CN103760337B - The Indirect cELISA detection method of phthalic acid (2-ethyl hexyl) ester - Google Patents
The Indirect cELISA detection method of phthalic acid (2-ethyl hexyl) ester Download PDFInfo
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- CN103760337B CN103760337B CN201310675076.XA CN201310675076A CN103760337B CN 103760337 B CN103760337 B CN 103760337B CN 201310675076 A CN201310675076 A CN 201310675076A CN 103760337 B CN103760337 B CN 103760337B
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- ester
- ethyl hexyl
- phthalic acid
- acid
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- DJDSLBVSSOQSLW-UHFFFAOYSA-N mono(2-ethylhexyl) phthalate Chemical compound CCCCC(CC)COC(=O)C1=CC=CC=C1C(O)=O DJDSLBVSSOQSLW-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 238000001514 detection method Methods 0.000 title claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims abstract description 39
- GJIZLOIXPDWCSM-UHFFFAOYSA-N 5-amino-2-(2-ethylhexoxycarbonyl)benzoic acid Chemical compound C(C)C(COC(C=1C(C(=O)O)=CC(=CC=1)N)=O)CCCC GJIZLOIXPDWCSM-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000002965 ELISA Methods 0.000 claims abstract description 14
- 239000007924 injection Substances 0.000 claims abstract description 11
- 238000002347 injection Methods 0.000 claims abstract description 11
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 230000002163 immunogen Effects 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 51
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 46
- 239000000427 antigen Substances 0.000 claims description 44
- 102000036639 antigens Human genes 0.000 claims description 44
- 108091007433 antigens Proteins 0.000 claims description 44
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 39
- 230000003053 immunization Effects 0.000 claims description 30
- 238000011534 incubation Methods 0.000 claims description 25
- 238000002360 preparation method Methods 0.000 claims description 22
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 20
- 239000002671 adjuvant Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 18
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 16
- 108010058846 Ovalbumin Proteins 0.000 claims description 15
- 239000012153 distilled water Substances 0.000 claims description 15
- 238000002649 immunization Methods 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 229940092253 ovalbumin Drugs 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 15
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 claims description 13
- 241001465754 Metazoa Species 0.000 claims description 12
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 12
- 238000013016 damping Methods 0.000 claims description 12
- 239000012530 fluid Substances 0.000 claims description 12
- 230000036039 immunity Effects 0.000 claims description 12
- 238000005406 washing Methods 0.000 claims description 12
- SLBQXWXKPNIVSQ-UHFFFAOYSA-N 4-nitrophthalic acid Chemical compound OC(=O)C1=CC=C([N+]([O-])=O)C=C1C(O)=O SLBQXWXKPNIVSQ-UHFFFAOYSA-N 0.000 claims description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 9
- ZNDIUQDGDYEAPT-UHFFFAOYSA-N 2-(2-ethylhexoxycarbonyl)-5-nitrobenzoic acid Chemical compound C(C)C(COC(C=1C(C(=O)O)=CC(=CC=1)[N+](=O)[O-])=O)CCCC ZNDIUQDGDYEAPT-UHFFFAOYSA-N 0.000 claims description 8
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 8
- 239000000872 buffer Substances 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims description 7
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 7
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 7
- 239000008363 phosphate buffer Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 241000283707 Capra Species 0.000 claims description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 6
- 239000004202 carbamide Substances 0.000 claims description 6
- 230000002860 competitive effect Effects 0.000 claims description 6
- 239000012043 crude product Substances 0.000 claims description 6
- CBMPTFJVXNIWHP-UHFFFAOYSA-L disodium;hydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].OP([O-])([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O CBMPTFJVXNIWHP-UHFFFAOYSA-L 0.000 claims description 6
- 239000001257 hydrogen Substances 0.000 claims description 6
- 229910052739 hydrogen Inorganic materials 0.000 claims description 6
- 230000028993 immune response Effects 0.000 claims description 6
- 230000001105 regulatory effect Effects 0.000 claims description 6
- 239000011734 sodium Substances 0.000 claims description 6
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- 238000000605 extraction Methods 0.000 claims description 5
- 230000031700 light absorption Effects 0.000 claims description 5
- 239000000376 reactant Substances 0.000 claims description 5
- 239000012488 sample solution Substances 0.000 claims description 5
- 239000012086 standard solution Substances 0.000 claims description 5
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims description 4
- 241001494479 Pecora Species 0.000 claims description 4
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 4
- 238000011587 new zealand white rabbit Methods 0.000 claims description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 4
- YIWUKEYIRIRTPP-UHFFFAOYSA-N 2-ethylhexan-1-ol Chemical compound CCCCC(CC)CO YIWUKEYIRIRTPP-UHFFFAOYSA-N 0.000 claims description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 3
- 229920000742 Cotton Polymers 0.000 claims description 3
- 230000009471 action Effects 0.000 claims description 3
- 238000010241 blood sampling Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 230000008878 coupling Effects 0.000 claims description 3
- 238000010168 coupling process Methods 0.000 claims description 3
- 238000005859 coupling reaction Methods 0.000 claims description 3
- 238000002425 crystallisation Methods 0.000 claims description 3
- 230000008025 crystallization Effects 0.000 claims description 3
- 230000006837 decompression Effects 0.000 claims description 3
- 238000004821 distillation Methods 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 238000001953 recrystallisation Methods 0.000 claims description 3
- 230000004044 response Effects 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 238000005728 strengthening Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 210000003462 vein Anatomy 0.000 claims description 3
- 238000010171 animal model Methods 0.000 claims description 2
- 229910021538 borax Inorganic materials 0.000 claims 2
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 claims 2
- 239000004328 sodium tetraborate Substances 0.000 claims 2
- 241000283973 Oryctolagus cuniculus Species 0.000 abstract description 5
- 239000002537 cosmetic Substances 0.000 abstract description 5
- 150000002148 esters Chemical class 0.000 abstract description 5
- 229940088597 hormone Drugs 0.000 abstract description 5
- 239000005556 hormone Substances 0.000 abstract description 5
- XNGIFLGASWRNHJ-UHFFFAOYSA-N phthalic acid Chemical compound OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 abstract description 5
- -1 2-ethyl hexyl Chemical group 0.000 abstract description 4
- 238000012360 testing method Methods 0.000 abstract description 4
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 230000036541 health Effects 0.000 abstract description 3
- 239000000463 material Substances 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 239000004033 plastic Substances 0.000 description 5
- 229920003023 plastic Polymers 0.000 description 5
- CDMADVZSLOHIFP-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane;decahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 CDMADVZSLOHIFP-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 4
- 239000004803 Di-2ethylhexylphthalate Substances 0.000 description 3
- BJQHLKABXJIVAM-UHFFFAOYSA-N bis(2-ethylhexyl) phthalate Chemical compound CCCCC(CC)COC(=O)C1=CC=CC=C1C(=O)OCC(CC)CCCC BJQHLKABXJIVAM-UHFFFAOYSA-N 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 239000003547 immunosorbent Substances 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- CVOFKRWYWCSDMA-UHFFFAOYSA-N 2-chloro-n-(2,6-diethylphenyl)-n-(methoxymethyl)acetamide;2,6-dinitro-n,n-dipropyl-4-(trifluoromethyl)aniline Chemical compound CCC1=CC=CC(CC)=C1N(COC)C(=O)CCl.CCCN(CCC)C1=C([N+]([O-])=O)C=C(C(F)(F)F)C=C1[N+]([O-])=O CVOFKRWYWCSDMA-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003344 environmental pollutant Substances 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 231100000719 pollutant Toxicity 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 235000002639 sodium chloride Nutrition 0.000 description 2
- 235000010288 sodium nitrite Nutrition 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229940076134 benzene Drugs 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 239000004568 cement Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000015165 citric acid Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 239000003777 experimental drug Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 238000009408 flooring Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 210000004392 genitalia Anatomy 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 229960000443 hydrochloric acid Drugs 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000010687 lubricating oil Substances 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000005022 packaging material Substances 0.000 description 1
- 239000004014 plasticizer Substances 0.000 description 1
- 229920002689 polyvinyl acetate Polymers 0.000 description 1
- 239000011118 polyvinyl acetate Substances 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 238000003822 preparative gas chromatography Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 238000002470 solid-phase micro-extraction Methods 0.000 description 1
- 230000019100 sperm motility Effects 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- 239000000057 synthetic resin Substances 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000004454 trace mineral analysis Methods 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to the Indirect cELISA detection method of a kind of phthalic acid (2-ethyl hexyl) ester, Indirect cELISA belongs to luminosity enzyme-linked immuno assay, utilize the specific reaction of antigen-antibody, can efficient testing environment hormone phthalic acid (2-ethyl hexyl) ester rapidly.First to synthesize 4-aminophthalic acid (2-ethyl hexyl) ester that is easy and protein bound, then protein bound is got on, obtained immunogene, then immunogen injection is obtained antibody in White Rabbit body.Then set up Indirect cELISA and detect phthalic acid (2-ethyl hexyl) ester.Phthalic acid (2-ethyl hexyl) ester is the environmental hormone of harm biosome health, and Indirect cELISA is set up, and efficiently can detect river rapidly, lake, food, the content of phthalic acid (2-ethyl hexyl) ester in the article such as cosmetics.
Description
Technical field
The present invention relates to the Indirect cELISA detection method of a kind of phthalic acid (2-ethyl hexyl) ester.
Background technology
Phthalate material is mainly used in pvc material, makes Polyvinylchloride become resilient plastic cement from hard plastic glue, plays the effect of plastifier.It is widely used in toy, packaging material for food, Medical blood bag and hundreds of product such as sebific duct, vinyl flooring and wallpaper, detersive, lubricating oil, personal-care supplies.Due to phthalic ester as can plastifier be added in plastics-production process time do not combine with plastic molecules, this kind of material can ooze out from plastic products, the mankind by skin contact, suction, directly this type of material of picked-up, thus cause great harm to the health of human body in the process using plastic product.
Phthalic ester plays the effect of similar female hormone in human body and animal body, can disturbance endocrine, and man semen amount and sperm quantity are reduced, Sperm Motility is low, sperm morphology is abnormal, and serious meeting causes carcinoma of testis, is to cause man's reproductive problems " arch-criminal ".In cosmetics, the phthalic acid ester content of nail polish is the highest, and the fragrance ingredient of a lot of cosmetics is also containing this material.This material in cosmetics can be entered in body by the respiratory system of women and skin, if excessive use, can increase the probability that women suffers from breast cancer, and also can jeopardize the reproductive system of the boy baby of their following fertility.
Phthalic acid (2-ethyl hexyl) ester (DEHP) is the most widely used plastifier, except cellulose acetate, polyvinyl acetate, all has good compatibility with the synthetic resin of the industrial use of the overwhelming majority and rubber.The similar artificial hormone of plasticiser DEHP effect, long-term accumulated high dose in body, may cause the entanglement of child's sex, comprise genitals shorten little, sex character is not obvious, though whether carcinogenicly cannot confirm the mankind at present, can produce carcinogenic reaction to animal.
So seem very important to the detection of phthalate.The detection method of current phthalate mainly contains vapor-phase chromatography, high performance liquid chromatography, gas chromatography-mass spectrography and Liquid Chromatography-Tandem Mass Spectrometry, Solid-phase Microextraction etc., and what wherein the most often adopt is gas phase and high performance liquid chromatography.These methods measure that pollutant sensitivity is higher, and degree of accuracy is good, but by pollutant to be measured separation and Extraction to remove the processing procedure of impurity interference very complicated from sample in this kind of analytical approach system, loaded down with trivial details during operating cost, instrument and analysis cost expensive.Have important practical significance so develop a kind of trace analysis methods that is simple, quick, that be applicable to on-site supervision.
Immuno analytical method carries out based on the specific reaction of antigen and antibody a kind of technological means of detecting, a kind of using antibody as biological chemistry detecting device, the materials such as compound, enzyme or protein carried out to the analytical technology of quantitative and qualitative analysis.Indirect enzyme-linked immunosorbent reaction utilizes the specific reaction of antigen-antibody to realize, first obtained immunogene, again by this immunogen injection in rabbit body, obtained antibody with high specificity, utilize Indirect cELISA by standard items and treat that test sample competes the reaction with antibody, the enzyme of ELIAS secondary antibody body can impel substrate solution luminous, records absorbance, can try to achieve and treat test sample concentration by Criterion curve.
Indirect cELISA belongs to luminosity enzyme-linked immuno assay, utilizes the specific reaction of antigen-antibody, can efficient testing environment hormone phthalic acid (2-ethyl hexyl) ester rapidly.
Indirect cELISA is the one of enzyme-linked immunosorbent analytical technique.Enzyme-linked immunosorbent assay (ELISA) has been widely used in the analysis fields such as clinical medicine, animal quarantine, medicament residue.Current Enzyme-linked immunosorbent assay phthalate has some reports, but also not yet reports the enzyme connection analysis of phthalic acid (2-ethyl hexyl) ester.Measure phthalic acid (2-ethyl hexyl) ester mainly contain phase chromatography, high performance liquid chromatography etc., these class methods from sample separation and Extraction and remove impurity interference processing procedure very complicated, loaded down with trivial details during operating cost.So we seek the Indirect cELISA that can detect phthalic acid (2-ethyl hexyl) ester quickly and easily at this.
Summary of the invention
The object of the present invention is to provide the Indirect cELISA detection method of a kind of phthalic acid (2-ethyl hexyl) ester, solve phthalic acid (2-ethyl hexyl) ester and belong to haptens, can not it be made to produce the problem of antibody by direct effect biosome.
First to synthesize 4-aminophthalic acid (2-ethyl hexyl) ester that is easy and protein bound, then protein bound is got on, obtained immunogene, then immunogen injection is obtained antibody in White Rabbit body.Then set up Indirect cELISA and detect phthalic acid (2-ethyl hexyl) ester.
Concrete technical scheme is as follows:
A kind of Indirect cELISA detection method of phthalic acid (2-ethyl hexyl) ester, comprises the steps:
(1) 4-aminophthalic acid (2-ethyl hexyl) ester that is easy and protein bound is synthesized;
(2) protein bound is got on, obtained immunogene;
(3) antibody is obtained by immunogen injection to experimental animals;
(4) set up Indirect cELISA and detect phthalic acid (2-ethyl hexyl) ester.
Further, comprise the steps: further in step (4)
(4-1) quilt is wrapped: envelope antigen DEHAP-OVA is diluted certain multiple bag by 96 hole ELISA Plate, incubation also washs;
(4-2) close: add OVA solution, close the unnecessary part not having envelope antigen, incubation also washs;
(4-3) compete: every hole adds phthalic acid (2-ethyl hexyl) ester standard solution or sample solution and phthalic acid (2-ethyl hexyl) ester antibody, and make it competitive reaction occurs, incubation also washs;
(4-4) enzyme-added: every hole adds the goat anti-rabbit igg of HRP mark, and incubation also washs;
(4-5) detect: add substrate solution colour developing, after incubation, add stop buffer cessation reaction, multi-functional microplate reader measures light absorption value.
Further,
In step (4-1), envelope antigen DEHAP-OVA is diluted certain multiple bag by 96 hole ELISA Plate, every hole 100 μ L, after placing incubator 37 DEG C of incubation 2h, taking-up cleansing solution PBST washs 3 times, each 3min; And/or,
Add 1%OVA solution in step (4-2), every hole 150 μ L, close the unnecessary part not having envelope antigen, avoid the non-specific adsorption of antibody in ELISA Plate, after 37 DEG C of incubation 30min, take out the same washing 3 times;
And/or,
In step (4-3), every hole adds 50 μ L phthalic acid (2-ethyl hexyl) ester standard solution or sample solution and 50 μ L phthalic acid (2-ethyl hexyl) ester antibody, makes it competitive reaction occurs, at 37 DEG C after incubation 2.5h, and the same washing;
And/or,
In step (4-4), every hole adds the goat anti-rabbit igg that 100 μ L HRP mark, and takes out the same washing 3 times at 37 DEG C after incubation 2h; And/or,
Add substrate solution colour developing in step (4-5), every hole 100 μ L, after incubation 30min, adds stop buffer cessation reaction, multi-functional microplate reader measures light absorption value.
Further, first synthesize 4-nitrophthalic acid (2-ethyl hexyl) ester by 4-nitrophthalic acid in step (1), restore and obtain haptens 4-aminophthalic acid (2-ethyl hexyl) ester.
Further, get on BSA coupling in step (2) obtained immunizing antigen, is expelled in animal body by immunizing antigen.
Further, in step (3), animal obtains antibody through certain hour growth response.
Further, set up by the idiosyncrasy between antigen, antibody and ELIAS secondary antibody the method measuring phthalic acid (2-ethyl hexyl) ester in step (4).
Further,
The preparation of described saturated ammonium sulfate solution adopts following steps: get 500mL distilled water, be heated to 70 ~ 80 DEG C, by soluble in water for 400g ammonium sulfate pulvis, stir 20min, at the bottom of ammonium sulfate crystallization is sunken to bottle, supernatant is saturated ammonium sulfate, filters, then get 28% ammoniacal liquor saturated ammonium sulfate is adjusted to pH7.0 after room temperature cooling with absorbent cotton; And/or
The preparation of physiological saline adopts following steps: 0.85gNaCl adding distil water is to 100mL;
And/or
The preparation of 0.01mol/LpH7.2 phosphate buffer (PB) adopts following steps: 0.2mol/LNa
2hPO
436.0mL, 0.2mol/LNaH
2pO
414.0mL mixes, and distilled water constant volume is to 1000mL;
And/or
The preparation that bag is buffered liquid adopts following steps: take 1.59gNa2CO3,2.94gNaHCO3, distilled water constant volume is to 1000mL;
And/or
The preparation of confining liquid adopts following steps: prepare 1%(w/v with PBS) ovalbumin (OVA) solution;
And/or
The preparation of dilution adopts following steps: take 8.0gNaCl, 2.96gNa
2hPO
412H
2o, 0.29gNaH
2pO
42H
2o, 0.1gKCl, distilled water constant volume is to 1000mL;
And/or
The preparation of cleansing solution adopts following steps: add 0.05% Tween-20 in PBS;
And/or
The preparation of substrate solution adopts following steps: o-phenylenediamine 4mg is dissolved in 10mL citrate-phosphate disodium hydrogen damping fluid, adds 15 μ L30% hydrogen peroxide before use;
And/or
The preparation of citrate-phosphate disodium hydrogen damping fluid adopts following steps: take Na
2hPO
412H
2o1.84g, citric acid 0.51g, be then settled to 50mL with distilled water;
And/or
Stop buffer is 2mol/L sulfuric acid solution.
Further, the preparation of described antigen specifically adopts following steps:
Excess thionyl chloride (SOCl is added in 4-nitrophthalic acid
2), nitrogen protection, stirring reaction 4 hours, then excess thionyl chloride decompression is taken away, obtained 4-nitrophthalic acid diacid chloride, then after adding 2-Ethylhexyl Alcohol ice bath reaction 30min,
40 DEG C are reacted 12 hours, obtain 4-nitrophthalic acid (2-ethyl hexyl) ester;
4-nitrophthalic acid (2-ethyl hexyl) ester is dissolved in benzene, adds pure zinc powder, after adding concentrated hydrochloric acid, again add pure zinc powder, stirring at room temperature 12h;
In reaction system, add cold water, and be neutralized to alkalescence by NaOH solution, isolate benzene layer, water layer benzene extracts again;
Combining extraction liquid, dry after washing;
Distillation removing benzene, obtains yellow solid, obtains 4-aminophthalic acid (2-ethyl hexyl) ester with ethyl alcohol recrystallization;
Take 4-aminophthalic acid (2-ethyl hexyl) ester to be dissolved in a small amount of concentrated hydrochloric acid, then to add intermediate water, under 4 DEG C of low temperature stir, instillation NaNO
2solution;
After reaction 30min, add a small amount of urea and remove unreacted NaNO2;
BSA is dissolved in sodium tetraborate decahydrate damping fluid, then dropwise adds in above-mentioned mixed liquid, and about regulating pH value of solution to 9.2, orange solution occurs, continue reaction 4h, obtain antigen crude product;
Reactant liquor is loaded in bag filter, put in intermediate water and dialyse 5 days, obtain phthalic acid (2-ethyl hexyl) ester immunity holoantigen; Take 4-aminophthalic acid (2-ethyl hexyl) ester to be dissolved in a small amount of concentrated hydrochloric acid, then to add intermediate water, under 4 DEG C of low temperature stir, instillation NaNO
2solution;
After reaction 30min, add a small amount of urea and remove unreacted NaNO2;
OVA is dissolved in sodium tetraborate decahydrate damping fluid, then dropwise adds in above-mentioned mixed liquid, and about regulating pH value of solution to 9.2, orange solution occurs, continue reaction 4h, obtain antigen crude product;
Further, the preparation of described antibody specifically adopts following steps:
Adjuvant: prior to antigen or with in the miscible rear injection animal body of antigen, can non-specifically change or strengthen the immune response of body, plays booster action;
By whiteruss and the mixing of sheep oil certain volume ratio, indiffusion on the water surface, is now incomplete Freund's adjuvant, and add Bacille Calmette-Guerin is Freund's complete adjuvant later;
Phthalic acid (2-ethyl hexyl) ester immunizing antigen is diluted to finite concentration, miscible with adjuvant 1:1, repeatedly immunity is carried out to 4 new zealand white rabbits;
First time immunity be by immunizing antigen and Freund's complete adjuvant miscible, booster immunization be afterwards by immunizing antigen and incomplete Freund's adjuvant miscible;
Select White Rabbit as immunization, adopt the method for dorsal sc multi-point injection;
Booster immunization after immune 3 weeks of first time, later every 2 weeks booster immunizations again, from second time booster immunization, ear vein blood sampling in the 7th day after each immunity, measuring serum titer, when no longer changing when tiring, strengthening primary immune response, Culling heart blood after 1 week, purifying, obtains antibody.
Compared with currently available technology, the present invention efficiently can detect river rapidly, lake, food, the content of phthalic acid (2-ethyl hexyl) ester in the article such as cosmetics.Indirect cELISA measures phthalic acid (2-ethyl hexyl) ester, and the range of linearity is 0.0001-1000ng/mL, and recovery of standard addition is between 95%-105%, and accuracy is good, reliable results.The method can measure environmental water sample, food after setting up, and phthalic acid (2-ethyl hexyl) ester content in commodity, also can be used for commodity production, obtains economic benefit.
Embodiment
Be a kind of preferred embodiment in numerous embodiments of the present invention below.
It is good that Indirect cELISA measures phthalic acid (2-ethyl hexyl) ester specificity, highly sensitive, fast and easy, has obtained the specific antibody of phthalic acid (2-ethyl hexyl) ester first, detects phthalic acid (2-ethyl hexyl) ester by the mode of antigen-antibody reaction.First 4-nitrophthalic acid (2-ethyl hexyl) ester has been synthesized by 4-nitrophthalic acid, restore and obtain haptens 4-aminophthalic acid (2-ethyl hexyl) ester, get on BSA coupling obtained immunizing antigen, be expelled to by immunizing antigen in animal body, animal obtains antibody through certain hour growth response.The method measuring phthalic acid (2-ethyl hexyl) ester is set up by the idiosyncrasy between antigen, antibody and ELIAS secondary antibody.
1. experimental drug and instrument
(1) medicine
4-nitrophthalic acid, thionyl chloride, zinc powder, concentrated hydrochloric acid, benzene, bovine serum albumin, ovalbumin, whiteruss, sheep oil, ammonium sulfate, sodium dihydrogen phosphate, sodium hydrogen phosphate, citric acid, 30% hydrogen peroxide, Tween-20, sodium chloride, ammoniacal liquor, absolute ethyl alcohol, fresh liquid BCG vaccine, 25% glutaraldehyde, the goat anti-rabbit igg of HRP mark, o-phenylenediamine.
(2) experimental apparatus
Multi-functional microplate reader, SCQ50 ultrasonic cleaner, TGL-16G high speed tabletop centrifuge, 96 hole ELISA Plate.
(3) preparation of solution
1) saturated ammonium sulfate solution: get 500mL distilled water, is heated to 70 ~ 80 DEG C, and by soluble in water for 400g ammonium sulfate pulvis, stir 20min, at the bottom of ammonium sulfate crystallization is sunken to bottle, supernatant is saturated ammonium sulfate, filters after room temperature cooling with absorbent cotton.Get 28% ammoniacal liquor again and saturated ammonium sulfate is adjusted to pH7.0.
2) physiological saline: 0.85gNaCl adding distil water is to 100mL.
3) 0.01mol/L pH7.2 phosphate buffer (PB): 0.2mol/LNa
2hPO
436.0mL, 0.2mol/LNaH
2pO
414.0mL mixes, and distilled water constant volume is to 1000mL.
4) bag is buffered liquid (0.05mol/L pH9.6 carbonate buffer solution, CB): take 1.59g Na
2cO
3, 2.94g NaHCO
3, distilled water constant volume is to 1000mL.
5) confining liquid: prepare 1%(w/v with PBS) ovalbumin (OVA) solution.
6) dilution (0.01mol/L pH7.4 phosphate buffer, PBS): take 8.0g NaCl, 2.96g Na
2hPO
412H
2o, 0.29g NaH
2pO
42H
2o, 0.1g KCl, distilled water constant volume is to 1000mL.
7) cleansing solution (0.01mol/L pH7.4, PBST): add 0.05% Tween-20 in PBS.
8) substrate solution: o-phenylenediamine 4mg is dissolved in 10mL citrate-phosphate disodium hydrogen damping fluid, adds 15 μ L30% hydrogen peroxide before use.
Citrate-phosphate disodium hydrogen damping fluid: take Na
2hPO
412H
2o1.84g, citric acid 0.51g.Then 50mL is settled to distilled water.
9) stop buffer: 2mol/L sulfuric acid solution.
2. experimental technique
(1) preparation of antigen
Excess thionyl chloride (SOCl2) is added in 4-nitrophthalic acid; nitrogen protection; stirring reaction 4 hours; again excess thionyl chloride decompression is taken away; obtained 4-nitrophthalic acid diacid chloride; after adding 2-Ethylhexyl Alcohol ice bath reaction 30min again, 40 DEG C are reacted 12 hours, obtain 4-nitrophthalic acid (2-ethyl hexyl) ester.4-nitrophthalic acid (2-ethyl hexyl) ester is dissolved in benzene, adds pure zinc powder, after adding concentrated hydrochloric acid, again add pure zinc powder, stirring at room temperature 12h.In reaction system, add cold water, and be neutralized to alkalescence by NaOH solution, isolate benzene layer, water layer benzene extracts again.Combining extraction liquid, dry after washing.Distillation removing benzene, obtains yellow solid, obtains 4-aminophthalic acid (2-ethyl hexyl) ester with ethyl alcohol recrystallization.
Take 4-aminophthalic acid (2-ethyl hexyl) ester to be dissolved in a small amount of concentrated hydrochloric acid, then to add intermediate water, under 4 DEG C of low temperature stir, instillation NaNO
2solution.After reaction 30min, add a small amount of urea and remove unreacted NaNO
2.BSA is dissolved in sodium tetraborate decahydrate damping fluid, then dropwise adds in above-mentioned mixed liquid, and about regulating pH value of solution to 9.2, orange solution occurs, continue reaction 4h, obtain antigen crude product.Reactant liquor is loaded in bag filter, put in intermediate water and dialyse 5 days, obtain phthalic acid (2-ethyl hexyl) ester immunity holoantigen.
Take 4-aminophthalic acid (2-ethyl hexyl) ester to be dissolved in a small amount of concentrated hydrochloric acid, then to add intermediate water, under 4 DEG C of low temperature stir, instillation NaNO
2solution.After reaction 30min, add a small amount of urea and remove unreacted NaNO
2.OVA is dissolved in sodium tetraborate decahydrate damping fluid, then dropwise adds in above-mentioned mixed liquid, and about regulating pH value of solution to 9.2, orange solution occurs, continue reaction 4h, obtain antigen crude product.Reactant liquor is loaded in bag filter, put in intermediate water and dialyse 5 days, obtain phthalic acid (2-ethyl hexyl) ester bag by holoantigen.
(2) preparation of antibody
Adjuvant: prior to antigen or with in the miscible rear injection animal body of antigen, can non-specifically change or strengthen the immune response of body, plays booster action.By whiteruss and the mixing of sheep oil certain volume ratio, indiffusion on the water surface, is now incomplete Freund's adjuvant, and add Bacille Calmette-Guerin is Freund's complete adjuvant later.
Phthalic acid (2-ethyl hexyl) ester immunizing antigen is diluted to finite concentration, miscible with adjuvant 1:1, repeatedly immunity is carried out to 4 new zealand white rabbits.First time immunity be by immunizing antigen and Freund's complete adjuvant miscible, booster immunization be afterwards by immunizing antigen and incomplete Freund's adjuvant miscible.Here we select 3 months sizes, and the male rabbit of New Zealand Journal of Health Physical Education of 1-2kg, as immunization, adopts the method for dorsal sc multi-point injection.Booster immunization after immune 3 weeks of first time, later every 2 weeks booster immunizations again, from second time booster immunization, ear vein blood sampling in the 7th day after each immunity, measuring serum titer, when no longer changing when tiring, strengthening primary immune response, Culling heart blood after 1 week, purifying, obtains antibody.
3. indirect competitive enzyme-linked immunosorbent measures the foundation of phthalic acid (2-ethyl hexyl) ester method
(1) bag quilt: envelope antigen DEHAP-OVA is diluted certain multiple bag by 96 hole ELISA Plate, every hole 100 μ L, after placing incubator 37 DEG C of incubation 2h, taking-up cleansing solution PBST washs 3 times, each 3min.
(2) close: add 1%OVA solution, every hole 150 μ L, close the unnecessary part not having envelope antigen, avoid the non-specific adsorption of antibody in ELISA Plate, after 37 DEG C of incubation 30min, take out the same washing 3 times.
(3) compete: every hole adds 50 μ L phthalic acid (2-ethyl hexyl) ester standard solution or sample solution and 50 μ L phthalic acid (2-ethyl hexyl) ester antibody, makes it competitive reaction occurs.At 37 DEG C after incubation 2.5h, the same washing.
(4) enzyme-added: every hole adds the goat anti-rabbit igg that 100 μ L HRP mark, and takes out the same washing 3 times at 37 DEG C after incubation 2h.
(5) detect: add substrate solution colour developing, every hole 100 μ L, after incubation 30min, adds stop buffer cessation reaction, multi-functional microplate reader measures light absorption value.
Linear equation
Y=0.741-0.0378lg X(Y-absorbance, DEHP concentration ng/mL in X-liquid to be measured), related coefficient: R
2=0.9015, range of linearity 0.0001-1000ng/mL.
Indirect cELISA measures phthalic acid (2-ethyl hexyl), and ester is highly sensitive, selectivity good, high specificity, simple to operation, high flux can be realized and detect.The method can measure environmental water sample, food after setting up, and phthalic acid (2-ethyl hexyl) ester content in the article such as commodity, practical, application prospect is good.
Claims (9)
1. an Indirect cELISA detection method for phthalic acid (2-ethyl hexyl) ester, comprises the steps:
(1) 4-aminophthalic acid (2-ethyl hexyl) ester that is easy and protein bound is synthesized; (2) protein bound is got on, obtained immunogene; (3) antibody is obtained by immunogen injection to experimental animals; (4) set up Indirect cELISA and detect phthalic acid (2-ethyl hexyl) ester; It is characterized in that, adopt following steps to prepare antigen:
Excess thionyl chloride (SOCl is added in 4-nitrophthalic acid
2), nitrogen protection, stirring reaction 4 hours, again excess thionyl chloride decompression is taken away, obtained 4-nitrophthalic acid diacid chloride, then after adding 2-Ethylhexyl Alcohol ice bath reaction 30min, 40 DEG C are reacted 12 hours, obtain 4-nitrophthalic acid (2-ethyl hexyl) ester;
4-nitrophthalic acid (2-ethyl hexyl) ester is dissolved in benzene, adds pure zinc powder, after adding concentrated hydrochloric acid, again add pure zinc powder, stirring at room temperature 12h;
In reaction system, add cold water, and be neutralized to alkalescence by NaOH solution, isolate benzene layer, water layer benzene extracts again;
Combining extraction liquid, dry after washing;
Distillation removing benzene, obtains yellow solid, obtains 4-aminophthalic acid (2-ethyl hexyl) ester with ethyl alcohol recrystallization;
Take 4-aminophthalic acid (2-ethyl hexyl) ester to be dissolved in a small amount of concentrated hydrochloric acid, then to add intermediate water, under 4 DEG C of low temperature stir, instillation NaNO
2solution;
After reaction 30min, add a small amount of urea and remove unreacted NaNO
2;
BSA is dissolved in sodium tetraborate damping fluid, then dropwise adds in above-mentioned mixed liquid, and about regulating pH value of solution to 9.2, orange solution occurs, continue reaction 4h, obtain antigen crude product;
Reactant liquor is loaded in bag filter, put in intermediate water and dialyse 5 days, obtain phthalic acid (2-ethyl hexyl) ester immunity holoantigen; Take 4-aminophthalic acid (2-ethyl hexyl) ester to be dissolved in a small amount of concentrated hydrochloric acid, then to add intermediate water, under 4 DEG C of low temperature stir, instillation NaNO
2solution;
After reaction 30min, add a small amount of urea and remove unreacted NaNO
2;
OVA is dissolved in sodium tetraborate damping fluid, then dropwise adds in above-mentioned mixed liquid, and about regulating pH value of solution to 9.2, orange solution occurs, continue reaction 4h, obtain antigen crude product;
Reactant liquor is loaded in bag filter, put in intermediate water and dialyse 5 days, obtain phthalic acid (2-ethyl hexyl) ester bag by holoantigen.
2. the Indirect cELISA detection method of phthalic acid (2-ethyl hexyl) ester as claimed in claim 1, it is characterized in that, step comprises the steps: in (4) further
(4-1) quilt is wrapped: envelope antigen DEHAP-OVA is diluted certain multiple bag by 96 hole ELISA Plate, incubation also washs;
(4-2) close: add OVA solution, close the unnecessary part not having envelope antigen, incubation also washs;
(4-3) compete: every hole adds phthalic acid (2-ethyl hexyl) ester standard solution or sample solution and phthalic acid (2-ethyl hexyl) ester antibody, and make it competitive reaction occurs, incubation also washs;
(4-4) enzyme-added: every hole adds the goat anti-rabbit igg of HRP mark, and incubation also washs;
(4-5) detect: add substrate solution colour developing, after incubation, add stop buffer cessation reaction, multi-functional microplate reader measures light absorption value.
3. the Indirect cELISA detection method of phthalic acid (2-ethyl hexyl) ester as claimed in claim 2, is characterized in that,
In step (4-1), envelope antigen DEHAP-OVA is diluted certain multiple bag by 96 hole ELISA Plate, every hole 100 μ L, after placing incubator 37 DEG C of incubation 2h, taking-up cleansing solution PBST washs 3 times, each 3min; And/or,
Add 1%OVA solution in step (4-2), every hole 150 μ L, close the unnecessary part not having envelope antigen, avoid the non-specific adsorption of antibody in ELISA Plate, after 37 DEG C of incubation 30min, take out the same washing 3 times;
And/or,
In step (4-3), every hole adds 50 μ L phthalic acid (2-ethyl hexyl) ester standard solution or sample solution and 50 μ L phthalic acid (2-ethyl hexyl) ester antibody, makes it competitive reaction occurs, at 37 DEG C after incubation 2.5h, and the same washing;
And/or,
In step (4-4), every hole adds the goat anti-rabbit igg that 100 μ L HRP mark, and takes out the same washing 3 times at 37 DEG C after incubation 2h; And/or,
Add substrate solution colour developing in step (4-5), every hole 100 μ L, after incubation 30min, adds stop buffer cessation reaction, multi-functional microplate reader measures light absorption value.
4. the Indirect cELISA detection method of phthalic acid (2-ethyl hexyl) ester according to any one of claim 1-3, it is characterized in that, first synthesize 4-nitrophthalic acid (2-ethyl hexyl) ester by 4-nitrophthalic acid in step (1), restore and obtain haptens 4-aminophthalic acid (2-ethyl hexyl) ester.
5. the Indirect cELISA detection method of phthalic acid (2-ethyl hexyl) ester according to any one of claim 1-3, is characterized in that, get on BSA coupling in step (2) obtained immunizing antigen, is expelled in animal body by immunizing antigen.
6. the Indirect cELISA detection method of phthalic acid (2-ethyl hexyl) ester according to any one of claim 1-3, is characterized in that, in step (3), animal obtains antibody through certain hour growth response.
7. the Indirect cELISA detection method of phthalic acid (2-ethyl hexyl) ester according to any one of claim 1-3, it is characterized in that, in step (4), set up the method measuring phthalic acid (2-ethyl hexyl) ester by the idiosyncrasy between antigen, antibody and ELIAS secondary antibody.
8. the Indirect cELISA detection method of phthalic acid (2-ethyl hexyl) ester according to any one of claim 1-3, is characterized in that,
Adopt following steps preparation saturated ammonium sulfate solution: get 500mL distilled water, be heated to 70 ~ 80 DEG C, by soluble in water for 400g ammonium sulfate pulvis, stir 20min, at the bottom of ammonium sulfate crystallization is sunken to bottle, supernatant is saturated ammonium sulfate, filters, then get 28% ammoniacal liquor saturated ammonium sulfate is adjusted to pH7.0 after room temperature cooling with absorbent cotton; And/or
Adopt following steps preparing normal saline: 0.85gNaCl adding distil water is to 100mL; And/or
The preparation of 0.01mol/LpH7.2 phosphate buffer (PB) adopts following steps: 0.2mol/LNa
2hPO
436.0mL, 0.2mol/LNaH
2pO
414.0mL mixes, and distilled water constant volume is to 1000mL; And/or
Following steps preparation bag is adopted to be buffered liquid: to take 1.59gNa
2cO
3, 2.94gNaHCO
3, distilled water constant volume is to 1000mL; And/or
Adopt following steps preparation confining liquid: prepare 1%(w/v with PBS) ovalbumin (OVA) solution; And/or
Adopt following steps prepared and diluted liquid: take 8.0gNaCl, 2.96g Na
2hPO
412H
2o, 0.29g NaH
2pO
42H
2o, 0.1gKCl, distilled water constant volume is to 1000mL; And/or
Adopt following steps preparation cleansing solution: in PBS, add 0.05% Tween-20; And/or
Adopt following steps preparation substrate solution: o-phenylenediamine 4mg is dissolved in 10mL citrate-phosphate disodium hydrogen damping fluid, adds 15 μ L30% hydrogen peroxide before use; And/or
Adopt following steps preparation citrate-phosphate disodium hydrogen damping fluid: take Na
2hPO
412H
2o 1.84g, citric acid 0.51g, be then settled to 50mL with distilled water; And/or
Stop buffer is 2mol/L sulfuric acid solution.
9. the Indirect cELISA detection method of phthalic acid (2-ethyl hexyl) ester according to any one of claim 1-3, it is characterized in that, the preparation of described antibody specifically adopts following steps:
Adjuvant: prior to antigen or with in the miscible rear injection animal body of antigen, non-specifically can strengthen the immune response of body, plays booster action;
By whiteruss and the mixing of sheep oil certain volume ratio, indiffusion on the water surface, is now incomplete Freund's adjuvant, and add Bacille Calmette-Guerin is Freund's complete adjuvant later;
Phthalic acid (2-ethyl hexyl) ester immunizing antigen is diluted to finite concentration, miscible with adjuvant 1:1, repeatedly immunity is carried out to 4 new zealand white rabbits;
First time immunity be by immunizing antigen and Freund's complete adjuvant miscible, booster immunization be afterwards by immunizing antigen and incomplete Freund's adjuvant miscible;
Select new zealand white rabbit as immunization, adopt the method for dorsal sc multi-point injection;
Booster immunization after immune 3 weeks of first time, later every 2 weeks booster immunizations again, from second time booster immunization, ear vein blood sampling in the 7th day after each immunity, measuring serum titer, when no longer changing when tiring, strengthening primary immune response, Culling heart blood after 1 week, purifying, obtains antibody.
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