CN103288723B - Sulfonamide hapten and its preparation method and application - Google Patents
Sulfonamide hapten and its preparation method and application Download PDFInfo
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- CN103288723B CN103288723B CN201210053206.1A CN201210053206A CN103288723B CN 103288723 B CN103288723 B CN 103288723B CN 201210053206 A CN201210053206 A CN 201210053206A CN 103288723 B CN103288723 B CN 103288723B
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- 229940124530 sulfonamide Drugs 0.000 title claims abstract description 48
- 150000003456 sulfonamides Chemical class 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 241001465754 Metazoa Species 0.000 claims abstract description 14
- 238000012360 testing method Methods 0.000 claims abstract description 7
- 239000003640 drug residue Substances 0.000 claims abstract description 4
- 239000003814 drug Substances 0.000 claims description 45
- 229940079593 drug Drugs 0.000 claims description 39
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 23
- 239000007788 liquid Substances 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 22
- 238000006243 chemical reaction Methods 0.000 claims description 13
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000012224 working solution Substances 0.000 claims description 11
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 238000002965 ELISA Methods 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 9
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- 230000001900 immune effect Effects 0.000 claims description 9
- SEEPANYCNGTZFQ-UHFFFAOYSA-N sulfadiazine Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=NC=CC=N1 SEEPANYCNGTZFQ-UHFFFAOYSA-N 0.000 claims description 9
- 229960004306 sulfadiazine Drugs 0.000 claims description 9
- 235000012907 honey Nutrition 0.000 claims description 8
- 238000012544 monitoring process Methods 0.000 claims description 7
- -1 phthaloyl sulphur thiazole Chemical compound 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 235000013336 milk Nutrition 0.000 claims description 6
- 239000008267 milk Substances 0.000 claims description 6
- 210000004080 milk Anatomy 0.000 claims description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical class CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 6
- 229940092253 ovalbumin Drugs 0.000 claims description 6
- 239000003208 petroleum Substances 0.000 claims description 6
- 239000008363 phosphate buffer Substances 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 235000018102 proteins Nutrition 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 239000012086 standard solution Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 210000002700 urine Anatomy 0.000 claims description 6
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 5
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims description 5
- WMPXPUYPYQKQCX-UHFFFAOYSA-N Sulfamonomethoxine Chemical compound C1=NC(OC)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 WMPXPUYPYQKQCX-UHFFFAOYSA-N 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 5
- 229940005654 nitrite ion Drugs 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 5
- 229950003874 sulfamonomethoxine Drugs 0.000 claims description 5
- JNMRHUJNCSQMMB-UHFFFAOYSA-N sulfathiazole Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=NC=CS1 JNMRHUJNCSQMMB-UHFFFAOYSA-N 0.000 claims description 5
- 229960001544 sulfathiazole Drugs 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 4
- 108010058846 Ovalbumin Proteins 0.000 claims description 4
- 239000004793 Polystyrene Substances 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 4
- 239000000385 dialysis solution Substances 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 239000012071 phase Substances 0.000 claims description 4
- 229920002223 polystyrene Polymers 0.000 claims description 4
- JLKIGFTWXXRPMT-UHFFFAOYSA-N sulphamethoxazole Chemical compound O1C(C)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 JLKIGFTWXXRPMT-UHFFFAOYSA-N 0.000 claims description 4
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 claims description 3
- ZCIFWRHIEBXBOY-UHFFFAOYSA-N 6-aminonicotinic acid Chemical compound NC1=CC=C(C(O)=O)C=N1 ZCIFWRHIEBXBOY-UHFFFAOYSA-N 0.000 claims description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 3
- 239000007832 Na2SO4 Substances 0.000 claims description 3
- 241001494479 Pecora Species 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000003810 ethyl acetate extraction Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 3
- 241000251468 Actinopterygii Species 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 2
- 241000238557 Decapoda Species 0.000 claims description 2
- 241000287828 Gallus gallus Species 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000005018 casein Substances 0.000 claims description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 2
- 235000021240 caseins Nutrition 0.000 claims description 2
- 239000012074 organic phase Substances 0.000 claims description 2
- 235000011837 pasties Nutrition 0.000 claims description 2
- 235000015277 pork Nutrition 0.000 claims description 2
- 238000002203 pretreatment Methods 0.000 claims description 2
- 239000012460 protein solution Substances 0.000 claims description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 238000010189 synthetic method Methods 0.000 claims description 2
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims 3
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 claims 1
- 238000003763 carbonization Methods 0.000 claims 1
- RAABOESOVLLHRU-UHFFFAOYSA-N diazene Chemical compound N=N RAABOESOVLLHRU-UHFFFAOYSA-N 0.000 claims 1
- 229910000071 diazene Inorganic materials 0.000 claims 1
- 150000002148 esters Chemical class 0.000 claims 1
- 230000002163 immunogen Effects 0.000 claims 1
- 238000012805 post-processing Methods 0.000 claims 1
- 239000003755 preservative agent Substances 0.000 claims 1
- 229960004906 thiomersal Drugs 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 13
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical group NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 11
- 239000000427 antigen Substances 0.000 description 10
- 102000036639 antigens Human genes 0.000 description 10
- 108091007433 antigens Proteins 0.000 description 10
- 238000002835 absorbance Methods 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 7
- 102000014914 Carrier Proteins Human genes 0.000 description 6
- 108010078791 Carrier Proteins Proteins 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 230000002860 competitive effect Effects 0.000 description 4
- 229960002135 sulfadimidine Drugs 0.000 description 4
- ASWVTGNCAZCNNR-UHFFFAOYSA-N sulfamethazine Chemical compound CC1=CC(C)=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 ASWVTGNCAZCNNR-UHFFFAOYSA-N 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- NHZLNPMOSADWGC-UHFFFAOYSA-N 4-amino-N-(2-quinoxalinyl)benzenesulfonamide Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=CN=C(C=CC=C2)C2=N1 NHZLNPMOSADWGC-UHFFFAOYSA-N 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 239000005864 Sulphur Substances 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 150000001718 carbodiimides Chemical class 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
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- 210000002966 serum Anatomy 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- ZZORFUFYDOWNEF-UHFFFAOYSA-N sulfadimethoxine Chemical compound COC1=NC(OC)=CC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 ZZORFUFYDOWNEF-UHFFFAOYSA-N 0.000 description 2
- 229960000973 sulfadimethoxine Drugs 0.000 description 2
- QPPBRPIAZZHUNT-UHFFFAOYSA-N sulfamerazine Chemical compound CC1=CC=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 QPPBRPIAZZHUNT-UHFFFAOYSA-N 0.000 description 2
- 229960003097 sulfaquinoxaline Drugs 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- CBQDCAAOUQVIJL-UHFFFAOYSA-N 4-amino-n-(4,6-dimethylpyrimidin-2-yl)benzenesulfonamide;4-amino-n-(4-methylpyrimidin-2-yl)benzenesulfonamide;4-amino-n-pyrimidin-2-ylbenzenesulfonamide Chemical compound C1=CC(N)=CC=C1S(=O)(=O)NC1=NC=CC=N1.CC1=CC=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1.CC1=CC(C)=NC(NS(=O)(=O)C=2C=CC(N)=CC=2)=N1 CBQDCAAOUQVIJL-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- GPKMZACUUQFPGH-UHFFFAOYSA-N C=NS(=O)=O Chemical compound C=NS(=O)=O GPKMZACUUQFPGH-UHFFFAOYSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- BIGPRXCJEDHCLP-UHFFFAOYSA-N ammonium bisulfate Chemical compound [NH4+].OS([O-])(=O)=O BIGPRXCJEDHCLP-UHFFFAOYSA-N 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 150000003851 azoles Chemical class 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- AOGYCOYQMAVAFD-UHFFFAOYSA-N chlorocarbonic acid Chemical compound OC(Cl)=O AOGYCOYQMAVAFD-UHFFFAOYSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
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- 229940012952 fibrinogen Drugs 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
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- DCKVNWZUADLDEH-UHFFFAOYSA-N sec-butyl acetate Chemical compound CCC(C)OC(C)=O DCKVNWZUADLDEH-UHFFFAOYSA-N 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
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- 150000004867 thiadiazoles Chemical class 0.000 description 1
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- 238000011282 treatment Methods 0.000 description 1
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 description 1
- 229960001082 trimethoprim Drugs 0.000 description 1
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Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of haptens and its preparation method and application, and in particular to a kind of sulfonamide hapten.The invention also discloses the preparation method and applications of the haptens.Based on the kit quick detection product that sulfonamide hapten is set up, easy to use, testing cost is low, detection method is efficient, it is accurate, quick, can simultaneously detect large batch of sample, it is adaptable to the on-site supervision of sulfa drug residue and the examination of great amount of samples in animal derived sample.
Description
Technical field
The present invention relates to a kind of haptens and its preparation method and application, and in particular to sulfonamide hapten and its system
Preparation Method and application.
Technical background
Sulfa drugs (Sulfonamides, SAs) be a class there is P-aminobenzene-sulfonamide structure, for preventing and controlling
Treat the chemotherapeutic agent of bacterial infection disease.Because having wide spectrum, stabilization, economic, easy-to-use and joint Trimethoprim effect
The characteristics of fruit can improve decades of times, the medicine of Chang Yiya treatment concentrations carrys out prophylactic generation as feed addictive, improves
The conversion ratio of feed and promote growth of animal, wherein conventional SAs just has ten several.But there are serious side effects in such medicine,
Long-term presence can cause many bacteriums to produce drug resistance to sulfa drugs in human body, and have potential carcinogenicity, therefore draw
Great attention both domestic and external is played.Total sulfanilamide (SN) and single in the national regulation such as China and the U.S., European Union animal food
The MRL (MRLs) of sulfanilamide (SN) is 0.1mg/kg, and the wherein MRLs of sulfamethazine (SM2) is 0.025 μ g/ml.
At present, the detection method of sulfa drug residue mainly has microbiology method, physico-chemical analysis method and immunization etc..
Microbiology method is easy, expense is low, but during operating cost and sensitiveness, specificity are all poor, is all had difficulties on qualitative, quantitative,
The need for modern residue detection can not be met, or even some testing results do not reach the requirement of MRL also；Physics and chemistry method
Be used to determining medicament residue in animal tissue frequently as confirmation method, such as gas-chromatography (GC), high performance liquid chromatography (HPLC),
Compounds GC-MS (GC-MS), liquid-mass chromatography (HPLC-MS) etc., these methods are sensitive, accurate, and high specificity, separating degree are good, can
To determine multi-medicament simultaneously, but the expensive instrument, complex pretreatment of sample, cumbersome time-consuming, detection relatively costly is needed,
Execute-in-place is unable to, and needs professional to operate, so limiting its application；By comparison, enzyme-linked immunoassay method has spy
Different in nature good, sensitivity is high, easy to operate, testing cost is low, the advantages of be suitable for the selective mechanisms of batch sample, and required set
It is standby few, it is simple to operate easy to learn, it is with low cost, can preferably meet China's livestock and poultry cultivation family, slaughterhouse, food enterprise, government
Functional supervision department etc. carries out detection work.
The content of the invention
It is an object of the invention to provide a kind of sulfonamide hapten and preparation method thereof.
The sulfonamide hapten that the present invention is provided, molecular structural formula is：
The preparation method of the sulfonamide hapten that the present invention is provided, comprises the following steps：
A) 6- amino-nicotinic acids, ethanol and 12mol/L hydrochloric acid are added in 25ml single port bottles, is heated to reflux, react 8h, TLC
Monitoring, reaction is finished, and removal of solvent under reduced pressure is dissolved in saturation NaHCO3The aqueous solution, ethyl acetate extraction, anhydrous Na2SO4Dry, post
Chromatography (ethyl acetate/petroleum ether, 2/1, v/v) purifying；
B) a) products therefrom, triethylamine (Et are added in 25ml single port bottles3) and CH N2Cl2, after stirring and dissolving, addition is urged
Agent DMAP (DMAP).The dichloromethane solution of 4- ASCs is slowly dropped into, TLC is monitored, 5h,
Process after completion of the reaction, dry, column chromatography purifying (ethyl acetate/petroleum ether, 1/10, v/v)；
C) b) products therefrom and the 2mol/L NaOH aqueous solution are added in 25ml single port bottles, is heated to reflux, TLC monitorings, instead
10h is answered, is finished, adjust pH4~5, there is solid to separate out, as sulfonamide hapten.
Another object of the present invention is application of the above-mentioned sulfonamide hapten in immune detection, specifically include by
The sulfa drugs antigen that the sulfonamide hapten is obtained with carrier protein couplet, and by gained sulfa drugs antigen
The sulfa drugs antibody that immune animal prepares.
Wherein described carrier protein can be mouse haemocyanin, thyroprotein, bovine serum albumin(BSA), rabbit serum proteins, people
Haemocyanin, ovalbumin, hemocyanin or fibrinogen,
The antibody is sulfa drugs monoclonal antibody.
The present invention is also provided by the enzyme linked immunological kit of above-mentioned sulfa drugs Antibody preparation, and its in detection animal sources
The application of sulfa drug residue in property sample.
The sulfonamide hapten that the present invention is provided both farthest had remained the chemical constitution of sulfanilamide (SN) parent nucleus, led to again
Cross chemical synthesis transformation introduce can with protein molecule-COOH, synthetic method is simple, and purity, yield are higher；With this
Used as raw material, preparation is suitable to the immune animal of antigen system of animal immune to haptens, the potency of gained antibody, specificity, affine
Power is all relatively good；The antibody of gained be used for enzyme linked immunological kit, can detect simultaneously sulphadiazine, daimeton,
MS-53, sulphathiazole, phthaloyl sulphur thiazole, ayerlucil, seven kinds of medicines of sulfapryidine, easy to use, inspection
Survey low cost, detection method are efficient, accurate, quick, can simultaneously detect large batch of sample, are suitable to sulfanilamide (SN) in animal derived sample
The on-site supervision of class medicament residue and the examination of great amount of samples.Inspection of the sulfonamide hapten of the invention in sulfa drugs
Played a significant role in survey.
Brief description of the drawings
Fig. 1：Sulfonamide hapten synthetic route chart
Fig. 2：Sulfonamide hapten hydrogen nuclear magnetic resonance spectrogram
Fig. 3：Sulfa drugs enzyme linked immunological kit standard curve
Specific embodiment
The present invention is expanded on further with reference to specific embodiment.It should be understood that these embodiments are merely to illustrate this
Invention, and it is not limited to the scope of the present invention.
The synthesis and identification of the sulfonamide hapten of embodiment 1
First, the synthesis (synthetic route such as Fig. 1) of sulfonamide hapten
A) 6- amino-nicotinic acid 0.87g, ethanol 20ml, 12mol/L hydrochloric acid 3.7ml are added in 25ml single port bottles, is heated back
Stream, reacts 8h, and TLC monitorings, reaction is finished, and removal of solvent under reduced pressure is dissolved in saturation NaHCO3The aqueous solution, ethyl acetate extraction, nothing
Water Na2SO4Dry, column chromatography (ethyl acetate/petroleum ether, 2/1, v/v) purifying；
B) a) products therefrom 0.89g, triethylamine (Et are added in 25ml single port bottles3N) 1.6ml, CH2Cl210ml, stirring
After dissolving, catalyst DMAP (DMAP) is added.It is slowly dropped into the 2ml dichloromethane of 4- ASCs
Solution, TLC monitorings, 5h is processed after completion of the reaction, is dried, column chromatography purifying (ethyl acetate/petroleum ether, 1/10, v/v)；
C) b) products therefrom 1g, 2mol/L NaOH aqueous solution 120ml are added in 25ml single port bottles, is heated to reflux, TLC
Monitoring, reacts 10h, finishes, and adjusts pH4~5, has solid to separate out, as sulfonamide hapten.
2nd, the identification of sulfonamide hapten
The sulfonamide hapten of synthesis is taken through nuclear magnetic resonance hydrogen spectruming determining, as shown in Fig. 2 6.7 in collection of illustrative plates~
Three peaks of 8.2ppm or so are the H signal peak on pyridine ring, unimodal N-H peaks on sulfanilamide (SN) key of 11ppm or so,
The unimodal of 13.1ppm or so is carboxyl peak, illustrates hapten synthesis success.
The sulfa drugs antigen of embodiment 2
Sulfonamide hapten and carrier protein be coupled and obtains sulfa drugs antigen.
First, the preparation of immunogene --- sulfonamide hapten-bovine serum albumin(BSA) conjugate synthesis
12mg sulfonamide haptens are taken, is dissolved in 1ml DMFs (DMF), obtain solution I；Take
15mg carbodiimides (EDC) 0.2ml water is fully dissolved in I is added, and 24h is stirred at room temperature, obtains solution II；Weigh
Bovine serum albumin(BSA) (BSA) 40mg, is allowed to be substantially dissolved in 3ml PBS (pH 7.2), and solution II is dropwise slowly dropped to
In protein solution, and 24h is stirred at room temperature；Dialysed 3 days for 4 DEG C with 0.01mol/L PBS, 3 dialyzates are changed daily, to remove
Unreacted small-molecule substance is removed, that is, obtains sulfa drugs immunogene；Packing, saves backup in -20 DEG C.
2nd, the preparation of coating antigen --- sulfonamide hapten-ovalbumin conjugate synthesis
15mg sulfonamide hapten 0.8ml DMF dissolvings are taken, 10 DEG C are cooled to, solution I is obtained；Take chloro-carbonic acid different
The μ l of butyl ester 10 are added in I, 10 DEG C of stirring reaction 30min；Take 30mg ovalbumin 2.2ml 50mmol/L Na2CO3Dissolving, 10
DEG C reaction 4h, then 4 DEG C overnight；Dialysed 3 days for 4 DEG C with 0.01mol/L PBS, 3 dialyzates are changed daily, to remove unreacted
Small-molecule substance, that is, obtain sulfa drugs coating antigen；Packing, saves backup in -20 DEG C.
3rd, the identification of sulfa drugs antigen
By carrier protein, sulfonamide hapten, sulfonamide hapten-carrier protein couplet thing with pH7.4's
PBS is made into the solution of 0.5mg/ml, with 0.01mol/L pH7.4PBS return to zero, with ultraviolet specrophotometer wavelength 200~
Scanned in the range of 800nm, obtain carrier protein, sulfonamide hapten, sulfonamide hapten-carrier protein couplet thing
Absorption curve, and calculate its combine than.Result finds that different absorption curves occurs in three, shows that sulfa drugs half is anti-
Former successful with carrier protein couplet, the combination ratio of haptens and bovine serum albumin(BSA) is (18~22):1, the knot with ovalbumin
Composition and division in a proportion is (16~21):1.
The sulfa drugs monoclonal antibody of embodiment 3
First, the preparation of sulfa drugs monoclonal antibody
Animal immune：Immunogene is injected into Balb/c Mice Bodies, immunizing dose is 150 μ g/, its is produced many grams
Grand antibody serum.
Cell fusion and cloning：After mice serum measurement result is higher, its splenocyte is taken, by 8:1 ratio and SP2/0 bones
Myeloma cells are merged, and cell supernatant, the positive hole of screening are determined using indirect competitive ELISA.Using limiting dilution assay to the positive
Hole carries out cloning, the hybridoma cell strain until obtaining secrete monoclonal antibody.
Cell cryopreservation and recovery：The monoclonal hybridoma strain of sulfa drugs is made 1 × 10 with frozen stock solution9Individual/
The cell suspension of ml, preserves for a long time in liquid nitrogen.Cryopreservation tube is taken out during recovery, 37 DEG C of water-bath middling speeds is immediately placed in and is melted, centrifugation is gone
After frozen stock solution, culture culture in glassware is moved into.
The production of monoclonal antibody and purifying：By Balb/c mouse peritoneals injection sterilizing paraffin oil 0.5ml/ only, abdomen after 7 days
The monoclonal hybridoma strain 6 × 10 of chamber injection sulfa drugs5Individual/only, gather ascites after 7 days.With octanoic acid-saturation sulfuric acid
Ammonium method carries out ascites purifying, -20 DEG C of preservations.
2nd, the measure of antibody titer
The potency for determining antibody with indirect competitive ELISA method is 1:(50000~100000).
Indirect competitive ELISA method：With sulfonamide hapten-ovalbumin conjugate coated elisa plate, sulphur is added
Amic metadiazine standard solution, ELIAS secondary antibody and monoclonal antibody working solution, 25 DEG C of reaction 30min, pour out liquid in hole, use PBST
Cleaning solution is washed 3~5 times, is patted dry with blotting paper；Substrate nitrite ion is added, after 25 DEG C of reaction 15min, adds terminate liquid to terminate anti-
Should；Setting ELIASA is determined per hole absorbance at wavelength 450nm.
3rd, the specificity of monoclonal antibody
Antibody specificity refers to the ability and the ratio with such antigen-analogues ability of its homospecificity antigen binding
Compared with conventional cross reacting rate is used as evaluation criterion.Cross reaction is smaller, and the specificity of antibody is then higher.
This experiment is by sulphadiazine, daimeton, MS-53, sulphathiazole, phthaloyl sulphur thiazole, sulphur
Amine first thiadiazoles, sulfapryidine, sulfamethazine, sulfadimethoxine, sulfaquinoxaline, 11 kinds of sulfamethyldiazine
Sulfa drugs is serially diluted, and carries out indirect competitive ELISA with monoclonal antibody respectively, makes standard curve, and analysis is obtained
IC50, then it is calculated as follows cross reacting rate：
Result shows that the cross reacting rate of each sulfa drugs is：Sulphadiazine 100.0%, daimeton
97.6%th, MS-53 169.0%, sulphathiazole 188.0%, phthaloyl sulphur thiazole 62.8%, ayerlucil
103%th, sulfapryidine 51.9%, sulfamethazine ＜ 1%, sulfadimethoxine ＜ 1%, sulfaquinoxaline ＜ 1%,
Sulfamethyldiazine ＜ 1%.Antibody of the present invention can simultaneously detect sulphadiazine, daimeton, methylene sulfonamide Yi Evil
Azoles, sulphathiazole, phthaloyl sulphur thiazole, ayerlucil, 7 kinds of sulfa drugs of sulfapryidine.
The enzyme linked immunological kit that embodiment 4 is prepared by sulfa drugs monoclonal antibody
First, the composition of enzyme linked immunological kit
(1) it is coated with the ELISA Plate of sulfonamide hapten-carrier protein couplet thing；
(2) with the sheep anti mouse antiantibody of horseradish peroxidase-labeled；
(3) sulfa drugs monoclonal antibody working solution；
(4) 6 bottles of sulphadiazine standard solution, concentration is respectively 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5
μg/L、40.5μg/L；
(5) substrate nitrite ion is made up of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine；
(6) terminate liquid is 2mol/L sulfuric acid；
(7) concentrated cleaning solution is pH7.2, containing 0.8%~1.2% Tween-20,0.01 ‰~0.03 ‰ thimerosal anti-corrosions
Agent, the carbonate buffer solution of 0.1~0.3mol/L, the percentage are percent weight in volume；
(8) it is pH7.6, the phosphate buffer containing 8%~12% casein, 0.1~0.3mol/L, institute that liquid is redissolved in concentration
Percentage is stated for percent weight in volume.
The main agents of this kit are provided in the form of working solution, and the method for inspection is convenient and easy, with specific high, spirit
The features such as sensitivity is high, accuracy is high, the degree of accuracy is high, while using one-step method, saving detection time.
2nd, enzyme linked immunological kit detects the application of actual sample
1. sample pre-treatments
(1) fresh milk pre-treating method
50 μ l fresh milk samples are taken, is added in 450 μ l redissolution working solutions, mixed；50 μ l are taken for analyzing
(2) tissue (chicken, pork, fish, shrimp) pre-treating method
After with homogenizer homogeneous structure sample, in weighing 2.0 ± 0.05g homogeneous thing to 50ml polystyrene centrifuge tubes, plus
Enter the phosphate buffer whirling motion of 4ml pH=6 into pasty state, add 6ml ethyl acetate, vibrate 1min, 4000rpm room temperatures
(20-25 DEG C/68-77 ℉) is centrifuged 5min；In taking 3ml supernatants to the clean teat glasses of 10ml, in 50~60 DEG C of water-bath nitrogen
Flow down drying；1ml n-hexanes are added, with vortex instrument whirling motion 30s, 1ml is added and is redissolved working solution, whirling motion 10s, 4000rpm rooms
Warm (20-25 DEG C/68-77 ℉) centrifugation 5min；Upper strata n-hexane phase is removed, the μ l of layer water phase 50 is removed for analyzing.
(3) honey pre-treating method
In weighing 1.0 ± 0.05g honey sample to 50ml polystyrene centrifuge tubes；1ml 1mol/L hydrochloric acid solutions are added,
Vibrated to honey with oscillator and all dissolved；It is put into 37 DEG C of incubators and is incubated 30min；Take out, add 1ml 1mol/L hydrogen-oxygens
Change the phosphate buffer of sodium solution and 1ml pH=6, then be separately added into 2ml acetonitriles and 6ml ethyl acetate, vibrated with oscillator
1min, 4000rpm room temperature (20-25 DEG C/68-77 ℉) are centrifuged 10min；Take 4ml upper organic phases to the clean teat glasses of 10ml
In, in drying under 50~60 DEG C of water-bath nitrogen streams；Plus 0.5ml redissolves working solution dissolving；50 μ l are taken for analyzing.
(4) urine pre-treating method
50 μ l urine specimens are taken, is added in 150 μ l wash operating solutions, mixed；50 μ l are taken for analyzing.
2. detected with kit
Sulphadiazine standard solution or sample are added to being coated with the ELISA Plate micropore of sulfa drugs envelope antigen
The μ l of solution 50, add the μ l/ holes of sheep anti mouse antiantibody 50 of horseradish peroxidase-labeled immediately, add sulfa drugs Dan Ke
The μ l/ holes of grand antibody working solution 50, with cover plate membrane cover plate after, put lucifuge reaction 30min in 25 DEG C of insulating boxs, pour out liquid in hole,
250 μ l cleaning solutions are added per hole, liquid in hole is poured out after 30s, repeat operation board-washing 5 times, patted dry with blotting paper；Add per hole
Enter the μ l of substrate nitrite ion A liquid urea peroxide 50, substrate nitrite ion B liquid tetramethyl benzidine (TMB) 50 μ l, gently vibration is mixed,
With lucifuge colour developing 15min in the rearmounted 25 DEG C of insulating boxs of cover plate membrane cover plate, the μ l of terminate liquid 2mol/L sulfuric acid 50 are added per hole, gently shaken
Mixing is swung, is set at 450nm with ELIASA wavelength, determined per hole absorbance (OD values).
3. Analysis of test results
With the absorbance values (B) of the standard solution of each concentration for being obtained divided by first standard solution (0
Standard) absorbance (B0) multiplied by with 100%, obtaining percentage absorbance.With sulphadiazine standard concentration (μ g/L)
Logarithm value is X-axis, and percentage absorbance is Y-axis, draws canonical plotting, as shown in Figure 3.It is molten sample to be calculated with same method
The percentage absorbance of liquid, from standard curve read sample corresponding to concentration, be multiplied by its corresponding extension rate, multiplied by with
The corresponding cross reacting rate of every kind of medicine, the actual concentrations of sulfa drugs as in sample.
3rd, the determination of enzyme linked immunological kit technical parameter
LDL：20 parts of dummies are detected, is found from standard curve corresponding to each percentage absorbance
The concentration of value, test limit is represented with 20 parts of average values of sample sulfa drugs concentration plus 3 times of standard deviations, as a result obtains the method
Lowest detection to milk sample is limited to 5 μ g/L, and the lowest detection to animal tissue, honey sample is limited to 0.5 μ g/kg, to urine
The lowest detection of liquid sample is limited to 2 μ g/L.
The degree of accuracy and precision：The degree of accuracy that ELISA is determined represents that precision is represented with the coefficient of variation with the rate of recovery.Take
Dummy, recovery test is added to it with 50,100,200 μ g/kg, tri- sulfa drugs of concentration, and (addition concentration surpasses
Detected again after going out curved measurement scope, it is necessary to sample is taken the circumstances into consideration dilution), as a result obtain recovery of the method to milk sample
Rate is 85% ± 15%, is 75% ± 10% to the rate of recovery of animal tissue's sample, to the rate of recovery of honey sample for 90% ±
10%, the rate of recovery to urine specimen is 95% ± 15%, variation within batch coefficient ＜ 15%, interassay coefficient of variation ＜ 25%.
Kit of the present invention can at least be preserved 12 months at 2~8 DEG C after measured.
Claims (3)
1. a kind of sulfa drugs enzyme linked immunological kit detects the application of sulfa drug residue in animal derived sample, including
Step：
(1) sample pre-treatments：
Fresh milk pre-treating method：Take 50 μ l fresh milk samples, in adding 450 μ l to redissolve working solution, mix, take 50 μ l for point
Analysis；
Chicken, pork, fish, shrimp pre-treating method：After with homogenizer homogeneous structure sample, 2.0 ± 0.05g homogeneous thing is weighed extremely
In 50ml polystyrene centrifuge tubes, add the phosphate buffer whirling motion of 4ml pH=6 into pasty state, add 6ml ethyl acetate,
Vibration 1min, 4000rpm room temperatures centrifugation 5min, in taking 3ml supernatants to the clean teat glasses of 10ml, in 50~60 DEG C of water-bath nitrogen
Dried up under air-flow, add 1ml n-hexanes, with vortex instrument whirling motion 30s, added 1ml and redissolve working solution, whirling motion 10s, 4000rpm
Room temperature is centrifuged 5min, removes upper strata n-hexane phase, removes the μ l of layer water phase 50 for analyzing；
Honey pre-treating method：Weigh in 1.0 ± 0.05g honey sample to 50ml polystyrene centrifuge tubes, add 1ml 1mol/
L hydrochloric acid solutions, are vibrated to honey with oscillator and all dissolved, and are put into 37 DEG C of incubators and are incubated 30min, are taken out, and are added
The phosphate buffer of 1ml1mol/L sodium hydroxide solutions and 1ml pH=6, then it is separately added into 2ml acetonitriles and 6ml acetic acid second
Ester, 1min, 4000rpm room temperatures centrifugation 10min are vibrated with oscillator, in taking 4ml upper organic phases to the clean teat glasses of 10ml,
Working solution dissolving is redissolved in drying under 50~60 DEG C of water-bath nitrogen streams, plus 0.5ml, 50 μ l is taken for analyzing；
Urine pre-treating method：50 μ l urine specimens are taken, is added in 150 μ l wash operating solutions, mixed, take 50 μ l for analyzing；
(2) detected with kit；
(3) Analysis of test results；
It is characterized in that the kit is the enzyme linked immunological kit for detecting sulfa drugs, it contains：
(1) it is coated with the ELISA Plate of sulfonamide hapten-carrier protein couplet thing；
(2) with the sheep anti mouse antiantibody of horseradish peroxidase-labeled；
(3) sulfa drugs monoclonal antibody working solution；
(4) 6 bottles of sulphadiazine standard solution, concentration is respectively 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/
L、40.5μg/L；
(5) substrate nitrite ion is made up of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine；
(6) terminate liquid is 2mol/L sulfuric acid；
(7) concentrated cleaning solution is pH7.2, containing 0.8%~1.2% Tween-20,0.01 ‰~0.03 ‰ thiomersal preservatives, 0.1
The carbonate buffer solution of~0.3mol/L, the percentage is percent weight in volume；
(8) it is pH7.6, the phosphate buffer containing 8%~12% casein, 0.1~0.3mol/L, described hundred that liquid is redissolved in concentration
Divide than being percent weight in volume；
The preparation method of the sulfonamide hapten-carrier protein couplet thing comprises the following steps：Take 15mg sulfonamides
Thing haptens 0.8ml DMFs dissolve, and are cooled to 10 DEG C, obtain solution I；Take the μ l of isobutyl chlorocarbonate 10
In addition I, 10 DEG C of stirring reaction 30min；Take 30mg ovalbumin 2.2ml 50mmol/L Na2CO3Dissolving, 10 DEG C of reactions
4h, then 4 DEG C overnight；Dialysed 3 days for 4 DEG C with 0.01mol/L PBS, 3 dialyzates are changed daily, that is, obtain sulfa drugs bag
It is former；
The synthetic method of the sulfonamide hapten is：A) 6- amino-nicotinic acid 0.87g, ethanol are added in 25ml single port bottles
20ml, 12mol/L hydrochloric acid 3.7ml, are heated to reflux, and react 8h, and TLC monitorings, reaction is finished, and removal of solvent under reduced pressure is dissolved in saturation
NaHCO3The aqueous solution, ethyl acetate extraction, anhydrous Na2SO4Dry, ethyl acetate:Petroleum ether=2:1(v:V) column chromatography purifying；
B) a) products therefrom 0.89g, triethylamine 1.6ml, CH are added in 25ml single port bottles2Cl210ml, after stirring and dissolving, addition is urged
Agent DMAP, is slowly dropped into the 2ml dichloromethane solutions of 4- ASCs, and TLC is monitored, 5h, instead
Post processing should be finished, is dried, ethyl acetate:Petroleum ether=1:10(v:V) column chromatography purifying；C) added b) in 25ml single port bottles
Products therefrom 1g, 2mol/L NaOH aqueous solution 120ml, are heated to reflux, TLC monitorings, react 10h, finish, and adjust pH4~5, have
Solid is separated out, as sulfonamide hapten, and its molecular structural formula is：
2. application as claimed in claim 1, it is characterised in that the sulfa drugs monoclonal antibody is by sulfa drugs
Animal prepares immunogen immune, can detect simultaneously sulphadiazine, daimeton, MS-53,
Sulphathiazole, phthaloyl sulphur thiazole, ayerlucil, 7 kinds of sulfa drugs of sulfapryidine.
3. application as claimed in claim 2, it is characterised in that the preparation method of the sulfa drugs immunogene includes as follows
Step：
12mg sulfonamide haptens are taken, is dissolved in 1ml DMFs, obtain solution I；Take 15mg carbonizations
Diimine is added in I after fully being dissolved with 0.2ml water, and 24h is stirred at room temperature, obtains solution II；Weigh bovine serum albumin(BSA)
40mg, is allowed to be substantially dissolved in the PBS of 3ml pH 7.2, and solution II is dropwise slowly dropped in protein solution, and in room
The lower stirring 24h of temperature；Dialysed 3 days for 4 DEG C with 0.01mol/L PBS, 3 dialyzates are changed daily, that is, obtained sulfa drugs and be immunized
It is former.
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| CN110330488A (en) * | 2019-06-20 | 2019-10-15 | 深圳市易瑞生物技术股份有限公司 | A kind of piroxicam haptens and its preparation method and application |
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| 磺胺母核结构半抗原的合成与表征;段振娟 等;《天津科技大学学报》;20070630;第22卷(第2期);第24-28页 * |
| 磺胺类药物残留酶免疫分析的研究;房超;《中国优秀硕士学位论文全文数据库 农业科技辑》;20041215(第4期);第12页1.3.2-1.3.3 * |
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