CN105017180A - Preparation method of ractopamine haptens and antigens and application of ractopamine haptens and antigens in chemiluminescence immunoassay kit - Google Patents
Preparation method of ractopamine haptens and antigens and application of ractopamine haptens and antigens in chemiluminescence immunoassay kit Download PDFInfo
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Abstract
The invention discloses a preparation method of ractopamine haptens and antigens and a special chemiluminescence immunoassay kit of the ractopamine haptens and the antigens. The haptens are compounds shown in a formula (I). The invention further relates to a conjugate (which is an immunogen or a coating antigen) of the compounds shown in the formula (I) and carrier protein. The invention further relates to the chemiluminescence immunoassay kit used for detecting ractopamine. The chemiluminescence immunoassay kit comprises a chemiluminescence micro-hole plate coated with the conjugates (which serve as coating antigens). The kit can also comprise antibodies obtained with the conjugates being immunogens. The kit is easy and convenient to operate, high in specificity, high in sensitivity, excellent in various performance and very suitable for being applied and popularized. (Please see the formula in the specification).
Description
Technical field
The present invention relates to a kind of Ractopamine hydrochloride haptens and antigen and chemiluminescence immunoassay kit special thereof.
Background technology
Ractopamine hydrochloride is a kind of medical material, has physiological effect widely, the cardiac tonic of available treatment congestive heart failure disease, and is usually used in the treatment of bronchial asthma, bronchospasm and obstetric conditions.When its consumption is increased to the 5-10 times of quantity, can increase muscle growth, reducing lipopexia, is good nutrient distribution agent and growth stimulant.U.S. FDA, approval in 2000, may be used for Animal nutrition ingredients again, is widely used for livestock industry and aquaculture.The day weight gain of animal can be improved simultaneously, improve efficiency of feed utilization, improve the protein content of animal.But its in animal body residual once enter human body through food chain, significant damage can be produced to eater, larger to patient's harm such as heart trouble, diabetes, hypertension, hyperthyroidism, glaucoma, prostatomegaly especially, even dead, as clenbuterol hydrochloride (Ractopamine hydrochloride is the one in " clenbuterol hydrochloride medicine ") once caused the poisoning of hundreds of people in Shanghai; In Taiwan owing to containing clenbuterol hydrochloride in the pork of imported from America, almost provoke a political controversial issue.Current China forbids the medicines such as beta-2-agonists to use as Animal feed-additive.But for a long time, the various poisoning caused because of illegal use beta-2-agonists happens occasionally.For hitting unsanctioned use, the health and safety of Protection of consumer, in the urgent need to sound relevant detection method.
At present, the method that detection of veterinary drugs in food is conventional has the physico-chemical analysis methods such as gas-chromatography, high performance liquid chromatography and gas chromatography mass spectrometry.Although these method high specificities, highly sensitive, sample pre-treatments complex operation step, cost is higher, is not also suitable for the selective mechanisms of batch samples.Immunochemical analyses in view of in the qualitative, quantitative of antigen-antibody uniqueness advantage and easy and simple to handle fast, cost is low, sensitivity is higher, analyzing samples amount is large advantage compensate for the deficiency of physico-chemical analysis, plays a part more and more important in the residue detection of Ractopamine hydrochloride.
The basic factor affecting immunochemical analyses quality is specificity and the affinity of antibody, these character are decided by again the structure of immune hapten molecule, and therefore immune haptenic molecular designing and synthesis are the steps of the most basic and most critical producing specific antibody and set up small molecules residue of veterinary drug Fast Detection Technique.
Summary of the invention
The object of this invention is to provide a kind of Ractopamine hydrochloride haptens and antigen and chemiluminescence immunoassay kit special thereof.
Haptens provided by the invention is the compound shown in formula I;
formula I.
Compound shown in formula I specifically can be the compound prepared as follows: (1) is got 700mg Ractopamine hydrochloride and is dissolved in 50ml ethanol, add 258mg epoxy chloropropane, 963mg salt of wormwood mixes, stirring at room temperature reaction 90h, cross and filter insolubles, filtrate is carried out vacuum-drying, and the dry-matter obtained is the compound shown in formula I.
The present invention also protects the conjugate of the compound shown in formula I and carrier proteins (this conjugate is immunogen or coating antigen).Described carrier proteins specifically can be BSA or OVA.
The present invention also protects a kind of test kit for detecting Ractopamine hydrochloride, comprises described conjugate.Described test kit also can comprise the antibody obtained for immunogen with described conjugate.Described antibody specific can be rabbit source polyclonal antibody.
The present invention also protects a kind of chemiluminescence immunoassay kit for detecting Ractopamine hydrochloride, comprises the chemoluminescence microwell plate (conjugate is as coating antigen) being coated with described conjugate.Described test kit also can comprise the antibody obtained for immunogen with described conjugate.Described antibody specific can be rabbit source polyclonal antibody.
The preparation method of described " being coated with the chemoluminescence microwell plate of described conjugate " is specific as follows: (1) gets described conjugate, be buffered liquid dilution (bag is buffered the carbonate buffer solution that liquid specifically can be pH9.6,0.03M) with bag, obtain coating antigen diluent (protein concentration specifically can be 2ng/mL); (2) coating antigen diluent is added chemoluminescence microwell plate (specifically can 100 μ L/ holes), hatch (specifically can 37 DEG C hatch 16 hours), washing, pats dry; (3), after completing steps (2), carry out closing that (every hole can add 200 μ L confining liquids, 37 DEG C of incubation 2h; The concrete formula of confining liquid: by the mixing of the phosphate buffered saline buffer of 10g bovine serum albumin, 0.1mL proclin 300 and 1000mL pH7.4,0.01M, obtain confining liquid), the liquid inclined in hole, dry.
Whether the present invention also protects above arbitrary described test kit detecting in sample to be tested containing the application in Ractopamine hydrochloride.
Test kit provided by the invention is easy and simple to handle, specificity good, highly sensitive, properties is excellent, is very suitable for applying.
Accompanying drawing explanation
Fig. 1 is canonical plotting.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.Stirring in embodiment 1 all adopts magnetic stirring apparatus to carry out.
Dimethyl formamide (DMF): sigma, model: D4551.Other reagent, all purchased from traditional Chinese medicines group, are analytical pure.
The structural formula of Ractopamine hydrochloride is as follows:
The preparation of embodiment 1, immunogen and coating antigen
One, haptenic preparation
1, get 700mg Ractopamine hydrochloride and be dissolved in 50ml ethanol, add 258mg epoxy chloropropane, 963mg salt of wormwood mixes, stirring at room temperature reaction 90h;
2, get the solution that step 1 obtains, cross and filter insolubles, filtrate is carried out vacuum-drying, and the dry-matter obtained is the compound shown in formula I.
Haptenic structural formula is shown in formula I.
formula I.
Two, immunogenic preparation
1, take the haptens of 16mg step one preparation, be dissolved in 2ml DMF;
2, get 50mg BSA, be dissolved in 5ml 0.1M sodium carbonate buffer;
3, added to by the dropwise that step 1 obtains in the solution that step 2 obtains, stirred overnight at room temperature, then loads dialysis tubing, carries out dialyse (3 days, change liquid every day 2 times) in PBS damping fluid (pH7.4,0.01M) 4 DEG C;
4, dialyzate is aseptically crossed the filter membrane in 0.22 μm of aperture, the solution obtained is immunogen solution, is sub-packed in ampere bottle ,-20 DEG C of preservations.
Immunogenic structural formula is shown in formula II.
formula II.
Three, the preparation of coating antigen
Replace BSA with OVA, other same step 2, obtains coating antigen solution.
The preparation of embodiment 2, polyclonal antibody
Immunogen solution prepared by Example 1, adopts the PBS damping fluid dilution of pH7.4,0.01M, obtains immunogen diluent, for the preparation of polyclonal antibody.Adopt new zealand white rabbit as immune animal.
Immunologic process following (immunizing dose is with albumen gauge):
First immunisation: immunogen diluent and isopyknic Freund's complete adjuvant are mixed and made into emulsifying agent, neck dorsal sc multi-point injection, immunizing dose is 1mg/kgb.w.;
Booster immunization: after first immunisation 4 weeks, 8 weeks afterwards and after 12 weeks, respectively carries out a booster immunization, by immunogen diluent and isopyknic Freund's incomplete adjuvant mixing and emulsifying, and neck dorsal sc multi-point injection, single immunization dosage is 1mg/kgb.w.;
Final immunization: first immunisation carried out final immunization after 16 weeks, direct neck dorsal sc multi-point injection immunogen diluent, immunizing dose is 1mg/kgb.w..
Final immunization is after 1 week, and blood sampling is separation of serum also, is polyclonal antibody corresponding to immunogen (being called for short polyclonal antibody first).
The assembling of embodiment 3, chemiluminescence immune detection reagent kit
Chemiluminescence immune detection reagent kit comprises following assembly:
1, bag is by the chemoluminescence microwell plate of coating antigen
Coating antigen solution prepared by Example 1, is buffered liquid dilution (bag is buffered the carbonate buffer solution of liquid and pH9.6,0.03M) with bag, obtains the coating antigen diluent that protein concentration is 2ng/mL.By the mixing of the phosphate buffered saline buffer of 10g bovine serum albumin, 0.1mL proclin 300 and 1000mL pH7.4,0.01M, obtain confining liquid.
(1) coating antigen diluent is added chemoluminescence microwell plate (100 μ L/ hole), hatch 16 hours for 37 DEG C.
(2), after completing steps (1), the liquid inclined in hole, washing, pats dry.
(3), after completing steps (2), every hole adds 200 μ L confining liquids, 37 DEG C of incubation 2h.
(4), after completing steps (3), the liquid inclined in hole, preserves with the vacuum-sealing of aluminium film after dry.
2, polyclonal antibody working fluid
Get 10mg bovine serum albumin, be settled to 1000mL with the PBS buffer solution of pH7.4,0.02M, obtain antibody diluent.
Polyclonal antibody first antibody diluent embodiment 2 prepared is diluted to 1200000 times of volumes, obtains primary antibodie working fluid first.
3, two anti-working fluids
The sheep anti mouse two of horseradish peroxidase-labeled is anti-is 115-035-003 purchased from Jackson ImmunoResearch Laboratories Inc. catalog number.
Antibody diluent in the anti-step 2 of sheep anti mouse two of horseradish peroxidase-labeled is diluted to 1000 times of volumes, obtains two anti-working fluids.
4, standard solution
Ractopamine hydrochloride is dissolved in the phosphate buffered saline buffer of pH7.4,0.05M, obtains the standard solution that concentration is 0.06ng/mL, 0.18ng/mL, 0.54ng/mL, 1.62ng/mL and 4.86ng/mL respectively.Using the negative control solution of the phosphate buffered saline buffer of pH7.4,0.05M as standard solution, be called 0 solution.
5, luminous substrate liquid
Luminescent solution is made up of A liquid and B liquid, A liquid and each one bottle of B liquid, 4mL/ bottle.
The preparation method of A liquid: get 0.2g luminol,3-aminophthalic acid cyclic hydrazide list sodium salt, 0.1g to iodophenol, 0.16g sodium-chlor and 0.18g EDTA-Na
2, be settled to 1000mL with the Tris-HCl buffer solution of pH8.4,0.1M.
B liquid: containing 0.3mM H
2o
2, 5mM EDTA-Na
2the Tris-HCl damping fluid of pH8.4,0.1M.
6,20 × concentrated cleaning solution
The phosphate buffered saline buffer of 1000mL pH7.4,0.2M is mixed with 1mL proclin 300, obtains 20 × concentrated cleaning solution.
The using method of the chemiluminescence immune detection reagent kit of embodiment 4, embodiment 3 preparation
Standard solution or sample to be tested solution (50 μ L/ hole) is added by the chemoluminescence microwell plate of coating antigen to bag, add primary antibodie working fluid first (50 μ L/ hole) and two anti-working fluids (50 μ L/ hole) again, room temperature reaction 20min, abandon supernatant, (each washing process is all as follows: add 250 μ L washingss in every hole, abandon supernatant after 30 seconds to wash four times; 20 × concentrated cleaning solution is diluted with water to 20 times of volumes, be washings), pat dry with thieving paper, every hole adds 100 μ L Fresh luminous substrate liquid (A liquid and the mixing of B liquid equal-volume), detects the number of photons in every hole with Chemiluminescence Apparatus (sky, Shenzhen crowd reaches).Five multiple holes are set.The mean value (B) of the luminous intensity of the standard solution of each concentration is divided by the mean value (B of the luminous intensity of 0 solution
0), then be multiplied by 100%, i.e. combination rate.Calculation formula: combination rate (%)=B/B
0× 100%.With the Ractopamine hydrochloride concentration (ng/mL) in standard solution for X-axis, B/B
0for Y-axis, drawing standard graphic representation (see figure 1).The concentration of Ractopamine hydrochloride in sample to be tested solution can be obtained according to the regression equation of typical curve.In the present invention, the analysis of detected result can utilize professional software, can realize the real-time analysis of great amount of samples, and whole testing process only needs just can complete for 30 minutes.Combination rate (B/B
0) Ractopamine hydrochloride concentration in standard solution corresponding when being 50% is IC
50value.According to canonical plotting, IC
50=0.059ng/mL.
The Cleaning Principle of test kit: when the conjugate of haptens pre-coated on chemoluminescence microwell plate and carrier proteins, add sample to be tested solution, add primary antibodie and ELIAS secondary antibody subsequently, coating antigen Ractopamine hydrochloride residual in sample to be tested solution and Chemiluminescent plate wrapping quilt competes primary antibodie, then with ELIAS secondary antibody further combined with, add Chemoluminescent substrate, luminous intensity becomes negative correlation with the content of Ractopamine hydrochloride in sample to be tested solution, compares the residual quantity that can draw Ractopamine hydrochloride in sample to be tested solution with typical curve.
The preparation of comparative example, immunogen contrast and coating antigen contrast
One, immunogen contrast is prepared
1, get 10mg Ractopamine hydrochloride, be dissolved in the 1ml 0.2mol/L HCl aqueous solution, be placed in ice bath and add 10mg Sodium Nitrite, then stirring at room temperature 30min.
2, get 50mg BSA, be dissolved in 5ml 1mol/L aqueous sodium carbonate.
3, added by the dropwise that step 1 obtains in the solution that step 2 obtains, stirring at room temperature 5h, then loads dialysis tubing, carry out dialysing in physiological saline (3 days, change liquid every day 2 times), then carry out filtering and collecting filtrate with the filter membrane in 0.22 μm of aperture, be immunogen contrast solution.
Two, coating antigen contrast is prepared
1, get 6mg Ractopamine hydrochloride, be dissolved in the 1ml 0.2mol/L HCl aqueous solution, to be in ice bath and to add 6mg Sodium Nitrite, stirring at room temperature 30min.
2, get 30mg OVA, be dissolved in 5ml 1mol/L aqueous sodium carbonate.
3, added by the dropwise that step 1 obtains in the solution that step 2 obtains, stirring at room temperature 5h, then loads dialysis tubing, carry out dialysing in physiological saline (3 days, change liquid every day 2 times), then carry out filtering and collecting filtrate with the filter membrane in 0.22 μm of aperture, be coating antigen contrast solution.
Three, the preparation of polyclonal antibody second
Replace immunogen solution to carry out embodiment 2 with immunogen contrast solution, obtain polyclonal antibody corresponding to immunogen contrast (being called for short polyclonal antibody second).
Four, coating antigen contrast and polyclonal antibody second detection Ractopamine hydrochloride is applied
Replace coating antigen solution to carry out the step 1 of embodiment 3 with coating antigen contrast solution, obtain control wells plate.
Replace polyclonal antibody first to carry out the step 2 of embodiment 3 by polyclonal antibody second, obtain primary antibodie working fluid second.
Replace " bag is by the chemoluminescence microwell plate of coating antigen " with control wells plate, replace " primary antibodie working fluid first " by primary antibodie working fluid second, the other the same as in Example 4, IC
50value=0.16ng/mL.
The specificity of the chemiluminescence immune detection reagent kit of embodiment 5, embodiment 3 preparation
Detect the cross reacting rate of 4 kinds of medicines to be measured (with Ractopamine hydrochloride structure or functionally similar 4 kinds of medicines) and Ractopamine hydrochloride respectively.
1, medicine to be measured is dissolved in the phosphate buffered saline buffer of pH7.4,0.05M, obtains the solution of different concns.
2, to wrapping in the quilt chemoluminescence microwell plate of coating antigen the solution (50 μ L/ hole) adding step 1 and prepare, add primary antibodie working fluid first (50 μ L/ hole) and two anti-working fluids (50 μ L/ hole) again, room temperature reaction 20min, abandon supernatant, (each washing process is all as follows: add 250 μ L washingss in every hole, abandon supernatant after 30 seconds to wash four times; 20 × concentrated cleaning solution is diluted with water to 20 times of volumes, is washings), pat dry with thieving paper, every hole adds 100 μ L Fresh luminous substrate liquid (A liquid and the mixing of B liquid equal-volume), detects the number of photons in every hole with Chemiluminescence Apparatus.
Cross reacting rate (%)=(IC of Ractopamine hydrochloride
50the IC of value/medicine to be measured
50value) × 100%.The results are shown in Table 1, the specificity of test kit is good.
Table 1 cross reacting rate result
| Medicine name | Purchase approach | Cross reacting rate (%) |
| Ractopamine hydrochloride | China Veterinary Drugs Supervisory Inst. | 100 |
| Salbutamol | China Veterinary Drugs Supervisory Inst. | ＜0.1 |
| Clenbuterol | China Veterinary Drugs Supervisory Inst. | ＜0.1 |
| Bromine Boot sieve | China Veterinary Drugs Supervisory Inst. | ＜0.1 |
| Cimaterol | China Veterinary Drugs Supervisory Inst. | ＜0.1 |
The performance of the test kit of embodiment 6, embodiment 3 preparation
One, the method (preparing dummy) of Sample pretreatment
Urine sample (pig urinates, and ox urinates, and sheep urinates)
Get fresh urine sample or-20 DEG C of frozen urines immediately after gathering, by centrifugal for urine 4000r/min 5 min, accurately measure in supernatant urine sample 1000 μ L to 2mL centrifuge tube, and with 1 mol/L sodium hydroxide solution, sample pH value is adjusted to 10.0, add 1ml ethyl acetate-isopropylcarbinol mixing solutions (blending ratio is 3:7), whirling motion instrument vortex 1 min, get in organic phase 500 μ L to 2 mL centrifuge tube, under 56 DEG C of water bath condition, nitrogen dries up, redissolve with 500 μ L sample diluting liquids, vortex 1 min gets 50 μ L after also fully dissolving and detects.
Tissue (pork, pork liver, beef)
Get flesh tissue or-20 DEG C of frozen tissue samples immediately after gathering, sample is shredded and grinds to form paste-like with super grinder, accurately take 1.0 g samples in 50 mL tool plug centrifuge tubes, add 9 mL ethyl acetate-acetonitrile mixing solutionss (blending ratio is 1:9), whirling motion instrument vortex 3 min, , centrifugal 10 min of whizzer 4000 r/min, get in organic phase 8 mL to 10 mL centrifuge tube, under 56 DEG C of water bath condition, nitrogen dries up, redissolve with 2 mL sample diluting liquids, vortex 1 min also adds 2 mL normal hexanes after fully dissolving, centrifugal 5 min of whizzer 4000 r/min after vortex 1 min, water phase separated, get 50 μ L to detect.
Two, test kit preservation period experiment
Test kit preservation condition is 2-8 DEG C, preserves after 6 months, measures the IC of Ractopamine hydrochloride
50value, zero hole number of photons.Consider in transport and use procedure, have improper preservation condition and occur, test kit is placed 6 days under 37 DEG C of preservation conditions, carries out hot Acceleration study.Result shows that this test kit indices meets the requirements completely.
Table 2 cryopreservation experimental result
| Time (d) | 0 | 10 | 20 | 30 | 60 | 90 | 120 | 150 | 180 |
| Number of photons (× 10 4） | 102.1 | 108.9 | 104.3 | 105.8 | 98.9 | 101.8 | 98.1 | 106.3 | 101.9 |
| IC 50(ng/mL) | 0.066 | 0.061 | 0.063 | 0.062 | 0.068 | 0.067 | 0.069 | 0.063 | 0.066 |
The hot Acceleration study of table 3
| Time (d) | 1 | 2 | 3 | 4 | 5 | 6 |
| Number of photons (× 10 4） | 108.1 | 109.8 | 107.1 | 103.5 | 103.6 | 105.7 |
| IC 50(ng/mL) | 0.062 | 0.061 | 0.068 | 0.069 | 0.071 | 0.068 |
Three, the sensitivity of test kit, precision, accuracy
1, sensitivity
Sensitivity index using lowest detectable limit as test kit of the present invention.Get 20 parts of dummies, detect by the using method of embodiment 3, detected signal value, calculate the mean value of dummy number of photons (RLU), and this mean value is brought into the testing concentration that typical curve obtains correspondence, calculate the standard deviation (SD) of each corresponding concentration value, add by mean value the lowest detectable limit (LOD) that three times of standard deviations are this sample, the results are shown in Table 4.
The lowest detectable limit of table 4 Ractopamine hydrochloride in urine sample and tissue
| Sample | Pig urinates | Ox urinates | Sheep urinates | Pork | Pork liver | Beef |
| LOD（ng/mL） | 0.02 | 0.02 | 0.05 | 0.01 | 0.02 | 0.01 |
2, accuracy
Its accuracy of interpolation experiment reflection of test kit.Get some batches of urine samples (pig urine, ox urine, sheep urine) dummy, addition is respectively: 0.1 ng/mL, 0.2 ng/mL, 0.5 ng/mL, 1 ng/mL, each concentration do five parallel, do altogether five batches add recovery tests.The working sample rate of recovery, variation within batch coefficient and interassay coefficient of variation, with the accuracy of evaluation method and precision.Result is as shown in table 5, and the variation within batch coefficient of each sample is all less than 15%, and interassay coefficient of variation is all less than 20%.Accuracy and the precision of this pre-treating process and detection method meet the requirements.
RAC TIANZHU XINGNAO Capsul and the variation coefficient in table 5 pig urine, ox urine, sheep urine
Get some batches of tissues (pork, pork liver, beef) dummy, addition is respectively: 0.05 ng/mL, 0.1 ng/mL, 0.2 ng/mL, 0.5 ng/mL, each concentration do five parallel, do altogether five batches add recovery tests.The working sample rate of recovery, variation within batch coefficient and interassay coefficient of variation, with the accuracy of evaluation method and precision.Result is as shown in table 6, and the variation within batch coefficient of each sample is all less than 15%, and interassay coefficient of variation is all less than 20%.Accuracy and the precision of this pre-treating process and detection method meet the requirements.
Ractopamine hydrochloride TIANZHU XINGNAO Capsul and the variation coefficient in table 6 pork, pork liver, beef
Claims (9)
1. the compound shown in formula I;
formula I.
2. the compound shown in formula I and the conjugate of carrier proteins.
3. conjugate as claimed in claim 2, is characterized in that: described carrier proteins is BSA or OVA.
4., for detecting a test kit for Ractopamine hydrochloride, comprise conjugate described in Claims 2 or 3.
5. test kit as claimed in claim 4, is characterized in that: described test kit also comprises the antibody obtained for immunogen with conjugate described in Claims 2 or 3.
6., for detecting a chemiluminescence immunoassay kit for Ractopamine hydrochloride, comprise the chemoluminescence microwell plate being coated with conjugate described in Claims 2 or 3.
7. test kit as claimed in claim 6, is characterized in that: described test kit also comprises the antibody obtained for immunogen with conjugate described in Claims 2 or 3.
8. whether test kit described in claim 6 or 7 is detecting in sample to be tested containing the application in Ractopamine hydrochloride.
9. whether test kit described in claim 4 or 5 is detecting in sample to be tested containing the application in Ractopamine hydrochloride.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108640866A (en) * | 2018-06-01 | 2018-10-12 | 中国农业大学 | Fluorobenzene insect amide antigen and the preparation method and application thereof |
| CN110950960A (en) * | 2019-11-26 | 2020-04-03 | 中国农业大学 | Preparation method of small molecule compound antibody based on high-throughput sequencing and hybrid hybridoma technology |
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| US20060223132A1 (en) * | 2004-11-10 | 2006-10-05 | Randox Laboratories Limited | Phenethanolamine-derived haptens, immunogens, antibodies and conjugates |
| CN102040568A (en) * | 2010-11-05 | 2011-05-04 | 河北科技大学 | Synthesis method of low-molecular weight epoxy resin |
| CN102718670A (en) * | 2012-05-31 | 2012-10-10 | 中国农业大学 | Ractopamine hapten, artificial antigen and preparation methods for Ractopamine hapten and artificial antigen |
| CN103012592A (en) * | 2011-09-21 | 2013-04-03 | 北京勤邦生物技术有限公司 | Preparation and applications of ractopamine monoclonal antibody |
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2014
- 2014-04-29 CN CN201410175283.3A patent/CN105017180A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060223132A1 (en) * | 2004-11-10 | 2006-10-05 | Randox Laboratories Limited | Phenethanolamine-derived haptens, immunogens, antibodies and conjugates |
| CN102040568A (en) * | 2010-11-05 | 2011-05-04 | 河北科技大学 | Synthesis method of low-molecular weight epoxy resin |
| CN103012592A (en) * | 2011-09-21 | 2013-04-03 | 北京勤邦生物技术有限公司 | Preparation and applications of ractopamine monoclonal antibody |
| CN102718670A (en) * | 2012-05-31 | 2012-10-10 | 中国农业大学 | Ractopamine hapten, artificial antigen and preparation methods for Ractopamine hapten and artificial antigen |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108640866A (en) * | 2018-06-01 | 2018-10-12 | 中国农业大学 | Fluorobenzene insect amide antigen and the preparation method and application thereof |
| CN110950960A (en) * | 2019-11-26 | 2020-04-03 | 中国农业大学 | Preparation method of small molecule compound antibody based on high-throughput sequencing and hybrid hybridoma technology |
| CN110950960B (en) * | 2019-11-26 | 2021-05-14 | 中国农业大学 | Preparation method of small molecule compound antibody based on high-throughput sequencing and hybrid hybridoma technology |
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