CN103018447A - Enzyme-linked immunoassay kit for salbutamol detection, and applications thereof - Google Patents
Enzyme-linked immunoassay kit for salbutamol detection, and applications thereof Download PDFInfo
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- CN103018447A CN103018447A CN2011102789600A CN201110278960A CN103018447A CN 103018447 A CN103018447 A CN 103018447A CN 2011102789600 A CN2011102789600 A CN 2011102789600A CN 201110278960 A CN201110278960 A CN 201110278960A CN 103018447 A CN103018447 A CN 103018447A
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Abstract
The present invention provides an enzyme-linked immunoassay kit for salbutamol detection. The enzyme-linked immunoassay kit comprises an enzyme label plate coated with coating antigen, special salbutamol antibody, an enzyme marker, salbutamol standard solutions, a substrate coloration solution, a termination solution, a concentration washing solution and a sample dilution solution. The present invention provides a salbutamol enzyme-linked immunoassay kit detection method, which mainly comprises: carrying out a sample pretreatment, adopting the kit to detect, and finally analyzing a detection result. With the detection kit, salbutamol residue in animal tissues (muscles and livers), urine and feed samples can be detected, characteristics of simple and rapid operation, high accuracy, high sensitivity, low cost and the like are provided, and the kit and the method can be applicable for screening and on-site monitoring of a large number of samples.
Description
Technical field
The present invention relates to the enzyme linked immunosorbent detection technology, be specifically related to a kind of enzyme linked immunological kit and detection method that detects salbutamol in animal tissue's (muscle, liver), urine, the feed.
Background technology
Salbutamol is a kind of potent selectivity beta-receptor stimulant substance, the respiratory diseases such as bronchial spasm that are usually used in clinically treating bronchial astehma, asthmatic bronchitis and pulmonary emphysema patient.Therefore medicine can improve lean meat percentage, reduces fat deposition and promotes growth of animal, is illegally used as cultivation promoter by some livestock-raising enterprises.Its residual quantity can cause that the human muscle trembles, palpitaition, neuroticism, headache, dizzy, the symptom such as feel sick, vomit, have a fever, tremble, the healthy utmost point to the consumer has harm, the beta-stimulants such as " veterinary drug and other compound inventory of food animal forbidding " clear salbutamol of China Ministry of Agriculture issue all purposes that are under an embargo are forbidden all food animals.
At present, for residue detection mainly contain the detection methods such as high performance liquid chromatography, liquid chromatography～mass spectrometry analytic approach, vapor-phase chromatography, By Capillary Zone Electrophoresis, enzymoimmunoassay, the instrument detection method has advantages of that the degree of accuracy of detection is high, but its instrumentation degree is high, sample preparation is complicated, testing cost is high, be not suitable for use in the detection of gross sample, immunological detection method is simple to operate, quick, sensitive, can detect simultaneously most samples, be desirable rapid screening means.
Summary of the invention
The purpose of this invention is to provide a kind of salbutamol enzyme linked immunological kit and application thereof.
A kind of salbutamol ELISA diagnostic kit, it comprises ELISA Plate, salbutamol specific antibody, enzyme labeling thing, salbutamol standard solution, substrate nitrite ion, stop buffer, concentrated cleaning solution, the sample dilution that is coated with coating antigen, described coating antigen is salbutamol medicine antigen, and described enzyme labeling thing is the enzyme labeling antiantibody.
Salbutamol enzyme linked immunological kit provided by the present invention, described salbutamol medicine antigen is the conjugate of salbutamol haptens and carrier protein, and described carrier protein is bovine serum albumin(BSA), mouse haemocyanin, rabbit anteserum albumen, thyroprotein, ovalbumin, hemocyanin or human serum albumins.
Described salbutamol specific antibody is the salbutamol monoclonal antibody, obtains with salbutamol immunogen immune animal.Described salbutamol monoclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, and described salbutamol monoclonal antibody is preferably the salbutamol mouse monoclonal antibody.
The marker enzyme of described enzyme labeling thing is horseradish peroxidase, and the enzyme labeling antiantibody is to adopt the sodium periodate method that marker enzyme and the coupling of sheep anti mouse antiantibody are obtained.
In order to carry out more easily great amount of samples examination and on-site supervision, described kit also comprises: salbutamol standard solution, substrate nitrite ion, stop buffer, concentrated cleaning solution, sample dilution.
Described standard solution is serial salbutamol standard solution, the 1mL/ bottle, and concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L.Described substrate solution A liquid is urea peroxide solution, substrate solution B liquid is the tetramethyl biphenyl amine aqueous solution, described stop buffer is the sulfuric acid solution of 1～2mol/L, described concentrated cleaning solution is the 0.15～0.2mol/LpH7.4 phosphate buffer that contains 1% Tween-20, and described sample dilution is that pH is that the content of Tween-20 is that the formulated dilution of 0.5% mixed solution dilutes in the phosphate buffer of 7.4 0.02mol/L.
The wherein said ELISA Plate carbonate buffer solution that used coated damping fluid is the 0.05mol/L of pH9.6 in preparation process, sealing damping fluid are 10% calf serum solution.
The preparation of ELISA Plate of the present invention is mainly: with coated damping fluid coating antigen is diluted to 0.2～0.3 μ g/ml, every hole adds 150 μ l, lucifuge is hatched 2h or 4 ℃ and is spent the night in 37 ℃ of environment, the liquid in the hole that inclines, cleansing solution washing 1～2 time pats dry, add 150 μ l confining liquids in every hole, 37 ℃ of lucifuges are hatched 2h, and liquid pats dry in the hole of inclining, and preserve with the vacuum seal of aluminium film.
Coating antigen and immunogenic building-up process are among the present invention:
1. haptens synthesizes (synthetic route such as Fig. 1)
(1) gets the 1-3g salbutamol, dissolve among the 20-50ml DMF, then add the TEMPO of 10% molar equivalent and the tetrabutylammonium chloride of 10% molar equivalent, the NaHCO3 aqueous solution of 20-50ml 0.5M stirred under the room temperature after 30 minutes, added the NCS of 1.1-1.5 molar equivalent, after room temperature reaction 2-5 hour, remove most of solvent, ethyl acetate extraction, washing, dry, column chromatography purification after the desolventizing obtains white solid, is salbutamol list oxide.
(2) 1-2g step (1) gained salbutamol oxide, dissolve among the 20-30ml DMF, the ethylenediamine that adds the 2-5 molar equivalent, room temperature to 100 ℃ reaction 5-20 hour, desolventizing, recrystallization in the alcohol-water obtains white solid, is the ethylenediamine condensation product of salbutamol list oxide.
2. immunogenic synthetic
(1) gets the salbutamol haptens 18mg water-soluble solution of 1.5ml.
(2) get 50% GA8 μ l and add in (1) stirring reaction 18h under the room temperature.
(3) getting BSA100mg adds in the mentioned solution with the dilution of 5.5ml water is rear.
(4) add 24mgNaBH behind the reaction 24h
4Reaction 2h.
(5) with tri-distilled water dialysis 48 hours, namely get immunogene.
3. coating antigen is synthetic
(1) gets the salbutamol haptens 15mg water-soluble solution of 1.5ml.
(2) get 50% GA8 μ l and add in (1) stirring reaction 18h under the room temperature.
(3) getting OVA30mg adds in the mentioned solution with the dilution of 4.5ml water is rear.
(4) add 24mgNaBH behind the reaction 24h
4Reaction 2h.
(5) with tri-distilled water dialysis 48 hours, namely get coating antigen.
4. the preparation of monoclonal antibody
Animal immune: salbutamol haptens and carrier protein couplet thing immunity 8-10 Bal b/c mouse in age in week.
Fusion of Cells and cloning: get the mice spleen cell after the immunity, merge under the effect of fusion agent polyglycol (PEG) 4000 with SP2/0 myeloma cell, screening obtain can the stably excreting monoclonal antibody hybridoma cell strain.
Cell cryopreservation and recovery: hybridoma is made 1 * 10 with cryopreserving liquid
9The cell suspension of individual/ml is preserved in liquid nitrogen for a long time.Take out cryopreservation tube during recovery, put into immediately 37 ℃ of water-bath middling speeds and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
The present invention also provides a kind of method that detects salbutamolum residue in the sample with the salbutamol enzyme linked immunological kit, adopt this method that the salbutamol in animal tissue, feed, the urine specimen is carried out qualitative or quantitative detection, the Sample pretreatment process is simple, energy is fast detecting sample in enormous quantities simultaneously, kit has very high degree of accuracy and sensitivity, lower operative technique requires and of short duration detection time, detects the characteristics such as sample size is large, and key step comprises:
1) sample to be tested is carried out pre-treatment, obtain sample to be tested solution;
2) detect sample to be tested solution with enzyme linked immunological kit;
3) analyzing and testing result.
Description of drawings
Fig. 1: salbutamol haptens composite diagram
Fig. 2: salbutamol typical curve
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only are used for explanation the present invention, and be not used for limiting the scope of the invention.
Embodiment one: the preparation of antigen, antibody and enzyme labeling thing
1. the haptenic preparation of salbutamol
(1) gets the 1-3g salbutamol, dissolve among the 20-50ml DMF, then add the TEMPO of 10% molar equivalent and the tetrabutylammonium chloride of 10% molar equivalent, the NaHCO3 aqueous solution of 20-50ml 0.5M stirred under the room temperature after 30 minutes, added the NCS of 1.1-1.5 molar equivalent, after room temperature reaction 2-5 hour, remove most of solvent, ethyl acetate extraction, washing, dry, column chromatography purification after the desolventizing obtains white solid, is salbutamol list oxide.
(2) 1-2g step (1) gained salbutamol oxide, dissolve among the 20-30ml DMF, the ethylenediamine that adds the 2-5 molar equivalent, room temperature to 100 ℃ reaction 5-20 hour, desolventizing, recrystallization in the alcohol-water obtains white solid, is the ethylenediamine condensation product of salbutamol list oxide.
2. immunogenic preparation
(1) gets the salbutamol haptens 18mg water-soluble solution of 1.5ml.
(2) get 50% GA8 μ l and add in (1) stirring reaction 18h under the room temperature.
(3) getting BSA100mg adds in the mentioned solution with the dilution of 5.5ml water is rear.
(4) add 24mgNaBH behind the reaction 24h
4Reaction 2h.
(5) with tri-distilled water dialysis 48 hours, namely get immunogene.
3. the preparation of coating antigen
(1) gets the salbutamol haptens 15mg water-soluble solution of 1.5ml.
(2) get 50% GA8 μ l and add in (1) stirring reaction 18h under the room temperature.
(3) getting OVA30mg adds in the mentioned solution with the dilution of 4.5ml water is rear.
(4) add 24mgNaBH behind the reaction 24h
4Reaction 2h.
(5) with tri-distilled water dialysis 48 hours, namely get coating antigen.
4. the preparation of ELISA Plate
The described ELISA Plate carbonate buffer solution that used coated damping fluid is the 0.05mol/L of pH9.6 in preparation process, sealing damping fluid are 10% calf serum solution.
The preparation of ELISA Plate of the present invention is mainly: with coated damping fluid coating antigen is diluted to 0.2～0.3 μ g/ml, every hole adds 150 μ l, lucifuge is hatched 2h or 4 ℃ and is spent the night in 37 ℃ of environment, the liquid in the hole that inclines, cleansing solution washing 1～2 time pats dry, add 150 μ l confining liquids in every hole, 37 ℃ of lucifuges are hatched 2h, and liquid pats dry in the hole of inclining, and preserve with the vacuum seal of aluminium film.
5. the preparation of sheep anti mouse antiantibody
Take sheep as immune animal, take mouse source antibody as immunogen immune pathogen-free domestic sheep, obtain the sheep anti mouse antiantibody.
6. the preparation of enzyme labeling sheep anti mouse antiantibody (enzyme labeling antiantibody)
Adopt the sodium periodate method after the improvement to carry out coupling horseradish peroxidase and antiantibody, saved amino closed process, because can produce self amino amino reality that connects seldom; Horseradish peroxidase: the volumetric molar concentration ratio of antiantibody is 2: 1, and the method after the improvement is easier than traditional method, and the loss of the activity of enzyme is reduced.
7. the preparation method of monoclonal antibody
Animal immune: salbutamol haptens and carrier protein couplet thing immunity 8-10 Bal b/c mouse in age in week.
Fusion of Cells and cloning: get the mice spleen cell after the immunity, merge under the effect of fusion agent polyglycol (PEG) 4000 with SP2/0 myeloma cell, screening obtain can the stably excreting monoclonal antibody hybridoma cell strain.
Obtain the strain of salbutamol monoclonal hybridoma through screening, the generation salbutamol specific antibody that this hybridoma cell strain can be endless, this antibody specificity is for salbutamol, and sensitivity reaches 0.1 μ g/L.
Cell cryopreservation and recovery: hybridoma is made 1 * 10 with cryopreserving liquid
9The cell suspension of individual/ml is preserved in liquid nitrogen for a long time.Take out cryopreservation tube during recovery, put into immediately 37 ℃ of water-bath middling speeds and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
Embodiment two: the establishment of each component of salbutamol enzyme linked immunological kit
Set up the salbutamol enzyme linked immunological kit, comprise following each component:
(1) is coated with the ELISA Plate of coating antigen.
(2) enzyme labeling antiantibody: horseradish peroxidase-sheep anti mouse antiantibody.
(3) salbutamol monoclonal antibody working fluid.
(4) standard solution: adopt gradient dilution method preparation standard solution, obtain 6 bottles of series standard product, concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, and high concentration standard items 100 μ g/L, the 1mL/ bottle.
(5) substrate nitrite ion A liquid is urea peroxide solution, and substrate nitrite ion B liquid is the tetramethyl biphenyl amine aqueous solution.
(6) stop buffer is the sulfuric acid solution of 1～2mol/L.
(7) concentrated cleaning solution is the 0.15～0.2mol/LpH7.4 phosphate buffer that contains 1% Tween-20.
(8) the sample dilution is that pH is that the content of Tween-20 is that the formulated dilution of 0.5% mixed solution dilutes in the phosphate buffer of 7.4 0.02mol/L.
Embodiment three: detect residual salbutamol in the sample
1. the pre-treatment of sample
(1) tissue, liver pre-treating method
Take by weighing the equal pledge of 2.0+0.05g, add 2ml 3% trichloroacetic acid (take by weighing 15g trichloroacetic acid (solid), add 500ml deionized water dissolving mixing), whirling motion 5min, more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F).Get supernatant 500 μ l, add 500 μ l sample dilutions, fully mixing (pH value should between 7-9).Getting 50 μ l is used for analyzing.
(2) pre-treating method of urine sample
Get the limpid urine sample of 50ml directly measure (as urine sample is muddy must be by filtering or more than the 3000g, 15 ℃ of centrifugal 10min are until limpid), the sample that wouldn't use should freezing preservation.
(3) pre-treating method of feed
Take by weighing 1.0g feed sample, add 10ml methyl alcohol, whirling motion 5min, more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F); Pipette 1ml upper organic phase (being equivalent to the 0.1g sample) to the 10ml glass tube, flow down in 50～60 ℃ of nitrogen and dry up; Add the 1ml normal hexane, whirling motion 30s adds 1ml 3% trichloroacetic acid, whirling motion 1min again; More than the 3000g, the centrifugal 5min of room temperature (20-25 ℃/68-77 °F) removes upper organic phase, takes off layer liquid 150 μ l, adds 450 μ l sample dilutions, fully mixing (pH value should between 7-9).Getting 50 μ l is used for analyzing.
2. detection method
(1) prepares: before using all reagent are taken out from cold storage environment with lath with needing, make temperature recovery to room temperature (20-25 ℃), immediately all reagent be put back to 2-8 ℃ after using.
(2) add standard items/sample: add standard items/sample 50 μ l in the micropore of correspondence.
(3) mixing of antibody working fluid and enzyme labeling antiantibody concentrate: antibody working fluid and enzyme labeling antiantibody concentrate are mixed and mixing by 10: 1 volume ratios.(annotate: this mixed liquor can not be preserved, and carries out at once application of sample after mixing)
(4) add the mixed liquor of antibody working fluid and enzyme labeling antiantibody concentrate: add the mixed liquor 50 μ l/ holes of antibody working fluid and enzyme labeling antiantibody concentrate, the mixing that vibrates gently is with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate.
(5) wash plate: carefully open the cover plate film, liquid in the hole is dried, with wash operating solution (20 * concentrated cleaning solution being diluted by 1: 19 volume ratio with deionized water) 250 μ l/ holes, fully washing is 4-5 time, every minor tick 10s pats dry (bubble that is not eliminated after patting dry can be poked with original rifle head) with thieving paper.
(6) colour developing: add substrate solution A liquid 50 μ l/ holes, add substrate solution B liquid 50 μ l/ holes again, the mixing that vibrates gently with the 15min that develops the color in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate, adds stop buffer 50 μ l/ holes, and mixing gently vibrates.
(7) measure: microplate reader is measured the absorbance in every hole, sets microplate reader in the 450nm place (suggestion detects with dual wavelength 450/630nm, runs through data in 5min).
3. Analysis of test results
The percentage absorptance of standard items or sample equals the mean value (diplopore) of the absorbance of standard items or sample divided by the absorbance of first standard (0 standard), multiply by 100% again, namely obtains the percentage absorptance.Take salbutamol standard items percentage absorptance as ordinate, take the logarithm of salbutamol standard items concentration (μ g/L) as horizontal ordinate, drawing standard curve map (such as Fig. 2).In the percentage absorptance substitution typical curve with sample, read the corresponding concentration of sample from typical curve, multiply by its corresponding extension rate and be salbutamol actual concentrations in the sample.
Embodiment four: the sensitivity of salbutamol kit, specificity, preci-sion and accuracy, storage life experiment
1. kit sensitivity determination
Measure the kit sensitivity test according to conventional method, kit typical curve minimum point is 0.1 μ g/L, and the scope of typical curve is 0.1 μ g/L～8.1 μ g/L, and salbutamol detects and is limited to 0.5 μ g/kg in tissue, detect in the urine and be limited to 0.3 μ g/L, detect in the feed and be limited to 5 μ g/kg.
2. kit accuracy and precision
Accuracy refers to the matching degree between measured value and true value, and the accuracy of the kit recovery commonly used represents; Precision is the reaction assay method is repeatedly measured acquired results to a certain specific sample repetition degree, and the coefficient of variation commonly used represents.Adding salbutamol to final concentration in blank pork, pork liver, pig urine, the feed respectively is 2 μ g/kg, 2 μ g/L, and 2 μ g/L, 10 μ g/kg repeat 5 times, get respectively three batches the kit calculating coefficient of variation, the results are shown in following table.
The accuracy of table 1 kit and precision are measured
The result shows, being added into final concentration in the pork is 2 μ g/kg, recovery scope is 77.5%～102.5%, being added into final concentration in the pork liver is 2 μ g/kg, recovery scope is 73.0%～101.0%, being added into final concentration in the pig urine is 2 μ g/kg, recovery scope is 61.5%～80.0%, being added into final concentration in the feed is 10 μ g/kg, recovery scope is 75.5%～100.5%, interassay coefficient of variation meets " Ministry of Agriculture's file " farming doctor [2005] No. 17 annex 2 kits and puts on record with reference to the regulation of the 4th preci-sion and accuracy in the judgment criteria less than 20% in batch.
3. cross reacting rate test
Select salbutamol as follows, clenbuterol, 3 kinds of medicines of Ractopamine to measure respectively cross reacting rate according to conventional method, the result is as shown in table 2.
The specificity of table 2 kit
4. storage life experiment
The kit preservation condition is 2-8 ℃, and through 12 months mensuration, the maximum absorbance value of kit, IC50 value, salbutamol added the practical measurement value all within normal range.Do simultaneously accelerated deterioration and refrigeration test, kit is placed in 37 ℃ ,-20 ℃ 6 days, measurement result shows that also the indices of kit is normal.Obtaining the salbutamol kit from above result can preserve 12 months at 2-8 ℃.
Claims (10)
1. salbutamol ELISA diagnostic kit, it comprises ELISA Plate, salbutamol specific antibody, enzyme labeling thing, salbutamol standard solution, substrate nitrite ion, stop buffer, concentrated cleaning solution, the sample dilution that is coated with coating antigen, described coating antigen is salbutamol medicine antigen, and described enzyme labeling thing is the enzyme labeling antiantibody.
2. kit as claimed in claim 1, it is characterized in that: described salbutamol medicine antigen is the conjugate of salbutamol haptens and carrier protein, and described carrier protein is bovine serum albumin(BSA), mouse haemocyanin, rabbit anteserum albumen, thyroprotein, ovalbumin, hemocyanin or human serum albumins.
3. such as the described kit of claim 1-2, it is characterized in that: described salbutamol is haptenic preparation method mainly comprise the steps:
(1) gets the 1-3g salbutamol, dissolve among the 20-50ml DMF, then add the TEMPO of 10% molar equivalent and the tetrabutylammonium chloride of 10% molar equivalent, the NaHCO of 20-50ml 0.5M
3Aqueous solution stirred under the room temperature after 30 minutes, added the NCS of 1.1-1.5 molar equivalent, after room temperature reaction 2-5 hour, remove most of solvent, ethyl acetate extraction, washing, drying, column chromatography purification after the desolventizing obtains white solid, is salbutamol list oxide.
(2) 1-2g step (1) gained salbutamol oxide, dissolve among the 20-30ml DMF, the ethylenediamine that adds the 2-5 molar equivalent, room temperature to 100 ℃ reaction 5-20 hour, desolventizing, recrystallization in the alcohol-water obtains white solid, is the ethylenediamine condensation product of salbutamol list oxide.
4. kit as claimed in claim 1, it is characterized in that: described salbutamol specific antibody is the salbutamol anti-drug monoclonal antibody.
5. kit as claimed in claim 1, it is characterized in that: described antiantibody is the sheep anti mouse antiantibody.
6. kit as claimed in claim 1, the marker enzyme that it is characterized in that described enzyme labeling thing is horseradish peroxidase; The enzyme labeling antiantibody is to adopt the sodium periodate method that marker enzyme and antiantibody coupling are obtained.
7. kit as claimed in claim 1, it is characterized in that: described substrate solution A liquid is urea peroxide solution, and substrate solution B liquid is the tetramethyl biphenyl amine aqueous solution, and described stop buffer is the sulfuric acid solution of 1～2mol/L.
8. kit as claimed in claim 1, it is characterized in that: described concentrated cleaning solution is the 0.15～0.2mol/LpH7.4 phosphate buffer that contains 1% Tween-20, and described sample dilution is that pH is that the content of Tween-20 is that the formulated dilution of 0.5% mixed solution dilutes in the phosphate buffer of 7.4 0.02mol/L.
9. kit as claimed in claim 1, it is characterized in that: described standard solution concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L.
10. detect the method for salbutamolum residue in the sample such as the described salbutamol enzyme linked immunological kit of claim 1-9, key step comprises:
1) sample to be tested is carried out pre-treatment, obtain sample to be tested solution;
2) detect sample to be tested solution with each described enzyme linked immunological kit of claim 1-9;
3) analyzing and testing result.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105301244A (en) * | 2014-06-03 | 2016-02-03 | 北京勤邦生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting acid orange and application of enzyme linked immunosorbent assay kit |
| CN107561269A (en) * | 2017-08-23 | 2018-01-09 | 太原瑞盛生物科技有限公司 | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of salbutamol |
| CN110004117A (en) * | 2019-04-11 | 2019-07-12 | 中抗生物医药(杭州)有限公司 | Secrete hybridoma cell strain, monoclonal antibody and the application of salbutamol monoclonal antibody |
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