CN106084059B - The general specific antibody of anti-Capsaicinoids, test strips and kitchen waste grease immunochromatography method for quick identification - Google Patents
The general specific antibody of anti-Capsaicinoids, test strips and kitchen waste grease immunochromatography method for quick identification Download PDFInfo
- Publication number
- CN106084059B CN106084059B CN201610361935.1A CN201610361935A CN106084059B CN 106084059 B CN106084059 B CN 106084059B CN 201610361935 A CN201610361935 A CN 201610361935A CN 106084059 B CN106084059 B CN 106084059B
- Authority
- CN
- China
- Prior art keywords
- capsaicinoids
- pad
- kitchen waste
- sample
- waste grease
- 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
- 238000012360 testing method Methods 0.000 title claims abstract description 88
- 239000004519 grease Substances 0.000 title claims abstract description 70
- 239000010806 kitchen waste Substances 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000003317 immunochromatography Methods 0.000 title claims abstract description 18
- YKPUWZUDDOIDPM-SOFGYWHQSA-N capsaicin Chemical compound COC1=CC(CNC(=O)CCCC\C=C\C(C)C)=CC=C1O YKPUWZUDDOIDPM-SOFGYWHQSA-N 0.000 claims abstract description 116
- 238000001514 detection method Methods 0.000 claims abstract description 71
- 239000000427 antigen Substances 0.000 claims abstract description 52
- 102000036639 antigens Human genes 0.000 claims abstract description 52
- 108091007433 antigens Proteins 0.000 claims abstract description 52
- 150000001875 compounds Chemical class 0.000 claims abstract description 14
- 230000036039 immunity Effects 0.000 claims abstract description 11
- 241001465754 Metazoa Species 0.000 claims abstract description 6
- 239000000523 sample Substances 0.000 claims description 70
- 239000007788 liquid Substances 0.000 claims description 41
- 239000000243 solution Substances 0.000 claims description 39
- 229960002504 capsaicin Drugs 0.000 claims description 38
- 239000000123 paper Substances 0.000 claims description 37
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 31
- 239000011248 coating agent Substances 0.000 claims description 29
- 238000000576 coating method Methods 0.000 claims description 29
- 238000002360 preparation method Methods 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 239000010931 gold Substances 0.000 claims description 26
- XJQPQKLURWNAAH-UHFFFAOYSA-N dihydrocapsaicin Chemical compound COC1=CC(CNC(=O)CCCCCCC(C)C)=CC=C1O XJQPQKLURWNAAH-UHFFFAOYSA-N 0.000 claims description 24
- RBCYRZPENADQGZ-UHFFFAOYSA-N dihydrocapsaicin Natural products COC1=CC(COC(=O)CCCCCCC(C)C)=CC=C1O RBCYRZPENADQGZ-UHFFFAOYSA-N 0.000 claims description 24
- 229910052737 gold Inorganic materials 0.000 claims description 21
- 229920001220 nitrocellulos Polymers 0.000 claims description 20
- RGOVYLWUIBMPGK-UHFFFAOYSA-N nonivamide Chemical compound CCCCCCCCC(=O)NCC1=CC=C(O)C(OC)=C1 RGOVYLWUIBMPGK-UHFFFAOYSA-N 0.000 claims description 20
- 239000000020 Nitrocellulose Substances 0.000 claims description 19
- 238000010521 absorption reaction Methods 0.000 claims description 19
- 239000000872 buffer Substances 0.000 claims description 19
- 239000000126 substance Substances 0.000 claims description 17
- 239000003365 glass fiber Substances 0.000 claims description 16
- 239000012528 membrane Substances 0.000 claims description 16
- 239000002671 adjuvant Substances 0.000 claims description 14
- 239000012488 sample solution Substances 0.000 claims description 14
- 210000004369 blood Anatomy 0.000 claims description 13
- 239000008280 blood Substances 0.000 claims description 13
- 239000007921 spray Substances 0.000 claims description 12
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 11
- 238000004587 chromatography analysis Methods 0.000 claims description 10
- 238000002965 ELISA Methods 0.000 claims description 9
- 238000002649 immunization Methods 0.000 claims description 8
- 239000006193 liquid solution Substances 0.000 claims description 7
- 238000005507 spraying Methods 0.000 claims description 7
- 230000003053 immunization Effects 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 239000013642 negative control Substances 0.000 claims description 6
- 238000004321 preservation Methods 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 210000002966 serum Anatomy 0.000 claims description 5
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 238000004945 emulsification Methods 0.000 claims description 4
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 4
- 230000002163 immunogen Effects 0.000 claims description 4
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 4
- 240000007594 Oryza sativa Species 0.000 claims description 3
- 235000007164 Oryza sativa Nutrition 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000012850 discrimination method Methods 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 235000009566 rice Nutrition 0.000 claims description 3
- 238000007920 subcutaneous administration Methods 0.000 claims description 3
- 240000007711 Peperomia pellucida Species 0.000 claims description 2
- GEWYFWXMYWWLHW-UHFFFAOYSA-N azanium;octanoate Chemical compound [NH4+].CCCCCCCC([O-])=O GEWYFWXMYWWLHW-UHFFFAOYSA-N 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims description 2
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 claims 6
- 241000283977 Oryctolagus Species 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 230000006978 adaptation Effects 0.000 abstract description 5
- 239000012086 standard solution Substances 0.000 abstract description 4
- 239000013641 positive control Substances 0.000 abstract description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 24
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 24
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 22
- 239000003921 oil Substances 0.000 description 19
- 235000019198 oils Nutrition 0.000 description 19
- 239000006228 supernatant Substances 0.000 description 17
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 13
- 238000003756 stirring Methods 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 12
- 239000011780 sodium chloride Substances 0.000 description 12
- 239000001390 capsicum minimum Substances 0.000 description 11
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 11
- 239000001103 potassium chloride Substances 0.000 description 11
- 235000011164 potassium chloride Nutrition 0.000 description 11
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 10
- 235000002566 Capsicum Nutrition 0.000 description 9
- 241000208293 Capsicum Species 0.000 description 9
- 239000007864 aqueous solution Substances 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 8
- 235000019796 monopotassium phosphate Nutrition 0.000 description 8
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 8
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 7
- 230000001376 precipitating effect Effects 0.000 description 7
- 235000008534 Capsicum annuum var annuum Nutrition 0.000 description 6
- 240000008384 Capsicum annuum var. annuum Species 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000000502 dialysis Methods 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 description 6
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 6
- 102000014914 Carrier Proteins Human genes 0.000 description 5
- 108010078791 Carrier Proteins Proteins 0.000 description 5
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 5
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 5
- 229930006000 Sucrose Natural products 0.000 description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000003960 organic solvent Substances 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 5
- 239000011591 potassium Substances 0.000 description 5
- 229910052700 potassium Inorganic materials 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 125000000440 benzylamino group Chemical group [H]N(*)C([H])([H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000008157 edible vegetable oil Substances 0.000 description 4
- 238000005374 membrane filtration Methods 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- PUDMGOSXPCMUJZ-UHFFFAOYSA-N (4-hydroxy-3-methoxyphenyl)methylazanium;chloride Chemical compound Cl.COC1=CC(CN)=CC=C1O PUDMGOSXPCMUJZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 235000019483 Peanut oil Nutrition 0.000 description 3
- 239000004698 Polyethylene Substances 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 3
- 239000004327 boric acid Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 3
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 description 3
- 239000000312 peanut oil Substances 0.000 description 3
- -1 polyethylene Polymers 0.000 description 3
- 229920000573 polyethylene Polymers 0.000 description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine Chemical compound CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 description 3
- WRPWWVNUCXQDQV-UHFFFAOYSA-N vanillylamine Chemical compound COC1=CC(CN)=CC=C1O WRPWWVNUCXQDQV-UHFFFAOYSA-N 0.000 description 3
- 229940053939 vanillylamine Drugs 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 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 2
- TZRXHJWUDPFEEY-UHFFFAOYSA-N Pentaerythritol Tetranitrate Chemical compound [O-][N+](=O)OCC(CO[N+]([O-])=O)(CO[N+]([O-])=O)CO[N+]([O-])=O TZRXHJWUDPFEEY-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 238000003760 magnetic stirring Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000000050 nutritive effect Effects 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 2
- 235000015112 vegetable and seed oil Nutrition 0.000 description 2
- 239000008158 vegetable oil Substances 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- CCVCIMGLFOOBCM-PLNGDYQASA-N COC1=C(C=CC(=C1)CNC(=O)/C=C\C(=O)O)O Chemical compound COC1=C(C=CC(=C1)CNC(=O)/C=C\C(=O)O)O CCVCIMGLFOOBCM-PLNGDYQASA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 231100000678 Mycotoxin Toxicity 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 1
- 235000019484 Rapeseed oil Nutrition 0.000 description 1
- XQFMGJYRWLQZEQ-UHFFFAOYSA-N S(=O)(=O)(O)O.N.C(CCCCCCC)(=O)O Chemical compound S(=O)(=O)(O)O.N.C(CCCCCCC)(=O)O XQFMGJYRWLQZEQ-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 235000019486 Sunflower oil Nutrition 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 244000290333 Vanilla fragrans Species 0.000 description 1
- 235000009499 Vanilla fragrans Nutrition 0.000 description 1
- 235000012036 Vanilla tahitensis Nutrition 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 235000017663 capsaicin Nutrition 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 235000013409 condiments Nutrition 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 239000002636 mycotoxin Substances 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 239000002600 sunflower oil Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
- G01N33/03—Edible oils or edible fats
-
- 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
- G01N33/558—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
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to the general specific antibody of anti-Capsaicinoids, test strips and kitchen waste grease immunochromatography method for quick identification.The anti-general specific antibody of Capsaicinoids, it is characterised in that: it is that animal is immunized in the general artificial immunity antigen compound of the Capsaicinoids as described in formula I, and the general specific antibody of anti-Capsaicinoids of acquisition is freeze-dried after isolating and purifying.Kitchen waste grease immunochromatography immunity test strip prepared therefrom can be used for the total amount that kitchen waste grease can be used for quickly detecting Capsaicinoids in kitchen waste grease；Detection sensitivity is high, pH wide adaptation range；Single step operation, it is simple and convenient.Capsaicinoids standard solution is not needed as positive control.
Description
Technical field
The present invention relates to the general specific antibody of anti-Capsaicinoids, test strips and kitchen waste grease immunochromatography are fast
Fast discrimination method belongs to immunochemical technique field.
Background technique
Gutter oil is one and refers to concept, is the general designation to all kinds of poor oils, generally comprises swill oil, frying waste oil, food
The waste grease that product and relevant enterprise generate, and the swill oil from catering trade is one of primary raw material source of gutter oil.?
In China's cooking culture, the capsicum containing capsaicine is one of essential condiment.Capsaicinoid compounds be present in it is peppery
The dominant chemical for making it have pungent stimulation in green pepper fruit, that has verified structure at present has more than 20 kinds.Capsaicine
Substance nutritive value is high, is widely used and food processing field.Synthetic capsaicin also has and natural capsicum element substance phase
As structure, also have pungent feature, be a member in Capsaicinoids, be mainly used for industrial circle, but because of its preparation cost
Low, some criminals are substituted natural capsicum element, are applied to field of food.Capsaicine, Dihydrocapsaicin and synthesis are peppery
Green pepper element is the Major Members in Capsaicinoids.Capsaicinoids nutritive value is high, is widely used and leads with food processing
Domain.Capsaicine element substance have the characteristics that it is fat-soluble it is strong, stability is good, boiling point is high.Current gutter oil processing technology is difficult
Completely removing has the characteristics that such substance.Multiple studies have shown that normal edible oil is substantially free of capsaicine, and contacted capsicum
Kitchen waste grease be difficult to avoid that containing this constituents.Therefore capsicum alkali components, which can be used as, identifies kitchen waste grease
Characteristic index can differentiate kitchen abandoned oil whether is impregnated in edible vegetable oil by detecting the content of Capsaicinoids
Rouge.Currently, the method for Capsaicinoids mainly has high performance liquid chromatography, chromatograph-mass spectrometer coupling in detection edible vegetable oil
Equal modern instruments detection method, high sensitivity, accuracy are good, but instrument and equipment is expensive, require height to experimental situation, and want
Ask professional operator, it is difficult to realize quickly detection.The immunoassay method developed rapidly in recent years due to overcoming on
The shortcomings that stating method, and have high specificity, high sensitivity, analysis capacity it is big, it is convenient and efficient, low in cost, be suitable for scene batch
The advantages that amount detection, it is widely used in the every field such as medicine, agronomy.
Immunochromatography technique based on colloidal gold labeled monoclonal antibody and antigentic specificity association reaction is due to its testing result meat
For eye as it can be seen that not needing large-scale instrument and equipment, testing cost is low, and analysis time is short, micro- in mycotoxin, pesticide residue etc. in recent years
It measures and is widely applied in qualitative, online, the quick detection of residue.This method is answered however, not having also both at home and abroad at present
In detection for kitchen waste grease Capsaicinoids.The core element of colloidal gold immunochromatographimethod technology is the anti-of specificity
Body, and kitchen waste grease matrix components are complicated, the pH of sample extracting solution is difficult to determine, it will usually drop specific antibody activity
Low, detection sensitivity reduces.Therefore, there is an urgent need to develop, affinity is high, Idiotype is good and organic solvent-resistant, pH adaptation range
Wide anti-Capsaicinoids specific antibody, and colloidal gold immuno-chromatography test paper strip detection method is established using the antibody, it is used for
Identify kitchen waste grease.
Summary of the invention
The problem to be solved by the invention is to provide a kind of anti-Capsaicinoids specific antibodies, kitchen waste grease glue
Body gold immuno-chromatographic test paper strip, kitchen waste grease immunochromatography method for quick identification.Anti- capsaicine class object provided by the invention
Matter specific antibody affinity height, specific good and organic solvent-resistant, pH wide adaptation range.The immunity-chromatography test made of it
Detection of the paper slip for Capsaicinoids in kitchen waste grease has easy to operate, low in cost, high sensitivity, pH suitable
Answer the feature that range is wide.
In order to solve the above technical problems, the technical solution adopted by the present invention are as follows:
The general artificial immunity antigen compound of Capsaicinoids is provided, structural formula is as described in formula I:
The preparation method of the general artificial immunity antigen compound of above-mentioned Capsaicinoids is provided, including is walked in detail below
It is rapid: with the hydrochloric acid in sodium hydroxide and in Vanillylamine hydrochloride, III compound of formula is obtained, it is then anti-with maleic anhydride
It answers, obtains artificial semiantigen compound, Capsaicinoids artificial semiantigen is passed through into N, N '-dicyclohexylcarbodiimide DCC
Activate with n-hydroxysuccinimide NHS, or activated by tri-n-butylamine and isobutyl chlorocarbonate, then with carrier protein BSA into
Row coupling obtains.
The synthetic route of artificial semiantigen compound is as follows:
The preparation method of the above-mentioned general artificial immunity antigen compound of Capsaicinoids specifically includes the following steps:
(1) by molar ratio be 1:(0.7~1.1) Vanillylamine hydrochloride and sodium hydroxide it is soluble in water, be stirred at room temperature
10-30min obtains white precipitate Vanillylamine (IV compound of formula) after filtering；
Thoroughly dry Vanillylamine and maleic anhydride are dissolved in chloroform with molar ratio for 1:0.8-1.2,
1-2h is reacted at room temperature, up to Capsaicinoids artificial semiantigen after filtering.
(2) by the general artificial semiantigen of Capsaicinoids in N,N-dimethylformamide with n-hydroxysuccinimide
(NHS) 30min-2h is reacted at room temperature, the n,N-Dimethylformamide solution of dicyclohexylcarbodiimide, room temperature reaction is then added
It 4-6 hours, stands overnight, capsaicine, Dihydrocapsaicin and the synthetic capsaicin artificial semiantigen solution for taking supernatant to activate,
The molar ratio of the n-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide is 1:1-1.2；
Or the general artificial semiantigen of Capsaicinoids is dissolved in N,N-dimethylformamide, three positive fourths are sequentially added
Amine and isobutyl chlorocarbonate react at room temperature 0.5-3h hours, and gained reaction solution is the Capsaicinoids artificial semiantigen activated
The molar ratio of solution, the tri-n-butylamine and isobutyl chlorocarbonate is 1:1-1.2.
(3) the general artificial semiantigen supernatant of Capsaicinoids activated obtained by above-mentioned (1) is added drop-wise to carrier protein
In the phosphate buffer of carrier protein of the concentration more than or equal to 2mg/mL, room temperature condition reacts 4-8h, in which: the step
(1) molar ratio of the general artificial semiantigen of Capsaicinoids and carrier protein in is greater than 10:1, and then dialysis obtains capsicum
Plain artificial antigen.
According to the above scheme, the phosphate buffer of the carrier protein is by the phosphoric acid of carrier protein 0.2M pH8.0
What salt buffer dissolved.
According to the above scheme, the dialysis is the 72h that dialysed with 0.01M PBS buffer solution, during which, the primary dialysis of 4-8h replacement
Liquid.
The anti-general specific antibody of Capsaicinoids is the general artificial immunity of the Capsaicinoids as described in formula I
Animal is immunized in antigen compound, and the general specific antibody of anti-Capsaicinoids of acquisition is freeze-dried after isolating and purifying；
According to the above scheme, described to be immunized to be repeated using being spaced after Freund's complete adjuvant or incomplete Freund's adjuvant emulsification
Immune animal, in which: initial immunity Freund's complete adjuvant, follow-up immunization incomplete Freund's adjuvant, until antiserum titre reaches
To 10000 or more, arteria carotis is taken a blood sample to its death, after the centrifugal blood adopted, antiserum is purified to obtain Capsaicinoids
Antibody.The antiserum titre obtained with antigen-immunized animal provided by the invention up to 320000, obtained antibody to capsaicine,
The 503nhibiting concentration IC of Dihydrocapsaicin and synthetic capsaicin50It is 0.27-0.41 μ g/ under conditions of pH value is 5.0-8.0
mL.Show the antibody can specificity be reacted with capsaicine, Dihydrocapsaicin and synthetic capsaicin, and can be used as capsaicine,
Dihydrocapsaicin and synthetic capsaicin universal antibody, affinity is good, high sensitivity, and stability is good, including tolerance organic solvent and
Salt ionic concentration ability is strong, pH is applied widely.
According to the above scheme, the immune dosage of the general artificial immunity antigen compound of Capsaicinoids described in the formula I
0.2-1.0mg is calculated as with OVA albumen therein；The immune number of plies is 4-6 times；First immunisation uses isometric Freund's complete adjuvant
Emulsified immunogen, the multi-point injection under rabbit；It is spaced after first immunisation and carries out within 2-4 weeks being immunized for second, it is for the second time and later
Immunization interval 2-3 weeks, by isometric incomplete Freund's adjuvant emulsified immunogen, in the subcutaneous multi-point injection of rabbit back.From the 4th
Secondary beginning is immunized every time after a week in 6-10 days, and the non-competing indirect ELISA method of rabbit ear edge vein exploitating blood measures serum titer,
After potency reaches 10000, i.e., rabbit arteria carotis is taken a blood sample to its death, and after the centrifugal blood adopted, antiserum is utilized octanoic acid-sulfuric acid
Ammonium method is purified, and Capsaicinoids antibody is obtained.
Kitchen waste grease immuno-chromatographic test paper strip (see Fig. 3), including cardboard, the one side of cardboard are successively pasted from top to bottom
Water absorption pad, detecting pad, gold-labelled pad and sample pad, adjacent each pad is in the overlapping connection in junction, and the detecting pad is with nitrocellulose
Film is base wad, and lateral nature controlling line is arranged from top to bottom on nitrocellulose filter and detection line, the nature controlling line are coated with rabbit-anti
Mouse polyclonal antibody is coated with Capsaicinoids envelope antigen in the detection line；The gold-labelled pad is laterally coated with nanometer
The above-mentioned anti-general specific antibody of Capsaicinoids of gold label.
According to the above scheme, the long 10~15mm of the water absorption pad, wide 3~4mm, detecting pad long 25~30mm, wide 3~4mm；
Gold-labelled pad grows 6~9mm, wide 3~4mm；Sample pad grows 12~15mm, wide 3~4mm, and the adjacent overlapping length respectively padded is 1~3mm.
According to the above scheme, the water absorption pad is blotting paper.
According to the above scheme, the spacing on edge is 8~20mm on the detection line on the detecting pad and nitrocellulose filter, described
Detection line and the spacing of nature controlling line are 5~10mm.
The molecular structural formula of the Capsaicinoids envelope antigen is as shown in formula II:
According to the above scheme, in the detecting pad detection line it is per cm required for envelope antigen package amount be 0.1~0.6
μg；On nature controlling line it is per cm required for the package amount of rabbit-anti mouse polyclonal antibody be 0.1~0.6 μ g.
According to the above scheme, the partial size of nanogold used in the gold-labelled pad is 15~20nm.
According to the above scheme, in the gold-labelled pad it is per cm spraying length needed for nano gold mark anti-Capsaicinoids
The dosage of general specific antibody is 0.40~0.98 μ g.
The preparation method of the colloidal gold immuno-chromatography test paper strip of Capsaicinoids is quickly detected as described above, including following
Step:
(1) preparation of water absorption pad
Blotting paper is cut out up to water absorption pad；
(2) preparation of detecting pad
The coating of detection line: Capsaicinoids envelope antigen is configured to the coating that concentration is 0.17~1.0mg/mL
Its transverse direction is coated on nitrocellulose filter along the position of 8~20mm on away from nitrocellulose filter with line spray mode by liquid,
Detection line is obtained, the package amount of the general artificial complete antigen-envelope antigen of Capsaicinoids needed for detection line per cm is 0.1
~0.6 μ g is then 30~60 minutes dry under the conditions of 37 DEG C；
The coating of nature controlling line: by rabbit-anti mouse polyclonal antibody be made into concentration be 0.17~1.0mg/mL coating buffer, in away from
Its transverse direction is coated on nitrocellulose filter with line spray mode, obtains nature controlling line, matter per cm by the position of 5~10mm of detection line
The package amount of rabbit-anti mouse polyclonal antibody needed for controlling line is 0.1~0.6 μ g, then 30~60 points dry under the conditions of 37 DEG C
Clock；
(3) preparation of sample pad
Glass fibre membrane is put into confining liquid and is soaked, is taken out, it is 6~12 hours dry under the conditions of 37~40 DEG C, obtain sample
Then product pad sets room temperature preservation in drier；
(4) preparation of gold-labelled pad
Glass fibre membrane is put into confining liquid and is soaked, is taken out, it is 6~12 hours dry under the conditions of 37~40 DEG C, in
On dry glass fibre membrane, the anti-capsicum of nano gold mark is laterally sprayed on the glass fibre membrane dried with spray mode
The plain general Anti-TNF-α liquid solution of substance, it is per cm spraying length needed for nano gold mark anti-Capsaicinoids it is general
The dosage of polyclonal antibody is 0.4~0.98ng, and then vacuum freeze drying 2~6 hours, set room temperature preservation in drier；
(5) assembling of test strips
Water absorption pad, detecting pad, gold-labelled pad and sample pad are successively pasted from top to bottom in the one side of cardboard, and adjacent each pad is even
The overlapping connection in place is connect, overlapping length is 1~3mm to get kitchen waste grease immune chromatography test paper.
According to the above scheme, Capsaicinoids envelope antigen ((2Z) -4- [(4- hydroxy-3-methoxy) benzyl amino] -4- carbonyl
Base -2- olefin(e) acid-OVA) coating buffer used in coating buffer are as follows: contain ovalbumin OVA 0.1g, nitrine in every 10mL
Change sodium 0.002g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g；
Coating buffer used in the rabbit-anti mouse polyclonal antibody coating buffer are as follows: contain bovine serum albumin(BSA) in every 10mL
0.1g, sodium azide 0.002g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, biphosphate
Potassium 0.002g；
According to the above scheme, contain in the every 100mL of confining liquid in the step (3) and step (4): oralbumin 1~
2g, 2~5g of sucrose, 0.02~0.05g of sodium azide, sodium chloride 0.8g, disodium hydrogen phosphate 0.29g, potassium chloride
0.02g, potassium dihydrogen phosphate 0.02g.
According to the above scheme, the general Anti-TNF-α liquid solution of the anti-Capsaicinoids of the nano gold mark is using insatiable hunger
It is prepared with labelling method, method particularly includes: taking 50.0mL commercial available quality concentration is 0.01% nano-Au solution, uses 0.5mL
0.1mol/L wet chemical adjust pH value, be slowly added to the anti-capsicum of the 0.1mg/mL of 2.0mL while stirring
The plain general polyclonal antibody aqueous solution of substance continues to stir 30min；It is 10% oralbumin (OVA) that mass concentration, which is added,
Aqueous solution to OVA end mass concentration is 1%, continues to stir 30min；After 4 DEG C of placement 2h, 3000r/min is centrifuged 15min, takes
Supernatant abandons precipitating；Supernatant 12000r/min is centrifuged 30min, discards supernatant liquid, 40.0mL label washing is added and saves
Liquid；30min is centrifuged with 12000r/min again, discards supernatant liquid, precipitating is saved into liquid with label washing and is resuspended, it is dense to obtain 5.0mL
It is spare to set 4 DEG C of refrigerators for contracting object.
According to the above scheme, the 0.1mol/L wet chemical are as follows: 13.8g potassium carbonate is dissolved in pure water and is settled to
1000mL, 0.22 μm of membrane filtration gained；The label washing saves liquid are as follows: 2.0g polyethylene glycol-20000,0.2g nitrine
Sodium, 0.1235g boric acid, pure water are settled to 1000mL, 0.22 μm of membrane filtration gained.
Kitchen waste grease immunochromatography method for quick identification, steps are as follows: pick-up kitchen waste grease sample to be tested, extracts
Capsaicinoids therein redissolve solubilizer after extracting solution drying, and the ratio of the sample to be tested and redissolution solvent is
20g:1ml obtains testing sample solution, then the testing sample solution (preferably 80-200 μ L) is added dropwise to meal as detection liquid
It is detected in the sample pad of kitchen waste grease immune chromatography test paper, is used as test strip, separately takes isometric methanol dense
Consistent methanol aqueous solution is spent as negative controls, and the sample pad of another kitchen waste grease immune chromatography test paper is added dropwise
On, it is used as control stripes item, test strips is will test after a period of time (10-20 minutes) and control stripes item carries out colour developing pair
According to:
Testing result:
(1) negative: when nature controlling line colour developing in test strip, and to detect detection line on line color and control stripes item
When color is close, show that Capsaicinoids capsaicine in testing sample solution, Dihydrocapsaicin or synthetic capsaicin content are low
In 3ng/mL, determine whether the sample is kitchen waste grease or mixed with the oil of kitchen waste grease in conjunction with authenticity detection method
Rouge sample；Content is lower than 3ng/mL, can determine whether that a possibility that sample to be tested is kitchen waste grease is little, need to be further with confirmation
Property detection method is determined；
(2) positive: when nature controlling line colour developing in test strip, and to detect line color than detection line in control test strips
When of light color, show that capsaicine in testing sample solution, Dihydrocapsaicin or synthetic capsaicin content are equal to or higher than 3ng/mL,
It then can determine that the sample is kitchen waste grease or the oil sample mixed with kitchen waste grease；
(3) invalid: when nature controlling line does not develop the color, no matter whether the detection line of test strip develops the color, which is judged to
In vain；
Work of the immuno-chromatographic test paper strip in the detection of kitchen waste grease capsaicine, Dihydrocapsaicin or synthetic capsaicin
Make principle: when testing sample solution is added in the sample pad of test strips lower end, testing sample solution edge through capillary action
Test strips are mobile to water absorption pad direction, when it is moved to gold-labelled pad, anti-capsaicine, Dihydrocapsaicin or the conjunction of nano gold mark
It is dissolved at the general polyclonal antibody of capsaicine.When containing capsaicine, Dihydrocapsaicin or synthetic capsaicin in sample, capsicum
Plain substance will combine and upward together with the general polyclonal antibody of the anti-Capsaicinoids of the nano gold mark in gold-labelled pad
Swimming, when reaching the detection line of fixed Capsaicinoids envelope antigen, antigen will be with capsaicine, Dihydrocapsaicin or conjunction
At limited antigen binding site on the general polyclonal antibody of anti-Capsaicinoids of capsaicine competitive binding nano gold mark,
Capsaicine, Dihydrocapsaicin or synthetic capsaicin content are higher in sample, the nanogold mark that the antigen in detection line can combine
Anti- capsaicine, Dihydrocapsaicin or the general polyclonal antibody of synthetic capsaicin of note will be fewer, the colour developing formed in detection line
Band color is more shallow.When the general polyclonal antibody of anti-Capsaicinoids that the antigen in detection line is combined is less than certain quantity
When, there will not be at detection line red lines to occur.No matter whether Capsaicinoids are contained in sample, on not tested survey line
Antigen intercept and capture nano gold mark anti-Capsaicinoids antibody or nano gold mark anti-Capsaicinoids antibody with it is peppery
Green pepper element, Dihydrocapsaicin or synthetic capsaicin conjugate will move on nature controlling line and with more grams of rabbit-anti mouse on nature controlling line
Grand antibody is combined and is developed the color by enrichment.Accordingly, will test respectively in test strips the detection line of Capsaicinoids envelope antigen with
Line color is accordingly detected on control stripes item and carries out colour developing control, and it is peppery to can get capsaicine in sample, Dihydrocapsaicin or synthesis
Green pepper cellulose content situation, to judge whether oil sample is kitchen waste grease.
According to the above scheme, the addition volumetric concentration into sample to be tested that is extracted as is 95% ethanol water, is mixed
It is even, it flows back 1 hour at 60~90 DEG C, cooling obtains extracting solution；The redissolution solvent is 10% methanol-PBS.
Beneficial effects of the present invention:
(1) present invention was both maximum by the way that the amide groups of vanillyl amine in capsaicine is carried out the artificial semiantigen that modification provides
Degree remains the feature structure (vanilla in capsaicine essential molecular structure of capsaicine, Dihydrocapsaicin and synthetic capsaicin
Base amine and partial fat chain group), and a big conjugated structure is formed at haptens linking arm, which can promote half to resist
Electronics flows in original structure, in addition, it is with certain rigidity, thus may all be conducive to active site in haptens
Exposure, and then thus the antigen compound being coupled with OVA can be used as antigenic determinant, and having can be with carrier
The active group that albumen is coupled, the antigen compound of acquisition, which makees immunizing antigen, can be immunized high, the high sensitivity that obtains affinity,
The capsaicine antibody of high specificity and organic solvent-resistant and salt ionic concentration, pH wide adaptation range.
(2) kitchen waste grease immunochromatography method for quick identification provided by the invention can detecte in kitchen waste grease
The total amount of Capsaicinoids；Detection sensitivity is high, pH wide adaptation range.
(3) kitchen waste grease immunochromatography method for quick identification provided by the invention is easy to operate: only kitchen need to be given up
Abandon oil sample treated detection solution be added dropwise in the sample pad of test strips, single step operation, it is simple and convenient.Inspection
Capsaicinoids standard solution is not needed when sample as positive control, it is only necessary to use blank sample as negative control.
And the testing result naked eyes of this method can interpretation.
Detailed description of the invention
Fig. 1 is the general artificial complete antigen ultraviolet spectrogram of anti-Capsaicinoids that the present invention synthesizes.
Fig. 2 is the general artificial complete antigen of anti-Capsaicinoids-envelope antigen ultraviolet spectrogram that the present invention synthesizes.
Fig. 3 is the structural schematic diagram of kitchen waste grease immuno-chromatographic test paper strip provided by the invention.1 cardboard；2 water suctions
Pad；5 detecting pads；6 gold-labelled pads；7 sample pads；3 nature controlling lines；4 detection lines.
Specific embodiment
Test method used in following examples is unless otherwise specified conventional method.
Material used in following examples, reagent etc., are commercially available unless otherwise specified.
The preparation of 1 Capsaicinoids artificial semiantigen of embodiment
(1) it weighs Vanillylamine hydrochloride 1g and is dissolved in 11.77ml ultrapure water, be vigorously stirred down and be added dropwise 2M's dropwise
NaOH2.12ml reacts at room temperature 10min.White precipitate is filtered to obtain, carries out the next step after white precipitate is thoroughly dried.
(2) it weighs step product 60mg to be dissolved in 30ml chloroform (after anhydrous sodium sulfate dehydration), room
Temperature stirs 40min, and 40mg maleic anhydride is added, and reacts at room temperature 1h.Gained precipitating is that Capsaicinoids are artificial after filtering
Haptens (2Z) -4- [(4- hydroxy-3-methoxy) benzyl amino] -4- carbonyl -2- olefin(e) acid ((2Z) -4- [(4-hydroxy-3-
methoxybenzyl)amino]-4-oxobut-2-enoic acid).Molecular formula is C12H13NO5。
The preparation of 2 Capsaicinoids artificial complete antigen of embodiment-immunizing antigen
Weigh above-mentioned Capsaicinoids artificial semiantigen 20.8mg (about 0.08mmol) and 11.6mg (about 0.1mmol)
NHS is added 400ulDMF and is dissolved in reaction flask, 30min is stirred at room temperature, weighs 20.6mg (about 0.1mmol) in reaction flask
DCC is dissolved in 50ulDMF, and DCC/DMF solution is added dropwise to above-mentioned reaction flask dropwise, and 4h is stirred at room temperature, and 4 DEG C stand overnight.
8000rpm/5min takes the active ester liquid of supernatant.
By the active ester liquid of supernatant, it is added drop-wise in the BSA solution of 6ml 7mg/ml and reacts dropwise, reaction buffer 0.2M
The phosphate buffer of pH8.0.Room temperature carries out 4h under magnetic stirring for reaction.Reaction solution is set in bag filter, 0.01M is used
4 DEG C of stirring dialysis, every 4h replace a dialyzate in the PBS of pH7.4, and dialyse 72h altogether.Capsaicine artificial antigen-is obtained to exempt from
Epidemic disease antigen.Ultraviolet-visible spectrum continuous scanning map is shown in that Fig. 1, qualification result show that artificial antigen is coupled successfully.
The preparation of 3 Capsaicinoids artificial complete antigen of embodiment-envelope antigen
It deserves to be called and states Capsaicinoids artificial semiantigen 4.52mg and be dissolved in 200uL anhydrous DMF, be then sequentially added into
4.27 μ L tri-n-butylamines, 2.34 μ L isobutyl chlorocarbonates, stirring is protected from light 1h at room temperature, and capsaicine, the dihydro for obtaining activation are peppery
Green pepper element and synthetic capsaicin haptens solution.
The capsaicine of activation, Dihydrocapsaicin and synthetic capsaicin haptens solution are added drop-wise to 10ml 4.5mg/ dropwise
It is reacted in the OVA solution of ml, reaction buffer is the phosphate buffer of 0.2M pH8.0.React Indoor Temperature under magnetic stirring
Degree carries out 4h.Reaction solution is set in bag filter, with 4 DEG C of stirring dialysis in the PBS of 0.01M pH7.4, every primary dialysis of 4h replacement
Liquid, dialyse 72h altogether.Obtain capsaicine artificial antigen-envelope antigen.Ultraviolet-visible spectrum continuous scanning map is shown in Fig. 2, mirror
Determine the result shows that artificial antigen is coupled successfully.
Embodiment 4: the preparation of Capsaicinoids specific antibody
The above-mentioned anti-Capsaicinoids artificial complete antigen (immunizing antigen) prepared is used to be immunized the day of 3 ㎏ or more
This big ear rabbit.Average each antigen dosage about 0.84mg (with the meter of protein).First immunisation is used by isometric Freund
The immunogene of Freund's complete adjuvant emulsification, the multi-point injection under rabbit；Head be spaced after exempting from 3 weeks, later every 2 weeks with by it is isometric not
The immunogene of family name's Freund's incomplete adjuvant emulsification, in the subcutaneous multi-point injection of rabbit back.Since the 4th time, latter week, rabbit are immunized every time
The non-competing indirect ELISA method of ear edge vein exploitating blood measures serum titer.After five exempt from, rabbit arteria carotis is taken a blood sample to its death.It adopts
Blood in 37 DEG C of placements about 1h, 4 DEG C are overnight, and centrifugation takes supernatant, mixes -20 DEG C of isometric glycerol on a small quantity and temporarily store for future use.Its
Remaining antiserum is purified using caprylic acid-ammonium, is finally obtained lyophilized antibodies, is stored for future use in -20 DEG C.
Embodiment 5: the measurement of Capsaicinoids specific antibody
1) Capsaicinoids specific antibody titres are detected using non-competing ELISA method indirectly
Coating: Capsaicinoids artificial complete antigen-envelope antigen is dilute with 0.1mol/L pH9.6 carbonate buffer solution
It releases to 1 μ g/mL, 100 holes μ l/, 4 DEG C of reactions are overnight.Incline coating buffer, PBST expires hole and washs 3 times, and button is dry.
Closing: 1%OVA PBST solution, 200 holes μ L/, 37 DEG C of incubations 1h, PBST expire hole and wash 3 times, and button is dry.
Antibody antigen specific reaction: by the Capsaicinoids specific antibody phosphate buffer of 0.01M PH7.4
Be configured to the solution of 1mg/mL, then by antibody-solutions since 1:10000 doubling dilution, and be added to the coating of each dilution
Kong Zhong, additional amount are 100 holes μ L/, and setting is negative and blank control wells, negative control sera dilute 2 000 times, and blank only adds
PBST, 37 DEG C of incubations 1h, PBST expire hole and wash 3 times, and button is dry；
Add secondary antibody: with PBST solution by 5000 times of sheep anti mouse ELIAS secondary antibody dilute, mix, additional amount be 100 holes μ L/, 37
DEG C incubate 1h, PBST expire hole wash 6 times, button do.
Colour developing: developing solution (0.5mL of tetramethyl benzidine containing 1mg/mL, citrate buffer solution 9.5mL, 1% hydrogen peroxide
32 μ L of urea) ready-to-use, 100 holes μ L/, it is protected from light 37 DEG C of incubation 15min.
It terminates: the H2SO4 of 2mol/L, 50 holes μ L/, immediately with light absorption value, that is, OD450 value at microplate reader measurement 450nm.
As a result interpretation: the highest extension rate of corresponding antibody-solutions is antibody when using OD450 value more than or equal to 1
ELISA potency is 320000.
2) Capsaicinoids specific antibody 503nhibiting concentration is detected using indirect competitive ELISA method
(1) working concentration of antibody is determined, with above-mentioned indirect ELISA method with OD450It is corresponding when being 1 or so to resist
Bulk concentration is most suitable working concentration.
(2) be coated with, wash and close: method operation is the same as indirect non-competing ELISA method.
(3) it prepares capsaicine standard solution: capsaicine, Dihydrocapsaicin and synthetic capsaicin is configured to methanol solution
The mother liquor of 10mg/ml (wherein the ratio of Capsaicinoids is 1:1:1).Before sample-adding, using 10% methanol/PBS
The 5 times of dilutions since 20 μ g/mL of (0.01mol/L pH7.4) solution, dilute 5 concentration altogether.50 μ L doubling dilutions are added in every hole
Capsaicine, Dihydrocapsaicin and synthetic capsaicin standard solution, it is 160000 that then every hole, which adds 50 μ L extension rates,
Antiserum, 37 DEG C of reaction 1h.
(4) add secondary antibody, colour developing, termination and reading: method operation is the same as indirect non-competing ELISA method.
(5) data processing: using the logarithm of capsaicine, Dihydrocapsaicin and each concentration of synthetic capsaicin as abscissa, capsicum
Element, Dihydrocapsaicin and the corresponding OD value of each concentration of synthetic capsaicin are ordinate, draw standard curve, calculate 50% and inhibit dense
Degree is 0.3 μ g/mL.
Change methanol concentration in standard dilutions, after detecting according to the above method, draws standard curve, calculate the antibody and exist
Methanol concentration is 10%, and under the conditions of 20%, 40%, 50% inhibition concentration distinguishes 0.31 μ g/mL, 0.36 μ g/mL, 0.34 μ g/mL.
Show that antibody tolerance organic solvent ability is strong.
Change the salt ionic concentration value of antibody diluent, after detecting according to the above method, draws standard curve, calculate the antibody
Sodium chloride concentration be 0.01M, 0.16M, 0.32M, under the conditions of, 50% inhibition concentration distinguish 0.28 μ g/mL, 0.33 μ g/mL,
0.37μg/mL.Show that the antibody can be resistance to higher by salt ionic concentration.
Change the pH value of standard dilutions, after detecting according to the above method, draws standard curve, calculate the antibody and exist
Under the conditions of pH5.0/6.0/7.0/7.4/8.0,50% inhibition concentration distinguish 0.41 μ g/mL, 0.33 μ g/mL, 0.29 μ g/mL,
0.27μg/mL,0.28μg/mL.Show that the antibody pH tolerant range is wide.
Embodiment 6: the preparation method of kitchen waste grease immune chromatography test paper, steps are as follows:
(1) preparation of water absorption pad
Blotting paper is cut out into growth 15mm, the specification of wide 4mm is to get water absorption pad；
(2) preparation of detecting pad
The coating of detection line: by Capsaicinoids envelope antigen ((2Z) -4- [(4- hydroxy-3-methoxy) benzyl amino] -
4- carbonyl -2- olefin(e) acid-OVA) it is configured to the coating buffer that concentration is 0.4mg/mL, along the position of 9mm on away from nitrocellulose filter,
Its transverse direction is coated on nitrocellulose filter with line spray mode, obtains detection line, capsaicine class object needed for detection line per cm
The package amount of matter envelope antigen is 0.24 μ g, then 30 minutes dry under the conditions of 37 DEG C；
Envelope antigen ((2Z) -4- [(4- hydroxy-3-methoxy) benzyl the amino] -4- carbonyl -2- olefin(e) acid-OVA) packet
The coating buffer used in liquid are as follows: contain ovalbumin OVA 0.1g, sodium azide 0.002g, sodium chloride in every 10mL
0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g；
The coating of nature controlling line: rabbit-anti mouse polyclonal antibody is made into the coating buffer that concentration is 0.5mg/mL, in away from detection line
Its transverse direction is coated on nitrocellulose filter by the position of 10mm with line spray mode, obtains nature controlling line, needed for nature controlling line per cm
The package amount of rabbit-anti mouse polyclonal antibody is 0.3 μ g, then 30 minutes dry under the conditions of 37 DEG C；
Coating buffer used in the rabbit-anti mouse polyclonal antibody coating buffer are as follows: contain cow's serum in every 10mL
Albumin 0.1g, sodium azide 0.002g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, phosphorus
Acid dihydride potassium 0.002g；
The nitrocellulose filter long 28mm, wide 4mm
(3) preparation of sample pad
Glass fibre membrane is cut out into growth 15mm, the specification of wide 4mm is put into confining liquid and soaks, and takes out, in 37 DEG C of conditions
Lower drying 8 hours, obtains sample pad, then sets room temperature preservation in drier.
Contain in the every 100mL of the confining liquid: oralbumin 1g, sucrose 2g, sodium azide 0.02g, sodium chloride
0.8g, disodium hydrogen phosphate 0.29g, potassium chloride 0.02g, potassium dihydrogen phosphate 0.02g.
(4) preparation of gold-labelled pad
The specification that glass fibre membrane is cut out to growth 8mm wide 4mm, is put into confining liquid and soaks, and takes out, under the conditions of 37 DEG C
It is 8 hours dry, on the glass fibre membrane dried, nanometer is laterally sprayed on the glass fibre membrane dried with spray mode
Gold label the general Anti-TNF-α liquid solution of anti-Capsaicinoids, it is per cm spraying length needed for nano gold mark resist it is peppery
The dosage of the general polyclonal antibody of green pepper element substance is 0.5 μ g, then vacuum freeze drying 6 hours, sets room temperature in drier and protects
It deposits；
Contain in the every 100mL of the confining liquid: oralbumin 1g, sucrose 2g, sodium azide 0.02g, sodium chloride
0.8g, disodium hydrogen phosphate 0.29g, potassium chloride 0.02g, potassium dihydrogen phosphate 0.02g.
The general Anti-TNF-α liquid solution of the anti-Capsaicinoids of the nano gold mark is using unsaturated label legal system
Standby, method particularly includes: taking 50.0mL commercial available quality concentration is 0.01% nano-Au solution, with 0.5mL0.1mol/L carbon
Sour aqueous solutions of potassium adjusts pH value, and the anti-Capsaicinoids for being slowly added to 2.0mL0.1mg/mL while stirring are general more
Clonal antibody aqueous solution continues to stir 30min；Mass concentration is added as 10% oralbumin (OVA) aqueous solution to OVA end matter
Measuring concentration is 1%, continues to stir 30min；After 4 DEG C of placement 2h, 3000r/min is centrifuged 15min, takes supernatant, abandons precipitating；It will
Supernatant 12000r/min is centrifuged 30min, discards supernatant liquid, and 40.0mL label washing is added and saves liquid；Again with 12000r/min
It is centrifuged 30min, discards supernatant liquid, precipitating is saved into liquid with label washing and is resuspended, 5.0mL concentrate is obtained, it is standby to set 4 DEG C of refrigerators
With.
The partial size of nanogold is 15nm in the nano-Au solution；
The 0.1mol/L wet chemical are as follows: 13.8g potassium carbonate is dissolved in pure water and is settled to 1000mL, 0.22 μm of filter
Film filtering gained；The label washing saves liquid are as follows: 2.0g polyethylene glycol-20000,0.2g Sodium azide, and 0.1235g boric acid,
Pure water is settled to 1000mL, 0.22 μm of membrane filtration gained.
(5) assembling of test strips
Water absorption pad, detecting pad, gold-labelled pad and sample pad are successively pasted from top to bottom in the one side of cardboard, and adjacent each pad is even
The overlapping connection in place is connect, overlapping length is 1mm to get kitchen waste grease immune chromatography test paper.See Fig. 1.
Above-mentioned colloidal gold immuno-chromatography test paper strip is quickly identifying the application in kitchen waste grease
The blind sample 20g of 3 edible vegetable oils is weighed respectively, and pH value is respectively 5.3,5.8,6.4, number 1,2,3.It is added
The ethanol water that 50ml volumetric concentration is 95% is mixed, is flowed back 1 hour at 90 DEG C, after cooling, by extracting solution vacuum rotating
It is dry, 10% methanol of 1ml-PBS solution is added and redissolves, obtains testing sample solution, then 200 μ L testing sample solutions is taken to make
It is added dropwise in the sample pad for quickly identifying kitchen waste grease colloidal gold immuno-chromatography test paper strip and is detected for detection liquid,
It separately takes the consistent methanol-PBS solution of isometric methanol concentration as negative controls as test strip, dropwise plus
In the sample pad for entering another quick identification kitchen waste grease colloidal gold immuno-chromatography test paper strip, be used as control stripes item, 10
It will test test strips after minute and control stripes item carry out colour developing control, read result:
Testing result: the nature controlling line of three sample detection test strips shows red stripes, and detection line does not develop the color, and can sentence
For positive findings, show that the content of Capsaicinoids is above 3ng/ml, 3 samples are kitchen waste grease sample.Through
After the detection of capsaicine immune affinity column-tablets by HPLC-MS, as the result is shown in 1,2, No. 3 samples capsaicine with
Dihydrocapsaicin total amount is respectively 36,86,1213ng/mL, be can determine that as kitchen waste grease.The result with it is provided by the invention
Kitchen waste grease colloidal gold immuno-chromatography test paper strip method for quick identification determines that result is consistent.
The standard of perfection of kitchen waste grease sample is mainly based upon what following reason was made in the present invention: logical to a large amount of
The capsaicin content of actual sample (including multiple peanut oil, soybean oil, sunflower oil, the samples such as rapeseed oil and waste grease) into
Row detection, detection method: capsaicine immune affinity column liquid matter method, the results are shown in Table 1.The result shows that: the food in addition to peanut oil
With the detection for having no Capsaicinoids in vegetable oil, Capsaicinoids are lower than 1.53ug/kg (1.53ng/ in peanut oil
), ml and in abandoned oil (gutter oil) content of Capsaicinoids is higher than 12.27ug/kg (12.7ng/ml), thus by capsicum
Plain substance content is equal to or more than 3ng/ml as identification sample for kitchen waste grease or mixed with the oil of kitchen waste grease
The screening standard of rouge sample.
Capsaicinoid compounds content in 1 vegetable oil of table
Note: '-' is not detected.
Embodiment 7: the preparation method of kitchen waste grease immune chromatography test paper, steps are as follows:
(1) preparation of water absorption pad
Blotting paper is cut out into growth 16mm, the specification of wide 3.8mm is to get water absorption pad；
(2) preparation of detecting pad
The coating of detection line: by Capsaicinoids envelope antigen ((2Z) -4- [(4- hydroxy-3-methoxy) benzyl amino] -
4- carbonyl -2- olefin(e) acid-OVA) it is configured to the coating buffer that concentration is 0.2mg/mL, along the position of 10mm on away from nitrocellulose filter
It sets, its transverse direction is coated on nitrocellulose filter with line spray mode, obtains detection line, capsaicine class needed for detection line per cm
The package amount of substance envelope antigen is 0.12 μ g, then 30 minutes dry under the conditions of 37 DEG C；
Envelope antigen ((2Z) -4- [(4- hydroxy-3-methoxy) benzyl the amino] -4- carbonyl -2- olefin(e) acid-OVA) packet
The coating buffer used in liquid are as follows: contain ovalbumin OVA 0.1g, sodium azide 0.002g, sodium chloride in every 10mL
0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g；
The coating of nature controlling line: rabbit-anti mouse polyclonal antibody is made into the coating buffer that concentration is 0.2mg/mL, in away from detection line
Its transverse direction is coated on nitrocellulose filter by the position of 8mm with line spray mode, obtains nature controlling line, needed for nature controlling line per cm
The package amount of rabbit-anti mouse polyclonal antibody is 0.12 μ g, then 30 minutes dry under the conditions of 37 DEG C；
Coating buffer used in the rabbit-anti mouse polyclonal antibody coating buffer are as follows: contain cow's serum in every 10mL
Albumin 0.1g, sodium azide 0.002g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, phosphorus
Acid dihydride potassium 0.002g；
The nitrocellulose filter long 25mm, wide 3.8mm
(3) preparation of sample pad
Glass fibre membrane is cut out into growth 13mm, the specification of wide 3.8mm is put into confining liquid and soaks, and takes out, in 37 DEG C of items
It is 8 hours dry under part, sample pad is obtained, room temperature preservation in drier is then set.
Contain in the every 100mL of the confining liquid: oralbumin 1g, sucrose 2g, sodium azide 0.02g, sodium chloride
0.8g, disodium hydrogen phosphate 0.29g, potassium chloride 0.02g, potassium dihydrogen phosphate 0.02g.
(4) preparation of gold-labelled pad
The specification that glass fibre membrane is cut out to growth 9mm wide 3.8mm, is put into confining liquid and soaks, and takes out, in 37 DEG C of conditions
Lower drying 8 hours, on the glass fibre membrane dried, with spray mode, laterally spraying is received on the glass fibre membrane dried
The general Anti-TNF-α liquid solution of anti-Capsaicinoids of rice gold label, nano gold mark needed for spraying length per cm it is anti-
The dosage of the general polyclonal antibody of Capsaicinoids is 0.6 μ g, and then vacuum freeze drying 6 hours, set room temperature in drier
It saves；
Contain in the every 100mL of the confining liquid: oralbumin 1g, sucrose 2g, sodium azide 0.02g, sodium chloride
0.8g, disodium hydrogen phosphate 0.29g, potassium chloride 0.02g, potassium dihydrogen phosphate 0.02g.
The general Anti-TNF-α liquid solution of the anti-Capsaicinoids of the nano gold mark is using unsaturated label legal system
Standby, method particularly includes: taking 50.0mL commercial available quality concentration is 0.01% nano-Au solution, with 0.5mL0.1mol/L carbon
Sour aqueous solutions of potassium adjusts pH value, and the anti-Capsaicinoids for being slowly added to 2.0mL0.1mg/mL while stirring are general more
Clonal antibody aqueous solution continues to stir 30min；Mass concentration is added as 10% oralbumin (OVA) aqueous solution to OVA end matter
Measuring concentration is 1%, continues to stir 30min；After 4 DEG C of placement 2h, 3000r/min is centrifuged 15min, takes supernatant, abandons precipitating；It will
Supernatant 12000r/min is centrifuged 30min, discards supernatant liquid, and 40.0mL label washing is added and saves liquid；Again with 12000r/min
It is centrifuged 30min, discards supernatant liquid, precipitating is saved into liquid with label washing and is resuspended, 5.0mL concentrate is obtained, it is standby to set 4 DEG C of refrigerators
With.
The partial size of nanogold is 15nm in the nano-Au solution；
The 0.1mol/L wet chemical are as follows: 13.8g potassium carbonate is dissolved in pure water and is settled to 1000mL, 0.22 μm of filter
Film filtering gained；The label washing saves liquid are as follows: 2.0g polyethylene glycol-20000,0.2g Sodium azide, and 0.1235g boric acid,
Pure water is settled to 1000mL, 0.22 μm of membrane filtration gained.
(5) assembling of test strips
Water absorption pad, detecting pad, gold-labelled pad and sample pad are successively pasted from top to bottom in the one side of cardboard, and adjacent each pad is even
The overlapping connection in place is connect, overlapping length is 1mm to get kitchen waste grease immune chromatography test paper.See Fig. 1.
Above-mentioned colloidal gold immuno-chromatography test paper strip is quickly identifying the application in kitchen waste grease
Weigh the kitchen waste grease sample and original each 20g of edible vegetable blend oil sample of collection, pH value point
Not Wei 5.4 and 7.2, be separately added into 50ml volumetric concentration be 95% ethanol water, mix, flow back 1 hour at 90 DEG C, it is cold
But after, extracting solution vacuum rotating is dry, 10% methanol of 1ml-PBS solution is added and redissolves, obtains testing sample solution, then take
180 μ L testing sample solutions are added dropwise to the colloidal gold immunochromatographimethod examination of quickly detection Capsaicinoids as detection liquid
It is detected in the sample pad of paper slip, is used as test strip, separately takes the consistent methanol-PBS of isometric methanol concentration molten
Liquid is added dropwise in the sample pad of another kitchen waste grease immune chromatography test paper as negative controls, as control examination
Paper slip, will test test strips after ten minutes and control stripes item carries out colour developing control, read result:
Testing result: the nature controlling line of original edible vegetable blend oil sample detection test strips shows red bar
Band, detection line is close with the detection of blank control test strips limit color, then is judged to negative findings, thus determines: being not used
Edible vegetable blend oil sample in Capsaicinoids content be lower than 3ng/mL, show the ready-mixed oil not mixed with or mixed with few
The kitchen waste grease sample of amount.The nature controlling line of the kitchen waste grease sample detection test strips of collection shows red stripes,
And detection line does not develop the color, then is judged to positive findings, shows the content of Capsaicinoids in the kitchen waste grease sample collected
More than 3ng/mL.
Claims (10)
1. the anti-general specific antibody of Capsaicinoids, it is characterised in that: it is that the Capsaicinoids as described in formula I are general
Animal is immunized in artificial immunity antigen compound, and the general specificity of anti-Capsaicinoids of acquisition is freeze-dried after isolating and purifying
Antibody；
2. the general specific antibody of anti-Capsaicinoids according to claim 1, it is characterised in that: described being immunized be
Immune animal is repeated using interval after Freund's complete adjuvant or incomplete Freund's adjuvant emulsification, in which: initial immunity Freund is complete
Full adjuvant, follow-up immunization incomplete Freund's adjuvant, until antiserum titre reaches 10000 or more, arteria carotis is taken a blood sample to its death,
After the centrifugal blood adopted, antiserum is purified to obtain Capsaicinoids antibody.
3. the general specific antibody of anti-Capsaicinoids according to claim 1, it is characterised in that: described in the formula I
The immune dosage of the general artificial immunity antigen compound of Capsaicinoids 0.2-1.0mg is calculated as with BSA albumen therein；Exempt from
Epidemic disease number is 4-6 times；First immunisation uses isometric Freund's complete adjuvant emulsified immunogen, the multi-point injection under rabbit；It is first
It is secondary it is immune after be spaced carry out within 2-4 weeks second it is immune, for the second time and later immunization interval 2-3 weeks, not by isometric Freund
Freund's complete adjuvant emulsified immunogen, in the subcutaneous multi-point injection of rabbit back, since the 4th time, after being immunized every time in 6-10 days, rabbit ear
The non-competing indirect ELISA method of edge venous blood collection measures serum titer, and after potency reaches 10000, i.e. rabbit arteria carotis is taken a blood sample to it
Death after the centrifugal blood adopted, antiserum is purified using caprylic acid-ammonium, obtains Capsaicinoids antibody.
4. kitchen waste grease immuno-chromatographic test paper strip, it is characterised in that: including cardboard, the one side of cardboard is successively glued from top to bottom
Water absorption pad, detecting pad, gold-labelled pad and sample pad are pasted, adjacent each pad is in the overlapping connection in junction, and the detecting pad is with cellulose nitrate
Plain film is base wad, and lateral nature controlling line is arranged from top to bottom on nitrocellulose filter and detection line, the nature controlling line are coated with rabbit
Anti- mouse polyclonal antibody is coated with Capsaicinoids envelope antigen in the detection line；The gold-labelled pad, which is laterally coated with, to be received
The anti-general specific antibody of Capsaicinoids described in claim 1 of rice gold label.
5. kitchen waste grease immuno-chromatographic test paper strip according to claim 4, it is characterised in that: the water absorption pad is long
10~15mm, wide 3~4mm, detecting pad long 25~30mm, wide 3~4mm；Gold-labelled pad grows 6~9mm, wide 3~4mm；Sample pad is long
12~15mm, wide 3~4mm, the adjacent overlapping length respectively padded are 1~3mm；
The water absorption pad is blotting paper.
6. kitchen waste grease immuno-chromatographic test paper strip according to claim 4, it is characterised in that: on the detecting pad
The spacing on edge is 8~20mm on detection line and nitrocellulose filter, and the spacing of the detection line and nature controlling line is 5~10mm；Institute
The partial size for stating nanogold used in gold-labelled pad is 15~20nm.
7. kitchen waste grease immuno-chromatographic test paper strip according to claim 4, it is characterised in that: the capsaicine class
The molecular structural formula of substance envelope antigen is as shown in formula II:
In the detecting pad detection line it is per cm required for envelope antigen package amount be 0.1~0.6 μ g；Every li on nature controlling line
The package amount of rabbit-anti mouse polyclonal antibody required for rice is 0.1~0.6 μ g；
In the gold-labelled pad it is per cm spraying length needed for nano gold mark the general specific antibody of anti-Capsaicinoids
Dosage be 0.40~0.98 μ g.
8. the preparation method of kitchen waste grease immuno-chromatographic test paper strip as claimed in claim 4, it is characterised in that: including following
Step:
(1) preparation of water absorption pad
Blotting paper is cut out up to water absorption pad；
(2) preparation of detecting pad
The coating of detection line: being configured to the coating buffer that concentration is 0.17~1.0mg/mL for Capsaicinoids envelope antigen, in
Away from the position on nitrocellulose filter along 8~20mm, its transverse direction is coated on nitrocellulose filter with line spray mode, is examined
Survey line, the package amount of the general artificial complete antigen-envelope antigen of Capsaicinoids needed for detection line per cm are 0.1~0.6 μ
G is then 30~60 minutes dry under the conditions of 37 DEG C；
The coating of nature controlling line: rabbit-anti mouse polyclonal antibody is made into the coating buffer that concentration is 0.17~1.0mg/mL, in away from detection
Its transverse direction is coated on nitrocellulose filter with line spray mode, obtains nature controlling line, nature controlling line per cm by the position of 5~10mm of line
The package amount of required rabbit-anti mouse polyclonal antibody is 0.1~0.6 μ g, then 30~60 minutes dry under the conditions of 37 DEG C；
(3) preparation of sample pad
Glass fibre membrane is put into confining liquid and is soaked, is taken out, it is 6~12 hours dry under the conditions of 37~40 DEG C, sample pad is obtained,
Then room temperature preservation in drier is set；
(4) preparation of gold-labelled pad
Glass fibre membrane is put into confining liquid and is soaked, is taken out, dry 6~12 hours under the conditions of 37~40 DEG C, in having dried
Glass fibre membrane on, the anti-capsaicine class of nano gold mark is laterally sprayed on the glass fibre membrane dried with spray mode
The general Anti-TNF-α liquid solution of substance, general more grams of anti-Capsaicinoids of nano gold mark needed for spraying length per cm
The dosage of grand antibody is 0.4~0.98 μ g, and then vacuum freeze drying 2~6 hours, set room temperature preservation in drier；
(5) assembling of test strips
Water absorption pad, detecting pad, gold-labelled pad and sample pad are successively pasted from top to bottom in the one side of cardboard, and adjacent each pad is in junction
Overlapping connection, overlapping length are 1~3mm to get kitchen waste grease immune chromatography test paper.
9. kitchen waste grease immunochromatography discrimination method, it is characterised in that: steps are as follows: pick-up kitchen waste grease waits for test sample
Product extract Capsaicinoids therein, by extracting solution it is dry after solubilizer methanol-PBS solution redissolve, the sample to be tested and
The ratio of redissolution solvent is 20g:1ml, obtains testing sample solution, then be added dropwise using testing sample solution as detection liquid
It is detected on to the sample pad of kitchen waste grease immuno-chromatographic test paper strip as claimed in claim 4, is used as Test paper
Item separately takes the consistent methanol-PBS solution of isometric methanol concentration as negative controls, another claim 4 is added dropwise
In the sample pad of the kitchen waste grease immuno-chromatographic test paper strip, it is used as control stripes item, will test after a period of time
Test strips and control stripes item carry out colour developing control:
Testing result:
(1) negative: when nature controlling line colour developing in test strip, and to detect the color of detection line on line color and control stripes item
When close, show that Capsaicinoids capsaicine in testing sample solution, Dihydrocapsaicin or synthetic capsaicin content are lower than
3ng/mL determines whether the sample is kitchen waste grease or mixed with the grease of kitchen waste grease in conjunction with authenticity detection method
Sample；Content is lower than 3ng/mL, can determine whether that a possibility that sample to be tested is kitchen waste grease is little, need to further use authenticity
Detection method is determined；
(2) positive: when nature controlling line colour developing in test strip, and to detect color of the line color than detection line in control test strips
When shallow, show that capsaicine in testing sample solution, Dihydrocapsaicin or synthetic capsaicin content are equal to or higher than 3ng/mL, then may be used
Determine the sample for kitchen waste grease or mixed with the oil sample of kitchen waste grease；
(3) invalid: when nature controlling line does not develop the color, no matter whether the detection line of test strip develops the color, which is judged in vain.
10. kitchen waste grease immunochromatography discrimination method according to claim 9, it is characterised in that:
The extracting method is that the ethanol water that volumetric concentration is 95% is added into sample to be tested, is mixed, 60~90
It flows back 1 hour at DEG C, cooling obtains extracting solution；The redissolution solvent is 10% methanol-PBS.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610361935.1A CN106084059B (en) | 2016-05-26 | 2016-05-26 | The general specific antibody of anti-Capsaicinoids, test strips and kitchen waste grease immunochromatography method for quick identification |
| KR1020170062643A KR101963023B1 (en) | 2016-05-26 | 2017-05-22 | General complete antigen hapten, specific antibody, test strip, and rapid immunochromatographic method for identification of a recovered oil from kitchen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610361935.1A CN106084059B (en) | 2016-05-26 | 2016-05-26 | The general specific antibody of anti-Capsaicinoids, test strips and kitchen waste grease immunochromatography method for quick identification |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106084059A CN106084059A (en) | 2016-11-09 |
| CN106084059B true CN106084059B (en) | 2019-10-25 |
Family
ID=57229324
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610361935.1A Active CN106084059B (en) | 2016-05-26 | 2016-05-26 | The general specific antibody of anti-Capsaicinoids, test strips and kitchen waste grease immunochromatography method for quick identification |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR101963023B1 (en) |
| CN (1) | CN106084059B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108982862A (en) * | 2018-08-01 | 2018-12-11 | 山西公大智检科技有限公司 | It is a kind of for detecting the immuno-chromatographic test paper strip and preparation method thereof of capsaicine in gutter oil |
| CN109991412B (en) * | 2018-12-10 | 2019-11-19 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | A kind of method of capsicum alkali composition in detection grease |
| CN109856318A (en) * | 2019-01-30 | 2019-06-07 | 扬州工业职业技术学院 | A kind of test paper of quick detection gutter oil |
| CN110045108B (en) * | 2019-05-28 | 2021-10-29 | 江南大学 | Capsaicin index-based illegal cooking oil enzyme-linked immunosorbent assay kit and detection method thereof |
| CN111579768A (en) * | 2019-06-25 | 2020-08-25 | 山西康健恩生物科技有限公司 | Anti-mullerian hormone detection kit and preparation method thereof |
| CN110981744B (en) * | 2019-11-08 | 2020-09-22 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Capsaicin hapten and artificial antigen for illegal cooking oil detection and preparation method and application thereof |
| CN112415186B (en) * | 2020-11-19 | 2022-11-04 | 北京科技大学 | Reverse phase transfer extraction detection process and application of capsaicin compounds in grease |
| CN113087638B (en) * | 2021-03-11 | 2022-06-14 | 杭州同舟生物技术有限公司 | Pregabalin artificial hapten, pregabalin artificial antigen, and preparation method and application thereof |
| CN113063938B (en) * | 2021-03-13 | 2023-09-12 | 河南省农业科学院 | High-sensitivity gradient semi-quantitative immunochromatography detection test strip and detection method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103951577A (en) * | 2014-04-25 | 2014-07-30 | 中国农业科学院油料作物研究所 | Artificial hapten and artificial antigen of capsaicine, as well as preparation methods thereof |
| CN104383889A (en) * | 2014-11-21 | 2015-03-04 | 中国农业科学院油料作物研究所 | Dihydrocapsaicin polyclonal antibody immunoadsorbent, immunoaffinity column and preparation method and application thereof |
| CN104447383A (en) * | 2014-11-21 | 2015-03-25 | 中国农业科学院油料作物研究所 | Dihydrocapsaicin artificial hapten and artificial antigen as well as preparation methods thereof |
| CN105548553A (en) * | 2016-02-04 | 2016-05-04 | 中国农业科学院油料作物研究所 | Colloidal gold immunochromatography test strip for rapidly detecting capsaicinoids as well as preparation method and application thereof |
| CN105541655A (en) * | 2016-02-04 | 2016-05-04 | 中国农业科学院油料作物研究所 | Universal artificial hapten and artificial complete antigen for capsaicins and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3479100B2 (en) * | 1993-06-02 | 2003-12-15 | 帝国臓器製薬株式会社 | Simple semi-quantitative immunochemical method and apparatus |
| KR101726181B1 (en) * | 2014-03-20 | 2017-04-13 | 주식회사 수젠텍 | Immunochromatography Analysis Device |
-
2016
- 2016-05-26 CN CN201610361935.1A patent/CN106084059B/en active Active
-
2017
- 2017-05-22 KR KR1020170062643A patent/KR101963023B1/en active IP Right Grant
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103951577A (en) * | 2014-04-25 | 2014-07-30 | 中国农业科学院油料作物研究所 | Artificial hapten and artificial antigen of capsaicine, as well as preparation methods thereof |
| CN104383889A (en) * | 2014-11-21 | 2015-03-04 | 中国农业科学院油料作物研究所 | Dihydrocapsaicin polyclonal antibody immunoadsorbent, immunoaffinity column and preparation method and application thereof |
| CN104447383A (en) * | 2014-11-21 | 2015-03-25 | 中国农业科学院油料作物研究所 | Dihydrocapsaicin artificial hapten and artificial antigen as well as preparation methods thereof |
| CN105548553A (en) * | 2016-02-04 | 2016-05-04 | 中国农业科学院油料作物研究所 | Colloidal gold immunochromatography test strip for rapidly detecting capsaicinoids as well as preparation method and application thereof |
| CN105541655A (en) * | 2016-02-04 | 2016-05-04 | 中国农业科学院油料作物研究所 | Universal artificial hapten and artificial complete antigen for capsaicins and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20170134217A (en) | 2017-12-06 |
| CN106084059A (en) | 2016-11-09 |
| KR101963023B1 (en) | 2019-03-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106084059B (en) | The general specific antibody of anti-Capsaicinoids, test strips and kitchen waste grease immunochromatography method for quick identification | |
| CN103951577B (en) | Artificial hapten and artificial antigen of capsaicine, as well as preparation methods thereof | |
| CN101413955B (en) | ELISA test box for detecting zearalenone and preparing and detecting method thereof | |
| CN104447383B (en) | A kind of Dihydrocapsaicin artificial semiantigen, artificial antigen and preparation method thereof | |
| CN111187346B (en) | Colloidal gold test strip for detecting fipronil and metabolites thereof and preparation method thereof | |
| CN101661043A (en) | Method for detecting fumonisin by colloidal gold immunochromatographic test | |
| CN105548553B (en) | The colloidal gold immuno-chromatography test paper strip of quick detection Capsaicinoids, preparation method and applications | |
| CN112666347B (en) | Immunofluorescence chromatography kit and method for synchronously detecting contents of aflatoxin B1 and zearalenone in vegetable oil | |
| CN103575887B (en) | A kind of test card and application thereof detecting aflatoxin B1 | |
| CN101650368A (en) | Method for testing zearalenone toxin by using colloidal gold immunochromatography assay | |
| CN107271665B (en) | Test strip for detecting salbutamol and application thereof | |
| CN101407480A (en) | Preparation of semicarbazide derivative hapten, antigen and antibody | |
| CN104109112B (en) | Cyproheptadine semiantigen, artificial antigen, antibody, preparation methods of cyproheptadine semiantigen and artificial antigen, and application of artificial antigen and antibody | |
| CN103513035B (en) | A kind of test strips detecting Aflatoxins M1 and method | |
| CN113109560A (en) | Test strip for quickly testing flonicamid pesticide, preparation method and detection method thereof | |
| CN104387467A (en) | Detection kit and detection paper for Beta-adrenergic receptor stimulant multiresidue detection | |
| CN108828205B (en) | Ochratoxin A hapten, artificial antigen, preparation method and kit of artificial antigen, and ochratoxin A detection method | |
| CN103163296A (en) | Immune colloidal gold test strip for detection of ractopamine residue in swine urine | |
| CN109061156A (en) | A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting pendimethalin | |
| CN108802378A (en) | A kind of test strips that the nature controlling line of test strips is coated with solution and is prepared with this solution | |
| CN112142756A (en) | Preparation of aflatoxin M1 hapten and holoantigen as well as preparation and application of rapid transfer quantitative kit | |
| CN113582923A (en) | Quinaldine hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN104215763B (en) | A kind of test strips detecting T-2 toxin and application thereof | |
| CN101962343A (en) | Hapten, artificial antigen and antibody of basic orange as well as preparation method and application thereof | |
| CN113501781B (en) | Hapten, artificial antigen and antibody for detecting chlorpheniramine maleate, and preparation methods and applications thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |