CN117069580B - Triclosan hapten as well as preparation method and application thereof - Google Patents
Triclosan hapten as well as preparation method and application thereof Download PDFInfo
- Publication number
- CN117069580B CN117069580B CN202311352084.0A CN202311352084A CN117069580B CN 117069580 B CN117069580 B CN 117069580B CN 202311352084 A CN202311352084 A CN 202311352084A CN 117069580 B CN117069580 B CN 117069580B
- Authority
- CN
- China
- Prior art keywords
- triclosan
- formula
- hapten
- compound
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- XEFQLINVKFYRCS-UHFFFAOYSA-N Triclosan Chemical compound OC1=CC(Cl)=CC=C1OC1=CC=C(Cl)C=C1Cl XEFQLINVKFYRCS-UHFFFAOYSA-N 0.000 title claims abstract description 106
- 229960003500 triclosan Drugs 0.000 title claims abstract description 106
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 28
- 239000000427 antigen Substances 0.000 claims abstract description 22
- 102000036639 antigens Human genes 0.000 claims abstract description 22
- 108091007433 antigens Proteins 0.000 claims abstract description 22
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 21
- 150000001875 compounds Chemical class 0.000 claims description 20
- 238000006243 chemical reaction Methods 0.000 claims description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 14
- 229940098773 bovine serum albumin Drugs 0.000 claims description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 102000014914 Carrier Proteins Human genes 0.000 claims description 11
- 108010078791 Carrier Proteins Proteins 0.000 claims description 11
- 108010058846 Ovalbumin Proteins 0.000 claims description 9
- 229940092253 ovalbumin Drugs 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 238000006460 hydrolysis reaction Methods 0.000 claims description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- RMXGTMRDXKUUDJ-UHFFFAOYSA-N methyl 2-[4-(bromomethyl)phenyl]benzoate Chemical compound COC(=O)C1=CC=CC=C1C1=CC=C(CBr)C=C1 RMXGTMRDXKUUDJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 239000012312 sodium hydride Substances 0.000 claims description 5
- 229910000104 sodium hydride Inorganic materials 0.000 claims description 5
- 239000003513 alkali Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 230000007062 hydrolysis Effects 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- 230000008878 coupling Effects 0.000 claims description 3
- 238000010168 coupling process Methods 0.000 claims description 3
- 238000005859 coupling reaction Methods 0.000 claims description 3
- 238000010791 quenching Methods 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 101000800130 Bos taurus Thyroglobulin Proteins 0.000 claims description 2
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 2
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 2
- 239000007810 chemical reaction solvent Substances 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 238000006386 neutralization reaction Methods 0.000 claims description 2
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 2
- 230000000171 quenching effect Effects 0.000 claims description 2
- 241000251468 Actinopterygii Species 0.000 abstract description 5
- 241000272525 Anas platyrhynchos Species 0.000 abstract description 5
- 241000287828 Gallus gallus Species 0.000 abstract description 5
- 235000013601 eggs Nutrition 0.000 abstract description 5
- 235000015277 pork Nutrition 0.000 abstract description 5
- 238000008157 ELISA kit Methods 0.000 abstract description 2
- 239000003640 drug residue Substances 0.000 abstract description 2
- 238000001308 synthesis method Methods 0.000 abstract description 2
- 229920002430 Fibre-reinforced plastic Polymers 0.000 abstract 1
- 239000002131 composite material Substances 0.000 abstract 1
- 239000011151 fibre-reinforced plastic Substances 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 25
- 239000007788 liquid Substances 0.000 description 18
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 15
- 239000011248 coating agent Substances 0.000 description 13
- 238000000576 coating method Methods 0.000 description 13
- 239000011324 bead Substances 0.000 description 11
- 239000000523 sample Substances 0.000 description 11
- 238000002965 ELISA Methods 0.000 description 10
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 10
- 239000002250 absorbent Substances 0.000 description 9
- 230000002745 absorbent Effects 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 238000003756 stirring Methods 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 7
- 238000002156 mixing Methods 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 6
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 235000013305 food Nutrition 0.000 description 6
- 230000003053 immunization Effects 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 230000002163 immunogen Effects 0.000 description 5
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 5
- 235000017557 sodium bicarbonate Nutrition 0.000 description 5
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 239000000598 endocrine disruptor Substances 0.000 description 4
- 238000007885 magnetic separation Methods 0.000 description 4
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- RMZZAEDPBSKZOM-UHFFFAOYSA-N 1,2,3-trichlorobutane Chemical compound CC(Cl)C(Cl)CCl RMZZAEDPBSKZOM-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 206010003445 Ascites Diseases 0.000 description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000007910 cell fusion Effects 0.000 description 3
- 230000037029 cross reaction Effects 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 231100000049 endocrine disruptor Toxicity 0.000 description 3
- 235000019688 fish Nutrition 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 2
- 239000012496 blank sample Substances 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000009260 cross reactivity Effects 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000003208 petroleum Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 239000012089 stop solution Substances 0.000 description 2
- NLYDHBBTVWMLFD-UHFFFAOYSA-N 2,4-dichloro-1-(4-chloro-2-methoxyphenoxy)benzene Chemical compound COC1=CC(Cl)=CC=C1OC1=CC=C(Cl)C=C1Cl NLYDHBBTVWMLFD-UHFFFAOYSA-N 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- AUYYCJSJGJYCDS-LBPRGKRZSA-N Thyrolar Chemical class IC1=CC(C[C@H](N)C(O)=O)=CC(I)=C1OC1=CC=C(O)C(I)=C1 AUYYCJSJGJYCDS-LBPRGKRZSA-N 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 229940106691 bisphenol a Drugs 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000000861 blow drying Methods 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 239000012304 carboxyl activating agent Substances 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- RGLYKWWBQGJZGM-ISLYRVAYSA-N diethylstilbestrol Chemical compound C=1C=C(O)C=CC=1C(/CC)=C(\CC)C1=CC=C(O)C=C1 RGLYKWWBQGJZGM-ISLYRVAYSA-N 0.000 description 1
- 229960000452 diethylstilbestrol Drugs 0.000 description 1
- 210000000750 endocrine system Anatomy 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012065 filter cake Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 238000002222 matrix solid-phase dispersion Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 210000002200 mouth mucosa Anatomy 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000000899 pressurised-fluid extraction Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000033458 reproduction Effects 0.000 description 1
- 238000013215 result calculation Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000005495 thyroid hormone Substances 0.000 description 1
- 229940036555 thyroid hormone Drugs 0.000 description 1
- ICUTUKXCWQYESQ-UHFFFAOYSA-N triclocarban Chemical compound C1=CC(Cl)=CC=C1NC(=O)NC1=CC=C(Cl)C(Cl)=C1 ICUTUKXCWQYESQ-UHFFFAOYSA-N 0.000 description 1
- 229960001325 triclocarban Drugs 0.000 description 1
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C65/21—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing ether groups, groups, groups, or groups
- C07C65/24—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing ether groups, groups, groups, or groups polycyclic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a triclosan hapten, a preparation method and application thereof, wherein the structure of the triclosan hapten is shown as a formula I:(I) A. The invention relates to a method for producing a fibre-reinforced plastic composite The triclosan hapten and the triclosan antigen provided by the invention have the advantages of simple synthesis method, high purity and high yield, and have great value for the preparation of triclosan antibodies and the detection of triclosan drug residues. The detection limit of the triclosan ELISA kit developed by the invention in chicken is 0.0091 mug/kg, the detection limit in duck is 0.0099 mug/kg, the detection limit in pork is 0.0090 mug/kg, the detection limit in fish is 0.0092 mug/kg, and the detection limit in eggs is 0.0094 mug/kg.
Description
Technical Field
The invention belongs to the field of purification and detection of endocrine disruptors in foods, and relates to triclosan hapten and antigen as well as a preparation method and application thereof.
Background
Endocrine disruptors (Environmental endocrine disrupting chemicals, EDCs), which are exogenous chemical substances that exist in the environment and interfere with the endocrine system of humans or animals, enter the body of the living body through the food chain, cause the interference of the organism's own hormones, accumulate in the blood circulation and fat, and cause normal physiological system abnormalities such as reproduction, development, immunity, behavioral activity, etc. of the organism. Triclosan (TCS) is one of endocrine disruptors, and is widely used as a high-efficiency broad-spectrum antibacterial agent in personal care products and daily chemical products, and can be introduced into the human body through skin contact, oral mucosa absorption and the like. TCS can reduce thyroid hormone levels in pregnant rats and can cause DNA damage to human hepatocytes with some genotoxicity. Thus, the human exposure level of TCS has attracted extensive attention from the academy.
It is highly necessary to accurately determine the residual level of triclosan in foods. The existing detection methods of triclosan mainly comprise gas chromatography, gas-mass spectrometry, liquid chromatography, liquid-mass spectrometry and the like. The sample pretreatment method mainly comprises liquid-liquid extraction, solid phase extraction, matrix solid phase dispersion extraction, accelerated solvent extraction and the like. The methods have the defects of poor selectivity, large use amount of organic solvent, complex operation, poor reproducibility among different matrixes and the like, and have complex operation and expensive instruments. The immunochemistry analysis method has unique advantages in qualitative and quantitative aspects of antigen and antibody, and has the advantages of simple and rapid operation, low cost, higher sensitivity and large analysis sample size, and overcomes the defects of physicochemical analysis.
The fundamental factors affecting the quality of immunochemistry analysis are the specificity and affinity of antibodies, which in turn are determined by the structure of the immune hapten molecules, so the molecular design and synthesis of immune hapten is the most fundamental and critical step in the generation of specific antibodies and in the establishment of rapid detection techniques for small molecule substance residues.
The prior art reports the use of triclosan haptens of different chemical structures for the preparation of artificial antigens for the detection of triclosan in a variety of complex matrices, particularly foods. CN105085204a discloses a triclosan hapten compound, which has the following structure:
。
the triclosan hapten compound disclosed in CN109956848A has the following structure:
。
the triclosan hapten compound disclosed in CN110286240A has the following structure:
。
the above patent discloses triclosan hapten with different structures, but has the problem of insufficient sensitivity and food matrix tolerance. Therefore, to meet the detection requirement of trace triclosan in food samples, it is also necessary to develop a triclosan hapten compound with better matrix tolerance and sensitivity for detection in complex matrices.
Disclosure of Invention
In order to solve the technical problems, the invention provides a triclosan hapten, and a preparation method and application thereof.
The first object of the invention is to provide a triclosan hapten, which has a structure shown in a formula I:
formula I.
The second object of the present invention is to provide a method for preparing triclosan hapten as shown in formula I, comprising the following steps:
(S1) triclosan and 4' -bromomethyl biphenyl-2-carboxylic acid methyl ester react in the presence of sodium hydride to prepare an intermediate compound of formula (II);
(S2) the compound of formula (II) is hydrolyzed and neutralized in sequence to obtain the compound of formula (I).
The preparation method of the triclosan hapten comprises the following synthetic routes:
。
further, the reaction conditions of step (S1) are: reacting for 5-10h at 50-70 ℃, wherein the mol ratio of triclosan to 4' -bromomethyl biphenyl-2-carboxylic acid methyl ester is 1-3:1, the dosage of sodium hydride is 5-10wt% of the mass of triclosan; the reaction solvent is at least one of DMF and DMSO, and the ratio of the volume consumption to triclosan is 1mL:30-50mg; after the reaction is finished, cooling to 4-8 ℃, carrying out water quenching reaction, extracting, drying, filtering, concentrating under reduced pressure, and purifying by a silica gel chromatographic column to obtain an intermediate (II). Further, the silica gel chromatographic column is purified by loading ethyl acetate, petroleum ether: ethyl acetate=20-50:1, and the product was concentrated under reduced pressure as an oily product, which was the intermediate (II) compound.
Further, in the step (S2), the hydrolysis is performed in the presence of C1-3 alcohol and alkali, wherein the C1-3 alcohol is at least one of methanol, ethanol and propanol, the alkali is at least one of NaOH and KOH, and the hydrolysis reaction condition is 40-50 ℃ to complete hydrolysis; the neutralization is to adjust the pH to 3-5 by 0.5-2M HCl solution, precipitate solid, filter, wash and dry to obtain the compound shown in the formula (I).
The third object of the invention is to provide a triclosan antigen, which is obtained by coupling the compound of formula (I) and a carrier protein.
Further, the carrier protein is at least one selected from bovine thyroglobulin, bovine serum albumin, rabbit serum albumin, human serum albumin and ovalbumin. Preferably Bovine Serum Albumin (BSA) or Ovalbumin (OVA).
Wherein the molar ratio of the triclosan hapten (formula I) to the carrier protein is 10-15:1, preferably 11-13:1.
In one embodiment of the invention, the triclosan antigen is prepared by a preparation method comprising the steps of:
(1) Dissolving triclosan hapten in a formula (I) in Dimethylformamide (DMF), then adding a carboxyl activating agent for activation, and magnetically stirring at 20-25 ℃ for reaction for 2-3 h to obtain a solution A;
the activator is a compound of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Further, the ratio of the triclosan hapten, the Dimethylformamide (DMF), the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and the N-hydroxysuccinimide (NHS) in the formula (I) is 25-35 mg:1.5-2 mL:21.5-25 mg:13-15 and mg.
(2) Placing the carrier protein into 0.1-0.2M sodium bicarbonate buffer solution, stirring and fully dissolving to obtain solution B; the ratio of the carrier protein to the sodium bicarbonate buffer solution is 33.6-50 mg:3.5 mL;
preferably, when the carrier protein is Bovine Serum Albumin (BSA), the ratio of the Bovine Serum Albumin (BSA) to the sodium bicarbonate buffer is 45-50 mg:3.5 mL; when the carrier protein is Ovalbumin (OVA), the ratio of the Ovalbumin (OVA) to the carbonic acid buffer is 33.6-35mg:3.5 And (3) mL.
(3) Dropwise adding the solution A into the solution B under the condition of 0-4 ℃ and stirring at 500-1000 rpm, and stirring at 500-1000 rpm for reaction 24 h to obtain a solution III;
(4) And (3) stirring and dialyzing the solution III for 3-5 days at 4-10 ℃ by using phosphate buffer solution to obtain the triclosan antigen.
Preferably, the pH of the phosphate buffer is 7-7.5, such as 7.1, 7.2, 7.3 or 7.4.
The fourth object of the present invention is to provide a triclosan antibody obtained by immunizing an animal with the triclosan antigen.
Further, the antibody is selected from monoclonal antibodies, polyclonal antibodies or antisera
A fifth object of the present invention is to provide any one of the following uses of triclosan hapten represented by the above formula (I):
(i) Use in the preparation of triclosan antigen or triclosan antibody;
(ii) Use in the preparation of a device for detecting triclosan;
(iii) Use in the detection of triclosan.
The device for detecting triclosan comprises, but is not limited to, a kit, a reagent tube, an immunoaffinity column and a colloidal gold detection card.
A sixth object of the present invention is to provide a method for detecting triclosan using the above-described triclosan antibody.
Further, the detection method comprises an immunoassay method, a biological sensing method and surface enhanced Raman scattering; still further, the immunoassay method includes immunoaffinity column chromatography, enzyme-linked immunoassay, colloidal gold immunochromatography, chemiluminescent immunoassay, and suspension array.
Further, the detection method comprises the combination of any one or two of ultra-high performance liquid chromatography and mass spectrometry.
The triclosan hapten and the triclosan antigen provided by the invention have the advantages of simple synthesis method, high purity and high yield, and have great value for the preparation of triclosan antibodies and the detection of triclosan drug residues.
Drawings
FIG. 1 is a mass spectrum of triclosan hapten represented by formula (I) obtained in example 1;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of triclosan hapten represented by formula (I) obtained in example 1;
FIG. 3 is a MALDI-TOF-MAS diagram of BSA in example 2;
FIG. 4 is a MALDI-TOF-MAS diagram of the triclosan-BSA complex of example 2.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The carboxyl magnetic beads are magnetic beads with carboxyl groups and particle diameters of 3 mu m, and are purchased from Soy micro-tech Co., ltd., number: magnesar MP3-COOH.
Example 1 preparation of triclosan hapten
(S1) to a 100 mL reaction flask were added 864mg triclosan (3 mmol) and 20 mL N, N-Dimethylformamide (DMF) and dissolved with stirring, then 50mg sodium hydride (HNa) was added and reacted at room temperature for 30 min; then, 608mg of 4' -bromomethyl biphenyl-2-carboxylic acid methyl ester (2 mmol) was added to the reaction mixture, and the reaction mixture was allowed to cool to 4-8 ℃ after completion of the reaction at 60 ℃ for 6 h; adding 50 mL water to quench the reaction, adding 50 mL ethyl acetate for extraction, adding 10 g anhydrous sodium sulfate to the organic phase for drying, filtering, and concentrating the filtrate under reduced pressure; the residue was dissolved by adding 10 mL ethyl acetate, stirred with 2 times by weight of silica gel, dried by rotary evaporation, and chromatographed using a 300 mesh silica gel column using petroleum ether: eluting with ethyl acetate=50:1, collecting main product, and concentrating under reduced pressure to obtain oily product as intermediate compound of formula (II);
(S2) dissolving the intermediate compound of the formula (II) by using 10 mL methanol, adding 2 times of NaOH (2M) with the molar weight of the product, and heating at 40 ℃ for reaction until the hydrolysis reaction is complete; adjusting pH to 3.0 with 2M HCl, precipitating white solid, filtering, washing filter cake with distilled water, and drying in a forced air drying oven at 40deg.C to obtain the compound shown in formula I.
FIG. 1 is a mass spectrum of triclosan hapten shown in formula (I) obtained in example 1, and the result shows that the chemical structural formula of the triclosan hapten is shown in formula I. The initial material triclosan contains only one phenolic hydroxyl group, the molecular weight of the target hapten is 498.02, and the molecular ion peak M+Na, M/z 521 and the strong peak appears on a mass spectrum chart M/z 521.
FIG. 2 shows the nuclear magnetic resonance hydrogen spectrum (Nuclear Magnetic Resonance, NMR) of the triclosan hapten represented by the formula (I) obtained in example 1, to identify a synthesized hapten. The results of FIGS. 1 and 2 show that the hapten structure is correct.
Example 2 preparation and Structure identification of triclosan Artificial antigen
1. Synthesis of immunogens
(1) 25. 25 mg triclosan hapten is dissolved in 1.5 mL DMF and stirred at 200 rpm for 10 min, 21.5mg EDC is added to dissolve and 13 mg NHS is added thereto and stirred at room temperature (500 rpm) to activate 2-3 h.
(2) 50mg of BSA was weighed and dissolved in 3.5 mL of 0.1M sodium bicarbonate solution, stirred at 200 rpm for 10 min to dissolve it sufficiently, cooled in an ice bath to 0-4℃and stirred at 1000 rpm, the reaction solution from step 1 was added dropwise (1 mL/min) and stirred at 500 rpm for 24 h.
(3) The reaction product was put into a distilled water washing dialysis bag (10 cm), dialyzed 3 d against 1L of 0.01M PBS (1X, pH 7.2) with stirring (100 rpm) at 4℃and 3 times daily (each time in the morning and evening) for 9 times, the dialyzed product was centrifuged at 5000rpm for 6 min,1.5 mL/tube was packed, and the antigen was numbered and stored at-20℃for use.
2. Synthesis of coating Material
(1) 25. 25 mg triclosan hapten is dissolved in 1.5 mL of DMF and stirred at 200 rpm for 10 min, 21.5mg of EDC is added to dissolve, then NHS 13 mg is added thereto, and the mixture is stirred at room temperature (500 rpm) to activate 2-3 h.
(2) OVA 33.6. 33.6 mg was weighed and dissolved in 3.5 mL of 0.1M sodium bicarbonate solution, stirred at 200 rpm for 10 min to allow the solution to be fully dissolved, cooled in an ice bath to 0-4 ℃ and stirred at 1000 rpm, the reaction solution obtained in step 1 was added dropwise (1 mL/min) and stirred at 500 rpm for reaction 24 h.
(3) The reaction product was put into a distilled water washing dialysis bag (10 cm), dialyzed 3 d with 1L 0.01M PBS (1X, pH 7.2) stirred (100 rpm) at 4℃and 3 times daily (each time in the morning, in the evening) and 9 times total, the dialyzed product was centrifuged at 5000rpm for 6 min,1.5 mL/tube was packed, and antigen was numbered, stored at-20℃for further use.
3. Identification of antigens
(1) The concentrations of the synthesized immunogens and coating precursors were determined by UV absorption. The OD280 nm and OD260 nm of the protein solution were measured using a 0.01M PBS as a blank and an ultraviolet spectrophotometer (NanoDrop 2000), and then the protein concentration was calculated according to the following formula: protein concentration (mg/mL) =1.45×od 280 nm - 0.74 × OD 260 nm 。
The results of the immunogen and the coating antigen were measured as follows: 7.92 mg/mL and 9.57. 9.57 mg/mL.
(2) The immunogen is identified by a matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) method, and the identification result is shown in FIG. 3 (MALDI-TOF detection result of carrier protein BSA) and FIG. 4 (MALDI-TOF detection result of immunogen triclosan-BSA); the molar ratio of coupling of triclosan hapten (formula I) to Bovine Serum Albumin (BSA) was calculated to be: r= (70046.390-64503.246)/498.02 =11.13.
EXAMPLE 3 preparation of monoclonal antibodies by immunization of animals with triclosan artificial antigen
1. Immunization of animals
The immunogen (TCS-BSA) prepared in example 2 was dissolved in 100. Mu.g/dose of physiological saline and mixed with Freund's complete adjuvant in equal volumes, 6-8 week old Balb/c females (8 total) were subcutaneously immunized in the nuchal, and the immunogen was mixed with Freund's incomplete adjuvant in equal volumes at days 7, 14 and 28 after the primary immunization, each additional immunization was performed once, 100. Mu.g/dose of the immune complex was administered 3 days before the fusion, and no Freund's adjuvant was added.
2. Screening of antisera
On day 7 after the fourth immunization, the mice were placed through the orbital veins Cong Caixie at room temperature for 2 hours, centrifuged at 4000 rpm for 10 min, and then the upper antiserum was taken for detection; determining the optimal working concentration of the coating antigen and the antibody by adopting an indirect ELISA method, and then adopting indirect competition ELISThe method A detects the specificity of the antibody. According to the result, the serum titer is selected to be high and IC 50 Low mice were used for cell fusion. As can be seen from table 1, the serum titer of the mouse # 5 was relatively high and the inhibitory effect was optimal, so that the mouse was selected for the subsequent cell fusion test.
TABLE 1 serum titers and inhibition of mice
。
3. Cell fusion and cloning
Mixing spleen cells of 5# immunized mice with mouse myeloma cells (SP 2/0) in logarithmic phase, slowly adding preheated fusion agent (PEG 4000) in 45 s for fusion, suspending uniformly with HAT medium, adding appropriate amount of feeder cells, culturing in 96-well culture plate at 37deg.C, and adding 5% CO 2 Culturing in incubator, half-changing with HT medium after 5 days, and full-changing on 9 th day.
When the cells grow to 1/4 of the area of the culture hole, the step-by-step screening method is adopted to screen the hybridoma cells. Cell culture supernatants were initially tested by indirect ELISA, and the results showed that 30 cell culture plates (96 wells/plate) shared positive well 1064 wells, with strong positive wells (titer>9000,OD max >1.8 217 holes; the screened strong positive holes are detected by an indirect competition ELISA method, the cell culture holes with strong positive and high inhibition rate are selected for subcloning screening, the positive rate is 100% after multiple subcloning screening, and finally a plurality of hybridoma cell strains capable of secreting triclosan monoclonal antibodies are obtained, wherein the cell strain with higher secreted antibody titer and the best inhibition rate is 16F10.
Conclusion: cell line 16F10 cell culture supernatant titers were 1:27000, IC 50 Is 0.012 ng/mL.
4. Preparation and purification of monoclonal antibodies
The triclosan monoclonal antibody is prepared by adopting a mouse in-vivo induced abdominal water method. Injecting 0.5-mL/liquid paraffin into the abdominal cavity of 8-10-week-old Balb/c mice, and injecting hybridoma cells into the abdominal cavity for 1-2X 10 after 7-10 days 5 OnlyAnd (3) extracting ascites of the mice after 7-10 d, centrifuging at 4 ℃ for 10 min at 5000rpm, removing upper grease and lower sediment, purifying by an octanoic acid-saturated ammonium sulfate method to obtain triclosan monoclonal antibodies, and preserving at-20 ℃.
The purification method of the octanoic acid-saturated ammonium sulfate monoclonal antibody comprises the following steps:
(1) adding 10 mL 0.06M pH4.0 acetic acid buffer solution into 5 mL centrifuged ascites, and adjusting pH to 4.5 with NaOH; (2) the ratio of octanoic acid to ascites after dilution was 11. Mu.L: 1.1 mL, 165 mu L of octanoic acid is added dropwise under stirring at room temperature, the addition is completed within 30 min, the mixture is kept stand at 4 ℃ for 2 h, and the mixture is centrifuged at 10000 rpm for 30 min; (3) adjusting pH of the supernatant to 7.4 with NaOH or HAc, stirring, adding saturated ammonium sulfate to 45% saturation at 4deg.C, standing for 30 min, and standing for 2 h; (4) centrifuging at 10000 rpm for 30 min; (5) the precipitate was dissolved in an equal volume of 0.01M PBS (pH 7.4), dialyzed against 2 d in a refrigerator at 4℃and the dialysate was changed twice daily; (6) centrifuging at 4000 rpm for 10 min, and collecting supernatant; after measuring the antibody concentration, the antibody was stored at-20℃until use.
5. Determination of monoclonal antibody concentration
The OD280 nm and OD260 nm of the purified triclosan monoclonal antibody solution were measured by an ultraviolet spectrophotometer (NanoDrop 2000) using 0.01M PBS as a blank, and then calculated according to the following formula:
protein concentration (mg/mL) =1.45×od 280 nm - 0.74 × OD 260 nm 。
Results: the concentration of the triclosan monoclonal antibody prepared by the invention is 2.49 mg/mL.
6. Specificity test
The cross-reaction rate of the monoclonal antibody prepared by the invention on triclosan structural/functional analogues is tested by an indirect competition ELISA method, and the method comprises the following steps: methyl triclosan, triclocarban, bisphenol a, 2', 4' -tetrabromodiphenyl ether, diethylstilbestrol. The results are shown in Table 2. Cross-reaction rate (%) =ic 50 (triclosan) / IC 50 (analogue) × 100%。
TABLE 2 results of triclosan monoclonal antibody Cross-reaction test
。
As can be seen from the data in table 2, the antibodies against triclosan hapten provided by the present invention have very low cross-reactivity against other analogues, in addition to methyltrichlorohydrin. However, the high cross-reactivity of methyltrichlorohydrin is not a bad factor, and thus the methyltrichlorohydrin can be used as a triclosan-derived antibody for a spectrum.
EXAMPLE 4 preparation of immunoaffinity magnetic beads
The triclosan monoclonal antibody prepared by the invention is coupled to magnetic particles by EDC/NHS method and is used for purifying and enriching triclosan in complex matrixes.
1) Taking 100 mL carboxyl magnetic beads (10 mg/mL), putting the beads into a magnetic rack for magnetic separation for 15 min, clarifying the liquid, and discarding the liquid;
2) Adding 100 mL MEST (0.1M, pH 6.0) solution, mixing thoroughly, magnetically separating, and discarding the liquid;
3) To 100 mL MEST (0.1M, pH 6.0), 1.0 g NHS and 1.0 g EDC were added, dissolved by ultrasound (2 min), shaken, and then added to the above-mentioned washed beads, and reacted at room temperature with shaking for 1.0. 1.0 h;
4) Adding 8 mL triclosan antibody (2.49 mg/mL) into the magnetic bead solution, and performing oscillation reaction at room temperature for 2.0 h;
5) After magnetic separation, the liquid was discarded, 100 mL MEST (0.1M, pH 6.0) containing 0.1% BSA was added, and the reaction was carried out at room temperature with shaking for 2.0. 2.0 h;
6) After magnetic separation, the liquid is discarded, and washed once by 100 mL MEST (0.1M, pH 6.0), the volume is fixed to 100 mL by using a magnetic bead antibody complex solution after the liquid is discarded by magnetic separation, and the liquid is preserved at 2-8 ℃ in a dark place for standby.
Example 5 establishment of an Indirect competitive enzyme-Linked immunosorbent assay method for triclosan
The optimal dosage of the coating antigen and the triclosan monoclonal antibody and the optimal coating liquid are determined by a chessboard method. The specific operation is as follows:
1) Antigen coating: the coating raw TCS-OVA was diluted with coating liquid to a series of concentrations (1:1000, 1:3000,1:9000,1:27000,1:81000, 1:243000), added to 96-well plates at 100. Mu.L/well, respectively, and incubated at 37℃for 2 h; wherein, test and screen two kinds of coating liquid: 0.05 M carbonate buffer (pH 9.6) and 0.01. 0.01M phosphate buffer (pH 7.2).
2) Washing: the liquid in the wells was decanted, washed 3 times with wash (PBST), 270. Mu.L/well, and dried on absorbent paper.
3) Closing: blocking solution 200. Mu.L/well was added, incubated at 37℃for 2 h, washed 3 times with wash solution (PBST) after blocking, and dried on absorbent paper at 270. Mu.L/well.
4) Sample adding: adding 50 mu L/well of triclosan standard substances (0.00 ng/mL, 0.005 ng/mL, 0.015 ng/mL, 0.045 ng/mL, 0.135ng/mL, 0.405 ng/mL and 1.215 ng/mL) with serial concentrations, adding 50 mu L/well of proper diluted triclosan monoclonal antibody, and incubating at 37 ℃ for 30 min; wherein the dilution gradient of the tested triclosan monoclonal antibody comprises: 1:1000,1:3000,1:9000,1:27000,1:81000,1:243000.
5) Washing: the liquid in the wells was decanted, washed 3 times with wash (PBST), 270. Mu.L/well, and blotted dry on absorbent paper.
6) Adding enzyme horseradish peroxidase labeled secondary antibody: HRP-goat anti-mouse IgG (1:5000 dilution) was added to 100. Mu.L/well and incubated at 37℃for 30 min.
7) Washing: the liquid in the wells was decanted, washed 3 times with wash solution, 270. Mu.L/well, and dried on absorbent paper.
8) Color development: a freshly prepared TMB substrate solution, 100. Mu.L/well, was added and developed in the dark for 10 min.
9) And (3) terminating: stop solution, 50. Mu.L/well, was added.
10 Determination: the OD450 nm value of each well (dual wavelength: 630 nm is the reference filter wavelength) was read with a microplate reader.
Results: through chessboard test, the optimal coating solution is selected to be 0.05M carbonate buffer solution (pH 9.6), the optimal coating concentration of the coating original TCS-OVA is 350 ng/mL, and the optimal concentration of the triclosan monoclonal antibody is 31 ng/mL.
Example 6 use of triclosan immunomagnetic beads and ELISA detection methods
1. Sample pretreatment
For animal-derived samples (chicken, duck, pork, fish, eggs, etc.), accurately weighing 5.00 (+ -0.01) g of the samples into a 50 mL centrifuge tube after homogenizing, adding 20 mL acetonitrile, mixing for about 1 min, extracting by ultrasonic for 10 min, centrifuging at 12000 rpm for 5 min, and transferring the supernatant into another centrifuge tube; adding 20 mL acetonitrile into the precipitate, repeatedly extracting, mixing the two supernatants, blow-drying with nitrogen at 50deg.C, adding 5 mL 10 mM PBS (pH 8.0) mixed solution containing 5% methanol, and vortex dissolving. Adding 150 mu L of the prepared immunomagnetic beads into the extracting solution, carrying out a room temperature combination reaction for 10 min, and removing the supernatant by magnetic attraction; adding 5 mL 10 mM PBS (pH 8.0), vortex mixing for 30 s, magnetically sucking for 2 min, and discarding supernatant; 1mL of 20 mM PBS (pH 8.0) is added, the mixture is placed in a metal bath (100 ℃) for heating for 2 min after uniform mixing, and after magnetic attraction, the supernatant is transferred to another centrifuge tube for standby, and the test is carried out.
2. Detection step
(1) TCS-OVA (9.57 mg/mL) was diluted 1:27000 with 0.05M carbonate buffer, added to 96-well plates at 100. Mu.L/well, incubated 2 h at 37℃and the well removed by decanting, washing 3 times with wash (PBST), 270. Mu.L/well and blotted dry on absorbent paper.
(2) Adding 200. Mu.L/well of blocking solution, incubating at 37deg.C for 2 h, discarding the liquid in the well after blocking, washing with washing solution (PBST) for 3 times, and beating on absorbent paper at 270. Mu.L/well.
(3) Adding 50 mu L of triclosan standard working solution (or sample solution to be detected) with each concentration into corresponding standard (or sample hole to be detected), adding 50 mu L of triclosan monoclonal antibody (31 ng/mL) into each hole, shaking and mixing uniformly, and incubating for 30 min at 37 ℃; the positions of each standard and sample were recorded and double well parallelism was recommended.
(4) The liquid in the wells was discarded, washed 3 times with wash (PBST), 270. Mu.L/well, and dried on absorbent paper.
(5) HRP-goat anti-mouse IgG (1:5000 dilution) was added to 100. Mu.L/well and incubated at 37℃for 30 min.
(6) The liquid in the wells was discarded, washed 3 times with wash (PBST), 270. Mu.L/well, and dried on absorbent paper.
(7) The cover plate film is covered, the ELISA plate 10 s is gently shaken, fully mixed and reacted for 30 min at room temperature (25+/-2 ℃), and in the absence of light.
(8) The wells were emptied of liquid, 260. Mu.L of wash working fluid was added to each well and washed thoroughly 4 times, 15-30 per immersion s.
(9) Pouring out the liquid in the plate hole, inverting the ELISA plate on the absorbent paper, and drying.
(10) A freshly prepared TMB substrate solution, 100. Mu.L/well, was added and developed in the dark for 10 min.
(11) And (3) terminating: stop solution, 50. Mu.L/well, was added.
(12) And (3) measuring: the OD450 nm value of each well (dual wavelength: 630 nm is the reference filter wavelength) was read with a microplate reader.
(13) Result calculation or determination
(1) The average absorbance value of each standard (or sample to be detected) is divided by the absorbance value of a zero standard (standard with the concentration of 0 ng/mL) and multiplied by 100, so that the percentage of absorbance corresponding to each standard, namely the percentage absorbance value, can be obtained; (2) drawing a standard curve by taking the percentage absorbance value of each standard as an ordinate and the corresponding triclosan concentration as an abscissa; (3) substituting the percentage absorbance value of the sample to be measured into a standard curve equation to obtain the corresponding concentration of the sample to be measured, and multiplying the concentration by the dilution multiple of the corresponding sample to obtain the actual content of triclosan in the original sample to be measured.
Example 7 evaluation and application of triclosan immunomagnetic beads and ELISA method
1. Minimum detection limit measurement
And (3) taking 20 blank samples for detection, calculating a measured value according to a standard curve, calculating an average value of the measured values, and adding 3 times of standard deviation to obtain the minimum detection limit. The results are shown in Table 3 below.
Table 3 statistical table of blank sample measurement results (μg/kg)
。
As shown in Table 3, the detection limit of the triclosan ELISA kit developed by the present invention was 0.0091. Mu.g/kg in chicken, 0.0099. Mu.g/kg in duck, 0.0090. Mu.g/kg in pork, 0.0092. Mu.g/kg in fish and 0.0094. Mu.g/kg in egg. The detection limit is obviously better than that of the antibodies obtained by the triclosan hapten known in the prior art.
3. Method accuracy and precision determination
Accuracy refers to the degree of agreement between measured and actual values, and in ELISA assays accuracy is often expressed as recovery and precision is often expressed as coefficient of variation. Taking blank chicken, duck, pork, fish and egg samples, and adding 5 samples in parallel according to 0.001 mug/kg and 0.002 mug/kg triclosan standard substances. The addition recovery rate and the intra-batch variation coefficient were calculated by measuring with a kit of 3 batches. The results are shown in Table 4 below.
TABLE 4 accuracy and precision test results
/>
。
The result shows that the recovery rate of each added concentration of chicken, duck, pork, fish and egg samples is 85.98% -114.60%, the intra-batch variation coefficient is 3.35% -9.59%, and the inter-batch variation coefficient is 7.04% -11.43%.
Claims (7)
1. Triclosan hapten is characterized by having a structure as shown in formula I:
(I)。
2. the method for preparing triclosan hapten represented by formula (I) according to claim 1, wherein the synthetic route is as follows:
。
3. the preparation method according to claim 2, characterized by comprising the steps of:
(S1) triclosan and 4' -bromomethyl biphenyl-2-carboxylic acid methyl ester react in the presence of sodium hydride to prepare an intermediate compound of formula (II);
(S2) the compound of formula (II) is hydrolyzed and neutralized in sequence to obtain the compound of formula (I).
4. A process according to claim 3, wherein the reaction conditions of step (S1) are: reacting for 5-10h at 50-70 ℃, wherein the mol ratio of triclosan to 4' -bromomethyl biphenyl-2-carboxylic acid methyl ester is 1-3:1, the dosage of sodium hydride is 5-10wt% of the mass of triclosan; the reaction solvent is at least one of DMF and DMSO, and the ratio of the volume consumption to triclosan is 1mL:30-50mg; cooling to 4-8deg.C after the reaction, water quenching, extracting, drying, filtering, concentrating under reduced pressure, and purifying with silica gel chromatographic column to obtain intermediate (II); and/or
In the step (S2), the hydrolysis is carried out in the presence of C1-3 alcohol and alkali, wherein the C1-3 alcohol is at least one of methanol, ethanol and propanol, the alkali is at least one of NaOH and KOH, and the hydrolysis reaction condition is 40-50 ℃ to complete hydrolysis; the neutralization is to adjust the pH to 3-5 by 0.5-2M HCl solution, precipitate solid, filter, wash and dry to obtain the compound shown in the formula (I).
5. Triclosan antigen, characterized in that it is obtained by coupling a compound of formula (I) according to claim 1 with a carrier protein.
6. Triclosan antigen according to claim 5, characterized in that the carrier protein is selected from at least one of bovine thyroglobulin, bovine serum albumin, rabbit serum albumin, human serum albumin and ovalbumin;
the molar ratio of the compound of formula (I) to the carrier protein is 10-15:1.
7. Use of any one of the triclosan haptens of formula (I) according to claim 1:
(i) Use in the preparation of triclosan antigen or triclosan antibody;
(ii) Use in the preparation of a device for detecting triclosan;
(iii) Use in the detection of triclosan.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311352084.0A CN117069580B (en) | 2023-10-19 | 2023-10-19 | Triclosan hapten as well as preparation method and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311352084.0A CN117069580B (en) | 2023-10-19 | 2023-10-19 | Triclosan hapten as well as preparation method and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117069580A CN117069580A (en) | 2023-11-17 |
CN117069580B true CN117069580B (en) | 2024-02-23 |
Family
ID=88713925
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311352084.0A Active CN117069580B (en) | 2023-10-19 | 2023-10-19 | Triclosan hapten as well as preparation method and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117069580B (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105085204A (en) * | 2015-05-18 | 2015-11-25 | 江苏大学 | Triclosan artificial antigen, synthesis method thereof and antibody preparation method |
CN109956848A (en) * | 2017-12-22 | 2019-07-02 | 北京维德维康生物技术有限公司 | A kind of triclosan haptens and antigen and its preparation method and application |
CN110286240A (en) * | 2019-07-17 | 2019-09-27 | 北京市疾病预防控制中心 | A kind of purposes of triclosan monoclonal antibody and/or triclocarban monoclonal antibody in detection triclosan and/or triclocarban |
-
2023
- 2023-10-19 CN CN202311352084.0A patent/CN117069580B/en active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105085204A (en) * | 2015-05-18 | 2015-11-25 | 江苏大学 | Triclosan artificial antigen, synthesis method thereof and antibody preparation method |
CN109956848A (en) * | 2017-12-22 | 2019-07-02 | 北京维德维康生物技术有限公司 | A kind of triclosan haptens and antigen and its preparation method and application |
CN110286240A (en) * | 2019-07-17 | 2019-09-27 | 北京市疾病预防控制中心 | A kind of purposes of triclosan monoclonal antibody and/or triclocarban monoclonal antibody in detection triclosan and/or triclocarban |
Non-Patent Citations (3)
Title |
---|
多氯酚的样品前处理及分析检测研究;刘育坚等;《化学世界》;第58卷(第4期);第243-251页 * |
环境中三氯生分布及其检测方法研究进展;韦薇等;《江苏农业科学》;第44卷(第10期);第21-26页 * |
环境介质与生物样品中三氯生残留检测方法研究进展;陈安诺等;《化学分析计量》;第32卷(第4期);第93-101页 * |
Also Published As
Publication number | Publication date |
---|---|
CN117069580A (en) | 2023-11-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107561273A (en) | A kind of time-resolved fluorescence test strips for detecting Aflatoxins M1 and its application | |
CN104109112B (en) | Cyproheptadine semiantigen, artificial antigen, antibody, preparation methods of cyproheptadine semiantigen and artificial antigen, and application of artificial antigen and antibody | |
CN109180519B (en) | Olaquindox metabolite antigen, antibody, enzyme-linked immunosorbent assay kit and detection method | |
CN111175511A (en) | Vomitoxin fluorescence immunochromatographic test strip and preparation method and application thereof | |
CN103288661A (en) | Preparation method and application of malachite green hapten | |
CN110938011B (en) | Benzocaine hapten, artificial antigen and antibody, and preparation and application thereof | |
CN117069580B (en) | Triclosan hapten as well as preparation method and application thereof | |
CN117362172B (en) | Nonylphenol hapten as well as preparation method and application thereof | |
CN110746286B (en) | Eugenol hapten, artificial antigen, preparation method and application thereof | |
CN106867970B (en) | Hybridoma cell strain secreting anti-malachite green monoclonal antibody and application thereof | |
CN107083368B (en) | hybridoma cell strains secreting monoclonal antibodies against total aflatoxin and application thereof | |
CN115746015B (en) | Patinopecten yessoensis toxin hapten, artificial antigen and antibody as well as preparation methods and application thereof | |
CN115124443B (en) | Nicarbazin metabolite hapten and artificial antigen as well as preparation methods and applications thereof | |
CN112462047B (en) | Nicarbazin detection kit and application thereof | |
CN111154000A (en) | Anti-cimaterol monoclonal antibody and application thereof | |
CN113636954B (en) | Fipronil artificial antigen, preparation method and application | |
CN116375663B (en) | Febuxostat hapten, artificial antigen and application thereof | |
CN114644572B (en) | Bromhexine hapten, artificial antigen, antibody, and preparation method and application thereof | |
CN109735502B (en) | Hybridoma cell strain secreting salinomycin-resistant monoclonal antibody and application thereof | |
CN114685387B (en) | Flutriafol hapten, antigen, antibody, detection device, preparation and application thereof | |
CN116640076A (en) | Thiamphenicol hapten, artificial antigen, antibody, and preparation and application thereof | |
CN110818599B (en) | Sulfonamide hapten, artificial antigen and application thereof in immunodetection | |
CN109336779B (en) | Preparation and application of metalaxyl hapten and antigen | |
CN116375578A (en) | Benzopyrene hapten, artificial antigen and benzopyrene detection kit | |
CN116903478A (en) | Gallic acid hapten, artificial antigen, monoclonal antibody and application |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |