CN109270260A - A kind of detection aflatoxin M1Method - Google Patents
A kind of detection aflatoxin M1Method Download PDFInfo
- Publication number
- CN109270260A CN109270260A CN201811346302.9A CN201811346302A CN109270260A CN 109270260 A CN109270260 A CN 109270260A CN 201811346302 A CN201811346302 A CN 201811346302A CN 109270260 A CN109270260 A CN 109270260A
- Authority
- CN
- China
- Prior art keywords
- aflatoxin
- added
- phosphate buffer
- concentration
- holes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 67
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 title abstract description 6
- 229930195730 Aflatoxin Natural products 0.000 title abstract description 5
- 239000005409 aflatoxin Substances 0.000 title abstract description 5
- 239000002108 aflatoxin M1 Substances 0.000 claims abstract description 96
- 238000000034 method Methods 0.000 claims abstract description 69
- MJBWDEQAUQTVKK-IAGOWNOFSA-N aflatoxin M1 Chemical compound C=1([C@]2(O)C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O MJBWDEQAUQTVKK-IAGOWNOFSA-N 0.000 claims abstract description 60
- 238000002965 ELISA Methods 0.000 claims abstract description 53
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 claims abstract description 33
- 239000000427 antigen Substances 0.000 claims abstract description 27
- 102000036639 antigens Human genes 0.000 claims abstract description 26
- 108091007433 antigens Proteins 0.000 claims abstract description 26
- 238000002296 dynamic light scattering Methods 0.000 claims abstract description 12
- 230000002860 competitive effect Effects 0.000 claims abstract description 11
- 238000013459 approach Methods 0.000 claims abstract description 5
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 51
- 230000036961 partial effect Effects 0.000 claims description 49
- 239000007788 liquid Substances 0.000 claims description 37
- 239000000243 solution Substances 0.000 claims description 37
- 239000008363 phosphate buffer Substances 0.000 claims description 36
- 108010015776 Glucose oxidase Proteins 0.000 claims description 29
- 239000004366 Glucose oxidase Substances 0.000 claims description 29
- 229940116332 glucose oxidase Drugs 0.000 claims description 29
- 235000019420 glucose oxidase Nutrition 0.000 claims description 29
- 238000006243 chemical reaction Methods 0.000 claims description 27
- 238000005406 washing Methods 0.000 claims description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 16
- 239000002115 aflatoxin B1 Substances 0.000 claims description 16
- 239000008103 glucose Substances 0.000 claims description 16
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 15
- 238000010790 dilution Methods 0.000 claims description 13
- 239000012895 dilution Substances 0.000 claims description 13
- 239000002245 particle Substances 0.000 claims description 13
- 239000003053 toxin Substances 0.000 claims description 13
- 231100000765 toxin Toxicity 0.000 claims description 13
- 241000228197 Aspergillus flavus Species 0.000 claims description 12
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 12
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 12
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 12
- 239000007790 solid phase Substances 0.000 claims description 11
- 101710120037 Toxin CcdB Proteins 0.000 claims description 9
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 7
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 7
- 229940098773 bovine serum albumin Drugs 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 238000012544 monitoring process Methods 0.000 claims description 5
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 4
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 claims description 4
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 4
- 235000009754 Vitis X bourquina Nutrition 0.000 claims description 3
- 235000012333 Vitis X labruscana Nutrition 0.000 claims description 3
- 240000006365 Vitis vinifera Species 0.000 claims description 3
- 235000014787 Vitis vinifera Nutrition 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 3
- 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 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 2
- 150000001412 amines Chemical class 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 238000009835 boiling Methods 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000011248 coating agent Substances 0.000 claims description 2
- 238000000576 coating method Methods 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 230000005284 excitation Effects 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000005457 ice water Substances 0.000 claims description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 2
- 239000001509 sodium citrate Substances 0.000 claims description 2
- 238000000967 suction filtration Methods 0.000 claims description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 2
- 229940038773 trisodium citrate Drugs 0.000 claims description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims 2
- 101710128063 Carbohydrate oxidase Proteins 0.000 claims 1
- -1 Methylaminopropyl Chemical group 0.000 claims 1
- 238000000502 dialysis Methods 0.000 claims 1
- 231100000614 poison Toxicity 0.000 claims 1
- 239000002574 poison Substances 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 10
- 239000000523 sample Substances 0.000 description 31
- 102000004190 Enzymes Human genes 0.000 description 25
- 108090000790 Enzymes Proteins 0.000 description 25
- 229940088598 enzyme Drugs 0.000 description 25
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 238000012360 testing method Methods 0.000 description 16
- 235000020247 cow milk Nutrition 0.000 description 12
- 239000012488 sample solution Substances 0.000 description 11
- 239000002105 nanoparticle Substances 0.000 description 9
- 239000000758 substrate Substances 0.000 description 9
- 239000010931 gold Substances 0.000 description 8
- 229910052737 gold Inorganic materials 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 7
- 238000010521 absorption reaction Methods 0.000 description 7
- 230000021615 conjugation Effects 0.000 description 7
- 238000002203 pretreatment Methods 0.000 description 7
- 239000012224 working solution Substances 0.000 description 7
- 239000007864 aqueous solution Substances 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 230000009514 concussion Effects 0.000 description 6
- 238000004090 dissolution Methods 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 239000013049 sediment Substances 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- QRARGUIFAGCOOA-UHFFFAOYSA-N aspertoxin Chemical compound O1C2=C(C3(C=COC3O3)O)C3=CC(OC)=C2C(=O)C2=C1C=CC=C2OC QRARGUIFAGCOOA-UHFFFAOYSA-N 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 125000003051 glycosyloxy group Chemical group 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- 241000228230 Aspergillus parasiticus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 244000170916 Paeonia officinalis Species 0.000 description 1
- 235000006484 Paeonia officinalis Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 229910052785 arsenic Inorganic materials 0.000 description 1
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 231100000299 mutagenicity Toxicity 0.000 description 1
- 230000007886 mutagenicity Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical class OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/02—Investigating particle size or size distribution
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/02—Investigating particle size or size distribution
- G01N15/0205—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging
- G01N15/0211—Investigating a scatter or diffraction pattern
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G01N15/01—
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N2015/0038—Investigating nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/02—Investigating particle size or size distribution
- G01N15/0205—Investigating particle size or size distribution by optical means, e.g. by light scattering, diffraction, holography or imaging
- G01N15/0211—Investigating a scatter or diffraction pattern
- G01N2015/0222—Investigating a scatter or diffraction pattern from dynamic light scattering, e.g. photon correlation spectroscopy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/02—Investigating particle size or size distribution
- G01N2015/0277—Average size only
Abstract
The present invention relates to a kind of detection aflatoxin Ms1Method, the method is the aflatoxin B marked with enzyme1As competition antigen, and it is based on dynamic light scattering technique, detects aflatoxin M with competitive ELISA analytic approach1Method.This approach reduces competition antigen and aflatoxin Ms1The affinity of monoclonal antibody overcomes competition antigen and is not easy the problem that sensitivity is low caused by being competed by target antigen；The detection for being carried out output signal by means of dynamic light scattering method simultaneously can be improved detection sensitivity compared to traditional competitive ELISA analytic approach, realize aflatoxin M1Trace detection.
Description
Technical field
The invention belongs to Antigen Detection Techniques fields, and in particular to a kind of detection aflatoxin M1Method.
Background technique
Aflatoxin M1Mainly generated by Aspergillus flavus and aspergillus parasiticus bacterium, be aflatoxin analogue it
One, toxicity is higher than potassium cyanide and arsenic, belongs to high extremely toxic substance, and have strong carcinogenicity and mutagenicity, research shows that liver
Aflatoxin M in the disease incidence and Urine in Patients of high cancer incidence area1It is closely related.Mammal is taken in by aflatoxin B1
After the feed or food of pollution, B1It can be exchanged into M in animal's liver1, to pollute cow's milk.Therefore, monitoring food especially cream system
Aflatoxin M in product1It is of great significance.
Due to aflatoxin M1Pollution mostly occur in milk and milk products, and aflatoxin M1It is relatively stable, therefore
Formulate stringent aflatoxin M1Limit standard not only facilitates the management of Dairy Industry quality, and can be to greatest extent
Ground guarantees that consumer buys the food of nutrient safe.It is directed to aflatoxin M at present1Quantitative detecting method have physical chemistry
Method (such as thin-layered chromatography, liquid chromatography), immuno-chemical method (such as transmitting immunization, enzyme-linked immunization).But compared to
Other methods, enzyme-linked immunization is easy to operate, at low cost, and sensitivity is relatively high, is suitble to the screening and generaI investigation of high throughput sample,
Suitable for quickly detecting.With the development of science and technology, in the world to aflatoxin M in dairy products1Limit standard it is increasingly stringent,
Requirement for detection sensitivity is also higher and higher, and conventional enzyme-linked immunization is no longer satisfied trace aflatoxin M1's
Detection.Therefore need sensitiveer, quick and convenient and fast methods and techniques to aflatoxin M1It is monitored.
CN107688087A discloses a kind of aflatoxin M1Detection gel column and aflatoxin M1Detection side
Method, aflatoxin M1Detection gel column include open tubular column and the detection layers being filled in open tubular column and Quality Control layer.Detection
When, by the aflatoxin M of sample to be tested and chemiluminescent labels label1Enzyme-labelled antigen is added to the aflatoxin M1's
In detection gel column and flow through detection layers and Quality Control layer, the aflatoxin M of sample to be tested and chemiluminescent labels label1Enzyme
Mark the aflatoxin M in the common competition detection layers of both antigen1Monoclonal antibody, according to the color of detection layers and Quality Control layer
Color judges whether contain aflatoxin M in sample to be tested1.Above-mentioned aflatoxin M1Detection gel column it is easy to carry, fit
The quick detection of live great amount of samples is closed, but sensitivity is not high, is not able to satisfy trace aflatoxin M1Testing requirements.
CN103091494B discloses a kind of aflatoxin M1Chemical luminescence ELISA detection kit and use
Method, the application method of the kit include the following steps: the pre-treatment of (1) sample to be tested；(2) aflatoxin is sequentially added
M1Standard solution or sample, aflatoxin M1Antibody is added enzyme mark antiantibody, is eventually adding chemiluminescence after competitive reaction
Liquid carries out aflatoxin M by chemical illumination immunity analysis instrument1Quantitative detection；(3) result treatment and analysis.The invention mentions
The detection kit of confession is not able to satisfy trace aflatoxin M still1Testing requirements.
CN104569381A discloses aflatoxin M in a kind of test sample1The method and ELISA reagent of content
Box, the invention use direct competive ELISA detection pattern, and the coating of ELISA Plate is carried out using envelope antigen, after improvement
Periodates oxidizing process carry out ELISA Plate label, enzyme is directly marked on aflatoxin M1On specific antibody, Jiang Huangqu
Mould toxin M1Specific antibody is combined into one with two kinds of most important reactants of enzyme, improves labeling effciency, is saved enzyme and is resisted
The dosage of body, enzyme is greatly reduced with good activity without being reconfigured antibody in kit with antibody after guaranteeing label
The cost of kit, but detection sensitivity is not high, is not able to satisfy trace aflatoxin M1Testing requirements.
To sum up, a kind of sensitiveer, quick and convenient and fast detection aflatoxin M is developed1Method be necessary
's.
Summary of the invention
In view of the deficiencies of the prior art, the present invention intends to provide a kind of detection aflatoxin M1New side
Method.
In order to achieve that object of the invention, the invention adopts the following technical scheme:
The present invention provides a kind of detection aflatoxin M1Method, the method is the aflatoxin B marked with enzyme1
As competition antigen, and it is based on dynamic light scattering technique, detects aflatoxin M with competitive ELISA analytic approach1's
Method.
In the present invention, it the described method comprises the following steps:
(1) by aflatoxin M1Monoclonal antibody is coated on solid carrier, forms solid phase antibody；
(2) it is added in the solid phase antibody obtained to step (1) and contains aflatoxin M1Sample to be tested and grape glycosyloxy
Change the aflatoxin B of enzyme label1, it is uniformly mixed, reaction；
(3) glucose solution is added in the mixed liquor obtained to step (2), is uniformly mixed, reaction；
(4) horseradish peroxidase, tyrasamine, colloidal gold solution, reaction are added in the mixed liquor obtained to step (3)；
(5) the average aquation partial size for detecting colloidal gold, according to aflatoxin M1The standard curve that standard items are established calculates
Obtain aflatoxin M in sample to be tested1Content.
The average aquation partial size of the detection colloidal gold is to use dynamic light scattering particle size based on dynamic light scattering technique
Instrument measures.
Aflatoxin M1Monoclonal antibody is to aflatoxin B1Cross reacting rate be 54.17%, the present invention use enzyme
The aflatoxin B of label1As competition antigen, competition antigen and aflatoxin M are on the one hand reduced1Monoclonal antibody
Affinity solves competition antigen and is not easy to be competed by target antigen, caused by the low problem of sensitivity；Due to aflatoxin
M1The price of standard items is more expensive, therefore also reduces aflatoxin M1The cost of detection；On the other hand, due to the presence of hydroxyl,
Synzyme marks aflatoxin M1Difficulty increase, therefore, the present invention select aflatoxin B1Conjugate with enzyme is as competing
Strive antigen.
Gold nanoparticle molar extinction coefficient with higher and apparent surface plasma characteristic, can be by changing it
Size, shape, component and dispersity change solution colour, but need a large amount of gold nanoparticles that could be tied with naked eye to experiment
Fruit carries out interpretation, and error is larger, and dynamic scattering analysis technology can distinguish the dimerization of single nanoparticle, nanoparticle
Aflatoxin M may be implemented compared to traditional substrate development process in body, oligomer or polymer1Trace detection.
The aflatoxin B that the present invention uses glucose oxidase to mark1As competition antigen, competes and tie with determinand
Together on ELISA Plate, by addition glucose, it is made to generate gluconic acid and hydrogen peroxide under the action of glucose oxidase, it is double
Oxygen water generates hydroxy radical under the action of horseradish peroxidase, and tyrasamine forms under the action of hydroxy radical and has amino
Reticular structure, pass through electrostatic interaction, induction with carboxyl lemon acid surfaces gold nanoparticle aggregation, measure Jenner's grain of rice
The average aquation partial size of son, by drawing aflatoxin M1The standard curve of standard items and average aquation partial size obtains determinand
Concentration and gold nanoparticle are averaged the linear relationship between aquation partial size, to calculate aflatoxin M1Content.
When the aflatoxin M to dissociate in determinand1It is more, due to lacking corresponding enzyme and subsequent catalysis reaction, only
There is minimal amount of hydrogen peroxide, the aggregation extent of gold nanoparticle is lower, and gold nanoparticle is caused to be averaged aquation partial size with regard to smaller；
Conversely, then the average aquation partial size of gold nanoparticle is bigger.
In the present invention, step (1) is described by aflatoxin M1Monoclonal antibody is coated in specific on solid carrier
Method are as follows:
(I) buffer diluted protein G is used, and dilution is added in ELISA Plate, is stood；
(II) liquid in ELISA Plate, washing are removed, and is added with the diluted aspergillus flavus resisting toxin M of buffer1Monoclonal
Antibody is stood；
(III) liquid in ELISA Plate is removed, washing adds the closing of bovine serum albumin(BSA) confining liquid, removes confining liquid.
In the present invention, step (I) buffer is 0.04-0.06mol/L (such as 0.04mol/L, 0.045mol/
L, 0.05mol/L, 0.055mol/L or 0.06mol/L etc.) pH=9.4-9.8 (such as 9.4,9.5,9.6,9.7 or 9.8 etc.)
Carbonate buffer solution.
Preferably, the concentration after the dilution of step (I) Protein G is 18-22 μ g/mL, for example, 18 μ g/mL, 19 μ g/mL,
20 μ g/mL, 21 μ g/mL, 22 μ g/mL or 23 μ g/mL etc..
Preferably, step (I) ELISA Plate is 96 hole elisa Plates.
Preferably, it is 100 holes μ L/ that step (I) described dilution, which is added to the amount in ELISA Plate,.
Preferably, the time of step (I) described standing be 8-12h, such as 8h, 9h, 9.5h, 10h, 10.5h, 11h,
11.5h or 12h etc..
Preferably, the temperature of step (I) described standing is 0-8 DEG C, such as 0 DEG C, 1 DEG C, 2 DEG C, 3 DEG C, 4 DEG C, 5 DEG C, 6 DEG C, 7
DEG C or 8 DEG C etc..
In the present invention, step (II) it is described washing for containing 0.01-0.06% (such as 0.01%, 0.02%,
0.03%, 0.04%, 0.05% or 0.06% etc.) phosphate buffer of Tween-20 is washed.
Preferably, the concentration of the phosphate buffer is 0.01mol/L.
Preferably, the pH value of the phosphate buffer is 7.0-7.5, such as 7.0,7.1,7.2,7.3,7.4 or 7.5 etc..
Preferably, step (II) buffer is the phosphate buffer of 0.01mol/L pH=7.4.
Preferably, step (II) the aspergillus flavus resisting toxin M1Concentration after monoclonal antibody dilution is 0.1-1 μ g/mL,
Such as 0.1 μ g/mL, 0.2 μ g/mL, 0.3 μ g/mL, 0.4 μ g/mL, 0.5 μ g/mL, 0.6 μ g/mL, 0.7 μ g/mL, 0.8 μ g/mL,
0.9 μ g/mL or 1 μ g/mL etc..
Preferably, the time of step (II) described standing is 1-3h, such as 1h, 1.5h, 2h, 2.2h, 2.5h, 2.8h or 3h
Deng.
Preferably, the temperature of step (II) described standing is 35-39 DEG C, such as 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C or 39 DEG C
Deng.
In the present invention, step (III) it is described washing for containing 0.01-0.06% (such as 0.01%, 0.02%,
0.03%, 0.04%, 0.05% or 0.06% etc.) phosphate buffer of Tween-20 is washed.
Preferably, the concentration of the phosphate buffer is 0.01mol/L.
Preferably, the pH value of the phosphate buffer is 7.0-7.5, such as 7.0,7.1,7.2,7.3,7.4 or 7.5 etc..
Preferably, step (III) the closed time is 1-3h, such as 1h, 1.5h, 2h, 2.5h or 3h etc..
Preferably, step (III) the closed temperature is 35-39 DEG C, such as 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C or 39 DEG C
Deng.
Preferably, it is washed after step (1) the formation solid phase antibody.
Preferably, step (1) it is described washing for containing 0.01-0.06% (such as 0.01%, 0.02%, 0.03%,
0.04%, 0.05% or 0.06% etc.) phosphate buffer of Tween-20 is washed.
Preferably, the concentration of the phosphate buffer is 0.01mol/L.
Preferably, the pH value of the phosphate buffer is 7.0-7.5, such as 7.0,7.1,7.2,7.3,7.4 or 7.5 etc..
In the present invention, the aflatoxin B of step (2) the glucose oxidase label1The preparation method comprises the following steps:
(A) by aflatoxin B1It is dissolved in pyridine, is protected from light to aflatoxin B with Carboxvmethoxv amine hydrochlorate1
By complete oximate, aflatoxin B is obtained1Oxime；
(B) aflatoxin B for obtaining step (A)1Oxime is dissolved in tetrahydrofuran, and N- hydroxysuccinimide is added
With 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride, it is protected from light；
(C) product that step (B) obtains is centrifuged, collects solid, remove tetrahydrofuran and be dissolved in N, N-
Dimethylformamide is added into the sodium bicarbonate solution containing glucose oxidase later, is protected from light, and is obtained described
The aflatoxin B of glucose oxidase label1。
In the present invention, step (A) time being protected from light is 2-10h, for example, 2h, 3h, 4h, 5h, 6h, 7h, 8h,
9h or 10h etc..
Preferably, step (A) is described is protected from light the monitoring reacted with thin-layered chromatography.
Preferably, by aflatoxin B after the completion of step (A)1Oxime ethyl acetate is extracted and is purified.
Preferably, step (B) is described is protected from light the monitoring reacted with thin-layered chromatography.
Preferably, the speed of step (C) described centrifugation be 3000-8000rpm, such as 3000rpm, 4000rpm,
5000rpm, 5500rpm, 6000rpm, 6500rpm, 7000rpm or 8000rpm etc..
Preferably, the time of step (C) described centrifugal filtration be 10-20min, such as 10min, 11min, 12min,
13min, 14min, 15min, 16min, 17min, 18min, 19min or 20min etc..
Preferably, step (C) glucose oxidase and aflatoxin B1Molar ratio be 1:(5-50), such as 1:
5,1:6,1:8,1:10,1:12,1:15,1:20,1:25,1:30,1:40,1:45 or 1:50 etc., preferably 1:10.
Preferably, step (C) time being protected from light is 1-5h, such as 1h, 2h, 3h, 4h or 5h etc..
Preferably, the aflatoxin B marked obtained glucose oxidase after the completion of step (C)1Dialyse pure
Change, the aflatoxin B of the glucose oxidase label purified1。
In the present invention, the additional amount of step (2) described sample to be tested is 40~60 holes μ L/, such as 40 holes μ L/, 45 μ L/
Hole, 50 holes μ L/, 55 holes μ L/ or 60 holes μ L/ etc., preferably 50 holes μ L/.
Preferably, the aflatoxin B of step (2) the glucose oxidase label1Additional amount be 40~60 μ L/
Hole, such as 40 holes μ L/, 45 holes μ L/, 50 holes μ L/, 55 holes μ L/ or 60 holes μ L/ etc., preferably 50 holes μ L/.
Preferably, the time of step (2) described reaction be 20-80min, such as 20min, 30min, 40min, 50min,
60min, 70min or 80min etc..
Preferably, the temperature of step (2) described reaction is 35-39 DEG C, such as 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C or 39 DEG C
Deng.
Preferably, it is washed after step (2) described reaction.
Preferably, step (2) it is described washing for containing 0.01-0.06% (such as 0.01%, 0.02%, 0.03%,
0.04%, 0.05% or 0.06% etc.) phosphate buffer of Tween-20 is washed.
Preferably, the concentration of the phosphate buffer is 0.01mol/L.
Preferably, the pH value of the phosphate buffer is 7.0-7.5, such as 7.0,7.1,7.2,7.3,7.4 or 7.5 etc..
Preferably, the time of step (3) described reaction be 40-90min, such as 40min, 50min, 60min, 70min,
80min or 90min etc..
Preferably, the temperature of step (3) described reaction is 35-39 DEG C, such as 35 DEG C, 36 DEG C, 37 DEG C, 38 DEG C or 39 DEG C
Deng.
Preferably, step (4) described colloidal gold is prepared by the following method:
Gold chloride is heated to boiling, then citric acid three sodium solution is added thereto, continues to heat, become to solution colour
Peony, cooling with ice water, Purification by suction filtration obtains the colloidal gold.
Preferably, the concentration of the gold chloride and trisodium citrate ratio be 1:(30-50), such as 1:30,1:32,1:35,
1:37,1:40,1:42,1:45,1:48 or 1:50 etc..
Preferably, the colloidal gold preparation is completed to be placed at 4 DEG C to save.
Preferably, the partial size of the colloidal gold is 13nm.
Preferably, the additional amount of step (4) described colloidal gold is the hole 90-110 μ L/, such as 90 holes μ L/, 95 holes μ L/, 100
The hole μ L/, 105 holes μ L/ or 110 holes μ L/ etc., preferably 100 holes μ L/.
Preferably, the additional amount of step (4) described horseradish peroxidase is the hole 40-60 μ L/, such as 40 holes μ L/, 45 μ
The hole L/, 50 holes μ L/, 55 holes μ L/ or 60 holes μ L/ etc., preferably 50 holes μ L/.
Preferably, the additional amount of step (4) described tyrasamine is the hole 40-60 μ L/, such as 40 holes μ L/, 45 holes μ L/, 50 μ L/
Hole, 55 holes μ L/ or 60 holes μ L/ etc., preferably 50 holes μ L/.
Preferably, the temperature of step (4) described reaction is 20-30 DEG C, such as 20 DEG C, 21 DEG C, 22 DEG C, 23 DEG C, 24 DEG C, 25
DEG C, 26 DEG C, 27 DEG C, 28 DEG C, 29 DEG C or 30 DEG C etc..
Preferably, the time of step (4) described reaction be 1-10min, such as 1min, 2min, 3min, 4min, 5min,
6min, 7min, 8min, 9min or 10min etc..
Preferably, the average aquation partial size of step (5) the detection colloidal gold is detected using laser particle analyzer.
Preferably, a length of 633nm of the excitation light wave of the detection, detection angles are 173 °.
Preferably, described by aflatoxin M1The standard curve that standard items are established obtains by the following method: choosing
Take the aflatoxin M of 5-10 various concentration1Standard solution, according to step described in claim 2 to glue under each standard items
The average aquation partial size of body gold is measured, using the value of average aquation partial size as ordinate, aflatoxin M1Concentration be cross
Coordinate establishes standard curve, finds out the equation of standard curve.
As the preferred technical solution of the present invention, the method specifically includes the following steps:
(1) by aflatoxin M1Monoclonal antibody is coated in 96 hole elisa Plates, is formed solid phase antibody, is used 0.01mol/
The phosphate buffer containing 0.01-0.06% Tween-20 of L pH=7.0-7.5 is washed；
(2) it is added in the solid phase antibody obtained to step (1) and contains aflatoxin M1Sample to be tested and grape glycosyloxy
Change the aflatoxin B of enzyme label1, it is uniformly mixed, 20-80min is reacted at 35-39 DEG C, with 0.01mol/L pH=7.0-
7.5 phosphate buffer containing 0.01-0.06% Tween-20 is washed；
(3) glucose solution is added in the mixed liquor obtained to step (2), is uniformly mixed, reacts 40- at 35-39 DEG C
90min；
(4) horseradish peroxidase, tyrasamine, the colloidal gold that partial size is 13nm are added in the mixed liquor obtained to step (3)
Solution reacts 1-10min at 20-30 DEG C；
(5) the average aquation partial size that colloidal gold is detected with laser particle analyzer, according to aflatoxin M1What standard items were established
Aflatoxin M in sample to be tested is calculated in standard curve1Content.
Compared with the existing technology, the invention has the following advantages:
The present invention uses the aflatoxin B of glucose oxidase label1As competition antigen, reduce competition antigen with
Aflatoxin M1The affinity of monoclonal antibody solves competition antigen and is not easy sensitivity caused by being competed by target antigen
Low problem；
The present invention uses aflatoxin B1Instead of aflatoxin M1It is coupled with enzyme as competition antigen, solves Huang Qu
Mould toxin M1Standard items price, at high cost, aflatoxin M1The difficult problem with enzyme coupling；
The present invention carries out the detection of output signal by means of dynamic light scattering method, can be with compared to traditional substrate development process
Detection sensitivity is improved, realizes aflatoxin M1Trace detection.
Detailed description of the invention
Fig. 1 is the schematic illustration of detection method of the present invention；
Fig. 2 is average aquation partial size percentage-concentration of the detection method described in embodiment 1 in 0-32pg/mL concentration range
Relational graph；
Fig. 3 is the canonical plotting of detection method described in embodiment 1；
Fig. 4 is the canonical plotting of detection method described in embodiment 2；
Fig. 5 is the canonical plotting of detection method described in embodiment 3；
Fig. 6 is the canonical plotting of detection method described in embodiment 4；
Fig. 7 is the canonical plotting of detection method described in comparative example 1；
Fig. 8 is the canonical plotting of detection method described in comparative example 2.
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright
, the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention.
Aflatoxin M involved in following embodiment1The Huang of monoclonal antibody and horseradish peroxidase-labeled is bent
Mould toxin B1Working solution comes from Wuxi Zhongde Bore Bioisystech Co., Ltd, aflatoxin M1、B1Purchased from Sigma company.
The preparation method of phosphate buffer involved in following embodiment (PBS, 0.01M, pH7.4) are as follows: by 40g
NaCl、13.5g Na2HPO4、1.0g KH2PO4, 1.0g KCl be dissolved in 1L ultrapure water, with 0.1M NaOH tune pH value to 8.0-
9.0。
Embodiment 1
The present embodiment provides one kind with glucose oxidase and aflatoxin B1Hapten conjugation is used as competition antigen, with
Glucose detects aflatoxin M in cow's milk based on the competitive ELISA absorption method of dynamic light scattering as substrate1
The method of residual quantity.The specific operation method is as follows:
(1) formal test:
With the μ g/mL of carbonate buffer solution diluted protein G to 20 of 0.05mol/L pH=9.6, and by dilution with 100 μ
The hole L/ is added in 96 hole elisa Plates, stands 12h at 4 DEG C；The liquid in ELISA Plate is removed, with the polysorbas20 containing 0.05%
0.01mol/L PBS washing ELISA Plate washed once again with the PBS of 0.01mol/L afterwards three times；It is added and is used with 100 holes μ L/
The diluted concentration of the phosphate buffer of 0.01mol/L pH=7.4 is the aspergillus flavus resisting toxin M of 0.1 μ g/mL1Monoclonal is anti-
Body stands 1h at 37 DEG C；The liquid in ELISA Plate is removed, is washed with the 0.01mol/L PBS of the polysorbas20 containing 0.05%
ELISA Plate washed once with the PBS of 0.01mol/L again afterwards three times；The bovine serum albumin(BSA) of 10mg/mL is added with 340 holes μ L/
Confining liquid is incubated for 120min at 37 DEG C, confining liquid is removed, with the 0.01mol/L PBS detersive enzyme of the polysorbas20 containing 0.05%
Target washed once with the PBS of 0.01mol/L again afterwards three times.
Respectively to being coated with aflatoxin M114 kinds of various concentrations are added with 50 holes μ L/ in the ELISA Plate of monoclonal antibody
(concentration be respectively 0pg/mL, 0.008pg/mL, 0.016pg/mL, 0.032pg/mL, 0.063pg/mL, 0.13pg/mL,
0.25pg/mL、0.5pg/mL、1.00pg/mL、2.00pg/mL、4.00pg/mL、8.00pg/mL、16.00pg/mL、
32.00pg/mL) aflatoxin M1Standard items, then the aflatoxin B that glucose oxidase marks is added with 50 holes μ L/1
Working solution is uniformly mixed, 30min is reacted at 37 DEG C, with the 0.01mol/L PBS detersive enzyme mark of the polysorbas20 containing 0.05%
Plate washed once with the PBS of 0.01mol/L again afterwards three times.
The glucose solution of 0.5mol/L is added with 100 holes μ L/, is uniformly mixed, reacts 60min at 37 DEG C.Respectively with
Horseradish peroxidase, tyrasamine and 13nm colloidal gold solution is added in 50 holes μ L/, 50 holes μ L/, 100 holes μ L/, reacts at 25 DEG C
5min；Measure the average aquation partial size of colloidal gold.
(2) standard curve is established:
Average aquation partial size percentage is first calculated according to the following formula:
Average aquation partial size percentage (%)=D/D0× 100%
Wherein, D0For the aflatoxin M of 0pg/mL1The average aquation particle size values measured under standard items, D are remaining 13 kinds
Concentration aflatoxin M1The average aquation particle size values measured under standard items.
Using the average aquation partial size percentage of colloidal gold as ordinate, aflatoxin M1The logarithm of the concentration of standard items
For abscissa, standard curve is established, standard curve is y=-18.6ln (x)+49.87, R2=0.9985, such as the institute of attached drawing 2 and 3
Show.With reference to the accompanying drawings 2 and 3 it is found that the detection method IC50Value is 0.99pg/mL, wherein IC50It is defined as average aquation partial size
Percentage aflatoxin M corresponding when being 50%1Concentration.
(3) contain aflatoxin M1Sample detection:
Sample pre-treatments: by 75% methanol aqueous solution dissolution extraction of cow's milk, concussion reaction 30min, is mixed at room temperature
It closes uniformly, is then centrifuged and is separated by solid-liquid separation under the conditions of 5000rpm, 10min, discard sediment, take supernatant to retain spare.
The step of repeating above-mentioned (2) formal test, only aflatoxin M1Standard items are replaced with containing aflatoxin M1
Above-mentioned sample solution, after the average aquation partial size for measuring colloidal gold, average aquation partial size fraction values are substituted into standard curve
In, aflatoxin M in sample solution is calculated1Concentration value, by its multiplied by the diluted multiple of solution i.e. obtain in sample
Aflatoxin M1Actual concentrations.
Embodiment 2
The present embodiment provides one kind with glucose oxidase and aflatoxin B1Hapten conjugation is used as competition antigen, with
Glucose detects aflatoxin M in cow's milk based on the competitive ELISA absorption method of dynamic light scattering as substrate1
The method of residual quantity.The specific operation method is as follows:
(1) formal test:
With the μ g/mL of carbonate buffer solution diluted protein G to 18 of 0.04mol/L pH=9.4, and by dilution with 100 μ
The hole L/ is added in 96 hole elisa Plates, stands 8h at 0 DEG C；The liquid in ELISA Plate is removed, with the polysorbas20 containing 0.01%
0.01mol/L PBS washing ELISA Plate washed once again with the PBS of 0.01mol/L afterwards three times；It is added and is used with 100 holes μ L/
The diluted concentration of the phosphate buffer of 0.01mol/L pH=7.4 is the aspergillus flavus resisting toxin M of 0.5 μ g/mL1Monoclonal is anti-
Body stands 2h at 35 DEG C；The liquid in ELISA Plate is removed, is washed with the 0.01mol/L PBS of the polysorbas20 containing 0.01%
ELISA Plate washed once with the PBS of 0.01mol/L again afterwards three times；The bovine serum albumin(BSA) of 10mg/mL is added with 340 holes μ L/
Confining liquid is incubated for 60min at 35 DEG C, confining liquid is removed, with the 0.01mol/L PBS detersive enzyme of the polysorbas20 containing 0.01%
Target washed once with the PBS of 0.01mol/L again afterwards three times.
Respectively to being coated with aflatoxin M114 kinds of various concentrations are added with 40 holes μ L/ in the ELISA Plate of monoclonal antibody
(concentration be respectively 0pg/mL, 0.008pg/mL, 0.016pg/mL, 0.032pg/mL, 0.063pg/mL, 0.13pg/mL,
0.25pg/mL、0.5pg/mL、1.00pg/mL、2.00pg/mL、4.00pg/mL、8.00pg/mL、16.00pg/mL、
32.00pg/mL) aflatoxin M1Standard items, then the aflatoxin B that glucose oxidase marks is added with 40 holes μ L/1
Working solution is uniformly mixed, 20min is reacted at 35 DEG C, with the 0.01mol/L PBS detersive enzyme mark of the polysorbas20 containing 0.01%
Plate washed once with the PBS of 0.01mol/L again afterwards three times.
The glucose solution of 0.5mol/L is added with 100 holes μ L/, is uniformly mixed, reacts 40min at 35 DEG C.Respectively with
Horseradish peroxidase, tyrasamine and 13nm colloidal gold solution is added in 40 holes μ L/, 40 holes μ L/, 90 holes μ L/, reacts at 20 DEG C
10min；Measure the average aquation partial size of colloidal gold.
(2) standard curve is established:
Average aquation partial size percentage is first calculated according to the following formula:
Average aquation partial size percentage (%)=D/D0× 100%
Wherein, D0For the aflatoxin M of 0pg/mL1The average aquation particle size values measured under standard items, D are remaining 13 kinds
Concentration aflatoxin M1The average aquation particle size values measured under standard items.
Using the average aquation partial size percentage of colloidal gold as ordinate, aflatoxin M1The logarithm of the concentration of standard items
For abscissa, standard curve is established, standard curve is y=-19.01ln (x)+52.178, R2=0.991, as shown in Fig. 4.
With reference to the accompanying drawings 4 it is found that the detection method IC50Value is 1.121pg/mL, wherein IC50It is defined as average aquation partial size percentage
Rate aflatoxin M corresponding when being 50%1Concentration.
(3) contain aflatoxin M1Sample detection:
Sample pre-treatments: by 75% methanol aqueous solution dissolution extraction of cow's milk, concussion reaction 30min, is mixed at room temperature
It closes uniformly, is then centrifuged and is separated by solid-liquid separation under the conditions of 5000rpm, 10min, discard sediment, take supernatant to retain spare.
The step of repeating above-mentioned (2) formal test, only aflatoxin M1Standard items are replaced with containing aflatoxin M1
Above-mentioned sample solution, after the average aquation partial size for measuring colloidal gold, average aquation partial size fraction values are substituted into standard curve
In, aflatoxin M in sample solution is calculated1Concentration value, by its multiplied by the diluted multiple of solution i.e. obtain in sample
Aflatoxin M1Actual concentrations.
Embodiment 3
The present embodiment provides one kind with glucose oxidase and aflatoxin B1Hapten conjugation is used as competition antigen, with
Glucose detects aflatoxin M in cow's milk based on the competitive ELISA absorption method of dynamic light scattering as substrate1
The method of residual quantity.The specific operation method is as follows:
(1) formal test:
With the μ g/mL of carbonate buffer solution diluted protein G to 22 of 0.06mol/L pH=9.8, and by dilution with 100 μ
The hole L/ is added in 96 hole elisa Plates, stands 12h at 8 DEG C；The liquid in ELISA Plate is removed, with the polysorbas20 containing 0.06%
0.01mol/L PBS washing ELISA Plate washed once again with the PBS of 0.01mol/L afterwards three times；It is added and is used with 100 holes μ L/
The diluted concentration of the phosphate buffer of 0.01mol/L pH=7.4 is the aspergillus flavus resisting toxin M of 1 μ g/mL1Monoclonal antibody,
3h is stood at 39 DEG C；The liquid in ELISA Plate is removed, with the 0.01mol/L PBS detersive enzyme mark of the polysorbas20 containing 0.06%
Plate washed once with the PBS of 0.01mol/L again afterwards three times；The bovine serum albumin(BSA) closing of 10mg/mL is added with 340 holes μ L/
Liquid is incubated for 180min at 39 DEG C, removes confining liquid, washs ELISA Plate with the 0.01mol/L PBS of the polysorbas20 containing 0.06%
It washed once again with the PBS of 0.01mol/L afterwards three times.
Respectively to being coated with aflatoxin M114 kinds of various concentrations are added with 60 holes μ L/ in the ELISA Plate of monoclonal antibody
(concentration be respectively 0pg/mL, 0.008pg/mL, 0.016pg/mL, 0.032pg/mL, 0.063pg/mL, 0.13pg/mL,
0.25pg/mL、0.5pg/mL、1.00pg/mL、2.00pg/mL、4.00pg/mL、8.00pg/mL、16.00pg/mL、
32.00pg/mL) aflatoxin M1Standard items, then the aflatoxin B that glucose oxidase marks is added with 60 holes μ L/1
Working solution is uniformly mixed, 80min is reacted at 39 DEG C, with the 0.01mol/L PBS detersive enzyme mark of the polysorbas20 containing 0.06%
Plate washed once with the PBS of 0.01mol/L again afterwards three times.
The glucose solution of 0.5mol/L is added with 100 holes μ L/, is uniformly mixed, reacts 90min at 39 DEG C.Respectively with
Horseradish peroxidase, tyrasamine and 13nm colloidal gold solution is added in 60 holes μ L/, 60 holes μ L/, 110 holes μ L/, reacts at 30 DEG C
1min；Measure the average aquation partial size of colloidal gold.
(2) standard curve is established:
Average aquation partial size percentage is first calculated according to the following formula:
Average aquation partial size percentage (%)=D/D0× 100%
Wherein, D0For the aflatoxin M of 0pg/mL1The average aquation particle size values measured under standard items, D are remaining 13 kinds
Concentration aflatoxin M1The average aquation particle size values measured under standard items.
Using the average aquation partial size percentage of colloidal gold as ordinate, aflatoxin M1The logarithm of the concentration of standard items
For abscissa, standard curve is established, standard curve is y=-18.2ln (x)+49.93, R2=0.9971, as shown in Fig. 5.Root
According to attached drawing 5 it is found that the IC of the detection method50Value is 0.9961pg/mL, wherein IC50It is defined as average aquation partial size percentage
Corresponding aflatoxin M when being 50%1Concentration.
(3) contain aflatoxin M1Sample detection:
Sample pre-treatments: by 75% methanol aqueous solution dissolution extraction of cow's milk, concussion reaction 30min, is mixed at room temperature
It closes uniformly, is then centrifuged and is separated by solid-liquid separation under the conditions of 5000rpm, 10min, discard sediment, take supernatant to retain spare.
The step of repeating above-mentioned (2) formal test, only aflatoxin M1Standard items are replaced with containing aflatoxin M1
Above-mentioned sample solution, after the average aquation partial size for measuring colloidal gold, average aquation partial size fraction values are substituted into standard curve
In, aflatoxin M in sample solution is calculated1Concentration value, by its multiplied by the diluted multiple of solution i.e. obtain in sample
Aflatoxin M1Actual concentrations.
Embodiment 4
The present embodiment provides one kind with glucose oxidase and aflatoxin B1Hapten conjugation is used as competition antigen, with
Glucose detects aflatoxin M in cow's milk based on the competitive ELISA absorption method of dynamic light scattering as substrate1
The method of residual quantity.The specific operation method is as follows:
(1) formal test:
With the μ g/mL of carbonate buffer solution diluted protein G to 20 of 0.05mol/L pH=9.4, and by dilution with 100 μ
The hole L/ is added in 96 hole elisa Plates, stands 8h at 4 DEG C；The liquid in ELISA Plate is removed, with the polysorbas20 containing 0.05%
0.01mol/L PBS washing ELISA Plate washed once again with the PBS of 0.01mol/L afterwards three times；It is added and is used with 100 holes μ L/
The diluted concentration of the phosphate buffer of 0.01mol/L pH=7.4 is the aspergillus flavus resisting toxin M of 0.3 μ g/mL1Monoclonal is anti-
Body stands 2h at 35 DEG C；The liquid in ELISA Plate is removed, is washed with the 0.01mol/L PBS of the polysorbas20 containing 0.05%
ELISA Plate washed once with the PBS of 0.01mol/L again afterwards three times；The bovine serum albumin(BSA) of 10mg/mL is added with 340 holes μ L/
Confining liquid is incubated for 60min at 37 DEG C, confining liquid is removed, with the 0.01mol/L PBS detersive enzyme of the polysorbas20 containing 0.05%
Target washed once with the PBS of 0.01mol/L again afterwards three times.
Respectively to being coated with aflatoxin M114 kinds of various concentrations are added with 50 holes μ L/ in the ELISA Plate of monoclonal antibody
(concentration be respectively 0pg/mL, 0.008pg/mL, 0.016pg/mL, 0.032pg/mL, 0.063pg/mL, 0.13pg/mL,
0.25pg/mL、0.5pg/mL、1.00pg/mL、2.00pg/mL、4.00pg/mL、8.00pg/mL、16.00pg/mL、
32.00pg/mL) aflatoxin M1Standard items, then the aflatoxin B that glucose oxidase marks is added with 50 holes μ L/1
Working solution is uniformly mixed, 20min is reacted at 37 DEG C, with the 0.01mol/L PBS detersive enzyme mark of the polysorbas20 containing 0.05%
Plate washed once with the PBS of 0.01mol/L again afterwards three times.
The glucose solution of 0.5mol/L is added with 100 holes μ L/, is uniformly mixed, reacts 40min at 35 DEG C.Respectively with
Horseradish peroxidase, tyrasamine and 13nm colloidal gold solution is added in 50 holes μ L/, 50 holes μ L/, 100 holes μ L/, reacts at 25 DEG C
10min；Measure the average aquation partial size of colloidal gold.
(2) standard curve is established:
Average aquation partial size percentage is first calculated according to the following formula:
Average aquation partial size percentage (%)=D/D0× 100%
Wherein, D0For the aflatoxin M of 0pg/mL1The average aquation particle size values measured under standard items, D are remaining 13 kinds
Concentration aflatoxin M1The average aquation particle size values measured under standard items.
Using the average aquation partial size percentage of colloidal gold as ordinate, aflatoxin M1The log concentration value of standard items is
Abscissa, establishes standard curve, and standard curve is y=-20.31ln (x)+51.2, R2=0.993, as shown in Fig. 6.According to
Attached drawing 6 it is found that the detection method IC50Value is 1.06pg/mL, wherein IC50Being defined as average aquation partial size percentage is
Corresponding aflatoxin M when 50%1Concentration.
(3) contain aflatoxin M1Sample detection:
Sample pre-treatments: by 75% methanol aqueous solution dissolution extraction of cow's milk, concussion reaction 30min, is mixed at room temperature
It closes uniformly, is then centrifuged and is separated by solid-liquid separation under the conditions of 5000rpm, 10min, discard sediment, take supernatant to retain spare.
The step of repeating above-mentioned (2) formal test, only aflatoxin M1Standard items are replaced with containing aflatoxin M1
Above-mentioned sample solution, after the average aquation partial size for measuring colloidal gold, average aquation partial size fraction values are substituted into standard curve
In, aflatoxin M in sample solution is calculated1Concentration value, by its multiplied by the diluted multiple of solution i.e. obtain in sample
Aflatoxin M1Actual concentrations.
Comparative example 1
The present embodiment provides one kind with horseradish peroxidase and aflatoxin B1Hapten conjugation is used as competition antigen,
Competitive ELISA absorption method using TMB as substrate detects aflatoxin M in cow's milk1The method of residual quantity.Tool
Body operating method is as follows:
(1) formal test:
Aflatoxin M is coated with according to the method preparation in embodiment 11The ELISA Plate of monoclonal antibody.
Respectively into solid phase antibody with 50 holes μ L/ be added 8 kinds of various concentrations (concentration be respectively 0ng/mL, 0.05ng/mL,
0.25ng/mL, 0.5ng/mL, 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL) aflatoxin M1Standard items, then with 50
The aflatoxin B of the hole μ L/ addition horseradish peroxidase-labeled1Working solution is uniformly mixed, reacts 45min at 37 DEG C, go
Except liquid in hole, with 340 hole μ L/ of wash operating solution, sufficiently washing 4-5 times, every minor tick 10s is patted dry with blotting paper.
TMB developing solution is added with 100 holes μ L/, is uniformly mixed, reacts 15min at 37 DEG C, is added and is terminated with 50 holes μ L/
Liquid reacts 5min at 25 DEG C；Absorbance value is detected at 450 nm with microplate reader.
(2) standard curve is established:
Mean percent absorptance is first calculated according to the following formula:
Mean percent absorptance (%)=B/B0× 100%
Wherein, B0For the aflatoxin M of 0ng/mL1The mean absorbance values measured under standard items, B are remaining 7 kinds of concentration
Aflatoxin M1The mean absorbance values measured under standard items.
Using mean percent absorbance value as ordinate, aflatoxin M1The logarithm of the concentration of standard items is abscissa,
Standard curve is established, standard curve is y=-13.39ln (x)+41.64, R2=0.9954, as shown in Fig. 7.With reference to the accompanying drawings 7
It is found that the IC of the detection method50Value is 0.54ng/mL, wherein IC50It is right when mean percent absorptance is 50% to be defined as
The aflatoxin M answered1Concentration.
(3) contain aflatoxin M1Sample detection:
Sample pre-treatments: by 75% methanol aqueous solution dissolution extraction of cow's milk, concussion reaction 30min, is mixed at room temperature
It closes uniformly, is then centrifuged and is separated by solid-liquid separation under the conditions of 5000rpm, 10min, discard sediment, take supernatant to retain spare.
The step of repeating above-mentioned (2) formal test, only aflatoxin M1Standard items are replaced with containing aflatoxin M1
Above-mentioned sample solution, measure absorbance value after, by mean percent absorbance value substitute into standard curve in, it is molten that sample is calculated
Aflatoxin M in liquid1Concentration value, it is obtained into aflatoxin M in sample multiplied by the diluted multiple of solution1Reality
Concentration.
Comparative example 2
The present embodiment provides one kind with glucose oxidase and aflatoxin B1Hapten conjugation is used as competition antigen, with
Glucose detects aflatoxin M in cow's milk based on the enzyme linked immunosorbent assay of colloidal gold colorimetric as substrate1Residual quantity
Method.The specific operation method is as follows:
(1) formal test:
With the μ g/mL of carbonate buffer solution diluted protein G to 20 of 0.05mol/L pH=9.4, and by dilution with 100 μ
The hole L/ is added in 96 hole elisa Plates, stands 8h at 4 DEG C；The liquid in ELISA Plate is removed, with the polysorbas20 containing 0.05%
0.01mol/L PBS washing ELISA Plate washed once again with the PBS of 0.01mol/L afterwards three times；It is added and is used with 100 holes μ L/
The diluted concentration of the phosphate buffer of 0.01mol/L pH=7.4 is the aspergillus flavus resisting toxin M of 0.3 μ g/mL1Monoclonal is anti-
Body stands 2h at 35 DEG C；The liquid in ELISA Plate is removed, is washed with the 0.01mol/L PBS of the polysorbas20 containing 0.05%
ELISA Plate washed once with the PBS of 0.01mol/L again afterwards three times；The bovine serum albumin(BSA) of 10mg/mL is added with 340 holes μ L/
Confining liquid is incubated for 60min at 37 DEG C, confining liquid is removed, with the 0.01mol/L PBS detersive enzyme of the polysorbas20 containing 0.05%
Target washed once with the PBS of 0.01mol/L again afterwards three times.
Respectively to being coated with aflatoxin M114 kinds of various concentrations are added with 50 holes μ L/ in the ELISA Plate of monoclonal antibody
(concentration be respectively 0pg/mL, 0.008pg/mL, 0.016pg/mL, 0.032pg/mL, 0.063pg/mL, 0.13pg/mL,
0.25pg/mL、0.5pg/mL、1.00pg/mL、2.00pg/mL、4.00pg/mL、8.00pg/mL、16.00pg/mL、
32.00pg/mL) aflatoxin M1Standard items, then the aflatoxin B that glucose oxidase marks is added with 50 holes μ L/1
Working solution is uniformly mixed, 20min is reacted at 37 DEG C, with the 0.01mol/L PBS detersive enzyme mark of the polysorbas20 containing 0.05%
Plate washed once with the PBS of 0.01mol/L again afterwards three times.
The glucose solution of 0.5mol/L is added with 100 holes μ L/, is uniformly mixed, reacts 40min at 35 DEG C.Respectively with
Horseradish peroxidase, tyrasamine and 13nm colloidal gold solution is added in 50 holes μ L/, 50 holes μ L/, 100 holes μ L/, reacts at 25 DEG C
10min；Record the ultravioletvisible absorption map of 400-700nm.
(2) standard curve is established:
First inhibiting rate is calculated according to the following formula:
Percent inhibition (%)=B/B0× 100%
Wherein, B0For the aflatoxin M of 0ng/mL1The colloidal gold solution measured under standard items is at 520nm and 630nm
The ratio of ultraviolet light absorption angle value, B are remaining 7 kinds of concentration aflatoxin M1The colloidal gold solution measured under standard items is in 520nm
With the ratio of ultraviolet light absorption angle value at 630nm.
With percent inhibition (B/B0) it is ordinate, aflatoxin M1The logarithm of the concentration of standard items is abscissa, is built
Day-mark directrix curve, standard curve are y=-21.29ln (x)+117.83, R2=0.9903, as shown in Fig. 8.It with reference to the accompanying drawings 8 can
Know, the IC of the detection method50Value is 24.19pg/mL, wherein IC50It is defined as Huang corresponding when percent inhibition is 50%
Aspertoxin M1Concentration.
(3) contain aflatoxin M1Sample detection:
Sample pre-treatments: by 75% methanol aqueous solution dissolution extraction of cow's milk, concussion reaction 30min, is mixed at room temperature
It closes uniformly, is then centrifuged and is separated by solid-liquid separation under the conditions of 5000rpm, 10min, discard sediment, take supernatant to retain spare.
The step of repeating above-mentioned (2) formal test, only aflatoxin M1Standard items are replaced with containing aflatoxin M1
Above-mentioned sample solution, measure colloidal gold 400-700nm absorbance value after, by percent inhibition value substitute into standard curve in, meter
Calculation obtains aflatoxin M in sample solution1Concentration value, it is obtained into aspergillus flavus in sample multiplied by the diluted multiple of solution
Toxin M1Actual concentrations.
To sum up, the standard curve that the detection method as described in embodiment 1-4 and comparative example 1-2 obtains is it is found that with comparative example phase
Than using detection method described in embodiment i.e. with glucose oxidase and aflatoxin B1Hapten conjugation is anti-as competition
Original, using glucose as substrate, standard curve I C that the competitive ELISA absorption method based on dynamic light scattering obtains50Value
It significantly reduces, detection sensitivity significantly improves, and realizes aflatoxin M1Trace detection.
The Applicant declares that the present invention is explained by the above embodiments detection aflatoxin M of the invention1Method,
But the present invention is not limited to the above embodiments, that is, does not mean that the present invention must rely on above-described embodiment and could implement.It is affiliated
Those skilled in the art it will be clearly understood that any improvement in the present invention, equivalence replacement to each raw material of product of the present invention and
Addition, selection of concrete mode of auxiliary element etc., all of which fall within the scope of protection and disclosure of the present invention.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
Claims (10)
1. a kind of detection aflatoxin M1Method, which is characterized in that the method is the aflatoxin B marked with enzyme1Make
To compete antigen, and it is based on dynamic light scattering technique, detects aflatoxin M with competitive ELISA analytic approach1Side
Method.
2. the method as described in claim 1, which is characterized in that the described method comprises the following steps:
(1) by aflatoxin M1Monoclonal antibody is coated on solid carrier, forms solid phase antibody；
(2) it is added in the solid phase antibody obtained to step (1) and contains aflatoxin M1Sample to be tested and glucose oxidase mark
The aflatoxin B of note1, it is uniformly mixed, reaction；
(3) glucose solution is added in the mixed liquor obtained to step (2), is uniformly mixed, reaction；
(4) horseradish peroxidase, tyrasamine, colloidal gold solution, reaction are added in the mixed liquor obtained to step (3)；
(5) the average aquation partial size for detecting colloidal gold, according to aflatoxin M1Standard items establish standard curve be calculated to
Aflatoxin M in sample1Content.
3. method according to claim 2, which is characterized in that step (1) is described by aflatoxin M1Monoclonal antibody coating
In on solid carrier method particularly includes:
(I) buffer diluted protein G is used, and dilution is added in ELISA Plate, is stood；
(II) liquid in ELISA Plate, washing are removed, and is added with the diluted aspergillus flavus resisting toxin M of buffer1Monoclonal antibody,
It stands；
(III) liquid in ELISA Plate is removed, washing adds the closing of bovine serum albumin(BSA) confining liquid, removes confining liquid.
4. method as claimed in claim 3, which is characterized in that step (I) buffer is 0.04-0.06mol/L pH=
The carbonate buffer solution of 9.4-9.8；
Preferably, the concentration after step (I) the Protein G dilution is 18-22 μ g/mL；
Preferably, step (I) ELISA Plate is 96 hole elisa Plates；
Preferably, it is 100 holes μ L/ that step (I) described dilution, which is added to the amount in ELISA Plate,；
Preferably, the time of step (I) described standing is 8-12h；
Preferably, the temperature of step (I) described standing is 0-8 DEG C.
5. the method as claimed in claim 3 or 4, which is characterized in that step (II) washing is with containing 0.01-0.06%
The phosphate buffer of Tween-20 is washed；
Preferably, the concentration of the phosphate buffer is 0.01mol/L；
Preferably, the pH value of the phosphate buffer is 7.0-7.5；
Preferably, step (II) buffer is the phosphate buffer of 0.01mol/L pH=7.4；
Preferably, step (II) the aspergillus flavus resisting toxin M1Concentration after monoclonal antibody dilution is 0.1-1 μ g/mL；
Preferably, the time of step (II) described standing is 1-3h；
Preferably, the temperature of step (II) described standing is 35-39 DEG C.
6. such as the described in any item methods of claim 3-5, which is characterized in that step (III) washing is with containing 0.01-
The phosphate buffer of 0.06% Tween-20 is washed；
Preferably, the concentration of the phosphate buffer is 0.01mol/L；
Preferably, the pH value of the phosphate buffer is 7.0-7.5；
Preferably, step (III) the closed time is 1-3h；
Preferably, step (III) the closed temperature is 35-39 DEG C；
Preferably, it is washed after step (1) the formation solid phase antibody；
Preferably, step (1) washing is to be washed with the phosphate buffer containing 0.01-0.06% Tween-20；
Preferably, the concentration of the phosphate buffer is 0.01mol/L；
Preferably, the pH value of the phosphate buffer is 7.0-7.5.
7. method according to claim 2, which is characterized in that the aspergillus flavus poison of step (2) the glucose oxidase label
Plain B1The preparation method comprises the following steps:
(A) by aflatoxin B1It is dissolved in pyridine, is protected from light to aflatoxin B with Carboxvmethoxv amine hydrochlorate1It is complete
Full oximate, obtains aflatoxin B1Oxime；
(B) aflatoxin B for obtaining step (A)1Oxime is dissolved in tetrahydrofuran, and N- hydroxysuccinimide and 1- (3- bis- is added
Methylaminopropyl) -3- ethyl-carbodiimide hydrochloride, it is protected from light；
(C) product that step (B) obtains is centrifuged, collects solid, remove tetrahydrofuran and be dissolved in N, N- diformazan
Base formamide, is added into the sodium bicarbonate solution containing glucose oxidase later, is protected from light, and obtains the grape
The aflatoxin B of carbohydrate oxidase label1。
8. the method for claim 7, which is characterized in that step (A) time being protected from light is 2-10h；
Preferably, step (A) is described is protected from light the monitoring reacted with thin-layered chromatography；
Preferably, by aflatoxin B after the completion of step (A)1Oxime ethyl acetate is extracted and is purified；
Preferably, step (B) is described is protected from light the monitoring reacted with thin-layered chromatography；
Preferably, the speed of step (C) described centrifugation is 3000-8000rpm；
Preferably, the time of step (C) described centrifugal filtration is 10-20min；
Preferably, step (C) glucose oxidase and aflatoxin B1Molar ratio be 1:(5-50), preferably 1:10；
Preferably, step (C) time being protected from light is 1-5h；
Preferably, the aflatoxin B marked obtained glucose oxidase after the completion of step (C)1Dialysis purification is carried out, is obtained
The aflatoxin B marked to the glucose oxidase of purifying1。
9. method according to claim 2, which is characterized in that the additional amount of step (2) described sample to be tested is 40-60 μ L/
Hole, preferably 50 holes μ L/；
Preferably, the aflatoxin B of step (2) the glucose oxidase label1Additional amount be the hole 40-60 μ L/, preferably
50 holes μ L/；
Preferably, the time of step (2) described reaction is 20-80min；
Preferably, the temperature of step (2) described reaction is 35-39 DEG C；
Preferably, it is washed after step (2) described reaction；
Preferably, step (2) washing is to be washed with the phosphate buffer containing 0.01-0.06% Tween-20；
Preferably, the concentration of the phosphate buffer is 0.01mol/L；
Preferably, the pH value of the phosphate buffer is 7.0-7.5；
Preferably, the time of step (3) described reaction is 40-90min；
Preferably, the temperature of step (3) described reaction is 35-39 DEG C；
Preferably, step (4) described colloidal gold is prepared by the following method:
Gold chloride is heated to boiling, then citric acid three sodium solution is added thereto, continues to heat, becomes dark red to solution colour
Color, cooling with ice water, Purification by suction filtration obtains the colloidal gold；
Preferably, the concentration of the gold chloride and trisodium citrate ratio is 1:(30-50)；
Preferably, the colloidal gold preparation is completed to be placed at 4 DEG C to save；
Preferably, the partial size of the colloidal gold is 13nm；
Preferably, the additional amount of step (4) described colloidal gold is the hole 90-110 μ L/, preferably 100 holes μ L/；
Preferably, the additional amount of step (4) described horseradish peroxidase is the hole 40-60 μ L/, preferably 50 holes μ L/；
Preferably, the additional amount of step (4) described tyrasamine is the hole 40-60 μ L/, preferably 50 holes μ L/；
Preferably, the temperature of step (4) described reaction is 20-30 DEG C；
Preferably, the time of step (4) described reaction is 1-10min；
Preferably, the average aquation partial size of step (5) the detection colloidal gold is detected using laser particle analyzer；
Preferably, a length of 633nm of the excitation light wave of the detection, detection angles are 173 °；
Preferably, described by aflatoxin M1The standard curve that standard items are established obtains by the following method: choosing 5-10
The aflatoxin M of a various concentration1Standard solution, according to step described in claim 2 to colloidal gold under each standard items
Average aquation partial size is measured, using the value of average aquation partial size as ordinate, aflatoxin M1Concentration be abscissa, build
Day-mark directrix curve finds out the equation of standard curve.
10. method as claimed in any one of claims 1-9 wherein, which is characterized in that the method specifically includes the following steps:
(1) by aflatoxin M1Monoclonal antibody is coated in 96 hole elisa Plates, solid phase antibody is formed, with 0.01mol/L pH
The phosphate buffer containing 0.01-0.06% Tween-20 of=7.0-7.5 is washed；
(2) it is added in the solid phase antibody obtained to step (1) and contains aflatoxin M1Sample to be tested and glucose oxidase mark
The aflatoxin B of note1, it is uniformly mixed, 20-80min is reacted at 35-39 DEG C, with containing for 0.01mol/L pH=7.0-7.5
There is the phosphate buffer of 0.01-0.06% Tween-20 to be washed；
(3) glucose solution is added in the mixed liquor obtained to step (2), is uniformly mixed, reacts 40- at 35-39 DEG C
90min；
(4) horseradish peroxidase, tyrasamine, the colloidal gold solution that partial size is 13nm are added in the mixed liquor obtained to step (3),
1-10min is reacted at 20-30 DEG C；
(5) the average aquation partial size that colloidal gold is detected with laser particle analyzer, according to aflatoxin M1The standard that standard items are established is bent
Line computation obtains aflatoxin M in sample to be tested1Content.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811346302.9A CN109270260A (en) | 2018-11-13 | 2018-11-13 | A kind of detection aflatoxin M1Method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811346302.9A CN109270260A (en) | 2018-11-13 | 2018-11-13 | A kind of detection aflatoxin M1Method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109270260A true CN109270260A (en) | 2019-01-25 |
Family
ID=65193569
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811346302.9A Pending CN109270260A (en) | 2018-11-13 | 2018-11-13 | A kind of detection aflatoxin M1Method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109270260A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111398577A (en) * | 2020-06-08 | 2020-07-10 | 南京颐兰贝生物科技有限责任公司 | Quantitative immunoassay method |
| CN112067815A (en) * | 2020-09-03 | 2020-12-11 | 南昌大学 | Homogeneous immunization method for detecting small molecule hapten |
| CN117723750A (en) * | 2024-02-07 | 2024-03-19 | 南昌大学 | Dynamic light scattering immune detection method based on streptavidin-biotin reaction |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20140034004A (en) * | 2012-09-11 | 2014-03-19 | 대한민국(농촌진흥청장) | Labeling agent for aflatoxin b1 detection and the kit for detecting aflatoxin b1 comprising thereof |
| CN105759045A (en) * | 2016-03-18 | 2016-07-13 | 南昌大学 | Method for detecting aflatoxin B1 |
| CN106546748A (en) * | 2016-11-02 | 2017-03-29 | 南昌大学 | A kind of aflatoxin B with quantum dot fluorescence microsphere as competition antigen vectors1Detection method |
| KR20170036630A (en) * | 2015-09-23 | 2017-04-03 | 한양대학교 에리카산학협력단 | High-sensitive lateral flow immunoassay strip based on a surface-enhanced raman scattering and method using the same |
| CN107344966A (en) * | 2017-07-05 | 2017-11-14 | 河南省农业科学院 | A kind of method that ZER monoclonal antibody is prepared based on analogue cross reactivity |
-
2018
- 2018-11-13 CN CN201811346302.9A patent/CN109270260A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20140034004A (en) * | 2012-09-11 | 2014-03-19 | 대한민국(농촌진흥청장) | Labeling agent for aflatoxin b1 detection and the kit for detecting aflatoxin b1 comprising thereof |
| KR20170036630A (en) * | 2015-09-23 | 2017-04-03 | 한양대학교 에리카산학협력단 | High-sensitive lateral flow immunoassay strip based on a surface-enhanced raman scattering and method using the same |
| CN105759045A (en) * | 2016-03-18 | 2016-07-13 | 南昌大学 | Method for detecting aflatoxin B1 |
| CN106546748A (en) * | 2016-11-02 | 2017-03-29 | 南昌大学 | A kind of aflatoxin B with quantum dot fluorescence microsphere as competition antigen vectors1Detection method |
| CN107344966A (en) * | 2017-07-05 | 2017-11-14 | 河南省农业科学院 | A kind of method that ZER monoclonal antibody is prepared based on analogue cross reactivity |
Non-Patent Citations (5)
| Title |
|---|
| XITUI CHEN 等: "A colorimetric immunoassay based on glucose oxidase-induced AuNP aggregation for the detection of fumonisin B1", 《TALANTA》 * |
| YI LIANG 等: "Plasmonic ELISA for naked-eye detection of ochratoxin A based on the tyramine-H2O2 amplification system", 《SENSORS AND ACTUATORS B:CHEMICAL》 * |
| YING XIONG 等: "Plasmonic ELISA based on enzyme-assisted etching of Au nanorods for the highly sensitive detection of aflatoxin B1 in corn samples", 《SENSORS AND ACTUATORS B:CHEMICAL》 * |
| 孙清 等: "黄曲霉毒素M1和黄曲霉毒素B1总量酶联免疫试剂盒的研制及应用", 《环境化学》 * |
| 胡梁斌 等: "《黄曲霉毒素危害及其控制》", 31 July 2018, 中国轻工业出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111398577A (en) * | 2020-06-08 | 2020-07-10 | 南京颐兰贝生物科技有限责任公司 | Quantitative immunoassay method |
| CN112067815A (en) * | 2020-09-03 | 2020-12-11 | 南昌大学 | Homogeneous immunization method for detecting small molecule hapten |
| CN117723750A (en) * | 2024-02-07 | 2024-03-19 | 南昌大学 | Dynamic light scattering immune detection method based on streptavidin-biotin reaction |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109270260A (en) | A kind of detection aflatoxin M1Method | |
| Li et al. | Preparing monoclonal antibodies and developing immunochromatographic strips for paraquat determination in water | |
| CN110862881B (en) | Special cleaning solution or diluent for full-automatic chemiluminescence determinator and preparation method thereof | |
| CN103364568B (en) | Laminin nano-magnetic particle chemiluminescent immunity quantitative detection kit and preparation method thereof | |
| CN102998467B (en) | β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof | |
| CN105759045B (en) | One kind is directed to aflatoxin B1Detection method | |
| CN102520196A (en) | Neonatus thyroid stimulating hormone/free thyroxine double-tagging detection kit and corresponding detection method | |
| CN105842441B (en) | A kind of detection method for ochratoxin A | |
| CN102998442A (en) | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for aldosterone, and preparation method of kit | |
| Wang et al. | In situ synthesis of fluorescent copper nanoclusters for rapid detection of ascorbic acid in biological samples | |
| CN110372632A (en) | A kind of fluorescent probe molecule and its preparation method and application of quick identification hypochlorite ion | |
| CN103091494A (en) | Chemiluminescence enzyme-linked immune detection kit of aflatoxin M1 and using method | |
| CN103308683B (en) | CA19-9 time-resolved fluoroimmunoassay and kit | |
| CN105115968A (en) | Method for quickly detecting acrylamide in heat-processed food by use of nano-gold colorimetric biosensor | |
| CN101643436A (en) | Sudan hapten and antigen as well as preparation method and application thereof | |
| EP0249357A2 (en) | Methods of immunoassay | |
| CN112683885B (en) | 5-methyltetrahydrofolate chemiluminescence detection kit and preparation method thereof | |
| CN111285836A (en) | Preparation and application of near-infrared fluorescent probe | |
| CN106290326A (en) | The detection colorimetric sensor of lipopolysaccharide, its preparation method and application | |
| CN112710645A (en) | Method for detecting dopamine and alkaline phosphatase in real time through permanganate-initiated in-situ fluorescence reaction and enzyme-linked immunosorbent assay (ELISA) application thereof | |
| Grafnetter et al. | Note on Slot's method for the specific determination of creatinine | |
| CN105785019B (en) | A kind of detection method for prostate specific antigen | |
| CN1474185A (en) | Luminol chemiluminescence immunological analysis detecting method for cardiac muscle calcium protein | |
| CN110045123A (en) | A kind of carbohydrate antigen 242 magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof | |
| CN108152257B (en) | Method for measuring zilpaterol by utilizing CdTe quantum dots |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190125 |