CN101655498B - Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method - Google Patents
Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method Download PDFInfo
- Publication number
- CN101655498B CN101655498B CN 200910093010 CN200910093010A CN101655498B CN 101655498 B CN101655498 B CN 101655498B CN 200910093010 CN200910093010 CN 200910093010 CN 200910093010 A CN200910093010 A CN 200910093010A CN 101655498 B CN101655498 B CN 101655498B
- Authority
- CN
- China
- Prior art keywords
- malachite green
- concealed malachite
- colloidal gold
- concealed
- preparation
- 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.)
- Active
Links
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 66
- 238000012360 testing method Methods 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- WZKXBGJNNCGHIC-UHFFFAOYSA-N Leucomalachite green Chemical compound C1=CC(N(C)C)=CC=C1C(C=1C=CC(=CC=1)N(C)C)C1=CC=CC=C1 WZKXBGJNNCGHIC-UHFFFAOYSA-N 0.000 title abstract 5
- 238000004519 manufacturing process Methods 0.000 title description 4
- 238000001514 detection method Methods 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 241001494479 Pecora Species 0.000 claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 8
- 238000012216 screening Methods 0.000 claims abstract description 3
- 239000000020 Nitrocellulose Substances 0.000 claims abstract 2
- 229920001220 nitrocellulos Polymers 0.000 claims abstract 2
- FDZZZRQASAIRJF-UHFFFAOYSA-M malachite green Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](C)C)C=C1 FDZZZRQASAIRJF-UHFFFAOYSA-M 0.000 claims description 84
- 229940107698 malachite green Drugs 0.000 claims description 84
- 229910052737 gold Inorganic materials 0.000 claims description 28
- 239000010931 gold Substances 0.000 claims description 28
- 239000000084 colloidal system Substances 0.000 claims description 23
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 15
- 230000036039 immunity Effects 0.000 claims description 14
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 210000004027 cell Anatomy 0.000 claims description 12
- 241000907663 Siproeta stelenes Species 0.000 claims description 9
- 210000004408 hybridoma Anatomy 0.000 claims description 9
- 239000013078 crystal Substances 0.000 claims description 8
- FDPIMTJIUBPUKL-UHFFFAOYSA-N pentan-3-one Chemical compound CCC(=O)CC FDPIMTJIUBPUKL-UHFFFAOYSA-N 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 claims description 7
- 239000002245 particle Substances 0.000 claims description 7
- 230000002829 reductive effect Effects 0.000 claims description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 6
- 102000014914 Carrier Proteins Human genes 0.000 claims description 6
- 108010078791 Carrier Proteins Proteins 0.000 claims description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 6
- 230000008878 coupling Effects 0.000 claims description 6
- 238000010168 coupling process Methods 0.000 claims description 6
- 238000005859 coupling reaction Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 230000000274 adsorptive effect Effects 0.000 claims description 5
- 206010003445 Ascites Diseases 0.000 claims description 4
- 239000000427 antigen Substances 0.000 claims description 4
- 102000036639 antigens Human genes 0.000 claims description 4
- 108091007433 antigens Proteins 0.000 claims description 4
- DIKBFYAXUHHXCS-UHFFFAOYSA-N bromoform Chemical compound BrC(Br)Br DIKBFYAXUHHXCS-UHFFFAOYSA-N 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 239000012043 crude product Substances 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 239000000376 reactant Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000000967 suction filtration Methods 0.000 claims description 4
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 4
- 241000283707 Capra Species 0.000 claims description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 3
- 239000012954 diazonium Substances 0.000 claims description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-O diazynium Chemical compound [NH+]#N IJGRMHOSHXDMSA-UHFFFAOYSA-O 0.000 claims description 3
- 150000002148 esters Chemical class 0.000 claims description 3
- 239000012467 final product Substances 0.000 claims description 3
- 230000004927 fusion Effects 0.000 claims description 3
- 239000003292 glue Substances 0.000 claims description 3
- 230000002163 immunogen Effects 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 3
- HOGDNTQCSIKEEV-UHFFFAOYSA-N n'-hydroxybutanediamide Chemical compound NC(=O)CCC(=O)NO HOGDNTQCSIKEEV-UHFFFAOYSA-N 0.000 claims description 3
- 229940038773 trisodium citrate Drugs 0.000 claims description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 2
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical compound [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 claims description 2
- 241000692870 Inachis io Species 0.000 claims description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 2
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 claims description 2
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 claims description 2
- 229910052794 bromium Inorganic materials 0.000 claims description 2
- 229950005228 bromoform Drugs 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 238000004821 distillation Methods 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 235000019253 formic acid Nutrition 0.000 claims description 2
- 238000000338 in vitro Methods 0.000 claims description 2
- 238000009413 insulation Methods 0.000 claims description 2
- 239000010813 municipal solid waste Substances 0.000 claims description 2
- TXOUDPQJASQYBZ-UHFFFAOYSA-N n,n'-dihexylmethanediimine Chemical compound CCCCCCN=C=NCCCCCC TXOUDPQJASQYBZ-UHFFFAOYSA-N 0.000 claims description 2
- 239000012074 organic phase Substances 0.000 claims description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 2
- 238000000247 postprecipitation Methods 0.000 claims description 2
- 239000006152 selective media Substances 0.000 claims description 2
- 239000004575 stone Substances 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 238000003786 synthesis reaction Methods 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000001179 sorption measurement Methods 0.000 abstract 2
- 239000003550 marker Substances 0.000 abstract 1
- 239000012528 membrane Substances 0.000 abstract 1
- 239000000047 product Substances 0.000 description 19
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 7
- 239000002671 adjuvant Substances 0.000 description 6
- 238000002649 immunization Methods 0.000 description 6
- 241000251468 Actinopterygii Species 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000003053 immunization Effects 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 241000238557 Decapoda Species 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 235000019263 trisodium citrate Nutrition 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 244000061458 Solanum melongena Species 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 2
- 229940097572 chloromycetin Drugs 0.000 description 2
- STJMRWALKKWQGH-UHFFFAOYSA-N clenbuterol Chemical compound CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 STJMRWALKKWQGH-UHFFFAOYSA-N 0.000 description 2
- 229960001117 clenbuterol Drugs 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- -1 fish body Chemical compound 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- PLHJDBGFXBMTGZ-WEVVVXLNSA-N furazolidone Chemical compound O1C([N+](=O)[O-])=CC=C1\C=N\N1C(=O)OCC1 PLHJDBGFXBMTGZ-WEVVVXLNSA-N 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229960002135 sulfadimidine Drugs 0.000 description 2
- 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 2
- 230000002277 temperature effect Effects 0.000 description 2
- 239000000273 veterinary drug Substances 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 208000037088 Chromosome Breakage Diseases 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 206010064571 Gene mutation Diseases 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
- 241001465754 Metazoa Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- CMEWLCATCRTSGF-UHFFFAOYSA-N N,N-dimethyl-4-nitrosoaniline Chemical compound CN(C)C1=CC=C(N=O)C=C1 CMEWLCATCRTSGF-UHFFFAOYSA-N 0.000 description 1
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 1
- 235000011613 Pinus brutia Nutrition 0.000 description 1
- 241000018646 Pinus brutia Species 0.000 description 1
- 206010068676 Pneumoretroperitoneum Diseases 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 208000005727 Retropneumoperitoneum Diseases 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- MUUGCDGGIVCSDT-UHFFFAOYSA-N [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O Chemical compound [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O MUUGCDGGIVCSDT-UHFFFAOYSA-N 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000005886 chromosome breakage Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000021393 food security Nutrition 0.000 description 1
- 229960001625 furazolidone Drugs 0.000 description 1
- 238000007499 fusion processing Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 210000004013 groin Anatomy 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 210000004754 hybrid cell Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000008279 sol Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- AAAQKTZKLRYKHR-UHFFFAOYSA-N triphenylmethane Chemical compound C1=CC=CC=C1C(C=1C=CC=CC=1)C1=CC=CC=C1 AAAQKTZKLRYKHR-UHFFFAOYSA-N 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000003805 vibration mixing Methods 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 239000007762 w/o emulsion Substances 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses an immune colloidal gold test stripe for detecting leucomalachite green in aquatic products and a preparation method; the colloidal gold test stripe provided by the invention detects leucomalachite green with a direct competition method, and comprises a sample pad, a colloidal gold combined pad, a nitrocellulose membrane (NC), a water adsorption pad and a PVC back lining, one end of the PVC back lining is sequentially adhered with the sample pad and the colloidal gold combined pad, and the middle thereof is adhered with the NC layer, and the other end thereof is sequentially adhered with water adsorption pad; the immune colloidal gold test stripe has the characteristics that colloidal gold combined pad is enveloped with colloidal gold marker which is monoclonal antibody of the specificity of the leucomalachite green, and the NC is adsorbed with leucomalachite green-OVA and sheep anti-mouse IgG. The invention has high detection sensitivity and low price and is simple and rapid, the whole reaction only needs 30 minutes; the immune colloidal gold test stripe can be used for large-batch screening of samples and can rapidly detect the residual leucomalachite green in the aquatic products at site.
Description
Technical field
The invention belongs to and relate to immune colloid gold and detection of veterinary drugs in food field in the food security field.Particularly, the present invention relates to a kind of being applicable to and detect the residual immunity colloidal gold test paper strip of concealed malachite green in the aquatic products.
Background technology
Malachite green (Malachite green) molecular formula is: C
23H
25ClN
2It is a kind of industrial triphenylmethane triphenylmehtane class dyestuff, owing to have the effect of antibiotic and resisting gram-positive bacteria, and low price, so be widely used in culturing saprolegniasis, branchiomycosis, ich of the prevention fish of fishery etc., and in order to make the impaired fish of squama prolong life, deposit in the pond in the transportation neutralization, also often use malachite green.When culturing water when containing malachite green, meeting fast Absorption malachite green such as fish body, shrimp and then metabolism become concealed malachite green (Le μ comalachite green, LMG), i.e. N, accelerine, molecular formula: C
23H
26N
2, the procrypsis malachite green is water insoluble, and residual toxicity is stronger than malachite green, can hoard to reach the several months in muscle.The toxicological study of relevant this type of dyestuff is found in recent years: malachite green and major metabolite concealed malachite green thereof have multiple toxicity, comprise carcinogenicity, cause gene mutation, cause chromosome breakage, teratogenesis tire and respiratory tract toxicity.Given this, many countries all classify malachite green as the aquaculture forbidden drugs.
Nutrition is complete, the characteristics of balance because aquatic products (flesh of fish, shrimp etc.) have, and more and more is subjected to people's favor.By 1996, China's output of aquatic products surpassed 2,000 ten thousand tons, accounts for 1/3 of national meat food production, leaps to the No. 1 in the world.Along with the raising of living standards of the people, the quality and quantity of aquatic products there has been the requirement of renewal.Yet China fishery development is faced with a series of new challenges, and wherein food-safety problem has seriously restricted the industrialized development of China's fishery to a certain extent, and illegal interpolation malachite green is distinct issues at present.China lists malachite green in May, 2002 veterinary drug of forbidding.
The Information Office of the Ministry of Agriculture has issued the monitoring result of the aquatic product quality safety first time in 2007.In last year in the concrete the first tenday period of a month in time, the Ministry of Agriculture organizes relevant quality inspection organization that malachite green pollution condition in 22 municipal water products of China has been carried out the routine monitor first time in the current year, and 10 cities all have sample to detect malachite green.Because the driving of huge interests is difficult to the use that malachite green is stopped in real control effectively, this just means that also to malachite green detection demand be long-term.
At present in the world the detection technique route of malachite green and concealed malachite green is mainly contained two: one is to be the chemical detection technique of core with look (matter) spectral technology, and another is the quick bio detection technique.High performance liquid chromatography cost height, the running time is long, and step is many and complicated, and the scene truly of therefore can't realizing is detected.And advantages such as the colloidal gold strip detection method is highly sensitive, high specificity, good stability, easy and simple to handle, on-the-spot detection soon, grasped by basic unit easily and the large tracts of land popularization, the field quick detection that is suitable for batch samples, do not need specific instrument equipment, do not need specific other to detect reagent yet, reduced the burden that detects unit, can fast detecting obtain preliminary testing result, being fit to China's current social economic and technique level, therefore is the main developing direction that following malachite green detects.
Summary of the invention
The object of the present invention is to provide a kind of residual colloidal gold strip of golden mark technology field quick detection concealed malachite green that adopts.
The invention provides the residual colloidal gold strip of a kind of fast detecting concealed malachite green, the monoclonal antibody that produces is the anti-concealed malachite green monoclonal antibody of specificity.Described concealed malachite green monoclonal antibody is that concealed malachite green haptens and carrier protein couplet thing make by immune animal (for example rabbit, mouse) as immunogene.Described concealed malachite green haptens is to adopt chemical method to make it contain reactive group, and described carrier protein is the blue albumen (KLH) of bovine serum albumin(BSA), human serum albumins or key hole copper.Described concealed malachite green-carrier protein couplet thing adopts couplings such as mixed anhydride method, diazonium method or glutaraldehyde method to obtain.Described colloid gold label can be used commercial gold chloride, makes by gold chloride and the coupling of concealed malachite green monoclonal antibody that produces.
Be convenient on-the-spot the detection and the great amount of samples examination, the immunity colloidal gold test paper strip of described concealed malachite green comprises: NC film, sample pad, glue gold pad, thieving paper, trisodium citrate, PVC backing and pad, PVC backing one end sticks sample pad, collaurum pad, centre successively and sticks the NC rete, the other end sticks adsorptive pads successively, it is characterized in that collaurum pad bag by anti-concealed malachite green monoclonal antibody specific-colloid gold label thing, has adsorbed concealed malachite green-OVA and sheep or rabbit anti-mouse igg on the NC film.
The immune colloid gold reagent method that preparation the present invention detects product concealed malachite green in the water is:
(1) concealed malachite green haptens and holoantigen are synthetic by at phenyl ring contraposition coupling one reactive group-COOH, synthesize the concealed malachite green haptens; Concealed malachite green haptens and carrier protein are pressed 1: 10～30 mol ratio mixings, and synthetic stealthy malachite green-carrier protein couplet things such as glutaraldehyde or diazonium method obtain concealed malachite green hapten-carrier protein conjugate after separation and purification;
(2) Monoclonal Antibody is repeatedly immune Balb/c mouse inbred lines of immunogene with haptens-BSA, gets the external fusion of mouse boosting cell and myeloma cell and produces hybridoma, obtains the positive hybridoma cell strain through the selective medium screening.Hybridoma cell strain is injected mouse peritoneal obtain methods such as ascites or cell in vitro cultivation collection supernatant, prepare anti-concealed malachite green monoclonal antibody in a large number;
(3) the monoclonal antibody colloid gold label is reduced into gold chloride with reductive agents such as trisodium citrates the colloid gold particle of 20～40nm; Then collaurum and anti-concealed malachite green monoclonal antibody are pressed 1: 0.005～0.015 (volume mass ratio) mixing, make it in conjunction with forming stable colloid gold particle, the anti-recessive peacock stone monoclonal antibody of purified and concentrated generation-colloid gold label thing;
(4) the anti-mouse IgG preparation of sheep (rabbit) is obtained antiserum with many immune mouses of haptens-OVA, and immune goat or rabbit obtain the anti-mouse IgG of sheep (rabbit) after separation and purification again;
(5) preparation of collaurum pad is coated on concealed malachite green specific monoclonal antibody-colloid gold label thing on the collaurum pad, concealed malachite green-OVA conjugate and the anti-mouse IgG of sheep (rabbit) are adsorbed on detection zone and the control zone of NC film, fully dry at 37 ℃;
(6) colloidal gold strip assembling: NC film, collaurum pad, sample pad and adsorptive pads etc. are sticked on the PVC backing successively, the PVC material that glues is cut into the test strips of certain width, be assembled at last in a certain size the plastic casing and get final product.
Beneficial effect
Detection concealed malachite green immunity colloidal gold test paper strip of the present invention adopts the direct competitive immunization, with colloid gold label concealed malachite green specific monoclonal antibody, can detect by fast qualitative, the result is accurate, need not reagent wash and standard control, can be in batches or single sample in time detect.
Detection concealed malachite green immunity colloidal gold test paper strip of the present invention uses starting material to produce, and the production flow process is simple, and cost is low, and testing cost only is about 1/5 of gas phase and liquid phase chromatogram, is 1/3 of enzyme marking reagent box.For the user saves time, reduce the error cause because operation steps is miscellaneous, advantage such as the reagent holding time is long, execute-in-place is convenient can play a significant role in aquatic products detect.
Description of drawings
Fig. 1 is stealthy malachite green immunity colloidal gold test paper strip structural drawing
Fig. 2 is colloidal gold solution visible light scanning spectra
Fig. 3 detects data and curves figure the stealthy malachite green immunity colloidal gold test paper strip shelf-life
Concrete to implement to provide following embodiment be in order further to understand the present invention better, and never content of the present invention and protection domain are constituted any restriction.
Embodiment
The invention will be further described below in conjunction with specific embodiment, but not as a limitation of the invention.
Embodiment 1 haptens and comlete antigen (immunogene) are synthetic
1.1 the preparation to the concealed malachite green ethyl ketone
In the 100mL three-necked bottle, add 20mL methylene chloride, 18g (0.135mol) aluminum trichloride (anhydrous).Cryosel is bathed and is cooled to-10 ℃, drips the mixed liquor of 7.1g (0.07mol) aceticanhydride and 21.61g (0.065mol) concealed malachite green, and temperature is no more than-5 ℃ in the control.After dropwising, continue to stir 15h, solution becomes kermesinus from yellow.In the potpourri with reactant liquor impouring 50mL concentrated hydrochloric acid and 50g trash ice, divide water-yielding stratum with separating funnel then, and with twice of dichloromethane extraction.Merge organic phase, wash to neutrality anhydrous sodium sulfate drying with water.The distillation desolventizing gets yellow oil 9.8g.Crude product can be directly used in bromoform reaction.
1.2 the preparation to concealed malachite green formic acid
NaOH 6.3g is dissolved in the 54mL water, is cooled to-5 ℃.Dripping bromine 6.4g (0.04mol), temperature is no more than 0 ℃ in the control.To drop in the reactant liquor concealed malachite green ethyl ketone crude product 5.04g then, temperature is no more than 10 ℃ in the control.Dropwise, insulation 1h is then in stirring at room 2h.Standing demix divides and to remove the bromofom that generates.Water layer transfers to pH1～2 with concentrated hydrochloric acid, separates out white solid.Suction filtration is washed to neutrality.Solid is dissolved in the 20mL sodium hydroxide solution (5%), filters, light yellow clarifying liquid body and function concentrated hydrochloric acid transfers to pH1～2, separates out white crystal.Suction filtration is washed to neutrality, and oven dry gets white crystal 1.7g, yield 81% (by concealed malachite green).m.p.117～120℃。HNMR(300MHz，CDCl
3)，δ：1.28(d，6H，J＝6.9Hz)；2.99(m，1H，J＝6.9Hz)；7.33(d，2H，J＝8.3Hz)；8.05(d，2H，J＝8.3Hz)。
1.3 it is immunogenic synthetic
Active ester method prepares immunogene and is achieved in that concealed malachite green haptens and the N-hydroxy-succinamide (NHS) of getting equimolar amounts, ring dihexyl carbodiimide (DCC), (DFM) dissolves potpourri with dimethyl formamide, the lucifuge reaction is spent the night under the room temperature, centrifugal place to go post precipitation, get the supernatant drying, getting its 225 μ L adding contains in the 8mL carbonate buffer solution (Ph9.6 contains 5% methyl alcohol) of 250mgBSA, potpourri stirred 2 hours at 4 ℃ of lower magnetic forces, dialysed overnight 4 times (phosphate buffer of pH7.4) under 4 ℃ of conditions is carried out full wavelength scanner through ultraviolet scanner and is identified the coupling result.
Fig. 2 is the UV scanning figure of the haptenic conjugate of BSA-(immunogene) of employing active ester method preparation, three kinds of materials are respectively carrier protein BSA, the haptenic conjugate of carrier protein BSA-, haptenic ultraviolet absorpting spectrum from top to bottom among the figure, carrier protein BSA characteristic absorption peak is at the 278nm place as can be seen from Figure, the haptens characteristic absorption peak is at the 260nm place, the haptenic conjugate characteristic absorption peak of carrier protein BSA-is at the 255nm place, and the conjugate characteristic peak drifts about.
Embodiment 2 holoantigen MONOCLONAL ANTIBODIES SPECIFIC FOR and the detection of tiring
2.1 MONOCLONAL ANTIBODIES SPECIFIC FOR
Be immunogene with the BSA-haptens, 4 BALB/C mice of immunity, every mouse is got 100 μ g immunogenes, and is even with equal-volume Freund's complete adjuvant mixing and emulsifying, injects in the abdominal cavity film along groin.After 4 week, booster immunization, dosage is constant, and adjuvant changes incomplete Freund into.Behind the booster immunization three times, blood sampling is surveyed and is tired, treat that serum titer no longer rises, antigen with two multiple doses does not add the adjuvant immunity mouse, get spleen cell and murine myeloma cell after three days in 5-10 under aseptic condition: 1 ratio is mixed in the 50mL centrifuge tube, adds 30mL serum-free IPMI1640 nutrient culture media, and 1200r/min is centrifugal, and 10min abandons supernatant, with the cell mass pine that shakes gently, place 37 ℃ of water-baths.1mL 50%PEG-4000 is slowly added in the cell, in 1min, drip off, stir bottom settlings gently simultaneously, leave standstill 1min.Slowly add serum free medium along tube wall and stop fusion process.Slowly at the uniform velocity added in preceding 30 seconds behind the 1mL and to add 2mL in 30 seconds, add the 27mL serum free medium then fast, the centrifugal 10min of 1200r/min abandons supernatant.Cell after the fusion screens in HAT selectivity nutrient solution earlier, changes the HT nutrient solution after 5 days into, treats that the hybrid cell quantity hole in reaches 300 when above, carries out multiple hole with the cell culture supernatant of ELISA and detects, the definite result of duplicate detection next day.Cell in the strong positive hole is cloned cultivation with limiting dilution assay, and track record, to cultivate and detect through the clone more than 3 times, the hole inner cell that all is positive is the hybridoma of secrete monoclonal antibody.Hybridoma is cultivated through enlarging, selected 4 multiparity BALB/C mice, lumbar injection saxol 0.5mL/, 7 days pneumoretroperitoneum injection hybridomas 5 * 10
5-10
6/ only, after 10 days, treat to collect when mouse web portion obviously expands ascites.Come purifying ascites with caprylic acid-ammonium sulfate precipitation method, the albumen that obtains through the preliminary judgement of nucleic acid-protein ultraviolet scanner analysis of protein is IgG albumen.
2.2 detection antibody titer
By every hole 100 μ L coated elisa plates, 4 ℃ of bags are spent the night, and wash 5 times with 1 μ g/mL concentration, pat dry, and seal 12h down for 4 ℃ by every hole 200 μ L confining liquids, wash 3 times, pat dry.Adding the antiserum extension rate by every hole 100 μ L is 2000,10000,250000,50000,250000,1250000, and negative serum and blank (do not add antiserum, only add its dilution) room temperature effect 30min washs five times, pats dry.Add every hole 100 μ L enzymes mark goat-anti rabbit (mouse) antibody, room temperature effect 30min washs five times, pats dry.Add every hole 100 μ L colour developing liquid, 37 ℃ of lucifuge effect 15min.Add every hole 50 μ L stop buffer cessation reactions, microplate reader detects A value (450nm).The antiserum dilutability corresponding with the serum OD value that doubles negative serum OD value is antiserum titre.Testing result sees Table 1:
The table 1 monoclonal antibody testing result of tiring
| Extension rate | 2000 | 10000 | 15000 | 30000 | 50000 | 250000 | Negative serum | Blank |
| The OD1 value | 0.988 | 0.692 | 0.415 | 0.324 | 0.317 | 0.362 | 0.172 | 0.024 |
| The OD1 value | 0.975 | 0.673 | 0.442 | 0.398 | 0.365 | 0.354 | 0.207 | 0.02 |
| The OD1 value | 1.136 | 0.943 | 0.754 | 0.503 | 0.387 | 0.351 | 0.193 | 0.032 |
| The OD1 value | 0.982 | 0.669 | 0.411 | 0.376 | 0.344 | 0.351 | 0.223 | 0.03 |
Can infer that from the data of table 1 the tiring of monoclonal antibody that the present invention prepares reaches 15000.
The preparation of the anti-mouse IgG of sheep (rabbit) among embodiment 3 the present invention
Choosing healthy male new zealand white rabbit or goat, is that immunogene and equivalent Freund's complete adjuvant are mixed into water in oil emulsion by syringe to the method for taking out with the OVA-haptens, carries out first immunisation by the amount of 1mg/kg body weight, takes the subcutaneous multi-point injection in back.Every two all booster immunizations once, replace Freund's complete adjuvant with incomplete Freund, the same first immunisation of dosage and method.From immunity for the third time, back 10 days of each immunity, auricular vein is got blood 1mL, carrying out antibody titer detects, when antibody titer no longer raises, do not add adjuvant and carry out for the last time (the 7th time) immunity, the leg muscle injection, rear neck artery bloodletting in 7 days, room temperature is solidified behind the 2h 4 ℃ and is spent the night, and centrifugal 10 minutes of 8000r/min removes clot, serum partly precipitates with 50% saturated ammonium sulfate solution, the centrifugal supernatant that goes, precipitation is resuspended with phosphate buffer, precipitates twice with 33% saturated ammonium sulfate solution again, sediment is with the dissolving of the least possible phosphate buffer, through dialyse concealed malachite green-anti-mouse IgG of OVA sheep (rabbit).
The preparation of colloidal gold strip among embodiment 4 the present invention
4.1 the preparation of collaurum
The ultimate principle of immune colloidal gold technique is, gold chloride is under the effect of reductive agent, and polymerizable is a certain size gold grain, form electronegative, because electrostatic interaction and stable hydrophobic sol solution.Our company adopts this reductive agent reducing process of trisodium citrate to prepare collaurum, detailed process is as follows: get 0.01% gold chloride 100mL aqueous solution and be heated to and boil, stir and accurately add 1% citrate three sodium aqueous solution 0.7mL down, flavous gold chloride became aubergine in 2 minutes, continue to boil 15min, original volume is recovered with distilled water in the cooling back, is the colloidal gold solution of preparation.Whether this colloidal gold solution meets the production demand, except the visual inspection color need be for the aubergine, also need to adopt the ultra-violet and visible spectrophotometer analysis, colloidal gold solution needs at visible range 535nm the highest absorption peak (see figure 2) is arranged, simultaneously, Electronic Speculum figure show preparation the colloid gold particle homogeneity better, grain size is about 40m.
4.2 the preparation of colloid gold label antibody
Regulate colloidal gold solution pH value to 8.2, evenly stir with constant speed stirrer, dropwise add the monoclonal antibody of anti-concealed malachite green simultaneously, and add other protective agent, after all adding, continue to stir 15 minutes.Centrifugal acquisition homogeneity gold labeling antibody precipitation by twice different rotating speeds.Add resuspended liquid again, move in the clean beaker standby behind the mixing.
4.3 the preparation of malachite green colloidal gold immune chromatography rapid detecting test paper strip
Successively thieving paper, the NC film that is sprayed with amino procrypsis malachite green-BSA (detection line) and the anti-mouse IgG of sheep (rabbit) (nature controlling line), the gold pad that is sprayed with anti-procrypsis malachite green antibody and sample pad are superimposed with each other and are fixed on the PVC base plate, be cut into test strips again, be contained in the plastic module, make the colloidal gold immunochromatographimethod rapid detection card.
The residual detection of concealed malachite green in embodiment 5 samples
5.1 kit of the present invention detects principle
Antibody labeling is on colloid gold particle; collaurum and sample are mobile at the NC film simultaneously; in mobile process; malachite green in the sample or procrypsis malachite green and the specific reaction of antibody that is marked on the colloid gold particle; when the malachite green in the sample or procrypsis malachite green content are lower than the detectability of test strips; then immune gold grain meeting and be sprayed on the detection tape that antigen line reaction on the NC film forms a redness, otherwise, coloured detection tape then can not be formed.
5.2 sample preparation
Getting 5g sample (flesh of fish/shrimp) rubbing inserts in the 50mL centrifuge tube, add 10mL ethyl acetate homogeneous, with 4000g centrifugal 5 minutes, get the 5mL supernatant and insert in the glass tube, under 60 ℃, dry up with nitrogen, in this glass tube, add 1mL normal hexane and 1mL distilled water, vibration mixing 2 minutes, centrifugal 10 minutes of room temperature 4000g gets 100 μ L subnatants, through 5 times of sample diluting liquid dilutions, to be measured.
Embodiment 6 colloidal gold strip sensitivity tests
Get 3ng/ml and suppress product fully, handle the stealthy malachite green mark of adding product in the sample at buffer solution and 5 negative aquatic products, make its final concentration be respectively 0.1,1,2,3,4,5,6,7,10,30ng/mL.Every kind of sample repeats 5 times, judges the detection sensitivity of test strips, the results are shown in following table:
Table 2 the present invention adopts the colloidal gold strip sensitivity test of monoclonal antibody
As can be seen from the above results, the detection threshold of concealed malachite green colloid gold immune quick detection test paper bar is 3ng/mL.
The specificity test of embodiment 7 colloidal gold strips of the present invention
(ELISA is determined as feminine gender) adds malachite green, recessive crystal violet, crystal violet, Clenbuterol, sulfadimidine, chloromycetin, furazolidone respectively in the aquatic products of feminine gender, and making its final concentration is 1,5,10,50,100,500ng/mL aquatic products Treatment Solution.The standard method that detects with test strips detects, and judges the specificity that test strips detects, and the aquatic products of every kind of concentration are handled sample and done 5 repetitions.
Concealed malachite green immunity colloidal gold test paper strip specificity experimental result in table 3 aquatic products
Experimental result is as shown in table 3, and recessive crystal violet, crystal violet, Clenbuterol, sulfadimidine, chloromycetin, these analogues of furazolidone do not produce cross reaction in stealthy malachite green colloid gold immune quick detection test paper bar.
The accuracy test of embodiment 8 colloidal gold strips
Stealthy malachite green colloid gold immune quick detection test paper bar is qualitative, rather than quantitative card.This product all shows as the positive to the sample of every 3ng/mL of equaling or exceeding.
When adding stealthy malachite green and handling in the sample to negative aquatic products, without any leaching process, only need directly aquatic products are handled sample and get final product in well with the vertical 3 bubble-free samples to be checked (about 70ul) that drip of plastic suction pipe.Therefore when adding concealed malachite green 〉=3ng/mL, then detection line does not develop the color, and namely is judged to the positive, thus the recovery test that is not similar to ELISA as a result the accuracy rate of this test card greater than 96%.
The test of embodiment 9 colloidal gold strip storage lives
The kit preservation condition is 2-8 ℃, and through 6 months mensuration, concealed malachite green added the actual sample measured value all within normal range.Consideration has improper preservation condition and occurs in transportation and use, and kit was placed 6 days under 37 ℃ of preservation conditions, carries out the accelerated stability experiment, and the result shows that the every index of this colloidal gold strip meets the requirements fully.
Claims (1)
1. the preparation method of a fast detecting concealed malachite green immunity colloidal gold test paper strip, wherein, described test strips comprises: sample pad, collaurum pad, nitrocellulose filter NC, adsorptive pads and PVC backing; Described PVC backing one end sticks sample pad, collaurum pad, NC rete, adsorptive pads successively; Described collaurum pad bag has been adsorbed concealed malachite green-OVA or concealed malachite green-BSA and sheep or rabbit anti-mouse igg by anti-concealed malachite green monoclonal antibody specific-colloid gold label thing on the NC film;
The preparation method who it is characterized in that described test strips may further comprise the steps:
(1) the concealed malachite green holoantigen is synthetic: by at phenyl ring contraposition coupling one reactive group-COOH, synthesize the concealed malachite green haptens; Concealed malachite green haptens and carrier protein are pressed 1: 10-30 mol ratio mixing, adopt the synthetic concealed malachite green of glutaraldehyde or diazonium method-carrier protein couplet thing, after separation and purification, obtain concealed malachite green hapten-carrier protein conjugate;
(2) preparation of concealed malachite green monoclonal antibody specific: be repeatedly immune Balb/c mouse inbred lines of immunogene with haptens-BSA, get the external fusion of mouse boosting cell and myeloma cell and produce hybridoma, obtain the positive hybridoma cell strain through the selective medium screening; Described hybridoma cell strain is injected mouse peritoneal obtain ascites or cell in vitro cultivation collection supernatant, prepare anti-concealed malachite green monoclonal antibody in a large number;
(3) acquisition of sheep or rabbit anti-mouse igg: with many immune mouses of haptens-OVA, obtain antiserum, immune goat or rabbit obtain sheep or rabbit anti-mouse igg after separation and purification again;
(4) monoclonal antibody specific Preparation of Colloidal Gold: the colloid gold particle that gold chloride is reduced into 20～40nm with the trisodium citrate reductive agent; Collaurum and anti-concealed malachite green monoclonal antibody mass ratio 1: 0.005～0.015 mixing by volume, make it in conjunction with forming stable colloid gold particle, the anti-recessive peacock stone monoclonal antibody of purified and concentrated generation-colloid gold label thing then;
(5) preparation of collaurum pad: concealed malachite green specific monoclonal antibody-colloid gold label thing is coated on the collaurum pad, concealed malachite green-OVA conjugate and sheep or rabbit anti-mouse igg are adsorbed on detection zone and the control zone of NC film, fully dry at 37 ℃;
(6) colloid gold reagent bar assembling: with sample pad, collaurum pad, NC rete, adsorptive pads sticks on the PVC backing successively, and the PVC material that glues is cut into the reagent strip of certain width, is assembled at last in a certain size the plastic casing to get final product;
Wherein, the synthesis step of haptens and comlete antigen is as follows in the step (1):
(a) to the preparation of concealed malachite green ethyl ketone
In the 100mL three-necked bottle, add 20mL methylene chloride, 18g aluminum trichloride (anhydrous); Cryosel is bathed and is cooled to-10 ℃, drips the mixed liquor of 7.1g aceticanhydride and 21.61g concealed malachite green, and temperature is no more than-5 ℃ in the control; After dropwising, continue to stir 15h, solution becomes kermesinus from yellow; In the potpourri with reactant liquor impouring 50mL concentrated hydrochloric acid and 50g trash ice, divide water-yielding stratum with separating funnel then, and with twice of dichloromethane extraction; Merge organic phase, wash to neutrality anhydrous sodium sulfate drying with water; The distillation desolventizing gets yellow oil 9.8g; Crude product is directly used in bromoform reaction;
(b) to the preparation of concealed malachite green formic acid
NaOH 6.3g is dissolved in the 54mL water, is cooled to-5 ℃; Dripping bromine 6.4g, temperature is no more than 0 ℃ in the control; To drop in the reactant liquor concealed malachite green ethyl ketone crude product 5.04g then, temperature is no more than 10 ℃ in the control; Dropwise, insulation 1h is then in stirring at room 2h; Standing demix divides and to remove the bromofom that generates; Water layer transfers to pH1～2 with concentrated hydrochloric acid, separates out white solid; Suction filtration is washed to neutrality; It is in 5% the sodium hydroxide solution that solid is dissolved in 20mL concentration, filters, and light yellow clarifying liquid body and function concentrated hydrochloric acid transfers to pH1～2, separates out white crystal; Suction filtration is washed to neutrality, and oven dry gets white crystal 1.7g, is 81% by its yield of concealed malachite green;
(c) immunogenic synthetic
Active ester method prepares immunogene and is achieved in that concealed malachite green haptens and the N-hydroxy-succinamide NHS that gets equimolar amounts, ring dihexyl carbodiimide DCC, DFM dissolves potpourri with dimethyl formamide, the lucifuge reaction is spent the night under the room temperature, centrifugal removal post precipitation, get the supernatant drying, getting its 225 μ L adding contains in the 8mL methyl alcohol carbonate buffer solution of 250mgBSA, potpourri stirred 2 hours at 4 ℃ of lower magnetic forces, at 4 ℃, pH is dialysed overnight 4 times under the condition of 7.4 phosphate buffer, carries out full wavelength scanner through ultraviolet scanner and identifies the coupling result; Wherein, the pH of described methyl alcohol carbonate buffer solution is 9.6, and it contains 5% methyl alcohol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910093010 CN101655498B (en) | 2009-09-25 | 2009-09-25 | Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910093010 CN101655498B (en) | 2009-09-25 | 2009-09-25 | Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101655498A CN101655498A (en) | 2010-02-24 |
| CN101655498B true CN101655498B (en) | 2013-07-03 |
Family
ID=41709871
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200910093010 Active CN101655498B (en) | 2009-09-25 | 2009-09-25 | Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101655498B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102213723A (en) * | 2010-04-02 | 2011-10-12 | 北京库尔科技有限公司 | Doxycycline detection kit and preparation method thereof |
| CN103399153B (en) * | 2013-07-24 | 2015-05-20 | 泰州康正生物技术有限公司 | Hybridoma cell strain, anti-leucomalachite green monoclonal antibody generated by same and application thereof |
| CN104977407A (en) * | 2015-05-20 | 2015-10-14 | 集美大学 | Colloidal gold immunochromatography test paper strip for detecting tetracycline drugs, and preparation method thereof |
| CN104914222A (en) * | 2015-05-20 | 2015-09-16 | 集美大学 | Colloidal gold immunochromatography assay test strip for detecting malachite green and preparation method thereof |
| CN106770232A (en) * | 2016-12-03 | 2017-05-31 | 无锡艾科瑞思产品设计与研究有限公司 | A kind of malachite green crystal violet detection method and detection card |
| CN108918850B (en) * | 2018-07-12 | 2021-02-26 | 北京市药品检验所 | Method for rapidly detecting aflatoxin in medicinal materials |
| CN116698551B (en) * | 2023-08-09 | 2023-10-13 | 苏州快捷康生物技术有限公司 | Sample pretreatment method for leucomalachite green colloidal gold method detection in aquatic tissues |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101308140A (en) * | 2008-06-30 | 2008-11-19 | 江苏省苏微微生物研究有限公司 | Concealed malachite green gold mark detection test paper box and method for making same |
| CN101451999A (en) * | 2008-01-16 | 2009-06-10 | 北京中德大地食品安全技术开发有限责任公司 | Indirectly racing ELISA kit for detecting leuco malachite green |
-
2009
- 2009-09-25 CN CN 200910093010 patent/CN101655498B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101451999A (en) * | 2008-01-16 | 2009-06-10 | 北京中德大地食品安全技术开发有限责任公司 | Indirectly racing ELISA kit for detecting leuco malachite green |
| CN101308140A (en) * | 2008-06-30 | 2008-11-19 | 江苏省苏微微生物研究有限公司 | Concealed malachite green gold mark detection test paper box and method for making same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101655498A (en) | 2010-02-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101655498B (en) | Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method | |
| CN101451999B (en) | Directly competitive ELISA kit for detecting implicit malachite green | |
| CN100482787C (en) | Immune colloid gold test paper strip for detecting sulfadiazine residue and its preparation method | |
| CN103575893A (en) | Method for rapidly detecting shellfish toxin | |
| CN108362881B (en) | Colloidal gold rapid detection and test device for pyrazolone antipyretic analgesic drugs, and preparation method and application thereof | |
| CN102827076B (en) | Universal hapten, artificial antigen and broad-spectrum monoclonal antibody for fluoroquinolone medicines as well as preparation method and application of universal hapten | |
| CN102621329B (en) | Bird's-nest enzyme linked immunosorbent assay kit | |
| CN106940373A (en) | The immunochromatography time-resolved fluorescence kit of four kinds of mycotoxin composite pollutions such as synchronous detection fumonisin B1 and its application | |
| CN101995467B (en) | Malachite green collaurum detection card and production and application methods thereof | |
| CN104569404A (en) | Method for detecting olaquindox by directly-competing TRFIA (Time Resolved Fluorescence Immunoassay) | |
| CN105044365A (en) | Preparation method of time-resolved fluoroimmunoassay test paper for detection of Enrofloxacin residue | |
| CN103323593A (en) | Test paper for detecting fluoroquinolone drugs and its application | |
| CN102928413A (en) | Magnetic particle chemiluminescence kit for detecting tetracyclines, and applications thereof | |
| CN104610079B (en) | A kind of salbutamol hapten derivant, salbutamol artificial antigen and its preparation method and application | |
| CN104109112B (en) | Cyproheptadine semiantigen, artificial antigen, antibody, preparation methods of cyproheptadine semiantigen and artificial antigen, and application of artificial antigen and antibody | |
| CN101993486B (en) | Clenbuterol immunogen, coatingen and application thereof in colloidal gold test paper | |
| CN101592660A (en) | Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection kit | |
| CN105334323A (en) | Method and test strip for detecting zilpaterol, and application of test strip | |
| CN107012128A (en) | A kind of hybridoma cell strain for secreting aspergillus flavus resisting toxin B1 monoclonal antibodies and its application | |
| CN104569408A (en) | Fenoterol colloidal gold detection card and preparation method thereof | |
| CN102250238A (en) | Method for synthesizing swainsonine antigen | |
| CN102432684A (en) | Preparation method and application of clenbuterol monoclonal antibody | |
| CN111500546A (en) | Cell strain secreting four subtype antibodies against aflatoxin, antibody secreted by cell strain and immunochromatography detection card | |
| CN110927375A (en) | Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof | |
| CN103073635B (en) | Salbutamol artificial antigen synthesis method and application thereof to colloid gold immunity test strip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| DD01 | Delivery of document by public notice |
Addressee: Zhongde-Dadi Food Safety Technology Development Co., Ltd., Beijing Document name: Notification of Decision on Request for Restoration of Right |
|
| ASS | Succession or assignment of patent right |
Owner name: ZHONGDE BIO-ENGRG CO., LTD., JIANGXI Free format text: FORMER OWNER: ZHONGDE-DADI FOOD SAFETY TECHNOLOGY DEVELOPMENT CO., LTD., BEIJING Effective date: 20130104 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 100081 HAIDIAN, BEIJING TO: NANCHANG, JIANGXI PROVINCE |
|
| TA01 | Transfer of patent application right |
Effective date of registration: 20130104 Address after: High tech Development Zone, Jiangxi, Nanchang high and new way (Chang Jian Industrial Park) Applicant after: Zhongde Bio-Engrg Co., Ltd., Jiangxi Address before: 100081, room 107, Granville Technology Building, 8 Zhongguancun South Street, Beijing, Haidian District Applicant before: Zhongde-Dadi Food Safety Technology Development Co., Ltd., Beijing |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method Effective date of registration: 20160615 Granted publication date: 20130703 Pledgee: Bank of Beijing, Limited by Share Ltd, Nanchang branch Pledgor: Zhongde Bio-Engrg Co., Ltd., Jiangxi Registration number: 2016360000008 |
|
| PLDC | Enforcement, change and cancellation of contracts on pledge of patent right or utility model | ||
| CP03 | Change of name, title or address |
Address after: 330046 Jiangxi city in Nanchang Province, the provincial government compound Dongsilu dragon building C, Room 308 Patentee after: Jiangxi Sino German bioengineering Limited by Share Ltd Address before: High tech Development Zone, Jiangxi, Nanchang high and new way (Chang Jian Industrial Park) Patentee before: Jiangxi Zodogenes Biotechnology Co., Ltd. |
|
| CP03 | Change of name, title or address | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20170811 Granted publication date: 20130703 Pledgee: Bank of Beijing, Limited by Share Ltd, Nanchang branch Pledgor: Zhongde Bio-Engrg Co., Ltd., Jiangxi Registration number: 2016360000008 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method Effective date of registration: 20170811 Granted publication date: 20130703 Pledgee: Bank of Beijing, Limited by Share Ltd, Nanchang branch Pledgor: Jiangxi Sino German bioengineering Limited by Share Ltd Registration number: 2017990000738 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20180730 Granted publication date: 20130703 Pledgee: Bank of Beijing, Limited by Share Ltd, Nanchang branch Pledgor: Jiangxi Sino German bioengineering Limited by Share Ltd Registration number: 2017990000738 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method Effective date of registration: 20180730 Granted publication date: 20130703 Pledgee: Bank of Beijing, Limited by Share Ltd, Nanchang branch Pledgor: Jiangxi Sino German bioengineering Limited by Share Ltd Registration number: 2018990000621 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20190927 Granted publication date: 20130703 Pledgee: Bank of Beijing, Limited by Share Ltd, Nanchang branch Pledgor: Jiangxi Sino German bioengineering Limited by Share Ltd Registration number: 2018990000621 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |