CN108715611A - A kind of preparation method and its usage of BaP immunogene - Google Patents
A kind of preparation method and its usage of BaP immunogene Download PDFInfo
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- CN108715611A CN108715611A CN201810548486.0A CN201810548486A CN108715611A CN 108715611 A CN108715611 A CN 108715611A CN 201810548486 A CN201810548486 A CN 201810548486A CN 108715611 A CN108715611 A CN 108715611A
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- bap
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- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 210000000780 bap Anatomy 0.000 claims abstract description 118
- 238000000034 method Methods 0.000 claims abstract description 13
- 229910052740 iodine Inorganic materials 0.000 claims abstract description 10
- 239000011630 iodine Substances 0.000 claims abstract description 10
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 claims abstract description 9
- 238000003018 immunoassay Methods 0.000 claims abstract description 8
- 238000005859 coupling reaction Methods 0.000 claims abstract description 6
- 150000002148 esters Chemical class 0.000 claims abstract description 5
- 238000006146 oximation reaction Methods 0.000 claims abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims description 34
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 26
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 18
- 239000003208 petroleum Substances 0.000 claims description 13
- 239000002904 solvent Substances 0.000 claims description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 11
- 238000004809 thin layer chromatography Methods 0.000 claims description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 9
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 102000014914 Carrier Proteins Human genes 0.000 claims description 6
- 108010078791 Carrier Proteins Proteins 0.000 claims description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 6
- 230000009514 concussion Effects 0.000 claims description 6
- 239000012074 organic phase Substances 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- 229910001868 water Inorganic materials 0.000 claims description 6
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 5
- 230000008859 change Effects 0.000 claims description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- 239000012265 solid product Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000007795 chemical reaction product Substances 0.000 claims description 4
- 238000002955 isolation Methods 0.000 claims description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 239000011734 sodium Substances 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 4
- NQRKYASMKDDGHT-UHFFFAOYSA-N (aminooxy)acetic acid Chemical class NOCC(O)=O NQRKYASMKDDGHT-UHFFFAOYSA-N 0.000 claims description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical class CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 3
- 230000002378 acidificating effect Effects 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- 239000002994 raw material Substances 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 229910052708 sodium Inorganic materials 0.000 claims description 3
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical class C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000012043 crude product Substances 0.000 claims description 2
- 239000012153 distilled water Substances 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 238000000703 high-speed centrifugation Methods 0.000 claims description 2
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 claims description 2
- 239000013067 intermediate product Substances 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 238000001953 recrystallisation Methods 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 239000003643 water by type Substances 0.000 claims description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims 3
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 claims 2
- 239000000203 mixture Substances 0.000 claims 1
- 229910052763 palladium Inorganic materials 0.000 claims 1
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical class [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 claims 1
- 229960002317 succinimide Drugs 0.000 claims 1
- 230000002163 immunogen Effects 0.000 abstract description 15
- 108091003079 Bovine Serum Albumin Proteins 0.000 abstract description 12
- 229940098773 bovine serum albumin Drugs 0.000 abstract description 12
- 238000001514 detection method Methods 0.000 abstract description 12
- 238000007336 electrophilic substitution reaction Methods 0.000 abstract description 3
- 238000010189 synthetic method Methods 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 15
- 241000283973 Oryctolagus cuniculus Species 0.000 description 8
- 238000002965 ELISA Methods 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 238000002649 immunization Methods 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- GVEPBJHOBDJJJI-UHFFFAOYSA-N fluoranthene Chemical compound C1=CC(C2=CC=CC=C22)=C3C2=CC=CC3=C1 GVEPBJHOBDJJJI-UHFFFAOYSA-N 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- DXBHBZVCASKNBY-UHFFFAOYSA-N 1,2-Benz(a)anthracene Chemical compound C1=CC=C2C3=CC4=CC=CC=C4C=C3C=CC2=C1 DXBHBZVCASKNBY-UHFFFAOYSA-N 0.000 description 3
- HAXBIWFMXWRORI-UHFFFAOYSA-N Benzo[k]fluoranthene Chemical compound C1=CC(C2=CC3=CC=CC=C3C=C22)=C3C2=CC=CC3=C1 HAXBIWFMXWRORI-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- BBEAQIROQSPTKN-UHFFFAOYSA-N pyrene Chemical compound C1=CC=C2C=CC3=CC=CC4=CC=C1C2=C43 BBEAQIROQSPTKN-UHFFFAOYSA-N 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- MWPLVEDNUUSJAV-UHFFFAOYSA-N anthracene Chemical compound C1=CC=CC2=CC3=CC=CC=C3C=C21 MWPLVEDNUUSJAV-UHFFFAOYSA-N 0.000 description 2
- 238000005452 bending Methods 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 150000001735 carboxylic acids Chemical class 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- LHRCREOYAASXPZ-UHFFFAOYSA-N dibenz[a,h]anthracene Chemical compound C1=CC=C2C(C=C3C=CC=4C(C3=C3)=CC=CC=4)=C3C=CC2=C1 LHRCREOYAASXPZ-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 206010013786 Dry skin Diseases 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 240000007711 Peperomia pellucida Species 0.000 description 1
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical class [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- -1 amino, carboxyl Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000005605 benzo group Chemical group 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000005253 cladding Methods 0.000 description 1
- 239000003245 coal Substances 0.000 description 1
- 238000002485 combustion reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000008157 edible vegetable oil Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- JEGUKCSWCFPDGT-UHFFFAOYSA-N h2o hydrate Chemical compound O.O JEGUKCSWCFPDGT-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 150000003949 imides Chemical class 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical class [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000001819 mass spectrum Methods 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
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000007886 mutagenicity Effects 0.000 description 1
- 231100000299 mutagenicity Toxicity 0.000 description 1
- 239000003345 natural gas Substances 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 150000002923 oximes Chemical class 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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
- C07K14/76—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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
- G01N2333/765—Serum albumin, e.g. HSA
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of preparation method and its usages of BaP immunogene, including：First electrophilic substitution reaction is carried out with BaP standard items generate 6- iodine BaPs, the carbonyl derivative BaP=O i.e. BaP haptens of BaP is obtained by Heck coupling reactions again, then through oximation reaction by the carbonyl carboxylated of BaP haptens, it is used in combination active ester method to be coupled with bovine serum albumin (BSA), obtains BaP immunogenes BSA-BaP；Above-mentioned synthetic method is simple, quick, provides a kind of new way for the BaP immunogenes preparing high specificity, can be used for preparing BaP artificial polyclonal antibodies and establishes BaP immunoassay detection method.
Description
Technical field
The invention belongs to biochemical fields, and in particular to a kind of preparation method and its usage of BaP immunogene.
Background technology
BaP (BaP) is also known as 3,4- BaPs, the fuel such as coal, oil and natural gas in industrial production and life process
Imperfect combustion and food can all generate BaP in the high temperature cooking process such as smoking, toasting and frying, and have carcinogenicity, cause
Abnormal property and mutagenicity, are widely present in surrounding medium.The toxicity of BaP has a long-term and concealment characteristic, human contact or
It can accumulate after intake BaP, have compared with long latency before showing symptom in vivo.Since BaP is derived from a wealth of sources, and to human body
Harmfulness is larger, causes the extensive research of domestic and foreign scholars, at present both at home and abroad focuses mostly on the research of BaP detection in big
The food such as the surrounding mediums such as gas and water body and grain and oil, meat, most common detection method have gas-chromatography, gas chromatography-mass spectrum,
The instrument analytical methods such as high performance liquid chromatography.
Currently, rapid screening and detection of the enzyme linked immunosorbent assay (ELISA) for batch samples, are widely ground
Study carefully, such as Matschulat is prepared for BaP monoclonal antibodies, and the content of BaP in water, sensitivity are had detected using ELISA method
IC50For 24ng/mL；Pschenitza etc. utilizes the BaP in the method detection edible oil of Solid Phase Extraction combination ELISA, inspection
Survey lower limit is 0.63 μ g/mg.It is immunized in addition, biotin-avidin-enzyme linked immunosorbent assay (BA-ELISA) etc. is highly sensitive
Detection technique is constantly risen, be it is a kind of by the efficient catalytic performance of the specific recognition immune response and enzyme between antigen-antibody and
Biotin-avidin system is combined together and the heterogeneous immuno analytical method that grows up, i.e., by antibody or antigen biology
Elementization is combined using single-minded, the specific identification of the antigen or antibody of solid phase cladding, recycles streptavidin couple biotin
Change antibody, carrying out catalytic small molecule substrate finally by enzyme generates the recognizable color change of naked eyes, due to being combined on solid phase carrier
Enzyme amount and the amount of determinand a certain amount is presented than relationship, it can be achieved that qualitative or quantitative analysis.In BA-ELISA methods, one
The identification that 4 subunits in a avidin molecule can distinguish monospecific combines a biotin molecule, affinity costant
Up to 1015mol/L is improved by the enzyme quantity of solid phase binding, is reduced and is reacted with the non-specific identification between reagent, improves detection
The sensitivity of method.
Above-mentioned immunoassay method is all based on the detection technique of the specific recognition immune response between antigen-antibody, BaP
The preparation of immunogene is to establish the basis of detection BaP immuno analytical methods, and BaP molecules are without the activity that can be coupled with protein
Function matrix needs to introduce active function groups in BaP small molecules, so as to carrier protein couplet such as amino, carboxyl, carbonyl
Deng.Reported at present about the preparation method of BaP immunogenes less, and there are complicated for operation, yield be relatively low or required equipment requirement
Higher, related BaP immunogene preparation method types are less and single.
Invention content
The present inventor has found on the basis of early-stage study, first carries out electrophilic substitution reaction with BaP standard items
6- iodine BaPs are generated, then obtain the carbonyl derivative BaP=O of the i.e. BaP of BaP haptens by Heck coupling reactions, are then passed through
The carbonyl carboxylated of BaP haptens is used in combination active ester method to be coupled with bovine serum albumin (BSA), obtains BaP immunogenes by oximation reaction
BSA-BaP.The present invention provides a kind of new way for the BaP immunogenes preparing high specificity, synthetic method is simple, quick, can
It is used to prepare BaP artificial polyclonal antibodies and establishes the immunoassay detection method of BaP.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of BaP immunogene, shown in structural formula such as formula (I),
The second aspect of the present invention, the preparation method of above-mentioned BaP immunogene, shown in synthetic route such as formula (II),
Specifically, include the following steps：
(1) iodide reaction is carried out with BaP standard items, reacts 48h at room temperature and obtains intermediate product 6- iodine BaPs, and
It is methyl tertiary butyl ether by solvent:Petroleum ether=1:The thin-layer chromatography of 25 (v/v) monitors above-mentioned reaction until raw material disappears；
(2) under conditions of reaction temperature is 120 DEG C, gained 6- iodine BaPs carry out Heck coupling reactions in step (1)
15h, and be methyl tertiary butyl ether by solvent:Petroleum ether=1:The thin-layer chromatography of 2 (v/v) monitors above-mentioned reaction to having reacted
Entirely, then through leacheate it is methyl tertiary butyl ether:Petroleum ether=1:The silica gel chromatographic column isolation of purified of 2 (v/v), obtains BaP haptens；
(3) the carbonyl carboxylated of BaP haptens obtained by step (2) is obtained by BaP carboxy derivatives as oximation reaction,
And by solvent be chloroform:Methanol=9:The thin-layer chromatography of 1 (v/v) monitors above-mentioned reaction to the reaction was complete；Then it is with BSA
Carrier protein, with active ester method by above-mentioned BaP carboxy derivatives and BSA be coupled to get.
In a preferred embodiment of the invention, the specific preparation process of above-mentioned BaP immunogene is：
(1-1) takes 500mL three neck round bottom flask, be separately added into 0.4g BaPs, 0.42gNIS, 10g acidic alumina and
Reaction 48h is stirred at room temperature in 30mL toluene, and is methyl tertiary butyl ether by solvent:Petroleum ether=1:The thin-layer chromatography of 25 (v/v)
Above-mentioned reaction is monitored until raw material disappears；
The reaction product of (2-1) step (1-1) merges organic phase after remaining acid aluminium oxide is removed by filtration, then uses respectively
Respectively three times, gained organic phase is through anhydrous MgSO for extraction for saturated sodium bisulfite solution, saturation NaCl solution, ultra-pure water4Dry, mistake
Filter is concentrated under reduced pressure, and after dichloromethane and petroleum ether recrystallization purification, obtains yellow powdery solid object 6- iodine BaPs, yield
It is 76.2%；
(3-1) be separately added into 100mL single-necked flasks gained yellow powdery solid object in 0.27mg steps (2-1),
0.01g palladiums and 0.02gLiCl are added 20mLDMF dissolvings, sequentially add 70 μ L4- amylene -2- alcohol, 200 μ L triethylamines,
120 DEG C are stirred to react 15h, and are methyl tertiary butyl ether by solvent:Petroleum ether=1:The thin-layer chromatography monitoring of 2 (v/v) is above-mentioned
Reaction is to the reaction was complete；
Reaction product in step (3-1) is cooled to room temperature by (4-1), and 120mL toluene is added, molten with saturation NaCl respectively
Respectively three times, gained organic phase is through anhydrous MgSO for extraction for liquid, ultra-pure water4Crude product is dried, filters, being concentrated under reduced pressure and to obtain, then through elution
Liquid is methyl tertiary butyl ether:Petroleum ether=1:The silica gel chromatographic column isolation of purified of 2 (v/v) obtains light yellow powder product BaP
Haptens, molecular formula C27H20O, yield 86.2%；
(5-1) takes the above-mentioned light yellow powder products of 0.12g, half hydrochloride of 0.24g carboxymethoxylamines (CMO) to be dissolved in 15mL
In dry pyridine, concussion reaction under the conditions of being protected from light, and be chloroform by solvent:Methanol=9:The thin-layer chromatography of 1 (v/v) monitors
Above-mentioned reaction is to the reaction was complete；After complete reaction, 0.54g sodium borohydrides is taken to be dissolved in 4.5mL ddH2In O, it is added to above-mentioned anti-
It answers in solution, then room temperature concussion reaction 5h blows down removal solvent in nitrogen, residue is used to hydrochloric acid solution (pH2.0) respectively and is steamed
Distilled water washes twice, and is dried in 60 DEG C of baking ovens, obtains yellow solid product BaP carboxy derivatives, structural formula BaP-COOH；
(6-1) weighs the above-mentioned yellow solid products of 0.082g and is dissolved in 0.5mLDMF, and 0.023gN- hydroxysuccinimidyls acyl is sub-
After amine (NHS), 0.412g dicyclohexylcarbodiimides (DCC) are dissolved in 0.5mLDMF, it is added to 4 DEG C of stirrings in above-mentioned solution
React 8h；The high speed centrifugation 20min at 6000rpm, 4 DEG C again, taking supernatant that 5mLBSA solution is added, (16mg/mL is dissolved in
0.01M, pH7.40 phosphate buffer), it is stirred to react at 4 DEG C overnight；Repeated centrifugation, and by supernatant 0.01M, pH7.40
Phosphate buffer is dialysed 3d, preserve at -20 DEG C to get.
The third aspect of the present invention, application of the above-mentioned BaP immunogene in preparing BaP artificial polyclonal antibodies, and
Purposes as the detection of BaP immunoassay.
Compared with prior art, the beneficial effects of the present invention are：
In the preparation method of BaP immunogene of the present invention, electrophilic substitution reaction, Heck coupling reactions and oxime are passed sequentially through
Change reaction by the carbonyl carboxylated of BaP haptens, is used in combination active ester method to be coupled with bovine serum albumin (BSA), BaP immunogenes are made
BSA-BaP, synthetic method is simple, quick, is the new way for the BaP immunogenes for preparing high specificity, can be used for preparing BaP artificial
Polyclonal antibody and establish BaP immunoassay detection method.
Description of the drawings
Fig. 1 is the infrared spectrogram of BaP immunogenes in the present invention；
Fig. 2 is the hydrogen nuclear magnetic resonance spectrogram of BaP immunogenes in the present invention；
Fig. 3 is the ultraviolet spectrogram of BaP haptens, carrier protein and BaP immunogenes in the present invention；
Fig. 4 is that Coomassie Brilliant Blue measures BSA concentration standard curves in the present invention；
Fig. 5 is the variation of moderate resistance BaP antibody titers of the present invention.
Specific implementation mode
The technical solution that the invention will now be described in detail with reference to the accompanying drawings, but protection scope of the present invention is not limited to following realities
Example is applied, the experimental method in following embodiment is conventional method unless otherwise specified, and reagent is with material without the equal city of specified otherwise
Selling can obtain.
Embodiment 1：The preparation of BaP immunogenes
It takes 200mgBaP standard items to be dissolved in 30mL toluene with 420mgNIS, 5g acidic aluminas is slowly added to, in room temperature
It is stirred to react 48h, reaction solution is respectively with saturation Na2SO3Solution, saturated salt solution and ultra-pure water respectively extraction 3 times, through anhydrous MgSO4
After dry and concentration, purified to obtain pale yellow powder 6- iodine BaPs with petroleum ether and recrystallize with dichloromethane；6- iodine is weighed again
BaP, 20mg lithium chlorides and 10mg palladiums are dissolved in 20mLDMF, are slowly added to 200 μ L triethylamines and 70 μ L4- penta successively
Dilute -2- alcohol, 120 DEG C are stirred to react 15h；It waits for that 120mL toluene is added after reaction, mixed liquor uses saturated brine and ultrapure respectively
Water respectively extraction 3 times, through anhydrous MgSO4It is methyl tertiary butyl ether(MTBE) by leacheate after dry and concentration：Petroleum ether=1：2 (v/v's)
Silica gel column chromatography isolation of purified obtains yellow powder BaP haptens, molecular formula：C27H20O, yield：86.2%, use infrared light
Spectrum and NMR spectrum characterization, as shown in attached drawing 1,2.
The above-mentioned BaP haptens of 120mg and half hydrochloride of 240mg carboxymethoxylamines (CMO) is taken to be dissolved in the pyrrole of 15mL dryings again
Pyridine, concussion reaction under the conditions of being protected from light, and be chloroform by solvent：Methanol=9：The thin-layer chromatography of 1 (v/v) monitors above-mentioned reaction
To the reaction was complete；It takes 540mg sodium borohydrides to be dissolved in 4.5mL ultra-pure waters and is added in above-mentioned reaction solution, concussion is anti-at room temperature
5h, reaction solution is answered to be blown down in nitrogen and dry up extra solvent, residue uses hydrochloric acid solution (pH2.0), ultra-pure water water washing twice respectively,
It is dried in 60 DEG C of baking ovens, obtains yellow solid product BaP carboxylic acid derivates.
Finally take 82.3mg BaP carboxylic acid derivates to be dissolved in 0.5mLDMF, by n-hydroxysuccinimide (NHS,
23mg), N, N '-dicyclohexyl acid imide (DCC, 41.2mg) are dissolved in DMF (0.5mL) and are added afterwards in above-mentioned solution, stirred at 4 DEG C
Mix reaction 8h, be centrifuged at a high speed 20min at 6000rpm and 4 DEG C, take supernatant be added 5mLBSA solution (16mg/mL, it is molten
In 0.01M, pH7.40 phosphate buffer) it is stirred to react at 4 DEG C, overnight, above-mentioned centrifugally operated is repeated, supernatant is used
0.01M, pH7.40 phosphate buffer dialysis 3d, obtain BSA-BaP immunogenes, are identified with ultraviolet-uisible spectrophotometer,
As shown in Fig. 3.
The infared spectrum of BaP haptens is as shown in Fig. 1, IR (KBr) ν (cm-1)：3042 (C-H, Ar stretch), 2875,
2696 (C-H, stretch), 1696 (C=O stretch), 1502,1232 (C-C, phenyl ring skeletal vibrations), 769 (C-C, phenyl ring are monosubstituted
Peak, bending), 712 (C-H, bendings) illustrate there is apparent carbonyl group characteristic peak in BaP haptens.
The nuclear magnetic resonance spectroscopy of BaP haptens is as shown in Fig. 2,1HNMR (400MHzCDCl3)δ(ppm)：2.11-2.15
(m,2H,4′-H),2.16(s,3H,1′-H),2.58-2.62(m,2H,3′-H),3.69-3.73(m,2H,5′-H),7.75-
7.77(m,2H,8-H,9-H),7.88(dd,1H,1-H,2-H),7.91(d,1H,12-H),8.00(dd,1H,1-H),8.14
(dd,1H,3-H),8.21(d.1H,4-H),8.26(d,1H,5-H),8.53-8.55(m,1H,7-H),8.99(d,1H,11-
H), 9.04-9.06 (m, 1H, 10-H) further proves that BaP haptens is successfully made.
BSA-BaP immunogenes are scanned with ultraviolet-uisible spectrophotometer to be identified, as shown in Fig. 3, the spy of carrier protein BSA
Sign absorption peak is located at 227nm and 281nm, and wherein the absorption value at 281nm absorption peaks is relatively low；BaP haptens is located at 323nm
There is apparent characteristic absorption peak, the ultra-violet absorption spectrum between BaP haptens, carrier protein and conjugate is closed there is significant
System and difference not only separately include the characteristic absorption peak of BSA, at 361nm in addition, in the ultra-violet absorption spectrum of conjugate
The characteristic peak for observing BSA-BaP shows that BaP immunogenes are successfully made.
The concentration of protein in immunogene, the absorbance of BSA standard solution are measured using Coomassie brilliant blue G250 staining
The standard curve that OD values change with BSA concentration of standard solution is as shown in figure 4, calibration curve equation：Y=0.519x+0.0327, phase
Close coefficients R2=0.9964, according to the absorbance value of BaP haptens and protein at its maximum absorption band, calculate conjugate
The coupling ratio of BSA-BaP, the results are shown in Table 1.
Table 1：Artificial antigen protein content
Embodiment 2：The preparation of BaP polyclonal antibodies
Healthy new zealand white rabbit is carried out using the BaP immunogenes of above-mentioned preparation the BaP for preparing specificity is immunized for several times
Polyclonal antibody.When rabbit first immunisation, using the mixing (protein of Freund's complete adjuvant (CFA) and BaP immunogene equal proportions
A concentration of 1mg/mL), when subsequent booster immunization, is all made of incomplete Freund's adjuvant (IFA) and mixes (albumen with immunogene equal proportion
A concentration of 1mg/mL of matter), the immune concrete scheme of White Rabbit is as shown in table 2, since third time booster immunization, after being immunized every time
Time week from rabbit ear edge vein exploitating blood 1mL, detach and sero-fast potency measured using ic-ELISA methods after antiserum.
Using BSA-BaP as immunogene, No. 1, No. 2 two rabbits are immunized respectively, as shown in Fig. 5, with exempting from
The increase of epidemic disease number, antiserum titre gradually rise, and after the 8th time immune, the antiserum titre of No. 1 rabbit is up to 1:126000, No. 2
The antiserum titre of rabbit is up to 1:112000, No. 2 rabbits potency after the 6th booster immunization rises clearly, repeatedly reinforces
After immune, No. 1 is stablized with the antibody titer prepared by No. 2 rabbits 96000 or more, can carry out subsequent blood sampling and separation is anti-
Serum step, the polyclonal antibody prepared by two rabbits can meet follow-up immunization requirement of experiment.
Table 2：Animal immune scheme
Embodiment 3：The specificity of BaP polyclonal antibodies
The specificity of immunoassay is evaluated using the cross reaction (CR) of multiple BaP analogues.Respectively with benzo
[k] fluoranthene (CAS：207-08-9), (CAS：198-55-0), dibenzo [a, h] anthracene (CAS：53-70-3), anthracene (CAS：120-
12-7), fluoranthene (CAS：206-44-0), benzo [a] anthracene (CAS：56-55-3), pyrene (CAS：129-00-0) and benzo [ghi]
(CAS：191-24-2) as the analogue of BaP.The calculation formula of CR is as follows：
CR=(IC50ofBaP)/(IC50Ofanalygues) × 100% (1)；
The chemical constitutions of BaP analogs and cross reacting rate the results are shown in Table 3, it can be seen that BaP Anti-TNF-αs obtained
Cross reacting rate between the body compound similar with other structures is respectively less than 6.90%, shows that pAb-BaP has higher spy
The opposite sex can be used for the immunoassay detection of trace BaP.
Table 3：The cross reacting rate of BaP antibody and BaP analogues
The above is presently preferred embodiments of the present invention, but the present invention should not be limited to disclosed in the embodiment
Content.So every do not depart from the lower equivalent or modification completed of spirit disclosed in this invention, the model that the present invention protects is both fallen within
It encloses.
Claims (9)
1. a kind of BaP immunogene, shown in structural formula such as formula (I),
2. the preparation method of BaP immunogene as described in claim 1, which is characterized in that shown in synthetic route such as formula (II),
Include the following steps：
(1) iodide reaction is carried out with BaP standard items, reacts 48h at room temperature, obtain intermediate product 6- iodine BaPs；
(2) gained 6- iodine BaPs carry out Heck coupling reactions in step (1), and 15h is reacted under conditions of 120 DEG C, and separation is net
Change, obtains BaP haptens；
(3) the carbonyl carboxylated of BaP haptens obtained by step (2) is obtained, with BSA by BaP carboxy derivatives as oximation reaction
For carrier protein, activated ester process by above-mentioned BaP carboxy derivatives and BSA be coupled to get.
3. the preparation method of BaP immunogene as claimed in claim 2, which is characterized in that the BaP immunogene it is specific
Preparation process is：
(1-1) takes 500mL three neck round bottom flask, is separately added into 0.4g BaPs, 0.42gNIS, 10g acidic alumina and 30mL
Reaction 48h is stirred at room temperature in toluene, and is methyl tertiary butyl ether by solvent:Petroleum ether=1:The thin-layer chromatography of 25 (v/v) monitors
Above-mentioned reaction is until raw material disappears；Above-mentioned reaction product is merged into organic phase after filtering again, uses saturated sodium sulfite respectively
Solution, saturation NaCl solution and ultra-pure water respectively extract three times, by the organic phase through anhydrous MgSO4Dry, filtering is concentrated under reduced pressure
It is purified with recrystallization, obtains yellow powdery solid object；
(2-1) is separately added into yellow powdery solid object, 0.01g acetic acid in 0.27mg steps (1-1) into 100mL single-necked flasks
Palladium and 0.02gLiCl are added 20mLDMF dissolvings, sequentially add 70 μ L4- amylene -2- alcohol and 200 μ L triethylamines, 120 DEG C are stirred
Reaction 15h is mixed, and is methyl tertiary butyl ether by solvent:Petroleum ether=1:The thin-layer chromatography of 2 (v/v) monitors above-mentioned reaction to anti-
It should be complete；Reaction product is cooled to room temperature again and 120mL toluene is added, is respectively extracted with saturation NaCl solution and ultra-pure water respectively
Three times, the organic phase is through anhydrous MgSO4It is dry, filter and be concentrated under reduced pressure to obtain crude product, then through leacheate be methyl tertiary butyl ether:
Petroleum ether=1:The silica gel chromatographic column isolation of purified of 2 (v/v), obtains light yellow powder product；
It is dry that (3-1) takes light yellow powder product in 0.12g steps (2-1), half hydrochloride of 0.24g carboxymethoxylamines to be dissolved in 15mL
In dry pyridine, concussion reaction under the conditions of being protected from light, and be chloroform by solvent:Methanol=9:The thin-layer chromatography of 1 (v/v) monitors
Above-mentioned reaction is to the reaction was complete；After complete reaction, it takes 0.54g sodium borohydrides to be dissolved in 4.5mL ultra-pure waters, is added to above-mentioned anti-
It answers in liquid, room temperature concussion reaction 5h, the residue after nitrogen is blown is washed twice with pH2.0 hydrochloric acid solutions, distilled water respectively, 60 DEG C of baking ovens
Middle drying, obtains yellow solid product；It weighs the above-mentioned yellow solid products of 0.823g again to be dissolved in 0.5mLDMF, by 0.023gN- hydroxyls
After base succinimide, 0.412g dicyclohexylcarbodiimides are dissolved in 0.5mLDMF, it is anti-to be added to 4 DEG C of stirrings in above-mentioned solution
Answer 8h；The high speed centrifugation 20min at 6000rpm, 4 DEG C again takes supernatant that 5mLBSA solution is added, is stirred to react at 4 DEG C overnight；
Repeated centrifugation dialyses supernatant 3d with 0.01M, pH7.40 phosphate buffer, preserve at -20 DEG C to get.
4. the preparation method of BaP immunogene as claimed in claim 3, which is characterized in that yellow powder described in step (1-1)
Last shape solids is 6- iodine BaPs, and yield is 76.2%.
5. the preparation method of BaP immunogene as claimed in claim 3, which is characterized in that recrystallized described in step (1-1)
The solvent of purification is the mixture of dichloromethane and petroleum ether.
6. the preparation method of BaP immunogene as claimed in claim 3, which is characterized in that light yellow described in step (2-1)
Powdery product is BaP haptens, molecular formula C27H20O, and yield is 86.2%.
7. the preparation method of BaP immunogene as claimed in claim 3, which is characterized in that in step (3-1), the yellow is solid
Body product is BaP carboxy derivatives, structural formula BaP-COOH.
8. application of the BaP immunogene as described in claim 1 in preparing BaP artificial polyclonal antibodies.
9. the purposes that BaP immunogene as described in claim 1 is detected as trace BaP immunoassays.
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| CN103852581A (en) * | 2014-03-11 | 2014-06-11 | 河南工业大学 | 3,4-benzopyrene enzyme-linked immune detection kit |
| WO2016196638A2 (en) * | 2015-06-01 | 2016-12-08 | The Administrators Of The Tulane Educational Fund | Pah antibodies and uses thereof |
| CN105017411A (en) * | 2015-07-06 | 2015-11-04 | 浙江大学 | Synthetic method for artificially coupling antigen through aflatoxin B1-carrier proteins |
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