CN102816196A - Tildipirosin hapten and artificial antigen as well as preparation methods thereof - Google Patents
Tildipirosin hapten and artificial antigen as well as preparation methods thereof Download PDFInfo
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- CN102816196A CN102816196A CN2012103317989A CN201210331798A CN102816196A CN 102816196 A CN102816196 A CN 102816196A CN 2012103317989 A CN2012103317989 A CN 2012103317989A CN 201210331798 A CN201210331798 A CN 201210331798A CN 102816196 A CN102816196 A CN 102816196A
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- tildipirosin
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- 239000000427 antigen Substances 0.000 title claims abstract description 61
- 102000036639 antigens Human genes 0.000 title claims abstract description 61
- 108091007433 antigens Proteins 0.000 title claims abstract description 61
- 238000002360 preparation method Methods 0.000 title claims abstract description 44
- HNDXPZPJZGTJLJ-UEJFNEDBSA-N (4r,5s,6s,7r,9r,11e,13e,15r,16r)-6-[(2r,3r,4s,5s,6r)-4-(dimethylamino)-3,5-dihydroxy-6-methyloxan-2-yl]oxy-16-ethyl-4-hydroxy-5,9,13-trimethyl-7-(2-piperidin-1-ylethyl)-15-(piperidin-1-ylmethyl)-1-oxacyclohexadeca-11,13-diene-2,10-dione Chemical compound O([C@@H]1[C@@H](C)[C@H](O)CC(=O)O[C@@H]([C@H](/C=C(\C)/C=C/C(=O)[C@H](C)C[C@@H]1CCN1CCCCC1)CN1CCCCC1)CC)[C@@H]1O[C@H](C)[C@@H](O)[C@H](N(C)C)[C@H]1O HNDXPZPJZGTJLJ-UEJFNEDBSA-N 0.000 title abstract description 14
- 229960002659 tildipirosin Drugs 0.000 title abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 31
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 12
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 11
- 150000008064 anhydrides Chemical class 0.000 claims abstract description 8
- 150000002148 esters Chemical class 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 74
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 38
- 238000003756 stirring Methods 0.000 claims description 32
- 238000006243 chemical reaction Methods 0.000 claims description 28
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 25
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 25
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 24
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 23
- 229940098773 bovine serum albumin Drugs 0.000 claims description 23
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 239000000872 buffer Substances 0.000 claims description 16
- 230000008859 change Effects 0.000 claims description 16
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 16
- 239000002953 phosphate buffered saline Substances 0.000 claims description 16
- 230000000890 antigenic effect Effects 0.000 claims description 12
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims description 10
- 102000002322 Egg Proteins Human genes 0.000 claims description 10
- 108010000912 Egg Proteins Proteins 0.000 claims description 10
- 210000000991 chicken egg Anatomy 0.000 claims description 10
- 238000000502 dialysis Methods 0.000 claims description 10
- 235000014103 egg white Nutrition 0.000 claims description 10
- 230000003203 everyday effect Effects 0.000 claims description 10
- 238000004321 preservation Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- 229910002027 silica gel Inorganic materials 0.000 claims description 7
- 239000000741 silica gel Substances 0.000 claims description 7
- 229960001866 silicon dioxide Drugs 0.000 claims description 7
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 claims description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- HOGDNTQCSIKEEV-UHFFFAOYSA-N n'-hydroxybutanediamide Chemical compound NC(=O)CCC(=O)NO HOGDNTQCSIKEEV-UHFFFAOYSA-N 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine Chemical compound CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- KMOUUZVZFBCRAM-OLQVQODUSA-N (3as,7ar)-3a,4,7,7a-tetrahydro-2-benzofuran-1,3-dione Chemical compound C1C=CC[C@@H]2C(=O)OC(=O)[C@@H]21 KMOUUZVZFBCRAM-OLQVQODUSA-N 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 4
- SWXVUIWOUIDPGS-UHFFFAOYSA-N diacetone alcohol Natural products CC(=O)CC(C)(C)O SWXVUIWOUIDPGS-UHFFFAOYSA-N 0.000 claims description 4
- 238000010025 steaming Methods 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 2
- 238000013016 damping Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims 1
- 238000010168 coupling process Methods 0.000 abstract description 24
- 230000008878 coupling Effects 0.000 abstract description 22
- 238000005859 coupling reaction Methods 0.000 abstract description 22
- 241001465754 Metazoa Species 0.000 abstract description 12
- 238000001514 detection method Methods 0.000 abstract description 6
- 230000008901 benefit Effects 0.000 abstract description 5
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 abstract description 4
- -1 3-carboxyl propionyl Chemical group 0.000 abstract description 3
- 238000002965 ELISA Methods 0.000 abstract 2
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 238000013537 high throughput screening Methods 0.000 abstract 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 abstract 1
- 239000007790 solid phase Substances 0.000 abstract 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 12
- 230000006837 decompression Effects 0.000 description 9
- 238000002390 rotary evaporation Methods 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 6
- 238000012797 qualification Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 3
- 238000000119 electrospray ionisation mass spectrum Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 125000000896 monocarboxylic acid group Chemical group 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 0 *OC(c1ccccc1C(O)=O)=O Chemical compound *OC(c1ccccc1C(O)=O)=O 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 241000606856 Pasteurella multocida Species 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 229940051027 pasteurella multocida Drugs 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 241000606748 Actinobacillus pleuropneumoniae Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- YULMNMJFAZWLLN-UHFFFAOYSA-N C=C1CCCCC1 Chemical compound C=C1CCCCC1 YULMNMJFAZWLLN-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000606807 Glaesserella parasuis Species 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 241000606831 Histophilus somni Species 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
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- 238000010276 construction Methods 0.000 description 1
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- 230000008588 hemolysis Effects 0.000 description 1
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- 230000002163 immunogen Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 239000003182 parenteral nutrition solution Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
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- UBCKGWBNUIFUST-YHYXMXQVSA-N tetrachlorvinphos Chemical compound COP(=O)(OC)O\C(=C/Cl)C1=CC(Cl)=C(Cl)C=C1Cl UBCKGWBNUIFUST-YHYXMXQVSA-N 0.000 description 1
- 229940080752 zuprevo Drugs 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a tildipirosin hapten and an artificial antigen as well as preparation methods thereof, wherein the preparation method of the tildipirosin hapten comprises the following step of: introducing 3-carboxyl propionyl or 2-carboxyl benzoyl on the base of the base structure of the tildipirosin, so that the prepared tildipirosin hapten not only keeps the base structure of the tildipirosin but also is beneficial to the coupling of a carrier protein, is used for preparing the artificial antigen, and can be easily identified by an animal body; and the artificial antigen of the tildipirosin takes the tildipirosin hapten as a base, and is prepared by a mixed anhydride method or an active ester method, and the prepared tildipirosin hapten can be used for detecting the ELISA (enzyme-linked immuno sorbent assay) residual of the tildipirosin. Compared with the existing detection method for the joint use of solid phase extraction liquid-mass (SPE-HPLC-MS/MS) (solid phase extraction-high performance liquid chromatography-memory system/memory system), the preparation method has the advantages of being simple and convenient, quick, high in sensitivity, suitable for high throughput screening. The preparation method provided by the invention is used for preparing the tildipirosin hapten and the artificial antigen.
Description
Technical field
The invention belongs to field of immunology, be specifically related to a kind of safe ground Luo Xin haptin, artificial antigen and preparation method thereof.
Background technology
Safe ground Luo Xin (Tildipirosin) is a kind of novel Macrolide animal specific microbiotic by Intervet-Schering Plough animal health company exploitation; The CAS accession number is: 328898-40-4; Chemical name is: 20-Deoxo-23-deoxy-5-O-[3; 6-dideoxy-3-(dimethylamino)-β-D-glucopyranosyl]-20,23-di-1-piperidinyltylonolide, structural formula is following:
On March 8th, 2011, it is the market permission application of the aseptic parenteral solution (commodity are called Zuprevo) of staple that the medicine for animals council of European Union (CVMP) has permitted with safe ground Luo Xin.The respiratory tract infection of pigs that is mainly used on veterinary clinic that bovine respiratory that treatment causes by haemolysis mannheim bacterium, pasteurella multocida and Haemophilus somnus infects and causes by actinobacillus pleuropneumoniae, pasteurella multocida, segmental bronchus sepsis boss bacillus and haemophilus parasuis.
The residual meeting of safe ground Luo Xin in animal tissues threatens food safety, and human health is worked the mischief.Given this, European Union's medicine for animals council is to residual also the formulated MRL of safe ground Luo Xin in animal food.
At present; The method that detects safe ground Luo Xin residual quantity mainly is SPE liquid-matter coupling technique (SPE-HPLC-MS/MS), because complicated plant and instrument and loaded down with trivial details operating process and the examination that reviewer's high professional qualification required to cause being not suitable for on-site supervision and great amount of samples.Immunochemical analyses method particularly Enzyme Linked Immunoadsorbent Assay (ELISA) technology has advantages such as quick, simple to operate, highly sensitive, that flexibility is strong, is fit to the screening of high-throughput sample.Therefore, the ELISA method is for residual the have more advantage of rapid detection safe ground Luo Xin in animal tissues.
Summary of the invention
The technical problem that the present invention will solve provides a kind of safe ground Luo Xin haptin, artificial antigen and preparation method thereof.The synthetic haptin has not only kept the substruction of safe ground Luo Xin but also has helped and carrier protein couplet, is used for the preparation of artificial antigen, is easy to discerned by the animal body; Artificial antigen can be used for the ELISA residue detection of safe ground Luo Xin, compares with SPE liquid-matter coupling (SPE-HPLC-MS/MS) detection method of present use, has the advantage of easy, quick, highly sensitive, suitable high flux screening.
For solving the problems of the technologies described above, the technical scheme that the present invention taked is:
A kind of safe ground Luo Xin haptin, its molecular structural formula is:
The present invention also provides above-mentioned safe ground Luo Xin haptenic preparation method, and it carries out according to the following steps order:
(11) with acetone or methyl alcohol Thailand ground Luo Xin is dissolved, process solution A;
(12) under the whipped state, in solution A, drip the acetone soln of Succinic anhydried or Tetra hydro Phthalic anhydride, dropwise the continued stirring reaction,, get clear solution to reacting completely;
(13) steaming desolventizes, and obtains faint yellow amorphous powder, the dissolving of back adding methylene dichloride, and washing, with alkali lye adjusting pH value, the separate dichloromethane layer, drying is filtered, and steaming desolventizes, and gets thick product; Cross silicagel column, concentrate, obtain safe ground Luo Xin haptin.
As a kind of qualification of the present invention, among the above-mentioned preparation method:
1. the ratio of quality and the number of copies of safe ground Luo Xin and acetone or methyl alcohol is 1:10～15 in the described step (11);
2. the ratio of quality and the number of copies of the acetone soln of solution A and Succinic anhydried or Tetra hydro Phthalic anhydride is 1:1～2 in the described step (12); Dropwise back 25 ℃ of stirring reaction 15 h;
3. wash in the described step (13) to the pH value be 5.5～7.0;
Described alkali lye is the NaOH of 1mol/L, regulates pH value to 10 with it;
Cross the used elutriant of silicagel column and be volume ratio and be 1～4: 1 the ETHYLE ACETATE and the mixture of sherwood oil.
The present invention also provides a kind of safe ground Luo Xin artificial antigen, and its molecular structural formula is:
The present invention also provides above-mentioned safe ground Luo Xin artificial antigenic preparation method, and it is to adopt mixed anhydride method or active ester method to prepare described safe ground Luo Xin artificial antigen.
As another kind of qualification the of the present invention, said mixed anhydride method is carried out according to the following steps order:
(21) tri-n-butylamine with 0.8-1.0mmol safe ground Luo Xin haptin and equivalent is dissolved in the 10-20mL dioxan; After stirring 30 min, add the isobutyl chlorocarbonate of equivalent, regulate pH to 7.5-8.5 with the NaOH solution of 1 mol/L; Stirring reaction 1 h makes solution B;
(22) get the dioxan and the phosphate buffered saline buffer 50-100 mL of the carrier proteins of 10 mg/mL in addition, solution B is dropped in the damping fluid,, make solution C at 3-5 ℃ of following stirring reaction 24 h;
(23) with solution C in 4 ℃ with distill water dialysis 3 days, change liquid every day 4 times, lyophilize, safe ground Luo Xin artificial antigen M1 ,-20 ℃ of preservations.
As further qualification, the carrier proteins described in the above-mentioned steps (22) is bovine serum albumin or chicken egg white.
The present invention also has a kind of qualification, and said active ester method is carried out according to the following steps order:
(31) with the N of 0.8～1.0mmol; N ,-NSC 57182 are dissolved in 10～20mL dioxan, splash under the whipped state in the dioxan solution of safe ground Luo Xin haptin and N-hydroxy-succinamide of equivalent; Stirring at room is reacted 24 h, makes solution D;
(32) solution D is centrifugal, get in the dioxan and phosphate buffered saline buffer of 10mg/mL carrier proteins that supernatant drops to 50～100 mL, 3～5 ℃ of stirring reaction 24 h make solution E;
(33) with solution E in 4 ℃ with distill water dialysis 3 days, change liquid every day 4 times, lyophilize gets safe ground Luo Xin artificial antigen M2 ,-20 ℃ of preservations.
As further qualification, the carrier proteins described in the step (32) is bovine serum albumin or chicken egg white.
Owing to adopted above-mentioned technical scheme, the present invention compared with prior art, the technical progress that is obtained is:
The present invention is the i.e. structural derivative of safe ground Luo Xin of haptin that precursor has synthesized safe ground Luo Xin with safe ground Luo Xin; This verivate is introduced bridge construction and is used for the proteic reactive group of coupling carrier on the basis that keeps safe ground Luo Xin substruction; Help itself and carrier protein couplet; Fully expose the substruction of the less safe ground Luo Xin of molecular weight own after the coupling again; The constitutional features of promptly safe ground Luo Xin does not have the proteic space structure of suppressed by vector to cover, and avoids it to be buried by macromole and influences the identification of animal body.Prepared safe ground Luo Xin haptin has not only kept the substruction of safe ground Luo Xin but also has helped and carrier protein couplet, is used for the preparation of artificial antigen, is easy to discerned by the animal body; Its artificial antigen can be used for the ELISA residue detection of safe ground Luo Xin, compares with SPE liquid-matter coupling technique (SPE-HPLC-MS/MS) detection method of present use, has the advantage of easy, quick, highly sensitive, suitable high flux screening.Preparing method of the present invention is used for the preparation of safe ground Luo Xin haptin, artificial antigen.
The present invention below will combine specific embodiment to do further explain.
Embodiment
1 one kinds of safe ground of embodiment Luo Xin haptin, artificial antigen and their preparation method
1.
A kind of safe ground Luo Xin haptin and preparation method thereof
A kind of safe ground its structure of Luo Xin haptin is following:
The haptenic a kind of preparation method of above-mentioned safe ground Luo Xin, carry out according to the following steps order:
(11) at exsiccant N
2Under the condition, 1 part of safe ground Luo Xin with 10 parts of acetone solutions, is processed solution A 1;
(12) under the whipped state, continue to feed N
2, and in solution A 1, drip the acetone soln of 1.5 parts of Succinic anhydrieds, dropwise back 25 ℃ of stirring reaction 15 h, to reacting completely, clear solution;
(13) the decompression rotary evaporation removes and desolvates, and obtains faint yellow amorphous powder, and the back adds methylene dichloride dissolving, washing; Using the 1mol/L sodium hydroxide solution to be adjusted to the pH value is 10, and the separate dichloromethane layer is used anhydrous magnesium sulfate drying; Filter, the decompression rotary evaporation is removed solvent, gets thick product; Cross silicagel column, elutriant is that volume ratio is 2: 1 the ETHYLE ACETATE and the mixture of sherwood oil, and the product pipe is merged, and the decompression rotary evaporation gets safe ground Luo Xin haptin to doing.
Above-mentioned preparing method's reaction process is following:
The synthetic haptin is carried out the ESI mass spectrum identify to have [M-1]
-Molecular ion peak 833.5.
Ultimate analysis (C
45H
75N
3O
11) measured value (theoretical value, %): C 64.82 (64.80), and H 9.05 (9.06), and N 5.06 (5.04), and O 21.07 (21.10).Show in the safe ground Luo Xin hapten molecule to contain 45 C, 75 H, 3 N, 11 O, molecular weight are 834.09.
1HNMR(CDCl
3)δ(ppm):0.90(t,3H,CH
2C
H 3),0.96(d,3H,4-CH
3),1.16(d,3H,8-CH
3),1.18(d,3H,5-CH
3),1.20(d,1H,CH),1.35(d,2H,CH),1.45(d,1H,CH),1.53(m,8H,CH
2),
1.57(m,2H,CH
2),1.59(m,4H,CH
2),1.79(q,1H,CH),2.05(q,1H,CH),2.21(d,3H,CH
3),2.23(s,1H,CH),2.26(s,6H,N(CH
3)
2),2.27(s,1H,CH),2.43(t,2H,CH
2),2.45(t,8H,CH
2),2.48(d,1H,CH),2.52(d,1H,CH),2.52(t,1H,CH),2.54(t,2H,CH
2),2.81(t,1H,CH),2.83(t,2H,CH
2),2.99(t,1H,CH),3.40(t,1H,CH),3.55(t,1H,CH),3.58(s,1H,OH),3.60(s,1H,OH),3.84(t,1H,CH),3.85(d,1H,CH),3.98(t,1H,CH),4.62(t,1H,CH),5.44(t,1H,CH=C),5.54(d,1H,CH),6.33(d,1H,CO-CH=CH),7.40(d,1H,CO-CH=CH),?11.28(s,1H,COOH)。
2.
A kind of safe ground Luo Xin artificial antigen and preparation method thereof
A kind of safe ground its structure of Luo Xin artificial antigen is following:
Prepare the artificial antigenic method of above-mentioned safe ground Luo Xin, it adopts the active ester method preparation and gets, and it carries out according to the following steps order:
(31) with the N of 0.8mmol; N ,-NSC 57182 (DCC) is dissolved in the 20mL dioxan, splashes under the whipped state in the dioxan solution of above-mentioned safe ground Luo Xin haptin and N-hydroxy-succinamide of equivalent; Stirring at room is reacted 24 h, makes solution D 1;
(32) solution D 1 is centrifugal, get in the dioxan and phosphate buffered saline buffer of 10mg/mL bovine serum albumin that supernatant drops to 50 mL, 5 ℃ of stirring reaction 24 h make solution E 1;
(33) with solution E 1 in 4 ℃ with distill water dialysis 3 days, change liquid every day 4 times, lyophilize gets safe ground Luo Xin artificial antigen M21 ,-20 ℃ of preservations.
Dissolve haptin, bovine serum albumin (BSA), artificial antigen with phosphate buffered saline buffer; Be diluted to suitable concentration respectively; Carry out UV spectrum (200-400nm) scanning; The result shows that artificial antigen is compared with the UV spectrum of BSA noticeable change has taken place, and explains that haptin and BSA success coupling makes artificial antigen.Calculate its coupling ratio through the absorbance of three under same wavelength, through calculating, the coupling ratio of safe ground Luo Xin hapten molecule and BSA molecule is 18: 1.
Above-mentioned preparing method's reaction process is following:
The artificial antigenic preparation method of 2 one kinds of safe ground of embodiment Luo Xin
Like embodiment 1 described safe ground Luo Xin artificial antigen, it adopts the mixed anhydride method preparation and gets, and it carries out according to the following steps order:
(21) tri-n-butylamine of the above-mentioned safe ground of 1.0mmol Luo Xin haptin with equivalent is dissolved in the 15mL dioxan, stir 30 min after, add the isobutyl chlorocarbonate of equivalent, with 1mol/L NaOH solution adjusting pH to 7.5, stirring reaction 1 h makes solution B 1;
(22) solution B 1 is dropped in the dioxan and phosphate buffered saline buffer of chicken egg white (OVA) of 70 mL, 10 mg/mL, 5 ℃ of stirring reaction 24 h make solution C 1;
(23) with solution C 1 in 4 ℃ with distill water dialysis 3 days, change liquid every day 4 times, lyophilize gets safe ground Luo Xin artificial antigen M11 ,-20 ℃ of preservations.
Dissolve haptin, chicken egg white (OVA), artificial antigen with phosphate buffered saline buffer; Be diluted to suitable concentration respectively; Carry out UV spectrum (200-400nm) scanning; The result shows that artificial antigen is compared with the UV spectrum of OVA noticeable change has taken place, and explains that haptin and OVA success coupling makes artificial antigen.Calculate its coupling ratio through the absorbance of three under same wavelength, through calculating, the coupling ratio of safe ground Luo Xin hapten molecule and OVA molecule is 11: 1.
Above-mentioned preparing method's reaction process is following:
3 one kinds of safe ground of embodiment Luo Xin haptin, artificial antigen and their preparation method
1. a kind of safe ground Luo Xin haptin and preparation method thereof
A kind of safe ground its structure of Luo Xin haptin is following:
The haptenic preparation method of above-mentioned safe ground Luo Xin, it carries out according to the following steps order:
(11) at exsiccant N
2Under the condition, 1 part of safe ground Luo Xin with 15 parts of dissolve with methanol, is processed solution A 2;
(12) under the whipped state, continue to feed N
2, and in solution A 2, drip the methanol solution of 1.0 parts of Tetra hydro Phthalic anhydrides, and dropwise back 25 ℃ of stirring reaction 15 h, be clear solution;
(13) the decompression rotary evaporation removes and desolvates, and obtains faint yellow amorphous powder, and the back adds methylene dichloride dissolving, washing; Using the 1mol/L sodium hydroxide solution to be adjusted to the pH value is 10, and the separate dichloromethane layer is used anhydrous magnesium sulfate drying; Filter, the decompression rotary evaporation is removed solvent, gets thick product; Cross silicagel column, elutriant is that volume ratio is 4: 1 the ETHYLE ACETATE and the mixture of sherwood oil, and the product pipe is merged, and the decompression rotary evaporation gets safe ground Luo Xin haptin to doing.
Above-mentioned preparing method's reaction process is following:
The synthetic haptin is carried out the ESI mass spectrum identify to have [M-1]
-Molecular ion peak 881.5.
Ultimate analysis (C
49H
75N
3O
11) measured value (theoretical value, %): C 66.74 (66.72), and H 8.58 (8.57), and N 4.75 (4.76), and O 19.93 (19.95).Show in the safe ground Luo Xin hapten molecule to contain 49 C, 75 H, 3 N, 11 O, molecular weight are 882.13.
?1H?NMR?(?CDCl
3)δ(?ppm?):?0.90（t,3H,CH
2C
H 3）,0.96(d,3H,4-CH
3),1.16(d,3H,?8-CH
3),1.18(d,3H,5-CH
3),1.20(d,1H,CH),1.35(d,2H,CH
2),1.45(d,1H,CH),1.53(m,8H,CH
2),
1.57(m,2H,CH
2),1.59(m,4H,CH
2),1.79(q,1H,CH),2.05(m,1H,CH),2.21(d,3H,CH
3),2.23(s,1H,CH),2.26(s,6H,N(CH
3)
2),2.27(s,1H,CH),2.43(t,2H,CH
2),2.45(t,8H,CH
2),2.48(d,1H,CH),2.52(d,1H,CH),2.52(t,1H,CH),2.81(t,1H,CH),2.99(q,1H,CH),3.40(t,1H,CH),3.55(t,1H,CH),3.58(s,2H,OH),3.84(t,1H,CH),3.85(d,1H,CH),3.98(t,1H,CH),4.62(t,1H,CH),5.44(d,1H,CH=C),5.54(d,1H,CH),6.33(d,1H,CO-CH=C),7.40(d,1H,11-CH),7.86~8.32(m,4H,ArH),11.32(s,1H,COOH)。
2.
A kind of safe ground Luo Xin artificial antigen and preparation method thereof
A kind of safe ground its structure of Luo Xin artificial antigen is following:
Prepare the artificial antigenic method of above-mentioned safe ground Luo Xin, it adopts the active ester method preparation and gets, and it carries out according to the following steps order:
(31) with the N of 1.0mmol, N ,-NSC 57182 are dissolved in the 10mL dioxan, splash under the whipped state in the dioxan solution of above-mentioned safe ground Luo Xin haptin and N-hydroxy-succinamide of equivalent, and stirring at room is reacted 24 h, makes solution D 2;
(32) solution D 2 is centrifugal, get in the dioxan and phosphate buffered saline buffer of 10mg/mL bovine serum albumin that supernatant drops to 100 mL, 4 ℃ of stirring reaction 24 h make solution E 2;
(33) with solution E 2 in 4 ℃ with distill water dialysis 3 days, change liquid every day 4 times, lyophilize gets safe ground Luo Xin artificial antigen M22 ,-20 ℃ of preservations.
Dissolve haptin, bovine serum albumin (BSA), artificial antigen with phosphate buffered saline buffer; Be diluted to suitable concentration respectively; Carry out UV spectrum (200-400nm) scanning; The result shows that artificial antigen is compared with the UV spectrum of BSA noticeable change has taken place, and explains that haptin and BSA success coupling makes artificial antigen.Calculate its coupling ratio through the absorbance of three under same wavelength, through calculating, the coupling ratio of safe ground Luo Xin hapten molecule and BSA molecule is 24: 1.
Above-mentioned preparing method's reaction process is following:
The artificial antigenic preparation method of 4 one kinds of safe ground of embodiment Luo Xin
Like embodiment 3 described safe ground Luo Xin artificial antigens, it adopts the mixed anhydride method preparation and gets, and it carries out according to the following steps order:
(21) tri-n-butylamine of the above-mentioned safe ground of 0.8mmol Luo Xin haptin with equivalent is dissolved in the 20mL dioxan, stir 30 min after, add the isobutyl chlorocarbonate of equivalent, with 1mol/L NaOH solution adjusting pH to 8.5, stirring reaction 1 h makes solution B 2;
(22) solution B 2 is dropped in the dioxan and phosphate buffered saline buffer of chicken egg white of 100 mL, 10 mg/mL, 4 ℃ of stirring reaction 24 h make solution C 2;
(23) with solution C 2 in 4 ℃ with distill water dialysis 3 days, change liquid every day 4 times, lyophilize gets safe ground Luo Xin artificial antigen M12 ,-20 ℃ of preservations.
Dissolve haptin, chicken egg white (OVA), artificial antigen with phosphate buffered saline buffer; Be diluted to suitable concentration respectively; Carry out UV spectrum (200-400nm) scanning; The result shows that artificial antigen is compared with the UV spectrum of OVA noticeable change has taken place, and explains that haptin and BSA success coupling makes artificial antigen.Calculate its coupling ratio through the absorbance of three under same wavelength, through calculating, the coupling ratio of safe ground Luo Xin hapten molecule and OVA molecule is 8: 1.
Above-mentioned preparing method's reaction process is following:
5 one kinds of safe ground of embodiment Luo Xin haptin, artificial antigen and their preparation method
1. a kind of safe ground Luo Xin haptin and preparation method thereof
A kind of safe ground its structure of Luo Xin haptin is following:
The haptenic preparation method of above-mentioned safe ground Luo Xin, it carries out according to the following steps order:
(11) at exsiccant N
2Under the condition, 1 part of safe ground Luo Xin with 12 parts of acetone solutions, is processed solution A 3;
(12) under the whipped state, continue to feed N
2, and in solution A 3, drip the acetone soln of 2.0 parts of Succinic anhydrieds, dropwise back 25 ℃ of stirring reaction 15 h, clear solution;
(13) the decompression rotary evaporation removes and desolvates, and obtains faint yellow amorphous powder, and the back adds methylene dichloride dissolving, washing; Using the 1mol/L sodium hydroxide solution to be adjusted to the pH value is 10, and the separate dichloromethane layer is used anhydrous magnesium sulfate drying; Filter, the decompression rotary evaporation is removed solvent, gets thick product; Cross silicagel column, elutriant is that volume ratio is 1: 1 the ETHYLE ACETATE and the mixture of sherwood oil, and the product pipe is merged, and the decompression rotary evaporation gets safe ground Luo Xin haptin to doing.
The synthetic haptin is carried out the ESI mass spectrum identify to have [M-1]
-Molecular ion peak 833.4.
Ultimate analysis (C
45H
75N
3O
11) measured value (theoretical value, %): C 64.78 (64.80), and H 9.08 (9.06), and N 5.03 (5.04), and O 21.11 (21.10).Show in the safe ground Luo Xin hapten molecule to contain 45 C, 75 H, 3 N, 11 O, molecular weight are 834.09.
1H?NMR?(?CDCl
3?)δ(?ppm?):?0.90(t,3H,CH
2C
H 3),0.96(d,3H,4-CH
3),1.16(d,?3H,8-CH
3),1.18(d,3H,5-CH
3),1.20(d,1H,CH),1.35(d,2H,CH),1.45(d,1H,CH),1.53(m,8H,CH
2),
1.57(m,2H,CH
2),1.59(m,4H,CH
2),1.79(q,1H,CH),2.05(q,1H,CH),2.21(d,3H,CH
3),2.23(s,1H,CH),2.26(s,6H,N(CH
3)
2),2.27(s,1H,CH),2.43(t,2H,CH
2),2.45(t,8H,CH
2),2.48(d,1H,CH),2.52(d,1H,CH),2.52(t,1H,CH),2.54(t,2H,CH
2),2.81(t,1H,CH),2.83(t,2H,CH
2),2.99(t,1H,CH),3.40(t,1H,CH),3.55(t,1H,CH),3.58(s,1H,OH),3.60(s,1H,OH),3.84(t,1H,CH),3.85(d,1H,CH),3.98(t,1H,CH),4.62(t,1H,CH),5.44(t,1H,CH=C),5.54(d,1H,CH),6.33(d,1H,CO-CH=CH),7.40(d,1H,CO-CH=CH),?11.28(s,1H,COOH)。
2.
A kind of safe ground Luo Xin artificial antigen and preparation method thereof
A kind of safe ground its structure of Luo Xin artificial antigen is following:
The artificial antigenic preparation method of above-mentioned safe ground Luo Xin, it adopts the active ester method preparation and gets, and it carries out according to the following steps order:
(31) with the N of 0.9mmol, N ,-NSC 57182 are dissolved in the 15mL dioxan, splash under the whipped state in the dioxan solution of above-mentioned safe ground Luo Xin haptin and N-hydroxy-succinamide of equivalent, and stirring at room is reacted 24 h, makes solution D 3;
(32) solution D 3 is centrifugal, get in the dioxan and phosphate buffered saline buffer of 10mg/mL chicken egg white that supernatant drops to 70 mL, 3 ℃ of stirring reaction 24 h make solution E 3;
(33) with solution E 3 in 4 ℃ with distill water dialysis 3 days, change liquid every day 4 times, lyophilize gets safe ground Luo Xin artificial antigen M23 ,-20 ℃ of preservations.
Dissolve haptin, chicken egg white (OVA), artificial antigen with phosphate buffered saline buffer; Be diluted to suitable concentration respectively; Carry out UV spectrum (200-400nm) scanning; The result shows that artificial antigen is compared with the UV spectrum of OVA noticeable change has taken place, and explains that haptin and OVA success coupling makes artificial antigen.Calculate its coupling ratio through the absorbance of three under same wavelength, through calculating, the coupling ratio of safe ground Luo Xin hapten molecule and OVA molecule is 22: 1.
6 one kinds of safe ground of embodiment Luo Xin artificial antigen and preparation method thereof
Like the artificial antigenic preparation method of embodiment 1 described safe ground Luo Xin, it adopts the mixed anhydride method preparation and gets, and it carries out according to the following steps order:
(21) tri-n-butylamine of the above-mentioned safe ground of 0.9mmol Luo Xin haptin with equivalent is dissolved in the 10mL dioxan, stir 30 min after, add the isobutyl chlorocarbonate of equivalent, with 1mol/L NaOH solution adjusting pH to 8.0, stirring reaction 1 h makes solution B 3;
(22) solution B 3 is dropped in the dioxan and phosphate buffered saline buffer of bovine serum albumin of 50 mL, 10 mg/mL, 3 ℃ of stirring reaction 24 h make solution C 3;
(23) with solution C 3 in 3 ℃ with distill water dialysis 3 days, change liquid every day 4 times, lyophilize gets safe ground Luo Xin artificial antigen M13 ,-20 ℃ of preservations.
Dissolve haptin, bovine serum albumin (BSA), artificial antigen with phosphate buffered saline buffer; Be diluted to suitable concentration respectively; Carry out UV spectrum (200-400nm) scanning; The result shows that artificial antigen is compared with the UV spectrum of BSA noticeable change has taken place, and explains that haptin and BSA success coupling makes artificial antigen.Calculate its coupling ratio through the absorbance of three under same wavelength, through calculating, the coupling ratio of safe ground Luo Xin hapten molecule and BSA molecule is 15: 1.
Artificial antigen immune animal that embodiment 7 embodiment 1-6 make and ELISA competition inhibition test
Be the immunogen immune mouse with embodiment 1, embodiment 3 and embodiment 6 synthetic artificial antigens respectively, gather afterbody serum after 10 days since the each immunity of the booster immunization second time; Be that envelope antigen carries out indirect competition property elisa assay with embodiment 2, embodiment 4 and embodiment 5 synthetic artificial antigens respectively.The result shows, behind the 5th booster immunization, has obtained specific antiserum(antisera).The safe ground Luo Xin that in the antiserum(antisera) of doubling dilution, adds 10 μ g/mL suppresses, and the result compares according to absorbancy and significantly reduces, and along with the increase of serum diluting multiple, absorbancy is the gradient of significantly successively decreasing, and valence value is as shown in the table:
Claims (9)
1. safe ground Luo Xin haptin,
It is characterized in thatIts molecular structural formula is:
。
2. haptenic preparation method of safe ground Luo Xin as claimed in claim 1,
It is characterized in thatIt carries out according to the following steps order:
(11) with acetone or methyl alcohol Thailand ground Luo Xin is dissolved, process solution A;
(12) under the whipped state, in solution A, drip the acetone soln of Succinic anhydried or Tetra hydro Phthalic anhydride, dropwise the continued stirring reaction,, get clear solution to reacting completely;
(13) steaming desolventizes, and obtains faint yellow amorphous powder, adds the methylene dichloride dissolving then, and washing is regulated the pH value with alkali lye, the separate dichloromethane layer, and drying is filtered, and steaming desolventizes, and gets thick product; Cross silicagel column, concentrate, obtain safe ground Luo Xin haptin.
3. the haptenic preparation method of safe ground Luo Xin according to claim 2,
It is characterized in that:
1. the ratio of quality and the number of copies of safe ground Luo Xin and acetone or methyl alcohol is 1:10～15 in the described step (11);
2. the ratio of quality and the number of copies of the acetone soln of solution A and Succinic anhydried or Tetra hydro Phthalic anhydride is 1:1～2 in the described step (12); Dropwise back 25 ℃ of stirring reaction 15 h;
3. wash in the described step (13) to the pH value be 5.5～7.0;
Described alkali lye is the NaOH solution of 1mol/L, regulates pH value to 10 with it;
Cross silicagel column, used elutriant is that volume ratio is 1～4: 1 the ETHYLE ACETATE and the mixture of sherwood oil.
4. safe ground Luo Xin artificial antigen,
It is characterized in thatIts molecular structural formula is:
5. artificial antigenic preparation method of safe ground Luo Xin as claimed in claim 4,
It is characterized in that:It is to adopt mixed anhydride method or active ester method to prepare described safe ground Luo Xin artificial antigen.
6. the artificial antigenic preparation method of safe ground Luo Xin according to claim 5,
It is characterized in thatSaid mixed anhydride method is carried out according to the following steps order:
(21) tri-n-butylamine with 0.8～1.0mmol safe ground Luo Xin haptin and equivalent is dissolved in 10～20mL dioxan; After stirring 30 min, add the isobutyl chlorocarbonate of equivalent, regulate pH to 7.5～8.5 with the NaOH solution of 1mol/L; Stirring reaction 1 h makes solution B;
(22) get the dioxan and phosphate buffered saline buffer 50～100 mL of the carrier proteins of 10 mg/mL in addition, solution B is dropped in the damping fluid,, make solution C at 3～5 ℃ of following stirring reaction 24 h;
(23) with solution C in 4 ℃ with distill water dialysis 3 days, change liquid every day 4 times, lyophilize, safe ground Luo Xin artificial antigen M1 ,-20 ℃ of preservations.
7. the artificial antigenic preparation method of safe ground Luo Xin according to claim 6,
It is characterized in that:Carrier proteins described in the step (22) is bovine serum albumin or chicken egg white.
8. the artificial antigenic preparation method of safe ground Luo Xin according to claim 5,
It is characterized in thatSaid active ester method is according to carrying out in proper order according to the following steps:
(31) with the N of 0.8～1.0mmol; N ,-NSC 57182 are dissolved in 10～20mL dioxan, splash under the whipped state in the dioxan solution of safe ground Luo Xin haptin and N-hydroxy-succinamide of equivalent; Stirring at room is reacted 24 h, makes solution D;
(32) solution D is centrifugal, get in the dioxan and phosphate buffered saline buffer of 10mg/mL carrier proteins that supernatant drops to 50～100 mL, 3～5 ℃ of stirring reaction 24 h make solution E;
(33) with solution E in 4 ℃ with distill water dialysis 3 days, change liquid every day 4 times, lyophilize gets safe ground Luo Xin artificial antigen M2 ,-20 ℃ of preservations.
9. the artificial antigenic preparation method of safe ground Luo Xin according to claim 8,
It is characterized in that:Carrier proteins described in the step (32) is bovine serum albumin or chicken egg white.
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| CN104447919A (en) * | 2014-11-28 | 2015-03-25 | 武汉回盛生物科技有限公司 | Refining method of 20,23-dipiperidinyl-5-O-mycaminose-tylonolide bulk drug |
| CN104710488A (en) * | 2015-03-11 | 2015-06-17 | 江南大学 | Synthesis method of half-antigen and complete antigen of tulathromycin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104341409A (en) * | 2013-07-31 | 2015-02-11 | 北京勤邦生物技术有限公司 | Thiabendazole hapten preparation method and application |
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| CN104447919A (en) * | 2014-11-28 | 2015-03-25 | 武汉回盛生物科技有限公司 | Refining method of 20,23-dipiperidinyl-5-O-mycaminose-tylonolide bulk drug |
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| CN105467024A (en) * | 2015-11-17 | 2016-04-06 | 宁夏泰瑞制药股份有限公司 | Detection method of Tildipirosin content |
| CN108191934A (en) * | 2017-12-29 | 2018-06-22 | 武汉市农业科学院 | A kind of tylonolide hapten derivant and preparation method thereof and detection kit |
| CN108191934B (en) * | 2017-12-29 | 2020-02-07 | 武汉市农业科学院 | Tildipirosin hapten derivative and preparation method and detection kit thereof |
| CN110865134A (en) * | 2019-10-22 | 2020-03-06 | 河北远征药业有限公司 | Tildipirosin content detection method |
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Inventor after: Wei Lijuan Inventor after: Li Jinming Inventor after: Liu Yi Inventor after: Jia Guobin Inventor after: Wei Zhanyong Inventor after: Yang Guohui Inventor after: Guo Hongzhi Inventor after: Wang Degong Inventor after: Liu Dong Inventor before: Yue Yongbo Inventor before: Yang Guohui Inventor before: Li Weiling Inventor before: Yang Fang Inventor before: Guo Hongzhi Inventor before: Liu Bo Inventor before: Liu Yi Inventor before: Liu Hailiang Inventor before: Chen Ling Inventor before: Wei Zhanyong |
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Application publication date: 20121212 |