CN111875652B - Spinosad hapten, artificial antigen and antibody as well as preparation methods and applications thereof - Google Patents
Spinosad hapten, artificial antigen and antibody as well as preparation methods and applications thereof Download PDFInfo
- Publication number
- CN111875652B CN111875652B CN202010697076.XA CN202010697076A CN111875652B CN 111875652 B CN111875652 B CN 111875652B CN 202010697076 A CN202010697076 A CN 202010697076A CN 111875652 B CN111875652 B CN 111875652B
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- CN
- China
- Prior art keywords
- spinosad
- spinosyn
- hapten
- reaction
- artificial antigen
- 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.)
- Active
Links
- 239000005930 Spinosad Substances 0.000 title claims abstract description 76
- 229940014213 spinosad Drugs 0.000 title claims abstract description 76
- JFLRKDZMHNBDQS-UCQUSYKYSA-N CC[C@H]1CCC[C@@H]([C@H](C(=O)C2=C[C@H]3[C@@H]4C[C@@H](C[C@H]4C(=C[C@H]3[C@@H]2CC(=O)O1)C)O[C@H]5[C@@H]([C@@H]([C@H]([C@@H](O5)C)OC)OC)OC)C)O[C@H]6CC[C@@H]([C@H](O6)C)N(C)C.CC[C@H]1CCC[C@@H]([C@H](C(=O)C2=C[C@H]3[C@@H]4C[C@@H](C[C@H]4C=C[C@H]3C2CC(=O)O1)O[C@H]5[C@@H]([C@@H]([C@H]([C@@H](O5)C)OC)OC)OC)C)O[C@H]6CC[C@@H]([C@H](O6)C)N(C)C Chemical compound CC[C@H]1CCC[C@@H]([C@H](C(=O)C2=C[C@H]3[C@@H]4C[C@@H](C[C@H]4C(=C[C@H]3[C@@H]2CC(=O)O1)C)O[C@H]5[C@@H]([C@@H]([C@H]([C@@H](O5)C)OC)OC)OC)C)O[C@H]6CC[C@@H]([C@H](O6)C)N(C)C.CC[C@H]1CCC[C@@H]([C@H](C(=O)C2=C[C@H]3[C@@H]4C[C@@H](C[C@H]4C=C[C@H]3C2CC(=O)O1)O[C@H]5[C@@H]([C@@H]([C@H]([C@@H](O5)C)OC)OC)OC)C)O[C@H]6CC[C@@H]([C@H](O6)C)N(C)C JFLRKDZMHNBDQS-UCQUSYKYSA-N 0.000 title claims abstract description 73
- 239000000427 antigen Substances 0.000 title claims abstract description 29
- 102000036639 antigens Human genes 0.000 title claims abstract description 29
- 108091007433 antigens Proteins 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title abstract description 15
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 15
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 15
- 230000003053 immunization Effects 0.000 claims abstract description 14
- 241001465754 Metazoa Species 0.000 claims abstract description 12
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 7
- 230000008878 coupling Effects 0.000 claims abstract description 3
- 238000010168 coupling process Methods 0.000 claims abstract description 3
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- 229930185156 spinosyn Natural products 0.000 claims description 49
- 238000006243 chemical reaction Methods 0.000 claims description 35
- 238000000034 method Methods 0.000 claims description 26
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 16
- 238000002649 immunization Methods 0.000 claims description 11
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical compound Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- FVAUCKIRQBBSSJ-UHFFFAOYSA-M sodium iodide Chemical compound [Na+].[I-] FVAUCKIRQBBSSJ-UHFFFAOYSA-M 0.000 claims description 9
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 8
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 8
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- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 6
- NHQDETIJWKXCTC-UHFFFAOYSA-N 3-chloroperbenzoic acid Chemical group OOC(=O)C1=CC=CC(Cl)=C1 NHQDETIJWKXCTC-UHFFFAOYSA-N 0.000 claims description 5
- 229940124277 aminobutyric acid Drugs 0.000 claims description 5
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 claims description 5
- 229910000042 hydrogen bromide Inorganic materials 0.000 claims description 5
- 239000003054 catalyst Substances 0.000 claims description 4
- 150000002978 peroxides Chemical class 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000007259 addition reaction Methods 0.000 claims description 3
- 239000007789 gas Substances 0.000 claims description 3
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- 239000012071 phase Substances 0.000 claims description 3
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 3
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- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 235000009518 sodium iodide Nutrition 0.000 claims description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 2
- 230000009471 action Effects 0.000 claims description 2
- 229940098773 bovine serum albumin Drugs 0.000 claims description 2
- -1 bromo spinosad Chemical compound 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 108060003552 hemocyanin Proteins 0.000 claims description 2
- 238000010534 nucleophilic substitution reaction Methods 0.000 claims description 2
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 5
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 4
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- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
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- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
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- 239000000758 substrate Substances 0.000 description 2
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- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 description 1
- AZSNMRSAGSSBNP-UHFFFAOYSA-N 22,23-dihydroavermectin B1a Natural products C1CC(C)C(C(C)CC)OC21OC(CC=C(C)C(OC1OC(C)C(OC3OC(C)C(O)C(OC)C3)C(OC)C1)C(C)C=CC=C1C3(C(C(=O)O4)C=C(C)C(O)C3OC1)O)CC4C2 AZSNMRSAGSSBNP-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- SPBDXSGPUHCETR-JFUDTMANSA-N 8883yp2r6d Chemical compound O1[C@@H](C)[C@H](O)[C@@H](OC)C[C@@H]1O[C@@H]1[C@@H](OC)C[C@H](O[C@@H]2C(=C/C[C@@H]3C[C@@H](C[C@@]4(O[C@@H]([C@@H](C)CC4)C(C)C)O3)OC(=O)[C@@H]3C=C(C)[C@@H](O)[C@H]4OC\C([C@@]34O)=C/C=C/[C@@H]2C)/C)O[C@H]1C.C1C[C@H](C)[C@@H]([C@@H](C)CC)O[C@@]21O[C@H](C\C=C(C)\[C@@H](O[C@@H]1O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C1)[C@@H](C)\C=C\C=C/1[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\1)O)C[C@H]4C2 SPBDXSGPUHCETR-JFUDTMANSA-N 0.000 description 1
- 239000005660 Abamectin Substances 0.000 description 1
- 241000239290 Araneae Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 241001414720 Cicadellidae Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 239000005894 Emamectin Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
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- 240000001624 Espostoa lanata Species 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 231100000703 Maximum Residue Limit Toxicity 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
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- 108010071390 Serum Albumin Proteins 0.000 description 1
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- 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
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- 238000011091 antibody purification Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- RRZXIRBKKLTSOM-XPNPUAGNSA-N avermectin B1a Chemical compound C1=C[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 RRZXIRBKKLTSOM-XPNPUAGNSA-N 0.000 description 1
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- CXEGAUYXQAKHKJ-NSBHKLITSA-N emamectin B1a Chemical compound C1=C[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](NC)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 CXEGAUYXQAKHKJ-NSBHKLITSA-N 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
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- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
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- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 229960002418 ivermectin Drugs 0.000 description 1
- 150000002596 lactones Chemical group 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
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- 238000005259 measurement Methods 0.000 description 1
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- 210000000952 spleen Anatomy 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
-
- 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
- G01N33/18—Water
- G01N33/1826—Organic contamination in water
- G01N33/184—Herbicides, pesticides, fungicides, insecticides or the like
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Biomedical Technology (AREA)
- General Physics & Mathematics (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Pathology (AREA)
- Toxicology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a spinosad hapten, an artificial antigen and an antibody, as well as preparation methods and applications thereof, and the spinosad hapten provided by the invention not only furthest maintains the characteristic structure of spinosad, so that the immunogenicity of the spinosad hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the spinosad artificial antigen obtained by coupling the spinosad hapten and the carrier protein is used for immunizing animals, is more beneficial to stimulating animal immune response to generate antibodies with stronger specificity and higher sensitivity, has the sensitivity of 1 mug/L after detecting the spinosad antibodies, has low cross reaction rate with other macrolide pesticides, and provides a basis for the subsequent establishment of various immunity analysis methods of spinosad.
Description
Technical Field
The invention belongs to the field of food safety detection. More particularly, the invention relates to spinosad hapten, artificial antigen and antibody, and preparation methods and applications thereof.
Background
Spinosad is a secondary metabolite produced by aerobic fermentation of spinosyns, and its active ingredients are spinosad a and spinosad, collectively referred to as spinosad. The insecticidal composition has the characteristics of wide insecticidal spectrum, high insecticidal activity, safety to non-target animals, safety to decomposition products and the like, and has been widely used for controlling lepidoptera, diptera, leafhopper, spider mite, flea, lice and other pests on more than 150 crops such as cotton and the like, and especially has good control effect on lepidoptera pests. However, the residue exceeding the standard still can affect the food safety and the environmental safety, so the residue limit of spinosad in different foods is specified in national standard GB 2763-2019 of maximum residue limit of pesticide in food safety national standard food.
At present, the spinosad is detected at home and abroad mainly by adopting analysis methods such as gas chromatography, gas chromatography-mass spectrometry, liquid chromatography tandem mass spectrometry and the like, and the defects of complicated sample pretreatment, long detection time, expensive instruments and the like exist, so that the spinosad cannot be widely applied in China and does not meet the requirements of on-site detection on accurate detection and screening of a large number of samples in a short time at low cost. The immunological detection analysis technology has the advantages of high sensitivity, high specificity, rapidness, simple and convenient operation and the like, is widely applied to the field of drug residue detection, and has a lot of advantages compared with the detection methods such as instruments and the like. Therefore, the immunoassay provides a new analytical detection method for spinosyn residue research.
When an immunological detection method is established and the detection method is applied to detect the residual spinosyn, the key technology is that antibodies with strong specificity and high sensitivity can be obtained, and the aim is to be fulfilled if proper spinosyn hapten is synthesized and prepared. It has been found that the macrolide tetracyclic ring of spinosad is a characteristic structure and plays a very important role in immune response. The invention patent CN106967140A adopts the structure that the formosan sugar connected on the spinosad macrolide ring is replaced by a spacer arm with-COOH, and is further coupled with carrier protein, so that the modification site is closer to the characteristic structure of the spinosad molecule, the exposure of the characteristic structure of the hapten molecule is not facilitated, the recognition difficulty of the characteristic structure of the hapten molecule by the immune system of animals is increased, and the affinity and the specificity of the antibody are reduced. Therefore, to prepare highly specific antibodies, the modification site in the spinosad molecule should be far from the macrolide ring of its characteristic structure, so that the characteristic molecular structure part of the hapten can be fully exposed, and the shielding effect of the carrier protein on the characteristic structure of the hapten molecule is avoided.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a hapten which can keep the characteristic structure of spinosad to the greatest extent and has a connecting arm with a certain length and a preparation method of the hapten; an artificial antigen prepared by the hapten, and an antibody with high detection sensitivity and strong specificity; and the use of such haptens.
To achieve the object of the present invention, in a first aspect, the present invention provides a spinosyn hapten having the following structural formula:
the spinosad hapten provided by the invention introduces carboxyl active groups on the molecular structure of spinosad, so that the spinosad hapten can be coupled with carrier protein to obtain artificial antigen for immunization; the spinosad hapten keeps all characteristic groups of spinosad, the original structural characteristics of spinosad are changed at least, and after the spinosad hapten is coupled with carrier protein, the unique twelve-membered lactone ring part structure of spinosad is highlighted, so that a foundation is laid for generating antibodies with stronger specificity and higher sensitivity for stimulating animal immune response subsequently.
In a second aspect, the present invention provides a method for preparing the spinosyn hapten, which comprises the following steps:
1) Under the action of peroxide, spinosad and hydrogen bromide undergo addition reaction to obtain bromospinosad, said bromospinosad has a structural formula
2) Under alkaline condition, the bromo spinosad and the aminobutyric acid undergo nucleophilic substitution reaction to obtain spinosad hapten.
Further, in the step 1), the peroxide is m-chloroperoxybenzoic acid;
the reaction is carried out in a solvent, wherein the solvent is anhydrous carbon tetrachloride;
the reaction conditions are as follows: introducing dry hydrogen bromide gas into anhydrous carbon tetrachloride containing m-chloroperoxybenzoic acid, stirring for 1h, dropwise adding anhydrous carbon tetrachloride containing spinosad, and continuously stirring at room temperature for 2h;
after the reaction in the step 1), the method further comprises the step of post-treating the reaction system, and the method specifically comprises the following steps: adding water into the reaction system, sufficiently oscillating, standing, separating out water phase, and evaporating the organic phase to dryness to obtain the bromo-spinosad.
Further, in the step 2), the alkaline condition is provided by anhydrous potassium carbonate, and the mass ratio of the brominated spinosad to the aminobutyric acid is 1:5-6;
the reaction is carried out in a solvent, wherein the solvent is acetonitrile;
the reaction requires the participation of a catalyst, wherein the catalyst is anhydrous sodium iodide;
the reaction conditions are as follows: heating and refluxing in an oil bath for reaction for 4 hours;
after the reaction of the step 2), the method further comprises the step of post-treating the reaction system, and the method specifically comprises the following steps: adding water into the reaction system, adding ethyl acetate for extraction for 3 times, combining organic phases, evaporating to dryness, purifying by using a silica gel column, and eluting and separating by using a mixed solvent with the volume ratio of dichloromethane to methanol being 10:1 to obtain the spinosad hapten.
The preparation method of the hapten mainly comprises the following steps: (1) The existing structure or intermediate of the object to be detected is utilized to generate corresponding functional groups through oxidation, reduction, substitution, hydrolysis and other reactions; (2) The metabolite or the structural analogue is used as a lead, and is modified to obtain the needed hapten; (3) The raw materials and the reagent are used for resynthesis, and small molecules with functional groups are introduced at proper positions. According to the structural characteristics of spinosad, the invention generates the spacer arm with carboxyl through addition and substitution reaction, and compared with the two latter methods, the invention has the advantages of easy obtaining of raw materials, simple reaction operation, easy control of reaction conditions, and higher purity and yield of the prepared spinosad hapten.
In a third aspect, the present invention provides a spinosyn artificial antigen which is a conjugate of a carrier protein and a spinosyn hapten as described above. The spinosyn artificial antigen can be used as an immunogen or a coating antigen.
Further, the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin; human serum albumin and egg albumin are preferred.
More specifically, the following are: an immunogen formed from spinosad hapten-Human Serum Albumin (HSA); spinosad hapten-Ovalbumin (OVA) formed coating antigen.
The spinosyn hapten molecules are only immunoreactive and not immunogenic. Thus, in order to render a spinosad hapten molecule immunogenic, it is also necessary to couple and bind the spinosad hapten molecule to a suitable carrier protein molecule, thereby producing a spinosad artificial antigen that is both immunoreactive and immunogenic.
In a fourth aspect, the present invention provides a method for preparing the spinosyn artificial antigen, wherein the carrier protein is coupled to the carboxyl of the spinosyn hapten by using an active ester method.
In a fifth aspect, the present invention provides a spinosyn antibody, which is obtained by immunizing an animal with the above-mentioned spinosyn artificial antigen, and which is capable of specifically immunoreacting with spinosyn.
Further, the spinosyn antibody is a monoclonal antibody or a polyclonal antibody. In addition, for the spinosyn antibody, preparation may be carried out using methods conventional in the art.
In a specific embodiment, the spinosyn antibody is a murine monoclonal antibody directed specifically against a spinosyn artificial antigen of the spinosyn hapten described above.
The spinosyn antibody obtained by the spinosyn artificial antigen has better potency, specificity and affinity and low cross reaction rate with other macrolide pesticides.
In a sixth aspect, the present invention provides the use of a spinosyn antibody as described above for detecting spinosyn residues.
The invention induces immune animals to generate antibodies through the spinosad artificial antigen, thereby being used in spinosad immune detection analysis.
The spinosad immunoassay comprises, but is not limited to, a spinosad ELISA kit, a spinosad colloidal gold test strip and a spinosad time-resolved fluorescence test strip.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
the spinosad hapten provided by the invention not only maintains the characteristic structure of spinosad to the greatest extent, so that the immunogenicity of the spinosad hapten is obviously enhanced, but also has carboxyl which can be coupled with carrier protein; the spinosad artificial antigen obtained by coupling the spinosad hapten and the carrier protein is used for immunizing animals, is more beneficial to stimulating the immune response of the animals to generate antibodies with stronger specificity and higher sensitivity, and provides a foundation for the subsequent establishment of various immune analysis methods of spinosad.
The preparation method of the spinosad hapten has the advantages of easily available raw materials, simple reaction operation, easily controlled reaction conditions and higher purity and yield of the prepared spinosad hapten.
The spinosyn antibody obtained by the spinosyn artificial antigen has better potency, specificity and affinity, the sensitivity can reach 1 mug/L, and the cross reaction rate with other macrolide pesticides is low.
Drawings
FIG. 1 is a synthetic route to spinosyn hapten of the present invention
Detailed Description
The present invention will be further described in detail with reference to the following specific examples, which are only preferred embodiments of the present invention, and are not limiting thereof.
Example 1
A method for preparing spinosyn hapten, which comprises the following steps:
1) Taking 0.43g of m-chloroperoxybenzoic acid, adding 70mL of anhydrous carbon tetrachloride for dissolution, introducing dry hydrogen bromide gas, and stirring for 1h; dropwise adding 100mL of anhydrous carbon tetrachloride containing 0.73g of spinosad, continuously stirring at room temperature for 2 hours, stopping the reaction, adding 100mL of water, sufficiently oscillating, standing, separating out a water phase, and evaporating an organic phase to dryness to obtain 0.62g of bromospinosad;
2) Taking 0.62g of bromospinosad, adding 80mL of acetonitrile for dissolution, adding 0.31g of anhydrous sodium iodide, fully stirring, adding 0.53g of anhydrous potassium carbonate, adding 0.44g of aminobutyric acid, heating in an oil bath, carrying out reflux reaction for 4 hours, stopping the reaction, adding 200mL of water and 100mL of ethyl acetate for 3 times, combining organic phases, evaporating to dryness, purifying by a silica gel column, and eluting and separating by using a mixed solvent with the volume ratio of dichloromethane to methanol being 10:1 to obtain 0.37g of spinosad hapten.
Example 2
The preparation process of artificial spinosyn antigen includes the following steps:
taking 32mg of spinosyn hapten prepared in example 1, adding 2mL of N, N-Dimethylformamide (DMF) for dissolution, adding 17.2mg of N-hydroxysuccinimide (NHS) and 23.4mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), and reacting for 3 hours at room temperature to obtain hapten solution A; taking 50mg of Human Serum Albumin (HSA), and adding 0.05mol/L CB buffer for dissolution to obtain solution B; dripping the solution A into the solution B, reacting for 12 hours at 4 ℃, dialyzing and purifying for 3 days by using 0.02mol/L PBS, changing the solution 3 times a day to obtain the spinosad artificial antigen coupled with the human serum albumin, subpackaging, and preserving at-20 ℃.
Example 3
The preparation process of artificial spinosyn antigen includes the following steps:
taking 18mg of spinosad hapten prepared in example 1, adding 1mL of dimethyl sulfoxide (DMSO) for dissolution, adding 13.3mg of NHS and 16.4mg of EDC, and reacting for 3 hours at room temperature to obtain hapten solution A; taking 50mg of Ovalbumin (OVA), and adding 0.05mol/L CB buffer for dissolving to obtain solution B; dripping the solution A into the solution B, reacting for 12 hours at 4 ℃, dialyzing and purifying for 3 days by using 0.02mol/L PBS, changing the solution 3 times a day to obtain the spinosad artificial antigen coupled with the ovalbumin, subpackaging, and preserving at-20 ℃.
Example 4
A spinosyn antibody, its preparation method is:
1. immunization of animals
Taking 10 healthy female Balb/c mice (divided into two groups A and B, 5 mice in each group), performing subcutaneous multipoint injection on the back of the neck after primary immunization is emulsified by Freund's complete adjuvant, wherein the immunization dose of each mouse is 200 mug of spinosad artificial antigen coupled with human serum albumin; then boosting the immunity, carrying out subcutaneous multipoint injection once every two weeks on the back of the neck, and emulsifying with Freund's incomplete adjuvant; the last immunization uses physiological saline instead of Freund's incomplete adjuvant, and adopts intraperitoneal injection, and the injection dosage is the same as that of the previous times. The specific immunization procedure is shown in Table 1.
Table 1 mouse immunization procedure
The mice were bled at the tail end 7d after the third, fourth and booster immunizations, and the ELISA method was used to determine the serum titers of the mice as follows:
(1) Diluting the spinosyn artificial antigen coupled with ovalbumin by using 0.05mol/L carbonate buffer solution with pH of 9.6 by 1:1000, coating 100 mu L of an ELISA plate on each hole, incubating for 2 hours at 37 ℃, throwing away the coating liquid, washing for 1 time by using PBST, and beating to dry;
(2) Adding 150 mu L of sealing liquid into each hole, reacting for 2 hours at 37 ℃, pouring off the sealing liquid, and beating to dry;
(3) Adding 50 mu L of antiserum diluted by PBS (phosphate buffered saline) in each hole, reacting for 30min at 25 ℃, pouring out the reaction solution, washing for 3-5 times by PBST (poly (butylene glycol) ST), and drying at intervals of 30 s;
(4) Adding 100 mu L/hole of horseradish peroxidase-labeled goat anti-mouse antibody (1:1000) diluted by PBS, reacting for 30min at 25 ℃, washing for 3-5 times by PBST, and beating to dryness at intervals of 30s each time;
(5) Adding 50 mu L of substrate chromogenic solution A and 50 mu L of substrate chromogenic solution B into each hole, carrying out light-shielding reaction at 25 ℃ for 15min, and adding 50 mu L of 2mol/L of H into each hole 2 SO 4 Stopping the reaction by the solution;
(6) Measuring the OD value of the wavelength at 450nm by using an enzyme label instrument, and obtaining the OD value of a sample hole 450 A dilution factor close to 1 was used as the titer of positive serum.
2. Cell fusion
(1) Feeder cell preparation: the Balb/c mice with the age of 8-10 weeks are killed by neck breaking, soaked in 75% alcohol for 5min, then placed in an ultra-clean workbench, and placed in a plate or fixed on an anatomic plate with the abdomen facing upwards. The skin of the abdomen of the mouse is clamped by using ophthalmic forceps, a small opening is cut by using scissors, and the peritoneum is not cut by taking care so as to avoid outflow of abdominal cavity liquid and pollution. Then, the peritoneum was fully exposed by blunt dissection with scissors to the upper and lower sides. The peritoneum was sterilized by swabbing with alcohol cotton balls. 5mL of RPMI-1640 basal culture solution is sucked by a syringe, injected into the abdominal cavity of a mouse, the syringe is gently withdrawn, and the legs and the tail of the mouse are shaken for several times. The liquid in the abdominal cavity is pumped back by the original injector and injected into the centrifuge tube. The operation is repeated for 3 to 4 times. Centrifuging at 1000r/min for 10min, and discarding supernatant. The cells are resuspended in 20-50 mL of complete culture medium, 100. Mu.L/Kong Di are added to the culture plates and placed in an incubator for use.
(2) Spleen cell preparation: 3d after the immunization is enhanced, taking an immunized Balb/c mouse, taking the orbit, taking dislocation for sacrifice after blood sampling, taking spleen after disinfection in 75% alcohol, removing connective tissue, preparing spleen cell suspension, transferring into a 50mL centrifuge tube, adding RPMI-1640 to 30mL, centrifuging for 5min at 1500-2000 r/min, discarding the supernatant, adding RPMI-1640 to 30mL, and counting for later use.
(3) Preparation of myeloma cells: taking 3 bottles of myeloma cells with good growth state (the number of living cells is more than 95%), completely blowing down, transferring the myeloma cells into a 50mL centrifuge tube, adding RPMI-1640 to 30mL, centrifuging for 5min at 1500-2000 r/min, discarding the supernatant, adding RPMI-1640 to 30mL, and counting for later use.
(4) Cell mixing: spleen cells myeloma cells=8:1, mixed, centrifuged at 1500-2000 r/min for 5min.
(5) Cell fusion: centrifuging the mixed cells, pouring the supernatant, flicking the precipitated cell mass into paste, placing in a water bath at 37 ℃, adding 1mL of a fusion agent which is polyethylene glycol (PEG) 4000 in 1min, reacting for 2min, gently stirring the cells, adding 20mL of serum-free PEG nutrient solution in the following 4min, centrifuging for 10min at 1000r/min, and discarding the supernatant. The cells were resuspended in 20-50 mL of complete medium, plated onto 96-well cell culture plates containing feeder cells, 100. Mu.L per well, and placed in an incubator.
3. Cell strain selection
And (3) detecting the antibody when the cells grow to 1/3-1/2 of the bottom of the hole. Screening culture holes with hybridoma cell growth by ELISA method, wherein the screening comprises two steps: the first step is to screen out positive cell holes by indirect ELISA, and the second step is to use spinosad as a standard substance, and to measure the inhibition effect of positive cells by indirect competition ELISA. Selecting a hole with better inhibition on a spinosyn standard, subcloning by adopting a limiting dilution method, and detecting by adopting the same method. Repeating the steps for three times to obtain the cell strain capable of stably secreting the spinosad monoclonal antibody.
4. Ascites preparation
Liquid paraffin was injected into Balb/c mice at a concentration of 500. Mu.L/mouse for 6 to 8 weeks. After 10 days, hybridoma cells in logarithmic growth phase were collected with RPMI-1640 basal medium, counted with a hemocytometer and a microscope, and cell concentration was 1.0X10 6 ～1.5×10 6 In the range of individual/mL. 0.5mL of hybridoma cells per mouse was injected into the peritoneal cavity. Note that after one week, the abdomen of the mice was distended, and ascites were collected from the abdomen of the mice with a sterile syringe, once every one to two days, so that there were moreThe collection was repeated until the mice died naturally. Centrifuging at 4deg.C for 5min at 5000r/min, collecting supernatant, and removing fat and protein membrane floating on the upper layer of ascites.
5. Antibody purification
Monoclonal antibodies were purified using the octanoic acid-ammonium sulfate method.
6. Antibody titer determination
The antibody titer was determined by indirect ELISA, step reference 1. Serum titer determination for animal immunization. The result shows that the titer of the spinosad monoclonal antibody is more than or equal to 100000.
7. Antibody cross-reactivity assay
As a result of measurement by adopting an indirect competition ELISA method, the cross reaction rate of the spinosad monoclonal antibody to spinosad and other macrolide pesticides is found to be: the spinosad is 100%, and the avermectin, emamectin and ivermectin are all less than 1%. Thus, the prepared antibody has better specificity.
The above examples only show embodiments of the present invention, and the description thereof is more specific and detailed, but should not be construed as limiting the scope of the invention, but all technical solutions obtained by equivalent substitution or equivalent transformation shall fall within the scope of the invention.
Claims (9)
1. A spinosyn hapten characterized by the following structural formula:
2. the method for preparing spinosyn hapten according to claim 1, comprising the steps of:
1) Under the action of peroxide, spinosad and hydrogen bromide undergo addition reaction to obtain bromospinosad, said bromospinosad has a structural formula
2) Under alkaline condition, the bromo spinosad and the aminobutyric acid undergo nucleophilic substitution reaction to obtain spinosad hapten.
3. The method for preparing spinosyn hapten according to claim 2, wherein in step 1), the peroxide is m-chloroperoxybenzoic acid;
the reaction is carried out in a solvent, wherein the solvent is anhydrous carbon tetrachloride;
the reaction conditions are as follows: introducing dry hydrogen bromide gas into anhydrous carbon tetrachloride containing m-chloroperoxybenzoic acid, stirring for 1h, dropwise adding anhydrous carbon tetrachloride containing spinosad, and continuously stirring at room temperature for 2h;
after the reaction in the step 1), the method further comprises the step of post-treating the reaction system, and the method specifically comprises the following steps: adding water into the reaction system, sufficiently oscillating, standing, separating out water phase, and evaporating the organic phase to dryness to obtain the bromo-spinosad.
4. The method for preparing spinosyn hapten according to claim 2, wherein in the step 2), the alkaline condition is provided by anhydrous potassium carbonate, and the mass ratio of the brominated spinosyn and the aminobutyric acid is 1:5-6;
the reaction is carried out in a solvent, wherein the solvent is acetonitrile;
the reaction requires the participation of a catalyst, wherein the catalyst is anhydrous sodium iodide;
the reaction conditions are as follows: heating and refluxing in an oil bath for reaction for 4 hours;
after the reaction of the step 2), the method further comprises the step of post-treating the reaction system, and the method specifically comprises the following steps: adding water into the reaction system, adding ethyl acetate for extraction for 3 times, combining organic phases, evaporating to dryness, purifying by using a silica gel column, and eluting and separating by using a mixed solvent with the volume ratio of dichloromethane to methanol being 10:1 to obtain the spinosad hapten.
5. A spinosyn artificial antigen, characterized in that it is a conjugate obtained by coupling a carrier protein and a spinosyn hapten according to claim 1.
6. The spinosyn artificial antigen of claim 5, wherein said carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.
7. The method for preparing the spinosyn artificial antigen according to claim 5 or 6, wherein the carrier protein is coupled to the carboxyl group of the spinosyn hapten according to claim 1 by an active ester method.
8. A spinosyn antibody, characterized in that it is obtained from the spinosyn artificial antigen of claim 5 by immunization of an animal, which is capable of specific immune reaction with spinosyn.
9. Use of the spinosyn antibody of claim 8 for detecting spinosyn residues.
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| CN1191541A (en) * | 1995-06-14 | 1998-08-26 | 道农业科学公司 | Synthetic modification to spinosyn compounds |
| CN106967140A (en) * | 2017-03-17 | 2017-07-21 | 海南大学 | A kind of pleocidin haptens and its preparation method and application |
| CN107226830A (en) * | 2017-05-02 | 2017-10-03 | 北京理工大学 | A kind of chemical synthesis process of ethyl pleocidin |
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| CN1191541A (en) * | 1995-06-14 | 1998-08-26 | 道农业科学公司 | Synthetic modification to spinosyn compounds |
| CN106967140A (en) * | 2017-03-17 | 2017-07-21 | 海南大学 | A kind of pleocidin haptens and its preparation method and application |
| CN107226830A (en) * | 2017-05-02 | 2017-10-03 | 北京理工大学 | A kind of chemical synthesis process of ethyl pleocidin |
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