CN109180519A - A kind of olaquindox metabolite antigen, antibody and enzyme-linked immunologic detecting kit and detection method - Google Patents
A kind of olaquindox metabolite antigen, antibody and enzyme-linked immunologic detecting kit and detection method Download PDFInfo
- Publication number
- CN109180519A CN109180519A CN201810652749.2A CN201810652749A CN109180519A CN 109180519 A CN109180519 A CN 109180519A CN 201810652749 A CN201810652749 A CN 201810652749A CN 109180519 A CN109180519 A CN 109180519A
- Authority
- CN
- China
- Prior art keywords
- olaquindox metabolite
- olaquindox
- solution
- antibody
- metabolite
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- TURHTASYUMWZCC-UHFFFAOYSA-N Olaquindox [BAN:INN] Chemical class C1=CC=C2N([O-])C(C)=C(C(=O)NCCO)[N+](=O)C2=C1 TURHTASYUMWZCC-UHFFFAOYSA-N 0.000 title claims abstract description 154
- 239000000427 antigen Substances 0.000 title claims abstract description 56
- 102000036639 antigens Human genes 0.000 title claims abstract description 56
- 108091007433 antigens Proteins 0.000 title claims abstract description 56
- 238000001514 detection method Methods 0.000 title claims abstract description 43
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 36
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 36
- 230000001900 immune effect Effects 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 30
- 238000012360 testing method Methods 0.000 claims abstract description 23
- 238000002360 preparation method Methods 0.000 claims abstract description 19
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 7
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 7
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 63
- 239000007788 liquid Substances 0.000 claims description 50
- 239000000758 substrate Substances 0.000 claims description 36
- 238000002965 ELISA Methods 0.000 claims description 20
- 239000012224 working solution Substances 0.000 claims description 18
- 238000004140 cleaning Methods 0.000 claims description 17
- 238000002835 absorbance Methods 0.000 claims description 16
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 15
- 239000007853 buffer solution Substances 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 238000004458 analytical method Methods 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 239000008363 phosphate buffer Substances 0.000 claims description 7
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 claims description 6
- ZBQCCTCQUCOXBO-UHFFFAOYSA-N 4-(4-aminophenyl)-2,2,6,6-tetramethylcyclohex-3-en-1-amine Chemical compound CC1(C)C(N)C(C)(C)CC(C=2C=CC(N)=CC=2)=C1 ZBQCCTCQUCOXBO-UHFFFAOYSA-N 0.000 claims description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 3
- 239000007979 citrate buffer Substances 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
- 238000003018 immunoassay Methods 0.000 claims description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 abstract description 18
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 235000013305 food Nutrition 0.000 abstract description 5
- 238000011084 recovery Methods 0.000 abstract description 3
- 238000010998 test method Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 28
- 239000012086 standard solution Substances 0.000 description 24
- 239000011248 coating agent Substances 0.000 description 22
- 238000000576 coating method Methods 0.000 description 22
- 229950010210 olaquindox Drugs 0.000 description 16
- 241001465754 Metazoa Species 0.000 description 14
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 108010058846 Ovalbumin Proteins 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 239000003814 drug Substances 0.000 description 8
- 229940079593 drug Drugs 0.000 description 8
- 230000002401 inhibitory effect Effects 0.000 description 8
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 8
- 229940092253 ovalbumin Drugs 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 210000004408 hybridoma Anatomy 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 230000004060 metabolic process Effects 0.000 description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 6
- 229910019142 PO4 Inorganic materials 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 238000004321 preservation Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 239000004793 Polystyrene Substances 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 235000013330 chicken meat Nutrition 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 229920002223 polystyrene Polymers 0.000 description 4
- 235000015277 pork Nutrition 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- -1 thyroprotein (BCG) Proteins 0.000 description 4
- 206010003445 Ascites Diseases 0.000 description 3
- 241000287828 Gallus gallus Species 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 230000007910 cell fusion Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000002203 pretreatment Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- JFBYGMUJXBUWEO-UHFFFAOYSA-N 3-methylnaphthalene-2-carboxylic acid Chemical compound C1=CC=C2C=C(C(O)=O)C(C)=CC2=C1 JFBYGMUJXBUWEO-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000272525 Anas platyrhynchos Species 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 231100000703 Maximum Residue Limit Toxicity 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 229910006124 SOCl2 Inorganic materials 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000000861 blow drying Methods 0.000 description 2
- 229960000427 carbadox Drugs 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 239000006210 lotion Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- BPMVRAQIQQEBLN-OBPBNMOMSA-N methyl n-[(e)-(1-hydroxy-4-oxidoquinoxalin-4-ium-2-ylidene)methyl]iminocarbamate Chemical compound C1=CC=C2N(O)C(=C/N=NC(=O)OC)/C=[N+]([O-])C2=C1 BPMVRAQIQQEBLN-OBPBNMOMSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 230000020477 pH reduction Effects 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000003252 repetitive effect Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Substances ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- YEKPNMQQSPHKBP-UHFFFAOYSA-N 2-methyl-6-nitrobenzoic anhydride Chemical compound CC1=CC=CC([N+]([O-])=O)=C1C(=O)OC(=O)C1=C(C)C=CC=C1[N+]([O-])=O YEKPNMQQSPHKBP-UHFFFAOYSA-N 0.000 description 1
- BJPNADFNSANIPF-UHFFFAOYSA-N 3-methylquinoxaline-2-carboxylic acid Chemical compound C1=CC=C2N=C(C(O)=O)C(C)=NC2=C1 BJPNADFNSANIPF-UHFFFAOYSA-N 0.000 description 1
- 240000003291 Armoracia rusticana Species 0.000 description 1
- 235000011330 Armoracia rusticana Nutrition 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 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
- 244000118681 Iresine herbstii Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000007059 acute toxicity Effects 0.000 description 1
- 231100000403 acute toxicity Toxicity 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 239000005030 aluminium foil Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000011001 backwashing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000000105 evaporative light scattering detection Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000009920 food preservation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000011020 pilot scale process Methods 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- UPUZGXILYFKSGE-UHFFFAOYSA-N quinoxaline-2-carboxylic acid Chemical compound C1=CC=CC2=NC(C(=O)O)=CN=C21 UPUZGXILYFKSGE-UHFFFAOYSA-N 0.000 description 1
- 150000003252 quinoxalines Chemical class 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229920002379 silicone rubber Polymers 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- GOLXNESZZPUPJE-UHFFFAOYSA-N spiromesifen Chemical compound CC1=CC(C)=CC(C)=C1C(C(O1)=O)=C(OC(=O)CC(C)(C)C)C11CCCC1 GOLXNESZZPUPJE-UHFFFAOYSA-N 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C233/00—Carboxylic acid amides
- C07C233/64—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings
- C07C233/81—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
- C07C233/82—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
- C07C233/83—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom of an acyclic saturated carbon skeleton
-
- 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
-
- 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
- C07K14/745—Blood coagulation or fibrinolysis factors
- C07K14/75—Fibrinogen
-
- 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
- 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
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of olaquindox metabolite antigen, antibody and enzyme-linked immunologic detecting kit and detection methods.The present invention is using 4- (3- methyl -2- naphthoyl amido) butyric acid as olaquindox metabolite hapten, available olaquindox metabolite antigen is coupled after the haptens and carrier protein, the olaquindox metabolite antigen can be applied to preparation olaquindox metabolite specific antibody, which can be used for preparing the remaining enzyme linked immunological kit of olaquindox metabolite or colloidal gold test paper card in detection food again.The invention also discloses corresponding kit test methods.This method is easy, quick, its linear detection range is 0.2~16.2 ng/mL, sensitivity is 0.54 ng/mL, detection is limited to 0.32 ng/mL, the rate of recovery is 80%~109.8%, detection limit is low, high sensitivity, high specificity, stability are good, at low cost, is very suitable to a large amount of sample detections and field quick detection.
Description
Technical field
The invention belongs to drug residue detection technique fields.More particularly, to a kind of olaquindox metabolite antigen, antibody
And enzyme-linked immunologic detecting kit and detection method.
Background technique
Olaquindox is the early production of quinoxaline class drug, since the 1970s, once in worldwide extensively
Using, but disabled by the whole world acted in food promoting animal growth at present.Forbade carbadox and quinoline second in 1998 in European Union
The use of alcohol, China forbid olaquindox and carbadox being used for food promoting animal growth, and provide that olaquindox is residual in animal tissue
Stay the maximum residue limit of marker.In recent years, olaquindox drug mainly uses on Australia, Brazil, Japan and China and other places.
The toxicity that olaquindox generates animal body mainly includes acute toxicity and cumulative toxicity.Olaquindox excess intake can make
Animal stress is bled profusely and dead, also can be generated teratogenesis to animal or people when accumulating to a certain extent in animal body, be caused
Cancer, mutagenic " three-induced effect ".Therefore, in use, its use scope must strictly be pressed, dose concentration carries out the off-drug period.Mirror
Toxicity and existing potential hazard in olaquindox, thus people again evaluate the safety of olaquindox, to olaquindox
Use be also made that new regulation.Olaquindox itself is unstable, and short time metabolism in animal body has more than ten kinds of metabolism to produce
Object, most of not have testing conditions, wherein 3- methylquinoxaline-2-carboxylic acid (MQCA) is major metabolite, in vivo relatively
Stablize, is the mark residue that the state food code committee is assert, therefore usually using olaquindox metabolite MQCA as residual
The object of analysis and monitoring.The Ministry of Agriculture of China in 2003 defines the maximum residue limit difference of MQCA in muscle and liver organization
For 4 μ g/kg and 50 μ g/kg.
However, olaquindox drug is still largely illegally used or abused in production practice, the residual of drug is to consumption
Person's health and animal food outlet cause huge potential threat, to drive the research of related detecting method.These detection sides
Method has liquid chromatography-mass spectrometry, liquid chromatography, gas phase process, enzyme-linked immunization, colloidal gold absorption detection method, molecular engram
With method for biosensor etc..Wherein, analysis method is flowed based on liquid chromatography-mass spectrometry and enzyme-linked immunization.In contrast, exempting from
Epidemic disease analytic approach becomes and is worthy to be popularized often because at low cost, easy to operate, speed is fast, one-time detection sample size is big, instrumentation degree is low
Screening technique.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect of the above-mentioned prior art and deficiencies, provide a kind of olaquindox generation
Thank object antigen, antibody and enzyme-linked immunologic detecting kit and detection method.The enzyme linked immunological kit has accurate, sensitive, fast
Speed, can high-throughput detection olaquindox metabolite the advantages of, can efficient detection olaquindox metabolite, be that olaquindox metabolite is fast
The exploitation of fast testing product provides certain theoretical direction, is of great significance to the monitoring of drug safety.
The first purpose of the invention is to provide a kind of olaquindox metabolite haptens.
A second object of the present invention is to provide the olaquindox generations being prepared using above-mentioned olaquindox metabolite hapten
Thank to object antigen.
Third object of the present invention is to provide above-mentioned olaquindox metabolite antigens in preparation olaquindox metabolite antibody
Application.
Fourth object of the present invention is to provide the olaquindox metabolism being prepared using above-mentioned olaquindox metabolite antigen
Object antibody.
Fifth object of the present invention is to provide above-mentioned olaquindox metabolite antibody answering in detection olaquindox metabolite
With.
Sixth object of the present invention is to provide application above-mentioned olaquindox metabolite hapten, olaquindox metabolite antigen,
The enzyme linked immunological kit that olaquindox metabolite Antibody preparation obtains.
7th purpose of the invention, which is to provide, carries out olaquindox generation in test sample using above-mentioned enzyme linked immunological kit
Thank to the remaining method of object.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The present invention relates to a kind of olaquindox metabolite hapten, the haptens is 4- (3- methyl -2- naphthoyl amido) butyric acid,
Molecular structural formula is as the formula (1):
Formula (1).
The preparation method of haptens 4- (3- methyl -2- naphthoyl amido) butyric acid, comprising the following steps: by 3- methyl -
2- naphthoic acid is dissolved in SOCl2In, it is heated to 90~110 DEG C of 1~3 h of reaction under nitrogen protection；Solvent is removed under reduced pressure, it will be remaining
Object is dissolved in Isosorbide-5-Nitrae-dioxanes, under agitation, is added dropwise to amino acid and Na2CO3Mixed aqueous solution in；Room temperature
It is stirred overnight, after hydrochloric acid solution acidification is added, is extracted with EtOAC, after merging organic phase, washed with saturation NaCl solution,
After drying, purify to obtain 4- (3- methyl -2- naphthoyl amido) butyric acid, the as described haptens using silica gel column chromatography.
It is by the olaquindox metabolite hapten and carrier egg the invention further relates to a kind of olaquindox metabolite antigen
White coupling obtains.
Wherein, the carrier protein can be mouse serum albumin, thyroprotein (BCG), bovine serum albumin(BSA), rabbit anteserum
The common carriers albumen such as albumin, human serum albumins, ovalbumin (OVA), hemocyanin or fibrinogen.
Preferably, the carrier protein is ovalbumin.
The olaquindox metabolite antigen the preparation method comprises the following steps: using active ester method, by the haptens (4- (3- first
Base -2- naphthoyl amido) butyric acid) and carrier protein couplet, olaquindox metabolite antigen can be obtained.
The olaquindox metabolite antigen can be used as immunogene preparation olaquindox metabolite antibody, can also be used as coating
Original prepares ELISA Plate.
The antibody can be monoclonal antibody, polyclonal antibody or genetic engineering antibody, preferably monoclonal antibody.
The olaquindox metabolite polyclonal antibody the preparation method is as follows:
Above-mentioned olaquindox metabolite antigen is made into immunogen immune rabbit or mouse, prepares olaquindox metabolite polyclonal antibody,
Antiserum is finally collected, deposition and purification is plated with sad sulfuric acid and crosses affinity column and purify.
The olaquindox metabolite monoclonal antibody the preparation method is as follows:
Above-mentioned olaquindox metabolite antigen is made into immunogen immune mouse, is carried out with mouse boosting cell and SP2/0 myeloma cell
Fusion obtains hybridoma, and hybridoma is placed in female Balb/c mouse In vivo culture, obtains monoclonal containing high concentration
The ascites of antibody, and ascites is purified to obtain the anti-olaquindox metabolite monoclonal antibody of high specific.
Correspondingly, the antibody that is prepared using the olaquindox metabolite antigen and its in detection olaquindox metabolite
Application, also within protection scope of the present invention.
Using the olaquindox metabolite hapten, the olaquindox metabolite antigen, the olaquindox metabolite antibody
The enzyme linked immunological kit or colloidal gold test paper card being prepared, also within protection scope of the present invention.
Preferably, the enzyme linked immunological kit, contains: being coated with ELISA Plate, the quinoline second of olaquindox metabolite antigen
Alcohol metabolite antibody, ELIAS secondary antibody, olaquindox metabolite serial standards solution, substrate buffer solution and substrate working solution, concentration
Cleaning solution, redissolves liquid and sulfuric acid solution at terminate liquid.
Preferably, the coating buffer of the coating olaquindox metabolite antigen are as follows: by 1.69 g sodium carbonate and 2.95 g carbonic acid
Hydrogen sodium, which is dissolved in 1 L distilled water, to be obtained；The peridium concentration of olaquindox metabolite antigen is 0.125 μ g/L.
Preferably, the ELISA Plate can be the removable transparent ELISA Plate in 96 holes, and material is polystyrene, and being coated with can be with anti-quinoline
The olaquindox metabolite antigen that alcohol metabolism object antibody specificity combines, and the unadsorbed olaquindox metabolite in closed porosity surface is anti-
Former site.
The ELISA Plate for being coated with olaquindox metabolite antigen the preparation method comprises the following steps: with coating buffer by olaquindox metabolite
Antigen dilutes as needed, and coating buffer is added into hole, is put into 37 DEG C of environment and is incubated overnight, coating buffer of inclining uses cleaning solution
Then confining liquid is added in washing in every hole, 37 DEG C are incubated for, liquid in hole of inclining, and is saved after dry with aluminium film vacuum sealing.
Herein staying overnight refers to 8~16 h.
Wherein in ELISA Plate preparation process, confining liquid used is by 0.1 g BSA(bovine serum albumin(BSA)), the sweet ammonia of 5 g
Acid, 5 g sucrose are dissolved in 100 mL PBS(0.01 mol/L, pH 7.4) solution obtains.
Furthermore it is possible to which the substance as fixed olaquindox metabolite antigen solid phase carrier is more, such as polystyrene, nitric acid
The cruel glue of cellulose, polyethylene, polypropylene, polypropylene, cross-linked glucose, silicon rubber, Ago-Gel etc..The form of the carrier can
Think shrinkage pool, the scraps of paper, globule etc..
Preferably, the ELIAS secondary antibody is goat anti-rabbit igg.The marker enzyme of enzyme labelled antibody is horseradish peroxidase or alkalinity
Phosphate, preferably horseradish peroxidase.
Preferably, the olaquindox metabolite serial standards solution, the phosphorus for being when in use 5.4 with 0.2 mol/L, pH
Phthalate buffer, it is respectively 0,0.2,0.6,1.8,5.4,16.2 ng/ that olaquindox metabolite standard solution, which is diluted to concentration,
A series of olaquindox metabolite standard solutions of mL.
It is highly preferred that the formula of the phosphate buffer are as follows: the NaH of 2.9 g2PO4·12H2O, the NaCl of 8.5 g,
The KH of the KCl of 0.2 g, 0.2 g2PO4, it is settled to 1 L.
Preferably, it is 4.5~5.5 that the substrate working solution, which is pH, phosphoric acid-lemon containing hydrogen peroxide or urea peroxide
Acid buffer；The substrate buffer solution is that pH is 4.5~5.5, and the phosphoric acid-citric acid for containing 3,3,5,5- tetramethyl benzidines is slow
Rush solution.
It is highly preferred that the ratio that the substrate working solution and the substrate buffer solution are 1:1 by volume mix
To substrate solution.
Preferably, it is 7.0~7.8 that the concentrated cleaning solution, which is pH, contains 0.4%~0.6% Tween-20,0.3~0.5
The phosphate buffer of mol/L, the percentage are percent weight in volume.
It is highly preferred that the concentrated cleaning solution that the concentrated cleaning solution is 20 times；1 times is diluted to deionized water when use to wash
Wash liquid.
Preferably, the terminate liquid is 0.5 M H2SO4。
Preferably, the sulfuric acid solution is the sulfuric acid solution of 3 M.
Preferably, the redissolution liquid is 0.2 M PBS.
The invention further relates to carry out the remaining method of olaquindox metabolite in test sample with above-mentioned enzyme linked immunological kit.
Particularly preferably, the remaining method of olaquindox metabolite in the test sample, comprising the following steps:
S1. the enzyme linked immunological kit is taken out from cold storage environment, is placed in 15~35 DEG C of 30~45min of balance；
S2. ELISA Plate is taken out, the olaquindox metabolite serial standards solution of various concentration is added in gauge orifice, toward sample
Sample to be tested is added in hole, then every hole is separately added into ELIAS secondary antibody, olaquindox metabolite antibody working solution, is incubated for；Wherein, institute
It states the olaquindox metabolite quasi- product solution of mark series and is obtained by phosphate buffer dilution olaquindox metabolite standard items at various concentration
It arrives；
S3. the reaction solution in plate hole is absorbed, is washed with concentrated cleaning solution, ELISA Plate is patted dry；
S4. substrate working solution and substrate buffer solution is added in every hole, pats mixing, covers cover board film；
S5. terminate liquid is added in every hole after 10~20 min, measures the absorbance in each hole；
S6. analysis detection result: the content of olaquindox metabolite in sample is determined.
As a result it is calculated with inhibiting rate, inhibiting rate (%)=B/B0× 100(%)；In formula: B is various concentration olaquindox metabolite
Standard solution hole or sample to be tested hole absorbance value, B0For the absorbance of 0 concentration olaquindox metabolite standard solution
Value；Using inhibiting rate as ordinate, standard curve is drawn by abscissa of the logarithm of olaquindox metabolite standard solution concentration, from
And determine the content of olaquindox metabolite in sample.
Sample to be tested of the invention includes but is not limited to the animal tissues such as chicken, chicken gizzard, pork or pork liver.
Preferably, sample to be tested described in step S2 is chicken, duck, pork.
Preferably, sample pre-treatments are first carried out before being detected with the kit.The pre-treatment of sample mainly be
Olaquindox metabolite solution is obtained, from sample for subsequent detection.
Here is the pre-treating method of common several samples (chicken, duck, pork), comprising the following steps:
(1) sample is taken out and is thawed, weighed the good sample of homogeneous (2.00 ± 0.05) g to 50 mL polystyrene centrifuge tubes, be added
The distilled water of 2 mL sufficiently vibrates 2 min；
(2) 8 mL ethyl acetate, the H of 1 mL, 1.5 M is added2SO4, 1 min is shaken, in room temperature (20~25 DEG C) with 4000 r/
The revolving speed of min or more is centrifuged 5 min；
(3) supernatant for taking out 4 mL is dried with nitrogen or air blow drying for 50 DEG C into teat glass；
(4) 0.5 mL n-hexane is added, with 30 s of vortex instrument vortex whirling motion, adds 0.5 mL and redissolves liquid, with vortex instrument whirling motion
10 s, 4000 r/min or more, in (20~25 DEG C) 5 min of centrifugation of room temperature；
(5) upper layer n-hexane phase is removed, takes 50 μ L subnatants to be measured.
ELISA is to combine enzymic catalytic reaction with immune response, for detecting the one of trace antigen or antibody
Kind novel markings immunoassay.Its testing principle is: (1) so that antigen or antibody is integrated to certain surface of solid phase carriers, and
Keep its immunocompetence；(2) antigen or antibody and certain enzyme is made to connect into enzyme-labelled antigen or antibody, this enzyme-labelled antigen or antibody
Not only retain its immunocompetence, but also retain the activity of enzyme；(3) substrate is added after washing, enzymatic makes substrate generate color products, passes through
Dedicated instrument detects absorbance, and the amount of product is directly related with the amount of tested substance in sample, so that inverse goes out unknown antigen
Or the concentration of antibody.The outstanding advantages of the method show as high sensitivity, and analysis method is easy quickly, as a result stable, error is small,
Safety is good and validity period is long, and the Heterosis compared to instrument analytical method exists: being able to achieve mass detection, and more economical, province
When.
Compared with prior art, the beneficial effects of the present invention are:
(1) haptens of the invention plays a significant role in the remaining detection of olaquindox metabolite, uses the haptens as original
Animal is immunized in material, the antigen system that preparation is suitable for animal immune, and potency, specificity, the affinity of gained antibody are all relatively good；Institute
Antibody be used for enzyme linked immunological kit, it is easy to use, testing cost is low, detection method efficiently, it is accurate, quickly, can be simultaneously
Large batch of sample is detected, the screening suitable for the remaining on-site supervision and great amount of samples of olaquindox metabolite in animal tissue.
(2) enzyme linked immunological kit provided by the invention and its method, maximum linear detection range are 0.2~16.2
Ng/mL, sensitivity are 0.54 ng/mL, and detection is limited to 0.32 ng/mL, and the rate of recovery is 80%~109.8%, kit detection
Quickly, substantially reduce detection time, do not consider the influence of testing staff's skilled operation degree, entire detection process it is only necessary to
70 min or so can be completed, and detection limit is lower, sensitivity is higher；Meanwhile examination is improved using the coating plate of envelope antigen
The stability and precision of agent box detect more stable, more acurrate.
Detailed description of the invention
Fig. 1 is the standard curve of olaquindox metabolite enzyme linked immunological kit.
Specific embodiment
Further illustrate the present invention below in conjunction with specific embodiment, but embodiment the present invention is not done it is any type of
It limits.Unless stated otherwise, the present invention uses reagent, method and apparatus is the art conventional reagents, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
Reagent used in the embodiment of the present invention is as follows:
Coating buffer: 1.69 g sodium carbonate and 2.95 g sodium bicarbonates are dissolved in 1 L distilled water and are obtained.
20 times of concentrated cleaning solutions: pH 7.4, the phosphate buffer of 0.4 mol/L containing 0.5% Tween-20 use
When with deionized water be diluted to 1 times, which is percent weight in volume.
Confining liquid: take the BSA(bovine serum albumin(BSA) of 0.1 g), 5 g glycine, 5 g sucrose be dissolved in 100 mL PBS it is molten
Liquid (0.01 mol/L, pH7.4) obtains.
Olaquindox metabolite serial standards solution: olaquindox metabolite standard solution is diluted to hplc grade methanol
1 mg/mL is spare；Being diluted to concentration with the PBS that 0.2 mol/L, pH is 5.4 again is respectively 0 ng/mL, 0.2 ng/mL, 0.6
A series of olaquindox metabolite standard solutions of ng/mL, 1.8 ng/mL, 5.4 ng/mL, 16.2 ng/mL, 4 DEG C of preservations.
Substrate solution: substrate solution is made of A liquid (substrate working solution) and B liquid (substrate buffer solution), and A liquid is that pH is 5.0, is contained
Phosphoric acid-citrate buffer solution of urea peroxide；B liquid is that pH is 5.0, contains phosphoric acid-lemon of 3,3,5,5- tetramethyl benzidines
Lemon acid buffering solution；When use by A liquid and B liquid by volume 1:1 ratio mix, substrate solution can be obtained.
Olaquindox metabolite monoclonal antibody (2.5 mg/mL): preparation laboratory early period.
The goat anti-rabbit igg (10 mg/mL) of horseradish peroxidase label: bioengineering Co., Ltd is obtained purchased from Wuhan doctor.
The preparation of 1 olaquindox metabolite hapten of embodiment, immunogene, coating antigen and monoclonal antibody
1, the preparation of haptens, immunogene and coating antigen
Using active ester method, by haptens 4- (3- methyl -2- naphthoyl amido) butyric acid (being abbreviated as MNBA) respectively with carrier protein
Bovine serum albumin(BSA) (BSA) and ovalbumin (OVA) coupling, are prepared into immunogene MNBA-BSA and coating antigen MNBA-OVA.Tool
Body method is as follows:
(1) hapten synthesis:
3- methyl -2- naphthoic acid (1.3 mmol) is dissolved in SOCl2In (11 mmol), microwave heating to 100 under nitrogen protection
DEG C reaction 2 h；Solvent is removed under reduced pressure, and residue is dissolved in Isosorbide-5-Nitrae-dioxanes (5 mL)；Under agitation, by it
It is added dropwise to and is mixed with amino acid (1.3 mmol/L) and Na2CO3In the 5 mL aqueous solutions of (3.2 mmol/L)；It is stirred at room temperature
Overnight, it after 20 mL hydrochloric acid solutions (1 mol/L) acidification is added, is extracted with the mL of EtOAC(3 × 15)；After merging organic phase,
It is washed 3 times with saturation NaCl solution, after anhydrous sodium sulfate drying, solvent is evaporated off；Residue is purified using silica gel column chromatography, is obtained
To 4- (3- methyl -2- naphthoyl amido) butyric acid, as target haptens.
1H NMR (500 MHz, DMSO-d 6): δ 8.29-8.22 (m, 4H), 7.97-7.90 (m, 2H), 7.88
(d, J = 0.6 Hz, 1H), 7.88-7.81 (m, 3H), 7.61-7.53 (m, 4H), 3.30 (q, J = 7.1
Hz, 4H), 2.57 (s, 6H), 2.37 (t, J = 7.1 Hz, 4H), 1.89 (p, J = 7.1 Hz, 4H)。
(2) immunogene synthesizes:
0.32 g haptens 4- (3- methyl -2- naphthoyl amido) butyric acid is weighed, then weighs 0.131 g NHS and is added in haptens,
It is dissolved in reaction unit with 400 μ L DMF, carries out 10 h of reaction in room temperature under magnetic stirring；Whole liquid will be reacted and be placed in 4 DEG C
Cooling 2 h or more in refrigerator, are centrifuged 5 min through 1.2 ten thousand rpm, take supernatant (i.e. active ester liquid), and be slowly dropped to 5 mL 6
It is reacted in the BSA solution of mg/mL；Reaction buffer is the phosphate buffer (PBS liquid) of 0.2 mol/L pH7.4, in magnetic force
It carries out reacting 4 h(in room temperature under stirring clocking since being loaded end)；Whole liquid will be reacted to be placed in bag filter, in 0.01
4 DEG C of stirring dialysis, every 4~8 h change a dialyzate in the PBS solution of mol/L pH7.4, dialyse 3 days altogether；After dialysis,
By the protein solution (the as immunogene MNBA-BSA of MQCA) in bag filter, it is divided into several equal portions and is sub-packed in 1 mL centrifuge tube
In, it is stored in -20 DEG C of refrigerators, it is spare.
(3) coating antigen synthesizes: the synthetic method of coating antigen is identical as above-mentioned immunogen synthesis method process, only immunogene
Carrier protein BSA in synthesis is changed to OVA, and coating antigen MNBA-OVA can be obtained.
2, the preparation of olaquindox metabolite monoclonal antibody
(1) animal immune
With the above-mentioned immunogene prepared by 150 μ g/ only with physiological saline solution after, mixed in equal volume with Freund's complete adjuvant,
The Balb/c female mice of immune 6,8 week old is subcutaneously injected in the nape of the neck, after initial immunity the 7th, 14,28 day it is endless with immunogene and Freund
Full adjuvant mixes in equal volume, and each supplementary immunization is primary, merges first 3 days with 150 μ of immune complex g/, Freund's adjuvant is not added again
Supplementary immunization is primary.
(2) cell fusion
The splenocyte of immune mouse is taken to mix with the murine myeloma cell (SP2/0) in logarithmic growth phase, then in 45 s
The fusion agent (PEG4000) for being inside slowly added to preheating is merged, and is suspended uniformly with HAT culture medium, is added suitable raising
Cell is incubated at 96 well culture plates, at 37 DEG C, 5%CO2Cultivated in incubator, partly change liquid with HT culture medium after 5 days, at 9 days into
Row changes liquid entirely.
(3) screening of positive hybridoma
After cell fusion, when cell grows to the 1/4 of culture hole area, hybridoma is screened using substep screening method；Primary election
Using indirect ELISA method, (diluted in advance with its best peridium concentration of square matrix method conventional titration and positive serum with envelope antigen
Degree) coated elisa plate, be added measured hole culture supernatant, be incubated for, after cleaning be added sheep anti-mouse igg-HRP and IgM-HRP, OPD into
Row chromogenic reaction；The positive Kong Zaiyong indirect competitive ELISA method screening filtered out, first by cell conditioned medium and 100 μ g/mL
Olaquindox metabolite mixes in equal volume, and 37 DEG C of water-baths act on 30 min, is then added in the ELISA Plate being coated with；PBS is used simultaneously
Olaquindox metabolite is replaced to compare, remaining step is same as above；If the OD after olaquindox metabolite blocks450Nm value drops to pair
According to hole 50% hereinafter, be then judged to the positive, be all positive hole through 2,3 detections, be subcloned immediately with limiting dilution assay
Change；
(4) the expansion culture of hybridoma
The hybridoma that 2~3 subclones are built after strain is expanded into culture, supernatant is collected with indirect ELISA and measures potency, freeze
It deposits；And 0.5 mL/ of Balb/c mouse peritoneal injecting fluid paraffin for taking 8~10 week old is only, and hybridoma is injected intraperitoneally after 7~10 days
Cell 1~2 × 106/ only, mouse ascites are extracted after 7~10 days, centrifuging and taking supernatant measures potency, and freezes spare.
The foundation of 2 enzyme-linked immunosorbent assay olaquindox metabolite method of embodiment
(1) primary concentration of envelope and antibody concentration is preferred, comprising the following steps:
1) coating antigen is diluted according to the concentration of 15.6,8.9,5.0,2.5,1.25 ng/mL with coating buffer and is longitudinally coated with enzyme mark
Plate, 100 holes μ L/, 37 DEG C of overnight incubations are washed twice with cleaning solution, are patted dry on blotting paper；
2) prepared 170 hole μ L/ of confining liquid is added to be closed, 37 DEG C overnight, and drying is put into baking oven；
3) the olaquindox metabolite standard solution that 50 hole μ L/ PBS solutions have diluted is added；
4) be added the diluted different gradients of 50 hole μ L/ PBS solutions olaquindox metabolite monoclonal antibody (1:32000,1:
64000,1:96000,1:128000), 25 DEG C of 30 min of incubation board-washing 5 times, are patted dry on blotting paper；
5) ELIAS secondary antibody (sheep anti-mouse igg) that 100 holes μ L/ dilute 5000 times is added, 25 DEG C of 30 min of incubation board-washing 5 times, inhale
It is patted dry on water paper；
6) substrate solution now matched, 100 holes μ L/, 25 DEG C of 10 min of reaction are added；
7) terminate liquid, 50 holes μ L/, with microplate reader readings, at 450 nm/630 nm of 450 nm of Single wavelength or double wave are added
Read the absorbance value in each hole.
Experimental result shows that obtained coating antigen best effort concentration is 1.25 ng/mL, and antibody extension rate is 64000
Times.
(2) measurement of antibody sensitivity
It take coating antigen optium concentration as 1.25 ng/mL of peridium concentration, antibody extension rate is 64000 times, and it is sensitive to carry out antibody
The measurement of degree.The following steps are included:
1) 96 orifice plates are taken, by coating antigen diluted to 1.25 ng/mL, 100 μ L are added in every hole, and 37 DEG C are incubated overnight,
It is washed twice with cleaning solution, is patted dry on blotting paper；
2) prepared 170 hole μ L/ of confining liquid is added to be closed, 37 DEG C overnight, and drying is put into baking oven；
3) olaquindox metabolite standard solution and the dilution 64000 of the different gradients that 50 μ L/ hole PBS have diluted are separately added into
Antibody again, 25 DEG C of incubation 30min board-washing 5 times, are patted dry on blotting paper；
4) ELIAS secondary antibody (sheep anti-mouse igg) that 100 holes μ L/ dilute 5000 times is added, 25 DEG C of 30 min of incubation board-washing 5 times, inhale
It is patted dry on water paper；
5) substrate solution now matched, 100 holes μ L/, 25 DEG C of 10 min of reaction are added；
6) terminate liquid, 50 holes μ L/, with microplate reader readings, at 450 nm/630 nm of 450 nm of Single wavelength or double wave are added
Read each hole absorbance value；
As a result it is calculated with inhibiting rate, inhibiting rate (%)=B/B0× 100(%), half inhibits lower, illustrates that the sensitivity of antibody is got over
It is high；In formula, B is the absorbance of various criterion strength solution competition, B0For the extinction of 0 concentration olaquindox metabolite standard solution
Angle value, with 50% inhibiting rate (half-inhibitory concentration, IC50) sensitivity of olaquindox metabolite monoclonal antibody is calculated, it is 0.54
ng/mL。
The preparation of 3 olaquindox metabolite enzyme linked immunological kit of embodiment
1, prepare reagent
(1) it is coated with the ELISA Plate of olaquindox metabolite antigen
The removable ELISA Plate in 96 holes, has been coated with olaquindox metabolite antigen and confining liquid, and peridium concentration is 1.25 mg/L；The quinoline
Alcohol metabolism object is the coupling of olaquindox metabolite hapten 4- (3- methyl -2- naphthoyl amido) butyric acid and ovalbumin (OVA)
Object；
The coating of micropore of enzyme marker plate plate: envelope antigen is diluted to 1.25 mg/L with coating buffer, and 100 μ L coating is added in every hole
Liquid, 37 DEG C are incubated overnight, and liquid in hole of inclining, cleaning solution washs 2 times, pat dry；Then it is added 170 μ L confining liquids in every hole, 37
DEG C 3 h are incubated for, liquid in hole of inclining is placed in 37 DEG C of baking ovens after drying, and is saved with 4 DEG C of aluminium foil bag vacuum sealing.
(2) preparation of olaquindox metabolite standard solution
Olaquindox metabolite standard solution is accurately weighed, is diluted to 1 mg/mL with hplc grade methanol, then with 0.2 mol/L
(formula is the NaH of 2.9 g to PBS buffer solution2PO4·12H2O, the KH of the NaCl of 8.5 g, the KCl of 0.2 g, 0.2 g2PO4, constant volume
To 1 L) the olaquindox metabolite solution that compound concentration is 0,0.2,0.6,1.8,5.4,16.2 ng/mL respectively, 4 DEG C of preservations.
(3) olaquindox metabolite antibody working solution
Olaquindox metabolite MAb concentration is 1.25 mg/mL；Its working concentration is 1:64000, is diluted with P liquid.
(4) substrate solution
It is made of A liquid (substrate working solution) and B liquid (substrate buffer solution), by the ratio of A liquid and B liquid 1:1 by volume when use
Mixing, can be obtained substrate solution；It is 5.0 that wherein A liquid, which is pH, phosphoric acid-citrate buffer solution containing urea peroxide；B liquid is pH
It is 5.0, contains phosphoric acid-citric acid solution of 3,3,5,5- tetramethyl benzidines.
(5) 20 times of concentrated cleaning solutions
PH is 7.4, the phosphate buffer of 0.4 mol/L containing 0.5% Tween-20, is diluted to 1 with deionized water when use
Times, which is percent weight in volume.
2, reagent dispenses
By aseptic subpackaged, mL/ bottles of olaquindox metabolite antibody working solution 7 after each reagent measurement qualification；7 mL/ bottles of ELIAS secondary antibody；
1 mL/ bottles of olaquindox metabolite standard solution；7 mL/ bottles of substrate solution；7 mL/ bottles of substrate buffer solution；7 mL/ bottles of terminate liquid；
50 mL/ bottles of 3M sulfuric acid solution；Redissolve 50 mL/ bottles of liquid；20 times 50 mL/ bottles of concentrated cleaning solution.Using the transparent of different colours lid
It labels after plastic bottle packing, indicates lot number and validity period, 4 DEG C of preservations.
3, the assembling of kit
Respectively by above-mentioned 1 piece of the ELISA Plate for being coated with olaquindox metabolite antigen, olaquindox metabolite antibody working solution, horseradish mistake
The secondary antibody of oxidase label, substrate solution, substrate buffer solution, 20 times of concentrated cleaning solutions, terminate liquid, 3M sulfuric acid solution and to redissolve liquid each
1 bottle, 6 bottles and 1 part of operation instructions of olaquindox metabolite standard items working solution are placed in designated position in kit, and kit is examined
Qualified post package, 4 DEG C of preservations.
The application of 4 olaquindox metabolite enzyme-linked immunologic detecting kit of embodiment
1, it is detected using enzyme linked immunological kit of the present invention
(1) it is loaded: required amount of lath is fixed on grillage, 50 μ L samples or standard items are added into microwell plate, by 50 μ
L enzyme marker is added in micropore, adds 50 μ L antibody working solutions into microwell plate, good with cover board membrane cover, and sufficiently oscillation is mixed
It is even；
(2) it incubates: setting (25 DEG C) 30 min of incubation of room temperature；
(3) it washs: discarding liquid in hole, the washing lotion after dilution is filled into each hole, 30~60 s is stood, discards washing lotion in hole；Weight
It is patted dry after after backwashing 4 times；
(4) develop the color: 50 μ L of substrate buffer solution is added in every hole, adds 50 μ L of substrate working solution, and oscillation, which mixes, (or delays substrate
After fliud flushing and substrate working solution are mixed with 1:1 ratio, 100 μ L developing solutions are added), it sets (25 DEG C) of room temperature and incubates 10~15
min；
(5) terminate: 50 μ L of terminate liquid is added in every hole, pats mixing；
(6) readings: microplate reader readings is used, reads each hole absorbance at 450 nm/630 nm of 450 nm of Single wavelength or dual wavelength
Value.
2, testing result is calculated and is analyzed
Inhibiting rate (%)=B/B0× 100(%)
In formula: B is the standard solution hole of various concentration olaquindox metabolite or the absorbance value in sample to be tested hole, B0It is dense for 0
Spend the absorbance value of olaquindox metabolite standard solution.
Using inhibiting rate as ordinate, it is bent that standard is drawn using the logarithm of olaquindox metabolite standard solution concentration as abscissa
Line substitutes into above-mentioned standard curve the content for finding out olaquindox metabolite in sample to be tested with the light absorption value of olaquindox metabolite.
Do not consider the influence of testing staff's skilled operation degree, entire detection process it is only necessary to 70 min or so can be complete
At.The analysis of testing result can also be calculated and be analyzed using computer professional software.
3, standard curve
Olaquindox metabolite canonical plotting (as shown in Fig. 1), table are obtained by the Analysis of test results to standard solution
It is 0.2~16.2 ng/mL, sensitivity 0.54 to the linear detection range of olaquindox metabolite that kit of the present invention, which is illustrated,
Ng/mL, detection are limited to 0.32 ng/mL.
The application effect appraisal of 5 olaquindox metabolite enzyme-linked immunologic detecting kit of embodiment
Detection method is the same as embodiment 4.
1, sample to be tested pre-treatment
(1) sample is taken out and is thawed, weighed the good sample of homogeneous (2.00 ± 0.05) g to 50 mL polystyrene centrifuge tubes, be added
The distilled water of 2 mL sufficiently vibrates 2 min；
(2) 8 mL ethyl acetate, the H of 1 mL, 1.5M is added2SO4, shake 1 min, room temperature (20~25 DEG C) 4000 r/min with
On revolving speed be centrifuged 5min；
(3) supernatant for taking out 4 mL is dried with nitrogen or air blow drying for 50 DEG C into teat glass；
(4) 0.5 mL n-hexane is added, with 30 s of vortex instrument vortex whirling motion, liquid is redissolved 0.5 mL is added, with vortex instrument whirling motion
The revolving speed of 10 s, room temperature (20~25 DEG C) 4000 r/min or more are centrifuged 5 min；
(5) upper layer n-hexane phase is removed, takes 50 μ L subnatants to be measured.
2, the repetitive test of olaquindox metabolite standard solution
In the ELISA Plate prepared from 3 batches according to the method in embodiment 3,16 micropores of each random extraction, according to 4 pilot scale of embodiment
The absorbance value of the detection method measurement olaquindox metabolite standard solution of agent box, is repeated 16 times, calculates the coefficient of variation
(CV, %), the results are shown in Table 1.
1 olaquindox metabolite standard solution repetitive test of table
Variation within batch coefficient: with the coefficient of variation of each parallel samples in primary measurement.
Interassay coefficient of variation: same sample takes its average value in the coefficient of variation of different batches measurement result.
The result shows that the variation within batch coefficient range of kit standard product solution detection of the present invention is 2.45%~10.77%
Between, interassay coefficient of variation is 7.43%~14.56%.It knows CV < 15%, illustrates that this method accuracy is good, the kit is applicable
The detection of olaquindox metabolite in actual sample.
3, sample repeatability and accuracy test
Accuracy refers to the matching degree of measured value and true value, and in enzyme-linked immunosorbent assay, accuracy is often indicated with the rate of recovery, essence
Density is often indicated with the coefficient of variation.In blank sample, addition olaquindox metabolite to final concentration of 0.5,1,2 μ g/L, often
A concentration each 10 parallel, measures 3 batches.Calculate average value, TIANZHU XINGNAO Capsul and batch interior and interassay coefficient of variation.It the results are shown in Table 2.
2 sample repeatability of table and accuracy test result
The result shows that the average TIANZHU XINGNAO Capsul of chicken meat sample is between 90.4%~107.2%, variation within batch coefficient is 1.45%
~5.81%, between interassay coefficient of variation 1.93%~10.93%, meet country for the standard of kit indices.
4, cross reaction
The specificity of antibody is to measure the important indicator of antibody mass.More high then its identification energy to target antigen of antibody specificity
Power is stronger, smaller to the cross reaction of non target antigen, so that the probability that false positive occurs in detection is also smaller.Test has selected quinoline
Alcohol metabolism object analogue and functional analogue carry out antibody association reaction.It is smaller with the cross reacting rate of other drugs,
Illustrate that olaquindox metabolite enzyme-linked immunologic detecting kit is better to the detection specificity of olaquindox metabolite.It the results are shown in Table 3.
3 cross reaction result of table
Note: QCA is quinoxalin-2-carboxylic acid；ND representative can not detect.
The result shows that olaquindox metabolite detection kit is low with the cross reacting rate of other drugs, illustrate olaquindox generation
It is good to the detection specificity of olaquindox metabolite to thank to object enzyme-linked immunologic detecting kit.
5, kit storage life is tested
(1) kit prepared by embodiment 3 is placed in 2~8 DEG C, takes store 0,2,4,6,8,9,10,11 and 12 respectively
The kit of the moon, to the absorbance value, 50% inhibition concentration, TIANZHU XINGNAO Capsul, variation within batch of olaquindox metabolite standard solution
Each parameter of coefficient is measured.
(2) kit is placed 12 days under conditions of 37 DEG C of preservations, daily to olaquindox metabolite standard solution
Absorbance value, 50% inhibition concentration, TIANZHU XINGNAO Capsul, each parameter of variation within batch coefficient are measured.
(3) kit is saved 12 days in -20 DEG C of refrigerators, daily to the absorbance of olaquindox metabolite standard solution
Value, 50% inhibition concentration, TIANZHU XINGNAO Capsul, each parameter of variation within batch coefficient are measured.
The result shows that the absorbance value decline of olaquindox metabolite standard solution is small by three kinds of condition food preservation tests
In 10%, indices conform to quality requirements, and therefore, kit can save 12 months at 2~8 DEG C.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of olaquindox metabolite hapten, which is characterized in that the haptens is 4- (3- methyl -2- naphthoyl amido) fourth
Acid, molecular structural formula are as the formula (1):
Formula (1).
2. a kind of olaquindox metabolite antigen, which is characterized in that be by olaquindox metabolite hapten described in claim 1 with
Carrier protein couplet obtains.
3. application of the olaquindox metabolite antigen described in claim 2 in preparation olaquindox metabolite antibody.
4. the olaquindox metabolite antibody that olaquindox metabolite antigen described in application claim 2 is prepared.
5. application of the olaquindox metabolite antibody described in claim 4 in detection olaquindox metabolite.
6. olaquindox metabolite hapten described in application claim 1, olaquindox metabolite antigen, right described in claim 2
It is required that enzyme linked immunological kit or colloidal gold test paper card that the 4 olaquindox metabolite Antibody preparations obtain.
7. enzyme linked immunological kit as claimed in claim 6, which is characterized in that contain: being coated with olaquindox metabolite antigen
ELISA Plate, olaquindox metabolite antibody, ELIAS secondary antibody, olaquindox metabolite serial standards solution, substrate buffer solution and substrate
Working solution, terminate liquid, redissolves liquid and sulfuric acid solution at concentrated cleaning solution.
8. enzyme linked immunological kit according to claim 7, which is characterized in that the substrate working solution be pH be 4.5~
5.5, phosphoric acid-citrate buffer solution containing hydrogen peroxide or urea peroxide；The substrate buffer solution is that pH is 4.5~5.5, is contained
There is phosphoric acid-citric acid solution of 3,3,5,5- tetramethyl benzidine；The concentrated cleaning solution is that pH is 7.0~7.8, is contained
The phosphate buffer of 0.4%~0.6% Tween-20,0.3~0.5 mol/L.
9. a kind of remaining method of olaquindox metabolite in test sample, which is characterized in that with enzyme described in claim 7 or 8
Linked immunoassay reagent kit is detected.
10. the remaining method of olaquindox metabolite in test sample according to claim 9, which is characterized in that including following
Step:
S1. the enzyme linked immunological kit is taken out from cold storage environment, is placed in 15~35 DEG C of 30~45 min of balance；
S2. ELISA Plate is taken out, the olaquindox metabolite serial standards solution of various concentration is added in gauge orifice, toward sample
Sample to be tested is added in hole, then every hole is separately added into ELIAS secondary antibody, olaquindox metabolite antibody working solution, is incubated for；
S3. the reaction solution in plate hole is absorbed, is washed with concentrated cleaning solution, ELISA Plate is patted dry；
S4. substrate working solution and substrate buffer solution is added in every hole, pats mixing, covers cover board film；
S5. terminate liquid is added in every hole after 10~20 min, measures the absorbance in each hole；
S6. analysis detection result: the content of olaquindox metabolite in sample is determined.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810652749.2A CN109180519B (en) | 2018-06-22 | 2018-06-22 | Olaquindox metabolite antigen, antibody, enzyme-linked immunosorbent assay kit and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810652749.2A CN109180519B (en) | 2018-06-22 | 2018-06-22 | Olaquindox metabolite antigen, antibody, enzyme-linked immunosorbent assay kit and detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109180519A true CN109180519A (en) | 2019-01-11 |
| CN109180519B CN109180519B (en) | 2021-08-03 |
Family
ID=64948727
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810652749.2A Active CN109180519B (en) | 2018-06-22 | 2018-06-22 | Olaquindox metabolite antigen, antibody, enzyme-linked immunosorbent assay kit and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109180519B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110927382A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Time-resolved fluorescence immunoassay kit for detecting olaquindox and application thereof |
| CN110927383A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof |
| CN114539171A (en) * | 2021-12-31 | 2022-05-27 | 华南农业大学 | MAC type multi-cluster tandem linear hapten, artificial antigen, preparation method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102621322A (en) * | 2012-03-29 | 2012-08-01 | 北京维德维康生物技术有限公司 | Kit for detecting 3-methyl-quinoxaline-2-carboxylic acid |
| CN102659693A (en) * | 2012-03-29 | 2012-09-12 | 中国农业大学 | 3-methylquinoxaline-2-carboxylic acid artificial antigen and antibody obtained by the 3-methylquinoxaline-2-carboxylic acid artificial antigen |
| CN103304495A (en) * | 2012-03-13 | 2013-09-18 | 北京勤邦生物技术有限公司 | Preparation method and applications of olaquindox metabolite hapten |
| CN104558185A (en) * | 2014-12-26 | 2015-04-29 | 华中农业大学 | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting mequindox and olaquindox metabolites |
| CN104814931A (en) * | 2015-04-14 | 2015-08-05 | 华南农业大学 | Olaquindox slow release particle and preparing method and application thereof |
| CN105061339A (en) * | 2015-07-02 | 2015-11-18 | 中国兽医药品监察所 | Semiantigen, artificial antigen thereof, and application of semiantigen in detection of olaquindox residual marker |
| CN106770210A (en) * | 2016-11-22 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | A kind of olaquindox method for quick |
-
2018
- 2018-06-22 CN CN201810652749.2A patent/CN109180519B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103304495A (en) * | 2012-03-13 | 2013-09-18 | 北京勤邦生物技术有限公司 | Preparation method and applications of olaquindox metabolite hapten |
| CN102621322A (en) * | 2012-03-29 | 2012-08-01 | 北京维德维康生物技术有限公司 | Kit for detecting 3-methyl-quinoxaline-2-carboxylic acid |
| CN102659693A (en) * | 2012-03-29 | 2012-09-12 | 中国农业大学 | 3-methylquinoxaline-2-carboxylic acid artificial antigen and antibody obtained by the 3-methylquinoxaline-2-carboxylic acid artificial antigen |
| CN104558185A (en) * | 2014-12-26 | 2015-04-29 | 华中农业大学 | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting mequindox and olaquindox metabolites |
| CN104814931A (en) * | 2015-04-14 | 2015-08-05 | 华南农业大学 | Olaquindox slow release particle and preparing method and application thereof |
| CN105061339A (en) * | 2015-07-02 | 2015-11-18 | 中国兽医药品监察所 | Semiantigen, artificial antigen thereof, and application of semiantigen in detection of olaquindox residual marker |
| CN106770210A (en) * | 2016-11-22 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | A kind of olaquindox method for quick |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110927382A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Time-resolved fluorescence immunoassay kit for detecting olaquindox and application thereof |
| CN110927383A (en) * | 2019-10-08 | 2020-03-27 | 杭州佰昕科技有限公司 | Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof |
| CN114539171A (en) * | 2021-12-31 | 2022-05-27 | 华南农业大学 | MAC type multi-cluster tandem linear hapten, artificial antigen, preparation method and application thereof |
| CN114539171B (en) * | 2021-12-31 | 2023-05-05 | 华南农业大学 | MAC type multi-cluster tandem linear hapten and artificial antigen as well as preparation methods and applications thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109180519B (en) | 2021-08-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103018450B (en) | A kind of preparation method of chemical luminescence ELISA detection kit of chloromycetin | |
| CN103018454B (en) | A kind of chemical luminescence ELISA detection kit of sulfa drugs | |
| CN100445746C (en) | Method for detecting 19-nortestosterone and special enzyme-linked immune reagent kit thereof | |
| CN103575890B (en) | The chemical luminescence reagent kit of a kind of Ractopamine and application thereof | |
| CN101571539B (en) | Elisa kit for detecting cephalo-type medicine and application thereof | |
| CN101241134B (en) | ELISA kit for detecting ractopamine residue and method of use thereof | |
| CN104655847A (en) | Enzyme linked immunosorbent assay kit (ELISA kit) for detecting adprin and detection method thereof | |
| CN101256188A (en) | ELISA kit for detecting lincomycin medicine as well as usage thereof | |
| CN109180519A (en) | A kind of olaquindox metabolite antigen, antibody and enzyme-linked immunologic detecting kit and detection method | |
| CN109580959A (en) | A kind of ELISA kit detecting heparin-binding epidermal growth factor | |
| CN106226523A (en) | Detection method, reagent and the detection kit of a kind of immunological marker thing | |
| CN106226522A (en) | The detection method of a kind of GP73, detectable and detection kit | |
| CN110441512A (en) | A kind of colloidal gold immunochromatographimethod detection device of ethylmaltol haptens and ethylmaltol | |
| CN100489530C (en) | Method for assaying sulfaquinoxaline and special enzyme-linked immune reagent kit | |
| CN105445462A (en) | Enzyme linked immunosorbent assay kit for specific quantitative detection of foot and mouth disease O type Guangxi strain antigen and application thereof | |
| CN109212200A (en) | A kind of Nicarbazin haptens, artificial antigen and the preparation method and application thereof | |
| CN104950105B (en) | The preparation method and its application in chemiluminescence immunoassay kit of chloramphenicol haptens and antigen | |
| CN107677807A (en) | A kind of kitasamycin magnetic immunochemiluminescence detection kit | |
| CN103288661A (en) | Preparation method and application of malachite green hapten | |
| CN109956848A (en) | A kind of triclosan haptens and antigen and its preparation method and application | |
| CN100489532C (en) | Method for detecting salinomycin and spcial enzyme-linked immune reagent kit thereof | |
| CN103304495A (en) | Preparation method and applications of olaquindox metabolite hapten | |
| CN104897651A (en) | Chemiluminescent kit for aflatoxin M1 and application thereof | |
| CN109305963A (en) | A kind of ketoconazole haptens, artificial antigen and the preparation method and application thereof | |
| CN100489531C (en) | Method for detecting Dorlasporin and special enzyme-linked immune reagent kit thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 510642 No. five, 483 mountain road, Guangzhou, Guangdong, Tianhe District Patentee after: SOUTH CHINA AGRICULTURAL University Patentee after: WENS FOODSTUFF GROUP Co.,Ltd. Address before: 510642 No. five, 483 mountain road, Guangzhou, Guangdong, Tianhe District Patentee before: SOUTH CHINA AGRICULTURAL University Patentee before: GUANGDONG WENS FOODSTUFF GROUP Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230412 Address after: 527499 Floor 4, Building 5, Wenshi Research Institute, east side of Huineng North Road, Xinxing County, Yunfu City, Guangdong Province Patentee after: Guangdong Biaoyun Biotechnology Co.,Ltd. Patentee after: WENS FOODSTUFF GROUP Co.,Ltd. Address before: 510642 No. five, 483 mountain road, Guangzhou, Guangdong, Tianhe District Patentee before: SOUTH CHINA AGRICULTURAL University Patentee before: WENS FOODSTUFF GROUP Co.,Ltd. |