CN116375578A - Benzopyrene hapten, artificial antigen and benzopyrene detection kit - Google Patents
Benzopyrene hapten, artificial antigen and benzopyrene detection kit Download PDFInfo
- Publication number
- CN116375578A CN116375578A CN202310168932.6A CN202310168932A CN116375578A CN 116375578 A CN116375578 A CN 116375578A CN 202310168932 A CN202310168932 A CN 202310168932A CN 116375578 A CN116375578 A CN 116375578A
- Authority
- CN
- China
- Prior art keywords
- benzopyrene
- hapten
- artificial antigen
- solution
- structural formula
- 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
- FMMWHPNWAFZXNH-UHFFFAOYSA-N Benz[a]pyrene Chemical compound C1=C2C3=CC=CC=C3C=C(C=C3)C2=C2C3=CC=CC2=C1 FMMWHPNWAFZXNH-UHFFFAOYSA-N 0.000 title claims abstract description 279
- 239000000427 antigen Substances 0.000 title claims abstract description 88
- 102000036639 antigens Human genes 0.000 title claims abstract description 88
- 108091007433 antigens Proteins 0.000 title claims abstract description 88
- 238000001514 detection method Methods 0.000 title claims abstract description 43
- 239000011248 coating agent Substances 0.000 claims abstract description 39
- 238000000576 coating method Methods 0.000 claims abstract description 39
- 239000000203 mixture Substances 0.000 claims description 35
- 238000000034 method Methods 0.000 claims description 26
- 102000014914 Carrier Proteins Human genes 0.000 claims description 25
- 108010078791 Carrier Proteins Proteins 0.000 claims description 25
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 23
- 229940098773 bovine serum albumin Drugs 0.000 claims description 23
- 238000002360 preparation method Methods 0.000 claims description 15
- 108010063045 Lactoferrin Proteins 0.000 claims description 14
- 102100032241 Lactotransferrin Human genes 0.000 claims description 14
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 claims description 13
- 229940078795 lactoferrin Drugs 0.000 claims description 13
- 235000021242 lactoferrin Nutrition 0.000 claims description 13
- 230000002163 immunogen Effects 0.000 claims description 8
- 102000012498 secondary active transmembrane transporter activity proteins Human genes 0.000 claims description 5
- 108040003878 secondary active transmembrane transporter activity proteins Proteins 0.000 claims description 5
- 238000003745 diagnosis Methods 0.000 claims description 4
- 201000010099 disease Diseases 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
- 239000003814 drug Substances 0.000 abstract description 17
- 229940079593 drug Drugs 0.000 abstract description 14
- 230000005764 inhibitory process Effects 0.000 abstract description 14
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 abstract description 11
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 101
- 239000000243 solution Substances 0.000 description 83
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 60
- 239000012074 organic phase Substances 0.000 description 50
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 48
- 238000005406 washing Methods 0.000 description 45
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 41
- 238000001035 drying Methods 0.000 description 40
- 238000006243 chemical reaction Methods 0.000 description 39
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 30
- 239000007788 liquid Substances 0.000 description 30
- 238000002965 ELISA Methods 0.000 description 27
- 239000007787 solid Substances 0.000 description 27
- 238000010898 silica gel chromatography Methods 0.000 description 26
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 24
- 238000002156 mixing Methods 0.000 description 24
- 239000002904 solvent Substances 0.000 description 24
- 238000003756 stirring Methods 0.000 description 23
- 239000007853 buffer solution Substances 0.000 description 20
- 238000005859 coupling reaction Methods 0.000 description 20
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- 101710145634 Antigen 1 Proteins 0.000 description 17
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 16
- 230000008878 coupling Effects 0.000 description 16
- 238000010168 coupling process Methods 0.000 description 16
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 15
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 14
- 230000003213 activating effect Effects 0.000 description 14
- 238000007789 sealing Methods 0.000 description 14
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 12
- 239000000047 product Substances 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 230000015572 biosynthetic process Effects 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 10
- 238000003786 synthesis reaction Methods 0.000 description 10
- 230000003053 immunization Effects 0.000 description 9
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Natural products CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 8
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 8
- 238000001816 cooling Methods 0.000 description 8
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 238000002649 immunization Methods 0.000 description 8
- -1 lithium aluminum hydride Chemical compound 0.000 description 8
- 229910052757 nitrogen Inorganic materials 0.000 description 8
- 239000000843 powder Substances 0.000 description 8
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 8
- 238000010521 absorption reaction Methods 0.000 description 7
- 238000010009 beating Methods 0.000 description 7
- 239000003085 diluting agent Substances 0.000 description 7
- 238000000605 extraction Methods 0.000 description 7
- 238000010438 heat treatment Methods 0.000 description 7
- 230000001105 regulatory effect Effects 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- MZRVEZGGRBJDDB-UHFFFAOYSA-N N-Butyllithium Chemical compound [Li]CCCC MZRVEZGGRBJDDB-UHFFFAOYSA-N 0.000 description 6
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 6
- 230000037029 cross reaction Effects 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 235000013305 food Nutrition 0.000 description 6
- 229910052739 hydrogen Inorganic materials 0.000 description 6
- 239000008055 phosphate buffer solution Substances 0.000 description 6
- 229910000027 potassium carbonate Inorganic materials 0.000 description 6
- 238000010791 quenching Methods 0.000 description 6
- 241000283707 Capra Species 0.000 description 5
- XTHFKEDIFFGKHM-UHFFFAOYSA-N Dimethoxyethane Chemical compound COCCOC XTHFKEDIFFGKHM-UHFFFAOYSA-N 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 230000000171 quenching effect Effects 0.000 description 5
- 210000004989 spleen cell Anatomy 0.000 description 5
- 239000012089 stop solution Substances 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 229920001213 Polysorbate 20 Polymers 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 239000005457 ice water Substances 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- 239000011259 mixed solution Substances 0.000 description 4
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 description 4
- 239000003208 petroleum Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 4
- 239000003755 preservative agent Substances 0.000 description 4
- 230000002335 preservative effect Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000002390 rotary evaporation Methods 0.000 description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 239000002912 waste gas Substances 0.000 description 4
- FADYDSCBJSKXOO-UHFFFAOYSA-N 3-bromo-1-methoxy-2,4-dimethylbenzene Chemical compound COC1=CC=C(C)C(Br)=C1C FADYDSCBJSKXOO-UHFFFAOYSA-N 0.000 description 3
- 206010003445 Ascites Diseases 0.000 description 3
- 238000006646 Dess-Martin oxidation reaction Methods 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 3
- 229960000583 acetic acid Drugs 0.000 description 3
- 239000007640 basal medium Substances 0.000 description 3
- 125000005605 benzo group Chemical group 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000007910 cell fusion Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- NKLCNNUWBJBICK-UHFFFAOYSA-N dess–martin periodinane Chemical compound C1=CC=C2I(OC(=O)C)(OC(C)=O)(OC(C)=O)OC(=O)C2=C1 NKLCNNUWBJBICK-UHFFFAOYSA-N 0.000 description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
- WBKFWQBXFREOFH-UHFFFAOYSA-N dichloromethane;ethyl acetate Chemical compound ClCCl.CCOC(C)=O WBKFWQBXFREOFH-UHFFFAOYSA-N 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 239000012362 glacial acetic acid Substances 0.000 description 3
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 3
- 229940071870 hydroiodic acid Drugs 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 239000012280 lithium aluminium hydride Substances 0.000 description 3
- KPTRDYONBVUWPD-UHFFFAOYSA-N naphthalen-2-ylboronic acid Chemical compound C1=CC=CC2=CC(B(O)O)=CC=C21 KPTRDYONBVUWPD-UHFFFAOYSA-N 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 239000007800 oxidant agent Substances 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 239000012286 potassium permanganate Substances 0.000 description 3
- 239000012460 protein solution Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000001632 sodium acetate Substances 0.000 description 3
- 235000017281 sodium acetate Nutrition 0.000 description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- XJDNKRIXUMDJCW-UHFFFAOYSA-J titanium tetrachloride Chemical compound Cl[Ti](Cl)(Cl)Cl XJDNKRIXUMDJCW-UHFFFAOYSA-J 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical group C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- JGFBQFKZKSSODQ-UHFFFAOYSA-N Isothiocyanatocyclopropane Chemical compound S=C=NC1CC1 JGFBQFKZKSSODQ-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 108010058846 Ovalbumin Proteins 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- PWLNAUNEAKQYLH-UHFFFAOYSA-N butyric acid octyl ester Natural products CCCCCCCCOC(=O)CCC PWLNAUNEAKQYLH-UHFFFAOYSA-N 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 229940078916 carbamide peroxide Drugs 0.000 description 2
- 229910002091 carbon monoxide Inorganic materials 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 231100000357 carcinogen Toxicity 0.000 description 2
- 239000003183 carcinogenic agent Substances 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- HWSRCQCLMVFRQK-UHFFFAOYSA-N chloro-(methoxymethyl)-triphenyl-$l^{5}-phosphane Chemical compound C=1C=CC=CC=1P(Cl)(C=1C=CC=CC=1)(COC)C1=CC=CC=C1 HWSRCQCLMVFRQK-UHFFFAOYSA-N 0.000 description 2
- 238000002485 combustion reaction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 229940098779 methanesulfonic acid Drugs 0.000 description 2
- QAWFLJGZSZIZHO-UHFFFAOYSA-N methyl 4-bromobutanoate Chemical compound COC(=O)CCCBr QAWFLJGZSZIZHO-UHFFFAOYSA-N 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- UUIQMZJEGPQKFD-UHFFFAOYSA-N n-butyric acid methyl ester Natural products CCCC(=O)OC UUIQMZJEGPQKFD-UHFFFAOYSA-N 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 150000002894 organic compounds Chemical class 0.000 description 2
- 229940092253 ovalbumin Drugs 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000001953 recrystallisation Methods 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 description 2
- TZIAZLUAMDLDJF-UHFFFAOYSA-N 1-(2-bromophenyl)propan-2-one Chemical compound CC(=O)CC1=CC=CC=C1Br TZIAZLUAMDLDJF-UHFFFAOYSA-N 0.000 description 1
- IYDMICQAKLQHLA-UHFFFAOYSA-N 1-phenylnaphthalene Chemical compound C1=CC=CC=C1C1=CC=CC2=CC=CC=C12 IYDMICQAKLQHLA-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- WPDWOCRJBPXJFM-UHFFFAOYSA-N 2-bromo-1-phenylpropan-1-one Chemical compound CC(Br)C(=O)C1=CC=CC=C1 WPDWOCRJBPXJFM-UHFFFAOYSA-N 0.000 description 1
- MONMFXREYOKQTI-UHFFFAOYSA-N 2-bromopropanoic acid Chemical compound CC(Br)C(O)=O MONMFXREYOKQTI-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 208000032170 Congenital Abnormalities Diseases 0.000 description 1
- 208000001951 Fetal Death Diseases 0.000 description 1
- 206010055690 Foetal death Diseases 0.000 description 1
- 206010060919 Foetal malformation Diseases 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- SXQBHARYMNFBPS-UHFFFAOYSA-N Indeno[1,2,3-cd]pyrene Chemical compound C=1C(C2=CC=CC=C22)=C3C2=CC=C(C=C2)C3=C3C2=CC=CC3=1 SXQBHARYMNFBPS-UHFFFAOYSA-N 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 241000699667 Mus spretus Species 0.000 description 1
- 206010039744 Scrotal cancer Diseases 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 206010043275 Teratogenicity Diseases 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 238000011111 UV-scan method Methods 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 208000020560 abdominal swelling Diseases 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- TXVHTIQJNYSSKO-UHFFFAOYSA-N benzo[e]pyrene Chemical group C1=CC=C2C3=CC=CC=C3C3=CC=CC4=CC=C1C2=C34 TXVHTIQJNYSSKO-UHFFFAOYSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- JYYOBHFYCIDXHH-UHFFFAOYSA-N carbonic acid;hydrate Chemical compound O.OC(O)=O JYYOBHFYCIDXHH-UHFFFAOYSA-N 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- 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 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000003245 coal Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000008157 edible vegetable oil Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 210000000750 endocrine system Anatomy 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 239000000568 immunological adjuvant Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- SJFNDMHZXCUXSA-UHFFFAOYSA-M methoxymethyl(triphenyl)phosphanium;chloride Chemical compound [Cl-].C=1C=CC=CC=1[P+](C=1C=CC=CC=1)(COC)C1=CC=CC=C1 SJFNDMHZXCUXSA-UHFFFAOYSA-M 0.000 description 1
- 239000003345 natural gas Substances 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 231100000211 teratogenicity Toxicity 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C59/40—Unsaturated compounds
- C07C59/58—Unsaturated compounds containing ether groups, groups, groups, or groups
- C07C59/64—Unsaturated compounds containing ether groups, groups, groups, or groups containing six-membered aromatic rings
- C07C59/66—Unsaturated compounds containing ether groups, groups, groups, or groups containing six-membered aromatic rings the non-carboxylic part of the ether containing six-membered aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
-
- 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
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/02—Ortho- or ortho- and peri-condensed systems
- C07C2603/40—Ortho- or ortho- and peri-condensed systems containing four condensed rings
- C07C2603/42—Ortho- or ortho- and peri-condensed systems containing four condensed rings containing only six-membered rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/02—Ortho- or ortho- and peri-condensed systems
- C07C2603/52—Ortho- or ortho- and peri-condensed systems containing five condensed rings
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/50—Polyaromatic hydrocarbons (PAHs)
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a benzopyrene hapten, an artificial antigen and a benzopyrene detection kit, wherein the artificial antigen is obtained as shown in a structural formula (III) as a coating source, the artificial antigen is obtained as shown in a structural formula (IV) as an artificial coating source, the antibody has good sensitivity to benzopyrene, the half inhibition concentration is 4.07ng/mL, and the minimum detection limit is 0.38ng/mL. The cross reaction rate of the antibody to common polycyclic aromatic hydrocarbon compounds is lower than 5%, which indicates that the antibody has good specificity to benzopyrene, can effectively eliminate the interference of other medicines, and provides a core reagent for establishing an immune detection method of benzopyrene. The benzopyrene hapten, the artificial antigen and the antibody of the invention can be used for realizing the purpose of rapidly and accurately detecting the benzopyrene in the related sample.
Description
Technical Field
The invention relates to the technical field of food safety, in particular to a benzopyrene hapten, an artificial antigen and a benzopyrene detection kit.
Background
Benzopyrene is a common polycyclic aromatic hydrocarbon compound with teratogenicity, carcinogenicity and mutability, and is widely distributed in nature, and mainly comes from two aspects: firstly, the waste gas generated by incomplete combustion of organic compounds, such as coal, petroleum, natural gas and the like, and secondly, the waste gas is generated by baking food under the conditions of high Wen Xunzhi, and the content of the waste gas is 10 to 20 times that of normal food after the scorching phenomenon occurs.
The presence of benzopyrene poses a great threat to human health, is a strong carcinogen with the greatest toxicity of polycyclic aromatic hydrocarbon compounds, and is first focused on the onset of scrotal cancer in the 1775-year london chimney cleaner population. Benzopyrene can enter a human body to cause serious harm to the human body through environmental pollution, and the benzopyrene in waste gas generated by incomplete combustion of organic compounds can be adsorbed on atmospheric particles and easily adhered to the skin of the human body to be inhaled by the human body, so that the cancer is caused; the benzopyrene can be absorbed into intestinal tracts, and then spread over all parts of the body through blood circulation, so that the endocrine system of the human body is influenced, and the human body is diseased and cancerous. Researchers administer the drug to animals by means of gastric lavage, intravenous injection, inhalation and the like, and find that benzopyrene can cause various cancers such as gastric cancer, digestive tract cancer, lung cancer, bladder cancer and the like. Benzopyrene also has the characteristics of long-term property and concealment, when a human body contacts with low-dose benzopyrene, the body does not generate adverse reaction, but is continuously accumulated in the human body, so that the human body is cancerous, and animal experiment researches show that an expert can influence the next generation by the placenta through the matrix, so that fetal malformation and death are caused, the immune system function of offspring is reduced, and the survival and reproduction of the human are seriously endangered. As one of the most representative carcinogens for polycyclic aromatic hydrocarbons, many countries and organizations have strict requirements on their content in foods. According to GB 2762-2017 "pollutant limit in food safety national Standard food", the maximum residual limit of benzopyrene in grains, meats, aquatic animals and products thereof is 5 mug/kg, and the maximum residual limit of grease and products thereof is 10 mug/kg. Research shows that the trispecificity of benzopyrene causes visible serious threat to human survival and reproduction, so that the development of more advanced, effective and sensitive benzopyrene detection technology is particularly important.
At present, the detection methods of benzopyrene are few, and the traditional detection methods include High Performance Liquid Chromatography (HPLC), liquid chromatography and mass spectrometry (LC-MS/MS), gas chromatography and mass spectrometry (GC-MS/MS) and the like. Although the instrument detection technology has higher accuracy and good repeatability, the required instrument is expensive, the sample pretreatment is complex, the detection time is long, and the method has limitation in field detection. The ELISA technology utilizes the specific binding capacity of antigen and antibody, has the advantages of high sensitivity, rapidness, high flux and the like, and is widely applied to the detection of benzopyrene residues.
Matschult et al (Matschult, D, deng, A, niessner, R, et al development of a Highly Sensitive Monoclonal Antibody Based ELISA for Detection of Benzo [ a ] pyrene in Potablewter. Analyst,2005,130 (7): 1078-1086.) prepared monoclonal antibodies of benzopyrene, the content of benzopyrene in water was detected by ELISA based on the monoclonal antibodies, the IC50 was 24ng/mL, and the prepared monoclonal antibodies had the disadvantage of poor specificity, and had a cross reaction rate of more than 20% with other polycyclic aromatic hydrocarbon compounds. Shao Huifeng and the like, the detection limit of the ELISA method is 3.3ng/mL, (Shao Huifeng. Synthesis of benzopyrene artificial antigen and establishment of ELISA method [ D ]. Nanchang City: jiangxi Master university, 2014.) achieve 67% of cross reaction rate to indeno (1, 2, 3-cd) pyrene and more than 15% of cross reaction rate to other polycyclic aromatic hydrocarbon compounds. Pschenitza et al (Pschenitza, M., hackenberg, R., niessner, R., et al analysis of Benzo [ a ] pyrene in Vegetable Oils Using Molecularly Imprinted Solid Phase Extraction (MISSE) Coupled with Enzyme-Linked Immunosorbent Assay (ELISA). Sensors,2014,14 (6): 9720-9737) detected the benzopyrene content of the edible oil by a method of solid phase extraction combined with ELISA, the detection limit being 0.63. Mu.g/g.
At present, the preparation research reports of the monoclonal antibody of benzopyrene are few, the sensitivity and the specificity are poor, and the drug crossing rate is over 20 percent, so that the preparation of the specific monoclonal antibody aiming at the benzopyrene is needed, and the sensitive, quick and simple detection method of the benzopyrene is provided.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a benzopyrene hapten, an artificial antigen and a benzopyrene detection kit.
The first object of the present invention is to provide a benzopyrene hapten.
It is a second object of the present invention to provide another benzopyrene hapten.
A third object of the present invention is to provide a benzopyrene hapten.
It is a fourth object of the present invention to provide another benzopyrene hapten.
The fifth object of the invention is to provide an application of the benzopyrene hapten in preparation of benzopyrene artificial antigen.
It is a sixth object of the present invention to provide a composition for detecting benzopyrene.
The seventh object of the invention is to provide the benzopyrene hapten, the benzopyrene artificial antigen or the application of the composition in preparing a benzopyrene detection kit.
The eighth object of the invention is to provide a benzopyrene detection kit.
A ninth object of the present invention is to provide a method for detecting benzopyrene for the purpose of non-disease diagnosis.
In order to achieve the above object, the present invention is realized by the following means:
a benzopyrene hapten (benzopyrene hapten 1) has a structural formula shown in formula (I),
the preparation method of the compound with the structural formula shown as (I) comprises the following steps:
s1.2-bromo-4-methoxy-1, 3-dimethylbenzene and potassium permanganate are fully reacted in a mixed solution of methanol and water, then insoluble substances are removed, the pH is regulated to 4, then ethyl acetate is used for extraction, an organic phase is reserved, and the intermediate 1 is obtained by purification;
s2, fully reacting an anhydrous methanol solution of the intermediate 1 with concentrated sulfuric acid, extracting with ethyl acetate, retaining an organic phase, washing with a saturated sodium bicarbonate solution after washing with water, retaining the organic phase, and purifying to obtain an intermediate 2;
s3, mixing anhydrous tetrahydrofuran of the intermediate 2 with lithium aluminum hydride at the temperature of minus 78 ℃, sufficiently reacting, quenching, extracting with ethyl acetate, reserving an organic phase, washing the organic phase with water, washing with saturated sodium bicarbonate solution, reserving the organic phase, and purifying to obtain the intermediate 3;
S4, fully reacting a dichloromethane solution of the intermediate 3 and sodium acetate with a dess-martin oxidant, extracting with dichloromethane, retaining an organic phase, and purifying to obtain an intermediate 4;
s5, fully reacting the intermediate 4 and a methanol solution of 2-naphthalene boric acid with sodium bicarbonate, removing the solvent, extracting with dichloromethane, retaining an organic phase, and purifying; heating and dissolving the product by using an ethyl acetate-dichloromethane mixed solution, stirring and distilling out the solvent until the solute is separated out, and then adding petroleum ether for recrystallization to obtain an intermediate 5;
fully reacting tetrahydrofuran solution of (methoxymethyl) triphenyl phosphorus chloride with n-butyllithium, fully reacting with intermediate 5, removing solvent, extracting with dichloromethane, retaining organic phase, and purifying to obtain intermediate 6;
s7, fully reacting the dichloromethane solution of the intermediate 6 with methylsulfonic acid, adjusting ph to be neutral, extracting with dichloromethane, retaining an organic phase, and purifying to obtain an intermediate 7;
s8, fully reacting glacial acetic acid solution of the intermediate 7 with hydroiodic acid, cooling, separating solid from liquid, retaining solid, dissolving with ethyl acetate, adding water for extraction, washing with saturated sodium chloride, retaining an organic phase, and purifying to obtain an intermediate 8;
S9, fully reacting the intermediate 8, 4-bromomethyl butyrate, anhydrous potassium carbonate and N, N-dimethylformamide, extracting with dichloromethane, retaining an organic phase, and purifying to obtain an intermediate 9;
s10, fully reacting the methanol solution of the intermediate 9 with sodium hydroxide, adjusting the pH value to 6, and carrying out solid-liquid separation to keep solid, thus obtaining the product after homozygosity.
Preferably, the preparation method of the compound with the structural formula shown as (I) comprises the following steps:
s1.2-bromo-4-methoxy-1, 3-dimethylbenzene and potassium permanganate are dissolved in a mixed solvent of methanol and water in a round-bottomed flask, the mixture is fully reacted at 70 ℃, after the reaction is finished, insoluble matters are removed by filtration, the pH of filtrate is regulated to 4 by hydrochloric acid, the mixture is extracted by ethyl acetate, organic matters are combined and organic matters are dried to remove the solvent, and then silica gel column chromatography purification and freeze drying are carried out to obtain white solid powder intermediate 1;
s2, mixing an anhydrous methanol solution of the intermediate 1 with concentrated sulfuric acid, stirring for full reaction at 80 ℃, adding water for extraction after the reaction is finished, adding saturated sodium bicarbonate solution for washing an organic phase, combining the organic phases, drying and dehydrating the organic phase by using anhydrous sodium sulfate, and spin-drying to obtain a colorless oily intermediate 2;
s3, cooling the intermediate 2 in tetrahydrofuran solution to the temperature of minus 78 ℃, mixing the intermediate 2 with lithium aluminum hydride, fully reacting, dropwise adding ice water to quench the mixture after the reaction is finished, extracting the mixture with ethyl acetate, washing an organic phase with water, adding saturated sodium bicarbonate solution to wash the mixture, combining the organic phases, drying and dehydrating the combined organic phases with anhydrous sodium sulfate, and purifying the combined organic phases through silica gel column chromatography to obtain a colorless oily intermediate 3;
S4, mixing the intermediate 3 with a dichloromethane solution of sodium acetate, fully reacting with a dess-Martin oxidant, adding water after the reaction is finished, extracting with dichloromethane, mixing organic phases, filtering, drying, and purifying by silica gel column chromatography to obtain a white solid intermediate 4;
s5, fully mixing the intermediate 4 and a methanol solution of 2-naphthalene boric acid with a sodium bicarbonate solution, fully reacting at 80 ℃, removing the solvent by rotary evaporation, adding water, and extracting with dichloromethane; mixing the organic phases, drying and dehydrating the organic phases by using anhydrous sodium sulfate, purifying the organic phases by using silica gel column chromatography, heating the product to 80 ℃ to dissolve the product by using an ethyl acetate-dichloromethane mixed solution, stirring and distilling the solvent until the solute is separated out, and adding petroleum ether for recrystallization to obtain a white powder fixed intermediate 5;
s6, (methoxymethyl) triphenyl phosphorus chloride, drying, placing into a dry three-mouth bottle, sealing, replacing air with nitrogen, cooling to room temperature, adding tetrahydrofuran for dissolution, cooling to-78 ℃, dropwise adding n-butyllithium, fully mixing, dropwise adding a tetrahydrofuran solution of an intermediate 5 into the system, fully mixing, fully reacting after heating, removing a solvent by rotary evaporation after the reaction is finished, adding water and dichloromethane for extraction, drying and dehydrating an organic phase with anhydrous sodium sulfate, rotary drying, and purifying by silica gel column chromatography to obtain a colorless oily intermediate 6;
S7, dropwise adding methanesulfonic acid into a dichloromethane solution of the intermediate 6, fully reacting, adding water and sodium bicarbonate dilute solution to neutralize acidity after the reaction is completed, extracting with dichloromethane, combining organic phases, drying and dehydrating with anhydrous sodium sulfate, and purifying by silica gel column chromatography to obtain a pale yellow solid intermediate 7;
s8, mixing the glacial acetic acid solution of the intermediate 7 with hydroiodic acid, fully reacting at 80 ℃, cooling after the reaction is completed, mixing the system with water, separating out yellow solid, carrying out solid-liquid separation, washing the solid with water, dissolving with ethyl acetate, extracting with water, washing with saturated saline, drying and dehydrating an organic phase with anhydrous sodium sulfate, and spin-drying to obtain a pale yellow solid intermediate 8;
s9, mixing the intermediate 8, 4-bromomethyl butyrate, anhydrous potassium carbonate and N, N-dimethylformamide, fully reacting at 80 ℃, adding water after the reaction is finished, extracting with dichloromethane, combining organic phases, drying and dehydrating with anhydrous sodium sulfate, spin-drying the solvent, and purifying by silica gel column chromatography to obtain a brown oily intermediate 9;
s10, mixing a methanol solution of the intermediate 9 with a sodium hydroxide aqueous solution, fully reacting at 80 ℃, regulating the pH of the system to 6 after the reaction is completed, discarding a water phase, washing with methylene dichloride to precipitate a solid, and purifying by silica gel column chromatography to obtain a brown solid.
The invention also claims a benzopyrene artificial antigen (benzopyrene artificial antigen 1), which is a benzopyrene hapten (benzopyrene hapten 1) coupled carrier protein with the structural formula shown in the formula (I), the structural formula of the benzopyrene hapten is shown in the formula (III),
preferably, the carrier protein is one or more of Lactoferrin (LF), bovine Serum Albumin (BSA), ovalbumin (OVA), or hemocyanin (KLH).
The preparation method of the benzopyrene artificial antigen (benzopyrene artificial antigen 1) comprises the following steps:
the benzopyrene hapten activating carboxyl is shown in a structural formula (I) to obtain a benzopyrene hapten activating solution; and (3) fully reacting the benzopyrene hapten activating solution with BB buffer solution of carrier protein, and purifying to obtain the benzopyrene artificial antigen with the structural formula shown in formula (III).
Preferably, the preparation method of the benzopyrene artificial antigen (benzopyrene artificial antigen 1) comprises the following steps:
dissolving carrier protein in BB buffer solution (boric acid buffer solution) to obtain carrier protein solution; dissolving benzopyrene hapten with a structural formula shown in formula (I) in DMF (N, N-dimethylformamide), adding EDC (1-ethyl-3- (3-dimethylaminopropyl)) and NHS (N-hydroxysuccinimide) to react completely in a dark place to obtain an activation solution of the benzopyrene hapten with a structural formula shown in formula (I); slowly dripping an activating solution of benzopyrene hapten with a structural formula shown in formula (I) into a solution in which carrier protein is dissolved, uniformly stirring, and fully performing coupling reaction in a dark place; and (3) dialyzing the coupling reaction product to obtain the benzopyrene artificial antigen with the structural formula shown in the formula (III).
Preferably, the molar ratio of benzopyrene hapten to carrier protein shown in the structural formula (I) is 1:125.
A benzopyrene hapten (benzopyrene hapten 2) has a structural formula shown in formula (II),
the preparation method comprises the following steps:
s1.2-bromopropiophenone glycol dimethyl ether solution fully reacts with tetra (triphenylphosphine) palladium under the protection of nitrogen, then is mixed with 5-hydroxy-2-naphthalene boric acid and ethanol, after the full reaction, is mixed with sodium carbonate, is heated and refluxed, is cooled to remove solid, is washed by ethyl acetate to remove liquid phase, is dissolved in ethyl acetate, is washed by water, is dried and dehydrated by anhydrous sodium sulfate, is removed of organic solvent, and is purified to obtain an intermediate 1;
s2, under the protection of nitrogen, mixing a dichloromethane solution of the intermediate 1 with a dichloromethane solution of titanium tetrachloride, sufficiently reacting, quenching, extracting the mixture after the reaction by using ethyl acetate, merging organic phases, washing the organic phases by using water, drying and dehydrating the organic phases by using anhydrous sodium sulfate, removing the organic solvent, and purifying to obtain an intermediate 2;
s3, mixing the intermediate 2, 4-bromomethyl butyrate, anhydrous potassium carbonate and N, N-dimethylformamide, fully reacting, extracting with dichloromethane, merging organic phases, drying and dehydrating with anhydrous sodium sulfate, removing an organic solvent, and purifying to obtain a product white intermediate 3;
S4, fully reacting the methanol solution of the intermediate 3 with sodium hydroxide, then adjusting the pH of the system to 6, removing a water phase by solid-liquid separation, washing the solid with methylene dichloride, and purifying to obtain a brown solid.
Preferably, the preparation method of the compound with the structural formula shown as (II) comprises the following steps:
s1, mixing ethylene glycol dimethyl ether solution of 2-bromopropionic acid with tetra (triphenylphosphine) palladium under the protection of nitrogen, fully mixing the obtained solution, then mixing the obtained solution with 5-hydroxy-2-naphthalene boric acid and ethanol, fully mixing the obtained solution with sodium carbonate solution, and heating the mixture at 90 ℃ for fully refluxing; after the reaction was completed, the solution was cooled, the solid was also separated and kept in the strand and the liquid phase was washed with ethyl acetate to remove the solvent, and the residue was dissolved in ethyl acetate, washed with water, dried and dehydrated with anhydrous sodium sulfate, then the solvent was removed by evaporation under reduced pressure, and the residue was purified by a silica gel column chromatography to obtain a product, white intermediate 1;
s2, cooling a dichloromethane solution of the intermediate 1 to the temperature of minus 78 ℃ under nitrogen, dropwise adding a dichloromethane solution of titanium tetrachloride, heating to fully react after the dropwise adding, quenching with ice water after the reaction is finished, extracting the mixture with ethyl acetate, washing an organic phase with water, drying and dehydrating with anhydrous sodium sulfate, spin-drying the solvent, and purifying by silica gel column chromatography to obtain an intermediate 2;
S3, mixing intermediate 2, 4-bromomethyl butyrate, anhydrous potassium carbonate and N, N-dimethylformamide, fully reacting at 80 ℃, adding water after the reaction is finished, extracting with dichloromethane, combining organic phases, drying and dehydrating anhydrous sodium sulfate, spin-drying a solvent, and purifying a brown oily intermediate 3 by silica gel column chromatography;
s4, mixing the methanol solution of the intermediate 3 with a sodium hydroxide aqueous solution, fully reacting at 80 ℃, regulating the pH of the system to 6 after the reaction is finished, centrifuging, discarding a water phase, washing a solid with dichloromethane, and purifying by silica gel column chromatography to obtain a brown solid.
The invention also claims a benzopyrene artificial antigen (benzopyrene artificial antigen 2), which is a benzopyrene hapten-coupled carrier protein with the structural formula shown in the formula (II), the structural formula of the benzopyrene hapten-coupled carrier protein is shown in the formula (IV),
the preparation method of the benzopyrene artificial antigen with the structural formula shown in the formula (IV) comprises the following steps:
the benzopyrene hapten activating carboxyl is shown in a structural formula (II) to obtain a benzopyrene hapten activating solution; and (3) fully reacting the benzopyrene hapten activating solution with BB buffer solution of carrier protein, and purifying to obtain the benzopyrene artificial antigen with the structural formula shown in formula (IV).
Preferably, the preparation method of the benzopyrene artificial antigen with the structural formula shown in the formula (IV) comprises the following steps: dissolving carrier protein in BB buffer solution (carbonic acid buffer solution) to obtain carrier protein solution; dissolving benzopyrene hapten with a structural formula shown in formula (II) in DMF, adding EDC and NHS, and fully reacting in a dark place to obtain an activation solution of the benzopyrene hapten with a structural formula shown in formula (II); slowly dripping an activating solution of benzopyrene hapten with a structural formula shown in formula (II) into a carrier protein solution, uniformly stirring, and fully performing coupling reaction in a dark place; and (3) dialyzing the coupling reaction product to obtain the benzopyrene artificial antigen with the structural formula shown in the formula (IV).
Preferably, the molar ratio of hapten 2 to carrier protein is 1:125.
the application of one or more of the benzopyrene hapten in preparing the benzopyrene artificial antigen also belongs to the protection scope of the invention.
The invention also claims a composition for detecting benzopyrene, which contains the benzopyrene artificial antigen with the structural formula shown in the formula (III) and the benzopyrene artificial antigen with the structural formula shown in the formula (IV), wherein the benzopyrene artificial antigen with the structural formula shown in the formula (III) is used as an immunogen, and the benzopyrene artificial antigen with the structural formula shown in the formula (IV) is used as a coating antigen.
Preferably, the carrier protein of the benzopyrene artificial antigen shown in the structural formula (III) is lactoferrin as an immunogen, and the carrier protein of the benzopyrene artificial antigen shown in the structural formula (IV) is bovine serum albumin as a coating antigen.
Namely, the structural formula of the immunogen is shown as a formula (III-1),
the structural formula of the coating raw material is shown as a formula (IV-1),
the benzopyrene hapten, the benzopyrene artificial antigen or the application of the composition in preparation of a benzopyrene detection kit also belongs to the protection scope of the invention.
The invention also discloses a benzopyrene detection kit containing the composition.
Preferably, one or more of an enzyme conjugate, a substrate color developing solution, a stop solution, and/or a wash solution are also included.
As a specific example, the detection kit contains the following components:
(1) An ELISA plate coated with a coating antigen (benzopyrene artificial antigen shown in the structural formula (IV-1));
(2) Benzopyrene standard solution
(3) A benzopyrene monoclonal antibody prepared by using a benzopyrene artificial antigen with a structural formula shown in (III-1);
(4) Enzyme conjugate: horseradish peroxidase-labeled goat anti-mouse secondary antibody;
(5) Substrate color development liquid: the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(6) The stop solution is 10% H by volume 2 SO 4 ；
(7) The washing liquid is pH 7.4 and contains 0.5-1.0% Tween-20, 0.01-0.03% sodium azide preservative and 0.1-0.3 mol/L phosphate buffer solution, wherein the percentages are weight volume percentages.
The method of use is the same as the method for detecting benzopyrene for the purpose of non-disease diagnosis hereinafter.
The invention also claims a method for detecting benzopyrene for non-disease diagnosis, and the composition is used.
Preferably, the benzopyrene artificial antigen with the structural formula shown in the formula (IV) takes benzopyrene artificial antigen with carrier protein of bovine serum albumin as a coating antigen, the benzopyrene artificial antigen with the structural formula shown in the formula (III) takes benzopyrene artificial antigen of lactoferrin as carrier protein, and an antibody prepared by immunizing an animal with the carrier protein as an immunogen is used as a detection antibody for detection.
Specifically, the structural formula of the immunogen is shown as a formula (III-1),
the structural formula of the coating raw material is shown as a formula (IV-1),
preferably, the coating concentration is 1. Mu.g/mL and the benzopyrene antibody dilution is 8000-fold.
The immunoassay method includes, but is not limited to, one or more of enzyme immunoassay, immunochromatography, immunosensory, or immune colloidal gold.
As a specific example, the detection method is based on an indirect competition ELISA method, and the specific detection method is as follows:
(1) Benzopyrene artificial antigen with a structural formula shown in formula (IV-1) is used as a coating source, diluted to 1 mug/mL by carbonate buffer solution (CB, 0.1M pH=9.8), a 96-well ELISA plate is coated, 100 mug of the benzopyrene artificial antigen is added to each well, and the mixture is incubated overnight at 37 ℃ for 12 hours;
(2) Discarding the coating liquid, washing twice with a washing liquid (pH value is 7.4, the washing liquid contains 0.6 percent of Tween-20, 0.02 percent of sodium azide preservative and 0.2mol/L of phosphate buffer solution, and the percentages are weight and volume percentages), and beating to dryness;
(3) Adding 120 mu L of sealing liquid (namely 5% of skimmed milk powder by mass) into each hole, and sealing for 3 hours at 37 ℃;
(4) Discarding the sealing liquid, beating the plate, drying at 37 ℃ for 30min, taking out, and bagging for standby by self-sealing;
(5) Phosphate buffer (PBS, 0.01m, ph=7.4) 1: 8000-fold dilution of benzopyrene monoclonal antibody prepared by benzopyrene artificial antigen with structural formula shown in (III-1), and 2-fold gradient dilution of benzopyrene drug to be detected to 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.91ng/mL, 1.95ng/mL, 0.98ng/mL, 0.49ng/mL, 0.24ng/mL, 0.12ng/mL, 0.06ng/mL;
(6) Adding 50 mu L of benzopyrene drug diluent to be detected (three groups are parallel) into each row, adding 50 mu L of benzopyrene monoclonal antibody prepared by benzopyrene artificial antigen with a structural formula shown as (III-1), incubating for 40min at 37 ℃, washing five times with washing liquid, and beating to dryness;
(7) Adding goat anti-mouse secondary antibody-HRP diluted 5000 times by Tween phosphate buffer (PBST, 0.01M), incubating at 37deg.C for 30min, washing with washing solution for five times, and drying;
(8) Adding a color development solution, and incubating at 37 ℃ for 10min with 100 mu L of each hole;
(9) 50. Mu.L of 10% H was added 2 SO 4 The reaction was stopped and the OD was read at 450 nm.
(10) And (3) operating according to the steps (1) to (9), replacing the benzopyrene diluent to be detected in the step (6) with the extracted sample diluent to be detected, and combining the drawn standard curve to determine the content of the actual benzopyrene medicine in the unknown sample.
Compared with the prior art, the invention has the following beneficial effects:
the benzopyrene hapten with the structural formula shown in the formula (I) is prepared, and the coupling carrier protein of the benzopyrene hapten is coupled with the benzopyrene hapten to obtain an artificial antigen with the structural formula shown in the formula (III); the benzopyrene hapten with the structural formula shown in the formula (II) is prepared, the coupling carrier protein structural formula shown in the formula (IV) is used for obtaining an artificial antigen, the specific antibody for detecting the benzopyrene is further prepared, the artificial antigen with the structural formula shown in the formula (III) is used as a coating source, the artificial antigen with the structural formula shown in the formula (IV) is used as an artificial coating source, the antibody has good sensitivity to the benzopyrene, the half inhibition concentration is 4.07ng/mL, and the minimum detection limit is 0.38ng/mL. The cross reaction rate of the antibody to common polycyclic aromatic hydrocarbon compounds is lower than 5%, which indicates that the antibody has good specificity to benzopyrene, can effectively eliminate the interference of other medicines, and provides a core reagent for establishing an immune detection method of benzopyrene. The benzopyrene hapten, the artificial antigen and the antibody of the invention can be used for realizing the purpose of rapidly and accurately detecting the benzopyrene in the related sample.
Drawings
FIG. 1 is a synthetic route diagram of benzopyrene hapten 1 of example 1.
FIG. 2 is a mass spectrum identification chart of benzopyrene hapten 1 of example 1.
FIG. 3 is a UV scan of benzopyrene hapten 1, benzopyrene artificial antigen 1 (benzopyrene hapten 1-LF) and LF of example 2.
FIG. 4 is a scanning ultraviolet image of benzopyrene hapten 1, benzopyrene artificial antigen 2 (benzopyrene hapten 1-BSA) and BSA of example 3.
FIG. 5 is a synthetic route for benzopyrene hapten 2 of example 4.
FIG. 6 is a mass spectrum identification chart of benzopyrene hapten 2 of example 4.
FIG. 7 is a scanning ultraviolet image of benzopyrene hapten 2, benzopyrene artificial antigen 3 (benzopyrene hapten 2-BSA) and BSA of example 5.
FIG. 8 is a graph showing the inhibition curves of benzopyrene by antibodies prepared in example 8 using benzopyrene artificial antigen 1 (benzopyrene hapten 1-LF) as immunogen.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
EXAMPLE 1 Synthesis and identification of benzopyrene hapten 1
1. Experimental method
1. Synthesis of benzopyrene hapten 1 (synthetic route patterns see FIG. 1)
2-bromo-4-methoxy-1, 3-dimethylbenzene (40 g,0.186 mol) and potassium permanganate (73.5 g, 0.463mol) are weighed and dissolved in a mixed solvent of 100mL of methanol and 100mL of water in a 1L round bottom flask, the reaction is carried out overnight at 70 ℃, after the completion of the reaction, insoluble matters are removed by filtration, the pH of the filtrate is regulated to 4 by 6mol/L hydrochloric acid, after extraction by ethyl acetate, the solvents are removed by combining organic phases, after purification by silica gel column chromatography, the white solid powder intermediate 1 (2-bromo-4-methoxyisophthalic acid) is obtained by freeze drying,
intermediate 1 (25.2 g 91.62 mmol) was dissolved in 100mL dry methanol and 10mL concentrated sulfuric acid was added and the reaction stirred at 80℃overnight. Adding water after the reaction is finished, extracting with ethyl acetate, washing an organic phase for 3 times, adding saturated sodium bicarbonate solution for washing, reserving the organic phase, drying and dehydrating with anhydrous sodium sulfate, spin-drying to obtain a colorless oily intermediate 2 (2-bromo-4-methoxy dimethyl isophthalate),
dissolving intermediate 2 (24.25 g 80 mmol) in 200mL tetrahydrofuran, cooling to-78 ℃, slowly adding 1mol/L lithium aluminum hydride, heating to room temperature for reaction for 1h, dripping ice water for quenching after the reaction is completed, extracting with ethyl acetate, washing an organic phase for 3 times, adding saturated sodium bicarbonate solution for washing, retaining the organic phase, drying and dehydrating with anhydrous sodium sulfate, purifying by silica gel column chromatography to obtain colorless oily intermediate 3 (2-bromo-4-methoxy-1, 3-phenylene dimethanol),
Intermediate 3 (6.4 g 25.90 mmol) and sodium acetate (9.560g 116.555mmol) were dissolved in 200mL dichloromethane, and dess-Martin oxidant (32.957g 77.703mmol) was added thereto and stirred at room temperature for 1h. Adding water after the reaction is finished, extracting with dichloromethane for 3 times, combining organic phases, filtering, drying and dehydrating with anhydrous sodium sulfate, purifying by silica gel column chromatography to obtain a white solid intermediate 4 (2-bromo-4-methoxym-phthalaldehyde),
intermediate 4 (6.015g 24.750mmol) and 2-naphthaleneboronic acid (4.257g 24.750mmol) were taken and dissolved in 50mL of methanol. 1mol/L sodium bicarbonate solution was added to the system all under stirring, and reacted at 80℃for 2.5 hours. Removing solvent by rotary evaporation, adding appropriate amount of water, extracting with dichloromethane for 3 times, mixing organic phases, drying and dehydrating with anhydrous sodium sulfate, purifying by silica gel column chromatography, heating the product to 80deg.C with ethyl acetate-dichloromethane mixed solution with volume ratio of 1:1, stirring to evaporate solvent until solute is separated out, adding petroleum ether, recrystallizing to obtain white powder fixed intermediate 5 (4-methoxy-2- (naphthalene-2-yl) isophthalaldehyde),
(methoxymethyl) triphenylphosphine chloride (15.302g 44.640mmol) was weighed, dried and poured into a dry three-necked flask, and after rapid sealing, air was replaced with nitrogen. After cooling to room temperature, 50mL of tetrahydrofuran was added for dissolution. Dropping 2.5mol/L n-butyllithium (17.9mL 44.640mmol) to minus 78 ℃, stirring for 15min, weighing intermediate 5 (4.32 g14.880 mmol) and dissolving in 50mL tetrahydrofuran, dropping the mixture into the system, continuously stirring for 15min, taking out a three-port bottle, and reacting for 1h at room temperature. After the reaction is completed, the solvent is removed by rotary evaporation, water and methylene dichloride are added for extraction for 3 times, the organic phase is combined, dried and dried by rotary drying after being dried by anhydrous sodium sulfate, and colorless oily intermediate 6 (2- (3-methoxy-2- (E) -2-methoxyvinyl) -6- (Z) -2-methoxyvinyl) phenyl naphthalene) is obtained by silica gel column chromatography purification,
Intermediate 6 (650 mg 1.893 mmol) was dissolved in 5mL of dichloromethane, 78. Mu.L of methanesulfonic acid was added dropwise, and the mixture was stirred at room temperature for 1 hour, after the completion of the reaction, water and a dilute solution of sodium hydrogencarbonate were added to neutralize the acidity, extraction was performed 3 times with dichloromethane, and the organic phases were combined. Drying with anhydrous sodium sulfate, spin drying, purifying by silica gel column chromatography to obtain pale yellow solid intermediate 7 (1-methoxybenzo [ PQR ] tetraanisole),
intermediate 7 (375.9mg 1.331mmol) was dissolved in 20mL glacial acetic acid, and 20mL of 55% hydroiodic acid (mass fraction) was added thereto and reacted at 80℃for 3 hours. After the reaction was completed, the reaction mixture was cooled to room temperature, the system was poured into water to precipitate a yellow solid, 4 layers of filter paper were interposed, and after washing the cake with water, it was dissolved with ethyl acetate, extracted with water, and washed with saturated brine for 1 time. The organic phase is dehydrated by anhydrous sodium sulfate and then is spin-dried to obtain a light yellow solid intermediate 8 (benzo [ PQR ] tetraphenyl-1-ol),
intermediate 8 (367.7mg 1.370mmol), methyl 4-bromobutyrate (496 mg 2.741mmol) and anhydrous potassium carbonate (753 mg 4.111 mmol) were taken together and dissolved in 20mL DMF and reacted overnight at 80 ℃. After the reaction, adding a proper amount of water, extracting with dichloromethane for 3 times, combining the organic phases, drying the organic phases with anhydrous sodium sulfate, spin-drying the organic phases, purifying the organic phases by silica gel column chromatography to obtain a brown oily intermediate 9 (4- (benzo [ PQR ] tetraphenyl-1-yloxy) methyl butyrate),
Intermediate 9 (505 mg 1.370 mmol) was taken and reacted with 10mL of methanol as solvent, followed by addition of 5mL of 10% aqueous sodium hydroxide (mass fraction) at 80℃for 2h. After the reaction is finished, regulating the pH of the system to 6, centrifuging, discarding the water phase, washing the separated solid by using methylene dichloride, and purifying by using a silica gel column chromatography to obtain brown solid (4- (benzo [ PQR ] tetraphenyl-1-yloxy) butyric acid), wherein the specific method for purifying by using the silica gel column chromatography is as follows: dissolving the solid by using a small amount of solvent methanol, adding silica gel powder, stirring uniformly, spin-drying the solvent, loading the silica gel powder, and purifying by using silica gel column chromatography.
2. Identification of benzopyrene hapten 1
Mass spectrometry and nuclear magnetic resonance spectroscopy are carried out on the benzopyrene hapten 1 to determine the molecular weight and structural characteristics of the benzopyrene hapten.
2. Experimental results
Nuclear magnetic results of benzopyrene hapten 1: 1H NMR (600 MHz, DMSO). Delta.12.20 (s, 1H), 8.60 (s, 1H), 8.42 (d, J=9.1 Hz, 1H), 8.36-7.97 (m, 4H), 7.78 (d, J=2.5 Hz, 2H), 7.52 (dd, J=9.2, 2.5Hz, 3H), 4.27 (t, J=6.5 Hz, 2H), 2.39 (s, 2H), 2.23-1.94 (m, 2H).
The ESI-MS identification result of benzopyrene hapten 1 is shown in FIG. 2, and MS of benzopyrene hapten 1: c (C) 24 H 18 O 3 ：354.41，ESI-[M+H]+：355.1。
The structural formula of the benzopyrene hapten 1 is shown as a formula (I):
example 2 Synthesis and identification of benzopyrene Artificial antigen 1
1. Experimental method
1. Synthesis of benzopyrene artificial antigen 1
The benzopyrene hapten 1 prepared in the example 1 is used for preparing the benzopyrene artificial antigen 1 by coupling Lactoferrin (LF) through an active ester method, and the method comprises the following steps:
dissolving 14.2mg of benzopyrene hapten 1 prepared in example 1 in 0.5mL of DMF solution, stirring, adding 12mg of EDC and 10mg of NHS, and stirring at room temperature for reaction overnight in a dark place to obtain benzopyrene hapten 1 activating solution; dissolving 16mg of LF in 1.6mL of BB buffer solution with pH=9.0, stirring, adding 150 mu L of benzopyrene hapten 1 activating solution, uniformly stirring, and performing light-shielding coupling at room temperature for 4 hours to obtain a coupling mixture; dialyzing the coupling mixture with PBS buffer solution at 4deg.C for 3 days, changing the dialysate for 2 times per day to obtain benzopyrene artificial antigen 1 (benzopyrene hapten 1-LF), subpackaging the benzopyrene artificial antigen 1 at a concentration of 1mg/mL, and freezing in a refrigerator at-20deg.C.
2. Identification of benzopyrene artificial antigen 1
Ultraviolet scanning measurement (190-400 nm) is carried out on carrier protein Lactoferrin (LF), benzopyrene hapten 1 and benzopyrene artificial antigen 1.
2. Experimental results
The measurement result is shown in fig. 3, it can be seen from fig. 3 that the ultraviolet characteristic absorption peak of the complete antigen is different in degree relative to both the benzopyrene hapten and the carrier protein (LF), and the benzopyrene artificial antigen 1 is found to have the characteristic absorption peaks of the benzopyrene hapten and the LF at the same time, which indicates that the coupling of the benzopyrene hapten 1 and the LF is successful, and the benzopyrene artificial antigen 1 is successfully prepared, and the structural formula of the benzopyrene artificial antigen 1 is shown as the formula (III-1).
EXAMPLE 3 Synthesis and identification of benzopyrene Artificial antigen 2
1. Experimental method
1. Synthesis of benzopyrene artificial antigen 2
The benzopyrene hapten 1 prepared in the example 1 is used for preparing benzopyrene artificial antigen 2 by coupling Bovine Serum Albumin (BSA) through an active ester method, and the method comprises the following steps:
dissolving 14.2mg of benzopyrene hapten 1 prepared in example 1 in 0.5mL of DMF solution, stirring, adding 12mg of EDC and 10mg of NHS, and stirring at room temperature for reaction overnight in a dark place to obtain benzopyrene hapten 1 activating solution; dissolving 24mg BSA in 2.4mL BB buffer with pH=9.0, stirring, adding 225 mu L benzopyrene hapten 1 activating solution, stirring uniformly, and coupling for 4 hours at room temperature in a dark place to obtain a coupling mixture; dialyzing the coupling mixture with PBS buffer solution at 4deg.C for 3 days, changing the dialysate for 2 times per day to obtain benzopyrene artificial antigen 2 (benzopyrene hapten 1-BSA), subpackaging the benzopyrene artificial antigen 2 at a concentration of 1mg/mL, and freezing in a refrigerator at-20deg.C.
2. Identification of benzopyrene artificial antigen 2
The carrier proteins Bovine Serum Albumin (BSA), benzopyrene hapten 1 and benzopyrene artificial antigen 2 are subjected to ultraviolet scanning measurement (190-400 nm).
2. Experimental results
The measurement result is shown in fig. 4, it can be seen from fig. 4 that the ultraviolet characteristic absorption peak of the complete antigen is different in degree relative to both benzopyrene hapten and carrier protein (BSA), and the benzopyrene artificial antigen 2 is found to have the characteristic absorption peak of the benzopyrene hapten and the BSA at the same time, which indicates that the coupling of the benzopyrene hapten 1 and the BSA is successful, the benzopyrene artificial antigen 2 is successfully prepared, the structural formula of which is shown as the formula (III-2),
EXAMPLE 4 Synthesis and identification of benzopyrene hapten 2
1. Experimental method
1. Synthesis of benzopyrene hapten 2 (synthetic route pattern see FIG. 5)
2-bromophenylacetone (6.07 g, 28.488 mol) was weighed into 80mL of an ethylene glycol dimethyl ether solution, tetrakis (triphenylphosphine) palladium (330 mg, 0.284 mol) was added under nitrogen protection, the resulting solution was stirred at room temperature for 20 minutes, then 5-hydroxy-2-naphthalene boric acid (6.430 g,34.205 mmol) and 100mL of ethanol were added, after stirring at room temperature for 20 minutes, a sodium carbonate solution (2 mol/L,28.5 mL) was added, and the mixture was heated under reflux for 4 hours at 90 ℃. After the reaction was completed, the solution was cooled to room temperature to give a precipitate, and the precipitate was filtered off, the liquid phase was washed with ethyl acetate, the filtrate was evaporated to dryness, and the residue was dissolved in ethyl acetate, washed with water, dried and dehydrated with anhydrous sodium sulfate, and then the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography to give the product, white intermediate 1 (1- (2- (5-hydroxynaphthalen-2-yl) phenyl) -2-propanone),
to the dried flask was added a solution of intermediate 1 (2.760 g,9.988 mmol) in 85mL of dichloromethane under nitrogen, cooled to-78deg.C, and then titanium tetrachloride in dichloromethane (1 mol/L,10 mL) was added dropwise, and after the dropwise addition was completed, the reaction was allowed to proceed to room temperature for 3h. Quenching with ice water after the reaction, extracting the mixture with ethyl acetate, combining the organic phases, washing with water, drying and dehydrating with anhydrous sodium sulfate, spin-drying the solvent, purifying by silica gel column chromatography to obtain intermediate 2 (5-methyl Qu Ben-1-ol),
Intermediate 2 (938 mg,3.635 mmol), methyl 4-bromobutyrate (1.316 g,7.270 mmol) and anhydrous potassium carbonate (1.997 g, 10.015 mmol) were taken together in 10mL of N, N-dimethylformamide and reacted overnight at 80 ℃. After the reaction is completed, adding a proper amount of water, extracting with dichloromethane for 3 times, combining organic phases, drying and dehydrating the organic phases with anhydrous sodium sulfate, spin-drying the solvent, purifying the solvent by silica gel column chromatography to obtain a brown oily intermediate 3 (4- ((5-methyl Qu Huangxi-1-yl) oxy) methyl butyrate),
intermediate 3 (1.008 g,2.814 mmol) was taken and reacted at 80℃for 2h with 10mL methanol as solvent and 5mL 10% aqueous sodium hydroxide solution. And after the reaction is finished, regulating the pH of the system to 6, centrifuging, discarding the water phase, washing the solid with dichloromethane, and purifying by silica gel column chromatography to obtain brown solid (4- ((5-methyl Qu Huangxi-1-yl) oxy) butyric acid), thus obtaining the product.
2. Identification of benzopyrene hapten 2
And (3) carrying out mass spectrometry and nuclear magnetic resonance hydrogen spectrometry on the benzopyrene hapten 2 to determine the molecular weight and structural characteristics of the benzopyrene hapten.
2. Experimental results
Nuclear magnetic results of benzopyrene hapten 2: 1H NMR (600 MHz, DMSO). Delta.12.19 (s, 1H), 9.05-8.86 (m, 2H), 8.18-7.83 (m, 1H), 7.48 (s, 6H), 7.49-7.20 (m, 1H), 4.23 (dd, J=38.4, 32.1Hz, 2H), 2.86 (d, J=13.4 Hz, 3H), 2.42 (d, J=43.9 Hz, 2H), 2.10-1.98 (m, 2H).
ESI-MS identification results of benzopyrene hapten 2: as shown in fig. 6, MS of the benzopyrene hapten 2: c (C) 23 H 20 O 3 ：344.41，ESI-[M+H] + :：345.1。
The structural formula of the benzopyrene hapten 2 is shown as a formula (II):
EXAMPLE 5 Synthesis and identification of benzopyrene Artificial antigen 3
1. Experimental method
dissolving 14.2mg of benzopyrene hapten 2 prepared in example 4 in 0.5mL of DMF solution, stirring, adding 12mg of EDC and 10mg of NHS, and stirring at room temperature for reaction overnight in a dark place to obtain benzopyrene hapten 2 activating solution; dissolving 24mg BSA in 2.4mL BB buffer solution with pH=9.0, slowly dropwise adding benzopyrene hapten 2 activating solution under stirring, and coupling overnight at room temperature in a dark place after stirring uniformly to obtain a coupling mixture; dialyzing the coupling mixture with PBS buffer solution at 4deg.C for 3 days, changing the dialysate for 2 times per day to obtain benzopyrene artificial antigen 3 (benzopyrene hapten 2-BSA), subpackaging the benzopyrene artificial antigen 3 at a concentration of 1mg/mL, and freezing in a refrigerator at-20deg.C.
Ultraviolet absorption peak scanning measurement (190-400 nm) is carried out on carrier proteins Bovine Serum Albumin (BSA), benzopyrene hapten 2 and benzopyrene artificial antigen 3.
2. Experimental results
As shown in FIG. 7, the ultraviolet characteristic absorption peak of the benzopyrene complete antigen 3 is offset to different degrees relative to the benzopyrene hapten 2 and the carrier protein BSA, and the benzopyrene artificial antigen 3 is found to have the characteristic absorption peak of the benzopyrene hapten 2 and the BSA at the same time, which indicates that the benzopyrene hapten 2 and the BSA are successfully coupled, and the benzopyrene artificial antigen 3 is successfully prepared, and the structural formula of the benzopyrene artificial antigen 3 is shown as the formula (IV-1).
Example 6 preparation and identification of antibodies
1. Experimental method
1. Immunization of mice: 250 μl of benzopyrene artificial antigen 1 (benzopyrene hapten 1-LF) prepared and diluted in example 2 is uniformly emulsified with an equal amount of immunological adjuvant (Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant for the subsequent booster immunization) to immunize animals.
Bal b/c mice with the sizes of 6-7 weeks are immunized by adopting various injection modes of subcutaneous back, subcutaneous parts, abdominal cavity and feet, the immunization is 100 mu L/mouse, the immunization is performed for the second time after 2 weeks, and the immunization is enhanced every 2 weeks after the interval. Mice tail bled 1 week after the third boost and serum titers were determined using an indirect competition ELISA. When the potency no longer rises, 100. Mu.L of benzopyrene artificial antigen 1 with the concentration of 1mg/mL is taken for intraperitoneal injection for impact immunization.
2. Cell fusion: three days after the impact immunization, cell fusion was performed using PEG (polyethylene glycol) as follows:
a. mouse spleen cells were collected: killing a mouse by a cervical dislocation method, immediately soaking the mouse in 75% alcohol for sterilization, taking out the spleen of the mouse by aseptic operation, putting the spleen into a 200-mesh cell screen, moderately grinding the spleen by a rubber head of an aseptic injector, flushing the spleen with a basal medium to obtain spleen cell suspension, collecting, centrifuging (1000 rpm,7 min), washing the spleen cells with the basal medium for three times, and diluting the spleen cells to a certain volume and counting for later use after the last centrifuging;
b. collecting SP2/0 cells: 7-10 days before fusion, SP2/0 myeloma cells were cultured in complete medium at 5% CO 2 Culturing in an incubator. The number of SP2/0 tumor cells before fusion reaches 1 to 4 multiplied by 10 7 Ensuring SP2/0 tumor cells to be in logarithmic growth phase before fusion. During fusion, myeloma cells are collected and suspended in a basal medium for cell counting;
c. according to spleen cells: SP 2/0=5: 1, mixing the two cells in proportion, centrifuging, and discarding the supernatant to obtain mixed cells deposited at the bottom of the centrifuge tube;
d. fusion: slowly dripping 1mL of PEG into cells at the bottom of the centrifuge tube in the first minute; the second minute, keep the centrifuge tube to shake at a constant speed; thirdly, 1mL of preheated basic culture medium is dripped in; dropwise adding 3mL of preheated basic culture medium in the fourth minute; fifth, 8mL of preheated basic culture medium is dripped; sixthly, 8mL of preheated basic culture medium is dripped in; centrifuging (1000 rpm,7 min), discarding supernatant, re-suspending in HAT-containing screening culture medium, adding to 96-well cell plate at 200 μl/well, standing at 37deg.C, 5% CO 2 Culturing in an incubator.
3. Cell screening and cell strain establishment: half-changing the fused cells with HT medium on day 5 of cell fusion, full-changing on day 8, taking cell supernatant on day 10, screening by ic-ELISA, and determining the potency inhibition effect of positive cells. Cell holes with better inhibition on benzopyrene standard substances are selected, subcloning is carried out by adopting a limiting dilution method, and detection is carried out by adopting the same method. Repeating the steps for 4 to 5 times to obtain a cell strain E6.
4. Preparation and identification of monoclonal antibodies: taking a plurality of Bal b/c mice over 10 weeks old, and injecting 500 mu L of liquid paraffin into the abdominal cavity of each mouse; about 1×10 per mouse was intraperitoneally injected 7 days later 6 Hybridoma cells, collecting ascites after 7 days of abdominal swelling of the mice, and purifying the collected ascites by chromatography column method. Filling a column with 1mL protein G, adding 50mL of ascites diluted by PBS buffer solution, repeatedly loading the flow-through solution for 7-8 times, eluting with glycine, and timely adjusting the eluent to be neutral with Tris-HCL. Dialyzing and desalting to finally obtain the purified monoclonal antibody E6, subpackaging and then preserving at-20 ℃.
5. The potency and inhibition rate of the antibodies were determined by indirect competition ELISA, the specific method was:
(1) Coating: diluting the coating antigen to 1 mug/mL, 100 mug/hole with coating liquid, and coating in 37 ℃ water bath overnight;
(2) Closing: removing the coating liquid, washing the plate with the washing liquid for 2 times, beating to dry on the absorbent paper, adding 120 mu L of sealing liquid into each hole, incubating in water bath at 37 ℃ for sealing for 3 hours, spin-drying, and inverting in an oven at 37 ℃ for 1 hour for drying;
(3) Adding an antibody and a drug: monoclonal antibody E6 was diluted 1K (1000), 2K, 4K, 8K, 16K, 32K, 64K, etc. fold with PBS and benzopyrene standard was diluted to 10ng/mL with PBS for use.
The titers are as follows: adding 50 mu L of PBS buffer solution into each hole, sequentially adding the diluted monoclonal antibody E6 into the holes according to 50 mu L/hole, and adding PBS into the last hole to serve as a blank control;
inhibition column: adding 50 mu L of a medicament diluted by PBS buffer solution into each hole, sequentially adding the monoclonal antibody E6 diluted by the double ratio into the holes according to 50 mu L/hole, and adding PBS into the last hole to serve as a blank control;
incubation is carried out for 40min at 37 ℃, and the plate is washed by washing liquid for 5 times;
(4) Adding a secondary antibody: adding goat anti-mouse secondary antibody (100 mu L/hole) diluted 5000 times by PBST buffer solution, incubating at 37 ℃ for 30min, and washing the plate 5 times by using washing solution;
(5) Color development: mixing TMB substrate buffer A, B solution in equal volume to obtain substrate solution, adding substrate solution (100 μl/well), and incubating at 37deg.C for 10min;
(6) And (3) terminating: 10% H was added to the ELISA plate 2 SO 4 The reaction was stopped by the stop solution (50. Mu.L/well);
(7) Reading: the absorbance (OD) was read with a microplate reader at a wavelength of 450 nm. The dilution multiple of the antibody with the absorbance value within the range of 1.0-1.5 is selected as the antibody titer, and the drug recognition performance of the antibody is obtained by the inhibition rate. The inhibition rate is calculated by the formula 1.
2. Experimental results
Antibody titers and results are shown in table 1.
TABLE 1 antiserum titers and inhibition rates for different combinations of coating precursors
As can be seen from Table 1, the homologous coating showed a higher potency of 16K, but the corresponding inhibition was lower, only 41.21%. And after the heterologous coating is used, the antibody titer is 8K, and the inhibition rate is remarkably improved to 73.42%, so that the sensitivity of the detection method can be improved by using the heterologous coating. Meanwhile, the defect of lower antibody titer can be solved by further concentrating the antibody or increasing the coating concentration.
Example 7 coating concentration versus antibody dilution times versus IC 50 Influence of (2)
1. Experimental method
In the immune analysis method for detecting benzopyrene, a chessboard detection method is used for detecting the influence of the coating concentration and the benzopyrene monoclonal antibody concentration on benzopyrene detection.
The artificial antigen 3 (hapten 2-BSA) prepared in example 5 was used as a coating antigen, and the benzopyrene antibody (monoclonal antibody E6) prepared in example 6 was used as an antibody.
Coating the 96-well ELISA plates with coating sources with the concentration of 2, 1, 0.5 and 0.25 mug/mL respectively; diluting antibodies 1000, 2000, 4000, 8000, 16000, 32000 and 64000 times respectively, and preparing benzopyrene standard products into a solution with the concentration of 10 ng/mL; 50. Mu.L of a benzopyrene antibody (monoclonal antibody E6 prepared in example 6) diluted in a serial gradient and 50. Mu.L of a benzopyrene standard of 10ng/mL were added to a 96-well ELISA plate, and after indirect competition ELISA was performed according to the method of embodiment 5, the combination of antibody concentration and coating concentration with absorbance value of 1.0-1.5 was read out by an ELISA reader.
2. Experimental results
And drawing a standard curve by adopting a normalization method by taking the drug concentration as an abscissa and B/B0 as an ordinate, calculating half inhibition concentration IC50 according to the standard curve, selecting maximum absorbance Amax, and taking the minimum IC50 value and the maximum Amax/IC50 value as the optimal working concentration. The optimal coating concentration of this antibody (monoclonal antibody E6) was 1. Mu.g/mL and the dilution factor of the antibody was 8K.
Example 8 method for detecting benzopyrene
An indirect competition ELISA method for detecting benzopyrene, comprising the following steps:
(1) Benzopyrene artificial antigen 3 (benzopyrene hapten 2-BSA, with a structural formula shown as formula (IV-1)) prepared in example 5 is used as a coating source, diluted to 1 mug/mL by carbonate buffer (CB, 0.1M pH=9.8), coated with 96-well ELISA plate, added with 100 mug/well and incubated overnight (12 h) at 37 ℃;
(2) Removing the coating liquid, washing twice with a washing liquid (pH value is 7.4, the washing liquid contains 0.6% of Tween-20, 0.02% of sodium azide preservative and 0.2mol/L of phosphate buffer solution, wherein the percentages are weight and volume percentages), and beating to dry;
(3) Adding 120 mu L of sealing liquid (namely 5% of skimmed milk powder by mass) into each hole, and sealing for 3 hours at 37 ℃;
(4) Discarding the sealing liquid, beating the plate, drying at 37 ℃ for 30min, taking out, and bagging for standby by self-sealing;
(5) Phosphate buffer (PBS, 0.01m, ph=7.4) 1: 8000-fold dilution of the monoclonal antibody prepared in example 6, and 2-fold gradient dilution of benzopyrene drug to be detected to 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.91ng/mL, 1.95ng/mL, 0.98ng/mL, 0.49ng/mL, 0.24ng/mL, 0.12ng/mL, 0.06ng/mL;
(6) Adding 50 mu L of benzopyrene drug diluent to be detected (three groups are parallel) into each row, adding 50 mu L of monoclonal antibody diluent prepared in example 6 per hole, incubating for 40min at 37 ℃, washing five times by using a washing solution, and beating to dry;
(7) Adding enzyme conjugate (horseradish peroxidase-labeled goat anti-mouse secondary antibody) diluted 5000 times with Tween phosphate buffer (PBST, 0.01M), incubating at 37deg.C for 30min at 100 μl/well, washing with washing solution for five times, and drying;
(8) Adding a developing solution with the volume ratio of the solution A to the solution B being 1:1, and incubating at 37 ℃ for developing for 10min at 100 mu L of each hole;
(9) Add 50. Mu.L of stop solution (10% H) 2 SO 4 Solution) was stopped and the OD was read at 450 nm.
(10) And (3) operating according to the steps (1) to (9), replacing the benzopyrene diluent to be detected in the step (6) with the extracted sample diluent to be detected, and combining the drawn standard curve to determine the content of the actual benzopyrene medicine in the unknown sample.
2. Experimental results
An indirect competition ELISA standard curve of the antibody for detecting benzopyrene drug is shown in FIG. 8, and it can be seen from FIG. 8 that the half Inhibitory Concentration (IC) of the antibody for detecting benzopyrene drug 50 ) 4.07ng/mL, the quantitative detection range is 0.91-18.22 ng/mL, and the lowest limit of detection (LOD) is 0.38ng/mL; illustrating the anti-benzopyrene for detecting benzopyrene prepared by the inventionThe body sensitivity is high, and the detection requirement can be met.
Example 9A kit for detecting benzopyrene
1. Composition of the composition
A kit for detecting benzopyrene comprising the following parts:
(1) Preparing an ELISA plate coated with a coating source: the benzopyrene artificial antigen 3 (benzopyrene hapten 2-BSA, the structural formula of which is shown as formula (IV-1)) prepared in example 5 is used as a coating source, the coating source is diluted to 1 mug/mL by carbonate buffer solution (CB, 0.1M pH=9.8), 100 mug/mL is added to each hole, the mixture is incubated overnight at 37 ℃ in a dark place, liquid in the hole is poured out, the mixture is washed for 2 times by washing liquid, each time for 30 seconds, and then 120 mug of blocking solution is added to each hole, incubation is carried out for 2 hours at 37 ℃ in a dark place, the liquid in the hole is poured out, and the mixture is dried and stored in a vacuum sealing way by an aluminum film; wherein the coating buffer solution is carbonate buffer solution with the pH value of 9.6 and 0.05mol/L, the sealing solution is phosphate buffer solution with the pH value of 7.1-7.5, and the coating buffer solution contains casein with the mass ratio of 1-3% and phosphate buffer solution with the pH value of 0.1-0.3 mol/L;
(2) Benzopyrene standard solution: 15 concentration gradients of 1000ng/mL,500ng/mL,250ng/mL,125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.91ng/mL, 1.95ng/mL, 0.98ng/mL, 0.49ng/mL, 0.24ng/mL, 0.12ng/mL, 0.06ng/mL, respectively;
(3) Benzopyrene monoclonal antibody (monoclonal antibody E6) prepared in example 6;
(4) Enzyme conjugate: horseradish peroxidase-labeled goat anti-mouse secondary antibody;
(5) Substrate color development liquid: the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(6) The stop solution is 10% H by volume 2 SO 4 ；
(7) The washing solution has a pH value of 7.4 and contains 0.6% of Tween-20, 0.02% of sodium azide preservative and 0.2mol/L of phosphate buffer solution, wherein the percentages are weight volume percentages.
2. Application method
Same as in example 8.
Example 10 sensitivity to detection of benzopyrene and other common polycyclic aromatic hydrocarbons
1. Experimental method
Using the kit of example 9, 50. Mu.L of a series concentration of benzopyrene standard and 50. Mu.L of the benzopyrene antibody prepared in example 6 diluted with 8K were added to a 96-well ELISA plate, and absorbance (OD) was measured by an ELISA analyzer after an indirect competition ELISA reaction.
2. Experimental results
In B/B 0 The corresponding standard substance concentration is the abscissa, and the half inhibition amount concentration of the standard curve is IC by adopting a normalization method 50 In IC 10 For detection limit, IC is used 20 ～IC 80 Is the detection range. The standard curve of ELISA was established using benzopyrene as standard, the results are shown in FIG. 8, and the relevant standard curve parameters are shown in Table 2.
TABLE 2 detection parameters of benzopyrene antibody against benzopyrene
As can be seen from fig. 8 and table 2, the standard curve established using benzopyrene as a standard has a typical S-shaped curve, which demonstrates that the detection sensitivity by using the antibody of the present invention is good.
Example 11 specificity for detection of benzopyrene and other common polycyclic aromatic hydrocarbons
1. Experimental method
Benzopyrene and other common polycyclic aromatic hydrocarbon drugs were detected using the kit of example 9.
2. Experimental results
The results are shown in Table 3. The cross-reaction rate was calculated as shown in equation 2.
Table 3:
as is clear from Table 3, IC was obtained with the cross-reactivity of the antibody to benzopyrene being 100% 50 The value is 4.07ng/mL, and the cross reaction rate of other polycyclic aromatic hydrocarbon compounds is lower than 5%, which indicates that the antibody can specifically identify benzopyrene and further detect the content of benzopyrene.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (10)
1. A benzopyrene hapten is characterized in that the structural formula is shown in a formula (I),
2. A benzopyrene artificial antigen is characterized in that the benzopyrene hapten-coupled carrier protein of claim 1 has a structural formula shown in formula (III),
3. a benzopyrene hapten is characterized in that the structural formula is shown as a formula (II),
4. a benzopyrene artificial antigen is characterized in that the benzopyrene hapten-coupled carrier protein of claim 3 has a structural formula shown in formula (IV),
5. use of the benzopyrene hapten as claimed in claim 1 and/or 3 for the preparation of artificial benzopyrene antigen.
6. A composition for detecting benzopyrene, comprising the benzopyrene artificial antigen according to claims 2 and 4, the benzopyrene artificial antigen according to claim 2 as an immunogen, and the benzopyrene artificial antigen according to claim 4 as a coating antigen.
7. The composition of claim 6, wherein the benzopyrene artificial antigen carrier protein of claim 2 is lactoferrin as an immunogen and the benzopyrene artificial antigen carrier protein of claim 4 is bovine serum albumin as a coating antigen.
8. Use of the benzopyrene hapten as defined in claim 1 and/or 3, the benzopyrene artificial antigen as defined in claim 2 and/or 3, and/or the composition as defined in claim 6 for preparing a benzopyrene detection kit.
9. A benzopyrene detection kit, which is characterized by comprising the composition of claim 6.
10. A method for detecting benzopyrene for the purpose of non-disease diagnosis, characterized by using the composition of claim 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310168932.6A CN116375578A (en) | 2023-02-23 | 2023-02-23 | Benzopyrene hapten, artificial antigen and benzopyrene detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310168932.6A CN116375578A (en) | 2023-02-23 | 2023-02-23 | Benzopyrene hapten, artificial antigen and benzopyrene detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116375578A true CN116375578A (en) | 2023-07-04 |
Family
ID=86977755
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310168932.6A Pending CN116375578A (en) | 2023-02-23 | 2023-02-23 | Benzopyrene hapten, artificial antigen and benzopyrene detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116375578A (en) |
-
2023
- 2023-02-23 CN CN202310168932.6A patent/CN116375578A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110441512B (en) | Colloidal gold immunochromatography detection device for ethyl maltol hapten and ethyl maltol | |
| CN112574956B (en) | Hybridoma cell strain secreting propamocarb monoclonal antibody and application thereof | |
| CN116286669B (en) | Hybridoma cell strain secreting toraset Mi Shan clone antibody and application thereof | |
| CN110927382A (en) | Time-resolved fluorescence immunoassay kit for detecting olaquindox and application thereof | |
| CN116375578A (en) | Benzopyrene hapten, artificial antigen and benzopyrene detection kit | |
| CN113005097B (en) | Hybridoma cell strain secreting anti-carbamazepine monoclonal antibody and application thereof | |
| WO2018103630A1 (en) | Hybridoma cell strain c1 for secreting anti-paromomycin monoclonal antibody and use thereof | |
| CN114031528B (en) | Florfenicol hapten, artificial antigen, antibody and synthetic method and application thereof | |
| CN110927376B (en) | Magnetic immunochemistry luminescence detection kit of olaquindox and application thereof | |
| CN109061153B (en) | Time-resolved fluorescence immunochromatographic test strip for detecting iprodione and preparation method and application thereof | |
| CN114014774A (en) | Fluoroamidone artificial hapten, artificial antigen, and preparation method and application thereof | |
| CN106867970B (en) | Hybridoma cell strain secreting anti-malachite green monoclonal antibody and application thereof | |
| CN117069580B (en) | Triclosan hapten as well as preparation method and application thereof | |
| CN110927375A (en) | Fluorescent microsphere immunochromatography test strip for detecting olaquindox residue and application thereof | |
| CN117362172B (en) | Nonylphenol hapten as well as preparation method and application thereof | |
| CN111060690B (en) | Time-resolved fluoroimmunoassay kit for detecting olaquindox and application thereof | |
| CN107226795B (en) | Linezolid hapten and complete antigen as well as preparation method and application thereof | |
| CN115819404B (en) | Rapid detection device and detection method for pymetrozine in agricultural products | |
| CN111925321B (en) | Paraquat hapten, complete antigen, antibody, detection test paper and kit | |
| CN115611774B (en) | Fenpropathrin hapten, artificial antigen and fenpropathrin detection method | |
| CN115746015B (en) | Patinopecten yessoensis toxin hapten, artificial antigen and antibody as well as preparation methods and application thereof | |
| CN114317450B (en) | Hybridoma cell strain secreting Flurobendiamide monoclonal antibody and application thereof | |
| CN114317448B (en) | Hybridoma cell strain secreting anti-etoxazole monoclonal antibody and application thereof | |
| CN113896743B (en) | Dipterex hapten, preparation method thereof, antigen, antibody and application thereof | |
| CN113563292B (en) | Henbane hapten, artificial antigen, preparation methods of henbane hapten and artificial antigen, antibody and application of henbane hapten and artificial antigen |
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 |