CN100501410C - ELISA kit for detecting ractopamine in animal derived food - Google Patents
ELISA kit for detecting ractopamine in animal derived food Download PDFInfo
- Publication number
- CN100501410C CN100501410C CNB2005100867760A CN200510086776A CN100501410C CN 100501410 C CN100501410 C CN 100501410C CN B2005100867760 A CNB2005100867760 A CN B2005100867760A CN 200510086776 A CN200510086776 A CN 200510086776A CN 100501410 C CN100501410 C CN 100501410C
- Authority
- CN
- China
- Prior art keywords
- ractopamine
- antibody
- antiantibody
- kit
- liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 241001465754 Metazoa Species 0.000 title claims abstract description 17
- YJQZYXCXBBCEAQ-UHFFFAOYSA-N ractopamine Chemical compound C=1C=C(O)C=CC=1C(O)CNC(C)CCC1=CC=C(O)C=C1 YJQZYXCXBBCEAQ-UHFFFAOYSA-N 0.000 title claims description 109
- 229940074095 ractopamine Drugs 0.000 title claims description 108
- 235000013305 food Nutrition 0.000 title claims description 8
- 238000008157 ELISA kit Methods 0.000 title 1
- 239000007788 liquid Substances 0.000 claims abstract description 66
- 102000004190 Enzymes Human genes 0.000 claims abstract description 55
- 108090000790 Enzymes Proteins 0.000 claims abstract description 55
- 238000000034 method Methods 0.000 claims abstract description 55
- 239000000427 antigen Substances 0.000 claims abstract description 33
- 102000036639 antigens Human genes 0.000 claims abstract description 33
- 108091007433 antigens Proteins 0.000 claims abstract description 33
- 239000000243 solution Substances 0.000 claims abstract description 24
- 239000012086 standard solution Substances 0.000 claims abstract description 20
- 238000004140 cleaning Methods 0.000 claims abstract description 8
- 229940126619 mouse monoclonal antibody Drugs 0.000 claims abstract description 4
- 239000000523 sample Substances 0.000 claims description 42
- 238000002372 labelling Methods 0.000 claims description 30
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- 238000012360 testing method Methods 0.000 claims description 21
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 19
- 239000008363 phosphate buffer Substances 0.000 claims description 19
- 238000002360 preparation method Methods 0.000 claims description 19
- 238000002835 absorbance Methods 0.000 claims description 16
- 239000000758 substrate Substances 0.000 claims description 16
- 238000002965 ELISA Methods 0.000 claims description 15
- 239000000872 buffer Substances 0.000 claims description 14
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims description 14
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 10
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 10
- 230000008878 coupling Effects 0.000 claims description 10
- 238000010168 coupling process Methods 0.000 claims description 10
- 238000005859 coupling reaction Methods 0.000 claims description 10
- 102000014914 Carrier Proteins Human genes 0.000 claims description 9
- 108010078791 Carrier Proteins Proteins 0.000 claims description 9
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 9
- 241001494479 Pecora Species 0.000 claims description 9
- 238000011534 incubation Methods 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 8
- 239000003550 marker Substances 0.000 claims description 7
- 239000012488 sample solution Substances 0.000 claims description 7
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 230000036039 immunity Effects 0.000 claims description 6
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 5
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 5
- 238000002203 pretreatment Methods 0.000 claims description 5
- 150000008064 anhydrides Chemical class 0.000 claims description 4
- 239000012141 concentrate Substances 0.000 claims description 4
- NFJCQBGAUBIGKV-UHFFFAOYSA-N nitro dihydrogen phosphate Chemical compound OP(O)(=O)O[N+]([O-])=O NFJCQBGAUBIGKV-UHFFFAOYSA-N 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 claims description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- FDRCDNZGSXJAFP-UHFFFAOYSA-M sodium chloroacetate Chemical compound [Na+].[O-]C(=O)CCl FDRCDNZGSXJAFP-UHFFFAOYSA-M 0.000 claims description 3
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 230000002140 halogenating effect Effects 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 239000004615 ingredient Substances 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 10
- 239000003795 chemical substances by application Substances 0.000 abstract description 7
- 229940079593 drug Drugs 0.000 abstract description 7
- 230000008569 process Effects 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 2
- 230000006835 compression Effects 0.000 abstract 2
- 238000007906 compression Methods 0.000 abstract 2
- 239000012224 working solution Substances 0.000 abstract 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 20
- 229910052757 nitrogen Inorganic materials 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 238000004321 preservation Methods 0.000 description 9
- 238000011084 recovery Methods 0.000 description 9
- 210000002700 urine Anatomy 0.000 description 9
- 239000011248 coating agent Substances 0.000 description 8
- 238000000576 coating method Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 230000001900 immune effect Effects 0.000 description 8
- 239000012530 fluid Substances 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 210000004408 hybridoma Anatomy 0.000 description 6
- 235000015277 pork Nutrition 0.000 description 6
- 235000011121 sodium hydroxide Nutrition 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 230000002163 immunogen Effects 0.000 description 5
- 239000011734 sodium Substances 0.000 description 5
- 206010003445 Ascites Diseases 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000012074 organic phase Substances 0.000 description 4
- -1 polypropylene Polymers 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 3
- 206010068676 Pneumoretroperitoneum Diseases 0.000 description 3
- 208000005727 Retropneumoperitoneum Diseases 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 3
- 229910052782 aluminium Inorganic materials 0.000 description 3
- 239000004411 aluminium Substances 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 244000309466 calf Species 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000011587 new zealand white rabbit Methods 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 2
- 239000005662 Paraffin oil Substances 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000003636 conditioned culture medium Substances 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 230000001804 emulsifying effect Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- NBEMQPLNBYYUAZ-UHFFFAOYSA-N ethyl acetate;propan-2-one Chemical compound CC(C)=O.CCOC(C)=O NBEMQPLNBYYUAZ-UHFFFAOYSA-N 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000009313 farming Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000000760 immunoelectrophoresis Methods 0.000 description 2
- 239000003547 immunosorbent Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- 206010000087 Abdominal pain upper Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 235000016936 Dendrocalamus strictus Nutrition 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 208000000112 Myalgia Diseases 0.000 description 1
- 241000872931 Myoporum sandwicense Species 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- BHHGXPLMPWCGHP-UHFFFAOYSA-N Phenethylamine Chemical compound NCCC1=CC=CC=C1 BHHGXPLMPWCGHP-UHFFFAOYSA-N 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 208000001871 Tachycardia Diseases 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000006793 arrhythmia Effects 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229940125388 beta agonist Drugs 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000003177 cardiotonic effect Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- HGAZMNJKRQFZKS-UHFFFAOYSA-N chloroethene;ethenyl acetate Chemical compound ClC=C.CC(=O)OC=C HGAZMNJKRQFZKS-UHFFFAOYSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229960001399 clenbuterol hydrochloride Drugs 0.000 description 1
- OPXKTCUYRHXSBK-UHFFFAOYSA-N clenbuterol hydrochloride Chemical compound Cl.CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 OPXKTCUYRHXSBK-UHFFFAOYSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 229940035429 isobutyl alcohol Drugs 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 201000006938 muscular dystrophy Diseases 0.000 description 1
- 210000001087 myotubule Anatomy 0.000 description 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 229920002379 silicone rubber Polymers 0.000 description 1
- 230000012232 skeletal muscle contraction Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000006794 tachycardia Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses an enzyme immune agent box for detecting lake drugs of animal foodstuff; it also provides a method for using the agent to detect the lake drugs residue. The agent box comprises: enzyme mark plate which coats lake drugs antigen or antibody, lake drugs mouse monoclonal antibody or polyclonal antibody working solution, enzyme mark antibody or enzyme mark lake drugs antigen, lake drugs standard solution, base material color developing solution, compression cleaning liquid, ending solution and compression twin solution. The invention also discloses a method for applying the detecting method, which comprises: first doing sample front process, then using the agent box to detect, at last analyzing the detected result.
Description
Technical field
The present invention relates to the immunology detection field, specifically, provide a kind of enzyme linked immunological kit and detection method thereof that detects Ractopamine in the animal derived food.
Background technology
Ractopamine (Ractopamine) is a kind of phenyl ethyl amine medicine.Be used for the herding fishery, it be a kind of " nutrition reallocation agent ", belong to beta-agonist, Ractopamine is effective as the substitute of " clenbuterol hydrochloride ", the economy return height uses general; Be used for medicine, it is a kind of cardiotonic drug, can be used for treatment of obesity and muscular dystrophy.But people's accumulative total is taken in dosage and is surpassed certain value, or practical Ractopamine high residue is when viscera tissue, toxic reaction appears, symptom shows as Skeletal Muscle Contraction to be increased, destroy the fusion between quick muscle fiber and the slow switch fibers, cause muscular tremor, the muscle of four limbs and face is particularly evident, other toxicity symptom comprises tachycardia, arrhythmia cordis, stomachache, myalgia, feel sick and dizzy etc., therefore China bans use of, and China Ministry of Agriculture No. 176 bulletin regulation Ractopamine is the medicine of forbidding using in feed and animal drinking water.Therefore, detecting the residual quantity of Ractopamine in animal food is very important.
At present, be usually used in the method that Rct opamine residue detects and mainly contain microbial method and instrumental method.Though the microorganism detection method is economical, easy and simple to handle, when having other microbial inhibitors to exist in sample, its sensitivity and specificity are restricted; Simple instrument analytical methods such as high efficiency liquid phase chromatographic analysis method, gas spectrum, GC-MS method, though highly sensitive, sample pre-treatment and measurement operation are loaded down with trivial details, the expense height is unwell to the great amount of samples examination, can be used as residual conclusive evidence analysis.
Summary of the invention
(1) technical matters that will solve
The object of the invention is to provide a kind of simple in structure, easy to use, low price, the portable enzyme linked immunological kit that is used for the animal derived food Ractopamine, and a kind of efficient, accurate, easy, qualitative and quantitative analysis method of being suitable for the batch samples screening is provided.
(2) technical scheme
Enzyme linked immunological kit provided by the present invention comprises:
(1) bag is by the ELISA Plate of Ractopamine antigen or antiantibody;
(2) Ractopamine antibody;
(3) enzyme labeling thing;
(4) Ractopamine standard solution;
(5) substrate colour developing liquid;
(6) concentrated cleaning solution;
(7) concentrate redissolution liquid;
(8) stop buffer.
Wherein said bag by the ELISA Plate of Ractopamine antigen in the process of preparation, used coating antigen is to use active ester method (DCC, NHS) that Ractopamine and carrier protein couplet are obtained; The antiantibody of bag quilt can be sheep anti mouse antiantibody or goat-anti rabbit antiantibody; Used coating buffer is the carbonate buffer solution of pH value 9.6,0.05～0.1mol/L, and used confining liquid is the solution that contains 3～10% calf serums.
Wherein said Ractopamine antibody is in preparation process, used immunogene adopts active ester method (DCC, NHS) that Ractopamine and carrier protein are carried out coupling and obtains, and used antibody diluent is pH value 8.2,0.05～0.1mol/L, contain the phosphate buffer of 3～5% calf serums.The protein concentration of Ractopamine antibody is 0.5～5.0 μ g/L in the kit of the present invention.
The enzyme labeling thing is enzyme labeling antiantibody or enzyme labeling Ractopamine antigen, the enzyme labeling antiantibody is enzyme labeling sheep anti mouse antiantibody or enzyme labeling goat-anti rabbit antiantibody, the sheep anti mouse antiantibody of enzyme labeling or goat-anti rabbit antiantibody adopt glutaraldehyde method or sodium periodate method that marker enzyme and antiantibody are carried out coupling and obtain, and enzyme labeling Ractopamine antigen employing mixed anhydride method is carried out coupling with marker enzyme and Ractopamine haptens and obtained.Used marker enzyme can be horseradish peroxidase or bacterium is extracted alkaline phosphatase, and the present invention is preferably horseradish peroxidase.Horseradish peroxidase can adopt several different methods of the prior art that itself and antiantibody or Ractopamine antigen are carried out coupling, as glutaraldehyde method, sodium periodate method etc., the present invention creates through long-term work the sodium periodate method is improved, make it save time, reduce the concentration rate of horseradish peroxidase (HRP) and antiantibody or Ractopamine antigen, saved starting material.
The concentration of wherein said standard solution is: 0～40.5 μ g/L, can select 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L.
Wherein said substrate colour developing liquid is when marker enzyme is horseradish peroxidase, and substrate colour developing liquid A liquid is that hydrogen peroxide or urea peroxide, the substrate liquid B liquid that develops the color is o-phenylenediamine or tetramethyl benzidine; When marker enzyme was bacterium extraction alkaline phosphatase, substrate colour developing liquid was to the nitro phosphate buffer.
Wherein said concentrated cleaning solution is the phosphate buffer that contains 0.8～1.2% polysorbas20.
Wherein said concentrated redissolution liquid is the phosphate buffer that contains 50% methyl alcohol, pH8.0～8.6,0.1～0.2mol/L.
Wherein said stop buffer is sulfuric acid, hydrochloric acid or the 2mol/L sodium hydrate buffer solution of 1～2mol/L.
The material that can be used as fixing Ractopamine coupled antigen or antiantibody carrier is a lot, as polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber, Ago-Gel etc.The form of this carrier can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc.
The present invention also provides the method for using Rct opamine residue amount in the mentioned reagent box qualitative and quantitative analysis animal derived food, and it may further comprise the steps:
(1) sample pre-treatments;
(2) detect with kit;
(3) analyzing and testing result.
Preferably, sample treatment is:
A, pig urine
Urine sample adds anhydrous Na after regulating pH to 10～11 with NaOH solution
2SO
4, add ethyl acetate-acetone again, vibration back centrifugal (or standing demix).Getting organic phase flows down at nitrogen and dries up.Redissolution liquid dissolving with dilution.
B, tissue
Take by weighing tissue, add anhydrous acetonitrile earlier, add anhydrous Na again
2SO
4Fully vibration.Get supernatant, nitrogen flows down and dries up.Add damping fluid and mix, add isobutyl alcohol and anhydrous Na
2SO
4, oscillation extraction, centrifugal, take out upper organic phase, nitrogen flows down and dries up.Redissolution liquid dissolving with dilution can be analyzed.
C, feed sample
Accurately take by weighing feed in centrifuge tube.Add methyl alcohol and ethanol, fully vibration is centrifugal.Get supernatant, nitrogen dries up.Add methylene chloride, dissolution residual substance adds damping fluid again and fully mixes, and is centrifugal, gets upper strata liquid and adds the redissolution liquid that has diluted, and can analyze.
Preferably, wherein the kit detection is to add standard solution or sample solution in the ELISA Plate micropore that is coated with the Ractopamine coupled antigen, adds Ractopamine antibody again, and washing pats dry behind the incubation, develop the color, stop after adding the enzyme labeling antiantibody, measure absorbance with microplate reader.Perhaps, be in the ELISA Plate micropore that is coated with antiantibody, to add Ractopamine antibody, washing pats dry behind the incubation, add enzyme mark Ractopamine antigen and standard solution or sample solution again, washing pats dry behind the incubation, and colour developing, termination are measured absorbance with microplate reader.
Preferably, the testing result analytic process is: the absorbance mean value (B) of standard solution of using each concentration that is obtained is divided by the absorbance (B of first standard solution (0 standard)
0) multiply by 100% again, i.e. percentage absorbance.Computing formula is:
Percentage absorbance (%)=(B/B
0) * 100%
B is the mean light absorbency value of standard solution in the formula, B
0It is the mean light absorbency value of 0 μ g/L standard solution.Natural logarithm value with Ractopamine concentration is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard curve map.Percentage absorbance with same way calculation sample solution, the concentration of Ractopamine then can be read from typical curve in corresponding each sample, according to the depth of the color sample on the ELISA Plate, but with the comparison judgement sample of the standard solution color of series concentration in the concentration range of Ractopamine.
Preferably, the analysis of testing result also can be adopted regression equation method, calculates sample solution concentration.
Preferably, the analysis of testing result can also utilize computer professional software, the be more convenient for express-analysis of a large amount of samples of this method, and whole testing process only needed to finish in 1.5 hours, and lowest detection is limited to 0.5 μ g/L.
Wherein, the preparation method of antigen and antibody is:
(1) antigen is synthetic
Haptenic synthetic:
Adopt sodium chloroacetate to synthesize the Ractopamine haptens Ractopamine, adopt sodium chloroacetate to carry out halogenating reaction, the hydroxyl on the Ractopamine molecule is replaced, the synthetic carboxyl spacerarm that contains 2 carbon is prepared into the haptens of Ractopamine.
Coating antigen and immunogenic synthetic:
With Ractopamine haptens and bovine serum albumin(BSA) (BSA), human serum albumins (HSA), thyroprotein carrier proteins such as (BCG), adopt water-soluble carbodiimide method (EDC), mixed anhydride method (isobutyl chlorocarbonate), active ester method (DCC, NHS) to carry out coupling and obtain coating antigen and immunogene respectively.
Common immunogenic purity requirement is higher, and immunogenic purity is high more, and the antibody specificity of preparation is strong more, will reach at least more than 90%, and it is 98.5% that the immunogene that the present invention synthesizes adopts immunoelectrophoresis to measure its purity.
(2) preparation of enzyme labeling antiantibody
Adopt the sodium periodate method after improveing to carry out coupling antiantibody and horseradish peroxidase (HRP).The molar concentration rate of enzyme and antiantibody is 4:1 in traditional sodium periodate method requirement reflection system; Because horseradish peroxidase produces many sites that combine with antiantibody under the effect of strong oxidation, Huo Hua horseradish peroxidase molecule has served as the bridge that connects each molecule like this, reduced the enzymatic activity of enzyme labeling thing, make in the conjugate of preparation and be mixed with many condensates, in order to address this problem, we improve traditional method, that is:
1) saved amino closed process, because can produce self amino amino reality that connects seldom.
2) reduced horseradish peroxidase: the volumetric molar concentration ratio of antiantibody is to 2:1, and the method after the improvement is easier than traditional method, and the loss of the activity of enzyme is reduced.
(3) preparation of enzyme labeling Ractopamine antigen
Adopt mixed anhydride method that horseradish peroxidase and Ractopamine hapten conjugation are obtained enzyme labeling Ractopamine antigen.
(4) Ractopamine MONOCLONAL ANTIBODIES SPECIFIC FOR
The animal immune program: adopting the Balb/c mouse as immune animal, is that immunogene is carried out immunity to mouse with Ractopamine and carrier protein couplet thing, can obtain containing in the blood mouse spleen of Ractopamine specific antibody.
Fusion of Cells and cloning: the splenocyte and the myeloma cell that get immune Balb/c mouse are merged, and adopt the indirect competitive enzyme-linked immunosorbent method to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, obtain the hybridoma cell strain of monoclonal antibody.
Cell cryopreservation and recovery: get the hybridoma that is in exponential phase and make cell suspension, be sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen with cryopreserving liquid.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying: adopt in the body and induce method, Balb/c mouse (8 age in week) abdominal cavity is injected the sterilization paraffin oil, 7～14 days pneumoretroperitoneum injection hybridomas were gathered ascites after 7～10 days.Carry out the ascites purifying through sad-saturated ammonium sulfate method, bottle packing ,-20 ℃ of preservations.
(5) preparation of Ractopamine rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, with Ractopamine and carrier protein couplet thing is that immunogene is carried out immunity to new zealand white rabbit, repeatedly serum antibody titer is measured in the immunity back, and heart is taken a blood sample, and obtains the polyclonal antibody of purifying through ammonium sulfate precipitation.
Wherein the compound method of agents useful for same is:
A. Ractopamine standard solution: 6 bottles of Ractopamine series standard solution, 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L;
B. bag is cushioned liquid: pH9.6, the carbonate buffer solution of 0.05～0.1mol/L;
C. confining liquid: the solution that contains 3～10% calf serums;
D. concentrated cleaning solution: (pH7.4 0.012mol/L), is 15～25 times of normal working concentration to contain the phosphate buffer of 0.8～1.2% polysorbas20;
E. enzyme labeling thing: enzyme labeling antiantibody or enzyme labeling Ractopamine antigen;
F. substrate colour developing liquid A liquid: hydrogen peroxide or urea peroxide;
G. substrate colour developing liquid B liquid: o-phenylenediamine (OPD) or tetramethyl benzidine (TMB);
H. substrate colour developing liquid to nitro phosphate buffer: pH8.1, contain MgCl
20.01% 100mmolTris-HCl;
I. stop buffer: 1～2mol/L sulfuric acid, hydrochloric acid or 2mol/L sodium hydrate buffer solution;
J. concentrate to redissolve liquid: pH8.0～8.6,0.1～0.2mol/L, contain the phosphate buffer of 50% methyl alcohol, be 5～10 times of normal working concentration;
K. damping fluid: the deionized water solution that contains 48% natrium carbonicum calcinatum and 15% sodium chloride.
Wherein the preparation method of ELISA Plate is:
(1) is cushioned liquid with Ractopamine and the dilution of carrier protein couplet thing with bag, in the elisa plate micropore, add antigenic dilution, putting into 37 ℃ of environment hatches, put into 4 ℃ of environment again and spend the night (good stability of the elisa plate that obtains), the coating buffer that inclines washs with cleansing solution, in every hole, add confining liquid then, hatch for 37 ℃, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
(2) being cushioned liquid with bag dilutes antiantibody, in the elisa plate micropore, add the antiantibody dilution, putting into 37 ℃ of environment hatches, put into 4 ℃ of environment and spend the night (good stability of the elisa plate that obtains), the coating buffer that inclines washs with cleansing solution, in every hole, add confining liquid then, hatch for 37 ℃, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
The detection principle of kit of the present invention is:
(1) Ractopamine antigen is adsorbed on the solid phase carrier, add sample or Ractopamine standard solution and add Ractopamine antibody working fluid, the Ractopamine antigenic competition Ractopamine antibody of bag quilt on residual Ractopamine and the solid phase carrier in the testing sample, add the enzyme labeling antiantibody again and carry out the amplification of enzymatic activity, the colour developing back stops, the absorbance of working sample, the Rct opamine residue amount is negative correlation in this value and the sample, relatively can draw the Ractopamine concentration range with typical curve.
(2) antiantibody is adsorbed on the solid phase carrier, add Ractopamine specific antibody working fluid, add enzyme labeling Ractopamine antigen again and add sample or the Ractopamine standard solution, residual Ractopamine and enzyme labeling Ractopamine antigenic competition Ractopamine antibody in the testing sample, the colour developing back stops, the absorbance of working sample, the Rct opamine residue amount is negative correlation in this value and the sample, relatively can draw the Ractopamine concentration range with typical curve.
(3) beneficial effect
The kit that the present invention detects Ractopamine mainly adopts the residual quantity of Ractopamine in the samples such as the qualitative or detection by quantitative pork of indirect competitive enzyme-linked immunosorbent assay method, urine sample, feed; Pre-treatment requirement to sample is low, and sample pretreatment process is simple, simultaneously the fast detecting gross sample.
This kit of the present invention adopts the Ractopamine monoclonal antibody or the polyclonal antibody of high specific, main agents provides with the working fluid form, can reduce the operation steps of kit, for the user saves time and reduces the error that causes because of operation steps is miscellaneous, that the present invention has is highly sensitive, high specificity, pinpoint accuracy, pin-point accuracy, low to the instrument and equipment requirement, the reagent holding time is long, automaticity is high, "dead" isotopic contamination etc. advantage, can in the animal food Rct opamine residue detects, play a significant role.
Description of drawings
Fig. 1 Ractopamine examination criteria curve map.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
The preparation of embodiment 1 reagent constituents
1, antigen is synthetic
A. get Ractopamine haptens 2g and be dissolved in 20ml, in the sodium hydroxide solution of 0.5M.
B. getting 2g nitrogen hydroxyl succinyl methylamine is dissolved in the 8ml pure water and is added in the Ractopamine haptens solution stirring at room reaction 2.5 hours.
C. get human serum albumins (HSA) or thyroprotein (BCG) carrier protein 22g is dissolved in 75ml
In the pH9 carbonate buffer solution.
D. carrier protein being added drop-wise in the haptens solution 4 ℃ of stirrings spends the night.
E. synthetic artificial antigen was dialysed 7 days with the phosphate buffer of 0.2M, change liquid every day 3～4 times.At last concentrated preservation of antigen or freeze-drying preservation are obtained Ractopamine immunogene and coating antigen.
Common immunogenic purity requirement is higher, and immunogenic purity is high more, and the antibody specificity of preparation is strong more, will reach at least more than 90%, and it is 98.5% that the immunogene that the present invention synthesizes adopts immunoelectrophoresis to measure its purity.
2, the preparation of enzyme labeling antiantibody
Antiantibody and horseradish peroxidase (HRP) are carried out coupling, and the method for employing is the sodium periodate method of improvement, and method is as follows:
A.8mg horseradish peroxidase is dissolved in the 2mL distilled water.
B. the 100mmol/LNaIO that adds existing preparation
4Solution 0.4mL, stirring at room reaction 20 minutes.
C. use the 1mmol/L acetate buffer in 4 ℃ of dialysed overnight, remove unnecessary NaIO
4, make the enzyme reduction of self coupling simultaneously.
D. add 0.5mol/L phosphate buffer (pH8.6) 40 μ l and phosphate buffer (pH8.6, the 5mol/L) 2.0mL that contains 16mg IgG, stirring at room reaction 4 hours.
E. the NaBH that adds existing preparation
4Aqueous solution (1mol/L) 0.1mL, 4 ℃ were reacted 4 hours, with reduction Schiff alkali.
F. purification storage.
3, Ractopamine mouse monoclonal antibody preparation
Animal immune program: adopt the Balb/c mouse as immune animal, with Ractopamine and human serum albumin conjugate is immunogene, immunizing dose is 80 μ g/, Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, getting the same dose immunogene 3 weeks adds equivalent incomplete Freund mixing and emulsifying at interval, and booster immunization is once, four exempt from the pneumoretroperitoneum booster immunization once, extracting spleen cell after 3 days.
Fusion of Cells and cloning: get immune Balb/c mouse boosting cell, merge, adopt the indirect competitive enzyme-linked immunosorbent method to measure cell conditioned medium liquid, screen positive hole in 5:1 ratio and SP2/0 myeloma cell.Utilize limiting dilution assay that cloning is carried out in positive hole, up to the hybridoma cell strain that obtains the stably excreting monoclonal antibody.
Cell cryopreservation and recovery: get the hybridoma that is in exponential phase and make 5 * 10 with cryopreserving liquid
6The cell suspension of individual/ml is sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying: adopt in the body and induce method, Balb/c mouse (8 age in week) abdominal cavity is only injected sterilization paraffin oil 0.5ml/, 10 days pneumoretroperitoneum injection hybridomas 5 * 10
6Individual/as only, to gather ascites after 10 days.Carry out the ascites purifying with sad-saturated ammonium sulfate method, bottle packing ,-20 ℃ of preservations.
4, the preparation of Ractopamine rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, with Ractopamine and human serum albumin conjugate is immunogene, immunizing dose is 1mg/kg, Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, getting the same dose immunogene 3～4 weeks adds equivalent incomplete Freund mixing and emulsifying at interval, and booster immunization is once, immunity is 5 times altogether, does not add adjuvant for the last time.Serum antibody titer is measured in last immunity blood sampling after 10 days, and heart is taken a blood sample, and obtains the polyclonal antibody of purifying with ammonium sulfate precipitation.
5, the preparation of ELISA Plate
Be cushioned liquid with bag Ractopamine and thyroprotein conjugate are diluted to 0.1 μ g/ml, every hole adds 100 μ l, and 37 ℃ of incubation 2h put into 4 ℃ of environment spend the night (good stability of the elisa plate that obtains), coating buffer inclines, with cleansing solution washing 2 times, each 1min pats dry, in every hole, add 150 μ l confining liquids then, 37 ℃ of incubation 2h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of Ractopamine
Set up the enzyme linked immunological kit that detects Ractopamine, make it comprise following component:
(1) bag is by the ELISA Plate of Ractopamine antigen;
(2) protein concentration is the Ractopamine mouse monoclonal antibody of 0.5 μ g/L;
(3) the sheep anti mouse antiantibody of usefulness horseradish peroxidase-labeled;
(4) the Ractopamine standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L;
(5) substrate colour developing liquid A liquid is hydrogen peroxide, and substrate colour developing liquid B liquid is o-phenylenediamine;
(6) concentrated cleaning solution is the phosphate buffer that contains 0.8% polysorbas20;
(7) redissolution liquid is the phosphate buffer that contains 50% methyl alcohol, pH8.0,0.1mol/L;
(8) stop buffer is the sulfuric acid solution of 1mol/L.
Embodiment 3 detects the establishment of the enzyme linked immunological kit of Ractopamine
Set up the enzyme linked immunological kit that detects Ractopamine, make it comprise following component:
(1) bag is by the ELISA Plate of sheep anti mouse antiantibody;
(2) protein concentration is the Ractopamine monoclonal antibody of 5.0 μ g/L;
(3) Ractopamine of usefulness alkaline phosphate ester enzyme labeling;
(4) the Ractopamine standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L;
(5) substrate colour developing liquid is to the nitro phosphate buffer;
(6) concentrated cleaning solution is the phosphate buffer that contains 1.2% polysorbas20;
(7) redissolution liquid is the phosphate buffer that contains 50% methyl alcohol, pH8.6,0.2mol/L;
(8) stop buffer is the sodium hydroxide solution of 2mol/L.
The detection of Rct opamine residue in embodiment 4 samples
1, sample pre-treatments
A, pig urine
The 1ml urine sample adds the 1g anhydrous Na after regulating pH to 10 with 1mol/L NaOH
2SO
4, add 3ml ethyl acetate-acetone (V/V=3:1) again, vibration back 3000rpm, centrifugal 5min (or standing demix) more than 15 ℃.Getting the 1.5ml organic phase flows down at nitrogen and dries up.With the redissolution liquid dissolving that 1ml has diluted, get 50 μ l and analyze.
B, tissue
Take by weighing and organize 3g ± 0.1g, add anhydrous acetonitrile 9ml earlier and add the 2g anhydrous Na again
2SO
410min fully vibrates.Get supernatant 4ml, nitrogen flows down and dries up.Add 1ml normal hexane vibration dissolving, add the redissolution liquid that has diluted with 1ml again and mix vibration 1min, the above centrifugal 5min of 3000g removes upper organic phase.Behind this extract 100 μ l+100 μ l that take a sample redissolution dilution, get 50 μ l and analyze.
C, feed sample
Accurately take by weighing 2.0 ± 0.1g feed in the 40ml centrifuge tube.Add 10ml methyl alcohol: ethanol (V/V=1:1), the 10min that fully vibrates, the centrifugal 10min of 4000rpm.Get supernatant 2ml, nitrogen dries up.Add the 1ml methylene chloride, dissolution residual substance adds 1ml redissolution liquid again, fully mixes 5min, and the centrifugal 10min of 10000rpm gets upper strata liquid, analyzes with getting 50 μ l after the dilution of redissolution liquid.
2, detect with kit
In the ELISA Plate micropore of Ractopamine and thyroprotein conjugate bag quilt, add series standard product or sample solution 50 μ l, add antibody working fluid 50 μ l again,, react 30min in 37 ℃ of constant temperature ovens with cover plate film shrouding.Pour out liquid in the hole, every hole adds the cleansing solution that 250 μ l have diluted (pH7.4,0.01M, contain the phosphate buffer of 0.8% tween), pours out liquid in the hole after 30 seconds, and so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.Every hole adds enzyme labeling antiantibody 100 μ l with cover plate film shrouding, reacts 30min in 37 ℃ of constant temperature ovens.Substrate colour developing liquid A liquid (hydrogen peroxide) 50 μ l add B liquid (o-phenylenediamine) 50 μ l again, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 15～30min.Every hole adds stop buffer (2mol/L sulfuric acid) 50 μ l, and the mixing that vibrates is gently measured every hole absorbance (OD value) with microplate reader.
Interpretation of result:
Calculate percentage absorbance and drawing standard curve, the concentration of Ractopamine can be read from typical curve in corresponding each sample.Also can use regression equation method, calculate the residual quantity of Ractopamine in sample.Utilize computer professional software, the express-analysis of a large amount of samples of being more convenient for.
According to the depth of the color sample on the ELISA Plate, but with the comparison judgement sample of the standard solution color of series concentration in the concentration range of Ractopamine.
The test of experimental example 1 kit precision
1, the repeatable test of standard
From every batch of elisa plate according to the preparation of the method the embodiment 2 (3), each extracts 10 micropores out, measures the absorbance (OD value) of 4.5 μ g/L standard solution, repeats 3 times, calculates coefficient of variation CV%, the results are shown in Table 1.
The repeatable test of table 1 standard
The result shows that coefficient of variation scope is between 4.6～8.1%, met the coefficient of variation less than 20% regulation, illustrated that this kit standard items precision has reached " Ministry of Agriculture's file " farming doctor and sent out [2005] No. 17 annex 2 kits and put on record with reference to the precision standard of the 4th precision in the judgment criteria and accuracy.
2, the repeatable test of sample
Ractopamine with 2.5 μ g/kg concentration adds urine and pork, Ractopamine with 100 μ g/kg concentration adds feed, add in the sample, get respectively three different batches kit each five, each concentration repeats 5 times, calculate the coefficient of variation respectively, the results are shown in Table 2～4.
The repeatable test of table 2 urine sample
The repeatable test of table 3 pork sample
The repeatable test of table 4 feed sample
The result shows that urine sample Variation Lines number average is lower than 15%, the Variation Lines number average of pork sample is lower than 10%, the Variation Lines number average of feed sample is less than 20%, met the coefficient of variation and sent out [2005] No. 17 annex 2 kits less than " Ministry of Agriculture's file " farming doctor of 20% and put on record with reference to the precision regulation of the 4th precision in the judgment criteria and accuracy.
The accuracy test of experimental example 2 kits
Get the Ractopamine standard specimen of two concentration, sample added recovery test, each concentration do 4 parallel, respectively calculate recovery rate the results are shown in Table 5.
The test of table 5 accuracy determination
The result shows pork, and the recovery that pork liver adds is between 62.4～89.8%, and urine is added the recovery between 72.4～90.6%, adds the recovery in the feed between 69.8～86.1%.
The test of experimental example 3 kit storage lives
The kit preservation condition is 2～8 ℃, and through 6 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, Ractopamine added the practical measurement value all within normal range.Consideration has improper preservation condition and occurs in transportation and use, and kit was placed 6 days under the condition of 37 ℃ of preservations, carries out the accelerated deterioration experiment, and the result shows that the every index of this kit meets the requirements fully.Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 5 days, measurement result shows that also the every index of kit is normal fully.Can draw kit from above result can preserve more than 6 months at 2～8 ℃.
Claims (10)
1, a kind of kit that is used for detecting the animal derived food Ractopamine is characterized in that it comprises following ingredients:
(1) bag is by the ELISA Plate of Ractopamine antigen or antiantibody;
(2) Ractopamine antibody;
(3) enzyme labeling thing;
(4) Ractopamine standard solution;
(5) substrate colour developing liquid;
(6) concentrated cleaning solution;
(7) concentrate redissolution liquid;
(8) stop buffer,
Wherein, described Ractopamine antigen is by Ractopamine being adopted sodium chloroacetate carry out halogenating reaction, hydroxyl on the Ractopamine molecule is replaced, and the synthetic carboxyl spacerarm that contains 2 carbon is prepared into the haptens of Ractopamine, more described haptens and carrier protein couplet is obtained; Institute's book antibody is to obtain by described antigen preparation.
2, kit as claimed in claim 1 is characterized in that described antiantibody is is immune animal with the sheep, is that immunogene is carried out sheep anti mouse antiantibody or the goat-anti rabbit antiantibody that immunity obtains to the pathogen-free domestic sheep with mouse endogenous antibody or rabbit endogenous antibody.
3, kit as claimed in claim 1 is characterized in that Ractopamine antibody is mouse monoclonal antibody or rabbit polyclonal antibody.
4, kit as claimed in claim 1, it is characterized in that the enzyme labeling thing is the Ractopamine antigen of sheep anti mouse antiantibody, goat-anti rabbit antiantibody or the enzyme labeling of enzyme labeling, the enzyme labeling antiantibody adopts glutaraldehyde method or sodium periodate method that horseradish peroxidase or bacterium are extracted alkaline phosphatase to carry out coupling with antiantibody and obtain, and enzyme-labelled antigen is to adopt mixed anhydride method that horseradish peroxidase or bacterium extraction alkaline phosphatase and Ractopamine haptens are carried out coupling to obtain.
5, kit as claimed in claim 1 is characterized in that concentrated cleaning solution is the phosphate buffer that contains 0.8～1.2% polysorbas20; The liquid that concentrate to redissolve is pH8.0～8.6,0.1～0.2mol/L, contain 50% methyl alcohol phosphate buffer; When marker enzyme was horseradish peroxidase, substrate colour developing liquid A liquid was that hydrogen peroxide or urea peroxide, substrate colour developing liquid B liquid are that o-phenylenediamine or tetramethyl benzidine, stop buffer are sulfuric acid or the hydrochloride buffer of 1～2mol/L; When marker enzyme is a bacterium when extracting alkaline phosphatase, substrate colour developing liquid is for being the NaOH of 2mol/L to nitro phosphate buffer, stop buffer.
6, kit as claimed in claim 5 is characterized in that: the protein concentration of Ractopamine antibody is 0.5～5.0 μ g/L.
7, kit as claimed in claim 1 is characterized in that: the concentration of Ractopamine standard solution is respectively 0 μ g/L, 0.5 μ g/L, 1.5 μ g/L, 4.5 μ g/L, 13.5 μ g/L, 40.5 μ g/L.
8, the method for Rct opamine residue in a kind of test sample comprises step:
(1) sample pre-treatments;
(2) detect with the arbitrary described kit of claim 1～7;
(3) analyzing and testing result.
9, method as claimed in claim 8, wherein kit detects to add standard items or sample solution in the ELISA Plate micropore that is coated with Ractopamine antigen, add Ractopamine antibody again, washing pats dry behind the incubation, add the enzyme labeling antiantibody, washing pats dry behind the incubation, and colour developing, termination are measured absorbance with microplate reader.
10, method as claimed in claim 8, wherein kit detects to add Ractopamine antibody in the ELISA Plate micropore that is coated with antiantibody, washing pats dry behind the incubation, add enzyme labeling Ractopamine antigen and standard items or sample solution again, washing pats dry behind the incubation, colour developing, termination are measured absorbance with microplate reader.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100867760A CN100501410C (en) | 2005-11-03 | 2005-11-03 | ELISA kit for detecting ractopamine in animal derived food |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100867760A CN100501410C (en) | 2005-11-03 | 2005-11-03 | ELISA kit for detecting ractopamine in animal derived food |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1766630A CN1766630A (en) | 2006-05-03 |
| CN100501410C true CN100501410C (en) | 2009-06-17 |
Family
ID=36742634
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100867760A Expired - Fee Related CN100501410C (en) | 2005-11-03 | 2005-11-03 | ELISA kit for detecting ractopamine in animal derived food |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100501410C (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101241134B (en) * | 2008-01-18 | 2012-05-30 | 华南农业大学 | ELISA kit for detecting ractopamine residue and method of use thereof |
| CN101315379B (en) * | 2008-07-15 | 2012-08-08 | 天津九鼎生物工程有限公司 | Reagent kit for detecting Ractopamine and application thereof |
| CN101993488B (en) * | 2009-08-27 | 2014-05-21 | 深圳市三方圆生物科技有限公司 | Ractopamine immunogen, coating antigen and use thereof in colloid-gold test paper |
| CN103013925A (en) * | 2011-09-21 | 2013-04-03 | 北京勤邦生物技术有限公司 | Bispecific monoclonal antibody, preparation method and uses thereof |
| CN103012592A (en) * | 2011-09-21 | 2013-04-03 | 北京勤邦生物技术有限公司 | Preparation and applications of ractopamine monoclonal antibody |
| CN102539747A (en) * | 2011-12-28 | 2012-07-04 | 于洪侠 | Enzyme-linked immunoassay kit for detecting phenylethanolamine A by direct competition method |
| CN102520158A (en) * | 2011-12-28 | 2012-06-27 | 于洪侠 | Indirect-competition-law enzyme linked immunosorbent assay kit for detecting phenylethanolamine A |
| CN102778564B (en) * | 2012-05-31 | 2014-06-04 | 华中农业大学 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting ractopamine |
| CN102901813B (en) * | 2012-09-27 | 2015-10-28 | 江苏维赛科技生物发展有限公司 | Detect enzyme linked immunological kit and the using method thereof of residual benzene monoethanolamine A |
| CN103792328B (en) * | 2014-01-13 | 2015-06-10 | 宁波大学 | Method for detecting ractopamine content in food |
| CN106405089A (en) * | 2015-08-03 | 2017-02-15 | 镇江先创生物科技有限公司 | ELISA kit capable of rapid detection of diminazene aceturate residues in animal-derived food |
-
2005
- 2005-11-03 CN CNB2005100867760A patent/CN100501410C/en not_active Expired - Fee Related
Non-Patent Citations (6)
| Title |
|---|
| 猪肝和猪尿中沙丁胺醇和克伦特罗残留的酶联免疫吸附检测法研究. 王建平等.畜牧兽医学报，第36卷第4期. 2005 |
| 猪肝和猪尿中沙丁胺醇和克伦特罗残留的酶联免疫吸附检测法研究. 王建平等.畜牧兽医学报，第36卷第4期. 2005 * |
| 莱克多巴胺单克隆抗体的研制. 张雨梅等.扬州大学学报，第25卷第3期. 2004 |
| 莱克多巴胺单克隆抗体的研制. 张雨梅等.扬州大学学报，第25卷第3期. 2004 * |
| 莱克多巴胺残留检测方法研究进展. 于洪侠等.中国兽医杂志，第38卷第11期. 2004 |
| 莱克多巴胺残留检测方法研究进展. 于洪侠等.中国兽医杂志，第38卷第11期. 2004 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1766630A (en) | 2006-05-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100501410C (en) | ELISA kit for detecting ractopamine in animal derived food | |
| CN100403030C (en) | ELISA kit for detecting Sudan red medicines and detection method thereof | |
| CN100501409C (en) | ELISA kit for detecting chloramphenicols in animal derived food | |
| CN101013129B (en) | Enzyme linked immunosorbent reagent casing for detecting furacilin metabolite and uses thereof | |
| CN101571541B (en) | Enzyme-linked immunosorbent inspect kit for inspecting sulfa drugs and method thereof | |
| CN101256188B (en) | ELISA kit for detecting lincomycin medicine as well as usage thereof | |
| CN102079788B (en) | Method for detecting sulfanilamide medicine and special enzyme-linked immunoassay reagent kit thereof | |
| CN100397083C (en) | ELISA kit for detecting furazolidone metabolites and detection method thereof | |
| CN100501404C (en) | ELISA kit for detecting beta-stimulants and detection method thereof | |
| CN101571539B (en) | Elisa kit for detecting cephalo-type medicine and application thereof | |
| CN101241134B (en) | ELISA kit for detecting ractopamine residue and method of use thereof | |
| CN101021536B (en) | Enzyme-linked immunological kit for detecting neomycin drug and method | |
| CN101013130A (en) | Enzyme linked immunosorbent reagent casing for detecting furantoin metabolite and uses thereof | |
| CN101839918B (en) | Method for detecting spiramycin and special enzyme-linked immunoassay kit thereof | |
| CN1811436B (en) | Method for detecting Ennoxacin and its special enzyme-linked immune reagent kit | |
| CN101021535B (en) | Enzyme-linked immunalogical kit for detecting gentamicin medicine and method | |
| CN101013131B (en) | Furantoin metabolite enzyme linked immunosorbent analytical reagent casing and uses thereof | |
| CN101776685A (en) | Enzyme linked immunosorbent assay kit for detecting trimethoprim medicament and application thereof | |
| CN103421072A (en) | Dexamethasone semi-antigen, preparation method and applications thereof | |
| CN100489530C (en) | Method for assaying sulfaquinoxaline and special enzyme-linked immune reagent kit | |
| CN100501407C (en) | ELISA kit for detecting avermectins and detection method thereof | |
| CN100476439C (en) | ELISA kit for detecting quinolones in animal derived food | |
| CN100533148C (en) | Method for detecting tylosin and special enzyme-linked immune reagent kit thereof | |
| CN101358967A (en) | Method for detecting chlorpromazine and special ELISA kit thereof | |
| CN100582778C (en) | Method for detecting Ivermectin and special enzyme-linked immune reagent kit thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Effective date of registration: 20091009 Pledge (preservation): Pledge |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20100901 Granted publication date: 20090617 Pledgee: Zhongguancun Beijing science and technology Company limited by guarantee Pledgor: Beijing Wanger Biotechnology Co.,Ltd. Registration number: 2009110000623 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20090617 |