CN104311438A - Ractopamine semiantigen, artificial antigen and monoclonal antibody, and preparation methods and applications thereof - Google Patents
Ractopamine semiantigen, artificial antigen and monoclonal antibody, and preparation methods and applications thereof Download PDFInfo
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Abstract
The invention provides an immunodetection technology, and concretely relates to a ractopamine semiantigen and an artificial antigen, preparation methods thereof, and a monoclonal antibody prepared through immunizing mice by using the artificial antigen. The monoclonal antibody is secreted by a hybridoma cell strain 12B2, and the hybridoma cell is preserved China Center for Type Culture Collection with the preservation number of (CCTCC NO):C2014127. The ractopamine monoclonal antibody provided by the invention has the advantages of high titer, high sensitivity and strong specificity, and can be applied in the detection of the residual of ractopamine medicines.
Description
Technical field
The present invention relates to technical field of immunoassay, be specifically related to a kind of Ractopamine hydrochloride haptens, artificial antigen, monoclonal antibody and its preparation method and application.
Background technology
Ractopamine hydrochloride (RAC, structural formula is as Fig. 1) also known as a gram rumba peace, it is a kind of receptor,β agonist containing two phenylalkylamine structure, be a kind of can by producing the similar thing of natural catecholamine of the synthetic of corresponding physiological effect after combining to the beta receptor on cytolemma, its effect is similar with the effect of norepinephrine to suprarenin, be generally used for disease such as treatment bronchial asthma, bronchospasm etc. clinically, also can be used for treating muscular dystrophy, increase muscle, reduce lipopexia, and useful to fetus an d neonate growth.
When Ractopamine hydrochloride is used for feeding animals, the nutrition in animal body can be made to be reallocated, change the Nutrition and Metabolism mode of animal, reduce steatogenesis, improve lean ratio, more can cater to consumer demand thus considerable economic benefit can be brought.But, if beta-receptor agonist is accumulated in animal body for a long time, then can be threatened to the healthy of human consumer by food chain, the toxicity symptoms such as palpitaition, muscular tremor, nausea and vomiting can be caused time serious, especially to having a heart disease, diabetes or the harm of hypertensive patient body larger.
The Ministry of Agriculture of China promulgated stringent laws against in 1997 and uses beta-receptor agonist to promote growth of animal as animal feedstuff additive.The Ministry of Industry and Information Technology of the People's Republic of China (PRC), the Ministry of Agriculture, Department of Commerce, the Ministry of Health, State Administration for Industry and Commerce, State Administration for Quality Supervision and Inspection and Quarantine combine issue bulletin, from 5 days December in 2011, forbade production and selling Ractopamine hydrochloride within the border in China.
At present, 24 countries such as the U.S., Canada, Brazil still allow Ractopamine hydrochloride to be added in animal-feed promote growth of animal and improve lean ratio, this becomes illegal use, and person is the excuse trying to gain illegitimate benefits.Ractopamine hydrochloride is still active in another major reason in market, is that current detection method does not reach requirement.
Secondly the detection method mainly chromatogram analysis method of current Ractopamine hydrochloride is immune analysis method.Although chromatogram analysis method is sensitiveer, accuracy is high, needs large-scale instrument support, needs loaded down with trivial details sample pre-treatments and the human users of specialty, can not adapt to the requirement that market is quick to analytical procedure, easy.Although immune analysis method is quick, easy, high specificity, highly sensitive, the advantages such as the detection of sample pre-treatments that need not be loaded down with trivial details, applicable on-the-spot batch sample, but enzyme linked immunological kit is undesirable to detection Ractopamine hydrochloride specificity, the sensitivity of test kit is not high, therefore prepare highly sensitive, the Anti-ractopamine antibody of high specificity is crucial, and solves the clone that its key to the issue depends on immune haptenic molecular structure and secretory antibody.
Patent 201210177057.X provides the preparation method of a kind of Ractopamine hydrochloride haptens, artificial antigen, although haptenic synthetic method is simple, purity and productive rate higher, but this haptens synthesizes based on Ractopamine hydrochloride raw material, can increase Ractopamine hydrochloride detect cost.
Patent CN 102924601B provides a kind of preparation method of ractopamine monoclonal antibody, and its haptens also synthesizes based on Ractopamine hydrochloride structure, and not only production cost is relatively high, and its sensitivity preparing monoclonal antibody is low.
In order to solve the problem, key point of the present invention from the analogue of basic material ractopamine synthesis as haptens, reduce testing cost, and haptens is the upper original structure retaining Ractopamine hydrochloride to greatest extent, and optimize immunizing antigen and prepare hybridoma and secrete a large amount of antibody, improve the specificity of antibody, sensitivity, strengthen the recognition capability of antibody and antigen.
Summary of the invention
In order to overcome above-mentioned defect and the deficiency of prior art, the object of the present invention is to provide a kind of Ractopamine hydrochloride haptens, artificial antigen, monoclonal antibody and its preparation method and application, the Ractopamine hydrochloride utilizing the method to prepare detects monoclonal antibody and has high, highly sensitive, that high specificity, testing cost are cheap feature of tiring.
The present invention mainly contains following technical scheme and realizes:
A kind of Ractopamine hydrochloride haptens, its structural formula is following formula I:
The haptenic preparation method of a kind of Ractopamine hydrochloride, comprises the steps: that compound 1 obtains intermediate 2 by bromo-reaction; Described intermediate 2 is obtained by reacting intermediate 3 with ethyl propenoate; Described intermediate 3 obtains intermediate 4 through reduction reaction; Described intermediate 4 is obtained by reacting intermediate 6 with compound 5; Described intermediate 6 obtains described Ractopamine hydrochloride haptens 7 through hydrolysis reaction;
The haptenic preparation method of a kind of Ractopamine hydrochloride, it is characterized in that comprising the steps: that described compound 1 is dissolved in acetonitrile by (a), add N-bromo-succinimide, described compound 1 and the ratio of the add-on of described N-bromo-succinimide are 1:1 ~ 1.5 times equivalents, react 2-4h under room temperature, obtain described intermediate 2; B () adds [1 in described intermediate 2, two (diphenylphosphino) ferrocene of 1'-] palladium chloride, substitute nitrogen, add triethylamine, N, dinethylformamide and ethyl propenoate, again substitute nitrogen, reflux at 90-110 DEG C stirring reaction 15-20h, obtains described intermediate 3; C () adds methyl alcohol and catalyst P d/C in described intermediate 3, substitute hydrogen, reacts 3-6h under room temperature, obtains described intermediate 4; D described intermediate 4 is dissolved in methyl alcohol by (), add compound 5, triethylamine stirring reaction 10-30min, add triacetoxy borohydride hydrogen sodium, after the stirring reaction 20-30h that refluxes, obtains described intermediate 6 at 36-60 DEG C; E described compound 6 is dissolved in methyl alcohol by (), add NaOH, and reflux at 50-70 DEG C stirring reaction 5-7h, obtains described Ractopamine hydrochloride haptens RAC-CA.
A kind of ractopamine artificial antigen, its structural formula is as shown in the formula II:
A preparation method for ractopamine artificial antigen, is characterized in that: described Ractopamine hydrochloride haptens and bovine serum albumin coupling are obtained described ractopamine artificial antigen.
A kind of ractopamine monoclonal antibody prepared by a kind of ractopamine artificial antigen, it is characterized in that: described ractopamine monoclonal antibody is secreted by hybridoma cell strain 12B2, described hybridoma is preserved in China typical culture collection center, and deposit number (CCTCC NO) is: C2014127.
The monoclonal antibody of being secreted by above cell strain 12B2, preparation method comprises the following steps:
A. ractopamine synthesis haptens RAC-CA is as haptens;
B. haptens RAC-CA and bovine serum albumin coupling are prepared immunizing antigen RAC-CA-BSA;
C. haptens RAC-CA and oralbumin coupling are prepared envelope antigen RAC-CA-OVA;
D. animal immune injection: using 6-8 week age female Balb/C mouse as immune animal, immunity is carried out, abdominal injection booster immunization in abdominal cavity, subcutaneous alternate injection immunizing antigen;
E. animal immune serum is screened: detect immune mouse serum with indirect ELISA and indirect competitive ELISA experiment, screening is exempted from mouse for cytogamy;
F. prepare hybridoma: get Mouse spleen cells and Sp2/0 myeloma cell carries out cytogamy, obtain Absorbable organic halogens through subclone and secrete anti-ractopamine monoclonal antibody cell strain 12B2;
G. ascites is prepared: to mouse peritoneal injection hybridoma 12B2, gather ascites;
H. the purifying of ascites: the ascites of above-mentioned collection is carried out purifying, preparation monoclonal antibody, measures antibody titer, and competitive ELISA method measures the IC of antibody
50.
Wherein, the concrete steps of described step B and C are: first RAC-CA is dissolved in N, in dinethylformamide, add N-hydroxysuccinimide, dicyclohexylcarbodiimide, under room temperature condition, reaction overnight forms Acibenzolar, cross the solid filtering and produce in reaction, be more slowly added drop-wise to the 0.01MNaHCO of BSA/OVA under condition of ice bath
3solution in form conjugate, dialyse in PBS solution under ice bath.Within every 4 hours, change a dialyzate, repeat six times.Dialyse complete, be carefully transferred in sample bottle from dialysis tubing by reaction solution, preservative film is sealed, and covers bottle cap, precooling at-80 DEG C, and lyophilize obtains white solid powder RAC-CA-BSA and RAC-CA-OV, and uv-vis spectra measures coupling ratio.
The concrete steps of described step D and E are each immunization dosage is 100 μ g/, first immunisation adopts abdominal injection, described RAC-CA-BSA and equal-volume not after the emulsification of formula Freund's complete adjuvant as first time immunogen, after described RAC-CA-BSA and the not emulsification of formula Freund's incomplete adjuvant equal-volume, subcutaneous injection is as second time and the 4th immunity, the not formula Freund's incomplete adjuvant emulsification pneumoretroperitoneum of described RAC-CA-BSA and 1/3 volume is injected as third time and the 5th immunity, and the immunization interval phase is three weeks.Described immunity terminates rear 7-10 days, survey serum antibody titer with indirect ELISA experiment and indirect competitive ELISA experiment, choose the high mouse of described serum titer and carry out booster immunization, booster immunization is for merging first three sky, described RAC-CA-BSA does not add adjuvant abdominal injection, is 200 μ g/.
In described step F, described booster immunization terminates latter three days, get the Mouse spleen cells accepting booster immunization, using PEG1500 as fusogen, cytogamy is carried out by number ratio 8:1 with SP2/0 myeloma cell, choose positive colony and carry out subclone, obtaining can the hybridoma cell strain of stably excreting monoclonal antibody.
Described step G, by 0.5ml pristone/ abdominal injection Balb/c mouse, sensitization is after 7 days, by 1-2 × 10
6the above-mentioned sensitization mouse of a hybridoma/abdominal injection, ascites is gathered after 7-10 days, the centrifugal 10min of 1000r/min removes cell in ascites, get supernatant, the centrifugal 30min of 12000r/min removes cell debris in ascites, get supernatant again, containing a large amount of ractopamine monoclonal antibody albumen in this supernatant, survey OD
280with OD
260value tentatively demarcates total protein content, and a part is used for next step antibody purification, and rest part packing-80 ° is frozen for subsequent use.
Described step H, get ascites prepared by described step G, use ice-water bath precooling, 0.01M PBS dilutes liquid of preparing after (a ascites+three parts of 0.01M PBS (pH 7.4)), get 1ml protein G resin by 10mg ascites total protein to measure, be filled in antibody protein affinity column bed, balanced chromatography column with the precooling 0.01M PBS (pH 7.4) of resin 50 × volume.Make Monoclonal Antibodies in Mice Ascites albumen fully with protein G specific binding, fully wash chromatography column to remove the foreign protein in adhesion chromatography column with the precooling 0.01M PBS (pH7.4) of 50 × volume.
1.0M Tris-Cl (pH 9.6) 100 μ l is respectively added in advance in 1.5ml EP pipe, again the chromatography column of above-mentioned process is added frozen water precooling 0.1M glycine-HCl (pH 2.7) and carry out wash-out, filtrate presses 0.9ml/ pipe fraction collection in the EP pipe of above-mentioned process, and often pipe is got and surveyed OD a little
280protein peak, merges protein peak pipe, and amalgamation liquid is placed in dialysis tubing, dialysed overnight in ice-water bath 0.01M PBS (pH 7.4).
Get this treatment solution a little, carry out the indirect competitive ELISA analysis of indirect ELISA and free Ractopamine hydrochloride do competition antigen.
Described Ractopamine hydrochloride haptens, or the application of described ractopamine artificial antigen in the described ractopamine monoclonal antibody of preparation.
Described Ractopamine hydrochloride haptens, or the application of described ractopamine artificial antigen at Ractopamine hydrochloride in qualitative and detection by quantitative.
Beneficial effect of the present invention: 1. Ractopamine hydrochloride haptens provided by the invention Material synthesis based on para hydroxybenzene propionic acid, N-bromo-succinimide, instead of synthesize based on the structure of Ractopamine hydrochloride, Ractopamine hydrochloride raw material need not be purchased like this, reduce the haptenic cost of ractopamine synthesis; 2. Ractopamine hydrochloride haptens introduces the carboxyl chain of three carbon atoms by Heck reaction ortho position of phenyl ring phenolic hydroxyl group in Ractopamine hydrochloride original structure in building-up process, retain all hydroxyls and amido in former Ractopamine hydrochloride structure, namely retain the feature such as electron distributions, molecular polarity in its original molecular structure to greatest extent, make the final ractopamine monoclonal antibody obtained have higher specificity; 3. ractopamine monoclonal antibody is secreted by hybridoma cell strain 12B2, has the antibody that stably excreting is a large amount of, and the ractopamine monoclonal antibody IC after experiment proof purifying
50value is 0.35ppb, tires 800000, improves Ractopamine hydrochloride sensitivity and tire, and cross reaction experiment also demonstrates the high specificity of ractopamine monoclonal antibody; 4. the Ractopamine hydrochloride haptens of synthesis and ractopamine artificial antigen can be applied and be applied preparing in Ractopamine hydrochloride product in Ractopamine hydrochloride qualitative and quantitative analysis.
Accompanying drawing explanation
Fig. 1 is Ractopamine hydrochloride structural formula;
Fig. 2 is the haptenic preparation of Ractopamine hydrochloride of embodiment 1;
Fig. 3 is the ractopamine artificial antigen of embodiment 1 and the preparation of envelope antigen and detection;
Fig. 4 is the RAC-CA-BSA conjugate of embodiment 1, the uv-absorbing scanning optical spectrum schematic diagram of RAC-CA, BSA;
Fig. 5 is the RAC-CA-OVA conjugate of embodiment 1, the uv-absorbing scanning optical spectrum schematic diagram of RAC-CA, OVA;
Fig. 6 is the IC of the ractopamine monoclonal antibody of embodiment 1
50measure curve.
Embodiment
Below in conjunction with drawings and Examples, the present invention will be further described in detail.
Embodiment 1
One, the haptenic preparation of Ractopamine hydrochloride
Specific experiment step is:
(1) compound 1 (164mg, 1.0mmol, 1.0eq) is dissolved in 4mL acetonitrile, under stirring, adds NBS (N-bromo-succinimide) (200mg, 1.1mmol, 1.1eq), stirred at ambient temperature reaction 3h.React and added 25mL H
2o, EA (ethyl acetate) (3 × 10mL) extracts, and merges organic phase, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure, dry column chromatography, DCM (methylene dichloride) is eluent, normal pressure wash-out, obtains yellow oil 209mg and intermediate 2.Productive rate: 86%.1H?NMR(400MHz,CDCl
3)δ7.28(d,J＝1.5Hz,1H),7.02(dd,J＝8.3,1.6Hz,1H),6.91(d,J＝8.3Hz,1H),5.62(s,1H),2.79(d,J＝6.9Hz,2H),2.73(d,J＝7.0Hz,2H),2.14(s,3H)。
(2) intermediate 2 (1.75g, 7.29mmol, 1.0eq) is joined in dry two-mouth bottle, add [two (diphenylphosphino) ferrocene of 1,1'-] palladium chloride (970mg, 1.46mmol, 0.2eq), substitute N
2repeatedly, with syringe, TEA (triethylamine) 5mL, DMF (DMF) 29mL is injected two-mouth bottle, inject ethyl propenoate, again substitute N
2repeatedly, reflux at 100 DEG C stirring reaction 17h, and reaction is finished, and adds the dilution of 200mL water, EA (100mL × 3) extracts, merge organic phase, washing (3 × 150mL), anhydrous sodium sulfate drying, filter, concentrating under reduced pressure, dry column chromatography, eluent system is converted into DCM:MeOH system by PE:EA system.Product is yellow oil, puts refrigerator overnight and obtains faint yellow solid 887mg and intermediate 3.Productive rate: 47%.1H?NMR(400MHz,CDCl
3)δ7.97(d,J＝16.2Hz,1H),7.04(dd,J＝8.2,1.8Hz,1H),6.83(s,1H),6.76(d,J＝8.3Hz,1H),6.61(d,J＝16.1Hz,1H),4.27(q,J＝7.1Hz,2H),2.82(t,J＝7.0Hz,2H),2.74(t,J＝7.1Hz,2H),2.14(s,3H),1.34(t,J＝7.1Hz,3H)。
(3) join in eggplant-shape bottle by intermediate 3 (100mg, 0.38mmol, 1.0eq), add MeOH3mL, portion-wise with caution adds Pd/C (10%) (30mg, 0.3eq) on a small quantity and substitutes H
2repeatedly, 4h is reacted under room temperature.React, reaction solution diatomite filtration, MeOH washing leaching cake, concentrating under reduced pressure, obtained crude product 93mg and intermediate 4, directly throw next step.Productive rate: 92%.
(4) by intermediate 4 (1.1g, 4.16mmol, 1.0eq) be dissolved in 21mL MeOH, add compound 5 (794mg, 4.16mmol, 1.0eq), TEA (849mg, 8.32mmol, 2.0eq), stirring reaction 10min, add STAB-H (triacetoxy borohydride hydrogen sodium) (3.55g, 12.48mmol, 3.0eq), just add and had a large amount of bubble and emerge, reflux at 50 DEG C stirring reaction 24h, add STAB-H (1.18g, 4.16mmol, 1.0eq), continue stirring reaction to spend the night, second time mends STAB-H (1.18g, 4.16mmol, 1.0eq), continue stirring reaction 12h, 50mL water is added in reaction solution, EA (30mL × 3) extracts, merge organic phase, saturated aqueous ammonium chloride (3 × 30mL) washs organic phase (except TEA), organic phase is dry, concentrating under reduced pressure, dry column chromatography, eluent system: D:M=20:1-10:1, obtain yellow oil, DCM, ether is concentrated obtains white powder 496mg and intermediate 6.Productive rate: 30%.1H?NMR(400MHz,CD
3OD)δ7.24(t,J＝6.9Hz,2H),6.95(s,1H),6.91(d,J＝8.3Hz,1H),6.79(d,J＝8.3Hz,2H),6.69(d,J＝8.1Hz,1H),4.83(s,1H),4.07(q,J＝7.2Hz,2H),3.24(s,1H),3.08(d,J＝4.4Hz,2H),2.85(t,J＝7.5Hz,2H),2.69(s,1H),2.58(t,J＝7.6Hz,2H),2.54(s,1H),2.07(s,1H),1.81(dd,J＝13.8,9.1Hz,1H),1.38(t,J＝6.1Hz,3H),1.20(t,J＝7.2Hz,3H)。
(5) by intermediate 6 (354mg, 0.88mmol, 1.0eq) be dissolved in 19mL MeOH, add 1.28mL 1M NaOH (51.2mg, 1.28mmol, 1.45eq), reflux at 60 DEG C stirring reaction 6h, adds 1.28mL 1M HCl after reacting completely, and concentrating under reduced pressure is except desolventizing (comprising water), dry column chromatography, eluent system: D:M=20:1-5:1, obtains yellow oil, adds diethyl ether concentrating under reduced pressure repeatedly, vacuum-drying, obtains white solid powder 168mg and Ractopamine hydrochloride haptens 7.Productive rate: 51%.1H?NMR(400MHz,DMSO-d
6)δ9.50(s,1H),7.18(d,J＝8.0Hz,2H),6.91(s,1H),6.85(d,J＝7.6Hz,1H),6.76(d,J＝7.9Hz,2H),6.71(d,J＝7.8Hz,1H),5.76(s,1H),4.82(t,J＝10.9Hz,1H),3.14(d,J＝15.4Hz,2H),3.03–2.86(m,2H),2.70(t,J＝7.5Hz,2H),2.55(s,1H),2.44(t,J＝7.6Hz,2H),2.01(s,1H),1.68(s,1H),1.25(t,J＝7.1Hz,3H).13C?NMR(101MHz,CD
3OD)δ181.5,161.3,157.6,135.8,135.2,133.9,131.6,131.2,131.1,119.3,72.9,58.0,55.0,38.9,38.3,34.6,30.1,19.4,18.8.HRMS:[M
+H
+]:Found?m/z374.1963,Calcd?m/z?374.1889。
The synthetic route schematic diagram of step (1)-(5) as shown in Figure 2.
Two, the preparation of ractopamine artificial antigen and envelope antigen and detection
1. the preparation of ractopamine artificial antigen and detection
A. the preparation specific experiment step of ractopamine artificial antigen is:
(1) RAC-CA (7.0mg, 0.01mmol, 60eq) is taken, be dissolved in the DMF of 0.5mL, add DCC (2.2mg, 0.015mmol, 33eq), NHS (1.2mg, 0.015mmol, 33eq), stirred at ambient temperature reaction 12h, filter out the DCU of generation, obtain A liquid.
(2) take BSA (20mg, 1.0eq) and be dissolved in 1.2mL NaHCO
3in (0.1M, pH 8.5).Under ice bath, stir 5-10min, more dropwise drip A liquid wherein, action is wanted slowly, softly.Dropwise, remove ice bath, react under continuing room temperature and spend the night.
(3) reaction solution is carefully moved in dialysis tubing, dialyse in PBS (0.01M, pH 7.4) solution under ice bath (0-4 DEG C).Within every 4 hours, change a dialyzate, repeat six times.Dialyse complete, be carefully transferred in sample bottle from dialysis tubing by reaction solution, preservative film is sealed, and covers bottle cap, precooling at-80 DEG C.
(4) lyophilize obtains white solid powder RAC-CA-BSA.
B. the detection of ractopamine artificial antigen:
(1) adopting ultraviolet spectrophotometer to carry out full wavelength scanner, take PBS as blank, finds the maximum absorption wavelength λ of RAC-CA-BSA, and the absorbance A when concentration C (cubage with BSA).
(2) measure the absorbance of RAC-CA, BSA different concns at λ place, draw RAC-CA, BSA uv-absorbing typical curve, calculate the absorbance A1 of BSA when concentration C according to BSA uv-absorbing typical curve.
(3) concentration C 1 when being (A-A1) according to the uv-absorbing typical curve calculating absorbance of RAC-CA.
(4) coupling ratio of RAC-CA and BSA is calculated according to formula coupling ratio n=(C1/MRAC-CA)/(C/MBSA).RAC-CA, RAC-CA-BSA, BSA ultraviolet scanning spectrum as shown in Figure 4.Wherein RAC-CA, BSA strength of solution is 100 μ g/mL, and RAC-CA-BSA strength of solution is 605 μ g/mL.
Interpretation of result: the coupling ratio of RAC-CA and the BSA calculated is 33:1.
2. the preparation of envelope antigen and detection
A. the preparation specific experiment step of envelope antigen is:
(1) RAC-CA (5.6mg, 0.015mmol, 30eq) is taken, be dissolved in the DMF of 0.6mL, add DCC (3.4mg, 0.0165mmol, 33eq), NHS (1.9mg, 0.0165mmol, 33eq), stirred at ambient temperature reaction 12h, filter out the DCU of generation, obtain A liquid.
(2) take OVA (13.54mg, 1.0eq) and be dissolved in 1.5mL NaHCO
3in (0.1M, pH 8.5), first under ice bath, stir 5-10min, more dropwise drip A liquid wherein, action is wanted slowly, softly.Dropwise, remove ice bath, react under continuing room temperature and spend the night.
(3) reaction solution is carefully moved in dialysis tubing, dialyse in PBS (0.01M, pH 7.4) solution under ice bath (0-4 DEG C).Within every 4 hours, change a dialyzate, repeat six times.Dialyse complete, be carefully transferred in sample bottle from dialysis tubing by reaction solution, preservative film is sealed, and covers bottle cap, precooling at-80 DEG C.
(4) lyophilize obtains white solid powder RAC-CA-OVA.
B. the detection of envelope antigen
(1) adopting ultraviolet spectrophotometer to carry out full wavelength scanner, take PBS as blank, finds the maximum absorption wavelength λ of RAC-CA-OVA, and the absorbance A2 when concentration C 2 (calculating with OVA).
(2) measure the absorbance of RAC-CA, OVA different concns at λ place, draw RAC-CA, OVA uv-absorbing typical curve, calculate the absorbance A3 of BSA when concentration C according to BSA uv-absorbing typical curve.
(3) concentration C 3 when being (A2-A3) according to the uv-absorbing typical curve calculating absorbance of RAC-CA.
(4) coupling ratio of RAC-CA and BSA is calculated according to formula coupling ratio n=(C2/MRAC-CA)/(C1/MOVA).RAC-CA, RAC-CA-OVA, OVA ultraviolet scanning spectrum as shown in Figure 5.Wherein RAC-CA, OVA strength of solution is 100 μ g/mL, and RAC-CA-OVA strength of solution is 432.5 μ g/mL.
Interpretation of result: the coupling ratio of RAC-CA and the OVA calculated is 18:1.
The syntheti c route of immunizing antigen and envelope antigen as shown in Figure 3.
Three, mouse immune and antiserum(antisera) detect
Get the Balb/C Healthy female mouse in 6 6-8 all ages, every mouse is by follow procedure immunity: each immunization dosage is 100 μ g/, first immunisation adopts abdominal injection, RAC-CA-BSA and equal-volume not after the emulsification of formula Freund's complete adjuvant as first time immunogen, after RAC-CA-BSA and the not emulsification of formula Freund's incomplete adjuvant equal-volume, subcutaneous injection is as second time and the 4th immunity, the not formula Freund's incomplete adjuvant emulsification pneumoretroperitoneum of RAC-CA-BSA and 1/3 volume is injected as third time and the 5th immunity, and the immunization interval phase is three weeks.Immunity terminates rear 7-10 days, tail vein centrifuging and taking serum, chooses the high mouse of serum titer carry out booster immunization with indirect ELISA experiment, and booster immunization is for merging first three sky, and RAC-CA-BSA does not add adjuvant abdominal injection, is 200 μ g/.Immune programme for children sees the following form 1.
Table 1 immune programme for children
Indirect ELISA experiment detects the foundation of antiserum titre method:
(1) configuration of carbonate buffer solution: 0.05M Na
2cO
3/ NaHCO
3(pH 9.6): 1.59g Na
2cO
3, 2.93g NaHCO
3add tri-distilled water to 1000mL.
(2) antigen coated: envelope antigen RAC-CA-OVA (coupling ratio is 18:1) carbonate buffer solution dilution, wrapping by concentration is 27,9 respectively, 3,1,0.333,0.111,0.037 μ g/mL (being coated in the A-G line of elisa plate, 100 μ l/well).Envelope antigen contrasts: OVA, wraps by concentration: 27 μ g/mL (being coated in the H line of elisa plate, 100 μ l/well), wraps and is placed in 37 DEG C hatches 1-1.5h by complete, rearmounted 4 DEG C of refrigerator overnight.
(3) PBS solution of 0.01M pH7.4 is configured: 2.9009g Na
2hPO
412H
2o, 0.2964g NaH
2pO
42H
2o, 8.5g NaCl adds tri-distilled water to 1000mL.
(4) the thick OVA of 1% (1g/100mL PBS configures) is configured as confining liquid.
(5) wash: taken out from 4 DEG C of refrigerators by plate, get rid of liquid in hole, PBS washs 3 times (300 μ l/well), pats dry.
(6) close: confining liquid 340 μ l/well.Add and be placed in 37 DEG C and hatch 2.5h.
(7) configuration of PBST: add the Tween20 of 0.05% in the PBS solution of the 0.01M pH7.4 configured, mixing.
(8) primary antibodie diluent is configured: the OVA of 0.25-0.5mg/mL, PBS configure.
(9) antibody dilution:
The selection of antibody concentration: with primary antibodie diluent, serum is diluted to respectively: 1:1000,3000,9000,27000,81000 five extent of dilution, and make negative control with the normal mouse serum of 1:1000, (be added on the 1-2 of elisa plate respectively, 3-4 as hatching primary antibodie, 5-6, in 7-8,9-10,11-12 row).
(10) plate is taken out, get rid of liquid in hole, wash 3 times with PBST, pat dry.
(11) primary antibodie Incubating Solution is added, 100 μ l/well.Add and hatch 2h.
(12) two anti-Incubating Solutions are configured.The thick OVA PBS of 1% is diluted to the diluent of 0.1%, adds ELIAS secondary antibody (sheep anti-mouse igg ELIAS secondary antibody), be diluted to 1:500.
(13) plate is taken out, get rid of liquid in hole, wash 5 times with PBST, pat dry.
(14) two anti-Incubating Solutions (100 μ l/well) are added.Add, hatch 1.5h for 37 DEG C.
(15) 0.1M Citric acid configures: 0.19213g Citric acid adds tri-distilled water and dissolves and be settled to 10mL.
(16) 0.2M Na
2hPO
4configuration: 0.716g Na
2hPO
4add tri-distilled water dissolve and be settled to 10mL.
(17) 0.2M H
2sO
4configuration: dense H
2sO
4: H2O=1:8.
(18) ELISA nitrite ion: 4.86mL 0.1M Citric acid liquid and 5.14mL 0.2M Na
2hPO
4liquid mixes, and adds after 6mg OPD fully dissolves and adds 20 μ l H again
2o
2, lucifuge is for subsequent use.
(19) plate is taken out, get rid of liquid in hole, 4 PBST, 2 PBS.
(20) develop the color: lucifuge adds above-mentioned developer (100 μ l/well).0.2M H is added after 3min
2sO
4(50 μ l/well) termination reaction.
(21) absorption (OD value) of above-mentioned solution at 490nm wavelength place is surveyed.(note OF: exceed instrument detection limit)
Result judges: with the OD value in Sample serum hole about 1.0, and the antibody dilution corresponding to the hole of P/N value more than 2.1 be this serum tire that (P represents the OD value in measuring samples serum hole; N represents the OD value in negative control sera hole).
Four, cytogamy, filtering hybridoma and colonized culture
1. feeder layer cells preparation
The day before yesterday of cytogamy, getting 1 Kunming mouse draws neck to put to death, this mouse is soaked in 5-10min in 75% alcohol, fully mouse peritoneum is exposed from the aseptic skin of cutting off of mouse web portion in Bechtop, after disinfecting in alcohol, inject RPMI RPMI-1640 and eluriate mouse peritoneal, eluriate cell injection centrifuge tube centrifugal, supernatant discarded, lower sediment is feeder layer cells very, the resuspended above-mentioned feeder layer cells of 30mL complete culture solution, 100 μ l/Well drip in 96Well cell culture plate well, be placed in cell culture incubator to cultivate, within second day, tentatively check normally and continue to cultivate.
2. splenocyte preparation
Choose indirect ELISA detect serum titer the highest exempted from mouse, pluck eyeball blood sampling, antiserum(antisera) is used for antibody and analyzes further or do positive antibody contrast, after disconnected neck is put to death, asepticly after this mouse is soaked in 75% alcohol disinfecting get spleen, remove reticular tissue, prepare splenocyte suspension, be transferred in centrifuge tube, RPMI RPMI-1640 washing reticular tissue, merging is centrifugal afterwards abandons supernatant, adds RPMI RPMI-1640 to 30mL, counts for subsequent use.
3. myeloma cell's preparation
A. at fusion first about 10 days recovery Sp2/0 cells, go down to posterity by 1:4-5 with complete culture medium culturing 2-3 days, an about 100-300 cell is inoculated in 1 piece of 96 porocyte culture plate and cultivates about one week, microscopy its clone's number and clone cell upgrowth situation, as also shown that the substratum containing 20% foetal calf serum prepared effectively can support that Sp2/0 cell becomes mono-clonal to grow, for next step test lays the foundation the while that the good mono-clonal growth table clear-cells culture plate of 70% cell energy being qualified.
B. during passage, pass 2 bottles of cells by 1:10, one bottle adds 1 × HAT, and parallel test done by another bottle, and cultivate microscopy after 3 days, 1 × HAT substratum inner cell is all dead routinely, and parallel group of Growth of Cells is vigorous, illustrates that this cell strain has susceptibility to HAT.
C. first 2 days are merged, Sp2/0 cell is carried out the cultivation of 1:4-5 sub-bottle, within second day, mend the substratum that doubles to continue to cultivate, make cell be in logarithmic phase (guaranteeing that rich cells rate is greater than 98%) before fusion, therefrom select 6 bottles of cells, piping and druming cell is transferred in 50mL centrifuge tube, the centrifugal 10min of 1000r/min, remove substratum, add 10mL RPMI1640 substratum suspension cell, for subsequent use after counting.
4. cytogamy
By number ratio splenocyte: myeloma cell=8:1, gently after mixing, the centrifugal 10min of 1000r/min, removes substratum, and bullet pine cell precipitation block, puts centrifuge tube in 37 DEG C of sterilizing warm water bath.Drip the cytogamy agent PEG1500 of 1mL 37 DEG C of incubations, stir 90s, add 1mLRPMI 1640 substratum (stirring of limit edged) 60s, add 1mLRPMI 1640 substratum (stirring of limit edged) 60s, add 1.5mL RPMI1640 substratum (stirring of limit edged) 60s, add 1.5mLRPMI 1640 substratum (stirring of limit edged) 60s, add 5mLRPMI 1640 substratum (stirring of limit edged) 60s, the centrifugal 7min of 800r/min, remove substratum, bullet pine cell precipitation block, add the selective medium containing HAT gently, the above-mentioned selective medium containing HAT of 100mL joining 37 DEG C of incubations in advance gently after shake prepares fused cell suspension.
5. cell cultures
Being dripped by 100 μ l/Well by above-mentioned fused cell suspension has in 96 porocyte culture plates of feeder cell individual layer, in CO in length in advance
2cultivate in incubator, second day, 100 μ l HAT (2.5 ×) substratum are mended in every hole, 4th day, HAT (1 ×) substratum changes liquid once, the 7th day, and HT (1 ×) substratum changes liquid once, tenth day, HT (1 ×) substratum changes liquid, and generaI investigation cell growth state, formulates race-card, mark, is ELISA in time according to cell quantity and detects.
6. the screening of hybridoma
After fusion when hybridoma number to a certain extent, just can carry out ELISA and detect screening the hybridoma of secretory antibody.Generally 3-4 days after changing liquid last time, so that antibody accumulation.
7. the cloning of hybridoma
Adopt limiting dilution assay to carry out subclone to the positive hole detected, within 10-15 days, select mono-clonal hole to be ELISA and detect, secondary subclone is carried out to positive hole, until antibody positive rate reaches 100%.Selection antibody strong positive, the clone that Growth of Cells is good carry out amplification cultivation, frozen, conservation.
Five, ascites preparation
Choose the Balb/C Healthy female mouse in female 6-8 age in week, every only injection 500 μ l pristane sensitization, collect hybridoma after 7-10 days, RPMI 1640 substratum washes cell twice, gets 1 × 10
6individual cell infusion is in mouse abdominal cavity, and visible mouse abdominal cavity enlargement in 7-10 days, gathers ascites with asepsis injector in mouse abdominal cavity, centrifugal segregation cell upper-layer fat and lower confluent monolayer cells, goes sample segment to do ELISA mensuration and tires and IC
50, all the other packing are frozen for subsequent use.
Six, the mensuration of ascites purifying and antibody titer, antibody I C
50the mensuration of value
Get ascites prepared by step G, use ice-water bath precooling, to prepare after 0.01M PBS doubly dilutes (a ascites+three parts of 0.01M PBS (pH7.4)) liquid, get 1ml protein G resin by 10mg ascites total protein to measure, be filled in antibody protein affinity column bed, balanced chromatography column with the precooling 0.01M PBS (pH7.4) of resin 50 × volume.Make Monoclonal Antibodies in Mice Ascites albumen fully with protein G specific binding, fully wash chromatography column to remove the foreign protein in adhesion chromatography column with the precooling 0.01M PBS (pH7.4) of 50 × volume.100 μ l 1M Tris-Cl (pH9.6) are respectively added in advance in 1.5ml EP pipe, again the chromatography column of above-mentioned process is added frozen water precooling 0.1M glycine-HCl (pH 2.7) and carry out wash-out, filtrate presses 0.9ml/ pipe fraction collection in the EP pipe of above-mentioned process, and often pipe is got and surveyed OD a little
280protein peak, merges protein peak pipe, and amalgamation liquid is placed in dialysis tubing, dialysed overnight in ice-water bath 0.01M PBS (pH7.4).Get this treatment solution a little, carry out the indirect competitive ELISA analysis of indirect ELISA and free Ractopamine hydrochloride do competition antigen.
Seven, the qualification of ractopamine monoclonal antibody
1. indirect ELISA measures antibody titer
The technical parameter of chessboard ELISA: A-G line envelope antigen (BSA-RAC) concentration is respectively: 9000,3000,1000,333,111,37,12.3ng/ml.H line wraps and by contrast antigen (BSA) concentration is: 3000ng/ml.1-2,3-4,5-6,7-8,9-10 row add respectively 1:100000,200000,400000,800000,16,000,000,000 dilution monoclonal antibodies.The normal balb/c mouse serum that 11-12 row add 1:100000 dilution compares.Choose: 3000ng/ml envelope antigen (BSA-RAC) makes working concentration envelope antigen, 1:80000, the monoclonal antibody of 160000 times of dilutions is made working concentration primary antibodie and is carried out ELISA test.The monoclonal antibody measured is tired as 1:800000.The numerical value listed in table 2 is the numerical value measured at 450nm wavelength.
The mensuration that table 2 Rac monoclonal antibody is tired
| ? | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| A | ND | ND | ND | ND | 3.73 | 3.82 | 2.15 | 2.20 | 1.19 | 1.21 | 0.15 | 0.15 |
| B | ND | ND | ND | ND | 2.78 | 2.86 | 1.45 | 1.50 | 0.82 | 0.83 | 0.14 | 0.14 |
| C | ND | ND | 3.71 | 3.61 | 1.81 | 1.74 | 0.98 | 0.95 | 0.54 | 0.55 | 0.14 | 0.14 |
| D | 3.75 | 3.84 | 2.28 | 238 | 1.11 | 1.1 | 0.57 | 0.59 | 0.36 | 0.35 | 0.14 | 0.14 |
| E | 2.07 | 2.22 | 1.24 | 0.62 | 0.58 | 0.60 | 0.35 | 0.36 | 0.24 | 0.24 | 0.14 | 0.13 |
| F | 1.06 | 1.07 | 0.61 | 0.17 | 0.33 | 0.35 | 0.25 | 0.26 | 0.19 | 0.19 | 0.14 | 0.13 |
| G | 0.64 | 0.65 | 0.41 | 0.17 | 0.24 | 0.25 | 0.20 | 0.19 | 0.16 | 0.17 | 0.14 | 0.15 |
| H | 0.14 | 0.15 | 0.14 | 0.17 | 0.15 | 0.17 | 0.14 | 0.15 | 0.15 | 0.14 | 0.14 | 0.15 |
Note: ND:overflow.
2, indirect competitive ELISA measures IC
50
Main technical details: envelope antigen (BSA-RAC) working concentration 3000ng/ml.Contrast antigen (BSA) working concentration 3000ng/ml, primary antibodie working concentration 1:80000; The concentration of free Ractopamine hydrochloride: 0.125,0.25,0.5,1.0,2.0,4.0,8.0ng/ml.The numerical value listed in table 3 is the numerical value measured at 450nm wavelength.
Table 3 monoclonal antibody IC
50mensuration
| RAC concentration | OD value (G) | G-X | (G-X)/G (inhibiting rate) |
| 0 | 1.645 | ? | ? |
| 0.125 | 1.615 | 0.03 | 0.018237082 |
| 0.25 | 1.359 | 0.286 | 0.173860182 |
| 0.5 | 1.012 | 0.633 | 0.384802432 |
| 1 | 0.562 | 1.083 | 0.658358663 |
| 2 | 0.341 | 1.304 | 0.792705167 |
| 4 | 0.221 | 1.424 | 0.865653495 |
| 8 | 0.119 | 1.526 | 0.927659574 |
Inhibiting rate is the concentration of a half RAC of (G-X)/G, is the IC of this monoclonal antibody
50value; With the logarithm of concentration for X-coordinate, take inhibiting rate as ordinate zou, curve plotting, as shown in Figure 6.From curve: the IC of this monoclonal antibody
50=0.35ppb.
Eight, the specificity of antibody
The specificity of antibody refer to ability that its homospecificity antigen Ractopamine hydrochloride combines with such the comparing of analogue binding ability.General cross reacting rate is as judgement criteria.The compound cross reaction that antibodies specific is good is little.
Concentration gradient is become to carry out indirect ELISA competition experiments various beta-receptor stimulant standard substance doubling dilution respectively, drawing standard curve, calculates the monoclonal antibody of preparation to the cross reacting rate (see table 4: cross reacting rate experimental result) of various analogue with following formula.
Table 4 cross reacting rate experimental result
| Beta-receptor stimulant | Cross reacting rate (%) |
| Ractopamine hydrochloride | 100 |
| Salbutamol | <0.005 |
| Cimaterol | <0.005 |
| Mabuterol | <0.005 |
| Clenbuterol | <0.005 |
| Dopamine HCL | <0.005 |
Embodiment 2
The haptenic preparation of Ractopamine hydrochloride
Specific experiment step is:
(1) compound 1 (164mg, 1.0mmol, 1.0eq) is dissolved in 4mL acetonitrile, under stirring, adds NBS (N-bromo-succinimide) (240mg, 1.5mmol, 1.5eq), stirred at ambient temperature reaction 4h.React and added 25mL H
2o, EA (ethyl acetate) (3 × 10mL) extracts, and merges organic phase, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure, dry column chromatography, DCM (methylene dichloride) is eluent, normal pressure wash-out, obtains yellow oil 345mg and intermediate 2.Productive rate: 94.6%.1H?NMR(400MHz,CDCl
3)δ7.28(d,J＝1.5Hz,1H),7.02(dd,J＝8.3,1.6Hz,1H),6.91(d,J＝8.3Hz,1H),5.62(s,1H),2.79(d,J＝6.9Hz,2H),2.73(d,J＝7.0Hz,2H),2.14(s,3H)。
(2) intermediate 2 (0.88g, 3.6mmol, 1.0eq) is joined in dry two-mouth bottle, add [two (diphenylphosphino) ferrocene of 1,1'-] palladium chloride (485mg, 0.73mmol, 0.2eq), substitute N
2repeatedly, with syringe, TEA (triethylamine) 5mL, DMF (DMF) 29mL is injected two-mouth bottle, inject ethyl propenoate, again substitute N
2repeatedly, reflux at 110 DEG C stirring reaction 20h, and reaction is finished, and adds the dilution of 200mL water, EA (100mL × 3) extracts, merge organic phase, washing (3 × 150mL), anhydrous sodium sulfate drying, filter, concentrating under reduced pressure, dry column chromatography, eluent system is converted into DCM ︰ MeOH system by PE:EA system.Product is yellow oil, puts refrigerator overnight and obtains faint yellow solid 975mg and intermediate 3.Productive rate: 52%.1H?NMR(400MHz,CDCl
3)δ7.97(d,J＝16.2Hz,1H),7.04(dd,J＝8.2,1.8Hz,1H),6.83(s,1H),6.76(d,J＝8.3Hz,1H),6.61(d,J＝16.1Hz,1H),4.27(q,J＝7.1Hz,2H),2.82(t,J＝7.0Hz,2H),2.74(t,J＝7.1Hz,2H),2.14(s,3H),1.34(t,J＝7.1Hz,3H)。
(3) join in eggplant-shape bottle by intermediate 3 (80mg, 0.3mmol, 1.0eq), add MeOH3mL, portion-wise with caution adds Pd/C (10%) (60mg, 0.75eq) on a small quantity and substitutes H
2repeatedly, 6h is reacted under room temperature.React, reaction solution diatomite filtration, MeOH washing leaching cake, concentrating under reduced pressure, obtained crude product 70mg and intermediate 4, directly throw next step.Productive rate: 90%.
(4) by intermediate 4 (1.5g, 5.65mmol, 1.0eq) be dissolved in 21mL MeOH, add compound 5 (1.07g, 5.65mmol, 1.0eq), TEA (849mg, 8.32mmol, 2.0eq), stirring reaction 30min, add STAB-H (triacetoxy borohydride hydrogen sodium) (3.55g, 12.48mmol, 3.0eq), just add and had a large amount of bubble and emerge, reflux at 60 DEG C stirring reaction 30h, add STAB-H (1.60g, 5.66mmol, 1.0eq), continue stirring reaction to spend the night, second time mends STAB-H (1.60g, 5.66mmol1.0eq), continue stirring reaction 10h, 50mL water is added in reaction solution, EA (30mL × 3) extracts, merge organic phase, saturated aqueous ammonium chloride (3 × 30mL) washs organic phase (except TEA), organic phase is dry, concentrating under reduced pressure, dry column chromatography, eluent system: D:M=20:1-10:1, obtain yellow oil, DCM, ether is concentrated obtains white powder 700mg and intermediate 6.Productive rate: 31%.1H?NMR(400MHz,CD
3OD)δ7.24(t,J＝6.9Hz,2H),6.95(s,1H),6.91(d,J＝8.3Hz,1H),6.79(d,J＝8.3Hz,2H),6.69(d,J＝8.1Hz,1H),4.83(s,1H),4.07(q,J＝7.2Hz,2H),3.24(s,1H),3.08(d,J＝4.4Hz,2H),2.85(t,J＝7.5Hz,2H),2.69(s,1H),2.58(t,J＝7.6Hz,2H),2.54(s,1H),2.07(s,1H),1.81(dd,J＝13.8,9.1Hz,1H),1.38(t,J＝6.1Hz,3H),1.20(t,J＝7.2Hz,3H)。
(5) by intermediate 6 (118mg, 0.29mmol, 1.0eq) be dissolved in 10mL MeOH, add 0.5mL 1M NaOH (17mg, 0.43mmol, 1.45eq), reflux at 70 DEG C stirring reaction 7h, adds 1.0mL 1M HCl after reacting completely, and concentrating under reduced pressure is except desolventizing (comprising water), dry column chromatography, eluent system: D:M=20:1-5:1, obtains yellow oil, adds diethyl ether concentrating under reduced pressure repeatedly, vacuum-drying, obtains white solid powder 50mg and Ractopamine hydrochloride haptens 7.Productive rate: 46%.1H?NMR(400MHz,DMSO-d
6)δ9.50(s,1H),7.18(d,J＝8.0Hz,2H),6.91(s,1H),6.85(d,J＝7.6Hz,1H),6.76(d,J＝7.9Hz,2H),6.71(d,J＝7.8Hz,1H),5.76(s,1H),4.82(t,J＝10.9Hz,1H),3.14(d,J＝15.4Hz,2H),3.03–2.86(m,2H),2.70(t,J＝7.5Hz,2H),2.55(s,1H),2.44(t,J＝7.6Hz,2H),2.01(s,1H),1.68(s,1H),1.25(t,J＝7.1Hz,3H).13C?NMR(101MHz,CD
3OD)δ181.5,161.3,157.6,135.8,135.2,133.9,131.6,131.2,131.1,119.3,72.9,58.0,55.0,38.9,38.3,34.6,30.1,19.4,18.8.HRMS:[M
+H
+]:Found?m/z374.1963,Calcd?m/z?374.1889。
The synthetic route schematic diagram of step (1)-(5) as shown in Figure 2.
The preparation of ractopamine artificial antigen and envelope antigen and detection; Cytogamy, filtering hybridoma and colonized culture; Prepared by ascites; The mensuration of ascites purifying and antibody titer; Antibody I C
50the mensuration of value; The qualification of ractopamine monoclonal antibody; The specificity of antibody; Above-described method steps is all with embodiment 1.
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. a Ractopamine hydrochloride haptens, its structural formula is following formula I:
2. the haptenic preparation method of a kind of Ractopamine hydrochloride according to claim 1, comprises the steps: that compound 1 obtains intermediate 2 by bromo-reaction; Described intermediate 2 is obtained by reacting intermediate 3 with ethyl propenoate; Described intermediate 3 obtains intermediate 4 through reduction reaction; Described intermediate 4 is obtained by reacting intermediate 6 with compound 5; Described intermediate 6 obtains described Ractopamine hydrochloride haptens 7 through hydrolysis reaction;
3. the haptenic preparation method of a kind of Ractopamine hydrochloride according to claim 2, it is characterized in that comprising the steps: that described compound 1 is dissolved in acetonitrile by (a), add N-bromo-succinimide, described compound 1 and the ratio of the add-on of described N-bromo-succinimide are 1:1 ~ 1.5 times equivalents, react 2-4h under room temperature, obtain described intermediate 2; B () adds [1 in described intermediate 2, two (diphenylphosphino) ferrocene of 1'-] palladium chloride, substitute nitrogen, add triethylamine, N, dinethylformamide and ethyl propenoate, again substitute nitrogen, reflux at 90-110 DEG C stirring reaction 15-20h, obtains described intermediate 3; C () adds methyl alcohol and catalyst P d/C in described intermediate 3, substitute hydrogen, reacts 3-6h under room temperature, obtains described intermediate 4; D described intermediate 4 is dissolved in methyl alcohol by (), add compound 5, triethylamine stirring reaction 10-30min, add triacetoxy borohydride hydrogen sodium, after the stirring reaction 20-30h that refluxes, obtains described intermediate 6 at 36-60 DEG C; E described compound 6 is dissolved in methyl alcohol by (), add NaOH, and reflux at 50-70 DEG C stirring reaction 5-7h, obtains described Ractopamine hydrochloride haptens RAC-CA.
4., according to a kind of ractopamine artificial antigen prepared by claim 1, its structural formula is as shown in the formula II:
5. the preparation method of a kind of ractopamine artificial antigen according to claim 4, is characterized in that: described Ractopamine hydrochloride haptens and bovine serum albumin coupling are obtained described ractopamine artificial antigen.
6. a kind of ractopamine monoclonal antibody of preparing of a kind of ractopamine artificial antigen according to claim 4, it is characterized in that: described ractopamine monoclonal antibody is secreted by hybridoma cell strain 12B2, described hybridoma is preserved in China typical culture collection center, and deposit number (CCTCC NO) is: C2014127.
7. the application of Ractopamine hydrochloride haptens according to claim 1, or ractopamine artificial antigen according to claim 4 in the described ractopamine monoclonal antibody of preparation.
8. the application of Ractopamine hydrochloride haptens according to claim 1, or ractopamine artificial antigen according to claim 4 at Ractopamine hydrochloride in qualitative and detection by quantitative.
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