CN112824877A - Method for preparing fluorescent quantum dot immunochromatography detection card by using aflatoxin - Google Patents
Method for preparing fluorescent quantum dot immunochromatography detection card by using aflatoxin Download PDFInfo
- Publication number
- CN112824877A CN112824877A CN201911146742.4A CN201911146742A CN112824877A CN 112824877 A CN112824877 A CN 112824877A CN 201911146742 A CN201911146742 A CN 201911146742A CN 112824877 A CN112824877 A CN 112824877A
- Authority
- CN
- China
- Prior art keywords
- aflatoxin
- quantum dot
- preparing
- antibody
- solution
- 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 83
- 239000005409 aflatoxin Substances 0.000 title claims abstract description 58
- 229930195730 Aflatoxin Natural products 0.000 title claims abstract description 57
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 239000002096 quantum dot Substances 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000003317 immunochromatography Methods 0.000 title claims abstract description 24
- 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 43
- 229930020125 aflatoxin-B1 Natural products 0.000 claims abstract description 43
- 101100449517 Arabidopsis thaliana GRH1 gene Proteins 0.000 claims abstract description 33
- 101100434479 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) AFB1 gene Proteins 0.000 claims abstract description 33
- 238000002965 ELISA Methods 0.000 claims abstract description 12
- 239000004005 microsphere Substances 0.000 claims description 39
- 239000012528 membrane Substances 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 31
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 30
- 239000012498 ultrapure water Substances 0.000 claims description 30
- 239000000427 antigen Substances 0.000 claims description 25
- 108091007433 antigens Proteins 0.000 claims description 25
- 102000036639 antigens Human genes 0.000 claims description 25
- 238000002360 preparation method Methods 0.000 claims description 25
- 238000006243 chemical reaction Methods 0.000 claims description 24
- 238000012360 testing method Methods 0.000 claims description 23
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- 229940098773 bovine serum albumin Drugs 0.000 claims description 18
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 18
- 238000001914 filtration Methods 0.000 claims description 16
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims description 15
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 15
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 12
- 239000007987 MES buffer Substances 0.000 claims description 12
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 12
- 229910021538 borax Inorganic materials 0.000 claims description 12
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 12
- 238000005303 weighing Methods 0.000 claims description 12
- NPWGWQRXHVJJRD-UHFFFAOYSA-N Hydroxylamino-methan-carbonsaeure Natural products ONCC(O)=O NPWGWQRXHVJJRD-UHFFFAOYSA-N 0.000 claims description 10
- 241000699670 Mus sp. Species 0.000 claims description 10
- 239000002115 aflatoxin B1 Substances 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 10
- 210000002966 serum Anatomy 0.000 claims description 10
- 238000005507 spraying Methods 0.000 claims description 10
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 9
- 241000283707 Capra Species 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 9
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 9
- 239000004328 sodium tetraborate Substances 0.000 claims description 9
- 239000000020 Nitrocellulose Substances 0.000 claims description 7
- 239000002250 absorbent Substances 0.000 claims description 7
- 230000002745 absorbent Effects 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 7
- 210000004027 cell Anatomy 0.000 claims description 7
- 239000003365 glass fiber Substances 0.000 claims description 7
- 230000003053 immunization Effects 0.000 claims description 7
- 238000002649 immunization Methods 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 7
- 229920001220 nitrocellulos Polymers 0.000 claims description 7
- 206010003445 Ascites Diseases 0.000 claims description 6
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
- 230000003213 activating effect Effects 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- 239000003085 diluting agent Substances 0.000 claims description 6
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 6
- 210000004408 hybridoma Anatomy 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 6
- 229910000077 silane Inorganic materials 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- 230000002194 synthesizing effect Effects 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 239000012224 working solution Substances 0.000 claims description 6
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 5
- 230000007910 cell fusion Effects 0.000 claims description 5
- 238000001291 vacuum drying Methods 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 238000003908 quality control method Methods 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
- 239000004327 boric acid Substances 0.000 claims description 3
- 150000001718 carbodiimides Chemical class 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
- 229920002678 cellulose Polymers 0.000 claims description 3
- 239000001913 cellulose Substances 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 239000002274 desiccant Substances 0.000 claims description 3
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 239000013024 dilution buffer 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
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 239000003550 marker Substances 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 239000011734 sodium Substances 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
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 3
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 claims description 3
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 claims 4
- 238000002390 rotary evaporation Methods 0.000 claims 2
- BPFKSUPTSXDJBL-UHFFFAOYSA-N 2-(hydroxyamino)acetic acid;hydrochloride Chemical compound Cl.ONCC(O)=O BPFKSUPTSXDJBL-UHFFFAOYSA-N 0.000 claims 1
- 241000712431 Influenza A virus Species 0.000 claims 1
- 238000010166 immunofluorescence Methods 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 6
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 abstract description 5
- 238000004587 chromatography analysis Methods 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 230000035484 reaction time Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 11
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 9
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 9
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 6
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 238000001704 evaporation Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 231100000572 poisoning Toxicity 0.000 description 3
- 230000000607 poisoning effect Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 231100000111 LD50 Toxicity 0.000 description 2
- ZRWPUFFVAOMMNM-UHFFFAOYSA-N Patulin Chemical compound OC1OCC=C2OC(=O)C=C12 ZRWPUFFVAOMMNM-UHFFFAOYSA-N 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 238000007259 addition reaction Methods 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 241000712461 unidentified influenza virus Species 0.000 description 2
- HJTAZXHBEBIQQX-UHFFFAOYSA-N 1,5-bis(chloromethyl)naphthalene Chemical compound C1=CC=C2C(CCl)=CC=CC2=C1CCl HJTAZXHBEBIQQX-UHFFFAOYSA-N 0.000 description 1
- HJVUQIHMNQMRIV-LOLPMWEVSA-N 4-aminobutyl alpha-L-fucopyranoside Chemical compound C[C@@H]1O[C@@H](OCCCCN)[C@@H](O)[C@H](O)[C@@H]1O HJVUQIHMNQMRIV-LOLPMWEVSA-N 0.000 description 1
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 description 1
- 101100434480 Arabidopsis thaliana AFB2 gene Proteins 0.000 description 1
- 229910004613 CdTe Inorganic materials 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- VYLQGYLYRQKMFU-UHFFFAOYSA-N Ochratoxin A Natural products CC1Cc2c(Cl)cc(CNC(Cc3ccccc3)C(=O)O)cc2C(=O)O1 VYLQGYLYRQKMFU-UHFFFAOYSA-N 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 229920002535 Polyethylene Glycol 1500 Polymers 0.000 description 1
- BXFOFFBJRFZBQZ-QYWOHJEZSA-N T-2 toxin Chemical compound C([C@@]12[C@]3(C)[C@H](OC(C)=O)[C@@H](O)[C@H]1O[C@H]1[C@]3(COC(C)=O)C[C@@H](C(=C1)C)OC(=O)CC(C)C)O2 BXFOFFBJRFZBQZ-QYWOHJEZSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- WWSYXEZEXMQWHT-WNWIJWBNSA-N aflatoxin B2 Chemical compound C=1([C@@H]2CCO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O WWSYXEZEXMQWHT-WNWIJWBNSA-N 0.000 description 1
- 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 description 1
- 208000007502 anemia Diseases 0.000 description 1
- GOLCXWYRSKYTSP-UHFFFAOYSA-N arsenic trioxide Inorganic materials O1[As]2O[As]1O2 GOLCXWYRSKYTSP-UHFFFAOYSA-N 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- UHYPYGJEEGLRJD-UHFFFAOYSA-N cadmium(2+);selenium(2-) Chemical compound [Se-2].[Cd+2] UHYPYGJEEGLRJD-UHFFFAOYSA-N 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- LINOMUASTDIRTM-QGRHZQQGSA-N deoxynivalenol Chemical compound C([C@@]12[C@@]3(C[C@@H](O)[C@H]1O[C@@H]1C=C(C([C@@H](O)[C@@]13CO)=O)C)C)O2 LINOMUASTDIRTM-QGRHZQQGSA-N 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 1
- 231100000502 fertility decrease Toxicity 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000003008 fumonisin Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 206010019692 hepatic necrosis Diseases 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 231100000149 liver necrosis Toxicity 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 235000003715 nutritional status Nutrition 0.000 description 1
- RWQKHEORZBHNRI-BMIGLBTASA-N ochratoxin A Chemical compound C([C@H](NC(=O)C1=CC(Cl)=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 RWQKHEORZBHNRI-BMIGLBTASA-N 0.000 description 1
- DAEYIVCTQUFNTM-UHFFFAOYSA-N ochratoxin B Natural products OC1=C2C(=O)OC(C)CC2=CC=C1C(=O)NC(C(O)=O)CC1=CC=CC=C1 DAEYIVCTQUFNTM-UHFFFAOYSA-N 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 230000005476 size effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- VUYXVWGKCKTUMF-UHFFFAOYSA-N tetratriacontaethylene glycol monomethyl ether Chemical compound COCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO VUYXVWGKCKTUMF-UHFFFAOYSA-N 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- LINOMUASTDIRTM-UHFFFAOYSA-N vomitoxin hydrate Natural products OCC12C(O)C(=O)C(C)=CC1OC1C(O)CC2(C)C11CO1 LINOMUASTDIRTM-UHFFFAOYSA-N 0.000 description 1
- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 description 1
Images
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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
-
- 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
- G01N33/533—Production of labelled immunochemicals with fluorescent label
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- General Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Pathology (AREA)
- Hematology (AREA)
- Optics & Photonics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
A method for preparing a fluorescent quantum dot immunochromatography detection card by using aflatoxin. Relates to aflatoxin, in particular to a method for preparing a fluorescent quantum dot immunochromatography detection card by using the aflatoxin. The method for preparing the fluorescent quantum dot immunochromatography detection card by using the aflatoxin is convenient to process and improves the detection speed. The AFB1 immunofluorescence quantum dot detection card has strong specificity, and the results of detecting the aflatoxin and other fungomycin of the same family are negative. The reaction time is generally 3-5 min, compared with the traditional detection methods such as chromatography, ELISA and the like, the detection time is greatly shortened, the detection speed is improved, and the limit of large-scale equipment is thoroughly eliminated. The detection sensitivity of the detection card reaches 1ppb, which is higher than that of the same type of colloidal gold products.
Description
Technical Field
The invention relates to aflatoxin, in particular to a method for preparing a fluorescent quantum dot immunochromatography detection card by using aflatoxin.
Background
The immunochromatography technology of quantum dot labeling has received much attention because it has all the advantages of the colloidal gold immunochromatography detection technology and has higher sensitivity. The quantum dots are nanoparticles mainly composed of main group elements II-VI, III-V such as CdSe, CdTe and InP, and the particle size is generally 1-100 nm. Because electrons and holes are limited by quanta, the continuous energy band structure is changed into a discrete energy level structure with molecular characteristics, and fluorescence can be emitted after the structure is excited. The quantum dots have remarkable quantum size effect and surface effect, so that the quantum dots have light absorption characteristics which are not possessed by conventional materials, the application fields of the quantum dots are wider and wider, particularly the application values of the quantum dots in clinical laboratory and immunological detection research attract great attention of scientists, and the luminescent quantum dots serve as fluorescent probes to mark biomacromolecules and are one of important applications of nano materials in the field of bioanalysis in recent years.
By combining the existing immunological detection method, the quantum dot shows attractive prospect in the aspect of labeled immunoassay. The fluorescence analysis and the immunochromatography are combined and applied to the existing immunochromatography rapid detection, and the sensitivity of the immunochromatography rapid detection is greatly improved compared with that of colloidal gold. In addition, different quantum dots are marked with different antibodies by utilizing the characteristic that the quantum dots with different particle sizes have different colors, and the immunochromatography test strip is prepared, so that the test strip is expected to be used for simultaneously detecting different substances, and the efficiency of food safety detection is greatly improved.
Based on the advantages of quantum dots for immunochromatography rapid detection, the invention aims to apply the technology to rapid detection of Aflatoxin (AFB) in food and feed safety, and research and develop related detection test strips, thereby having better market prospect and wide social benefit.
Aflatoxin poisoning (Aflatoxicosis) is a major injury to the liver of animals, and the injured individuals vary with the age, sex and nutritional status of the animal species. Research results show that aflatoxin can cause liver function reduction, reduce milk yield and egg production rate, reduce immunity of animals and is easy to be infected by harmful microorganisms. In addition, long-term consumption of feed containing low concentrations of aflatoxin can also lead to intra-embryonic poisoning. Generally, young animals are more sensitive to aflatoxins, which are clinically manifested by dysfunction of the digestive system, reduced fertility, reduced feed utilization, anemia, and the like.
The median lethal dose of aflatoxin Bl is 0.36 mg/kg body weight, and belongs to a range of extremely virulent poisons (the median lethal dose of animals is less than 10 mg/kg = its toxicity is 10 times greater than that of potassium cyanide and 68 times greater than that of arsenic trioxide). It causes poisoning of human mainly by liver damage, hepatitis and liver cirrhosis, liver necrosis, etc. In conclusion, the development of the test strip for rapidly detecting aflatoxin is particularly key to the guarantee of the safety of grains and feeds.
Disclosure of Invention
Aiming at the problems, the invention provides the method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin, which is convenient to process and improves the detection speed.
The invention comprises the following steps: the method comprises the following steps:
1) selecting materials;
2) a fluorescent quantum dot reagent formula;
3) synthesizing an aflatoxin immune antigen;
4) preparing an aflatoxin specific antibody;
5) detecting the affinity constant and the titer of the monoclonal antibody;
6) synthesizing an aflatoxin detection antigen;
7) preparing an aflatoxin b1 antibody marked by fluorescent microspheres;
8) preparing an AFB1 antibody binding pad;
9) preparing an aflatoxin b1 fluorescent quantum dot detection test reagent strip;
10) and (4) finishing.
The materials in the step 1) comprise MES, NHS, carbodiimide, 2- (N-morpholino) ethanesulfonic acid, bovine serum albumin, Tween-20, Tritonx-100 and PVP-40;
HAT culture medium, fluorescent microspheres, goat anti-mouse secondary antibody, nitrocellulose membrane, glass fiber membrane and absorbent paper.
The step 2) comprises the following steps:
preparing borax buffer solution: 6.183g of boric acid, 6.864g of sodium borate; making the volume of ultrapure water to 860 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
0.1M, ph4.7mes buffer: 19.52g MES, 58.44g NaCl, ultrapure water to 1L, adjusted pH to 4.7 with NaOH, stored at 4 ℃ for no more than one week;
preparation of 10% BSA: 100.0g BSA; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% PVP-40: 100.0g PVP-40; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% Tween-20: 100.0mL Tween-20; diluting with ultrapure water to a constant volume of 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% sodium azide: 100.0g of sodium azide; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
glassware cleaning solution: 1000g of potassium dichromate and 2500mL of concentrated sulfuric acid, and adding distilled water to 10000 mL;
preparation of antibody marker dilution buffer: taking 50g of sucrose, adding 5mL of 10% BSA, 3mL of 10% Tween-20, 2mL of 10% PVP-40, 0.2M of PH7.4 PB to a volume of 100mL, and adding 200uL of 10% sodium azide;
preparing glass fiber gasket treating fluid: weighing 30.0g of trehalose, 5g of BSA and 15g of sucrose into a 1000mL reagent bottle, adding 100mL of 0.02mol/L PBS (pH7.4), 5mL of goat serum and 3.1mL of Triton-100, and adding ultrapure water to fix the volume to 1L;
preparation of sample pad treatment solution: weighing 1.8g of trehalose, 6g of BSA, 2.5g of casein, BRIJ-355 g of PVP-k405.4g and Na2HPO4 13.578g、NaH2PO4 2.8g and 4.015g of NaCl are put into a 1000mL reagent bottle, 5mL of Tween-20 is added, and ultrapure water is added to the solution to reach the constant volume of 1L;
preparation of antigen and secondary antibody diluent: weighing 2g of trehalose in a 250mL reagent bottle, adding 1mL of methanol and 5mL of 10% glycerol, adding 0.02mol/L of PBS and fixing the volume to 100 mL;
0.2 mol/LPB: weighing 2.3g Na2HPO4And 0.456g NaH2PO4Adding ultrapure water for dissolving, then fixing the volume to 1000mL, sterilizing at high pressure for 20min, and then storing at normal temperature, wherein the pH value of the working solution is 7.4;
17.4g NaCl, 2.3g Na were weighed into 0.02mol/L PBS2HPO4And 0.456g NaH2PO4Adding ultrapure water for dissolving, then fixing the volume to 1000mL, sterilizing at high pressure for 20min, and then storing at normal temperature, wherein the pH value of the working solution is 7.4;
5% solution of dichlorodipotassium silane in chloroform: 25mL of dichlorodipotassium silane was dissolved in 475mL of a chloroform solution and stored under a sealed condition.
Dissolving aflatoxin b1 in absolute methanol in the step 3), adding pyridine, then adding carboxymethyl hydroxylamine hemihydrochloride, rotationally evaporating the solvent, and purifying by a column to obtain hapten;
dissolving hapten in DMF, adding an activating agent for reaction overnight, adding the reaction solution into PBS dissolved with OVA, dialyzing with 0.01MPBS after reaction to obtain aflatoxin B1 immune antigen.
The step 4) comprises the following steps:
4.1) immunization of mice;
4.2) cell fusion;
4.3) screening hybridoma cells;
4.4) preparation and purification of ascites.
And 5) carrying out 2-time gradient dilution on the two monoclonal antibodies from 200 times by using a sample diluent by adopting an indirect ELISA method, and detecting.
Step 6), dissolving aflatoxin b1 in absolute ethyl alcohol, adding pyridine, then adding carboxymethyl hydroxylamine hemihydrochloride for reaction, rotationally evaporating the solvent, and purifying by a column to obtain hapten; dissolving hapten in DMF, adding an activating agent for reaction overnight, then adding the reaction solution into PBS dissolved with BSA, dialyzing with 0.01M PBS after reaction to obtain aflatoxin b1 detection antigen.
In the step 7), the fluorescent microsphere with the average diameter of 110nm and modified carboxyl and the AFB 1-resistant monoclonal antibody are used for preparing the fluorescent microsphere labeled A-type influenza virus labeled antibody according to the following method:
washing 5mg of the carboxyl modified fluorescent microspheres with MES buffer solution, centrifuging, then resuspending with 1ml of MES buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide to a final concentration of 5mM, adding NHS to a final concentration of 10mM, keeping the temperature away from the sun at room temperature, and reacting for half an hour to obtain activated carboxyl modified fluorescent microspheres;
washing the activated carboxyl modified fluorescent microspheres with 50mM borax buffer solution with pH8.5, and mixing 0.37mg of the AFB1 antibody to be marked and 5mg of the activated carboxyl modified fluorescent microspheres with 50mM borax buffer solution with pH8.5 to be fully and uniformly mixed;
reacting for 2 hours at room temperature in a dark place, and enabling the antibody and the fluorescent microsphere to form stable peptide bond covalent bonding to obtain a conjugate of the fluorescent microsphere and the AFB1 antibody;
after the reaction is finished, adding BSA solution with the final concentration of 1% to block the residual active carboxyl sites on the conjugate of the fluorescent microspheres and the AFB1 antibody, and reacting at room temperature in a dark place for 0.5 hour;
after completion, the cells were washed with 0.02M PBS buffer (pH7.4) and resuspended to obtain 5mg/ml fluorescent microsphere-labeled AFB1 antibody liquid, which was stored at 4 ℃ until use.
And 8) spraying the fluorescent microsphere-labeled fluorescent quantum dot-labeled AFB1 antibody mixed solution obtained in the step on a treated glass cellulose membrane by adopting an XYZ3050 membrane spraying system of Biodot according to the spraying amount of 2.5uL/cm to prepare an antibody binding pad, then transferring the antibody binding pad into a vacuum drying oven by using a grid tray, carrying out vacuum drying for 2 hours, adding a drying agent or allochroic silica gel, and sealing and storing in a refrigerator at 4 ℃.
Step 9), spraying the marked fluorescent quantum dot antibody compound on a bonding pad; detecting an antigen by adopting aflatoxin B1 and coating the antigen on the surface of an NC membrane as a detection line; coating goat anti-mouse IgG on the surface of the nitrocellulose membrane to serve as a quality control line; and finally assembling the combined pad, the absorbent paper and the treated sample pad into an aflatoxin B1 test strip.
The AFB1 immunofluorescence quantum dot detection card has strong specificity, and the results of detecting the aflatoxin and other fungomycin of the same family are negative. The reaction time is generally 3-5 min, compared with the traditional detection methods such as chromatography, ELISA and the like, the detection time is greatly shortened, the detection speed is improved, and the limit of large-scale equipment is thoroughly eliminated. The detection sensitivity of the detection card reaches 1ppb, which is higher than that of the same type of colloidal gold products.
Drawings
Fig. 1 is a schematic structural view of the present invention.
Detailed Description
The invention, as shown in fig. 1, comprises the following steps:
1) selecting materials;
2) a fluorescent quantum dot reagent formula;
3) synthesizing an aflatoxin immune antigen;
4) preparing an aflatoxin specific antibody;
5) detecting the affinity constant and the titer of the monoclonal antibody;
6) synthesizing an aflatoxin detection antigen;
7) preparing an aflatoxin b1 antibody marked by fluorescent microspheres;
8) preparing an AFB1 antibody binding pad;
9) preparing an aflatoxin b1 fluorescent quantum dot detection test reagent strip;
10) and (4) finishing.
The material in the step 1) comprises MES (2- (N-morpholine) ethanesulfonic acid), NHS (N-hydroxysuccinimide), carbodiimide (EDC), 2- (N-morpholino) ethanesulfonic acid, Bovine Serum Albumin (BSA), Tween-20, Tritonx-100 and Polyvinylpyrrolidone (PVP-40) which are products of sigma company;
HAT medium, positive serum (anti-aflatoxin b1 mouse serum) was purchased from Ha animal research, and negative serum was prepared in the laboratory.
Fluorescent microspheres were purchased from Bangs Laboratories, Inc. under the catalog number FC 02F/10930;
goat anti-mouse secondary antibody purchased from ABGAM-0500, changshabeu biotechnology limited;
nitrocellulose membrane (Nitrocellulose membrane) was purchased from Waterman and Schleicher & Schuell;
glass fiber membrane (glass fiber), absorbent paper (absorbent paper), and support plate are all available from Shanghai gold-labeled Biotech Co.
The step 2) comprises the following steps:
preparing borax buffer solution: 6.183g of boric acid, 6.864g of sodium borate; making the volume of ultrapure water to 860 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
0.1M, ph4.7mes buffer: 19.52g MES, 58.44g NaCl, ultrapure water to 1L, adjusted pH to 4.7 with NaOH, stored at 4 ℃ for no more than one week;
preparation of 10% BSA: 100.0g BSA; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% PVP-40: 100.0g PVP-40; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% Tween-20: 100.0mL Tween-20; diluting with ultrapure water to a constant volume of 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% sodium azide: 100.0g of sodium azide; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
glassware cleaning solution: 1000g of potassium dichromate and 2500mL of concentrated sulfuric acid, and adding distilled water to 10000 mL;
preparation of antibody marker dilution buffer: taking 50g of sucrose, adding 5mL of 10% BSA, 3mL of 10% Tween-20, 2mL of 10% PVP-40, 0.2M of PH7.4 PB to a volume of 100mL, and adding 200uL of 10% sodium azide;
preparing glass fiber gasket treating fluid: weighing 30.0g of trehalose, 5g of BSA and 15g of sucrose into a 1000mL reagent bottle, adding 100mL of 0.02mol/L PBS (pH7.4), 5mL of goat serum and 3.1mL of Triton-100, and adding ultrapure water to fix the volume to 1L;
preparation of sample pad treatment solution: weighing 1.8g of trehalose, 6g of BSA, 2.5g of casein, BRIJ-355 g of PVP-k405.4g and Na2HPO4 13.578g、NaH2PO4 2.8g and 4.015g of NaCl are put into a 1000mL reagent bottle, 5mL of Tween-20 is added, and ultrapure water is added to the solution to reach the constant volume of 1L;
preparation of antigen and secondary antibody diluent: weighing 2g of trehalose in a 250mL reagent bottle, adding 1mL of methanol and 5mL of 10% glycerol, adding 0.02mol/L PBS (pH7.4) and fixing the volume to 100 mL;
0.2 mol/LPB (pH 7.4): weighing 2.3g Na2HPO4And 0.456g NaH2PO4Adding ultrapure water for dissolving, then fixing the volume to 1000mL, and autoclavingStoring at normal temperature after 20min, wherein the pH value of the working solution is 7.4;
17.4g NaCl, 2.3g Na were weighed out in 0.02mol/L PBS (pH7.4)2HPO4And 0.456g NaH2PO4Adding ultrapure water for dissolving, then fixing the volume to 1000mL, sterilizing at high pressure for 20min, and then storing at normal temperature, wherein the pH value of the working solution is 7.4;
5% solution of dichlorodipotassium silane in chloroform: 25mL of dichlorodipotassium silane was dissolved in 475mL of a chloroform solution and stored under a sealed condition.
Dissolving aflatoxin b1 in absolute methanol in the step 3), adding pyridine, then adding carboxymethyl hydroxylamine hemihydrochloride, rotationally evaporating the solvent, and purifying by a column to obtain hapten;
dissolving hapten in DMF, adding an activating agent for reaction overnight, adding the reaction solution into PBS dissolved with OVA, dialyzing with 0.01MPBS after reaction to obtain aflatoxin B1 immune antigen.
The step 4) comprises the following steps:
4.1) immunization of mice;
4.2) cell fusion;
4.3) screening hybridoma cells;
4.4) preparation and purification of ascites.
The method comprises the following specific steps: 4.1) immunization of mice
Completely mixing 60 mu g of aflatoxin b1 immune antigen with an equal amount of Freund incomplete adjuvant to form emulsion, performing primary immunization on 4 female Balb/c mice numbered 1,2,3 and 4 for multiple times subcutaneously, mixing 130 mu g of aflatoxin b with an equal amount of Freund incomplete adjuvant after two weeks, and performing boosting immunization for 3 times. Collecting blood from tail vein of immunized mouse, standing at 37 deg.C for 1 hr, standing at 4 deg.C overnight, and collecting serum. And (3) carrying out indirect ELISA (enzyme-Linked immuno sorbent assay) to detect the titer of the immune serum, and setting a blank control and a negative control. Finally, Balb/c mice which can be fused with mouse myeloma cells SP/20 are determined, and 150 mu g of aflatoxin b is taken for intraperitoneal injection immunization.
4.2) cell fusion
Taking abdominal cavity macrophages of normal mice as feeder cells, taking spleen cells and SP/20 cells of immune mice, and adding a Chinese selective medium HAT. Cell fusion was performed at 37 ℃ under the action of PEG 1500. Adding serum-free IMDM to terminate fusion, centrifuging, adding newborn calf serum and prepared thymocyte, adding into semi-solid culture medium, mixing, pouring into culture dish, and adding into incubator for culture.
4.3) hybridoma cell selection
The fused cells were plated in a 96-well plate with 2. mu.g/mL of "aflatoxin b 1", and the culture supernatants of the wells in which hybridoma cells grew were aspirated and screened by the indirect ELISA described above. The negative control is SP/20 cell culture supernatant, the blank control is PBS, and the positive control is positive serum diluted 100 times with PBS. Screening was performed twice in total. Stop solution was added and absorbance was measured at two wavelengths (450 nm, 630 nm) using a 96-well plate.
4.4) preparation and purification of ascites
500 μ L of paraffin oil was intraperitoneally injected into 10-week-old Balb/c mice. Balb/c mice were injected with positive hybridoma cells 5X 105∽9×105And (4) respectively. After the abdominal cavity of the mouse is obviously enlarged, ascites is collected. Centrifuging the collected ascites fluid at 5000r/min for 10 min, collecting the supernatant, and purifying the supernatant with Protein A column. The supernatant was diluted and centrifuged, and sterilized by filtration using a 0.22 μm filter. The column was equilibrated with 10 column volumes of coupling buffer, maintaining tassel at 1 mL/min. And (4) loading, keeping the same flow rate, and collecting liquid. Then 5 times column volume of coupling buffer solution is used for passing through the column, and the antibody is eluted by the same amount of elution buffer solution. The pH was adjusted and the antibody was dialyzed overnight using PBS. And (5) subpackaging and freezing the antibody.
And 5) carrying out 2-time gradient dilution on the two monoclonal antibodies from 200 times by using a sample diluent by adopting an indirect ELISA method, and detecting.
Step 6), dissolving aflatoxin b1 in absolute ethyl alcohol, adding pyridine, then adding carboxymethyl hydroxylamine hemihydrochloride for reaction, rotationally evaporating the solvent, and purifying by a column to obtain hapten; dissolving hapten in DMF, adding an activating agent for reaction overnight, then adding the reaction solution into PBS dissolved with BSA, dialyzing with 0.01M PBS after reaction to obtain aflatoxin b1 detection antigen.
In the step 7), the fluorescent microsphere with the average diameter of 110nm and modified carboxyl and the AFB 1-resistant monoclonal antibody are used for preparing the fluorescent microsphere labeled A-type influenza virus labeled antibody according to the following method:
washing 5mg of the carboxyl modified fluorescent microspheres with MES buffer solution (0.1M, pH 4.7), centrifuging, then resuspending with 1ml of MES buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) to a final concentration of 5mM, adding NHS (N-hydroxysuccinimide) to a final concentration of 10mM, keeping the room temperature away from light, and reacting for half an hour to obtain activated carboxyl modified fluorescent microspheres;
washing the activated carboxyl modified fluorescent microspheres with 50mM borax buffer solution with pH8.5, and mixing 0.37mg of the AFB1 antibody to be marked and 5mg of the activated carboxyl modified fluorescent microspheres with 50mM borax buffer solution with pH8.5 to be fully and uniformly mixed;
reacting for 2 hours at room temperature in a dark place, and enabling the antibody and the fluorescent microsphere to form stable peptide bond covalent bonding to obtain a conjugate of the fluorescent microsphere and the AFB1 antibody;
after the reaction is finished, adding BSA solution with the final concentration of 1% (m/v) to seal the residual active carboxyl sites on the conjugate of the fluorescent microspheres and the AFB1 antibody, and reacting for 0.5 hour at room temperature in a dark place;
after completion, the cells were washed with 0.02M PBS buffer (pH7.4) and resuspended to obtain 5mg/ml fluorescent microsphere-labeled AFB1 antibody liquid, which was stored at 4 ℃ until use.
And 8) spraying the fluorescent microsphere-labeled fluorescent quantum dot-labeled AFB1 antibody mixed solution obtained in the step on a treated glass cellulose membrane by adopting an XYZ3050 membrane spraying system of Biodot according to the spraying amount of 2.5uL/cm to prepare an antibody binding pad, then transferring the antibody binding pad into a vacuum drying oven by using a grid tray, carrying out vacuum drying for 2 hours, adding a drying agent or allochroic silica gel, and sealing and storing in a refrigerator at 4 ℃.
Step 9), spraying the marked fluorescent quantum dot antibody compound on a bonding pad; detecting antigen by adopting aflatoxin B1 and coating the antigen on the surface of an NC membrane as a detection line (T line); coating goat anti-mouse IgG on the surface of a nitrocellulose membrane (NC) as a quality control line (C line); and finally assembling the combined pad, the absorbent paper and the treated sample pad into an aflatoxin B1 test strip.
After the dried envelope film with the detection line and the quality control line, the sample pad, the water absorption pad and the back plate are assembled in a 10 ten thousand clean and dry workshop in a matching way as shown in figure 1, a CM4000 cutting system of Biodot is adopted to cut the pasted paper plate, and the paper plate is put into a clamping piece for detection for standby.
Sensitivity results of AFB1 fluorescence quantum dot detection test paper strip:
the AFB1 detection antigen is subjected to antigen detection with different concentrations, and the established aflatoxin b1 fluorescent quantum dot detection test strip is used for detection, wherein the detection results are shown in the following.
TABLE 1 detection results of different sample concentrations of AFB1 fluorescent Quantum dot detection card
| Concentration (ppb) | 50 | 100 | 50 | 20 | 10 | 5 | 3 | 1 | 0.5 |
| Results | + | + | + | + | + | + | + | + | - |
Note: + indicates that the detection line is obvious after the sample addition reaction, -indicates that there is no detection line after the sample addition reaction.
The detection result shows that the sensitivity of AFB1 antigen detected by the AFB1 fluorescence quantum dot detection test paper strip is 1 ppb.
Specific results of the AFB1 fluorescent quantum dot detection test strip are as follows:
the established AFB1 immunofluorescence quantum dot detection card is adopted to detect various similar toxins such as AFB2, AFM1 and AFG, and detection results are negative. The detection results of other fungicins, such as vomitoxin, ochratoxin A, fumonisin, zearalenone, patulin and T-2 toxin, are negative.
Sampling detection test results:
the AFB1 immunofluorescence quantum dot detection card and the ELISA detection are carried out on 200 parts of collected test samples, and the detection results of the AFB1 immunofluorescence quantum dot detection card and the ELISA detection are shown in the table 2, which shows that the established method is high in usability.
TABLE 2 AFB1 immunofluorescent Quantum dot assay card sample assay results
Note: the ELISA method judges that the test result is positive when the calculated concentration is more than or equal to 1ppb, and judges that the test result is negative when the concentration is less than 1 ppb.
The reason for the false positive in the final sampling test of the invention is probably related to the test judgment mode, the lowest detection concentration of the AFB1 immunofluorescence quantum dot detection card is about 1ppb, in order to ensure the comparability of the two detection modes, the result of the ELISA method is converted into the concentration of aflatoxin B1, and the AFB1 detection card prepared by the test is judged to be positive when the concentration is more than or equal to 1ppb, and the test result according to the table 1 shows that the detection concentration of the AFB1 detection card prepared by the test is between 0.5 ppb and 1ppb, wherein a small amount of errors can exist.
The disclosure of the present application also includes the following points:
(1) the drawings of the embodiments disclosed herein only relate to the structures related to the embodiments disclosed herein, and other structures can refer to general designs;
(2) in case of conflict, the embodiments and features of the embodiments disclosed in this application can be combined with each other to arrive at new embodiments;
the above embodiments are only embodiments disclosed in the present disclosure, but the scope of the disclosure is not limited thereto, and the scope of the disclosure should be determined by the scope of the claims.
Claims (10)
1. A method for preparing a fluorescent quantum dot immunochromatography detection card by using aflatoxin is characterized by comprising the following steps:
1) selecting materials;
2) a fluorescent quantum dot reagent formula;
3) synthesizing an aflatoxin immune antigen;
4) preparing an aflatoxin specific antibody;
5) detecting the affinity constant and the titer of the monoclonal antibody;
6) synthesizing an aflatoxin detection antigen;
7) preparing an aflatoxin b1 antibody marked by fluorescent microspheres;
8) preparing an AFB1 antibody binding pad;
9) preparing an aflatoxin b1 fluorescent quantum dot detection test reagent strip;
10) and (4) finishing.
2. The method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin, according to claim 1, wherein the materials in the step 1) comprise MES, NHS, carbodiimide, 2- (N-morpholino) ethanesulfonic acid, bovine serum albumin, Tween-20, Tritonx-100 and PVP-40;
HAT culture medium, fluorescent microspheres, goat anti-mouse secondary antibody, nitrocellulose membrane, glass fiber membrane and absorbent paper.
3. The method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin according to claim 1, wherein the step 2) comprises the following steps:
preparing borax buffer solution: 6.183g of boric acid, 6.864g of sodium borate; making the volume of ultrapure water to 860 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
0.1M, ph4.7mes buffer: 19.52g MES, 58.44g NaCl, ultrapure water to 1L, adjusted pH to 4.7 with NaOH, stored at 4 ℃ for no more than one week;
preparation of 10% BSA: 100.0g BSA; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% PVP-40: 100.0g PVP-40; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% Tween-20: 100.0mL Tween-20; diluting with ultrapure water to a constant volume of 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
preparation of 10% sodium azide: 100.0g of sodium azide; keeping the volume of the ultrapure water to 1000.0 mL; filtering with a 0.22 mu m membrane, and standing at 4 ℃ for later use;
glassware cleaning solution: 1000g of potassium dichromate and 2500mL of concentrated sulfuric acid, and adding distilled water to 10000 mL;
preparation of antibody marker dilution buffer: taking 50g of sucrose, adding 5mL of 10% BSA, 3mL of 10% Tween-20, 2mL of 10% PVP-40, 0.2M of PH7.4 PB to a volume of 100mL, and adding 200uL of 10% sodium azide;
preparing glass fiber gasket treating fluid: weighing 30.0g of trehalose, 5g of BSA and 15g of sucrose into a 1000mL reagent bottle, adding 100mL of 0.02mol/L PBS (pH7.4), 5mL of goat serum and 3.1mL of Triton-100, and adding ultrapure water to fix the volume to 1L;
preparation of sample pad treatment solution: weighing 1.8g of trehalose, 6g of BSA, 2.5g of casein, BRIJ-355 g of PVP-k405.4g and Na2HPO4 13.578g、NaH2PO4 2.8g and 4.015g of NaCl are put into a 1000mL reagent bottle, 5mL of Tween-20 is added, and ultrapure water is added to the solution to reach the constant volume of 1L;
preparation of antigen and secondary antibody diluent: weighing 2g of trehalose in a 250mL reagent bottle, adding 1mL of methanol and 5mL of 10% glycerol, adding 0.02mol/L of PBS and fixing the volume to 100 mL;
0.2 mol/LPB: weighing 2.3g Na2HPO4And 0.456g NaH2PO4Adding ultrapure water for dissolving, diluting to 1000mL, autoclaving for 20min, and keeping at room temperatureStoring, wherein the pH value of the working solution is 7.4;
17.4g NaCl, 2.3g Na were weighed into 0.02mol/L PBS2HPO4And 0.456g NaH2PO4Adding ultrapure water for dissolving, then fixing the volume to 1000mL, sterilizing at high pressure for 20min, and then storing at normal temperature, wherein the pH value of the working solution is 7.4;
5% solution of dichlorodipotassium silane in chloroform: 25mL of dichlorodipotassium silane was dissolved in 475mL of a chloroform solution and stored under a sealed condition.
4. The method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin according to claim 1, wherein in the step 3), the aflatoxin b1 is dissolved in absolute methanol, pyridine is added, then carboxymethyl hydroxylamine hemihydrochloride is added, the solvent is removed by rotary evaporation, and the hapten is obtained by column purification;
dissolving hapten in DMF, adding an activating agent for reaction overnight, adding the reaction solution into PBS dissolved with OVA, dialyzing with 0.01MPBS after reaction to obtain aflatoxin B1 immune antigen.
5. The method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin according to claim 1, wherein the step 4) comprises the following steps:
4.1) immunization of mice;
4.2) cell fusion;
4.3) screening hybridoma cells;
4.4) preparation and purification of ascites.
6. The method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin, as claimed in claim 1, wherein the step 5) is to perform 2-fold gradient dilution of the two monoclonal antibodies with a sample diluent from 200-fold by using an indirect ELISA method for detection.
7. The method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin according to the claim 1, wherein the step 6) is that the aflatoxin b1 is dissolved in absolute ethyl alcohol, pyridine is added, then carboxymethyl hydroxylamine hydrochloride is added for reaction, the solvent is removed by rotary evaporation, and the hapten is obtained by column purification; dissolving hapten in DMF, adding an activating agent for reaction overnight, then adding the reaction solution into PBS dissolved with BSA, dialyzing with 0.01M PBS after reaction to obtain aflatoxin b1 detection antigen.
8. The method for preparing the fluorescence quantum dot immunochromatography detection card by using aflatoxin according to claim 1, wherein in the step 7), the fluorescence microsphere labeled influenza A virus labeled antibody is prepared by using carboxyl modified fluorescence microspheres with the average diameter of 110nm and anti-AFB 1 monoclonal antibodies according to the following method:
washing 5mg of the carboxyl modified fluorescent microspheres with MES buffer solution, centrifuging, then resuspending with 1ml of MES buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide to a final concentration of 5mM, adding NHS to a final concentration of 10mM, keeping the temperature away from the sun at room temperature, and reacting for half an hour to obtain activated carboxyl modified fluorescent microspheres;
washing the activated carboxyl modified fluorescent microspheres with 50mM borax buffer solution with pH8.5, and mixing 0.37mg of the AFB1 antibody to be marked and 5mg of the activated carboxyl modified fluorescent microspheres with 50mM borax buffer solution with pH8.5 to be fully and uniformly mixed;
reacting for 2 hours at room temperature in a dark place, and enabling the antibody and the fluorescent microsphere to form stable peptide bond covalent bonding to obtain a conjugate of the fluorescent microsphere and the AFB1 antibody;
after the reaction is finished, adding BSA solution with the final concentration of 1% to block the residual active carboxyl sites on the conjugate of the fluorescent microspheres and the AFB1 antibody, and reacting at room temperature in a dark place for 0.5 hour;
after completion, the cells were washed with 0.02M PBS buffer (pH7.4) and resuspended to obtain 5mg/ml fluorescent microsphere-labeled AFB1 antibody liquid, which was stored at 4 ℃ until use.
9. The method for preparing the fluorescence quantum dot immunochromatography detection card using aflatoxin according to claim 1, wherein in step 8), the fluorescence microsphere-labeled fluorescence quantum dot-labeled AFB1 antibody mixed solution obtained in the above step is sprayed onto the treated glass cellulose membrane by using XYZ3050 film spraying system of Biodot according to the spraying amount of 2.5uL/cm to prepare the antibody binding pad, and then the antibody binding pad is moved into a vacuum drying oven by using a grid tray, vacuum-dried for 2h, added with a drying agent or allochroic silica gel, sealed and stored in a refrigerator at 4 ℃.
10. The method for preparing the fluorescence quantum dot immunochromatography detection card by using the aflatoxin according to the claim 1, wherein in the step 9), the marked fluorescence quantum dot antibody compound is sprayed on the bonding pad; detecting an antigen by adopting aflatoxin B1 and coating the antigen on the surface of an NC membrane as a detection line; coating goat anti-mouse IgG on the surface of the nitrocellulose membrane to serve as a quality control line; and finally assembling the combined pad, the absorbent paper and the treated sample pad into an aflatoxin B1 test strip.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911146742.4A CN112824877A (en) | 2019-11-21 | 2019-11-21 | Method for preparing fluorescent quantum dot immunochromatography detection card by using aflatoxin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911146742.4A CN112824877A (en) | 2019-11-21 | 2019-11-21 | Method for preparing fluorescent quantum dot immunochromatography detection card by using aflatoxin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112824877A true CN112824877A (en) | 2021-05-21 |
Family
ID=75907424
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911146742.4A Pending CN112824877A (en) | 2019-11-21 | 2019-11-21 | Method for preparing fluorescent quantum dot immunochromatography detection card by using aflatoxin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112824877A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113721034A (en) * | 2021-11-02 | 2021-11-30 | 瑞博奥(广州)生物科技股份有限公司 | Time-resolved immunochromatography test strip and kit for detecting S100B protein, and preparation method and application thereof |
| CN115326951A (en) * | 2022-07-27 | 2022-11-11 | 河南省奶牛生产性能测定中心 | Method for detecting aflatoxin M1 in milk |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050277203A1 (en) * | 2004-06-15 | 2005-12-15 | Aimo Niskanen | Immunochemical filter device and methods for use thereof |
| CN104897863A (en) * | 2014-03-06 | 2015-09-09 | 北京勤邦生物技术有限公司 | Fluorescent microsphere immunochromatographic test strip for detecting aflatoxin M1, and method thereof |
| CN108241061A (en) * | 2016-12-23 | 2018-07-03 | 中粮集团有限公司 | Vomitoxin detects colloidal gold quick measuring card and kit and the method being detected to vomitoxin |
| CN108362892A (en) * | 2018-01-18 | 2018-08-03 | 河南省科学院生物研究所有限责任公司 | A kind of Procalcitonin colloid gold immune turbidimetry detection reagent |
| CN108508001A (en) * | 2018-03-30 | 2018-09-07 | 迈克生物股份有限公司 | Chemiluminescence detection kit |
| CN110108870A (en) * | 2019-04-30 | 2019-08-09 | 中国农业科学院油料作物研究所 | Synchronous detection Aspergillus flavus metabolin cyclopiazonic acid and the quantum dot fluorescence microballoon of aflatoxin composite pollution chromatograph kit |
-
2019
- 2019-11-21 CN CN201911146742.4A patent/CN112824877A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050277203A1 (en) * | 2004-06-15 | 2005-12-15 | Aimo Niskanen | Immunochemical filter device and methods for use thereof |
| CN104897863A (en) * | 2014-03-06 | 2015-09-09 | 北京勤邦生物技术有限公司 | Fluorescent microsphere immunochromatographic test strip for detecting aflatoxin M1, and method thereof |
| CN108241061A (en) * | 2016-12-23 | 2018-07-03 | 中粮集团有限公司 | Vomitoxin detects colloidal gold quick measuring card and kit and the method being detected to vomitoxin |
| CN108362892A (en) * | 2018-01-18 | 2018-08-03 | 河南省科学院生物研究所有限责任公司 | A kind of Procalcitonin colloid gold immune turbidimetry detection reagent |
| CN108508001A (en) * | 2018-03-30 | 2018-09-07 | 迈克生物股份有限公司 | Chemiluminescence detection kit |
| CN110108870A (en) * | 2019-04-30 | 2019-08-09 | 中国农业科学院油料作物研究所 | Synchronous detection Aspergillus flavus metabolin cyclopiazonic acid and the quantum dot fluorescence microballoon of aflatoxin composite pollution chromatograph kit |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113721034A (en) * | 2021-11-02 | 2021-11-30 | 瑞博奥(广州)生物科技股份有限公司 | Time-resolved immunochromatography test strip and kit for detecting S100B protein, and preparation method and application thereof |
| CN115326951A (en) * | 2022-07-27 | 2022-11-11 | 河南省奶牛生产性能测定中心 | Method for detecting aflatoxin M1 in milk |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111733141B (en) | Hybridoma cell capable of secreting monoclonal antibody against novel coronavirus N protein, monoclonal antibody and application | |
| US3553310A (en) | Immunologically reactive particles | |
| CN108956989B (en) | Detection reagent card for helicobacter pylori typing detection and preparation method thereof | |
| CN112824877A (en) | Method for preparing fluorescent quantum dot immunochromatography detection card by using aflatoxin | |
| CN108802381A (en) | Bovine viral diarrhea virus differentiates test strip and preparation method thereof | |
| CN114264827A (en) | Antibody detection kit for interleukin biological agent and preparation method thereof | |
| CN107365364A (en) | A kind of quick detection kit of Adenovirus Antigen preparation method and the detection adenovirus antibody prepared using the antigen | |
| CN101498730B (en) | Improved double-antigen sandwiching immunity detection method | |
| CN103739675B (en) | Strawberry veinbanding virus antibody and antigenic peptide, immunogen and application | |
| CN111781353A (en) | Colloidal gold test paper strip for detecting SARS-CoV-2 antibody and its preparation method and application | |
| CN105753981A (en) | Anti-human respiratory syncytial virus N protein antibodies and immunochromatographic kit using the same | |
| CN113024669A (en) | RBP antibody marked by amino-group or carboxyl-group-containing substance, human RBP immunochromatography detection kit and preparation method thereof | |
| KR101776232B1 (en) | Rapid Kit for Diagnosing Porcine Respiratory Reproductive Syndrome Virus Antigen | |
| CN108588030B (en) | Anti-human IgM monoclonal antibody, hybridoma cell strain and application thereof | |
| CN109280644A (en) | Anti-human igg monoclonal antibody, its hybridoma cell strain and application | |
| CN114560936A (en) | Fluorescent microsphere marking method and detection kit | |
| CN111220802B (en) | Clenbuterol hydrochloride small molecule hapten high sensitivity detection test paper based on nano antibody and preparation method thereof | |
| EP4075132A1 (en) | Immunoassay method and immunoassay device | |
| CN108531460B (en) | Anti-human IgM monoclonal antibody, hybridoma cell strain and application thereof | |
| KR101793267B1 (en) | Rapid Kit for Diagnosing Bovine Leukosis | |
| CN110501493A (en) | A kind of detection kit of hemadsorption virus type 1's IgM antibody | |
| CN114605531B (en) | Fluorescent microsphere labeled antibody and application thereof | |
| CN108152495A (en) | A kind of colloid gold test paper for detecting tyrosine | |
| CN103172525B (en) | Preparation method of ethyl vanillin antigen and antibody, and application of ethyl vanillin antigen and antibody | |
| CN117448279A (en) | Hybridoma cell strain secreting monoclonal antibody against human parainfluenza virus 3 type N protein, monoclonal antibody, application and kit |
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 |
Application publication date: 20210521 |
|
| RJ01 | Rejection of invention patent application after publication |