CN106442482A - Detection kit for melamine in food - Google Patents
Detection kit for melamine in food Download PDFInfo
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- CN106442482A CN106442482A CN201611025748.2A CN201611025748A CN106442482A CN 106442482 A CN106442482 A CN 106442482A CN 201611025748 A CN201611025748 A CN 201611025748A CN 106442482 A CN106442482 A CN 106442482A
- Authority
- CN
- China
- Prior art keywords
- tripolycyanamide
- magnetic bead
- solution
- detection kit
- melamine
- 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.)
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- 238000001514 detection method Methods 0.000 title claims abstract description 29
- 235000013305 food Nutrition 0.000 title claims abstract description 28
- 229920000877 Melamine resin Polymers 0.000 title claims abstract description 24
- JDSHMPZPIAZGSV-UHFFFAOYSA-N melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 title claims abstract description 23
- 239000011324 bead Substances 0.000 claims abstract description 45
- 102000004190 Enzymes Human genes 0.000 claims abstract description 27
- 108090000790 Enzymes Proteins 0.000 claims abstract description 27
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 22
- 239000000758 substrate Substances 0.000 claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims abstract description 14
- 238000004140 cleaning Methods 0.000 claims abstract description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 58
- 239000011780 sodium chloride Substances 0.000 claims description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 24
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 22
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 19
- 239000008213 purified water Substances 0.000 claims description 19
- 239000000126 substance Substances 0.000 claims description 19
- 238000002360 preparation method Methods 0.000 claims description 18
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 18
- 235000018102 proteins Nutrition 0.000 claims description 17
- 102000004169 proteins and genes Human genes 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 239000003513 alkali Substances 0.000 claims description 15
- 239000007853 buffer solution Substances 0.000 claims description 14
- 239000001509 sodium citrate Substances 0.000 claims description 14
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 12
- 239000007983 Tris buffer Substances 0.000 claims description 11
- 239000000872 buffer Substances 0.000 claims description 11
- 238000005259 measurement Methods 0.000 claims description 11
- 239000008363 phosphate buffer Substances 0.000 claims description 11
- 238000004321 preservation Methods 0.000 claims description 11
- 239000012530 fluid Substances 0.000 claims description 10
- BLSAPDZWVFWUTL-UHFFFAOYSA-N 2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound OS(=O)(=O)C1CC(=O)NC1=O BLSAPDZWVFWUTL-UHFFFAOYSA-N 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 230000004913 activation Effects 0.000 claims description 9
- 238000002372 labelling Methods 0.000 claims description 9
- 239000002075 main ingredient Substances 0.000 claims description 9
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 8
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 8
- 229940018557 citraconic acid Drugs 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 125000004836 hexamethylene group Chemical group [H]C([H])([*:2])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[*:1] 0.000 claims description 7
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 claims description 6
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 claims description 6
- 238000004587 chromatography analysis Methods 0.000 claims description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 6
- 239000000376 reactant Substances 0.000 claims description 6
- 239000012224 working solution Substances 0.000 claims description 6
- HNEGQIOMVPPMNR-IHWYPQMZSA-N citraconic acid Chemical compound OC(=O)C(/C)=C\C(O)=O HNEGQIOMVPPMNR-IHWYPQMZSA-N 0.000 claims description 5
- ZICQBHNGXDOVJF-UHFFFAOYSA-N diamantane Chemical compound C1C2C3CC(C4)CC2C2C4C3CC1C2 ZICQBHNGXDOVJF-UHFFFAOYSA-N 0.000 claims description 5
- 239000002808 molecular sieve Substances 0.000 claims description 5
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 5
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 claims description 4
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 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 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- 239000005018 casein Substances 0.000 claims description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 3
- 235000021240 caseins Nutrition 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 3
- 230000002255 enzymatic effect Effects 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 3
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 3
- 238000005829 trimerization reaction Methods 0.000 claims description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims 2
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims 1
- 125000002252 acyl group Chemical group 0.000 claims 1
- 235000019270 ammonium chloride Nutrition 0.000 claims 1
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims 1
- 159000000000 sodium salts Chemical class 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 10
- 239000007790 solid phase Substances 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 239000006249 magnetic particle Substances 0.000 abstract description 3
- 238000003018 immunoassay Methods 0.000 abstract 1
- 238000004020 luminiscence type Methods 0.000 abstract 1
- 230000035484 reaction time Effects 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 8
- 239000000523 sample Substances 0.000 description 7
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 6
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 6
- 102100031013 Transgelin Human genes 0.000 description 6
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 6
- 235000013336 milk Nutrition 0.000 description 5
- 239000008267 milk Substances 0.000 description 5
- 210000004080 milk Anatomy 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 102000009027 Albumins Human genes 0.000 description 4
- 108010088751 Albumins Proteins 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 230000002485 urinary effect Effects 0.000 description 3
- XZMCDFZZKTWFGF-UHFFFAOYSA-N Cyanamide Chemical compound NC#N XZMCDFZZKTWFGF-UHFFFAOYSA-N 0.000 description 2
- XYIPYISRNJUPBA-UHFFFAOYSA-N [3-(3'-methoxyspiro[adamantane-2,4'-dioxetane]-3'-yl)phenyl] dihydrogen phosphate Chemical compound O1OC2(C3CC4CC(C3)CC2C4)C1(OC)C1=CC=CC(OP(O)(O)=O)=C1 XYIPYISRNJUPBA-UHFFFAOYSA-N 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- FYFFGSSZFBZTAH-UHFFFAOYSA-N methylaminomethanetriol Chemical compound CNC(O)(O)O FYFFGSSZFBZTAH-UHFFFAOYSA-N 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 150000003457 sulfones Chemical class 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- -1 triazines nitrogen heterocyclic ring organic compound Chemical class 0.000 description 2
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical compound C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 1
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000790917 Dioxys <bee> Species 0.000 description 1
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 1
- 239000001263 FEMA 3042 Substances 0.000 description 1
- 208000000913 Kidney Calculi Diseases 0.000 description 1
- 239000004640 Melamine resin Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010029148 Nephrolithiasis Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- 208000006568 Urinary Bladder Calculi Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- UHOVQNZJYSORNB-UHFFFAOYSA-N benzene Substances C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000012459 cleaning agent Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003063 flame retardant Substances 0.000 description 1
- 235000013350 formula milk Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- ZFSLODLOARCGLH-UHFFFAOYSA-N isocyanuric acid Chemical compound OC1=NC(O)=NC(O)=N1 ZFSLODLOARCGLH-UHFFFAOYSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 235000008476 powdered milk Nutrition 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
- 229940033123 tannic acid Drugs 0.000 description 1
- 235000015523 tannic acid Nutrition 0.000 description 1
- 229920002258 tannic acid Polymers 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- KSGWLNWYCHAFIX-UHFFFAOYSA-N triazine-4,5-diamine Chemical compound NC1=CN=NN=C1N KSGWLNWYCHAFIX-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a detection kit for melamine in food, which comprises: 1) a melamine calibrator; 2) a melamine-carboxylate magnetic bead linker reagent; 3) an enzyme conjugate; 4) a chemiluminescent substrate; 5) cleaning solution. Through a method for directly and stably connecting melamine with the solid phase carrier, the detection kit ensures the stability in connection with the solid phase carrier, and the full exposure of the melamine molecules, and further enlarges the effective reaction area by introducing the magnetic particles, the melamine chemiluminescence immunoassay kit that is higher in sensitivity and shorter in the reaction time is provided. The kit can be applied to a variety of luminescence detectors.
Description
Technical field
The present invention relates in technical field of food detection, specifically a kind of food tripolycyanamide detection kit.
Background technology
Tripolycyanamide（Melamine）, melamine, extract of protein is commonly called as, is a kind of triazines nitrogen heterocyclic ring organic compound, is
Pure white monoclinic prism body.Tripolycyanamide is a kind of broad-spectrum Organic Chemicals, topmost purposes be as production
The raw material of melamine resin, is also used as fire retardant, water reducer, formaldehyde cleaning agent etc..
Kjeldahl's method is generally adopted in food industry, calculates protein content, tripolycyanamide by determining nitrogen content
Nitrogen content be 66%, be often added in food by lawless person is illegal, to cause protein content illusion up to standard.
However, this food for adding tripolycyanamide does not only really increase the content of protein, its toxic and side effects is anti-
And harm can be brought to our healthy.As tripolycyanamide has stronger stickiness, hydrolysis after entering human body generates three
Poly cyanamid, the easy material such as the absorption lithogenous oxalic acid of shape, tannic acid and calcium in vivo, and be deposited in urinary system.Take the photograph for a long time
Entering tripolycyanamide can cause urinary system to damage, and can damage reproduction, the urinary system of human body and animal, produce kidney, vesical calculuses.
2008, the event that Sanlu baby milk powder adds tripolycyanamide, the cause that the event is exposed were broken out in China
It is that having eaten the infant for adding tripolycyanamide milk powder generates renal calculuss disease.It can be seen that, tripolycyanamide mixes milk product
The practice has directly challenged food safety, and threatens our health.Therefore, in order to prevent to add tripolycyanamide in food,
A kind of method for needing tripolycyanamide in food to be detected.
Existing tripolycyanamide detection method mainly has high performance liquid chromatography, Liquid Chromatography-Tandem Mass Spectrometry, gas phase color
Spectrum-tandem mass spectrometry etc..Although these methods can realize the accurate detection of tripolycyanamide, its sensitivity, reaction efficiency are equal
There is certain deficiency.
Content of the invention
It is an object of the invention to provide the detection of tripolycyanamide is tried in the food that a kind of sensitivity is higher, the response time is short
Agent box.
For achieving the above object, the present invention provides following technical scheme：
The detection kit of tripolycyanamide in a kind of food, including：1）Tripolycyanamide calibration object；2）Tripolycyanamide-carboxyl magnetic bead
Junctional complex reagent；3）Enzyme conjugates；4）Chemical luminous substrate；5）Cleanout fluid.
As the further scheme of the present invention：The Concentraton gradient of described tripolycyanamide standard substance is 0ng/ml, 10ng/
ml、20ng/ml、40ng/ml、80ng/ml、200ng/ml.
As the further scheme of the present invention：The Main Ingredients and Appearance of described tripolycyanamide-carboxyl magnetic bead junctional complex reagent is
Tripolycyanamide is coupled carboxyl magnetic bead junctional complex.
As the further scheme of the present invention：The particle diameter of described carboxyl magnetic bead is 2～3 μm, and surface active groups are carboxylic
Base（-COOH）.
As the further scheme of the present invention：The Main Ingredients and Appearance of described enzyme conjugates is the trimerization of alkali phosphatase enzyme mark
Cyanamide associated proteins.
As the further scheme of the present invention：The Main Ingredients and Appearance of described chemical luminous substrate is (3- (2- spiral Buddha's warrior attendant
Alkane) -4- methoxyl group -4- (3- phosphorus oxygen acyl)-phenyl -1,2- dioxane disodium salt).
In described food, the preparation method of the detection kit of tripolycyanamide, comprises the following steps：
1）Prepare tripolycyanamide calibration object；
2）Tripolycyanamide is connected on carboxyl magnetic bead junctional complex is prepared, with magnetic bead buffer solution dilution junctional complex to finite concentration,
Prepare junctional complex reagent；
3）With enzyme labelling tripolycyanamide associated proteins, enzyme conjugates are prepared；
4）Prepare chemical luminous substrate；
5）Prepare cleanout fluid；
6）More than subpackage each reagent, constitutes finished product.
As the further scheme of the present invention：The preparation of described tripolycyanamide calibration object, comprises the following steps：
1）The preparation of calibration object buffer：Weigh 5g Tris alkali, 9g Sodium Chloride（NaCl）, 0.6g Proclin-300, add
900ml purified water, is adjusted pH value to 7.2 ± 0.1 with 2mol/L sodium citrate；5g albumin rabbit serum is added, is settled to
Repetition measurement pH value after 1000ml, is allowed to filter after 2～8 DEG C of preservations in 7.2 ± 0.1,0.22 μm；
2）By tripolycyanamide sterling powder calibration object buffer solution, preparing becomes the concentrated solution of 1 μ g/ml, subsequently dilute successively
5 concentration are disengaged, respectively：10ng/ml、20ng/ml、40ng/ml、80ng/ml、200ng/ml；Plus 0 point, totally 6 concentration
Calibration object Grad.
As the further scheme of the present invention：The preparation of described tripolycyanamide-carboxyl magnetic bead junctional complex reagent, including with
Lower step：
1）Tripolycyanamide 5mg is accurately weighed, is dissolved in pure water, final concentration of 2.5mg/ml, obtain melamine solution；
2）The phosphate buffer of the 25mmol/L of secure ph 7.0：Weigh NaH2PO4·H2O 1.5g、Na2HPO4·2H2O
2.5g, Sodium Chloride（NaCl）3g, adds 900ml purified water, is adjusted pH value to 7.0 with 2mol/L sodium citrate or sodium bicarbonate
± 0.1, repetition measurement pH value after 1000ml is settled to, is allowed to filter after 2～8 DEG C of preservations in 7.0 ± 0.1,0.22 μm；
3）Prepare sulfosuccinimide 4- [N- the citraconic acid] -1- carboxylic hexamethylene of 5mg/ml（SMCC）, with dimethyl Asia
Sulfone（DMSO）Dissolving；
4）Sulfosuccinimide 4- [N- the citraconic acid] -1- carboxylic ring of the 5mg/ml of 80 μ L is added toward in melamine solution
Hexane（SMCC）Activated, 20～22 DEG C are reacted 40 minutes, are fully mixed in course of reaction；
5）10mg carboxyl magnetic bead is measured, carboxyl magnetic bead is positioned on magnet stand 2 minutes are stood, supernatant is removed, with pH=7.0's
25mmol/L phosphate buffer washs three times and adds phosphate buffer preservation afterwards, makes magnetic bead concentration for 20mg/ml；
6）Melamine solution after activation is added in the carboxyl magnetic bead after cleaning, and magnetic bead concentration in reactant liquor is adjusted to
5mg/ml, 20～22 DEG C are reacted 35 minutes, are fully mixed in course of reaction；
7）Prepare magnetic bead buffer solution：Weigh 12g Tris alkali, 9g Sodium Chloride（NaCl）, 0.5ml tween 20,1g Proclin-
300,900ml purified water is added, and pH value is adjusted to 7.2 ± 0.1 with 2mol/L sodium citrate；Add 1g casein sodium, constant volume
The repetition measurement pH value to 1000ml, is allowed to filter after 2～8 DEG C of preservations in 7.2 ± 0.1,0.22 μm；
8）Supernatant is removed after the completion of reaction, is washed with 25mmol/L phosphate buffer three times and is added 1ml magnetic bead buffer solution to enter afterwards
Row is preserved；During use, magnetic bead buffer solution is diluted to the working solution that concentration is 0.03mg/ml.
As the further scheme of the present invention：The preparation of described enzyme conjugates, comprises the following steps：
1）Alkali phosphatase 1.0mg is taken, concentration is adjusted to 6mg/ml；
2）Weigh 5mg sulfosuccinimide 4- [N- citraconic acid] -1- carboxylic hexamethylene（SMCC）, use dimethyl sulfoxide
（DMSO）Being dissolved, final concentration of 15mg/ml, 5 μ L being added to alkali phosphatase, 20～22 DEG C are reacted 15 minutes；
3）With molecular sieve chromatography, purification is carried out according to the difference of molecular weight to reactant mixture, collect the alkaline phosphatase after activation
Enzymatic solution；
4）Sterling tripolycyanamide associated proteins 0.5mg is taken, is added in the alkaline phosphatase buffer after activation, 2～8 DEG C of reactions 4
Hour, finally with molecular sieve chromatography, the tripolycyanamide associated proteins of enzyme labelling are purified out；
5）Enzyme conjugates buffer：Weigh 12g trishydroxymethylaminomethane（Tris）, 9g Sodium Chloride（NaCl）, 6g glycerol,
0.6g Proclin-300, adds 900ml purified water, is adjusted pH value to 7.2 ± 0.1 with 2mol/L sodium citrate；Add 10g
Albumin rabbit serum, is settled to repetition measurement pH value after 1000ml, is allowed to filter in 7.2 ± 0.1,0.22 μm and protects after 2～8 DEG C
Deposit；
6）The tripolycyanamide associated proteins concentration that conjugate enzyme conjugates buffer is diluted to enzyme labelling is for 2 μ g/ml
Working solution.
As the further scheme of the present invention：The preparation of described chemical luminous substrate, comprises the following steps：Accurately weigh
3- (2- spiral diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus oxygen acyl)-phenyl -1,2- dioxane disodium salt（AMPPD）0.5g、
Trishydroxymethylaminomethane（Tris）18g, Sodium Chloride（NaCl）100g, hexadecyltrimethylammonium chloride（CTAC）0.02g is pure
Change water and 1000ml is settled to, adjustment chemical luminous substrate solution ph is 9.4 ± 0.05.
As the further scheme of the present invention：The preparation of described cleanout fluid, comprises the following steps：Weigh 1g trihydroxy methyl
Aminomethane（Tris）, 8.5g Sodium Chloride（NaCl）, 1g tween 20, add purified water about 900ml, mix to each reagent dissolve,
With 2mol/L sodium citrate solution adjustment solution ph to 7.8 ± 0.1, add purified water and 1000ml is settled to, mix after filtration
Stand-by.
The method for being detected using the detection kit of tripolycyanamide in described food, step is：
（1）50 μ L samples or calibration object is added in flat based tubes；
（2）50 μ L Magneto separate reagents are added in flat based tubes；
（3）40 μ L tripolycyanamide associated proteins alkaline phosphatase conjugate are added in flat based tubes；
（4）Flat based tubes are placed on vortex blending instrument and are mixed 30 seconds, be subsequently placed in 37 DEG C and react 20 minutes；
（5）Flat based tubes being placed on after 2 minutes being stood on magnet stand and outwell supernatant, pats dry, add 500 μ L of cleanout fluid, mix 20
Second；
（6）Repeat step（5）Twice；
（7）The flat based tubes for patting dry are put in immunity analysis instrument and are detected.
Compared with prior art, the invention has the beneficial effects as follows：The present invention is directly steady with solid phase carrier by tripolycyanamide
The method of fixed connection, can not only ensure the stability being connected with solid phase carrier but also can guarantee that melamine molecule is fully exposed,
While expanding effective affecting acreage also by magnetic particle is introduced, the melamine that sensitivity is higher, the response time is shorter is established
Amine chemiluminescence immune detection reagent kit, the test kit is applicable to various luminous detection instruments.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment obtained under the premise of creative work is not made by those of ordinary skill in the art, all
Belong to the scope of protection of the invention.
Embodiment 1
In the embodiment of the present invention, the detection kit of tripolycyanamide in a kind of food, including：1）Tripolycyanamide calibration object；2）Three
Poly cyanamid-carboxyl magnetic bead junctional complex reagent；3）Enzyme conjugates；4）Chemical luminous substrate；5）Cleanout fluid.Tripolycyanamide standard substance
Concentraton gradient is 0ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml, 200ng/ml.Tripolycyanamide-carboxyl magnetic bead is even
The Main Ingredients and Appearance for connecing thing reagent is that tripolycyanamide is coupled carboxyl magnetic bead junctional complex.The particle diameter of carboxyl magnetic bead is 2～3 μm, and surface is lived
Property group be carboxyl（-COOH）.The Main Ingredients and Appearance of enzyme conjugates is the tripolycyanamide associated proteins of alkali phosphatase enzyme mark.Chemistry
The Main Ingredients and Appearance of luminous substrate is (3- (2- spiral diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus oxygen acyl)-phenyl -1,2- dioxy ring second
Alkane disodium salt).
In described food, the preparation method of the detection kit of tripolycyanamide, comprises the following steps：
1）Tripolycyanamide calibration object is prepared, including：The preparation of calibration object buffer：Weigh 5g Tris alkali, 9g Sodium Chloride
（NaCl）, 0.6g Proclin-300, add 900ml purified water, with 2mol/L sodium citrate, pH value is adjusted to 7.2 ± 0.1；
5g albumin rabbit serum is added, repetition measurement pH value after 1000ml is settled to, be allowed to filter after 2～8 in 7.2 ± 0.1,0.22 μm
DEG C preserve；By tripolycyanamide sterling powder calibration object buffer solution, preparing becomes the concentrated solution of 1 μ g/ml, subsequently dilute successively
5 concentration are disengaged, respectively：10ng/ml、20ng/ml、40ng/ml、80ng/ml、200ng/ml；Plus 0 point, totally 6 concentration
Calibration object Grad；
2）Tripolycyanamide is connected on carboxyl magnetic bead junctional complex is prepared, with magnetic bead buffer solution dilution junctional complex to finite concentration,
Junctional complex reagent is prepared, including：Tripolycyanamide 5mg is accurately weighed, is dissolved in pure water, final concentration of 2.5mg/ml, obtain
Melamine solution；The phosphate buffer of the 25mmol/L of secure ph 7.0：Weigh NaH2PO4·H2O 1.5g、
Na2HPO4·2H2O 2.5g, Sodium Chloride（NaCl）3g, adds 900ml purified water, will with 2mol/L sodium citrate or sodium bicarbonate
PH value is adjusted to 7.0 ± 0.1, is settled to repetition measurement pH value after 1000ml, is allowed to filter after 2～8 in 7.0 ± 0.1,0.22 μm
DEG C preserve；Prepare sulfosuccinimide 4- [N- the citraconic acid] -1- carboxylic hexamethylene of 5mg/ml（SMCC）, with dimethyl Asia
Sulfone（DMSO）Dissolving；Sulfosuccinimide 4- [the N- methyl Malaysia of the 5mg/ml of 80 μ L is added toward in melamine solution
Acid] -1- carboxylic hexamethylene（SMCC）Activated, 20～22 DEG C are reacted 40 minutes, are fully mixed in course of reaction；Measure 10mg carboxylic
Base magnetic bead, carboxyl magnetic bead is positioned on magnet stand and stands 2 minutes, remove supernatant, with the 25mmol/L phosphate-buffered of pH=7.0
Liquid washs three times and adds phosphate buffer preservation afterwards, makes magnetic bead concentration for 20mg/ml；Melamine solution after activation is added
Enter in the carboxyl magnetic bead after cleaning, and magnetic bead concentration in reactant liquor is adjusted to 5mg/ml, 20～22 DEG C are reacted 35 minutes, reaction
During fully mix；Prepare magnetic bead buffer solution：Weigh 12g Tris alkali, 9g Sodium Chloride（NaCl）, 0.5ml tween 20,1g
Proclin-300, adds 900ml purified water, is adjusted pH value to 7.2 ± 0.1 with 2mol/L sodium citrate；Add 1g casein
Sodium, is settled to repetition measurement pH value after 1000ml, is allowed to filter after 2～8 DEG C of preservations in 7.2 ± 0.1,0.22 μm；Reaction is completed
After remove supernatant, washed with 25mmol/L phosphate buffer three times afterwards add 1ml magnetic bead buffer solution preserved；Use during use
Magnetic bead buffer solution is diluted to working solution of the concentration for 0.03mg/ml；
3）With enzyme labelling tripolycyanamide associated proteins, enzyme conjugates are prepared, including：Alkali phosphatase 1.0mg is taken, concentration is adjusted
For 6mg/ml；Weigh 5mg sulfosuccinimide 4- [N- citraconic acid] -1- carboxylic hexamethylene（SMCC）, use dimethyl sulfoxide
（DMSO）Being dissolved, final concentration of 15mg/ml, 5 μ L being added to alkali phosphatase, 20～22 DEG C are reacted 15 minutes；With point
Sub- sieve chromatography carries out purification according to the difference of molecular weight to reactant mixture, collects the alkaline phosphatase enzymatic solution after activation；Take
Sterling tripolycyanamide associated proteins 0.5mg, add activation after alkaline phosphatase buffer in, 2～8 DEG C react 4 hours, finally
With molecular sieve chromatography, the tripolycyanamide associated proteins of enzyme labelling are purified out；Enzyme conjugates buffer：Weigh 12g tri-
Hydroxymethyl aminomethane（Tris）, 9g Sodium Chloride（NaCl）, 6g glycerol, 0.6g Proclin-300, add 900ml purified water,
With 2mol/L sodium citrate, pH value is adjusted to 7.2 ± 0.1；10g albumin rabbit serum is added, is settled to repetition measurement pH after 1000ml
Value, is allowed to filter after 2～8 DEG C of preservations in 7.2 ± 0.1,0.22 μm；Conjugate enzyme conjugates buffer is diluted to enzyme
The tripolycyanamide associated proteins concentration of labelling is working solution for 2 μ g/ml；
4）Chemical luminous substrate is prepared, including：3- (2- spiral diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus oxygen acyl)-benzene is accurately weighed
Base -1,2- dioxane disodium salt（AMPPD）0.5g, trishydroxymethylaminomethane（Tris）18g, Sodium Chloride（NaCl）
100g, hexadecyltrimethylammonium chloride（CTAC）0.02g, purified water is settled to 1000ml, adjusts chemical luminous substrate solution
PH value is 9.4 ± 0.05；This chemical luminous substrate is the epidioxy ethane substrate for alkali phosphatase, needs at 2～8 DEG C
Under the conditions of keep in dark place, the used time slowly mixes；
5）Cleanout fluid is prepared, including：Weigh 1g trishydroxymethylaminomethane（Tris）, 8.5g Sodium Chloride（NaCl）, 1g tween-
20, add purified water about 900ml, mix and dissolve to each reagent, with 2mol/L sodium citrate solution adjustment solution ph to 7.8 ±
0.1, add purified water and 1000ml is settled to, mix after filtration stand-by；
6）More than subpackage each reagent, constitutes finished product.
The method for being detected using the detection kit of tripolycyanamide in described food, step is：
（1）50 μ L samples or calibration object is added in flat based tubes；
（2）50 μ L Magneto separate reagents are added in flat based tubes；
（3）40 μ L tripolycyanamide BP alkaline phosphatase conjugate are added in flat based tubes；
（4）Flat based tubes are placed on vortex blending instrument and are mixed 30 seconds, be subsequently placed in 37 DEG C and react 20 minutes；
（5）Flat based tubes being placed on after 2 minutes being stood on magnet stand and outwell supernatant, pats dry, add 500 μ L of cleanout fluid, mix 20
Second；
（6）Repeat step（5）Twice；
（7）The flat based tubes for patting dry are put in immunity analysis instrument and are detected.
The judgement of testing result：By the meansigma methodss of the standard substance for being obtained and sample luminous intensity values divided by first standard
（0 standard）Luminous intensity values be multiplied by 100 again, with suppression ratio as vertical coordinate, the logarithm of melamine concentration is marked for abscissa
Directrix curve, the concentration of each sample can be read from standard curve.
Relative luminous intensity（%）=RLU/RLU0, RLU is the luminous intensity that standard substance or sample solution are determined, RLU0It is
Blank（Concentration is 0 standard solution）Luminous intensity values.
The accuracy and precision of test kit
Accuracy refers to the matching degree between measured value and true value, and test kit accuracy is commonly used the response rate and represented.Precision also known as
Repeatability, the conventional coefficient of variation represents.
According to sample-pretreating method, with the tripolycyanamide of two concentration of 1.0ng/ml, 2.0ng/ml respectively to milk, milk
Powder sample is added reclaiming, and each concentration mensuration of every kind of sample 5 is parallel, is measured with three batches of test kits, calculates sample
The response rate and precision.Experimental result shows, in milk sample the TIANZHU XINGNAO Capsul scope of tripolycyanamide 85.6～
99.2%, in powdered milk sample, the TIANZHU XINGNAO Capsul scope of tripolycyanamide is 84.3～92.8%.Within-run and between-run analysis coefficient is all little
In 10%.
The specificity of test kit
Using tripolycyanamide as standard, if the cross reacting rate of tripolycyanamide is 100%, for antibody cross reaction Journal of Sex Research
Medicine is and tripolycyanamide structure or intimate medicine：Cyanuric acid, triazine, triazinediamine.By test kit step
Operation, but the competitor for adding is respectively different tripolycyanamide and is similar to thing, makes suppression curve, is calculated according to linear equation each
50% inhibition concentration of competitor（IC50）.Cross reacting rate（%CR）As IC of the antibody to tripolycyanamide50With antibody to melamine
The IC of amine competitor50Ratio percent.As a result show, each tripolycyanamide is similar to the equal ＜ 1% of cross reacting rate of thing, and this is described
Test kit has good specificity.
The method that the present invention is directly stably connected with solid phase carrier by tripolycyanamide, can both ensure to be connected with solid phase carrier
Stability can guarantee that melamine molecule is fully exposed again, while also by introduce magnetic particle expand effecting reaction
Area, establishes the tripolycyanamide chemiluminescence immune detection reagent kit that sensitivity is higher, the response time is shorter, and the test kit can
Suitable for various luminous detection instruments.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of spirit or essential attributes without departing substantially from the present invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit is required rather than described above is limited, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although this specification is been described by according to embodiment, not each embodiment is only wrapped
Containing an independent technical scheme, this narrating mode of description is only that those skilled in the art should for clarity
Using description as an entirety, the technical scheme in each embodiment can also form those skilled in the art through appropriately combined
Understandable other embodiment.
Claims (10)
1. in a kind of food tripolycyanamide detection kit, it is characterised in that include：1）Tripolycyanamide calibration object；2）Trimerization
Cyanamide-carboxyl magnetic bead junctional complex reagent；3）Enzyme conjugates；4）Chemical luminous substrate；5）Cleanout fluid.
2. in food according to claim 1 tripolycyanamide detection kit, it is characterised in that described tripolycyanamide
The Concentraton gradient of standard substance is 0ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml, 200ng/ml.
3. in food according to claim 1 tripolycyanamide detection kit, it is characterised in that described melamine
The Main Ingredients and Appearance of amine-carboxyl magnetic bead junctional complex reagent is that tripolycyanamide is coupled carboxyl magnetic bead junctional complex, described carboxyl magnetic bead
Particle diameter is 2～3 μm, and surface active groups are carboxyl（-COOH）.
4. in food according to claim 1 tripolycyanamide detection kit, it is characterised in that described enzyme conjugates
Main Ingredients and Appearance be alkali phosphatase enzyme mark tripolycyanamide associated proteins.
5. in food according to claim 1 tripolycyanamide detection kit, it is characterised in that described chemiluminescence
The Main Ingredients and Appearance of substrate is (3- (2- spiral diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus oxygen acyl)-phenyl -1,2- dioxane two
Sodium salt).
6., according to the preparation method of the detection kit of tripolycyanamide in the arbitrary described food of Claims 1 to 5, its feature exists
In comprising the following steps：
1）Prepare tripolycyanamide calibration object；
2）Tripolycyanamide is connected on carboxyl magnetic bead junctional complex is prepared, with magnetic bead buffer solution dilution junctional complex to finite concentration,
Prepare junctional complex reagent；
3）With enzyme labelling tripolycyanamide associated proteins, enzyme conjugates are prepared；
4）Prepare chemical luminous substrate；
5）Prepare cleanout fluid；
6）More than subpackage each reagent, constitutes finished product.
7. in food according to claim 6 the detection kit of tripolycyanamide preparation method, it is characterised in that described
Tripolycyanamide-carboxyl magnetic bead junctional complex reagent preparation, comprise the following steps：
1）Tripolycyanamide 5mg is accurately weighed, is dissolved in pure water, final concentration of 2.5mg/ml, obtain melamine solution；
2）The phosphate buffer of the 25mmol/L of secure ph 7.0：Weigh NaH2PO4·H2O 1.5g、Na2HPO4·2H2O
2.5g, Sodium Chloride 3g, add 900ml purified water, are adjusted pH value to 7.0 ± 0.1 with 2mol/L sodium citrate or sodium bicarbonate,
Repetition measurement pH value after 1000ml is settled to, is allowed to filter after 2～8 DEG C of preservations in 7.0 ± 0.1,0.22 μm；
3）Sulfosuccinimide 4- [N- the citraconic acid] -1- carboxylic hexamethylene of 5mg/ml is prepared, uses dmso solution；
4）Sulfosuccinimide 4- [N- the citraconic acid] -1- carboxylic ring of the 5mg/ml of 80 μ L is added toward in melamine solution
Hexane is activated, and 20～22 DEG C are reacted 40 minutes, are fully mixed in course of reaction；
5）10mg carboxyl magnetic bead is measured, carboxyl magnetic bead is positioned on magnet stand 2 minutes are stood, supernatant is removed, with pH=7.0's
25mmol/L phosphate buffer washs three times and adds phosphate buffer preservation afterwards, makes magnetic bead concentration for 20mg/ml；
6）Melamine solution after activation is added in the carboxyl magnetic bead after cleaning, and magnetic bead concentration in reactant liquor is adjusted to
5mg/ml, 20～22 DEG C are reacted 35 minutes, are fully mixed in course of reaction；
7）Prepare magnetic bead buffer solution：12g Tris alkali, 9g Sodium Chloride, 0.5ml tween 20,1g Proclin-300 is weighed, is added
900ml purified water, is adjusted pH value to 7.2 ± 0.1 with 2mol/L sodium citrate；1g casein sodium is added, is settled to 1000ml
Repetition measurement pH value, is allowed to filter after 2～8 DEG C of preservations in 7.2 ± 0.1,0.22 μm afterwards；
8）Supernatant is removed after the completion of reaction, is washed with 25mmol/L phosphate buffer three times and is added 1ml magnetic bead buffer solution to enter afterwards
Row is preserved；During use, magnetic bead buffer solution is diluted to the working solution that concentration is 0.03mg/ml.
8. in food according to claim 6 the detection kit of tripolycyanamide preparation method, it is characterised in that described
Enzyme conjugates preparation, comprise the following steps：
1）Alkali phosphatase 1.0mg is taken, concentration is adjusted to 6mg/ml；
2）5mg sulfosuccinimide 4- [N- citraconic acid] -1- carboxylic hexamethylene is weighed, is dissolved with dimethyl sulfoxide,
Final concentration of 15mg/ml, adds 5 μ L to alkali phosphatase, and 20～22 DEG C are reacted 15 minutes；
3）With molecular sieve chromatography, purification is carried out according to the difference of molecular weight to reactant mixture, collect the alkaline phosphatase after activation
Enzymatic solution；
4）Sterling tripolycyanamide associated proteins 0.5mg is taken, is added in the alkaline phosphatase buffer after activation, 2～8 DEG C of reactions 4
Hour, finally with molecular sieve chromatography, the tripolycyanamide associated proteins of enzyme labelling are purified out；
5）Enzyme conjugates buffer：Weigh 12g trishydroxymethylaminomethane, 9g Sodium Chloride, 6g glycerol, 0.6g
Proclin-300, adds 900ml purified water, is adjusted pH value to 7.2 ± 0.1 with 2mol/L sodium citrate；Add 10g Sanguis Leporis seu oryctolagi
Pure albumen, is settled to repetition measurement pH value after 1000ml, is allowed to filter after 2～8 DEG C of preservations in 7.2 ± 0.1,0.22 μm；
6）The tripolycyanamide associated proteins concentration that conjugate enzyme conjugates buffer is diluted to enzyme labelling is for 2 μ g/ml
Working solution.
9. in food according to claim 6 the detection kit of tripolycyanamide preparation method, it is characterised in that described
Chemical luminous substrate preparation, comprise the following steps：3- (2- spiral diamantane (obsolete)) -4- methoxyl group -4- (3- phosphorus oxygen is accurately weighed
Acyl)-phenyl -1,2- dioxane disodium salt 0.5g, trishydroxymethylaminomethane 18g, Sodium Chloride 100g, cetyl front three
Ammonium chloride 0.02g, purified water is settled to 1000ml, and adjustment chemical luminous substrate solution ph is 9.4 ± 0.05.
10. in food according to claim 6 the detection kit of tripolycyanamide preparation method, it is characterised in that institute
The preparation of the cleanout fluid that states, comprises the following steps：1g trishydroxymethylaminomethane, 8.5g Sodium Chloride, 1g tween 20 is weighed, plus
Enter purified water 900ml, mix and dissolve to each reagent, with 2mol/L sodium citrate solution adjustment solution ph to 7.8 ± 0.1, mend
Plus purified water is settled to 1000ml, mix after filtration stand-by.
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| CN107490574A (en) * | 2017-08-11 | 2017-12-19 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of melamine and preparation method thereof |
| CN107656072A (en) * | 2017-11-17 | 2018-02-02 | 南通伊仕生物技术股份有限公司 | Liver fatty acid binding protein detection kit |
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| CN105181680A (en) * | 2015-09-28 | 2015-12-23 | 成都博奥新景医学科技有限公司 | A magnetic bead separation chemiluminescent immunodetection method for melamine |
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| CN107490574A (en) * | 2017-08-11 | 2017-12-19 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of melamine and preparation method thereof |
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Application publication date: 20170222 |