CN113979994A - Imidacloprid hapten, complete antigen, synthesis and application thereof - Google Patents
Imidacloprid hapten, complete antigen, synthesis and application thereof Download PDFInfo
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- CN113979994A CN113979994A CN202111618682.9A CN202111618682A CN113979994A CN 113979994 A CN113979994 A CN 113979994A CN 202111618682 A CN202111618682 A CN 202111618682A CN 113979994 A CN113979994 A CN 113979994A
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- imidacloprid
- hapten
- complete antigen
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- 239000005906 Imidacloprid Substances 0.000 title claims abstract description 325
- YWTYJOPNNQFBPC-UHFFFAOYSA-N imidacloprid Chemical compound [O-][N+](=O)\N=C1/NCCN1CC1=CC=C(Cl)N=C1 YWTYJOPNNQFBPC-UHFFFAOYSA-N 0.000 title claims abstract description 325
- 229940056881 imidacloprid Drugs 0.000 title claims abstract description 325
- 239000000427 antigen Substances 0.000 title claims abstract description 104
- 102000036639 antigens Human genes 0.000 title claims abstract description 104
- 108091007433 antigens Proteins 0.000 title claims abstract description 104
- 230000015572 biosynthetic process Effects 0.000 title abstract description 10
- 238000003786 synthesis reaction Methods 0.000 title abstract description 10
- 239000000243 solution Substances 0.000 claims description 79
- 150000002148 esters Chemical class 0.000 claims description 48
- -1 Azolin-1-yl hexanoic acid Chemical compound 0.000 claims description 33
- 238000005303 weighing Methods 0.000 claims description 32
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 23
- 239000007853 buffer solution Substances 0.000 claims description 22
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
- 102000014914 Carrier Proteins Human genes 0.000 claims description 14
- 108010078791 Carrier Proteins Proteins 0.000 claims description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 claims description 12
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 11
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 claims description 11
- 239000012312 sodium hydride Substances 0.000 claims description 11
- 229910000104 sodium hydride Inorganic materials 0.000 claims description 11
- UOUFRTFWWBCVPV-UHFFFAOYSA-N tert-butyl 4-(2,4-dioxo-1H-thieno[3,2-d]pyrimidin-3-yl)piperidine-1-carboxylate Chemical class CC(C)(C)OC(=O)N1CCC(CC1)n1c(=O)[nH]c2ccsc2c1=O UOUFRTFWWBCVPV-UHFFFAOYSA-N 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 10
- YOETUEMZNOLGDB-UHFFFAOYSA-N 2-methylpropyl carbonochloridate Chemical compound CC(C)COC(Cl)=O YOETUEMZNOLGDB-UHFFFAOYSA-N 0.000 claims description 9
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 9
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims description 8
- 230000002194 synthesizing effect Effects 0.000 claims description 8
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 238000010511 deprotection reaction Methods 0.000 claims description 6
- HQABUPZFAYXKJW-UHFFFAOYSA-N butan-1-amine Chemical compound CCCCN HQABUPZFAYXKJW-UHFFFAOYSA-N 0.000 claims description 4
- 235000019260 propionic acid Nutrition 0.000 claims description 4
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 claims description 4
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 4
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 claims description 3
- 230000002378 acidificating effect Effects 0.000 claims description 3
- 230000008878 coupling Effects 0.000 claims description 3
- 238000010168 coupling process Methods 0.000 claims description 3
- 238000005859 coupling reaction Methods 0.000 claims description 3
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 claims description 3
- 229940005605 valeric acid Drugs 0.000 claims description 3
- JOHWNGGYGAVMGU-UHFFFAOYSA-N trifluorochlorine Chemical compound FCl(F)F JOHWNGGYGAVMGU-UHFFFAOYSA-N 0.000 claims 1
- 238000010189 synthetic method Methods 0.000 abstract description 23
- 238000001308 synthesis method Methods 0.000 abstract description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 61
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 48
- 229940098773 bovine serum albumin Drugs 0.000 description 35
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 30
- 238000003756 stirring Methods 0.000 description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 239000002994 raw material Substances 0.000 description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 238000001514 detection method Methods 0.000 description 13
- 238000008157 ELISA kit Methods 0.000 description 10
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 239000012074 organic phase Substances 0.000 description 10
- 239000000126 substance Substances 0.000 description 9
- 238000005406 washing Methods 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 8
- 238000001035 drying Methods 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 101710145634 Antigen 1 Proteins 0.000 description 6
- 241000283707 Capra Species 0.000 description 6
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 6
- 101710123661 Venom allergen 5 Proteins 0.000 description 6
- 230000002163 immunogen Effects 0.000 description 6
- 239000011148 porous material Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 241000607479 Yersinia pestis Species 0.000 description 5
- 239000012295 chemical reaction liquid Substances 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 5
- 238000001816 cooling Methods 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 5
- 238000004108 freeze drying Methods 0.000 description 5
- 239000002480 mineral oil Substances 0.000 description 5
- 235000010446 mineral oil Nutrition 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 101710135378 pH 6 antigen Proteins 0.000 description 5
- 238000010898 silica gel chromatography Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine Chemical compound CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 description 5
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 4
- UAWMVMPAYRWUFX-UHFFFAOYSA-N 6-Chloronicotinic acid Chemical compound OC(=O)C1=CC=C(Cl)N=C1 UAWMVMPAYRWUFX-UHFFFAOYSA-N 0.000 description 4
- 239000012043 crude product Substances 0.000 description 4
- 229960002715 nicotine Drugs 0.000 description 4
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 4
- 239000000575 pesticide Substances 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- 239000007858 starting material Substances 0.000 description 4
- WCXDHFDTOYPNIE-RIYZIHGNSA-N (E)-acetamiprid Chemical compound N#C/N=C(\C)N(C)CC1=CC=C(Cl)N=C1 WCXDHFDTOYPNIE-RIYZIHGNSA-N 0.000 description 3
- 239000005875 Acetamiprid Substances 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000005138 cryopreservation Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 239000002917 insecticide Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000008055 phosphate buffer solution Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000037029 cross reaction Effects 0.000 description 2
- 230000009260 cross reactivity Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 230000036046 immunoreaction Effects 0.000 description 2
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000011895 specific detection Methods 0.000 description 2
- 239000012089 stop solution Substances 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical group C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- WWZKQHOCKIZLMA-UHFFFAOYSA-N Caprylic acid Natural products CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- GONOPSZTUGRENK-UHFFFAOYSA-N benzyl(trichloro)silane Chemical compound Cl[Si](Cl)(Cl)CC1=CC=CC=C1 GONOPSZTUGRENK-UHFFFAOYSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229940078916 carbamide peroxide Drugs 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
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- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
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- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N n-hexanoic acid Natural products CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- BNWCETAHAJSBFG-UHFFFAOYSA-N tert-butyl 2-bromoacetate Chemical compound CC(C)(C)OC(=O)CBr BNWCETAHAJSBFG-UHFFFAOYSA-N 0.000 description 1
- RMWVUWLBLWBQDS-UHFFFAOYSA-N tert-butyl 3-bromopropanoate Chemical compound CC(C)(C)OC(=O)CCBr RMWVUWLBLWBQDS-UHFFFAOYSA-N 0.000 description 1
- HJEZRYIJNHAIGY-UHFFFAOYSA-N tert-butyl 4-bromobutanoate Chemical compound CC(C)(C)OC(=O)CCCBr HJEZRYIJNHAIGY-UHFFFAOYSA-N 0.000 description 1
- PSELCIMQRLODQE-UHFFFAOYSA-N tert-butyl 6-bromohexanoate Chemical compound CC(C)(C)OC(=O)CCCCCBr PSELCIMQRLODQE-UHFFFAOYSA-N 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
-
- 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
- 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
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/10—Insecticides
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Hematology (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides an imidacloprid hapten, a complete antigen and synthesis and application thereof, and comprises an imidacloprid hapten. The invention relates to an imidacloprid hapten and a synthesis method thereof, the synthesis method of the imidacloprid hapten is simple and efficient, compared with the prior art, the synthesis cost of the imidacloprid hapten can be saved, and the synthesis efficiency of the imidacloprid hapten is improved; the invention also relates to an imidacloprid complete antigen and two synthetic methods, wherein the imidacloprid complete antigen basically reserves the characteristic structure of imidacloprid.
Description
Technical Field
The invention relates to an imidacloprid hapten and complete antigen as well as synthesis and application thereof, belonging to the technical field of imidacloprid hapten and complete antigen.
Background
Imidacloprid (IMI) is a nitryl methylene systemic insecticide, belongs to chloronicotinyl insecticides, is also called neonicotinyl insecticides, has the characteristics of broad spectrum, high efficiency, low toxicity, low residue and the like, and has multiple effects of contact poisoning, stomach toxicity, systemic absorption and the like. After the pests contact the pesticide, normal conduction of central nerves is blocked, so that the pests die paralyzed, the pesticide can be mainly used for preventing and controlling the pests with piercing-sucking mouthparts, and the pests are not easy to generate resistance.
At present, pesticides containing imidacloprid are registered and used in more than 120 countries in the global scope, the application range relates to more than 60 crops such as grains, vegetables, fruit trees, tea leaves and the like, the control objects relate to more than 50 pests, the mass use of the pesticides containing imidacloprid has certain influence on the environment, on the one hand, a large amount of residues exist in the plants, and the residues can finally enter soil or human bodies and harm the environment or the human health.
At present, in order to research the relevant mechanism that imidacloprid antigen can cause immune response in animals, methods for preparing imidacloprid hapten and imidacloprid complete antigen by most experimenters have certain limitations, for example, an invention patent with application publication number CN109917126A discloses a preparation method of imidacloprid hapten, an invention patent with application publication number CN107805241A discloses a preparation method of imidacloprid hapten and complete antigen, the preparation methods for preparing imidacloprid hapten and complete antigen by two invention patents have long reaction time and complicated reaction conditions, and involve the steps of oil bath heating, repeated extraction, column loading and the like, so the invention relates to a novel imidacloprid hapten, complete antigen and relevant synthesis method, which can be applied to immunoassay technology and avoid the limitations.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide an imidacloprid hapten, a complete antigen and synthesis and application thereof, so as to solve the problems in the background technology.
In order to realize the first purpose, the invention provides an imidacloprid hapten, and the invention is realized by the following technical scheme.
An imidacloprid hapten with the structural formula as follows:
wherein n denotes the number of methylene groups and n = 1-6.
In order to realize the second purpose, the invention provides a method for synthesizing imidacloprid hapten.
A synthetic method of imidacloprid hapten is used for preparing the imidacloprid hapten and comprises the following steps:
s1, preparing imidacloprid intermediate
Dissolving imidacloprid in DMF, adding sodium hydride and halogenated carboxylic acid tert-butyl ester to obtain imidacloprid N-substituted carboxylic acid tert-butyl ester intermediate;
s2, preparing imidacloprid hapten
Under the acidic condition and the deprotection reagent environment, the imidacloprid N-substituted carboxylic acid tert-butyl ester intermediate is subjected to a tert-butyl removal reaction to obtain the imidacloprid hapten.
Preferably, the molar ratio of the imidacloprid to the sodium hydride to the tert-butyl halocarboxylate is 1:1-1.1: 1-3.
Preferably, the deprotection reagent is one of hydrochloric acid, trifluorohydrochloric acid and hydrobromic acid.
Preferably, the imidacloprid hapten is imidacloprid hapten derivatives such as 2- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminimimidazolin-1-yl ] acetic acid, 3- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminmidazolin-1-yl ] propionic acid, 4- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminmidazolin-1-yl ] butyric acid, 5- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminmidazolin-1-yl ] valeric acid, 6- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminmidazolin-1-yl ] valeric acid Imidazolidin-1-yl hexanoic acid.
In order to achieve the third purpose, the invention provides an imidacloprid complete antigen, and the invention is realized by the following technical scheme.
An imidacloprid complete antigen is prepared by coupling the imidacloprid hapten and carrier protein, and the structural formula is as follows:
wherein,
represents the carrier protein, n = 1-6.
In order to realize the fourth purpose, the invention provides a synthetic method of an imidacloprid complete antigen, and the method is realized by the following technical scheme.
A synthetic method of imidacloprid complete antigen is used for preparing the imidacloprid complete antigen and comprises the following steps:
s1, preparing imidacloprid hapten active ester solution
Dissolving imidacloprid hapten with a DMF solution, adding NHS and EDC hydrochloride, and reacting at room temperature for 16-24h to obtain an imidacloprid hapten active ester solution;
s2, preparing imidacloprid complete antigen
Weighing carrier protein, dissolving in 0.1M borate buffer solution, adjusting pH =8.7, slowly dropwise adding the imidacloprid hapten active ester solution obtained in the step S1, reacting for 24h at room temperature, and dialyzing with PBS buffer solution for three days at 4 ℃ to obtain the imidacloprid complete antigen.
In order to achieve the fifth object, the invention provides another synthetic method of imidacloprid complete antigen, and the invention is realized by the following technical scheme.
A synthetic method of imidacloprid complete antigen is used for preparing the imidacloprid complete antigen and comprises the following steps:
s1, preparing imidacloprid hapten active ester solution
Dissolving imidacloprid hapten with a DMF solution, adding n-butylamine and isobutyl chloroformate, and reacting for 1h to obtain an imidacloprid hapten active ester solution;
s2, preparing imidacloprid complete antigen
Weighing carrier protein, dissolving in 0.1M borate buffer solution, adjusting pH =8.7, slowly dropwise adding the imidacloprid hapten active ester solution obtained in the step S1, reacting for 24h at room temperature, and dialyzing with PBS buffer solution for three days at 4 ℃ to obtain the imidacloprid complete antigen.
In order to achieve the sixth object, the invention provides a compound, namely an imidacloprid specific antibody, and the invention is achieved by the following technical scheme.
An imidacloprid specific antibody is prepared from the imidacloprid hapten or the imidacloprid complete antigen.
In order to achieve the seventh object, the invention provides an application of an imidacloprid hapten, and the application is achieved through the following technical scheme.
An application of the imidacloprid hapten is to apply the imidacloprid hapten to the detection of imidacloprid or the preparation of imidacloprid detection products.
The invention has the beneficial effects that:
(1) the invention relates to an imidacloprid hapten and a synthesis method thereof, the synthesis method of the imidacloprid hapten is simple and efficient, compared with the prior art, the synthesis cost of the imidacloprid hapten can be saved, and the synthesis efficiency of the imidacloprid hapten is improved.
(2) The invention relates to an imidacloprid complete antigen and two synthetic methods, wherein the imidacloprid complete antigen basically reserves the characteristic structure of imidacloprid.
(3) The invention relates to an imidacloprid specific antibody, which is prepared by immunizing animals with an imidacloprid artificial antigen, can generate specific immunoreaction with imidacloprid and is a high-efficiency monoclonal or polyclonal antibody capable of generating the specific immunoreaction.
(4) The invention relates to an application of imidacloprid hapten, wherein the application of the imidacloprid hapten is an enzyme linked immunosorbent assay kit, qualitative and quantitative analysis and detection of imidacloprid can be performed, the imidacloprid hapten can also be used for immunoassay technology, and the imidacloprid hapten can be efficiently and accurately detected.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained by combining the specific implementation modes, and the experimental methods in the embodiments are all conventional methods if no special description is provided.
An imidacloprid hapten with the structural formula as follows:
wherein n denotes the number of methylene groups and n = 1-6.
The synthetic route of the imidacloprid hapten is as follows:
the synthetic method of the imidacloprid hapten comprises the following steps:
s1, preparing imidacloprid intermediate
Dissolving imidacloprid in DMF, adding sodium hydride and halogenated carboxylic acid tert-butyl ester to obtain imidacloprid N-substituted carboxylic acid tert-butyl ester intermediate, wherein the molar ratio of imidacloprid to sodium hydride to halogenated carboxylic acid tert-butyl ester is 1:1-1.1: 1-3;
s2, preparing imidacloprid hapten
Under the acidic condition and the environment of a deprotection reagent, carrying out a tert-butyl removal reaction on an imidacloprid N-substituted carboxylic acid tert-butyl ester intermediate to obtain an imidacloprid hapten, wherein the deprotection reagent is one of hydrochloric acid, trifluorohydrochloric acid and hydrobromic acid, and the imidacloprid hapten is an imidacloprid hapten derivative 2- [3- [ (6-chloropyridyl-3-yl) methyl ] -2-nitroiminimimidazolin-1-yl ] acetic acid, 3- [3- [ (6-chloropyridyl-3-yl) methyl ] -2-nitroiminmidazolin-1-yl ] propionic acid, 4- [3- [ (6-chloropyridyl-3-yl) methyl ] -2-nitroiminmidazolin-1-yl ] butyric acid, a derivative thereof, namely a derivative thereof, 5- [3- [ (6-chloropyridyl-3-yl) methyl ] -2-nitroiminoimidazol-1-yl ] pentanoic acid and 6- [3- [ (6-chloropyridyl-3-yl) methyl ] -2-nitroiminoimidazol-1-yl ] hexanoic acid.
An imidacloprid complete antigen is prepared by coupling imidacloprid hapten and carrier protein, and has a structural formula as follows:
wherein,
represents the carrier protein, n = 1-6.
The synthetic route of the imidacloprid complete antigen is as follows:
the imidacloprid complete antigen has two synthesis methods, wherein the first method comprises the following steps:
s1, preparing imidacloprid hapten active ester solution
Dissolving imidacloprid hapten with a DMF solution, adding NHS and EDC hydrochloride, and reacting at room temperature for 16-24h to obtain an imidacloprid hapten active ester solution, wherein the molar ratio of the imidacloprid hapten to the NHS to the EDC hydrochloride is 1:1-2: 1-2;
s2, preparing imidacloprid complete antigen
Weighing carrier protein, dissolving in 0.1M borate buffer solution, adjusting pH =8.7, slowly adding dropwise the imidacloprid hapten active ester solution obtained in step S1, reacting for 24h at room temperature, dialyzing for three days with PBS buffer solution at 4 ℃ to obtain imidacloprid complete antigen, wherein the carrier protein can be one of BSA (bovine serum albumin), KLH (keyhole limpet hemocyanin), LPH (hemocyanin), OVA (egg serum albumin) and HAS (human serum albumin), and other carrier proteins can be selected according to needs.
The second method for synthesizing imidacloprid complete antigen comprises the following steps:
s1, preparing imidacloprid hapten active ester solution
Dissolving imidacloprid hapten with a DMF (dimethyl formamide) solution, adding n-butylamine and isobutyl chloroformate, and reacting for 1h to obtain an imidacloprid hapten active ester solution, wherein the molar ratio of the imidacloprid hapten to the n-butylamine to the isobutyl chloroformate is 1:1.2: 1.2;
s2, preparing imidacloprid complete antigen
Weighing carrier protein, dissolving in 0.1M borate buffer solution, adjusting pH =8.7, slowly dropwise adding the imidacloprid hapten active ester solution obtained in the step S1, reacting for 24h at room temperature, and dialyzing with PBS buffer solution for three days at 4 ℃ to obtain the imidacloprid complete antigen.
Example 1
The imidacloprid hapten synthesized in this example is 2- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminoimidazol-1-yl ] acetic acid (hapten 1).
The embodiment relates to an imidacloprid hapten and a synthetic method thereof:
s1, preparing imidacloprid intermediate
Weighing 5g of imidacloprid, dissolving in 20ml of DMF, stirring, adding 0.469g of sodium hydride (60 percent in mineral oil) in batches, stirring for 30min at room temperature, dropwise adding 7.6344g of tert-butyl 2-bromoacetate, reacting and stirring overnight, slowly pouring the reaction liquid into 150ml of ice saturated ammonium chloride solution, extracting for 3 times by using 150ml of methyl tert-ether, combining organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, and vacuum concentrating to obtain an intermediate crude product;
s2, preparing imidacloprid hapten
Weighing 0.2g of the intermediate obtained in the step S1, slowly adding 50ml of 2M hydrochloric acid, stirring at room temperature overnight, adjusting the pH to be =6.0 by 1M NaOH, extracting for 3 times by 50ml of ethyl acetate, combining organic phases, drying by anhydrous sodium sulfate, concentrating, and performing silica gel column chromatography (ethyl acetate: methanol = 10: 1) to obtain a white solid 2- [3- [ (6-chloropyridyl-3-yl) methyl ] -2-nitroiminmidazolin-1-yl ] acetic acid, namely the imidacloprid hapten 1.
The imidacloprid hapten synthesized in this example is 2- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminoimidazol-1-yl ] acetic acid (hapten 1), n =2, and the structural formula is as follows:
example 2
The imidacloprid hapten synthesized in this example is 3- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminoimidazolin-1-yl ] propionic acid (hapten 2).
The embodiment relates to an imidacloprid hapten and a synthetic method thereof:
s1, preparing imidacloprid intermediate
Weighing 5g of imidacloprid, dissolving in 20ml of DMF, fully stirring, adding 0.469g of sodium hydride (60 percent in mineral oil) in batches, stirring for 30min at room temperature, dripping 8.183g of tert-butyl 3-bromopropionate, reacting and stirring overnight, slowly pouring the reaction liquid into 150ml of ice saturated ammonium chloride solution, extracting for 3 times by using 150ml of methyl tert-ether, combining organic phases, washing by using saturated saline solution, drying by using anhydrous sodium sulfate, and carrying out vacuum concentration to obtain a target product crude product, namely an intermediate;
s2, preparing imidacloprid hapten
0.2g of the intermediate obtained in step S1 was weighed, 50ml of 2M hydrochloric acid was added, the mixture was stirred at room temperature overnight, pH =6.0 was adjusted with 1M NaOH, the mixture was extracted 3 times with 50ml of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated and subjected to silica gel column chromatography (ethyl acetate: methanol = 10: 1) to obtain 3- [3- [ (6-chloropyridin-3-yl) methyl ] -2-nitroiminoimidazol-1-yl ] propionic acid as a white solid, i.e., imidacloprid hapten 2.
The imidacloprid hapten synthesized in this example is 3- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminmidazolin-1-yl ] propionic acid (hapten 2), n =3, and the structural formula is as follows:
example 3
The imidacloprid hapten synthesized in this example is 4- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminoimidazolin-1-yl ] butyric acid (hapten 3).
The embodiment relates to an imidacloprid hapten and a synthetic method thereof:
s1, preparing imidacloprid intermediate
Weighing 5g of imidacloprid, dissolving in 20ml of DMF, stirring, adding 0.469g of sodium hydride (60 percent in mineral oil) in batches, stirring for 30min at room temperature, dripping 8.732g of tert-butyl 4-bromobutyrate, reacting and stirring overnight, slowly pouring the reaction liquid into 150ml of ice saturated ammonium chloride solution, extracting for 3 times by using 150ml of methyl tert-ether, combining organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, and vacuum concentrating an intermediate crude product, namely an intermediate;
s2, preparing imidacloprid hapten
0.2g of the intermediate obtained in step S1 was weighed, 50ml of 2M hydrochloric acid was added, stirred at room temperature overnight, adjusted to pH =6.0 with 1M NaOH, extracted 3 times with 50ml of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography (ethyl acetate: methanol = 10: 1) to obtain 4- [3- [ (6-chloropyridin-3-yl) methyl ] -2-nitroiminoimidazol-1-yl ] butyric acid as a white solid, that is, imidacloprid hapten 3.
The imidacloprid hapten synthesized in this example is 4- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminoimidazolin-1-yl ] butyric acid (hapten 3), n =4, and the structural formula is as follows:
example 4
The imidacloprid hapten synthesized in this example was 5- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminoimidazoiin-1-yl ] pentanoic acid (hapten 4).
The embodiment relates to an imidacloprid hapten and a synthetic method thereof:
s1, preparing imidacloprid intermediate
Weighing 5g of imidacloprid, dissolving in 20ml of DMF, stirring, adding 0.469g of sodium hydride (60 percent in mineral oil) in batches, stirring for 30min at room temperature, dripping 8.183g of 5-tert-butyl bromovalerate, reacting and stirring overnight, slowly pouring the reaction liquid into 150ml of ice saturated ammonium chloride solution, extracting for 3 times by using 150ml of methyl tert-ether, combining organic phases, washing by using saturated saline solution, drying by using anhydrous sodium sulfate, and performing vacuum concentration on a crude intermediate product, namely an intermediate;
s2, preparing imidacloprid hapten
0.2g of the intermediate obtained in step S1 was weighed, 50ml of 2M hydrochloric acid was added, stirred at room temperature overnight, adjusted to pH =6.0 with 1M NaOH, extracted 3 times with 50ml of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated and subjected to silica gel column chromatography (ethyl acetate: methanol = 10: 1) to obtain 5- [3- [ (6-chloropyridin-3-yl) methyl ] -2-nitroiminoimidazol-1-yl ] pentanoic acid as a white solid, i.e., imidacloprid hapten 4.
The imidacloprid hapten synthesized in this example is 5- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminoimidazoiin-1-yl ] pentanoic acid (hapten 4), n =5, and the structural formula is:
example 5
The imidacloprid hapten synthesized in this example is 6- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminoimidazolin-1-yl ] hexanoic acid (hapten 5).
The embodiment relates to an imidacloprid hapten and a synthetic method thereof:
s1, preparing imidacloprid intermediate
Weighing 5g of imidacloprid, dissolving in 20ml of DMF, stirring, adding 0.469g of sodium hydride (60 percent in mineral oil) in batches, stirring for 30min at room temperature, dripping 9.83g of tert-butyl 6-bromohexanoate, reacting and stirring overnight, slowly pouring the reaction liquid into 150ml of ice saturated ammonium chloride solution, extracting for 3 times by using 150ml of methyl tert-ether, combining organic phases, washing by using saturated saline solution, drying by using anhydrous sodium sulfate, and vacuum-concentrating an intermediate crude product, namely an intermediate;
s2, preparing imidacloprid hapten
0.2g of the intermediate obtained in step S1 was weighed, 50ml of 2M hydrochloric acid was added, stirred at room temperature overnight, adjusted to pH =6.0 with 1M NaOH, extracted 3 times with 50ml of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated and subjected to silica gel column chromatography (ethyl acetate: methanol = 10: 1) to obtain 5- [3- [ (6-chloropyridin-3-yl) methyl ] -2-nitroiminoimidazol-1-yl ] pentanoic acid as a white solid, i.e., imidacloprid hapten 5.
The imidacloprid hapten synthesized in this example is 6- [3- [ (6-chloropyrid-3-yl) methyl ] -2-nitroiminoimidazolin-1-yl ] hexanoic acid (hapten 5), n =6, and the structural formula is as follows:
example 6
The imidacloprid complete antigen synthesized in this example was synthesized as imidacloprid complete antigen 1 using the imidacloprid hapten (hapten 1) synthesized in example 1 as a raw material.
The embodiment relates to an imidacloprid complete antigen and a synthetic method thereof:
s1, preparing imidacloprid hapten active ester solution
Weighing 10mg of hapten 1 prepared in example 1, dissolving in 200. mu.l of DMF, adding 4.4mg of NHS and 12.2mg of EDC hydrochloride, and stirring overnight at room temperature to obtain an active ester solution;
s2, preparing imidacloprid BSA complete antigen
Weighing 50mg of bovine serum albumin BSA, dissolving in 4ml of borate buffer solution, slowly dripping active ester solution into the BSA solution, standing overnight at room temperature, dialyzing for 3 days at 4 ℃, taking out dialysate, freeze-drying to obtain imidacloprid BSA complete antigen 1, and storing at-40 ℃ for later use.
The imidacloprid complete antigen synthesized in this example takes the imidacloprid hapten (hapten 1) synthesized in example 1 as a raw material to synthesize the imidacloprid complete antigen 1, wherein n =2, and the structural formula is as follows:
example 7
The imidacloprid complete antigen synthesized in this example was synthesized by using the imidacloprid hapten (hapten 1) synthesized in example 1 as a raw material and by another method different from example 6.
The embodiment relates to an imidacloprid complete antigen and a synthetic method thereof:
s1, preparing imidacloprid hapten active ester solution
Weighing 13.8mg of hapten 1 prepared in example 1, dissolving the hapten in 250 mu l of DMF, cooling the mixture in an ice bath, sequentially adding 10.3 mu l of tri-n-butylamine and 5.9 mu l of isobutyl chloroformate, and continuously reacting the mixture in the ice bath for 1 hour to obtain an imidacloprid hapten active ester solution;
s2, preparing imidacloprid BSA complete antigen
Weighing 25mg of bovine serum albumin BSA, dissolving the bovine serum albumin BSA in 2ml of carbonate buffer solution (pH = 9.6), dropwise adding an imidacloprid hapten active ester solution, reacting for 4 hours at room temperature in a dark place, and dialyzing the reaction solution in a refrigerator at 4 ℃ for three days to obtain an imidacloprid BSA artificial complete antigen 1.
Example 8
The imidacloprid complete antigen synthesized in this example was synthesized as imidacloprid complete antigen 2 using the imidacloprid hapten (hapten 2) synthesized in example 2 as a raw material.
The embodiment relates to an imidacloprid complete antigen and a synthetic method thereof:
s1, preparing imidacloprid hapten active ester solution
Weighing 10mg of hapten 2 prepared in example 2, dissolving in 200 mul of DMF, adding 12.0mg of EDC hydrochloride and 4.2mg of NHS, and stirring overnight at room temperature to obtain an active ester solution;
s2, preparing imidacloprid BSA complete antigen
Weighing 50mg of bovine serum albumin BSA, dissolving in 4ml of borate buffer solution, slowly dropwise adding the active ester solution into the BSA solution, standing overnight at room temperature, dialyzing for 3 days, taking out the dialysate after completion, freeze-drying to obtain imidacloprid BSA complete antigen 2, and storing at-40 ℃.
The imidacloprid complete antigen synthesized in this example takes the imidacloprid hapten (hapten 2) synthesized in example 2 as a raw material to synthesize the imidacloprid complete antigen 2, wherein n =3, and the structural formula is as follows:
example 9
The imidacloprid complete antigen synthesized in this example was synthesized by using the imidacloprid hapten (hapten 2) synthesized in example 2 as a raw material and by another method different from example 8.
The embodiment relates to an imidacloprid complete antigen and a synthetic method thereof:
s1, preparing imidacloprid hapten active ester solution
Weighing 7.2mg of hapten 2, dissolving in 200 mul of DMF, cooling in ice bath, sequentially adding 5.2 mul of tri-n-butylamine and 2.9 mul of isobutyl chloroformate, and continuing to react in ice bath for 1h to obtain an imidacloprid hapten active ester solution;
s2, preparing imidacloprid BSA complete antigen
Weighing 13mg of bovine serum albumin BSA, dissolving in 1ml of carbonate buffer solution (PH = 9.6), dripping imidacloprid hapten active ester solution, reacting at room temperature in a dark place for 4h, and dialyzing the reaction solution in a refrigerator at 4 ℃ for three days to obtain imidacloprid BSA artificial complete antigen 2.
Example 10
The imidacloprid complete antigen synthesized in this example was synthesized as imidacloprid complete antigen 3 using the imidacloprid hapten (hapten 3) synthesized in example 3 as a raw material.
The embodiment relates to an imidacloprid complete antigen and a synthetic method thereof:
s1, preparing imidacloprid hapten active ester solution
Weighing 10mg of hapten 3, dissolving in 200 mul of DMF, adding 12.0mg of EDC hydrochloride and 4.0mg of NHS, and stirring overnight at room temperature to obtain an active ester solution;
s2, preparing imidacloprid BSA complete antigen
Weighing 50mg of bovine serum albumin BSA, dissolving the bovine serum albumin BSA in 4ml of borate buffer solution, slowly dripping the active ester solution into the BSA solution, standing overnight at room temperature, dialyzing for 3 days, taking out the dialysate after the completion, freeze-drying to obtain imidacloprid BSA complete antigen 3, and storing at-40 ℃ for later use.
The imidacloprid complete antigen synthesized in this example takes the imidacloprid hapten (hapten 3) synthesized in example 3 as a raw material to synthesize the imidacloprid complete antigen 3, wherein n =4, and the structural formula is as follows:
example 11
The imidacloprid complete antigen synthesized in this example was synthesized by using the imidacloprid hapten (hapten 3) synthesized in example 3 as a raw material and by another method different from example 10.
The embodiment relates to an imidacloprid complete antigen and a synthetic method thereof:
s1, preparing imidacloprid hapten active ester solution
Weighing 7.5mg of hapten 3, dissolving in 200 mul of DMF, cooling in ice bath, sequentially adding 5.2 mul of tri-n-butylamine and 2.9 mul of isobutyl chloroformate, and continuing to react in ice bath for 1h to obtain an imidacloprid hapten active ester solution;
s2, preparing imidacloprid BSA complete antigen
Weighing 15mg of bovine serum albumin BSA, dissolving the bovine serum albumin BSA in 1ml of carbonate buffer solution (PH = 9.6), dropwise adding an imidacloprid hapten active ester solution, reacting for 4 hours at room temperature in a dark place, and dialyzing the reaction solution in a refrigerator at 4 ℃ for three days to obtain an imidacloprid BSA artificial complete antigen 3.
Example 12
The imidacloprid complete antigen synthesized in this example was synthesized as imidacloprid complete antigen 4 using the imidacloprid hapten (hapten 4) synthesized in example 4 as a raw material.
The embodiment relates to an imidacloprid complete antigen and a synthetic method thereof:
s1, preparing imidacloprid hapten active ester solution
Weighing 10mg of hapten 4, dissolving in 200 mul of DMF, adding 11.0mg of EDC hydrochloride and 3.8mg of NHS, and stirring overnight at room temperature to obtain an active ester solution;
s2, preparing imidacloprid BSA complete antigen
Weighing 50mg of bovine serum albumin BSA, dissolving in 4ml of borate buffer solution, slowly dropping an active ester solution into the BSA solution, standing overnight at room temperature, dialyzing for 3 days, taking out a dialysate after completion, freeze-drying to obtain imidacloprid BSA complete antigen 4, and storing at-40 ℃.
The imidacloprid complete antigen synthesized in this example takes the imidacloprid hapten (hapten 4) synthesized in example 4 as a raw material to synthesize the imidacloprid complete antigen 4, wherein n =5, and the structural formula is as follows:
example 13
The imidacloprid complete antigen synthesized in this example was synthesized by using the imidacloprid hapten (hapten 4) synthesized in example 4 as a raw material and by another method different from example 12.
The embodiment relates to an imidacloprid complete antigen and a synthetic method thereof:
s1, preparing imidacloprid hapten active ester solution
Weighing 8mg of hapten 4 prepared in example 4, dissolving the hapten 4 in 200 mu l of DMF, cooling the mixture in an ice bath, sequentially adding 5.3 mu l of tri-n-butylamine and 2.7 mu l of isobutyl chloroformate, and continuing to react in the ice bath for 1 hour to obtain an imidacloprid hapten active ester solution;
s2, preparing imidacloprid BSA complete antigen
Weighing 15mg of bovine serum albumin BSA, dissolving in 1ml of carbonate buffer solution (PH = 9.6), dripping imidacloprid hapten active ester solution, reacting at room temperature in a dark place for 4h, and dialyzing the reaction solution in a refrigerator at 4 ℃ for three days to obtain imidacloprid BSA artificial complete antigen 4.
Example 14
The imidacloprid complete antigen synthesized in this example was synthesized as imidacloprid complete antigen 5 using the imidacloprid hapten (hapten 5) synthesized in example 5 as a raw material.
The embodiment relates to an imidacloprid complete antigen and a synthetic method thereof:
s1, preparing imidacloprid hapten active ester solution
Weighing 10mg of hapten 5, dissolving in 200 mul of DMF, adding 10.3mg of EDC hydrochloride and 3.4mg of NHS, and stirring overnight at room temperature to obtain an active ester solution;
s2, preparing imidacloprid BSA complete antigen
Weighing 50mg of bovine serum albumin BSA, dissolving in 4ml of borate buffer solution, slowly dropping an active ester solution into the BSA solution, standing overnight at room temperature, dialyzing for 3 days, taking out a dialysate after completion, freeze-drying to obtain imidacloprid BSA complete antigen 5, and storing at-40 ℃.
The imidacloprid complete antigen synthesized in this example takes the imidacloprid hapten (hapten 5) synthesized in example 5 as a raw material to synthesize the imidacloprid complete antigen 5, wherein n =6, and the structural formula is as follows:
example 15
The imidacloprid complete antigen synthesized in this example was synthesized by using the imidacloprid hapten (hapten 5) synthesized in example 5 as a raw material and by another method different from example 14.
The embodiment relates to an imidacloprid complete antigen and a synthetic method thereof:
s1, preparing imidacloprid hapten active ester solution
Weighing 8.1mg of hapten 5, dissolving in 200 mul of DMF, cooling in ice bath, sequentially adding 5.4 mul of tri-n-butylamine and 2.9 mul of isobutyl chloroformate, and continuing to react in ice bath for 1h to obtain an imidacloprid hapten active ester solution;
s2, preparing imidacloprid BSA complete antigen
Weighing 15mg of bovine serum albumin BSA, dissolving in 1ml of carbonate buffer solution (PH = 9.6), dripping imidacloprid hapten active ester solution, reacting at room temperature in a dark place for 4h, and dialyzing the reaction solution in a refrigerator at 4 ℃ for three days to obtain imidacloprid BSA artificial complete antigen 5.
Example 16
This example relates to a compound and its synthesis, which is an imidacloprid monoclonal antibody.
1. Animal immunization
The immunogen obtained in example 6 was injected into Balb/c (8-week) mice at an immunization dose of 80. mu.g/mouse, and antiserum was generated.
2. Cell fusion and cloning
After the serum determination result of the mouse is higher, spleen cells of the mouse are taken and fused with FO myeloma cells according to the quantitative ratio of 7:1, indirect competition ELISA is adopted to determine cell supernatant, positive holes are screened, and the positive holes are cloned by using a limiting dilution method until a hybridoma cell strain secreting the imidacloprid monoclonal antibody is obtained.
3. Cell cryopreservation and recovery
Preparation of monoclonal hybridoma cell line into 1 × 10 frozen stock solution6And (3) preserving the cell suspension per mL in liquid nitrogen for a long time, taking out the cryopreservation tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast thawing, centrifuging to remove the cryopreservation liquid, and transferring the tube into a culture bottle for culture.
4. Production and purification of monoclonal antibodies
The method of inducing monoclonal antibody in animal body was adopted, ascites was collected after 7 days and purified by protein G purification column to obtain monoclonal antibody 1 prepared from complete antigen 1 prepared in example 6 as a raw material and stored at-20 ℃.
Example 17
Using the complete antigen 2 obtained in example 8 as a starting material, monoclonal antibody 2 was obtained by the same production method as in example 16 and stored at-20 ℃.
Example 18
Using the complete antigen 3 obtained in example 10 as a starting material, a monoclonal antibody 3 was obtained by the same production method as in example 16 and stored at-20 ℃.
Example 19
Using the complete antigen 4 obtained in example 12 as a starting material, a monoclonal antibody 4 was prepared by the same method as in example 16 and stored at-20 ℃.
Example 20
Using the complete antigen 5 obtained in example 14 as a starting material, a monoclonal antibody 5 was prepared by the same method as in example 16 and stored at-20 ℃.
Example 21
The embodiment relates to application of an imidacloprid hapten, and the imidacloprid hapten is applied to detection of imidacloprid by an enzyme labeling kit.
1. Preparation of enzyme-labeled Secondary antibody
A goat is used as an immune animal, the imidacloprid monoclonal antibody 1 prepared in the example 16 is used as an immunogen to immunize a goat without a pathogen to obtain an imidacloprid antibody 1, and the imidacloprid antibody 1 is coupled with horseradish peroxidase (HRP) to obtain an enzyme-labeled secondary antibody.
2. Preparation of ELISA plates
The imidacloprid BSA complete antigen prepared in example 6 is diluted to 2 μ g/mL by using a coating buffer (carbonate with pH = 9.6), 100 μ l of the complete antigen is added into each hole, the complete antigen is incubated for 16h at 4 ℃ in the dark, the liquid in the holes is poured off, the complete antigen is washed for 1 time by using a washing solution, the complete antigen is kept standing for 30s and patted dry, then 200 μ l of a sealing solution is added into each hole, the complete antigen is incubated for 2h at 37 ℃ in the dark, the liquid in the holes is poured off and patted dry, and the complete antigen is stored in an aluminum film vacuum seal mode after being dried.
3. Construction of enzyme linked immunosorbent assay kit for detecting imidacloprid
An enzyme linked immunosorbent assay kit for detecting imidacloprid is constructed, and comprises the following components:
(1) an enzyme label plate coated with imidacloprid coupled antigen;
(2) 6 bottles of imidacloprid standard substance concentrated solution (concentrated solution solvent methanol) with the concentration of 0mg/L, 1mg/L, 3mg/L, 9mg/L, 27mg/L and 81mg/L respectively, and when in use, the imidacloprid standard substance concentrated solution is diluted into standard substance solution by 0.02mol of phosphate buffer solution (pH7.2) 9:1, and the concentration after dilution is 0mg/L, 0.1mg/L, 0.3mg/L, 0.9mg/L, 2.7mg/L and 8.1mg/L respectively;
(3) an imidacloprid anti-antibody labeled by horseradish peroxidase;
(4) the substrate color development liquid consists of a liquid A and a liquid B, wherein the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(5) the stop solution is 2mol/L sulfuric acid;
(6) the washing solution has a pH value of 7.2, and contains 0.01% of tween-20, 3g/L of sodium azide preservative and 0.01mol/L of phosphate buffer solution, wherein the percentages are weight volume percentages;
(7) the compound solution is phosphate buffer solution with pH value of 7.0 and 0.02mol/L, and the percentage is weight volume percentage.
4. Detection of precision of imidacloprid kit
Adding 50 mul of standard solution/sample into corresponding micropores, adding 50 mul of antibody into the micropores, finally adding 50 mul of enzyme-labeled secondary antibody into the micropores, slightly oscillating and uniformly mixing, placing the cover plate in a dark environment at 25 ℃ for reaction for 45min, carefully uncovering the cover plate, drying liquid in the pores, using 250 mul of washing working solution into the pores, fully washing for 4-5 times at intervals of 10s each time, patting the pores with absorbent paper (the bubbles which are not cleared after patting the pores can be slightly punctured by an unused gun head), adding 50 mul of substrate solution A into the pores, adding 50 mul of substrate solution B into the pores, slightly oscillating and uniformly mixing, placing the cover plate in a dark environment at 25 ℃ for color development for 15 min. Adding 50 mul/hole of stop solution, lightly shaking and mixing, setting the detection wavelength of an enzyme-labeling instrument to be 450nm and the reference wavelength to be 620nm, and measuring the OD value of each hole.
And the percent absorbance of the standard substance or the sample is equal to the absorbance of the standard substance or the sample divided by the absorbance of the first standard substance (0 standard), and then multiplied by 100 percent to obtain the percent absorbance of the standard substance or the sample, the percent absorbance of the standard substance is taken as a vertical coordinate, the logarithm of the concentration (mg/L) of the imidacloprid standard substance is taken as a horizontal coordinate, a standard curve graph is drawn, the percent absorbance of the sample is substituted into a standard curve, the concentration corresponding to the sample is read from the standard curve, and the dilution multiple corresponding to the concentration is multiplied to obtain the actual concentration of the imidacloprid in the sample.
Example 22
Immunity was performed on a pathogen-free goat using the imidacloprid monoclonal antibody 2 prepared in example 17 as an immunogen, an ELISA plate was prepared using the imidacloprid BSA complete antigen prepared in example 8 to obtain the imidacloprid antibody 2, and imidacloprid detection was performed by the same method as in example 21.
Example 23
Immunity was performed on a pathogen-free goat using the imidacloprid monoclonal antibody 3 prepared in example 18 as an immunogen, an ELISA plate was prepared using the imidacloprid BSA complete antigen prepared in example 10 to obtain the imidacloprid antibody 3, and imidacloprid detection was performed by the same method as in example 21.
Example 24
The imidacloprid monoclonal antibody 4 prepared in example 19 was used as an immunogen to immunize a pathogen-free goat, an elisa plate was prepared using the imidacloprid BSA complete antigen prepared in example 12 to obtain the imidacloprid antibody 4, and imidacloprid detection was performed by the same method as in example 21.
Example 25
Immunity was performed on a pathogen-free goat using the imidacloprid monoclonal antibody 5 prepared in example 20 as an immunogen, an ELISA plate was prepared using the imidacloprid BSA complete antigen prepared in example 14 to obtain the imidacloprid antibody 5, and imidacloprid detection was performed by the same method as in example 21.
Comparative example 1
The imidacloprid hapten described in example 1 and the imidacloprid BSA complete antigen described in example 3, with the authorization publication number CN107805241B, were selected.
Test example 1 specific detection
Specific detection tests were carried out using the imidacloprid enzyme linked immunosorbent assay kit prepared in examples 21-25 and comparative example 1.
Respectively configuring different concentration standard curves of the acetamiprid, the nicotine and the 6-chloronicotinic acid, namely 0mg/L, 0.1mg/L, 0.3mg/L, 0.9mg/L, 2.7mg/L and 8.1mg/L, detecting by using an imidacloprid kit, and obtaining IC50 values of the acetamiprid, the nicotine and the 6-chloronicotinic acid according to absorbance values, wherein the calculation mode of the cross reaction rate is as follows:
the results of measuring the cross-reactivity rates of acetamiprid, nicotine and 6-chloronicotinic acid are shown in Table 1:
TABLE 1 Cross-reactivity
The above tests show that, compared with comparative example 1, the cross-reaction rates of imidacloprid, nicotine and 6-chloronicotinic acid measured by the five imidacloprid enzyme-linked immunosorbent assay kits prepared in examples 21-25 are all less than 1%, and are obviously smaller, which indicates that the imidacloprid enzyme-linked immunosorbent assay kit related by the invention has higher specificity and stronger imidacloprid complete antigen specificity.
Test example 2 detection of precision
The imidacloprid enzyme linked immunosorbent assay kit related in the examples 21-25 and the comparative example 1 is selected for precision detection test.
10 kits (i.e., 3 batches of each kit prepared for BSA complete antigen 1 through BSA complete antigen 5) were extracted from each of 3 different kit batches in each of the kits of examples 21-25, and the absorbance values of 10 microwell assay standard solutions (containing 5mg/L imidacloprid) were then extracted from each microplate, and the coefficients of variation were calculated, with the results shown in Table 2:
TABLE 2 Imidacloprid ELISA kit concentration and variation coefficient of standard solution
It can be seen from the above tests that the standard solutions (containing 5mg/L imidacloprid) tested by the imidacloprid enzyme linked immunosorbent assay kit prepared in examples 21-25 all have variation coefficients of 4.0-7.5%, which indicates that the imidacloprid enzyme linked immunosorbent assay kit prepared in examples 21-25 can effectively improve the accuracy of content detection compared with comparative example 1.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. An imidacloprid hapten which is characterized by the following structural formula:
wherein n denotes the number of methylene groups and n = 1-6.
2. A method for synthesizing imidacloprid hapten as claimed in claim 1, which comprises the following steps:
s1, preparing imidacloprid intermediate
Dissolving imidacloprid in DMF, adding sodium hydride and halogenated carboxylic acid tert-butyl ester to obtain imidacloprid N-substituted carboxylic acid tert-butyl ester intermediate;
s2, preparing imidacloprid hapten
Under the acidic condition and the deprotection reagent environment, the imidacloprid N-substituted carboxylic acid tert-butyl ester intermediate is subjected to a tert-butyl removal reaction to obtain the imidacloprid hapten.
3. The method for synthesizing imidacloprid hapten according to claim 2, which is characterized in that: the molar ratio of the imidacloprid to the sodium hydride to the halogenated carboxylic acid tert-butyl ester is 1:1-1.1: 1-3.
4. The method for synthesizing imidacloprid hapten according to claim 2, which is characterized in that: the deprotection reagent is one of hydrochloric acid, trifluoro hydrochloric acid and hydrobromic acid.
5. The method for synthesizing imidacloprid hapten according to claim 2, which is characterized in that: the imidacloprid hapten is imidacloprid hapten derivatives such as 2- [3- [ (6-chloropyridyl-3-yl) methyl ] -2-nitroiminimimidazolin-1-yl ] acetic acid, 3- [3- [ (6-chloropyridyl-3-yl) methyl ] -2-nitroiminmidazolin-1-yl ] propionic acid, 4- [3- [ (6-chloropyridyl-3-yl) methyl ] -2-nitroiminmidazolin-1-yl ] butyric acid, 5- [3- [ (6-chloropyridyl-3-yl) methyl ] -2-nitroiminmidazolin-1-yl ] valeric acid, and 6- [3- [ (6-chloropyridyl-3-yl) methyl ] -2-nitroiminmidazolin Azolin-1-yl hexanoic acid.
6. An imidacloprid complete antigen prepared by coupling the imidacloprid hapten and carrier protein according to claim 1, which is characterized by the following structural formula:
wherein,
represents the carrier protein, n = 1-6.
7. The method for synthesizing imidacloprid complete antigen of claim 6, which is characterized by comprising the following steps:
s1, preparing imidacloprid hapten active ester solution
Dissolving imidacloprid hapten with a DMF solution, adding NHS and EDC hydrochloride, and reacting at room temperature for 16-24h to obtain an imidacloprid hapten active ester solution;
s2, preparing imidacloprid complete antigen
Weighing carrier protein, dissolving in 0.1M borate buffer solution, adjusting pH =8.7, slowly dropwise adding the imidacloprid hapten active ester solution obtained in the step S1, reacting for 24h at room temperature, and dialyzing with PBS buffer solution for three days at 4 ℃ to obtain the imidacloprid complete antigen.
8. The method for synthesizing imidacloprid complete antigen of claim 6, which is characterized by comprising the following steps:
s1, preparing imidacloprid hapten active ester solution
Dissolving imidacloprid hapten with a DMF solution, adding n-butylamine and isobutyl chloroformate, and reacting for 1h to obtain an imidacloprid hapten active ester solution;
s2, preparing imidacloprid complete antigen
Weighing carrier protein, dissolving in 0.1M borate buffer solution, adjusting pH =8.7, slowly dropwise adding the imidacloprid hapten active ester solution obtained in the step S1, reacting for 24h at room temperature, and dialyzing with PBS buffer solution for three days at 4 ℃ to obtain the imidacloprid complete antigen.
9. An imidacloprid-specific antibody which is prepared from the imidacloprid hapten as claimed in claim 1 or the imidacloprid complete antigen as claimed in claim 6.
10. The application of the imidacloprid hapten is characterized in that: the imidacloprid hapten of claim 1 is applied to detecting imidacloprid or preparing an imidacloprid product.
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