CN102432684A - Preparation method and application of clenbuterol monoclonal antibody - Google Patents
Preparation method and application of clenbuterol monoclonal antibody Download PDFInfo
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- CN102432684A CN102432684A CN2011102587702A CN201110258770A CN102432684A CN 102432684 A CN102432684 A CN 102432684A CN 2011102587702 A CN2011102587702 A CN 2011102587702A CN 201110258770 A CN201110258770 A CN 201110258770A CN 102432684 A CN102432684 A CN 102432684A
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Abstract
The invention discloses a preparation method and an application of a specific monoclonal antibody. The invention relates to the technical field of detection. According to the invention, a conjugate of clenobuterol hydrochloride and bovine serum albumin is used and an immunogen for immunizing mice; hybridoma cells are obtained by using a cell fusion technology, and a specific clenbuterol monoclonal antibody is prepared; and the prepared clenbuterol monoclonal antibody is adopted as a core reagent for preparing a colloidal gold immunization test strip used for detecting clenbuterol. According to the invention, the prepared clenbuterol antibody is a high-specificity monoclonal antibody with characteristics of high specificity, high sensitivity, high accuracy, and the like. The test strip prepared with the high-specificity monoclonal antibody as a basis is used for detecting clenbuterol residue, and has characteristics of simple, fast, sensitive, accurate, and cheap. The application provided by the invention has a good prospect.
Description
Technical field:
The present invention relates to a kind of preparation of monoclonal antibody specific of clenbuterol, and be used for rapid determination, belong to the detection technique field feed and animal food clenbuterol.
Background technology:
Clenbuterol is NAB-365Cl again, belongs to receptor, agonist class (abbreviation beta-2-agonists) medicine, has the growth of the animal muscle of promotion; Reduce the trunk lipid content; Improving efficiency of feed utilization, significantly improve the effect of lean ratio, is one of the most practical beta-2-agonists.Good absorption in clenbuterol Domestic Animal and the human body, and compare with other beta-stimulants, its bioavailability is high, occurs poisoning so that eaten the pork that contains clenbuterol.As far back as the just ban of promulgation forbidding beta-2-agonists of Europe in 1986.China Ministry of Agriculture and State Administration of Quality Supervision, Inspection and Quarantine also completely forbid the use of such material.NAB-365Cl had once caused hundreds of people's poisoning in Shanghai.And,, almost provoke a political controversial issue owing in the pork of U.S.'s import, contain NAB-365Cl in Taiwan.
Hit the illegal dynamics of using NAB-365Cl though strengthened in recent years, under the ordering about of economic interests, still have a lot of illegal situation of NAB-365Cl of using to take place.In order to hit illegal use forbidden drug, except perfecting laws and regulations, set up outside the effective supervision and management system, also need perfect corresponding detecting method.The applicant finds through retrieval; The specificity of the clenbuterol monoclonal antibody that present domestic report is prepared is not strong; Be in the application of the prepared colloidal gold strip of core with the monoclonal antibody; Do not see the treatment process of different test sample and the report of minimal detectable concentration, and test strip false positive rate in the market is higher.Therefore the detection of clenbuterol can not satisfy far away fast, cheap, stablize and the requirement of aspect such as accurate.
Summary of the invention:
The object of the present invention is to provide a kind of clenbuterol MONOCLONAL ANTIBODIES SPECIFIC FOR method and the application on the detection clenbuterol thereof.
For realizing the object of the invention, technical scheme realizes through following mode:
A kind of clenbuterol MONOCLONAL ANTIBODIES SPECIFIC FOR method; It is characterized in that: the conjugate with Clenbuterol hydrochloride and bovine serum albumin is the immunogen immune mouse; Obtain hybridoma through cell-fusion techniques again, induce ascites and prepare the clenbuterol monoclonal antibody.
Said immunogen is through diazonium method synthetic.
A kind of application of clenbuterol monoclonal antibody is characterized in that: with the clenbuterol monoclonal antibody is the colloid gold immune test paper bar that the core reagent preparation detects clenbuterol.
Said colloid gold immune test paper bar is made up of PVC base plate, sample pad, Radioactive colloidal gold pad, nitrocellulose filter and several parts of absorbent pad; Wherein the Radioactive colloidal gold pad is coated with the colloid gold label thing of clenbuterol, is coated with the detection line (T) of clenbuterol-carrier protein couplet thing formation on the nitrocellulose filter and encapsulates the nature controlling line (C) that sheep anti-mouse igg constitutes.
Said colloid gold immune test paper bar, the minimum concentration that detects clenbuterol in the feed is 0.005mgkg
-1, the minimum concentration of clenbuterol is 1.6ngg in animal muscle and the liver
-1, the minimum concentration of clenbuterol is 1.5ngmL in the pig urine
-1The main foundation of as a result of judging with wire (C) and two chromogenic lines of detection line (T) during detection.
1. the preparation of artificial immunization antigen and envelope antigen
Take by weighing accurately quantitative Clenbuterol hydrochloride be dissolved in an amount of HCl while shaking to wherein slowly adding NaNO
2, and constantly dip in micropipet and get a little and starch potassium iodide paper and detect, just become bluish voilet up to the test paper color, stop to add NaNO
2, the continuation concussion reacts fully and has just obtained diazotizing clenbuterol solution, and diazonium compound is slowly added in bovine serum albumin and the ovalbumin respectively, shakes reaction.4 ℃ of refrigerators were dialysed 3 days with PBS in 4 ℃ of refrigerators after continuing to place 6h continuously.
2. clenbuterol Monoclonal Antibody and evaluation
(1) animal immune program: with 1100 μ gmL
-1The PBS solution of CL-BSA (0.22 μ m membrane filtration degerming) is mixed into emulsion with the equal-volume Freund's complete adjuvant, subcutaneous branch injection female BALB/C mice in healthy 6 ages in week, every 0.4mL (containing CL-BSAl00 μ g); Head exempts from the same dosage booster immunization in 2 week backs, and adjuvant is a Freund's incomplete adjuvant, exempts from altogether 3 times; Each 2 weeks at interval; Indirect elisa method monitoring serum antibody titer, the highest mouse of selecting to tire carries out cytogamy, merges first three sky and with CL-BSA mouse is carried out the booster immunization injection.
(2) cytogamy and cloning: splenocyte of the mouse of the booster immunization of learning from else's experience and myeloma cell carry out cytogamy, obtain the cell strain of monoclonal antibody 5G8 of stably excreting anti-clenbuterol through subclone repeatedly.
(3) Monoclonal Antibody, purifying and evaluation: get the BALB/c mouse in 8～10 ages in week, with sterilization paraffin mouse is carried out immunity, pneumoretroperitoneum was injected said cell strain of monoclonal antibody 5G8 in 10 days, and injection volume is 5 * 10
5Individual/as only, to treat that mouse web portion obviously expands, gather ascites, to the ascites purifying, promptly get the clenbuterol monoclonal antibody with sad-ammonium sulfate salting-out process and Protein A immunochromatographic method.
Identify that through hypotype the hypotype that draws monoclonal antibody of the present invention is the IgG1 type.Adopt indirect competitive ELISA measure antibodies specific and with the cross reactivity of similar other compound.
3. the development of clenbuterol colloid gold immune test paper bar
For the core reagent preparation detects clenbuterol colloid gold immune test paper bar, confirm the prescription and the production technique of all ingredients in the immunity colloidal gold test paper strip with the monoclonal antibody, the test strip performance is identified, and confirmed the pre-treating process of test sample.
The invention has the beneficial effects as follows: the clenbuterol monoclonal antibody that produces high specificity through mouse hybridoma cell pearl 5G8; Low with other type medicine cross reacting rate; Can mass production, can be used for preparing the colloidal gold strip that detects clenbuterol, have the characteristics of highly sensitive, high specificity and easy and simple to handle and good stability; Be suitable for the screening to a large amount of samples, application prospect is very wide.
Description of drawings:
Fig. 1 is clenbuterol monoclonal antibody SDS-PAGE electrophoretogram behind the purifying of the present invention;
Fig. 2 is the response curve diagrammatic sketch of monoclonal antibody of the present invention and clenbuterol standard substance;
Fig. 3 is the susceptibility diagrammatic sketch of colloidal gold strip of the present invention.
Embodiment:
For the present invention is elaborated, further specify below in conjunction with specific embodiment,
Synthesizing of embodiment 1 artificial immunization antigen and envelope antigen
The preparation of CL-BSA, CL-OVA (be reflected under 2～4 ℃ of conditions and carry out).
Accurately take by weighing CL 3.295mg (about 0.01mmol) and be dissolved in 400ul 0.1M HCl (0.04mmol), while shaking to the NaNO that wherein slowly adds 10mg/ml
2(about 0.01mmol), and constantly dip in micropipet and get a little and starch potassium iodide paper and detect, just become bluish voilet up to the test paper color, stop to add NaNO
2Continuation concussion 35min reacts fully and has just obtained diazotizing clenbuterol solution.The diazotization product is slowly added the protein solution (BSA and OVA4ml) that dissolves with 8 * PBS (PH8.6), and BSA5.0mg (0.08 * 10
-3Mmol); OVA5.0mg (0.11 * 10
-3Mmol).Making diazotizing clenbuterol and proteic molecular amounts ratio is about 50: 1.Inspection pH value, pH value 8.0 still more than, continue to shake and react 45min.After 4 ℃ of refrigerators continued to place 6h, with PBS (0.01mol/L), pH value 7.4 was dialysed 3 days continuously, changed liquid 8 times in 4 ℃ of refrigerators.
Diazonium salt generate azo cpd, and azo-group is a chromophoric group with a kind of aromatic amine or phenol chemical combination the time, the colour-change the when Clenbuterol hydrochloride behind the observation azo splashes into albumen confirms tentatively whether coupling is successful.
Calculated at last by the ultraviolet full wavelength scanner: CL and BSA coupling ratio were at 35: 1; CL and OVA coupling ratio were at 46: 1.
CL-BSA by the Gene5 detection system is 1.1mg/ml; CL-OVA concentration is 1.6mg/ml.
Embodiment 2 MONOCLONAL ANTIBODIES SPECIFIC FOR
With 1100 μ gmL
-1The PBS solution of CL-BSA (0.22 μ m membrane filtration degerming) is mixed into emulsion with the equal-volume Freund's complete adjuvant; 5 of subcutaneous branch injection female BALB/C mices in healthy 6 ages in week; Every 0.4mL (containing CL-BSA 100 μ g), head exempt from the same dosage booster immunization in 3 week of back back, and adjuvant is a Freund's complete adjuvant; Exempt from each 2 weeks at interval altogether 3 times.
Three detect mouse after exempting from antibody titer and blocking-up situation.
The square formation method confirms that ELISA detects antigen the best and encapsulates concentration, is 0.8 μ g/ml through testing the concentration that encapsulates of confirming envelope antigen CL-OVA; Negative control concentration is 1: 2560.
Detect the serum titer of 5 mouse again by indirect elisa method,
The CL-BSA mice serum to tire at 1: 10240 be 2,3, No. 4 mouse more than the extent of dilution, 1, No. 5 mouse serum is tired at 1: 20480 extent of dilution.
By above result's judgement No. 5 mouse are impacted immunity.
Get No. 5 mouse boosting cells and myeloma cell SP2/0 and merge, add the HAT substratum, 37 ℃, 6%CO
2Cultivate in the incubator.After 5 days with the fresh HAT substratum half the substratum that swaps out, after 8-10 days with the HT substratum HAT substratum that swaps out.Observe the growing state of hybridoma every day; Treat that it is distributed to hole floorage 2/5 when above; Get supernatant and adopt indirect ELISA screening specific antibody, carry out subclone 2-3 time with limiting dilution assay again, detect cells and supernatant; There is the hole positive rate of cell to reach 100%, obtains the hybridoma cell strain 5G8 of stably excreting monoclonal antibody.
Get 10 of the BALB/c mouses in 8～10 ages in week, with the paraffin of sterilizing mouse is carried out immunity, pneumoretroperitoneum was injected said cell strain of monoclonal antibody 5G8 in 10 days, and injection volume is 5 * 10
5Individual/0.5mL/ only treats that mouse web portion obviously expands, and gathers ascites, promptly contains a large amount of monoclonal antibodies in the ascites.
The method of indirect ELISA method that adopts in the above step and square formation experiment is carried out with reference to the method in fine works immunological experiment guide and Linchuan immunological technique.
Embodiment 3 Purification of Monoclonal Antibodies and evaluation thereof
1. antibody purification
Ascites adopts sad-ammonium sulfate salting-out process and ProteinA immunochromatographic method to carry out purifying, with SDS-PAGE electrophoresis purification Identification thing, the heavy chain of antibody band of 51KD and the light chain of antibody band (Fig. 1) of 26KD occur.
2. the foundation of TPPA method
The detection 2.1 monoclonal antibody 5G8 tires
By square formation test the concentration of coating antigen at 0.79ug, the antibody titer in this hole is 1: 20480 times of dilution.
2.2 monoclonal antibody 5G8 specific assay
Homologue salbutamol, Clenbuterol hydrochloride are mixed with 1000ng/ml, 100ng/ml, 50ng/ml, 10ng/ml, 5ng/ml, six concentration of 1ng/ml; Do the indirect competitive ELISA test with 5G8 antibody, judge the no cross reaction that has of antibody and homologue salbutamol, Ractopamine hydrochloride, sympathin and Racemic isoproterenol.(result such as table 1)
Table 1 monoclonal antibody 5G8 specific assay result
| The competition thing | IC50(ng/ml) | Cross reacting rate (%) |
| Clenbuterol | 1.2 | 100 |
| Ractopamine hydrochloride | ＞1000 | ＜0.01 |
| Salbutamol | ＞1000 | ＜0.01 |
| Sympathin | ＞1000 | ＜0.01 |
| Racemic isoproterenol | ＞1000 | ＜0.01 |
2.3 monoclonal antibody 5G8 measures the reaction sensitivity of CL
Adopt the ciELISA method to measure the blocking-up rate of antibody purified: with PBS dissolving CL standard substance; Redilution becomes a series of concentration gradients (100ng/ml, 10ng/ml, 5ng/ml, 1ng/ml, 0.50ng/ml), and every kind of concentration standard article 50 μ l add working concentration 5G8 antibody 50 μ l respectively, and 0 standard orifice and blank hole are set simultaneously; PBST washing three times (each 5min) is clapped and is done behind 37 ℃ of water-bath effect 30min; The sheep anti-mouse igg that adds the HRP mark of dilution in 1: 10000,100 μ l/ holes, the same washing is clapped and is done behind 37 ℃ of water-bath effect 30min; Add tmb substrate colour developing liquid 100 μ l/ holes, 37 ℃ of water-bath lucifuge reaction 10-15min.At last, every hole adds 2M H
2SO
4After the 50 μ l termination reactions, read OD
450Value.
The blocking-up rate is that 50% o'clock CL mass concentration is small molecules antigen amount one timing of IC50 in adding, and the blocking-up rate is high more, explains that the antigenic ability of antibody capture small molecules is strong more, and monoclonal antibody is high more to the antigenic sensitivity of small molecules.Otherwise the blocking-up rate is low more, explain that the antigenic ability of antibody capture small molecules more a little less than, monoclonal antibody sensitivity is low more.
The result can read the IC of monoclonal antibody 5G8 to CL from Fig. 2
50Be about 1.2ng/ml.
3. the somatotype of monoclonal antibody is identified
Getting cell conditioned medium by muroid monoclonal antibody subgroup identification test kit specification sheets operates.Contrast the different subclass two colour developing result under anti-, with OD
450Value is judged subclass under this cell strain excretory monoclonal antibody apparently higher than other hole person, and the subclass of monoclonal antibody 5G8 is IgG1 (result sees table 2).
| Antibody subtype | 5G8 |
| IgG1 | 0.985 |
| IgG2a | 0.028 |
| IgG2b | 0.034 |
| IgG3 | 0.057 |
| IgA | 0.041 |
| Blank | 0.065 |
The preparation and the application of embodiment 4 clenbuterol quick detection test paper bars
1. Preparation of Colloidal Gold
Adopt the trisodium citrate improved method to prepare Radioactive colloidal gold, prepare 1% chlorauric acid solution (the 1g hydrochloro-auric acid is dissolved in the 100mL distilled water).Get 3mL 1% chlorauric acid solution and add in the round-bottomed flask that the 297mL ultrapure water is housed, boil 2min, add 1% citric acid three sodium solution 8mL with the electric heating oven heated.When the color of solution becomes transparent wine red, to continue to stir and boil 10min, the cooling back is settled to 300mL with distilled water, and is subsequent use with 4 ℃ of storages behind the 0.22um filtering with microporous membrane.
2. the preparation of golden labeling antibody
Get the 10mL colloidal gold solution, use 0.2M K
2CO
3The pH value that solution is regulated colloidal gold solution is 8.2, slowly adds the clenbuterol monoclonal antibody of purifying, and the concentration that makes monoclonal antibody is 0.6 μ gmL
-1, continue to stir 30min; Adding 1%PEG is 0.1% to final concentration, and 30min is stirred in continuation, and adding 10%BSA solution to final concentration is 1%, continues to stir 30min; 4 ℃, the centrifugal 10min of 1000r/min get supernatant; 4 ℃, the centrifugal 30rain of 5000r/min, the careful suction removed supernatant, and deposition is with damping fluid (25% sucrose, 1.0%BSA, 0.1%PEG, 0.5%Tween20,0.1%NaN
30.01molL
-1PBS) resuspended.
3. the preparation of colloidal gold immune chromatography test
Leached glass fiber is placed 7% sucrose, 25 ℃ of dry 2h.The clenbuterol monoclonal antibody 5G8 point of colloid gold label is sprayed on the spun glass with 16 μ L/cm with the three-dimensional specking plateform system of BioDotXYZ3000 test strip, 25 ℃ of dry 2h are glue gold pad.With the three-dimensional specking plateform system of BioDotXYZ3000 test strip with 0.891 μ Lcm
-1With clenbuterol-OVA coupled antigen (0.3mgmL
-1) point be sprayed on the nitrocellulose filter, be detection line; Spraying sheep anti-mouse igg (0.5mgmL from detection line 4mm place
-1), be nature controlling line, 25 ℃ of dry 2h.Take out a PVC plate, the nitrocellulose filter that is sprayed with detection line and nature controlling line is sticked on central area; Respectively thieving paper and glue gold are filled up the above and below that is pasted on nitrocellulose filter, overlapping about 2mm between the two; The sticking glass fiber is assembled into colloidal gold immune chromatography test as sample pad below glue gold pad again.
4. detection method
Test strip is put on the clean smooth table top, draws testing sample solution, drip 1～2 on sample pad, wait for the appearance of red-purple band with dropper, read test result in the time of standing and reacting 5-10 minute, judgement later in 10 minutes is invalid.The result is judged as C line (nature controlling line) colour developing, and T line (detection line) does not develop the color positive: C, T line all develop the color negative; C, T line all do not develop the color and explain that then test strip is invalid.
5. the sensitivity of test strip and specificity
The preparation final concentration is 0,1,1.1,1.2,1.4,5,10,25,50ngmL
-1Clenbuterol solution and 100ngmL
-1Ractopamine hydrochloride, salbutamol solution.Get the sample pad of the above-mentioned solution adding of 50 μ L colloidal gold immune chromatography test respectively, judged result is 10min, and each concentration repeats 5 times.The result shows that colloidal gold immune chromatography experiment is to 1ngmL
-1Following clenbuterol solution detects negative, to 1.2ngmL
-1And the solution test positive of above clenbuterol, 100ngmL
-1Ractopamine hydrochloride and husky butanolamine solution detect negatively, show that colloidal gold immune chromatography experiment has good sensitivity and specificity, the result is as shown in Figure 3.
Test strip batch in and batch between repeated experiments
Will with batch detect 0,1,2,5,10,50 respectively with the test strip of different batches, 100ngmL
-1Clenbuterol solution standard substance, each concentration repeats 3 times, observes its repeatability.Empirical tests clenbuterol quick detection test paper bar batch in and batch between repeatability be 100%, false positive rate and false negative rate are 0.
8. the stable experiment of test strip
Packaged test strip is stored in 4 ℃ and 60 ℃ respectively, observes its stability.Be stored in 4 ℃ test strip every at a distance from 10 of taking-ups in 7 days detect 0,3,5,10,20,50 respectively, 100ngmL
-1The clenbuterol standard serial solution.The test strip that is stored in 60 ℃ is taken out 10 every day, detects 0,1,2,5,10,50 respectively, 100ngmL
-1The clenbuterol standard serial solution.At a distance from 7 days check in, the colour developing degree does not have obvious change to the test strip of 4 ℃ of preservations every, and the golden labeling antibody of gold mark pad release is more complete.A kind liquid transition time slows down, gold mark pad discharges phenomenons such as golden labeling antibody is incomplete, sensitivity reduction and the test strip of 60 ℃ of preservations occurred when detecting in the 15th day.Stablized for two weeks according to test strip at 60 ℃, be equivalent to stablize under the room temperature 1 year, can calculate that this test strip can preserve 12 months at normal temperatures at least.
9. sample treatment and lowest detection line are measured
(1) feed sample
Get 3g feed sample and add 9ml extracting solution (methyl alcohol and PBS damping fluid were by 4: 1 mixed), mixing is got supernatant and is added 450 μ lPBS damping fluids with the centrifugal 10min of the rotating speed of 4000rpm, gets 50 μ l samples behind the mixing and detects with colloidal gold immune chromatography experiment.In the feed sample of the feminine gender of confirming, add the clenbuterol of different concns during simulated determination respectively, draw that the clenbuterol minimal detectable concentration is 0.005mgkg in the feed sample
-1
(2) animal tissues's sample
Take by weighing sample 5g and place centrifuge tube, add acetonitrile 10mL, vibration 10min, the centrifugal 10min of 4000r/min; Get supernatant 3mL, nitrogen dries up under 50 ℃ of water-baths; Add normal hexane 1mL, whirling motion 30s; Add sample buffering working fluid 1mL again, whirling motion 1min, the centrifugal 5min of 4000r/min, muscle tissue is got subnatant 100 μ L and is mixed with sample buffering working fluid 100 μ L; Hepatic tissue is got subnatant 50 μ L and is mixed with sample buffering working fluid 150 μ L, gets 50 μ L and detects with colloidal gold immune chromatography experiment.In animal tissues's sample of the feminine gender of confirming, add the clenbuterol of different concns during simulated determination respectively, draw that the clenbuterol minimal detectable concentration is 1.6ngg in animal tissues's sample
-1
(3) urine or serum sample
Urine or blood can directly be tested, if urine is muddy, then need get supernatant again and test with the centrifugal 5min of 3000rpm.In the urine of the feminine gender of confirming, add the clenbuterol of different concns during simulated determination respectively, draw that the clenbuterol minimal detectable concentration is 1.5ngmL in the urine sample
-1
More than show and described ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; The present invention is not restricted to the described embodiments; That describes in the foregoing description and the specification sheets is merely preference of the present invention, is not used for limiting the present invention, under the prerequisite that does not break away from spirit and scope of the invention; The present invention also has various changes and modifications, and these variations and improvement all fall in the scope of the invention that requires protection.The present invention requires protection domain to be defined by appending claims and equivalent thereof.
Claims (4)
1. clenbuterol MONOCLONAL ANTIBODIES SPECIFIC FOR method; It is characterized in that: the conjugate with Clenbuterol hydrochloride and bovine serum albumin is the immunogen immune mouse; Obtain hybridoma through cell-fusion techniques again, induce ascites and prepare the clenbuterol monoclonal antibody.
2. clenbuterol MONOCLONAL ANTIBODIES SPECIFIC FOR method according to claim 1 is characterized in that: said immunogen is through diazonium method synthetic.
3. the application of a clenbuterol monoclonal antibody of preparing with the described preparation method of claim 1 is characterized in that: with the clenbuterol monoclonal antibody is the colloid gold immune test paper bar that the core reagent preparation detects clenbuterol.
4. the application of clenbuterol monoclonal antibody according to claim 3; It is characterized in that: said colloid gold immune test paper bar is made up of PVC base plate, sample pad, Radioactive colloidal gold pad, nitrocellulose filter and several parts of absorbent pad; Wherein the Radioactive colloidal gold pad is coated with the colloid gold label thing of clenbuterol, is coated with the detection line (T) of clenbuterol-carrier protein couplet thing formation on the nitrocellulose filter and encapsulates the nature controlling line (C) that sheep anti-mouse igg constitutes.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103361741A (en) * | 2013-07-04 | 2013-10-23 | 潍坊科技学院 | Phage antibody library and application thereof in content determination of clenbuterol hydrochloride |
| CN104678093A (en) * | 2015-03-20 | 2015-06-03 | 上海理工大学 | Preparation method of immobilized anti-clenbuterol hydrochloride monoclonal antibody |
| CN107015010A (en) * | 2017-06-09 | 2017-08-04 | 深圳大学 | It is a kind of for kit of testosterone residue detection and preparation method thereof and detection method |
| CN111220802A (en) * | 2020-01-19 | 2020-06-02 | 新乡医学院 | Clenbuterol hydrochloride small molecule hapten high-sensitivity detection test paper based on nano antibody and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361741A (en) * | 2013-07-04 | 2013-10-23 | 潍坊科技学院 | Phage antibody library and application thereof in content determination of clenbuterol hydrochloride |
| CN104678093A (en) * | 2015-03-20 | 2015-06-03 | 上海理工大学 | Preparation method of immobilized anti-clenbuterol hydrochloride monoclonal antibody |
| CN107015010A (en) * | 2017-06-09 | 2017-08-04 | 深圳大学 | It is a kind of for kit of testosterone residue detection and preparation method thereof and detection method |
| CN111220802A (en) * | 2020-01-19 | 2020-06-02 | 新乡医学院 | Clenbuterol hydrochloride small molecule hapten high-sensitivity detection test paper based on nano antibody and preparation method thereof |
| CN111220802B (en) * | 2020-01-19 | 2023-05-02 | 新乡医学院 | Clenbuterol hydrochloride small molecule hapten high sensitivity detection test paper based on nano antibody and preparation method thereof |
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Application publication date: 20120502 |