CN107188830A - A kind of pyrethrin pesticide haptens and its preparation method and application - Google Patents
A kind of pyrethrin pesticide haptens and its preparation method and application Download PDFInfo
- Publication number
- CN107188830A CN107188830A CN201710466703.7A CN201710466703A CN107188830A CN 107188830 A CN107188830 A CN 107188830A CN 201710466703 A CN201710466703 A CN 201710466703A CN 107188830 A CN107188830 A CN 107188830A
- Authority
- CN
- China
- Prior art keywords
- pyrethrin pesticide
- haptens
- monoclonal antibody
- pyrethrin
- pesticide
- 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.)
- Granted
Links
- VQXSOUPNOZTNAI-UHFFFAOYSA-N Pyrethrin I Natural products CC(=CC1CC1C(=O)OC2CC(=O)C(=C2C)CC=C/C=C)C VQXSOUPNOZTNAI-UHFFFAOYSA-N 0.000 title claims abstract description 53
- 239000000575 pesticide Substances 0.000 title claims abstract description 53
- HYJYGLGUBUDSLJ-UHFFFAOYSA-N pyrethrin Natural products CCC(=O)OC1CC(=C)C2CC3OC3(C)C2C2OC(=O)C(=C)C12 HYJYGLGUBUDSLJ-UHFFFAOYSA-N 0.000 title claims abstract description 53
- VJFUPGQZSXIULQ-XIGJTORUSA-N pyrethrin II Chemical compound CC1(C)[C@H](/C=C(\C)C(=O)OC)[C@H]1C(=O)O[C@@H]1C(C)=C(C\C=C/C=C)C(=O)C1 VJFUPGQZSXIULQ-XIGJTORUSA-N 0.000 title claims abstract description 53
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 150000002148 esters Chemical class 0.000 claims abstract description 14
- 241000723353 Chrysanthemum Species 0.000 claims abstract description 13
- 235000007516 Chrysanthemum Nutrition 0.000 claims abstract description 13
- 238000001514 detection method Methods 0.000 claims abstract description 10
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 6
- 230000028327 secretion Effects 0.000 claims abstract description 3
- 239000000427 antigen Substances 0.000 claims description 14
- 102000036639 antigens Human genes 0.000 claims description 14
- 108091007433 antigens Proteins 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 14
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 8
- 102000014914 Carrier Proteins Human genes 0.000 claims description 7
- 108010078791 Carrier Proteins Proteins 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 4
- CUDYUNNRMLWYTR-UHFFFAOYSA-N 1-amino-2,2-dimethylcyclopropane-1-carboxylic acid Chemical class CC1(C)CC1(N)C(O)=O CUDYUNNRMLWYTR-UHFFFAOYSA-N 0.000 claims description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 3
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000010828 elution Methods 0.000 claims description 3
- 239000012074 organic phase Substances 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229960001866 silicon dioxide Drugs 0.000 claims description 3
- 238000013517 stratification Methods 0.000 claims description 3
- 238000003809 water extraction Methods 0.000 claims description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims 3
- 210000004027 cell Anatomy 0.000 abstract description 19
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 abstract description 12
- OWZREIFADZCYQD-NSHGMRRFSA-N deltamethrin Chemical compound CC1(C)[C@@H](C=C(Br)Br)[C@H]1C(=O)O[C@H](C#N)C1=CC=CC(OC=2C=CC=CC=2)=C1 OWZREIFADZCYQD-NSHGMRRFSA-N 0.000 abstract description 5
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 abstract description 4
- 239000000460 chlorine Substances 0.000 abstract description 4
- 229910052801 chlorine Inorganic materials 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 abstract description 4
- 229960000490 permethrin Drugs 0.000 abstract description 4
- RLLPVAHGXHCWKJ-UHFFFAOYSA-N permethrin Chemical compound CC1(C)C(C=C(Cl)Cl)C1C(=O)OCC1=CC=CC(OC=2C=CC=CC=2)=C1 RLLPVAHGXHCWKJ-UHFFFAOYSA-N 0.000 abstract description 4
- 239000005936 tau-Fluvalinate Substances 0.000 abstract description 4
- INISTDXBRIBGOC-XMMISQBUSA-N tau-fluvalinate Chemical compound N([C@H](C(C)C)C(=O)OC(C#N)C=1C=C(OC=2C=CC=CC=2)C=CC=1)C1=CC=C(C(F)(F)F)C=C1Cl INISTDXBRIBGOC-XMMISQBUSA-N 0.000 abstract description 4
- ZXQYGBMAQZUVMI-RDDWSQKMSA-N (1S)-cis-(alphaR)-cyhalothrin Chemical compound CC1(C)[C@H](\C=C(/Cl)C(F)(F)F)[C@@H]1C(=O)O[C@@H](C#N)C1=CC=CC(OC=2C=CC=CC=2)=C1 ZXQYGBMAQZUVMI-RDDWSQKMSA-N 0.000 abstract description 3
- 239000005910 lambda-Cyhalothrin Substances 0.000 abstract description 3
- 229920000728 polyester Polymers 0.000 abstract description 3
- 239000007788 liquid Substances 0.000 description 21
- 241000699666 Mus <mouse, genus> Species 0.000 description 14
- 238000000034 method Methods 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 11
- 238000002965 ELISA Methods 0.000 description 9
- 239000011248 coating agent Substances 0.000 description 9
- 238000000576 coating method Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 206010003445 Ascites Diseases 0.000 description 8
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 239000012980 RPMI-1640 medium Substances 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 6
- 108010058846 Ovalbumin Proteins 0.000 description 5
- 229940092253 ovalbumin Drugs 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- -1 cyfloxylate Chemical compound 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 238000003018 immunoassay Methods 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 108010088751 Albumins Proteins 0.000 description 3
- 102000009027 Albumins Human genes 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 238000013019 agitation Methods 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 201000000050 myeloid neoplasm Diseases 0.000 description 3
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 210000004988 splenocyte Anatomy 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- 108010049003 Fibrinogen Proteins 0.000 description 2
- 102000008946 Fibrinogen Human genes 0.000 description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 108010071390 Serum Albumin Proteins 0.000 description 2
- 102000007562 Serum Albumin Human genes 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 150000008064 anhydrides Chemical class 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 229940012952 fibrinogen Drugs 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 235000021393 food security Nutrition 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 108060003552 hemocyanin Proteins 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229940005654 nitrite ion Drugs 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- AVMSWPWPYJVYKY-UHFFFAOYSA-N 2-Methylpropyl formate Chemical compound CC(C)COC=O AVMSWPWPYJVYKY-UHFFFAOYSA-N 0.000 description 1
- 241000256844 Apis mellifera Species 0.000 description 1
- SUZQPTKYCSADPP-UHFFFAOYSA-M C(CCCC)(=O)[O-].[Cl+] Chemical compound C(CCCC)(=O)[O-].[Cl+] SUZQPTKYCSADPP-UHFFFAOYSA-M 0.000 description 1
- 239000005635 Caprylic acid (CAS 124-07-2) Substances 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 101100203936 Mus musculus Srpra gene Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000393460 Peromyscus nasutus Species 0.000 description 1
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- MUUGCDGGIVCSDT-UHFFFAOYSA-N [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O Chemical compound [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O MUUGCDGGIVCSDT-UHFFFAOYSA-N 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 238000004500 asepsis Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- QQODLKZGRKWIFG-QSFXBCCZSA-N cyfluthrin Chemical compound CC1(C)[C@@H](C=C(Cl)Cl)[C@H]1C(=O)O[C@@H](C#N)C1=CC=C(F)C(OC=2C=CC=CC=2)=C1 QQODLKZGRKWIFG-QSFXBCCZSA-N 0.000 description 1
- 229960001591 cyfluthrin Drugs 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- GBIHOLCMZGAKNG-CGAIIQECSA-N flucythrinate Chemical compound O=C([C@@H](C(C)C)C=1C=CC(OC(F)F)=CC=1)OC(C#N)C(C=1)=CC=CC=1OC1=CC=CC=C1 GBIHOLCMZGAKNG-CGAIIQECSA-N 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000749 insecticidal effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 229960002446 octanoic acid Drugs 0.000 description 1
- 210000004279 orbit Anatomy 0.000 description 1
- 235000011837 pasties Nutrition 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000004454 trace mineral analysis Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C255/00—Carboxylic acid nitriles
- C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
- C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
- C07C255/38—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by esterified hydroxy groups
- C07C255/39—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by esterified hydroxy groups with hydroxy groups esterified by derivatives of 2,2-dimethylcyclopropane carboxylic acids, e.g. of chrysanthemumic acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The present invention relates to a kind of haptens and its preparation method and application, and in particular to a kind of pyrethrin pesticide haptens and its preparation method and application.The chrysanthemum esters monoclonal antibody hybridoma cell strain F 32 screened based on the pyrethrin pesticide haptens, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.13842.The chrysanthemum esters antibody titer of this cell line secretion reaches 2 × 105Cross reacting rate with decis is 100%, cross reacting rate with lambda-cyhalothrin is 50.9%, cross reacting rate with taufluvalinate is 103.6%, cross reacting rate with cyfloxylate is 70.7%, and the cross reacting rate with the polyester of chlorine penta is 74.4%, and the cross reacting rate with benzene Permethrin is 69.0%, condition being provided for the immune detection that remains pyrethrin pesticide, with actual application value more.
Description
Technical field
The present invention relates to a kind of haptens and its preparation method and application, and in particular to pyrethrin pesticide haptens and its system
Preparation Method and application, belong to food security technical field of immunological detection.
Background technology
Pyrethrin pesticide has that insecticidal spectrum is wide, drug effect rapid, to light and thermally stable, efficacy time is long the features such as, agricultural,
The fields such as forestry, public health are widely used, but it exists to the ecosystem and human health and endangered, especially to honeybee and aquatic
Biology has high toxicity.The World Health Organization (WHO) and FAO (Food and Agriculture Organization of the United Nation) (FAO) are residual on vegetables and fruits to pyrethrin pesticide
Stay and formulated strict limit standard, China has also formulated such agricultural chemicals limit standard (GB 2763-2016).
At present, the detection method of pyrethrin pesticide is mainly chromatography, including gas-chromatography (GC), high performance liquid chromatography
(HPLC), mass spectrography (MS) and gas chromatography-mass spectrography (GC-MS) or Liquid Chromatography-Mass Spectrometry (HPLC-MS) etc..
Instrument analytical method has the advantages that sensitivity is high, but expense is costly, and technical requirements are high, does not apply to on-site quick screening a large amount of
Sample.
Immunoassay can make up all of above shortcoming, immunoassay be with specific recognition between antigen and antibody and
Trace analysis methods based on invertibity association reaction, due to low with high specificity, sensitivity height, simple, quick, expense
The advantages of with being screened suitable for live batch samples, it is used widely in the analysis field such as environment and food security.Build
The key of the immunoassay method of vertical micromolecular compound is can to prepare to have high-affinity and height to micromolecular compound
Antibody selective, and the synthesis of haptens is the basis for preparing high-quality antibody.The key of hapten design is as much as possible
Retain the feature structure of former test substance, and introduce suitable linking arm and the activity with carrier protein couplet in position
Group.The introducing position of the linking arm of different structure, different coupling methods or linking arm, the antibody for all influenceing subsequently to prepare
Sensitivity and specificity.In addition, as the same clan of family compound, a variety of pyrethrin pesticides are with the cumulative toxicity in environment in food
Serious implicit risk, therefore synthesizing efficient valency, the general antigen with chrysanthemum esters structural features, preparing, there is wide spectrum to know
Other antibody turns into the emphasis that pyrethrin pesticide immunoassay is studied.
The content of the invention
It is an object of the invention to provide a kind of pyrethrin pesticide haptens and preparation method thereof, and the haptens and people
The monoclonal antibody that the conjugate that seralbumin coupling is obtained is prepared as immunogen immune animal.Verified through test data,
The monoclonal antibody of preparation is to decis, lambda-cyhalothrin, taufluvalinate, cyfloxylate, chlorine valerate, benzene Permethrin
6 kinds of pyrethrin pesticides have preferable detection sensitivity, can be for setting up the immunology detection side remained pyrethrin pesticide more
Method.
The purpose of the present invention is achieved by the following technical programs：
A kind of pyrethrin pesticide haptens, its molecular structural formula is：
The preparation method of above-mentioned pyrethrin pesticide haptens, comprises the following steps：
Take [cyano group-(3- phenoxy groups)-methyl] 2 bromacetate 1.0g, plus DMF dissolving, plus bicarbonate
Sodium 0.72g, is sufficiently stirred for after mixing, plus 1- amino -2,2- dimethyl cyclopropane carboxylic acids 0.45g, 60 DEG C of stirring reaction 4h；Stop
Reaction, the ethyl acetate that adds water extraction, stratification, organic phase anhydrous sodium sulfate drying is evaporated, upper silicagel column, petroleum ether/acetic acid
Ethyl ester=1:1 elution separation, obtains cyclopropane carboxyl chrysanthemum ester hapten 1.1g, as pyrethrin pesticide haptens, yield
97.35%.
Application of the above-mentioned pyrethrin pesticide haptens in immune detection, is specifically included by the pyrethrin pesticide haptens
The pyrethrin pesticide artificial antigen obtained with carrier protein couplet, and animal system is immunized by the pyrethrin pesticide artificial antigen of gained
Standby monoclonal antibody.
Wherein described carrier protein be mouse serum albumin, thyroprotein, bovine serum albumin(BSA), albumin rabbit serum,
Human serum albumins, ovalbumin, hemocyanin or fibrinogen.
The monoclonal antibody is thin for CGMCC No.13842 chrysanthemum esters monoclonal antibody hybridoma by deposit number
Born of the same parents' strain F-3-2 secretions are produced, many residual immune detections applied to pyrethrin pesticide.
The beneficial effects of the present invention are：
(1) the pyrethrin pesticide haptens that the present invention is provided both farthest had remained the chemistry of pyrethrin pesticide parent nucleus
Structure, transformed further through chemical synthesis introduce can with protein molecule-COOH, synthetic method is simple, purity, yield
It is higher；
(2) monoclonal antibody that animal obtains is immunized in the pyrethrin pesticide artificial antigen that the present invention is prepared, and potency reaches
To 2 × 105, illustrate that the antigen of the invention synthesized is intactly remained during having excellent immunogenicity, synthetic antigen
The prototype structure of hapten molecule；
(3) monoclonal antibody specificity that the present invention is provided is good, and the cross reacting rate with decis is 100%, with chlorine
The cross reacting rate of flucythrinate is 50.9%, and the cross reacting rate with taufluvalinate is 103.6%, with cyfloxylate
Cross reacting rate is 70.7%, and the cross reacting rate with the polyester of chlorine penta is 74.4%, and the cross reacting rate with benzene Permethrin is
69.0%, it can be applied to many residual immune detections of pyrethrin pesticide.
Biological material specimens preservation：One plant of chrysanthemum esters monoclonal antibody hybridoma cell strain, has been preserved in China Microbiological
Culture presevation administration committee common micro-organisms center, abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3,
Institute of Microorganism, Academia Sinica, preservation date 2017 year 05 month 05 day, deposit number is CGMCC No.13842.
Brief description of the drawings
Fig. 1 pyrethrin pesticide hapten synthesis route maps
Fig. 2 pyrethrin pesticide haptens hydrogen nuclear magnetic resonance spectrograms
Fig. 3 pyrethrin pesticide competitive ELISA canonical plottings
Embodiment
The present invention is further described with specific embodiment below in conjunction with the accompanying drawings, it should be appreciated that these embodiments are only used for
Illustrate the present invention, and be not limited to the scope of the present invention.Experimental method used in following embodiments unless otherwise specified,
It is conventional method；Used material, reagent etc., are the reagent and material commercially obtained unless otherwise specified.
Embodiment 1：Pyrethrin pesticide hapten synthesis and identification
The synthetic route of pyrethrin pesticide haptens is as shown in Figure 1.
Take [cyano group-(3- phenoxy groups)-methyl] 2 bromacetate 1.0g, plus DMF dissolving, plus bicarbonate
Sodium 0.72g, is sufficiently stirred for after mixing, plus 1- amino -2,2- dimethyl cyclopropane carboxylic acids 0.45g, 60 DEG C of stirring reaction 4h；Stop
Reaction, the ethyl acetate that adds water extraction, stratification, organic phase anhydrous sodium sulfate drying is evaporated, upper silicagel column, petroleum ether/acetic acid
Ethyl ester=1:1 elution separation, obtains cyclopropane carboxyl chrysanthemum ester hapten 1.1g, as pyrethrin pesticide haptens, yield
97.35%.
Take above-mentioned haptens to be identified through proton nmr spectra, as a result see accompanying drawing 2.1H-NMR(CDCl3,300MHz)δ：11.00
(1H, s, COOH), 7.41 (2H, dd, ArH), 7.17 (1H, dd, ArH), 7.14 (2H, dd, ArH), 7.08 (2H, dd, ArH),
7.34 (2H, dd, ArH), 6.25 (1H, s, CH), 3.51 (2H, s, CH2), 2.0 (1H, s, NH), 0.94 (6H, s, CH3), 0.57
(1H, d, CH), 0.33 (1H, d, CH).In collection of illustrative plates, chemical shift δ=11.0 for carboxyl hydrogen resonance absorbing peak in compound,
7.08~7.41 be the absworption peak of hydrogen on phenyl ring, and 0.94 is the absworption peak of two methyl hydrogens, and 0.33~0.57 is on three-membered ring
Two hydrogen absworption peaks, with reference to other absworption peaks, it was demonstrated that hapten synthesis success.
Embodiment 2：Pyrethrin pesticide artificial antigen synthesizes and identified
It is even with mixed anhydride method and human serum albumins (HSA) using the carboxyl of pyrethrin pesticide haptens as avtive spot
Connection, prepares immunogene；It is coupled with carbodlimide method and ovalbumin (OVA), prepares coating antigen.
1. mixed anhydride method prepares immunogene
Cyclopropane carboxyl chrysanthemum ester hapten 24mg, plus dimethyl sulfoxide (DMSO) 0.5mL dissolvings, plus the μ L of triethylamine 20 are taken, stirring is mixed
Even, the μ L of chlorination iso-butyl formate 20 stir 30min, obtain haptens activating solution A liquid；Take HSA 100mg, plus 0.1mol/L
Phosphate buffer dissolves, and obtains B liquid；A liquid is added drop-wise in B liquid dropwise, 4h is stirred at room temperature, 0.01mol/L phosphate delays
Fliud flushing is dialysed 3 days, liquid is changed daily 3 times, is obtained immunogene, -20 DEG C save backup.
2. carbodlimide method prepares coating antigen
Take cyclopropane carboxyl chrysanthemum ester hapten 10mg, plus DMF (DMF) 0.3mL dissolvings, plus the Asia of carbon two
Amine (EDC) 13mg, stirs 30min, plus n-hydroxysuccinimide (NHS) 13mg, stirs 30min, obtain haptens activating solution A
Liquid；Take OVA 100mg, plus 0.1mol/L phosphate buffer to dissolve, obtain B liquid；A liquid is added drop-wise in B liquid dropwise, room temperature
4h is stirred, 0.01mol/L phosphate buffer is dialysed 3 days, liquid changed daily 3 times, obtain coating antigen, -20 DEG C save backup.
Mouse serum albumin, thyroprotein, bovine serum albumin(BSA), the white egg of rabbit anteserum can also be used in the above method
In vain, hemocyanin or fibrinogen.
3. artificial antigen is identified
By carrier protein, pyrethrin pesticide haptens, pyrethrin pesticide hapten-carrier protein conjugate with pH's 7.4
PBS is made into 0.5mg/mL solution, is returned to zero with 0.01mol/L pH7.4 PBS, with ultraviolet specrophotometer wavelength 200~
Scanned in the range of 800nm, obtain carrier protein, pyrethrin pesticide haptens, pyrethrin pesticide hapten-carrier protein conjugate
Absorption curve, and calculate its combine than.As a result find, different absorption curves occurs in three, show that pyrethrin pesticide half is anti-
Former successful with carrier protein couplet, haptens and HSA combination ratio are (17~21):1, and OVA combination ratio is (15~20):
1。
Embodiment 3：Preparation and purification of monoclonal antibody and CHARACTERISTICS IDENTIFICATION
1. animal immune
Take 6~8 weeks female Balb/c mouse 10 (being divided into two groups of A and B, every group 5) of health, initial immunity Freund
Freund's complete adjuvant emulsifies collare dorsal sc multi-point injection, and every mouse immune dosage is 200 μ g immunogenes；Booster immunization is every afterwards
Two weeks subcutaneous multi-point injections of nape part once, emulsification incomplete Freund's adjuvant；Last time is immune to be replaced using physiological saline
Incomplete Freund's adjuvant, using intraperitoneal injection, injection dosage and above identical several times.Specifically immune step is shown in Table 1.
The mouse immune program of table 1
For the third time, four times, 7d after booster immunization, blood is taken to mouse docking, and ELISA method determines mice serum potency, tool
Body step is as follows：
(1) coating antigen is done 1 with 0.05mol/L pH9.6 carbonate buffer solution:1000 dilutions, the coating per the μ L of hole 100
ELISA Plate, 37 DEG C of incubation 2h, is got rid of coating buffer, is washed 1 time, patted dry with PBST；
(2) 150 μ L confining liquids are added per hole, 37 DEG C of reaction 2h hypsokinesis deblocking liquid are patted dry；
(3) 50 μ L are added per hole with the antiserum of PBS doubling dilutions, 25 DEG C of reaction 30min hypsokinesis dereaction liquid, with PBST
Washing 3~5 times, per minor tick 30s, is patted dry；
(4) ELIAS secondary antibody (1 of PBS dilutions is added:1000) 100 μ L/ holes, 25 DEG C of reaction 30min, 3~5 are washed with PBST
It is secondary, per minor tick 30s, pat dry；
(5) substrate nitrite ion A liquid and each 50 μ L of B liquid are added per hole, 25 DEG C of lucifuges react 15min, 50 μ are added per hole
L2mol/L H2SO4Solution terminating reaction；
(6) ELIASA determines OD value of the wavelength in 450nm, with sample well OD450Extension rate close to 1 is used as the positive
The potency of serum.
2. cell fusion
(1) prepared by feeder cells：Disconnected neck puts to death 8~10 week old Balb/c mouse, is immersed in 5min in 75% alcohol, immediately
It is put into superclean bench, belly is put in plate or be fixed on dissection plate upward.Mouse web portion skin is played with ophthalmic tweezers sub-folder
Skin, an osculum is cut with scissors, notes being sure not to break peritonaeum, in order to avoid peritoneal fluid outflow and pollution.Then scissors is used to both sides up and down
Blunt separation is done, peritonaeum is fully exposed.Peritonaeum is wiped with cotton ball soaked in alcohol to sterilize.5mL RPMI-1640 bases are drawn with syringe
Nutrient solution, injects mouse peritoneal, gently draws back syringe, rock mouse leg and afterbody several times.Abdominal cavity is drawn back with original annotation emitter
Interior liquid, injects centrifuge tube.So operate 3~4 times repeatedly.1000r/min centrifuges 10min, abandons supernatant.It is complete with 20~50mL
Cell is resuspended in nutrient solution, and 100 μ L/ holes are added drop-wise to culture plate, put incubator standby.
(2) prepared by splenocyte：3d after booster immunization, takes immune Balb/c mouse, dislocates and put to death after eye socket blood sampling, 75%
Spleen is taken after being sterilized in alcohol, connective tissue is removed, prepares splenocyte suspension, be transferred in 50mL centrifuge tubes, plus RPMI-1640
To 30mL, 1500~2000r/min centrifugation 5min, supernatant, plus RPMI-1640 to 30mL are abandoned, is counted stand-by.
(3) prepared by myeloma cell：(the viable count for taking 3 bottles of growth conditions good>95%) myeloma cell, by it is complete
Full blow down, be transferred in 50mL centrifuge tubes, plus RPMI-1640 to 30mL, 1500~2000r/min centrifugation 5min, supernatant is abandoned, plus
RPMI-1640 to 30mL, is counted stand-by.
(4) mixing with cells：Splenocyte:Myeloma cell=8:1, mixing, 1500~2000r/min centrifugations 5min.
(5) cell fusion：By the cell mixed centrifugation, dry supernatant, sedimentation cell block bullet into pasty state, puts 37 DEG C of water
Bath, adds 1mL fusion agents in 1min, and fusion agent is polyethylene glycol (PEG) 4000, acts on 2min, and is gently mixed cell,
The PEG nutrient solutions of 20mL serum-frees are added in subsequent 4min, 1000r/min centrifugation 10min abandon supernatant.It is complete with 20~50mL
Cell is resuspended in nutrient solution, and cover plant, per the μ L of hole 100, is put in incubator in 96 porocyte culture plates containing feeder cells.
3. cell line is screened
When the 1/2~1/3 of cell length to bottom hole, you can carry out antibody test.Using ELISA method to there is hybridoma thin
The culture hole of intracellular growth is screened, and screening is in two steps：The first step first filters out positive cell hole, second step with indirect ELISA
It is standard items from a variety of pyrethrin pesticides, inhibition measure is carried out to positive cell with indirect competitive ELISA.Select to many
Plant pyrethrin pesticide standard items and be respectively provided with the hole preferably suppressed, be subcloned, entered with same method using limiting dilution assay
Row detection.In triplicate, you can obtain the cell line F-3-2 of energy stably excreting chrysanthemum esters monoclonal antibody.The cell line in
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, ground within 05 05th, 2017
Location：Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCC
No.13842。
4. it is prepared by ascites
Atoleine is injected into 6~8 weeks Balb/c mouse, 500 μ L/ are only.By the hybridization in exponential phase after 10 days
Oncocyte CGMCC No.13842 are collected with RPMI-1640 basal mediums, and with blood counting chamber and microscopic counting, cell is dense
Degree is 1.0 × 106~1.5 × 106In the range of individual/mL.Every mouse 0.5mL hybridoma CGMCC No.13842 is expelled to
Abdominal cavity.Notice that observation mouse web portion after one week expands, ascites is gathered in mouse peritoneal with asepsis injector, every one to two days
Collection once, is so repeatedly gathered until mouse natural death repeatedly.5000r/min centrifuges 5min at 4 DEG C, collects supernatant, and
Remove the fat and albuminous membranae of ascites upper strata floating.
5. antibody purification
Monoclonal antibody is comprised the following steps that using octanoic acid-ammonium sulfate method purifying：
(1) ascites is taken out into thaw at RT from -20 DEG C of refrigerators.Ascites is filtered with double-layer filter paper, it is preliminary remove fatty piece, it is thin
Born of the same parents' fragment and other impurities.12000r/min centrifuges 15min, takes supernatant, abandons precipitation.Precise volume ascites volume；
The acetate buffer magnetic agitation of the ascites of (2) 1 parts of volumes and 3 parts of volumes is mixed, and pH is adjusted with 2mol/L HCl
To 4.5~4.8；
(3) caprylic acid is slowly added under magnetic agitation, 1mL ascites adds 33 μ L caprylic acids, adds rear room temperature magnetic agitation
30min, rearmounted 4 DEG C of standings 2h；
(4) 12000r/min centrifuges 5min, takes supernatant, and filtrate is collected in double-layer filter paper filtering；
(5) filtrate volume is measured, the 0.1mol/L pH7.4 of 1/10 volume PBS is added, with 2mol/L NaOH (records
NaOH volumes) adjust pH to 7.4；
(6) by supernatant ice bath precooling, plus ammonium sulfate solids are to 0.277g/mL, stirring while adding, and in being added in 30min,
Put 4 DEG C overnight；
(7) 12000r/min centrifuges 15min, abandons supernatant.Precipitation is dissolved with the 0.01mol/L PBS of certain volume.Use PB
Dialysis changes 0.01mol/L PBSs two days two days later, collects dialyzate, and 12000r/min centrifugation 15min take supernatant, put -20
DEG C preserve.
6. antibody titer is determined
Indirect ELISA method is used to determine the potency of antibody for 1:200000.Specific steps refer to above-mentioned 1. animal immune
In content.
7. antibody cross reaction is determined
Determined, concretely comprised the following steps using indirect competitive ELISA method：
(1) coating antigen is done 1 with 0.05mol/L pH9.6 carbonate buffer solution:1000 dilutions, the coating per the μ L of hole 100
ELISA Plate, 37 DEG C of incubation 2h, is got rid of coating buffer, is washed 1 time, patted dry with PBST；
(2) 150 μ L confining liquids are added per hole, 37 DEG C of reaction 2h hypsokinesis deblocking liquid are patted dry；
(3) first added per hole pyrethrin pesticide standard working solution that 50 μ L are serially diluted (concentration is respectively 0,1,3,9,27,
81 μ g/L), then add 50 μ L and do 1 with PBS:The monoclonal antibody of 200000 dilutions, 25 DEG C of reaction 30min hypsokinesis dereactions
Liquid, is washed 3~5 times with PBST, per minor tick 30s, is patted dry；
(4) ELIAS secondary antibody (1 of PBS dilutions is added:1000) 100 μ L/ holes, 25 DEG C of reaction 30min, 3~5 are washed with PBST
It is secondary, per minor tick 30s, pat dry；
(5) substrate nitrite ion A liquid and each 50 μ L of B liquid are added per hole, 25 DEG C of lucifuges react 15min, 50 μ are added per hole
L2mol/L H2SO4Solution terminating reaction；
(6) ELIASA determines OD value of the wavelength in 450nm.
Using the logarithm value of pyrethrin pesticide concentration as abscissa, with percentage absorbance (each concentration standards OD values with not
The percentage of the quasi- sample wells OD values of mark-on) standard curve is drawn for ordinate, see accompanying drawing 3.With each percentage absorbance of curve 50%
Mass concentration (IC50) cross reacting rate is calculated as follows, 2 are the results are shown in Table, cross reacting rate is smaller, antibody specificity is higher.
In formula：
Si--- cross reacting rate, %；
Y --- the IC of decis standard working solution curve50Value；
Z --- the IC of other pyrethrin pesticide standard working solution curves50Value.
The cross reaction measurement result of table 2
Note："-" represents curve torsional deformation, it is impossible to for calculating.
As shown in Table 2, pyrethrin pesticide monoclonal antibody is to decis, lambda-cyhalothrin, taufluvalinate, cyfluthrin
Chrysanthemum ester, the polyester of chlorine penta, benzene Permethrin have cross reaction, illustrate that the antibody can be used for the immune inspection of many residuals of pyrethrin pesticide
Survey.
Claims (6)
1. a kind of pyrethrin pesticide haptens, it is characterised in that molecular structural formula is：
2. the preparation method of the pyrethrin pesticide haptens described in claim 1, it is characterised in that comprise the following steps：
Take [cyano group-(3- phenoxy groups)-methyl] 2 bromacetate 1.0g, plus DMF dissolving, plus sodium acid carbonate
0.72g, is sufficiently stirred for after mixing, plus 1- amino -2,2- dimethyl cyclopropane carboxylic acids 0.45g, 60 DEG C of stirring reaction 4h；Stop anti-
Should, the ethyl acetate that adds water extraction, stratification, organic phase anhydrous sodium sulfate drying is evaporated, upper silicagel column, petroleum ether/acetic acid second
Ester=1:1 elution separation, obtains cyclopropane carboxyl chrysanthemum ester hapten, as pyrethrin pesticide haptens.
3. manually resisted as the pyrethrin pesticide that the pyrethrin pesticide haptens described in claim 1 is obtained with carrier protein couplet
It is former.
4. monoclonal antibody prepared by animal is immunized as the pyrethrin pesticide artificial antigen described in claim 3.
5. monoclonal antibody as claimed in claim 4, it is characterised in that it by deposit number is CGMCC No.13842 that it, which is,
The strain F-3-2 secretions of chrysanthemum esters monoclonal antibody hybridoma cell are produced.
6. application of the monoclonal antibody in immune detection is remained pyrethrin pesticide described in claim 5 more.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710466703.7A CN107188830B (en) | 2017-06-20 | 2017-06-20 | A kind of pyrethrin pesticide haptens and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710466703.7A CN107188830B (en) | 2017-06-20 | 2017-06-20 | A kind of pyrethrin pesticide haptens and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107188830A true CN107188830A (en) | 2017-09-22 |
| CN107188830B CN107188830B (en) | 2019-07-26 |
Family
ID=59878225
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710466703.7A Active CN107188830B (en) | 2017-06-20 | 2017-06-20 | A kind of pyrethrin pesticide haptens and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107188830B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106543163A (en) * | 2016-11-08 | 2017-03-29 | 佛山市飞时达新材料科技有限公司 | A kind of haptenic preparation method and applications of fluoxastrobin |
| CN108535474A (en) * | 2018-03-16 | 2018-09-14 | 北方工业大学 | Time-resolved fluorescence test strip for detecting permethrin and application thereof |
| CN108931646A (en) * | 2018-05-30 | 2018-12-04 | 中国烟草总公司郑州烟草研究院 | A kind of test strips and its preparation method and application detecting safrole |
| CN111812316A (en) * | 2020-06-03 | 2020-10-23 | 北京勤邦生物技术有限公司 | Application of fenpropathrin artificial antigen in enzyme linked immunosorbent assay kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434652A (en) * | 2008-12-05 | 2009-05-20 | 中国农业科学院油料作物研究所 | Pyrethroid pesticide artificial antigen, antibody and preparation thereof |
| CN102901811A (en) * | 2012-10-20 | 2013-01-30 | 江南大学 | Pyrethroid hapten design based on computer molecular simulation technique and application |
| CN105732788A (en) * | 2016-03-21 | 2016-07-06 | 贵州勤邦食品安全科学技术有限公司 | Preparation method and application of efficient cyhalothrin hapten |
-
2017
- 2017-06-20 CN CN201710466703.7A patent/CN107188830B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434652A (en) * | 2008-12-05 | 2009-05-20 | 中国农业科学院油料作物研究所 | Pyrethroid pesticide artificial antigen, antibody and preparation thereof |
| CN102901811A (en) * | 2012-10-20 | 2013-01-30 | 江南大学 | Pyrethroid hapten design based on computer molecular simulation technique and application |
| CN105732788A (en) * | 2016-03-21 | 2016-07-06 | 贵州勤邦食品安全科学技术有限公司 | Preparation method and application of efficient cyhalothrin hapten |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106543163A (en) * | 2016-11-08 | 2017-03-29 | 佛山市飞时达新材料科技有限公司 | A kind of haptenic preparation method and applications of fluoxastrobin |
| CN108535474A (en) * | 2018-03-16 | 2018-09-14 | 北方工业大学 | Time-resolved fluorescence test strip for detecting permethrin and application thereof |
| CN108931646A (en) * | 2018-05-30 | 2018-12-04 | 中国烟草总公司郑州烟草研究院 | A kind of test strips and its preparation method and application detecting safrole |
| CN108931646B (en) * | 2018-05-30 | 2021-04-27 | 中国烟草总公司郑州烟草研究院 | Test strip for detecting safrole and preparation method and application thereof |
| CN111812316A (en) * | 2020-06-03 | 2020-10-23 | 北京勤邦生物技术有限公司 | Application of fenpropathrin artificial antigen in enzyme linked immunosorbent assay kit |
| CN111812316B (en) * | 2020-06-03 | 2022-11-18 | 北京勤邦生物技术有限公司 | Application of fenpropathrin artificial antigen in enzyme linked immunosorbent assay kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107188830B (en) | 2019-07-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107188830B (en) | A kind of pyrethrin pesticide haptens and its preparation method and application | |
| CN1317302C (en) | 2, 4-dichlorophen artificial antigen, and its preparing method and use | |
| CN110498766B (en) | Fluazinam hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN106093381B (en) | Rod method toxin rod method phenol monomethyl ether colloidal gold immuno-chromatography test paper strip | |
| CN111333570B (en) | Haloxyfop hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN101962358A (en) | Ciprofloxacin hapten, artificial antigen and antibody and preparation method and application thereof | |
| CN101182356A (en) | Clenbuterol complete antigen and method for preparing monoclonal antibody thereof | |
| CN110642793A (en) | Azoxystrobin hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN106866568A (en) | Furaxone metabolite AOZ derivatizations haptens, the preparation method and applications of artificial antigen | |
| CN110627726B (en) | Prochloraz hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN106872706A (en) | A kind of FPIA method for detecting carbaryl | |
| CN107012128A (en) | A kind of hybridoma cell strain for secreting aspergillus flavus resisting toxin B1 monoclonal antibodies and its application | |
| CN103288965B (en) | Polychlorobiphenyl monoclonal antibody preparation method | |
| CN103539853A (en) | Specific antibody for resisting atrazine herbicide | |
| CN106831498A (en) | Furacilin metabolite SEM derivatizations haptens, the preparation method and applications of artificial antigen | |
| CN111943882B (en) | Pythium hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN100570323C (en) | A kind of ractopamine column and its production and use | |
| CN105754954B (en) | One plant of imidacloprid monoclonal antibody hybridoma cell strain YH5 and its application | |
| CN102924601B (en) | Method for preparing ractopamine monoclonal antibodies | |
| CN101962368A (en) | Cyanurate hapten, artificial antigen and antibody, and preparation method and applications thereof | |
| CN1966523A (en) | Dursban monoclonal antibody preparing method | |
| CN107988168A (en) | Anti-cocaine monoclonal antibody, the cell line and preparation method for secreting the antibody | |
| CN101498728B (en) | Reagent kit for detecting o-allyl phenol and its special antibody | |
| CN107083368A (en) | A kind of hybridoma cell strain for secreting anti-total aflatoxina monoclonal antibody and its application | |
| CN107513522A (en) | A kind of hybridoma cell strain for secreting anti-ochratoxin monoclonal antibody and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |