CN113980117B - 一种唑螨酯抗原及其制备方法与应用 - Google Patents
一种唑螨酯抗原及其制备方法与应用 Download PDFInfo
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- CN113980117B CN113980117B CN202111361024.6A CN202111361024A CN113980117B CN 113980117 B CN113980117 B CN 113980117B CN 202111361024 A CN202111361024 A CN 202111361024A CN 113980117 B CN113980117 B CN 113980117B
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- fenpyroximate
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- ethyl
- antigen
- methyl
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- YYJNOYZRYGDPNH-MFKUBSTISA-N fenpyroximate Chemical compound C=1C=C(C(=O)OC(C)(C)C)C=CC=1CO/N=C/C=1C(C)=NN(C)C=1OC1=CC=CC=C1 YYJNOYZRYGDPNH-MFKUBSTISA-N 0.000 title claims abstract description 73
- 239000005657 Fenpyroximate Substances 0.000 title claims abstract description 72
- 239000000427 antigen Substances 0.000 title claims abstract description 48
- 102000036639 antigens Human genes 0.000 title claims abstract description 48
- 108091007433 antigens Proteins 0.000 title claims abstract description 48
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 150000001875 compounds Chemical class 0.000 claims abstract description 43
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 24
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 24
- 238000005859 coupling reaction Methods 0.000 claims abstract description 17
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims abstract description 10
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims abstract description 8
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims abstract description 8
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims abstract description 8
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 5
- 125000004185 ester group Chemical group 0.000 claims abstract description 4
- AKGVMZJNWXUJBJ-UHFFFAOYSA-N n-[(1,3-dimethyl-5-phenoxypyrazol-4-yl)methylidene]hydroxylamine Chemical compound CC1=NN(C)C(OC=2C=CC=CC=2)=C1C=NO AKGVMZJNWXUJBJ-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000008157 ELISA kit Methods 0.000 claims abstract description 3
- 238000003149 assay kit Methods 0.000 claims abstract description 3
- 239000003547 immunosorbent Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 17
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 16
- 239000008055 phosphate buffer solution Substances 0.000 claims description 15
- 239000007853 buffer solution Substances 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 229940098773 bovine serum albumin Drugs 0.000 claims description 8
- 238000000502 dialysis Methods 0.000 claims description 6
- 229940092253 ovalbumin Drugs 0.000 claims description 6
- 239000012460 protein solution Substances 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- 108010058846 Ovalbumin Proteins 0.000 claims description 5
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 4
- 108060003552 hemocyanin Proteins 0.000 claims description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 3
- TWQLMAJROCNXEA-UHFFFAOYSA-N ethyl 4-(bromomethyl)benzoate Chemical compound CCOC(=O)C1=CC=C(CBr)C=C1 TWQLMAJROCNXEA-UHFFFAOYSA-N 0.000 claims description 3
- DQHSFNAHHRSSRY-UHFFFAOYSA-M 2-ethyl-2-iodobutanoate Chemical compound CCC(I)(CC)C([O-])=O DQHSFNAHHRSSRY-UHFFFAOYSA-M 0.000 claims description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 2
- 125000003277 amino group Chemical group 0.000 claims description 2
- NMEGSGKCIWQRDB-UHFFFAOYSA-N butyl 2-bromoacetate Chemical compound CCCCOC(=O)CBr NMEGSGKCIWQRDB-UHFFFAOYSA-N 0.000 claims description 2
- DKZLMRKANVHJGG-UHFFFAOYSA-N butyl 2-bromobutanoate Chemical compound CCCCOC(=O)C(Br)CC DKZLMRKANVHJGG-UHFFFAOYSA-N 0.000 claims description 2
- YJRGMUWRPCPLNH-UHFFFAOYSA-N butyl 2-chloroacetate Chemical compound CCCCOC(=O)CCl YJRGMUWRPCPLNH-UHFFFAOYSA-N 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 claims description 2
- WHNBIRCJWQMENU-UHFFFAOYSA-N ethyl 2-(4-bromophenyl)propanoate Chemical compound CCOC(=O)C(C)C1=CC=C(Br)C=C1 WHNBIRCJWQMENU-UHFFFAOYSA-N 0.000 claims description 2
- XIMFCGSNSKXPBO-UHFFFAOYSA-N ethyl 2-bromobutanoate Chemical compound CCOC(=O)C(Br)CC XIMFCGSNSKXPBO-UHFFFAOYSA-N 0.000 claims description 2
- QTEITWPJVHAOTN-UHFFFAOYSA-N ethyl 2-chlorobutanoate Chemical compound CCOC(=O)C(Cl)CC QTEITWPJVHAOTN-UHFFFAOYSA-N 0.000 claims description 2
- PQJJJMRNHATNKG-UHFFFAOYSA-N ethyl bromoacetate Chemical compound CCOC(=O)CBr PQJJJMRNHATNKG-UHFFFAOYSA-N 0.000 claims description 2
- VEUUMBGHMNQHGO-UHFFFAOYSA-N ethyl chloroacetate Chemical compound CCOC(=O)CCl VEUUMBGHMNQHGO-UHFFFAOYSA-N 0.000 claims description 2
- MFFXVVHUKRKXCI-UHFFFAOYSA-N ethyl iodoacetate Chemical compound CCOC(=O)CI MFFXVVHUKRKXCI-UHFFFAOYSA-N 0.000 claims description 2
- -1 ethyl m-bromobenzoate Chemical compound 0.000 claims description 2
- QHJOWSXZDCTNQX-UHFFFAOYSA-N methyl 2-(4-bromophenyl)acetate Chemical compound COC(=O)CC1=CC=C(Br)C=C1 QHJOWSXZDCTNQX-UHFFFAOYSA-N 0.000 claims description 2
- YDCHPLOFQATIDS-UHFFFAOYSA-N methyl 2-bromoacetate Chemical compound COC(=O)CBr YDCHPLOFQATIDS-UHFFFAOYSA-N 0.000 claims description 2
- UFQQDNMQADCHGH-UHFFFAOYSA-N methyl 2-bromobutanoate Chemical compound CCC(Br)C(=O)OC UFQQDNMQADCHGH-UHFFFAOYSA-N 0.000 claims description 2
- BHQQXAOBIZQEGI-UHFFFAOYSA-N methyl 2-chlorobutanoate Chemical compound CCC(Cl)C(=O)OC BHQQXAOBIZQEGI-UHFFFAOYSA-N 0.000 claims description 2
- JLEJCNOTNLZCHQ-UHFFFAOYSA-N methyl 2-chloropropanoate Chemical compound COC(=O)C(C)Cl JLEJCNOTNLZCHQ-UHFFFAOYSA-N 0.000 claims description 2
- BXXLTVBTDZXPTN-UHFFFAOYSA-N methyl 2-iodobenzoate Chemical compound COC(=O)C1=CC=CC=C1I BXXLTVBTDZXPTN-UHFFFAOYSA-N 0.000 claims description 2
- SVSKWGDTQMPPQE-UHFFFAOYSA-N methyl 2-iodobutanoate Chemical compound CCC(I)C(=O)OC SVSKWGDTQMPPQE-UHFFFAOYSA-N 0.000 claims description 2
- NLWBJPPMPLPZIE-UHFFFAOYSA-N methyl 4-(bromomethyl)benzoate Chemical compound COC(=O)C1=CC=C(CBr)C=C1 NLWBJPPMPLPZIE-UHFFFAOYSA-N 0.000 claims description 2
- LXNFVVDCCWUUKC-UHFFFAOYSA-N methyl 4-chlorobenzoate Chemical compound COC(=O)C1=CC=C(Cl)C=C1 LXNFVVDCCWUUKC-UHFFFAOYSA-N 0.000 claims description 2
- DYUWQWMXZHDZOR-UHFFFAOYSA-N methyl 4-iodobenzoate Chemical compound COC(=O)C1=CC=C(I)C=C1 DYUWQWMXZHDZOR-UHFFFAOYSA-N 0.000 claims description 2
- XMJHPCRAQCTCFT-UHFFFAOYSA-N methyl chloroformate Chemical compound COC(Cl)=O XMJHPCRAQCTCFT-UHFFFAOYSA-N 0.000 claims description 2
- NZHZYDSYLLWKOK-UHFFFAOYSA-N propyl 2-chlorobutanoate Chemical compound CCCOC(=O)C(Cl)CC NZHZYDSYLLWKOK-UHFFFAOYSA-N 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims 3
- 101710125507 Integrase/recombinase Proteins 0.000 claims 1
- 238000004587 chromatography analysis Methods 0.000 claims 1
- YDJDSUNRSIVGMI-UHFFFAOYSA-N ethyl 2-(4-iodophenyl)acetate Chemical compound CCOC(=O)CC1=CC=C(I)C=C1 YDJDSUNRSIVGMI-UHFFFAOYSA-N 0.000 claims 1
- RETLCWPMLJPOTP-UHFFFAOYSA-N ethyl 2-chlorobenzoate Chemical compound CCOC(=O)C1=CC=CC=C1Cl RETLCWPMLJPOTP-UHFFFAOYSA-N 0.000 claims 1
- LVFRSNCBCHABAM-UHFFFAOYSA-N ethyl 3-chlorobenzoate Chemical compound CCOC(=O)C1=CC=CC(Cl)=C1 LVFRSNCBCHABAM-UHFFFAOYSA-N 0.000 claims 1
- KMFJVYMFCAIRAN-UHFFFAOYSA-N methyl 3-bromobenzoate Chemical compound COC(=O)C1=CC=CC(Br)=C1 KMFJVYMFCAIRAN-UHFFFAOYSA-N 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 abstract description 7
- 238000003786 synthesis reaction Methods 0.000 abstract description 7
- 230000003053 immunization Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 238000001514 detection method Methods 0.000 description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000002965 ELISA Methods 0.000 description 7
- 239000003085 diluting agent Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 230000000895 acaricidal effect Effects 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
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- 239000011248 coating agent Substances 0.000 description 6
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- 239000002671 adjuvant Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 241000238876 Acari Species 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- 125000006615 aromatic heterocyclic group Chemical group 0.000 description 3
- 125000003118 aryl group Chemical group 0.000 description 3
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- 229940089960 chloroacetate Drugs 0.000 description 3
- 238000011161 development Methods 0.000 description 3
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- 239000003814 drug Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
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- 230000001988 toxicity Effects 0.000 description 3
- 241000283707 Capra Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 238000004896 high resolution mass spectrometry Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Chemical compound OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- CQQSQBRPAJSTFB-UHFFFAOYSA-N 4-(bromomethyl)benzoic acid Chemical compound OC(=O)C1=CC=C(CBr)C=C1 CQQSQBRPAJSTFB-UHFFFAOYSA-N 0.000 description 1
- KMZIZRHLLMNWNT-UHFFFAOYSA-N 5-phenoxy-1h-pyrazole Chemical compound C=1C=CC=CC=1OC=1C=CNN=1 KMZIZRHLLMNWNT-UHFFFAOYSA-N 0.000 description 1
- 241000256593 Brachycaudus schwartzi Species 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- ACTIUHUUMQJHFO-UHFFFAOYSA-N Coenzym Q10 Natural products COC1=C(OC)C(=O)C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UHFFFAOYSA-N 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
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- 108010010803 Gelatin Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 244000141359 Malus pumila Species 0.000 description 1
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- 206010034133 Pathogen resistance Diseases 0.000 description 1
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- 241000985245 Spodoptera litura Species 0.000 description 1
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- ACTIUHUUMQJHFO-UPTCCGCDSA-N coenzyme Q10 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UPTCCGCDSA-N 0.000 description 1
- 235000017471 coenzyme Q10 Nutrition 0.000 description 1
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- 239000002552 dosage form Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- YCBJOQUNPLTBGG-UHFFFAOYSA-N ethyl 4-iodobenzoate Chemical compound CCOC(=O)C1=CC=C(I)C=C1 YCBJOQUNPLTBGG-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
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- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
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- 231100000053 low toxicity Toxicity 0.000 description 1
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- YUHSMQQNPRLEEJ-UHFFFAOYSA-N methyl 3-(bromomethyl)benzoate Chemical compound COC(=O)C1=CC=CC(CBr)=C1 YUHSMQQNPRLEEJ-UHFFFAOYSA-N 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
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- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
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- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- NPCOQXAVBJJZBQ-UHFFFAOYSA-N reduced coenzyme Q9 Natural products COC1=C(O)C(C)=C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)C(O)=C1OC NPCOQXAVBJJZBQ-UHFFFAOYSA-N 0.000 description 1
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- LMBFAGIMSUYTBN-MPZNNTNKSA-N teixobactin Chemical compound C([C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](CCC(N)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H]1C(N[C@@H](C)C(=O)N[C@@H](C[C@@H]2NC(=N)NC2)C(=O)N[C@H](C(=O)O[C@H]1C)[C@@H](C)CC)=O)NC)C1=CC=CC=C1 LMBFAGIMSUYTBN-MPZNNTNKSA-N 0.000 description 1
- 229940035936 ubiquinone Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
本发明公开了一种唑螨酯抗原及其制备方法与应用,包括:将1,3‑二甲基‑5苯氧基‑1H‑吡唑‑4‑甲醛肟与含有酯基的化合物进行反应得到含有羧基的化合物;再和N‑羟基琥珀酰亚胺在二环己基碳二亚胺或1‑乙基‑(3‑二甲基氨基丙基)碳酰二亚胺盐酸盐存在的条件下进行偶联反应;再与载体蛋白经偶联反应得唑螨酯抗原;唑螨酯抗原在制备用于检测样品中唑螨酯的酶联免疫试剂盒、唑螨酯的发光免疫试剂盒或免疫亲和色谱柱中的应用。本发明的优点在于:能够方便、快捷地获得唑螨酯抗原,合成步骤简洁明了、合成成本低,效果好。通过唑螨酯抗原进行免疫,可以得到的特异性好、灵敏度高的唑螨酯抗体。
Description
技术领域
本发明涉及化合物制备与应用技术领域,特别涉及一种唑螨酯抗原及其制备方法与应用。
背景技术
唑螨酯(fenpyroximate)别名霸螨灵,是1985年日本农药株式会社开发研制的苯氧基吡唑类高效、广谱、非内吸性杀螨剂,由于唑螨酯结构中含有碳氮双键,所以存在同分异构体E体和Z体,其中E体比Z体杀螨活性高。该药对植食性螨类具有很强的选择性,作用方式以触杀为主,能够选择性抑制螨虫体内细胞线粒体泛醌过氧化物酶,以至线粒体氧化呼吸链断裂,迫使螨虫死亡。唑螨酯为中毒性杀螨剂,可有效防治斜纹夜蛾、红叶螨、桃蚜、全爪叶螨、小菜蛾等害虫,在柑橘、苹果等果树以及各种农作物上应用广泛。其化学结构式如下:
唑螨酯在1991年上市,2010年全球销售额为0.35亿美元。2016年PMRA批准了日本农药公司的杀虫/杀螨剂唑螨酯及其终端产品5%SC剂型唑螨酯的正式登记,之后产品在加拿大销售使用。2017年成为我国湖南公布主要作物用药推荐名录之一。作为杀螨剂中的优良品种,是主要的农药上市品种之一,市场占有率较高,已使之成为一种畅销全球的高效、广谱、低毒杀螨剂,在国际市场具有很强的竞争力。随着唑螨酯的大量使用,其残留问题也引起人们的关注。农业上施用的唑螨酯,直接或间接地进入土壤甚至地下水,其在土壤中降解缓慢,易造成环境污染。唑螨酯不具有内吸性,不与其他农药有交互抗性。作为一种中毒性农药,唑螨酯对多数哺乳动物毒性较弱,但对水生生物具有较大毒性,目前研究表明唑螨酯短期进入动植物体内几乎不发生代谢降解,大部分以母体化合物的形式排出,国内外对唑螨酯的毒性及安全性研究不多,风险评估报告数量也较少,对于不同的果蔬唑螨酯也可能存在不同的效应。
目前,唑螨酯在农产品中的残留检测方法主要是高效液相色谱法(HPLC)和高效液相色谱-串联质谱法(HPLC-MS),尽管仪器法检测具有灵敏度高、准确可靠等优点,但样品前处理繁琐,检测时间长,耗资大,技术性要求高,且仪器昂贵,不适合大量样品的快速检测,也不具备现场快速检测的能力。与仪器分析法相比,免疫分析法具有快速、简便、实时、易于进行现场检测、样品前处理简单、灵敏度高、选择性强、适合于高通量分析等优点,而且还能大幅度降低检测成本。
发明内容
本发明针对现有技术的缺陷,提供了一种唑螨酯抗原及其制备方法与应用,能有效的解决上述现有技术存在的问题。
为了实现以上发明目的,本发明采取的技术方案如下:
一种唑螨酯抗原,其结构通式如式A1/A2所示,
所述式A1/A2中,X为芳香环、芳香杂环化合物中的一种,n为0-6的整数,a为1-6的整数,b为0-6的整数;Protein表示载体蛋白,所述载体蛋白选自牛血清蛋白、卵清蛋白和血蓝蛋白中至少一种。
一种唑螨酯抗原的制备方法,包括如下步骤:
步骤1,将式B(1,3-二甲基-5苯氧基-1H-吡唑-4-甲醛肟)与式C1/C2所示含有酯基的化合物进行反应得到含有羧基的式D1/D2所示化合物;
所述式C1/C2和式D1/D2中,X为芳香环、芳香杂环化合物中的一种,Y为Cl、Br和I基团中的一种,n为0-6的整数,m为0-6的整数,a为1-6的整数,b为0-6的整数。
步骤2,式D1/D2所示化合物和N-羟基琥珀酰亚胺在二环己基碳二亚胺或1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐存在的条件下进行偶联反应得到式E1/E2所示化合物;
所述式E1/E2中,X为芳香环、芳香杂环化合物中的一种,n为0-6的整数,a为1-6的整数,b为0-6的整数。
步骤3,式E1/E2所示化合物与载体蛋白经偶联反应即得式A1/A2所示唑螨酯抗原;所述载体蛋白选自牛血清蛋白、卵清蛋白和血蓝蛋白中至少一种。
作为优选,步骤1中,式C1所示化合物可选自氯甲酸甲酯、氯甲酸乙酯、氯乙酸乙酯、氯乙酸丁酯、氯丙酸甲酯、氯丁酸甲酯、氯丁酸乙酯、氯丁酸丙酯、溴乙酸甲酯、溴乙酸乙酯、溴乙酸丁酯、溴丁酸甲酯、溴丁酸乙酯、溴丁酸丁酯、碘乙酸乙酯、碘丁酸甲酯、碘丁酸乙酯等中至少一种,式C2所示化合物可选自对氯苯甲酸甲酯、对氯苯乙酸乙酯、邻氯苯乙酸乙酯、间氯苯乙酸乙酯、对溴苯乙酸甲酯、对溴苯乙酸乙酯、对溴苯丙酸乙酯、对溴甲基苯甲酸甲酯、对溴甲基苯甲酸乙酯、间溴甲基苯甲酸甲酯、间溴苯甲酸甲酯、间溴苯甲酸乙酯、对碘苯甲酸甲酯、对碘苯甲酸乙酯、对碘苯乙酸乙酯、邻碘苯甲酸甲酯等中至少一种。
作为优选,所述步骤1中,式C1/C2化合物和式B化合物的投料摩尔配比可为(0.1-10):1;
反应的温度可为0~100℃,时间可为3~48小时;
反应的溶剂选自吡啶、N,N-二甲基甲酰胺、二甲亚砜和四氢呋喃中至少一种。
作为优选,所述步骤2中,式D1/D2所示化合物、N-羟基琥珀酰亚胺与二环己基碳二亚胺的摩尔份数比可为1:(1~5):(1~5);
式D1/D2所示化合物、N-羟基琥珀酰亚胺与1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐的摩尔份数比可为1:(1~5):(1~5);
所述偶联反应的温度可为0~50℃,时间可为4~24小时。
作为优选,步骤3中,式E1/E2所示化合物与所述载体蛋白的摩尔份数比可为(5~30):1;
所述偶联反应的温度可为0~50℃,时间可为4~36小时;所述偶联反应在pH值为5~9的条件下进行;
式E1/E2所示化合物在所述载体蛋白的溶液中进行偶联反应,所述载体蛋白的溶液是由所述载体蛋白加入至缓冲溶液中得到的,所述缓冲溶液选自碳酸盐缓冲液、磷酸盐缓冲液、硼酸盐缓冲液和4-羟乙基哌嗪乙磺酸缓冲液中至少一种,所述缓冲液的pH值均可为7.4;
作为优选,步骤3之后,所述方法还包括将所述偶联反应的反应体系进行透析的步骤;所述透析步骤中,所用透析液为pH值可为4~10、浓度可为0.01~0.2mol/L的磷酸盐缓冲溶液。
作为优选,所述唑螨酯抗原是式E1/E2化合物与载体蛋白通过酰胺键连接形成的偶联物;所述酰胺键是式D1/D2上的羧基通过活泼酯与载体蛋白上的氨基形成的。
一种基于上述唑螨酯抗原的应用,包括:在制备用于检测样品中唑螨酯的酶联免疫试剂盒、唑螨酯的发光免疫试剂盒或免疫亲和色谱柱中的应用。
作为优选,所述检测样品为水体、药品、食品或土壤。
与现有技术相比本发明的优点在于:能够方便、快捷地获得唑螨酯抗原,合成步骤简洁明了、合成成本低,效果好。本发明方法制备的唑螨酯抗原进行免疫得到的抗体的特异性好、最低检测限值低。本发明的制备唑螨酯抗原的方法及由该方法获得的唑螨酯抗原在唑螨酯的快速免疫检测应用中将有广阔的前景。
具体实施方式
为使本发明的目的、技术方案及优点更加清楚明白,以下列举实施例,对本发明做进一步详细说明。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
1,3-二甲基-5苯氧基-1H-吡唑-4-甲醛肟,4-溴甲基苯甲酸乙酯,二环己基碳二亚胺(DCC),1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC),N-羟基琥珀酰亚胺(NHS),弗氏完全佐剂,弗氏不完全佐剂,牛血清白蛋白和卵清白蛋白,羊抗小鼠IgG-HRP,单组分TMB显色液。
实施例1、唑螨酯-卵清白蛋白(唑螨酯-OVA)抗原的制备
合成路线:
1)式I所示化合物的合成
在50mL三口瓶中加入1g1,3-二甲基-5苯氧基-1H-吡唑-4-甲醛肟,加入40mL乙腈使其溶解,滴加入4-溴甲基苯甲酸乙酯1.16g,加入无水碳酸钾1.19g,搅拌加热至100度,反应24小时,旋转蒸发干后,用乙酸乙酯(3×50mL)萃取,无水硫酸镁干燥。旋转脱溶,柱层析得到式I所示化合物。
1H NMR(400MHz,DMSO)δ12.91(s,1H),7.89(d,J=8.2Hz,2H),7.79(s,1H),7.37(dt,J=7.8,7.4Hz,2H),7.35(d,J=8.2Hz,2H),7.15(t,J=7.4Hz,1H),6.95(d,J=7.8Hz,2H),4.99(s,2H),4.30(q,2H,J=7.1),3.55(s,3H),2.21(s,3H),1.32(t,3H,J=7.1).
13C NMR(101MHz,DMSO)δ166.00,156.81,147.71,146.00,143.68,141.36,129.50,128.58,124.24,116.02,99.47,74.78,61.14,34.48,14.62
HRMS:m/z calcd for C22H24N3O4(M+H+)394.1761,found 394.1768.
产物经1H-NMR、13C-NMR和高分辨质谱确证。
2)式Ⅱ所示化合物的合成
在50mL三口瓶中加入0.4g式I化合物,0.3g氢氧化锂,用20mL 甲醇和5mL水溶解,室温下搅拌反应8h。反应完毕后,旋转蒸发去除甲醇,然后加入20mL水,用浓盐酸调节pH值为3,过滤后烘干得到式Ⅱ所示化合物。
1H NMR(400MHz,DMSO)δ12.91(s,1H),7.88(d,J=8.2Hz,2H),7.80(s,1H),7.40(dt,J=7.9,7.4Hz,2H),7.35(d,J=8.2Hz,2H),7.15(t,J=7.4Hz,1H),6.95(d,J=7.9Hz,2H),4.99(s,2H),3.55(s,3H),2.22(s,3H).
13C NMR(101MHz,DMSO)δ167.56,156.82,147.72,146.00,143.24,141.29,130.61,129.69,128.48,124.25,116.03,99.48,74.87,34.47,14.55.
HRMS:m/z calcd for C20H20N3O4(M+H+)366.1448,found 366.1439.
产物经1H-NMR、13C-NMR和高分辨质谱确证。
3)偶联反应
方法(1):称取步骤2)得到的式Ⅱ所示唑螨酯半抗原(0.036mmol),NHS(0.047mmol),和DCC(0.030mmol)用1mL无水DMF溶解,在室温25℃搅拌反应6小时后,将反应液在8000转下离心5分钟,取上清液,得到式Ⅲ所示化合物;
方法(2):称取步骤1)得到的式Ⅱ所示唑螨酯半抗原(0.036mmol),NHS(0.047mmol),和EDC(0.030mmol)用1mL水溶解,在室温25℃搅拌反应6小时后,得到式Ⅲ所示化合物;
4)将步骤3)所得式Ⅲ所示化合物缓慢滴加入载体蛋白OVA溶液(该载体蛋白溶液是由20mg OVA溶于5mL pH值为7.4的磷酸盐(PBS)缓冲液混匀而得)中,式Ⅲ化合物与载体蛋白的投料摩尔比为20:1,在4℃搅拌过夜。
5)透析:将步骤4)所得反应液用pH值为7.4、浓度为0.01mol/L的PBS溶液透析三天,将透析完全的反应产物溶液(唑螨酯-OVA)稀释为1mg/mL的溶液,放于-40℃冻存待用。透析的作用在于去除未反应的唑螨酯半抗原和其它小分子,得到式Ⅳ-1所示唑螨酯与OVA的偶联物,也即式A所示唑螨酯抗原。
其中,该PBS溶液按照如下方法制备而得:将NaCl、KH2PO4和Na2HPO4·12H2O以质量比8.0:0.2:2.96的比例溶于水中,用水定容到1L。
实施例2、唑螨酯-牛血清白蛋白(唑螨酯-BSA)抗原的制备
1)式Ⅱ所示唑螨酯半抗原的合成及其活化与实施例1中无差别,在此不再赘述。
2)将实施例1中步骤3)所得式Ⅲ所示化合物缓慢滴加到载体蛋白溶液中(该载体蛋白溶液是由30mg BSA溶于5mL pH值为7.4的磷酸盐缓冲液(PBS)),式Ⅲ化合物与载体蛋白的投料摩尔比为20:1,在4℃搅拌过夜。
3)透析:将步骤2)所得反应液用pH值为7.4、浓度为0.01mol/L的PBS溶液透析三天,将透析完全的反应产物溶液(唑螨酯-BSA)稀释为1mg/mL的溶液,至于-40℃冻存待用。透析的作用在于去除未反应的唑螨酯半抗原和其它小分子,得到式Ⅳ-2所示唑螨酯与OVA的偶联物,也即式A所示唑螨酯抗原。
其中,该PBS溶液按照如下方法制备而得:将NaCl、KH2PO4和Na2HPO4·12H2O以质量比8.0:0.2:2.96的比例溶于水中,用水定容到1L。
实施例3、唑螨酯-牛血清白蛋白(唑螨酯-BSA)抗原的应用
一、利用唑螨酯-牛血清白蛋白(唑螨酯-BSA)抗原制备抗体
(1)取8-10周龄的Bal b/c小白鼠作为实验动物。
(2)基础免疫:由实施例2中得到稀释好的唑螨酯-BSA抗原溶液(浓度为1mg/mL),经无菌过滤器过滤后加入等体积弗氏完全佐剂,用磁力搅拌器充分搅拌乳化,直到滴入水中不扩散。用乳化好的完全抗原采用腹腔及背部皮下多点注射Bal b/c小鼠,注射剂量为0.1mg乳化抗原/只。
(3)加强免疫:基础免疫2周后,取1mL上述稀释好的唑螨酯-BSA抗原溶液,然后加入1mL弗氏不完全佐剂,用磁力搅拌器充分搅拌乳化,直到滴入水中不扩散。将乳化好的抗原采用腹腔及背部皮下多点注射Bal b/c小鼠,每只小鼠的注射剂量为0.1mg乳化稀释抗原(8周龄的Bal b/C小鼠体重约23-25g)。
加强免疫每隔15天免疫一次,从第三次加强免疫开始,每次免疫后第3-5天,从小鼠眼眶采血,测定抗体效价,包被原为1mg/mL唑螨酯-OVA稀释500倍用,待效价大于1:8000后(效价定义为零孔显色值为1时,血清的稀释倍数),眼球摘除采血,血液室温静置1小时后,再于4℃冰箱中静置2小时,然后于离心机中8000r/min离心5分钟后,分离出抗血清,即得到唑螨酯-BSA抗体。用于下述各实验。
二、抗体效果检测
下述实验中所用的各种缓冲液如下:
(1)包被缓冲液:0.05M、pH 9.6的碳酸盐缓冲液;
(2)磷酸盐缓冲液PBS(pH 7.4):称量4.0g NaCl、0.1g KH2PO4、1.48g Na2HPO4·12H2O用蒸馏水定容到500mL,浓度为0.01M、pH为7.4磷酸盐缓冲液;
(3)样品稀释液PBSTG:由0.5mL吐温20、0.5g明胶和500mL浓度为0.1M、pH为7.4的PBS缓冲液混合得到;
(4)终止缓冲液:2.0M的硫酸水溶液;
(5)洗涤液:由NaCl、KH2PO4、Na2HPO4·12H2O、Tween-20和水组成;NaCl在洗涤液中的浓度为8.0g/L,KH2PO4在洗涤液中的浓度为0.2g/L,Na2HPO4·12H2O在洗涤液中的浓度为2.96g/L,Tween-20在洗涤液中体积百分含量为1:1000。
(一)抗体抑制实验
1、唑螨酯-OVA包被抗原溶液的配制
将上述实施例1制备的稀释后的1mg/mL的唑螨酯-OVA抗原完全解冻后,用包被缓冲液按1:1000、1:2000、1:4000、1:8000进行梯度稀释,得到不同浓度的唑螨酯-OVA的包被抗原溶液。
2、唑螨酯标准品溶液的配制
(1)称取10mg唑螨酯标样,充分溶解于10mL无水甲醇中,即得到1.0mg/mL唑螨酯标准品溶液;
(2)用样品稀释液将上述步骤(1)的1.0mg/mL唑螨酯标准品溶液配成浓度为1000ng/mL的唑螨酯标准品溶液。
3、唑螨酯-BSA抗血清稀释液的配制
将上述步骤一制备的唑螨酯-BSA抗体用样品稀释液按1:1000、1:2000、1:4000、1:8000进行梯度稀释,得到唑螨酯-BSA抗血清稀释液。
4、抗原、抗体的棋盘格实验
包被:在96孔酶标板中每孔加入100μL步骤1制备得到的唑螨酯-OVA包被抗原溶液,37℃包被3小时,用洗涤液洗涤4次。
封闭:秤取5g脱脂奶粉,充分溶解于100mL样品稀释液中,即得到5%的封闭液。在96孔酶标板中每孔加入200μL封闭液,在37℃湿盒中封闭1h,弃封闭液,洗涤3次。
竞争:零孔每孔加50μl样品稀释液,抑制孔每孔加入50μl步骤2制备得到的唑螨酯标准品溶液。将上述步骤3得到的唑螨酯-BSA抗血清稀释液(从1×103倍到8×103倍)加入酶标板中(50μl/孔),置湿盒中37℃条件下30min,洗板4次。
加酶标二抗:将羊抗鼠酶标二抗(IgG-HRP,Jackson公司,产品目录号为79556)(0.1mg/mL)稀释1000倍,稀释液是0.01M,pH为7.4的PBSTG,每孔加100μL,置湿盒中37℃条件下30min,洗板4次。
显色:将单组分TMB显色液加入酶标板中,每孔100μl。避光显色15min。
终止:每孔加入50μL终止缓冲液,用酶标仪450nm处测定各孔的OD值。
效价的定义为零孔OD值为1时的血清稀释倍数。
结果如表1所示。
表1、抗唑螨酯小鼠的血清效价检测(单组分TMB显色液室温显色15min,1000ng标样抑制)
注:I表示酶标板中的抑制孔,C表示酶标板中的对照孔。
结果说明上述实施例2制备的唑螨酯-BSA可以作为免疫原制备出检测唑螨酯的抗体。
本领域的普通技术人员将会意识到,这里所述的实施例是为了帮助读者理解本发明的实施方法,应被理解为本发明的保护范围并不局限于这样的特别陈述和实施例。本领域的普通技术人员可以根据本发明公开的这些技术启示做出各种不脱离本发明实质的其它各种具体变形和组合,这些变形和组合仍然在本发明的保护范围内。
Claims (9)
1.一种唑螨酯抗原的制备方法,其特征在于,唑螨酯抗原结构通式如式A1或A2所示,
所述式A1、A2中,X为苯环,n为0-6的整数,a为1-6的整数,b为0-6的整数;Protein表示载体蛋白,所述载体蛋白选自牛血清蛋白、卵清蛋白和血蓝蛋白中至少一种;
通过以下制备步骤获得:
步骤(1)将式B所示的1,3-二甲基-5苯氧基-1H-吡唑-4-甲醛肟与式C1或C2所示含有酯基的化合物进行反应得到含有羧基的式D1或D2所示化合物;
Y-(CH2)n-COO-(CH2)m-CH3
式C1结构式
Y-(CH2)a-X-(CH2)b-COO-(CH2)m-CH3
式C2结构式
所述式C1、C2和式D1、D2中,X为苯环,Y为Cl、Br和I基团中的一种,n为0-6的整数,m为0-6的整数,a为1-6的整数,b为0-6的整数;
步骤(2)式D1或D2所示化合物和N-羟基琥珀酰亚胺在二环己基碳二亚胺或1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐存在的条件下进行偶联反应得到式E1/E2所示化合物;
所述式E1、E2中,X为苯环,n为0-6的整数,a为1-6的整数,b为0-6的整数;
步骤(3)式E1或E2所示化合物与载体蛋白经偶联反应即得式A1或A2所示唑螨酯抗原;所述载体蛋白选自牛血清蛋白、卵清蛋白和血蓝蛋白中至少一种。
2.根据权利要求1所述的一种唑螨酯抗原的制备方法,其特征在于:所述步骤(1)中,式C1所示化合物选自氯甲酸甲酯、氯甲酸乙酯、氯乙酸乙酯、氯乙酸丁酯、氯丙酸甲酯、氯丁酸甲酯、氯丁酸乙酯、氯丁酸丙酯、溴乙酸甲酯、溴乙酸乙酯、溴乙酸丁酯、溴丁酸甲酯、溴丁酸乙酯、溴丁酸丁酯、碘乙酸乙酯、碘丁酸甲酯、碘丁酸乙酯中至少一种;
式C2所示化合物选自对氯苯甲酸甲酯、对氯苯乙酸乙酯、邻氯苯乙酸乙酯、间氯苯乙酸乙酯、对溴苯乙酸甲酯、对溴苯乙酸乙酯、对溴苯丙酸乙酯、对溴甲基苯甲酸甲酯、对溴甲基苯甲酸乙酯、间溴甲基苯甲酸甲酯、间溴苯甲酸甲酯、间溴苯甲酸乙酯、对碘苯甲酸甲酯、对碘苯甲酸乙酯、对碘苯乙酸乙酯、邻碘苯甲酸甲酯中至少一种。
3.根据权利要求1所述的一种唑螨酯抗原的制备方法,其特征在于:所述步骤(1)中,式C1或C2化合物和式B化合物的投料摩尔配比为0.1-10:1;
反应的温度为0~100℃,时间为6~48小时;
反应的溶剂选自吡啶、N,N-二甲基甲酰胺、二甲亚砜和四氢呋喃中至少一种。
4.根据权利要求1所述的一种唑螨酯抗原的制备方法,其特征在于:所述步骤(2)中,式D1或D2所示化合物、N-羟基琥珀酰亚胺与二环己基碳二亚胺的摩尔份数比为1:1~5:1~5;
式D1或D2所示化合物、N-羟基琥珀酰亚胺与1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐的摩尔份数比为1:1~5:1~5;
所述偶联反应的温度为0~50℃,时间为4~24小时。
5.根据权利要求1所述的一种唑螨酯抗原的制备方法,其特征在于:所述步骤(3)中,式E1或E2所示化合物与所述载体蛋白的摩尔份数比为5~30:1;
所述偶联反应的温度为0~50℃,时间为8~36小时;所述偶联反应在pH值为5~9的条件下进行;
式E1或E2所示化合物在所述载体蛋白的溶液中进行偶联反应,所述载体蛋白的溶液是由所述载体蛋白加入至缓冲溶液中得到的,所述缓冲溶液选自碳酸盐缓冲液、磷酸盐缓冲液、硼酸盐缓冲液和4-羟乙基哌嗪乙磺酸缓冲液中至少一种,所述缓冲液的pH值均为7.4。
6.根据权利要求1所述的一种唑螨酯抗原的制备方法,其特征在于:步骤(3)之后,所述方法还包括将所述偶联反应的反应体系进行透析的步骤;所述透析步骤中,所用透析液为pH值为4~10、浓度为0.01~0.2mol/L的磷酸盐缓冲溶液。
7.根据权利要求1所述的一种唑螨酯抗原的制备方法,其特征在于:所述唑螨酯抗原是式E1或E2化合物与载体蛋白通过酰胺键连接形成的偶联物;所述酰胺键是式D1或D2上的羧基通过活泼酯与载体蛋白上的氨基形成的。
8.一种唑螨酯抗原,根据权利要求1到7任一项所述的一种唑螨酯抗原的制备方法所得。
9.根据权利要求8所述的唑螨酯抗原的应用,其特征在于,用于制备检测样品中唑螨酯的酶联免疫试剂盒、唑螨酯的发光免疫试剂盒或免疫亲和色谱柱。
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