CN117603095A - 灭幼脲半抗原、抗原、抗体、检测装置及其制备和应用 - Google Patents
灭幼脲半抗原、抗原、抗体、检测装置及其制备和应用 Download PDFInfo
- Publication number
- CN117603095A CN117603095A CN202311589425.6A CN202311589425A CN117603095A CN 117603095 A CN117603095 A CN 117603095A CN 202311589425 A CN202311589425 A CN 202311589425A CN 117603095 A CN117603095 A CN 117603095A
- Authority
- CN
- China
- Prior art keywords
- chlorbenzuron
- hapten
- reaction
- antibody
- antigen
- 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.)
- Pending
Links
- YPSCQJTUAKNUNF-UHFFFAOYSA-N 2-chloro-n-[(4-chlorophenyl)carbamoyl]benzamide Chemical compound C1=CC(Cl)=CC=C1NC(=O)NC(=O)C1=CC=CC=C1Cl YPSCQJTUAKNUNF-UHFFFAOYSA-N 0.000 title claims abstract description 121
- 238000001514 detection method Methods 0.000 title claims abstract description 65
- 239000000427 antigen Substances 0.000 title claims abstract description 25
- 102000036639 antigens Human genes 0.000 title claims abstract description 25
- 108091007433 antigens Proteins 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title abstract description 24
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 17
- 235000013311 vegetables Nutrition 0.000 claims abstract description 13
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 8
- 230000001900 immune effect Effects 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims description 47
- 239000000243 solution Substances 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 20
- 238000012360 testing method Methods 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 239000002904 solvent Substances 0.000 claims description 16
- 238000001704 evaporation Methods 0.000 claims description 15
- 239000012528 membrane Substances 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 13
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 12
- 238000003908 quality control method Methods 0.000 claims description 12
- 238000010521 absorption reaction Methods 0.000 claims description 11
- 230000003053 immunization Effects 0.000 claims description 11
- 238000002649 immunization Methods 0.000 claims description 11
- CTSLXHKWHWQRSH-UHFFFAOYSA-N oxalyl chloride Chemical compound ClC(=O)C(Cl)=O CTSLXHKWHWQRSH-UHFFFAOYSA-N 0.000 claims description 10
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 229940098773 bovine serum albumin Drugs 0.000 claims description 9
- 241000283707 Capra Species 0.000 claims description 8
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims description 7
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 6
- 239000004202 carbamide Substances 0.000 claims description 6
- ONIKNECPXCLUHT-UHFFFAOYSA-N 2-chlorobenzoyl chloride Chemical compound ClC(=O)C1=CC=CC=C1Cl ONIKNECPXCLUHT-UHFFFAOYSA-N 0.000 claims description 5
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 5
- HJEZRYIJNHAIGY-UHFFFAOYSA-N tert-butyl 4-bromobutanoate Chemical compound CC(C)(C)OC(=O)CCCBr HJEZRYIJNHAIGY-UHFFFAOYSA-N 0.000 claims description 5
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 4
- 102000014914 Carrier Proteins Human genes 0.000 claims description 4
- 108010078791 Carrier Proteins Proteins 0.000 claims description 4
- 241001465754 Metazoa Species 0.000 claims description 4
- 108010058846 Ovalbumin Proteins 0.000 claims description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 4
- 239000012074 organic phase Substances 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims description 3
- RBGDLYUEXLWQBZ-UHFFFAOYSA-N 2-chlorobenzamide Chemical compound NC(=O)C1=CC=CC=C1Cl RBGDLYUEXLWQBZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims description 3
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 3
- 229940092253 ovalbumin Drugs 0.000 claims description 3
- 238000000967 suction filtration Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 claims description 2
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 2
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 2
- 108060003552 hemocyanin Proteins 0.000 claims description 2
- 229940034208 thyroxine Drugs 0.000 claims description 2
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 239000000523 sample Substances 0.000 description 24
- 238000000576 coating method Methods 0.000 description 10
- 239000011248 coating agent Substances 0.000 description 9
- 240000007124 Brassica oleracea Species 0.000 description 8
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 8
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 8
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 8
- 239000003550 marker Substances 0.000 description 7
- 239000008055 phosphate buffer solution Substances 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 239000006143 cell culture medium Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 239000000020 Nitrocellulose Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 241000255925 Diptera Species 0.000 description 2
- 241000220225 Malus Species 0.000 description 2
- 235000011430 Malus pumila Nutrition 0.000 description 2
- 235000015103 Malus silvestris Nutrition 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000012055 fruits and vegetables Nutrition 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000575 pesticide Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- OOSZCNKVJAVHJI-UHFFFAOYSA-N 1-[(4-fluorophenyl)methyl]piperazine Chemical compound C1=CC(F)=CC=C1CN1CCNCC1 OOSZCNKVJAVHJI-UHFFFAOYSA-N 0.000 description 1
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 102000005469 Chitin Synthase Human genes 0.000 description 1
- 108700040089 Chitin synthases Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 101000609762 Gallus gallus Ovalbumin Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- HRYILSDLIGTCOP-UHFFFAOYSA-N N-benzoylurea Chemical compound NC(=O)NC(=O)C1=CC=CC=C1 HRYILSDLIGTCOP-UHFFFAOYSA-N 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- DGLRDKLJZLEJCY-UHFFFAOYSA-L disodium hydrogenphosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O DGLRDKLJZLEJCY-UHFFFAOYSA-L 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 238000003810 ethyl acetate extraction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 229940074545 sodium dihydrogen phosphate dihydrate Drugs 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 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 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
- C07C275/46—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups containing any of the groups, X being a hetero atom, Y being any atom, e.g. acylureas
- C07C275/48—Y being a hydrogen or a carbon atom
- C07C275/54—Y being a carbon atom of a six-membered aromatic ring, e.g. benzoylureas
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C201/00—Preparation of esters of nitric or nitrous acid or of compounds containing nitro or nitroso groups bound to a carbon skeleton
- C07C201/06—Preparation of nitro compounds
- C07C201/12—Preparation of nitro compounds by reactions not involving the formation of nitro groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C213/00—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton
- C07C213/02—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton by reactions involving the formation of amino groups from compounds containing hydroxy groups or etherified or esterified hydroxy groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C273/00—Preparation of urea or its derivatives, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
- C07C273/18—Preparation of urea or its derivatives, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups of substituted ureas
- C07C273/1809—Preparation of urea or its derivatives, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups of substituted ureas with formation of the N-C(O)-N moiety
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C273/00—Preparation of urea or its derivatives, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
- C07C273/18—Preparation of urea or its derivatives, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups of substituted ureas
- C07C273/1854—Preparation of urea or its derivatives, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups of substituted ureas by reactions not involving the formation of the N-C(O)-N- moiety
- C07C273/1863—Preparation of urea or its derivatives, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups of substituted ureas by reactions not involving the formation of the N-C(O)-N- moiety from urea
-
- 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
- C07K14/76—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
-
- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Food Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Nanotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了灭幼脲半抗原、抗原、抗体、检测装置及其制备和应用,涉及灭幼脲半抗原、灭幼脲抗原、灭幼脲抗体和灭幼脲胶体金层析检测装置及其制备和检测灭幼脲的应用。本发明应用免疫学检测方法,对样品中残留的灭幼脲进行专属检测,具有特异性好、灵敏度高、操作简便、可实现现场快速检测等优点,能更好的满足监管部门、检测机构现场监督执法的需要,为相关执法部门快速检测蔬菜、水果中是否残留灭幼脲提供技术支持。
Description
技术领域
本发明涉及食品安全的免疫学检测技术领域,更具体地,本发明涉及灭幼脲半抗原、抗原、抗体、检测装置及其制备和应用,其检测装置特别适用于蔬菜、水果中灭幼脲残留的快速检测。
背景技术
灭幼脲为苯甲酰脲类几丁质合成抑制剂,主要通过抑制昆虫表皮几丁质合成酶和尿核苷辅酶的活性,从而抑制昆虫几丁质合成导致其死亡,对蚊蝇幼虫有较高的活性,也可抑制双翅目成虫卵的孵化。然而,长期食用含灭幼脲的食品会对人体产生一定的损伤。国家标准GB2763-2021《食品安全国家标准食品中农药最大残留限量》,规定果蔬中灭幼脲的最大残留量分别为0.2-30mg/kg。因此,有必要对水果、蔬菜、谷物中灭幼脲的残留量进行控制。
由于蔬菜基质比较复杂,测定前需要对其进行前处理,目前采用的方法主要为固相萃取法,但用其分析复杂基质样品或不同性质多农药残留时,可能引起回收率偏低,主要由气相色谱仪或液相色谱仪进行检测,但过程较复杂,成本较高,很难达到快速检测的目的,不适用于现场快速检测,不能满足监管部门、检测机构现场监督执法的需要。
发明内容
本发明的目的在于提供了灭幼脲半抗原、抗原、抗体、检测装置及其制备和应用。
为解决上述的技术问题,本发明采用以下技术方案:
根据本发明的一个方面,提供了灭幼脲半抗原,其结构如式(Ⅰ)所示:
根据本发明的另一个方面,制备灭幼脲半抗原的方法,包括以下步骤:
S1.冰浴下在氨水溶液中滴加2-氯苯甲酰氯,反应2h后将白色沉淀抽滤、水洗烘干得到第一中间体2-氯苯甲酰胺;
S2.将对硝基苯酚、4-溴丁酸叔丁酯用DMF溶解,溶解后加入碳酸钾,80℃搅拌反应6h,反应结束后蒸干溶剂,加水稀释,乙酸乙酯萃取,有机相蒸干过柱纯化得第二中间体,第二中间体的结构式如式(Ⅱ)所示:
S3.用水合肼、Pd/C和三氯化铁加入甲醇溶解的第二中间体中,经过纯化,得到第三中间体,第三中间体的结构式如式(Ⅲ)所示:
S4.将第一中间体用1,2-二氯乙烷溶解,溶解后加入草酰氯,70℃搅拌反应6h后蒸干溶剂,再次加入1,2-二氯乙烷作为溶剂,滴加第三中间体,70℃保温反应过夜,反应后蒸干溶剂,重结晶后得到第四中间体,第四中间体的结构式如式(IV)所示:
S5.将第四中间体用二氯甲烷溶解,溶解后加入三氟乙酸,室温搅拌反应1h后蒸干溶剂,得到结构如式(Ⅰ)所示的半抗原。
在一些实施方式中,所述步骤S1中,2-氯苯甲酰氯与氨水的摩尔比为1:(1-10);
所述步骤S2中,对硝基苯酚、4-溴丁酸叔丁酯与碳酸钾的摩尔比为1:(1.2-2):(1.5-5)。在一些实施方式中,所述步骤S4中,所述步骤S4中,第一中间体与草酰氯、第三中间体的摩尔比为1:(1-1.2):(1-1.2)。
根据本发明的再一个方面,提供了灭幼脲抗原,包含灭幼脲半抗原以及与灭幼脲半抗原偶联的载体蛋白,载体蛋白为牛血清白蛋白、卵清蛋白、血蓝蛋白、甲状腺蛋白或人血清白蛋白中的一种。
根据本发明的第四个方面,提供了灭幼脲抗体,是由灭幼脲抗原经动物免疫制备得到,灭幼脲抗体为灭幼脲单克隆抗体。
根据本发明的第五个方面,提供了灭幼脲半抗原、灭幼脲抗原、灭幼脲抗体在灭幼脲的免疫学检测中的应用。
根据本发明的第六个方面,提供了灭幼脲胶体金层析检测装置,检测装置包括试纸条和微孔反应杯,试纸条包括反应膜、样品吸收垫、吸水垫和底板,反应膜设有检测线和质控线,检测线包被有灭幼脲抗原,质控线包被有羊抗鼠抗体,微孔反应杯中含有胶体金标记的灭幼脲抗体。
根据本发明的第七个方面,提供了一种检测样品中灭幼脲残留的方法,该方法为使用灭幼脲胶体金层析检测装置对样品中的灭幼脲进行检测,该方法包括以下步骤:
1)样品前处理;
2)用灭幼脲胶体金层析检测装置进行检测;
3)分析检测结果。
在一些实施方式中,所述样品为蔬菜或水果。
与现有技术相比,本发明的至少具有以下有益效果之一:
(1)本发明提供的灭幼脲半抗原的制备方法,使用的化学试剂容易得到、操作过程简单、合成步骤简洁有效、反应产率较高、并且检测成本较低。
(2)本发明利用层析式免疫胶体金原理,通过检测卡中检测线与质控线之间的比色来定性检测样本中是否残留有灭幼脲,应用时不需要使用液相色谱或质谱等大型仪器,达到快速检测的目的。
(3)本发明应用免疫学检测方法,对样品中残留的灭幼脲进行专属检测,具有特异性好、灵敏度高、操作简便、可实现现场快速检测等优点,能更好的满足监管部门、检测机构现场监督执法的需要,为相关执法部门快速检测蔬菜或水果中是否残留灭幼脲提供技术支持。
附图说明
图1为本发明的灭幼脲半抗原的质谱图。
图2为本发明的灭幼脲半抗原的合成流程图。
图3为本发明的灭幼脲胶体金层析检测装置的试纸条的结构示意图。
图4为本发明的灭幼脲胶体金层析检测装置的微孔反应杯的结构示意图。
图5为本发明的灭幼脲胶体金层析检测装置的结果判定示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1
灭幼脲半抗原的合成与鉴定
灭幼脲半抗原的制备方法,包括以下步骤:
S1.冰浴下100ml在单口烧瓶中加入30ml 10%氨水溶液,缓慢滴加2-氯苯甲酰氯5.0g,反应2h后将白色沉淀抽滤、水洗烘干得到第一中间体2-氯苯甲酰胺4.5g;
S2.将2.8g对硝基苯酚、4.5g 4-溴丁酸叔丁酯用DMF溶解,溶解后加入碳酸钾5.5g,80℃搅拌反应6h,反应结束后蒸干溶剂,加水稀释,乙酸乙酯萃取,有机相蒸干过柱纯化得第二中间体5.1g;
S3.称取2.5g水合肼、0.52gPd/C和0.52g三氯化铁,加入到50ml甲醇溶解5.2g第二中间体中,50℃搅拌反应2h,反应结束后,过滤,蒸干溶剂加水稀释后,乙酸乙酯萃取,有机相蒸干后经过纯化,得到第三中间体3.5g;
S4.将1.6g第一中间体用1,2-二氯乙烷溶解,溶解后加入1.4g草酰氯,70℃搅拌反应6h后蒸干溶剂,再次加入1,2-二氯乙烷作为溶剂,滴加2.5g第三中间体,70℃保温反应过夜,反应后蒸干溶剂,重结晶后得到第四中间体3.8g;
S5.将第四中间体用10ml二氯甲烷溶解,溶解后加入5ml三氟乙酸,室温搅拌反应1h后蒸干溶剂即得灭幼脲半抗原。
采用质谱法鉴定灭幼脲半抗原,所得到的质谱图见说明书附图图1。从质谱图可以看出,灭幼脲半抗原的分子离子峰为m/z 377.1[M+H]+,且为最高峰,其与该灭幼脲半抗原的分子量376.09相符,表明成功合成了式(Ⅰ)所示的灭幼脲半抗原。
实施例2
灭幼脲免疫用抗原、包被用抗原的制备与鉴定
2.1灭幼脲免疫用抗原的制备:称量实施例1中制备得到的灭幼脲半抗原25mg,溶于2ml N,N-二甲基甲酰胺(DMF)中,加入18mg N-羟基丁二酰亚胺(NHS)、26mg1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC.HCl),室温反应6h,制备得到活化液;60mg牛血清白蛋白(BSA)溶于2ml 0.05M pH9.0的硼酸缓冲液,加入1ml DMF,0.5ml上述活化液,室温反应4h后用PBS(0.02mol/L,pH=7.4的磷酸盐缓冲液)透析,每4h换液1次,换液7~8次,透析后4000转/min离心5min,取上清液,得到灭幼脲半抗原-BSA偶联物,即为灭幼脲免疫用抗原,分装,于-20℃保存。
2.2灭幼脲包被用抗原的制备:称取10mg实施例2中制备得到的灭幼脲半抗原溶于1ml无水N,N-二甲基甲酰胺(DMF)中,然后按顺序依次加入3μl二异丙基乙胺和5μl氯甲酸异丁酯,0℃反应30min,制备得到A液;将70mg鸡卵清白蛋白(OVA),0.1M硼酸缓冲液7ml溶解,得到B液;将A液滴加到B液中,室温反应4h后用PBS(0.02mol/L,pH=7.4的磷酸盐缓冲液)透析,每4h换液1次,换液7~8次,透析后4000转/min离心5min,取上清液,得到灭幼脲半抗原-OVA偶联物,即为灭幼脲包被用抗原,分装,于-20℃保存。
实施例3
灭幼脲单克隆抗体的制备及纯化
3.1动物免疫
选择健康的6~8周龄BALB/c小鼠进行免疫,将实施例2中制备得到的灭幼脲免疫用抗原与弗氏完全佐剂等量混合乳化后,对Balb/c小鼠进行颈背部皮下多点注射免疫(冲刺免疫除外)。首次免疫用完全弗氏佐剂,剂量为150μg/只;时隔4周后进行加强免疫,剂量为75μg/只,用不完全弗氏佐剂混合乳化,之后多次的加强免疫时间间隔3周;冲刺免疫时剂量再减半,为37.5μg/只,完全抗原用生理盐水稀释,对小鼠进行腹腔注射,小鼠第三次免疫后可进行断尾采血检测,通过间接竞争酶联免疫法(ic-ELISA)检测小鼠血清的效价和IC50,选择效价高、IC50低的小鼠进行融合。
3.2细胞融合和克隆化
取免疫BALB/c小鼠脾细胞,按9:1的数量比例与SP2/0骨髓瘤细胞融合,筛选得到稳定分泌灭幼脲单克隆抗体的灭幼脲单克隆杂交瘤细胞株。
3.3细胞冻存和复苏
将灭幼脲单克隆杂交瘤细胞用冻存液制成1×106个/mL的细胞悬液,在液氮中长期保存。复苏时取出冻存管,立即放入37℃水浴中速融,离心去除冻存液后,移入培养瓶内培养。
3.4单克隆抗体的制备与纯化
增量培养法:将灭幼脲单克隆杂交瘤细胞置于细胞培养基中,在37℃条件下进行培养,用辛酸-饱和硫酸铵法将得到的培养液进行纯化,得到灭幼脲单克隆抗体,-20℃保存。其中,细胞培养基为向RPMI-1640培养基中添加小牛血清和碳酸氢钠,小牛血清在细胞培养基中的质量百分含量为15~20%,碳酸氢钠在细胞培养基中的质量百分含量为0.1~0.2%,细胞培养基的pH为7.4。
实施例4
羊抗鼠抗体的制备
以羊作为免疫动物,以鼠源抗体为免疫原对无病原体羊进行免疫,得到羊抗鼠抗体。
实施例5
灭幼脲胶体金层析检测装置的制备
该灭幼脲胶体金层析检测装置的制备方法主要包括以下步骤:
1)制备冻干有灭幼脲单克隆抗体-胶体金标记物的微孔反应杯;
2)制备具有包被有灭幼脲半抗原-载体蛋白偶联物的检测线和包被有羊抗鼠抗体的质控线的反应膜;
3)将2)制备好的反应膜与样品吸收垫、吸水垫、底板组装成试纸;
4)将1)和3)制备好的冻干有灭幼脲单克隆抗体-胶体金标记物的微孔反应杯和试纸组装。
下面分步详细叙述:
5.1灭幼脲单克隆抗体-胶体金标记物的制备
(1)胶体金溶液的制备
用双蒸去离子水将质量分数1%的氯金酸溶液稀释成0.01%(质量分数),取100ml0.01%的氯金酸溶液置于锥形瓶中,用恒温电磁搅拌器加热至沸腾,在持续高温、持续搅拌下加入2.5ml 1%柠檬酸三钠溶液,继续匀速搅拌加热至溶液呈透亮的红色时停止,冷却至室温后用去离子水恢复到原体积,得到胶体金溶液,4℃保存。制备好的胶体金溶液外观纯净、透亮、无沉淀和漂浮物,在日光下观察颜色为酒红色。
(2)灭幼脲单克隆抗体-胶体金标记物的制备
在磁力搅拌下,用0.2mol/L碳酸钾溶液调节胶体金溶液的pH值至7.2,按每毫升胶体金溶液中加入20~60μg灭幼脲单克隆抗体的标准,向胶体金溶液中加入实施例4制备得到的灭幼脲单克隆抗体,继续搅拌混匀30min,静置10min后,加入10%牛血清白蛋白(BSA)溶液,使其在胶体金溶液中的体积百分含量为1%,静置10min。以12000rpm,在4℃条件下离心40min,弃上清液,用体积为初始胶体金溶液体积1/10的复溶缓冲液将沉淀重悬,制备得到灭幼脲单克隆抗体-胶体金标记物,置于4℃保存备用。
复溶缓冲液:含体积百分含量0.1%~0.3%牛血清白蛋白、质量百分含量0.1%~0.2%吐温-20、质量百分含量3%~6%海藻糖,pH=7.2的0.02mol/L磷酸盐缓冲液。
5.2微孔反应杯的制备
向微孔反应杯中加入100μl灭幼脲单克隆抗体-胶体金标记物,放入冷冻干燥机中,在冷阱温度为-50℃条件下,预冻3h后,再真空干燥6h,即可取出,得到冻干有灭幼脲单克隆抗体-胶体金标记物的微孔反应杯,密封保存,灭幼脲单克隆抗体-胶体金标记物冻干量为0.20~0.50μg/mL。
5.3样品吸收垫的制备
将样品吸收垫置于含牛血清白蛋白的0.05mol/L的磷酸盐缓冲液中浸泡2h,37℃烘干2h备用。0.02mol/L的磷酸盐缓冲液的pH为7.2,其中牛血清白蛋白的体积百分含量为0.5%。
5.4反应膜的制备
将灭幼脲包被用抗原(即灭幼脲半抗原-鸡卵清白蛋白偶联物)包被到反应膜上构成检测线,将羊抗鼠抗体包被在反应膜上构成质控线。
包被过程:用磷酸缓冲液将灭幼脲包被用抗原稀释到浓度为1mg/ml,用金标喷金点膜仪将其包被于硝酸纤维素膜上的检测区(T区),包被浓度为0.5mg/mL;用浓度0.01mol/L,pH=7.4的磷酸盐缓冲液将羊抗鼠抗体浓度稀释到200μg/mL,用金标喷金点膜仪将其包被于硝酸纤维素膜上的质控区(C区),包被浓度为0.5mg/mL。将包被好的反应膜置于37℃条件下干燥6h,作为生产备用。
5.5灭幼脲胶体金层析检测装置的制备
(1)试纸条的组装
将样品吸收垫、反应膜、吸水垫依次按顺序粘贴在底板上,其中,底板为PVC底板,样品吸收垫为吸滤纸,吸水垫为吸水滤纸,反应膜为硝酸纤维素膜。样品吸收垫的末端与反应膜的始端相连,反应膜的末端与吸水垫的始端相连,样品吸收垫的始端与底板的始端对齐,吸水垫的末端与底板的末端对齐。微孔反应杯上具有微孔塞。
(2)灭幼脲胶体金层析检测试纸盒的组装
将上述步骤(1)得到的试纸条与微孔反应杯组装成试纸盒,在2~8℃的环境中贮存,有效期12个月。
实施例6
一种检测样品中灭幼脲残留的方法
6.1样本提取液的配制
准确称取氯化钠9g、十二水合磷酸氢二钠6g、二水合磷酸二氢钠0.4g,用水溶解并定容至100ml,即为样品提取液,样品提取液为0.1mmol/L磷酸盐缓冲溶液,pH值为8.0。
6.2样品前处理
准确称取制备好的蔬菜试样2g(精确至0.01g)至15ml离心管中,加入8ml样品提取液,盖上盖子,涡旋混合器混匀或手动上下振荡混匀30s,静置分层或4000r/min离心1min,取上清液为待测液。
6.3测定步骤
吸取200μl上述待测液于微孔反应杯中,上下抽吸5~10次使混合均匀。室温温育3min,将试纸条插入到反应杯中,室温温育3min,取出试纸条,轻轻刮去试纸条下端的样品垫,并进行结果判读。
6.4结果判定
通过对比控制线(C线)和检测线(T线)的颜色深浅进行结果判定。
阳性:当质控区(C)显示出条带,检测区(T)不显色,判为阳性,即样品中有灭幼脲,用“+”表示;
阴性:当质控区和检测区均显示出条带,判为阴性,即样品中没有灭幼脲,用“-”表示;
无效:当质控区(C)不显示条带,试纸失效,具体如说明书附图图5所示。
实施例7
灭幼脲胶体金层析检测装置的灵敏度和假阴性率试验
选取经GB/T 20769-2008《水果和蔬菜中450种农药及相关化学品残留量的测定液相色谱-串联质谱法》测试的不含灭幼脲的甘蓝、白菜和苹果作为空白样品,依据GB 2763-2021中规定甘蓝、白菜和苹果最大残留限量(MRL)分别为3mg/kg,30mg/kg和2mg/kg,设定甘蓝的方法检出限为3mg/kg,白菜的方法检出限为30mg/kg,苹果的方法检出限为2mg/kg,即关注浓度。添加水平为1倍关注浓度、2倍关注浓度,考察灵敏度和假阴性率。两个添加浓度水平的样品,每个浓度水平各50份试样,按实施例6中检测方法进行检测。检测结果见下表1。
表1灭幼脲胶体金层析检测装置的灵敏度和假阴性率检测结果
本实施例中的检测方法对样品中灭幼脲残留的灵敏度≥95%,假阴性率为≤5%。
实施例8
灭幼脲胶体金层析检测装置的特异性和假阳性
选用甘蓝、白菜和苹果样品,采用空白基质加标的方式,制备成2个浓度水平(0.5倍检测限、空白基质)的样品各50份。采用实施例7中检测方法对样品进行检测,检测结果见下表2。
表2灭幼脲胶体金层析检测装置的特异性和假阳性检测结果
由表2可知,本实施例中的检测方法的特异性≥90%,假阳性率为≤10%。上述结果表明本发明的检测灭幼脲的试纸盒具有良好的特异性,能够准确地检测出蔬菜、水果样品中的灭幼脲,因此可以对蔬菜、水果样品中的灭幼脲残留进行快速检测。
实施例9
灭幼脲胶体金层析检测装置的保质期测定
用三批常规生产的产品分别做保质期实验,放置于室内室温环境保持,间隔1个月取12个装置,用质控样本检测,分别做阴性,1.5mg/kg,15mg/kg和1mg/kg样品,重复三次,观察产品显色变化,考察保质期时间。
阴性显色从13个月开始下降,这表明在1年时间内产品的品质无明显变化,因此确定保质期为1年。
尽管这里参照本发明的多个解释性实施例对本发明进行了描述,但是,应该理解,本领域技术人员可以设计出很多其他的修改和实施方式,这些修改和实施方式将落在本申请公开的原则范围和精神之内。更具体地说,在本申请公开、附图和权利要求的范围内,可以对主题组合布局的组成部件和/或布局进行多种变型和改进。除了对组成部件和/或布局进行的变形和改进外,对于本领域技术人员来说,其他的用途也将是明显的。
Claims (10)
1.灭幼脲半抗原,其特征在于,其结构如式(Ⅰ)所示:
2.制备权利要求1所述的灭幼脲半抗原的方法,其特征在于,包括以下步骤:
S1.冰浴下在氨水溶液中滴加2-氯苯甲酰氯,反应2h后将白色沉淀抽滤、水洗烘干得到第一中间体2-氯苯甲酰胺;
S2.将对硝基苯酚、4-溴丁酸叔丁酯用DMF溶解,溶解后加入碳酸钾,80℃搅拌反应6h,反应结束后蒸干溶剂,加水稀释,乙酸乙酯萃取,有机相蒸干过柱纯化得第二中间体,第二中间体的结构式如式(Ⅱ)所示:
S3.用水合肼、Pd/C和三氯化铁加入甲醇溶解的第二中间体中,经过纯化,得到第三中间体,第三中间体的结构式如式(Ⅲ)所示:
S4.将第一中间体用1,2-二氯乙烷溶解,溶解后加入草酰氯,70℃搅拌反应6h后蒸干溶剂,再次加入1,2-二氯乙烷作为溶剂,滴加第三中间体,70℃保温反应过夜,反应后蒸干溶剂,重结晶后得到第四中间体,第四中间体的结构式如式(IV)所示:
S5.将第四中间体用二氯甲烷溶解,溶解后加入三氟乙酸,室温搅拌反应1h后蒸干溶剂,得到结构如式(Ⅰ)所示的半抗原。
3.根据权利要求2的制备灭幼脲半抗原的方法,其特征在于,所述步骤S1中,2-氯苯甲酰氯与氨水的摩尔比为1:(1-10);
所述步骤S2中,对硝基苯酚、4-溴丁酸叔丁酯与碳酸钾的摩尔比为1:(1.2-2):(1.5-5)。
4.根据权利要求2的制备灭幼脲半抗原的方法,其特征在于,所述步骤S4中,第一中间体与草酰氯、第三中间体的摩尔比为1:(1-1.2):(1-1.2)。
5.灭幼脲抗原,其特征在于,灭幼脲抗原包含权利要求1所述的灭幼脲半抗原以及与所述灭幼脲半抗原偶联的载体蛋白,所述载体蛋白为牛血清白蛋白、卵清蛋白、血蓝蛋白、甲状腺蛋白或人血清白蛋白中的一种。
6.灭幼脲抗体,其特征在于:所述幼脲抗体是由灭幼脲抗原经动物免疫制备得到,所述灭幼脲抗体为灭幼脲单克隆抗体。
7.权利要求1所述的灭幼脲半抗原、权利要求5所述的灭幼脲抗原、权利要求6所述的灭幼脲抗体在灭幼脲的免疫学检测中的应用。
8.灭幼脲胶体金层析检测装置,所述检测装置包括试纸条和微孔反应杯,所述试纸条包括反应膜、样品吸收垫、吸水垫和底板,所述反应膜设有检测线和质控线,所述检测线包被有权利要求5所述的灭幼脲抗原,所述质控线包被有羊抗鼠抗体,所述微孔反应杯中含有胶体金标记的权利要求6所述的灭幼脲抗体。
9.一种检测样品中灭幼脲残留的方法,其特征在于,所述方法为使用权利要求8所述的灭幼脲胶体金层析检测装置对样品中的灭幼脲进行检测,所述方法包括以下步骤:
1)样品前处理;
2)用权利要求8所述的灭幼脲胶体金层析检测装置进行检测;
3)分析检测结果。
10.根据权利要求9所述的一种检测样品中灭幼脲残留的方法,所述样品为蔬菜或水果。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311589425.6A CN117603095A (zh) | 2023-11-24 | 2023-11-24 | 灭幼脲半抗原、抗原、抗体、检测装置及其制备和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311589425.6A CN117603095A (zh) | 2023-11-24 | 2023-11-24 | 灭幼脲半抗原、抗原、抗体、检测装置及其制备和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117603095A true CN117603095A (zh) | 2024-02-27 |
Family
ID=89954386
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311589425.6A Pending CN117603095A (zh) | 2023-11-24 | 2023-11-24 | 灭幼脲半抗原、抗原、抗体、检测装置及其制备和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117603095A (zh) |
-
2023
- 2023-11-24 CN CN202311589425.6A patent/CN117603095A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN101413955B (zh) | 一种检测玉米赤霉烯酮的elisa测试盒及制备及检测方法 | |
CN108508215A (zh) | 一种检测四环素类药物的时间分辨荧光免疫层析试纸条及其制备方法和应用 | |
CN111239399B (zh) | 一种检测丙溴磷的试纸条及方法 | |
CN108776219B (zh) | 一种快速检测细交链格孢酮酸的免疫层析试纸条 | |
CN110441512B (zh) | 一种乙基麦芽酚半抗原以及乙基麦芽酚的胶体金免疫层析检测装置 | |
CN106831498B (zh) | 呋喃西林代谢物sem衍生化半抗原、人工抗原的制备方法及其应用 | |
CN106918705B (zh) | 检测甲氰菊酯的试纸及其应用 | |
CN112067811B (zh) | 一种检测互隔交链孢霉素的试纸条及其应用 | |
CN117603095A (zh) | 灭幼脲半抗原、抗原、抗体、检测装置及其制备和应用 | |
CN113156125B (zh) | 一种检测米酵菌酸的试纸条及方法 | |
CN113267622B (zh) | 一种检测氯羟吡啶的试纸条及方法 | |
CN111751535B (zh) | 一种检测硫丹的试纸条及其应用 | |
CN108469517A (zh) | 一种检测敌百虫的时间分辨荧光试纸条及其应用 | |
CN110441517B (zh) | 一种检测6-苄基腺嘌呤的试纸条及方法 | |
CN211453649U (zh) | 基于量子点荧光微球的免疫层析试纸条 | |
CN114685387B (zh) | 粉唑醇半抗原、抗原、抗体、检测装置及其制备和应用 | |
CN109856091B (zh) | 一种检测久效磷的时间分辨荧光试纸条及其应用 | |
CN111253283A (zh) | 一种金刚烷胺半抗原、抗原及其化学发光酶联免疫检测试剂盒与应用 | |
CN111961010A (zh) | 一种糖精钠半抗原Ri、人工抗原、抗体及其制备方法和应用 | |
CN116120242B (zh) | 茶叶中喹螨醚快速检测装置及其制备和应用 | |
CN111289752A (zh) | 一种检测三氯杀螨醇的试纸条及方法 | |
CN111965359B (zh) | 一种检测溴虫腈的试纸条及方法 | |
CN115322089B (zh) | 一种覆盆子酮半抗原、人工抗原及其制备方法、抗体和应用 | |
CN116008554B (zh) | 一种检测兽药奥芬达唑的试纸条及方法 | |
CN116375606B (zh) | 降糖类药品和保健品中苯乙双胍的快速检测装置及其制备和应用 |
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 |