definite as follows
With the β of anti-SAL polyclonal antibody to variable concentrations 2-receptor stimulating agent calibration object suppresses, and with the calibration object of 100ppb, carries out 3 times of dilutions, is matrix with 0.01M PBS.Measure according to the ELISA running program.With β 2-receptor agonism agent concentration is horizontal ordinate, variable concentrations calibration object OD value, and the B/B0 value is ordinate, each OD value sees Table 4, draws the typical curve equation, sees Fig. 1.
Table 4 variable concentrations calibration object OD value
Concentration value Absorbance Entry name
100 0.321 OD
33.3 0.356 OD
11.1 0.452 OD
3.7 0.956 OD
1.23 1.211 OD
0.41 1.521 OD
0.137 1.686 OD
0.045 1.876 OD
0 1.871 OD
Conclusion (of pressure testing): as seen from Figure 1, with β 2-receptor agonism agent concentration is horizontal ordinate, and variable concentrations calibration object OD value B/B0 value is ordinate, draws the typical curve equation, is y=0.977x+0.0447, R 2Be 0.9669, calibration object is linear inhibition within the specific limits.
Detect example:
This kit can be used for the direct detection of pig urine, if pig urine samples muddiness is answered 4000 rev/mins, centrifugal 5 minutes or filter after get supernatant and detect.For fear of high background value, available sample extracting solution is done 5 times of dilutions.
According to the operation of kit, to containing series concentration β 2-receptor stimulating agent calibration object carry out test of many times, the competition curve of acquisition is seen Fig. 2, this curve becomes the S type as shown in Figure 2, the range of linearity is worked as β between 0.1-100ppb 2-receptor stimulating agent calibration object concentration can produce 50% and suppress when 3.4ppb, linearly dependent coefficient is 0.9697, and minimum detectability (IC10) is about 0.1ppb, and this result shows that anti-SAL polyclonal antibody also can present compatibility preferably in urine matrix.

Claims (9)

1. β 2-receptor stimulating agent enzyme linked immunological kit is characterized in that: its constituent and ratio are as follows:
Comprise that bag is by plate, calibration object, enzyme labeling thing working fluid, substrate solution A, substrate solution B, stop buffer, concentrated cleaning solution, concentrated extracting solution;
Described bag is coated with desertification butylamine alcohol polyclonal antibody at the bottom of for the hole by plate; Package amount is 5 μ g/mL, and bag is spent the night for 4 ℃ by temperature;
Described calibration object is the clenobuterol hydrochloride series calibration object of variable concentrations; Concentration is 0,0.1ppb, 0.3ppb, 0.9ppb, 2.7ppb, 8.1ppb;
Described enzyme labeling thing working fluid is the salbutamol haptens that contains underlined horseradish peroxidase, for the sodium periodate method with horseradish peroxidase with the combination of salbutamol haptens; Enzyme labeling thing enzyme diluted;
Be weight percentage 0.07% hydrogen peroxide urea solution of described substrate solution A;
Described substrate solution B be weight percentage 0.04% 3,3', 5,5'-tetramethyl biphenyl amine aqueous solution;
Described stop buffer is 2M sulfuric acid;
Described concentrated extracting solution is 7.4 phosphate buffer for 0.2mol/L PH;
Described concentrated cleaning solution is that to contain mass concentration be 1% polysorbas20, and 0.2mol/l pH is 7.4 phosphate buffer.
2. β according to claim 1 2-receptor stimulating agent enzyme linked immunological kit is characterized in that: described enzyme labeling thing working fluid is: with the salbutamol haptens dilution with the mark horseradish peroxidase of enzyme dilution, to the salbutamol haptens: the volume ratio of enzyme dilution is 1:1000; The enzyme dilution is to contain the phosphate buffer that mass concentration is 1% bovine serum albumin(BSA).
3. β according to claim 1 2-receptor stimulating agent enzyme linked immunological kit is characterized in that: described substrate solution A uses acetic acid-citrate buffer solution dilution of 0.1M; Described substrate solution B uses acetic acid-citrate buffer solution of 0.1M with 3,3', and 5,5'-tetramethyl benzidine is diluted to 3,3', and the mass concentration of 5,5'-tetramethyl benzidine is 0.04%.
4. β according to claim 1 2-receptor stimulating agent enzyme linked immunological kit is characterized in that: described concentrated cleaning solution is the 20X concentrated cleaning solution, and use preceding according to 0.2M phosphate tween damping fluid: the volume ratio of distilled water is used behind the mixing as the dilution proportion of 1:19; Described concentrated extracting solution 20X concentrated extracting solution, use preceding according to 0.2M PBS phosphate buffer: the volume ratio of distilled water is used behind the mixing as the dilution proportion of 1:19.
5. β according to claim 1 2-receptor stimulating agent enzyme linked immunological kit is characterized in that: described bag is as follows by step by the bag of plate:
⑴ antibody sandwich: with best the bag by the concentration coated elisa plate of desertification butylamine alcohol polyclonal antibody, 100uL/ hole, 4 ℃ are spent the night, and best package amount is 5 μ g/mL, and 0.05M pH9.6 carbonate buffer solution is used for bag by solid phase carrier as dilution;
⑵ wash plate: liquid in the hole is dried, and with cleansing solution 250 μ L/ holes washing 4 ~ 5 times, each 10s at interval pats dry with thieving paper;
⑶ sealing: will be that 1% bovine serum albumin(BSA) and massfraction are the confining liquid adding ELISA Plate that the phosphate buffer of 10% sucrose is formed by massfraction, 200uL/ hole, 37 ℃ of incubations 2 hours, liquid in the hole is dried, pat dry, vacuum drying adds the aluminium foil bag Vacuum Package.
6. as the described β of each claim of claim 1 to 5 2-receptor stimulating agent enzyme linked immunological kit detects β at fast quantification 2Application in the-receptor stimulating agent residual quantity.
7. application according to claim 6 is characterized in that: described β 2-receptor stimulating agent derives from urine, serum, musculature, feed or the milk.
8. according to claim 6 or 7 described application, it is characterized in that: described detection β 2The lowest detection of-receptor stimulating agent is limited to 0.1ng/mL.
9. one kind as each described β of claim 1 to 5 2The using method of-receptor stimulating agent enzyme linked immunological kit is characterized in that: step is as follows:
⑴ take out required reagent from cold storage environment, place more than the equilibrium at room temperature 30min, shakes up;
⑵ take out microwell plate, and no microwell plate is put into aluminide-coating bag, is stored in 2-8 ℃;
⑶ concentrated cleaning solution is returned to room temperature before use;
⑷ numbering: sample and the corresponding micropore of calibration object are numbered according to the order of sequence, and it is parallel that each sample and calibration object are done, and the position at record calibration hole and sample aperture place;
⑸ add calibration object/sample: add each 50 μ L of calibration object and sample in the micropore of correspondence, add enzyme labeling thing working fluid 50 μ L/ holes immediately, the vibration mixing is with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of shrouding film shrouding;
⑹ wash plate: open the shrouding film, liquid in the hole is dried, with concentrated cleaning solution 250 μ L/ holes washing 4 ~ 5 times, each 10s at interval pats dry;
⑺ colour developing: substrate A liquid and substrate B liquid is the 1:1 mixing by volume, and 100 μ L/ holes add in the microwell plate, and the vibration mixing behind shrouding film shrouding, is put in 25 ℃ of lucifuge environment and reacted 15min;
⑻ measure: add stop buffer 50 μ L/ holes, the mixing that vibrates is gently set microplate reader in the 450nm place, measures every hole OD value;
⑼ result: the mean value of the calibration object that obtains and sample absorbance multiply by 100 again divided by the absorbance of first calibration, can detect and draw β 2-receptor agonism agent dose.
CN2013101282706A 2013-04-15 2013-04-15 Beta 2-acceptor stimulant ELISA kit and usage method and application thereof Pending CN103235117A (en)

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CN105315166A (en) * 2014-06-27 2016-02-10 北京维德维康生物技术有限公司 Preparation methods of salbutamol hapten and salbutamol antigen, and application thereof in quantum dot immunofluorescence kit
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CN105628924A (en) * 2015-12-31 2016-06-01 苏州科铭生物技术有限公司 Kit and method for measuring content of corticosterone
CN106153952A (en) * 2016-07-28 2016-11-23 内蒙古蒙牛乳业（集团）股份有限公司 The qualitative checking method of beta receptor agonist content in milk
CN106706934A (en) * 2016-12-08 2017-05-24 河北省科学院生物研究所 ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting formoterol as well as preparation method and application of ELISA kit
CN106706934B (en) * 2016-12-08 2017-12-01 河北省科学院生物研究所 A kind of enzyme linked immunological kit for detecting Formoterol and preparation method and application
CN108181463A (en) * 2017-12-11 2018-06-19 华南农业大学 A kind of beta-2-agonists haptens and artificial antibody and its preparation method and application
CN108181463B (en) * 2017-12-11 2020-12-11 华南农业大学 Beta-agonist hapten and artificial antibody as well as preparation method and application thereof
CN108982712A (en) * 2018-10-08 2018-12-11 山东越品检验检测有限公司 Salmeterol, datro, the method for Ao Dateluo in a kind of measurement pork

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