CN111961010A - Saccharin sodium hapten Ri, artificial antigen, antibody and preparation method and application thereof - Google Patents
Saccharin sodium hapten Ri, artificial antigen, antibody and preparation method and application thereof Download PDFInfo
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- CN111961010A CN111961010A CN202010435981.8A CN202010435981A CN111961010A CN 111961010 A CN111961010 A CN 111961010A CN 202010435981 A CN202010435981 A CN 202010435981A CN 111961010 A CN111961010 A CN 111961010A
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- Prior art keywords
- saccharin sodium
- hapten
- saccharin
- sodium
- antigen
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- FTLYMKDSHNWQKD-UHFFFAOYSA-N (2,4,5-trichlorophenyl)boronic acid Chemical compound OB(O)C1=CC(Cl)=C(Cl)C=C1Cl FTLYMKDSHNWQKD-UHFFFAOYSA-N 0.000 title claims abstract description 146
- 229940085605 saccharin sodium Drugs 0.000 title claims abstract description 139
- 239000000427 antigen Substances 0.000 title claims abstract description 55
- 102000036639 antigens Human genes 0.000 title claims abstract description 55
- 108091007433 antigens Proteins 0.000 title claims abstract description 55
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 230000002163 immunogen Effects 0.000 claims abstract description 15
- 108010063045 Lactoferrin Proteins 0.000 claims abstract description 13
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 claims abstract description 13
- 229940078795 lactoferrin Drugs 0.000 claims abstract description 13
- 235000021242 lactoferrin Nutrition 0.000 claims abstract description 13
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229940014800 succinic anhydride Drugs 0.000 claims abstract description 12
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 6
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- 238000000576 coating method Methods 0.000 claims description 16
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 claims description 14
- 102100032241 Lactotransferrin Human genes 0.000 claims description 12
- 102000014914 Carrier Proteins Human genes 0.000 claims description 10
- 108010078791 Carrier Proteins Proteins 0.000 claims description 10
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 235000018102 proteins Nutrition 0.000 claims description 5
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- 101000609762 Gallus gallus Ovalbumin Proteins 0.000 claims description 4
- SSRKZHLPNHLAKM-UHFFFAOYSA-N 6-amino-1,1-dioxo-1,2-benzothiazol-3-one Chemical compound NC1=CC=C2C(=O)NS(=O)(=O)C2=C1 SSRKZHLPNHLAKM-UHFFFAOYSA-N 0.000 claims description 3
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- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims 1
- 125000003277 amino group Chemical group 0.000 claims 1
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 abstract description 6
- 241001465754 Metazoa Species 0.000 abstract description 5
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- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
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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 6
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- WNZQDUSMALZDQF-UHFFFAOYSA-N 2-benzofuran-1(3H)-one Chemical compound C1=CC=C2C(=O)OCC2=C1 WNZQDUSMALZDQF-UHFFFAOYSA-N 0.000 description 2
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 2
- 239000007832 Na2SO4 Substances 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
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- 239000001963 growth medium Substances 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 235000015110 jellies Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
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- 238000012544 monitoring process Methods 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 235000019204 saccharin Nutrition 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
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- 210000004989 spleen cell Anatomy 0.000 description 2
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- 229960004793 sucrose Drugs 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- MWGKAIKWBCTKHF-UHFFFAOYSA-N C1(=O)OCC2=CC=CC=C12.[Na] Chemical compound C1(=O)OCC2=CC=CC=C12.[Na] MWGKAIKWBCTKHF-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- UDIPTWFVPPPURJ-UHFFFAOYSA-M Cyclamate Chemical compound [Na+].[O-]S(=O)(=O)NC1CCCCC1 UDIPTWFVPPPURJ-UHFFFAOYSA-M 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
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- 208000032843 Hemorrhage Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000353135 Psenopsis anomala Species 0.000 description 1
- 241000983742 Saccharina Species 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- 240000008866 Ziziphus nummularia Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
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- 235000019789 appetite Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 210000004369 blood Anatomy 0.000 description 1
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- 239000000625 cyclamic acid and its Na and Ca salt Substances 0.000 description 1
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- 238000011161 development Methods 0.000 description 1
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- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
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- 238000000605 extraction Methods 0.000 description 1
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- 230000004907 flux Effects 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
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- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
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- 229940057995 liquid paraffin Drugs 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
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- 238000001819 mass spectrum Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 235000013615 non-nutritive sweetener Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
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- 230000028327 secretion Effects 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
- 239000004299 sodium benzoate Substances 0.000 description 1
- 229960003885 sodium benzoate Drugs 0.000 description 1
- 229960001462 sodium cyclamate Drugs 0.000 description 1
- 229940073490 sodium glutamate Drugs 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 206010043554 thrombocytopenia Diseases 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D275/00—Heterocyclic compounds containing 1,2-thiazole or hydrogenated 1,2-thiazole rings
- C07D275/04—Heterocyclic compounds containing 1,2-thiazole or hydrogenated 1,2-thiazole rings condensed with carbocyclic rings or ring systems
- C07D275/06—Heterocyclic compounds containing 1,2-thiazole or hydrogenated 1,2-thiazole rings condensed with carbocyclic rings or ring systems with hetero atoms directly attached to the ring sulfur atom
-
- 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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Abstract
The invention discloses a saccharin sodium hapten Ri, an artificial antigen, an antibody, and a preparation method and application thereof. The invention firstly provides a saccharin sodium hapten Ri, which has a structural formula shown as a formula (I):
the hapten Ri of saccharin sodium is prepared by taking an amino functional group on a benzene ring of saccharin sodium analog 6-saccharin as an arm extension site, reacting with succinic anhydride to generate amide, and reacting the obtained hapten Ri with to-be-treated saccharinThe overlapping degree of the skeleton structure of the saccharin sodium is high, the characteristic structure of the saccharin sodium is exposed, and a foundation is laid for generating a high-specificity antibody; further using the carboxyl terminal of the saccharin sodium hapten Ri and an artificial antigen obtained by coupling lactoferrin as an immunogen to immunize animals, preparing a hybridoma monoclonal antibody cell strain through cell fusion, and obtaining the saccharin sodium high-specificity monoclonal antibody. And the preparation method of the saccharin sodium hapten Ri, the artificial antigen and the antibody is simple and easy to implement, low in cost and wide in application prospect.
Description
Technical Field
The invention belongs to the technical field of biotechnology. More particularly, relates to saccharin sodium hapten Ri, an artificial antigen, an antibody, and a preparation method and application thereof.
Background
The phthalide sodium phthalide is commonly called saccharin sodium, the artificially synthesized non-nutritive sweetener which is applied at the earliest is dissolved in water, and the sweetness of the sweetener in a dilute solution is 200-500 times that of cane sugar. Because of its low cost, it is relatively stable in the production process of various foods, some manufacturers commonly use saccharin sodium to replace sucrose in beverages, jellies, preserved fruits, preserves, jams, cakes, and even in jellies and other foods intended for children to consume. But saccharin sodium is not metabolized and absorbed by human body, and has no nutritive value to human body except sweet feeling caused by taste. On the contrary, when more saccharin sodium is eaten, the normal secretion of digestive enzymes in intestines and stomach is influenced, and the absorption capacity of small intestines is reduced. Can reduce appetite. Research shows that a small number of people can cause thrombocytopenia and acute hemorrhage due to a large amount of saccharin sodium taken in a short time.
The use amount of the saccharin sodium is limited in many countries in the world, and the GB 2760-2014 of China clearly stipulates that the maximum use amount of the saccharin sodium in food is 0.15g/kg-5.0 g/kg. Meanwhile, the Ministry of health, namely the food additive sanitation management method, also stipulates that the artificial sweet agent, pigment, essence, sodium glutamate, unsuitable additives and the like are not required to be added into main and auxiliary foods specially used for infants except for the fact that the enhancer can be added according to the stipulation. In addition, saccharin sodium should not be used in patient foods and staple foods for large-scale consumption, such as steamed bread, steamed sponge cakes, etc. However, in production and operation activities, few enterprises use excessive saccharin sodium without law, so that the phenomenon that saccharin sodium exceeds the standard is serious. According to data, the food and drug administration of Sichuan province in 2018 organizes the detection of the saccharin sodium content of the cream melon seeds produced by the Heshi roasted food processing factory in the New City exceeding the standard in the special spot inspection of the Xinchun festival food; in 2015, the notice of Ministry of agriculture in China indicates that the food and drug administration of Hainan province found 3 tons of saccharin jujube with illegally added saccharin sodium, sodium cyclamate and sodium benzoate in the fruit wholesale market of Nantong and North China in Haikou. Therefore, a rapid, sensitive and efficient detection method is urgently needed to realize the rapid detection of the saccharin sodium. The existing common methods for detecting saccharin sodium comprise the following steps: the method mainly comprises the instrumental analysis methods such as high performance liquid chromatography, fluorescence spectroscopy, gas chromatography, ultraviolet spectroscopy, liquid chromatography tandem mass spectrometry and the like, but the instrumental analysis method is not suitable for screening a large number of samples due to the fact that the sample pretreatment and determination processes are complicated, the cost is high, and the operation of professional personnel is required. The enzyme-linked immunosorbent assay method has the advantages of high sensitivity, strong specificity, low cost, simple operation, high speed, large sample amount for one-time detection, low requirements on instruments and equipment, relatively simple pretreatment of samples and the like, and is suitable for market monitoring and field monitoring.
The immunoassay method based on antigen-antibody specific binding mainly takes monoclonal antibodies and polyclonal antibodies as main materials, has the characteristics of simple and convenient sample pretreatment, simple operation, rapidness, sensitivity, high flux and the like, is known as a rapid detection technology which has the greatest competition and challenge in the 21 st century, and has wide application prospect in the field of food safety. However, in the case of immunoassays, antibodies are used as a core material, and the effect of antibodies depends greatly on the structure of the antigen that causes an immune response in the corresponding animal. Therefore, the method for preparing the saccharin sodium salt has a stable antigen structure, can be used for preparing a high-specificity antibody, and has important significance in quickly, sensitively and accurately detecting the saccharin sodium salt.
Disclosure of Invention
The invention aims to overcome the defects of the existing saccharin sodium detection method and provide a saccharin sodium hapten Ri, an artificial antigen, an antibody, a preparation method and application thereof. The synthesis method of the saccharin sodium hapten Ri prepared by the invention is simpler, the molecular structural formula of saccharin sodium analog 6-saccharin is directly utilized for modification, and amino on a benzene ring on the molecular structural formula reacts with succinic anhydride to generate amide, so that the tail end of the amide has an active group-carboxyl.
The first purpose of the invention is to provide a saccharin sodium hapten Ri.
The second purpose of the invention is to provide a preparation method of the saccharin sodium hapten Ri.
The third purpose of the invention is to provide the application of the saccharin sodium hapten Ri in preparing the saccharin sodium artificial antigen.
The fourth purpose of the invention is to provide a saccharin sodium artificial antigen.
A fifth object of the invention is to provide a combination of an immunogen and a coating antigen for the detection of sodium saccharin.
The sixth purpose of the invention is to provide the application of the saccharin sodium hapten Ri, the saccharin sodium artificial antigen or the combination of the immunogen and the coating antigen in detecting saccharin sodium or preparing a saccharin sodium detection kit.
It is a seventh object of the present invention to provide an antibody to sodium saccharin.
The eighth purpose of the invention is to provide a preparation method of the saccharin sodium monoclonal antibody.
A ninth object of the present invention is to provide a method for detecting saccharin sodium.
The above purpose of the invention is realized by the following technical scheme:
the invention provides a saccharin sodium hapten Ri which has a structural formula shown as a formula (I):
the overlapping degree of the saccharin sodium hapten Ri and the saccharin sodium molecular skeleton structure is high, so that immune induction is facilitated, and the immunogenicity of the saccharin sodium hapten Ri is effectively improved.
The invention also provides a preparation method of the saccharin sodium hapten Ri, which is characterized in that an amino functional group on a benzene ring of saccharin sodium analog 6-saccharin is utilized to react with succinic anhydride to generate amide to obtain the saccharin sodium hapten.
Specifically, the preparation method of the saccharin sodium hapten specifically comprises the following steps:
s1: dissolving 6-aminosaccharide in anhydrous DMF, and reacting in the presence of catalyst K2CO3Reacts with succinic anhydride under the action of (1).
S2: extracting the reaction solution, and collecting an organic phase; extracting the water phase with ethyl acetate, and collecting the organic phase; the combined organic phases were washed with saturated NaCl solution and then with anhydrous Na2SO4Drying and filtering, removing the solvent from the filtrate by reduced pressure distillation, and purifying the residue by silica gel column chromatography to obtain a light yellow solid, wherein the light yellow solid is saccharin sodium hapten.
Wherein; preferably, 6-aminosaccharin, K, as described in step S12CO3The molar ratio of succinic anhydride to succinic anhydride is 1: 1-3: 1.5-3.
More preferably: 6-aminosaccharin, K, as described in step S12CO3And succinic anhydride in a molar ratio of 1:1.5:2.
Preferably, the extracting agents for the extraction in step S2 are ethyl acetate and water.
The invention also provides a saccharin sodium artificial antigen, the structural formula of which is shown as the formula (II):
When the saccharin sodium hapten Ri is coupled with a carrier Protein (Protein), the specific structure of the saccharin sodium hapten Ri protrudes out of the surface of the carrier Protein, and the saccharin sodium hapten Ri is used as an epitope of a carrier and is exposed to an animal immune system, so that a foundation is laid for obtaining an antibody with high specificity and high quality.
The invention also provides a preparation method of the saccharin sodium artificial antigen, which is prepared by coupling the hapten Ri with carrier protein by an active ester method, and the preparation method of the saccharin sodium artificial antigen specifically comprises the following steps:
s1: dissolving saccharin sodium hapten Ri in DMF, adding NHS and EDC, and stirring at 4 ℃ for reacting overnight to obtain solution A;
s2: weighing carrier protein, and dissolving in phosphate buffer solution to obtain solution B; the solution A is added into the solution B drop by drop and reacted for 8h at 4 ℃.
S3: dialyzing the reaction solution at 4 deg.C for 3 days, and changing the dialyzate for 2 times per day to obtain saccharin sodium artificial antigen.
Preferably, the dosage ratio of the saccharin sodium hapten, the NHS and the EDC in the step S1 is 1: 1-2: 1 to 2.
More preferably, the dosage ratio of the saccharin sodium hapten, the NHS and the EDC in the step S1 is 1:1.5: 1.5.
Preferably, the molar ratio of carrier protein to sodium saccharin hapten is 1: 60.
The saccharin sodium antigen is prepared by taking an artificial antigen obtained by coupling a hapten Ri and lactoferrin as an immunogen, and also is prepared by taking an artificial antigen obtained by coupling the hapten Ri and ovalbumin as an envelope antigen.
The saccharin sodium antibody can be a monoclonal antibody, a polyclonal antibody or a genetic engineering antibody, and is preferably a monoclonal antibody.
The preparation method of the saccharin sodium monoclonal antibody comprises the following steps:
immunizing a mouse by using the saccharin sodium antigen Ri-LF as an immunogen, fusing a mouse spleen cell with an SP2/0 myeloma cell to obtain a hybridoma cell, culturing the hybridoma cell in a female Balb/c mouse to obtain ascites containing a high-concentration monoclonal antibody, and purifying the ascites to obtain the high-specificity saccharin sodium resistant monoclonal antibody.
Correspondingly, the antibody prepared by using the saccharin sodium antigen and the application thereof in detecting saccharin sodium are also within the protection scope of the invention.
The enzyme linked immunosorbent assay kit prepared by the saccharin sodium hapten, the saccharin sodium antigen and the saccharin sodium antibody is also within the protection scope of the invention.
The invention also provides a method for detecting saccharin sodium, which takes a conjugate of the saccharin sodium hapten Ri and the chicken ovalbumin as a coating antigen and the saccharin sodium monoclonal antibody as a detection antibody and adopts an indirect competitive ELISA method for detection.
The invention has the following beneficial effects:
the invention firstly provides a saccharin sodium hapten Ri, the saccharin sodium hapten Ri has high overlapping degree with the framework structure of saccharin sodium to be detected, and the immunogenicity of the saccharin sodium hapten Ri is effectively improved; further using an artificial antigen obtained by coupling the saccharin sodium hapten Ri and lactoferrin as an immunogen to immunize a mouse, obtaining hybridoma cells through cell fusion, preparing ascites through in vivo inoculation of the hybridoma cells, and purifying to obtain the monoclonal antibody. The titer, the specificity and the affinity of the obtained antibody are good, the minimum detection limit of the antibody on saccharin sodium established by the antibody is 30.48ng/mL, the IC50 is 489.68ng/mL, and the linear range is 103.26-2322.01 ng/mL; the method has the characteristics of simplicity, convenience, rapidness, strong specificity, wide linear range and high sensitivity, and has good application prospect and wide development space in the rapid and effective detection of saccharin sodium.
Drawings
FIG. 1 is a mass spectrum of saccharin sodium hapten Ri
FIG. 2 is a standard graph of indirect competition ELISA based on monoclonal antibody.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 preparation of sodium saccharin hapten Ri
A preparation method of saccharin sodium hapten Ri comprises the following steps:
1mol of 6-aminosaccharide is dissolved in 50mL of anhydrous DMF solution, and 1.5mol of K is added2CO3Dropwise adding succinic anhydride DMF solution (2mol succinic anhydride is dissolved in 10ml anhydrous DMF), refluxing for 12h at 60 ℃ under magnetic stirring, extracting the reaction liquid with ethyl acetate and water, and collecting an organic phase; extracting the water phase with ethyl acetate, and collecting the organic phase; the combined organic phases were washed with saturated NaCl solution and then with anhydrous Na2SO4Drying and filtering, removing the solvent from the filtrate by reduced pressure distillation, and purifying the residue by silica gel column chromatography to obtain a light yellow solid, wherein the light yellow solid is saccharin sodium hapten Ri, and the structural formula of the light yellow solid is shown in formula (I):
the mass spectrogram of the saccharin sodium hapten Ri is shown in figure 1, wherein 296.9 is the negative ion molecular peak of the saccharin sodium hapten Ri, the calculated relative molecular mass is 298, and the calculated relative molecular mass is consistent with the actual relative molecular mass, which indicates that the saccharin sodium hapten Ri is successfully prepared.
The hapten Ri of the saccharin sodium takes an amino functional group on a benzene ring of the saccharin sodium analog 6-saccharin as an arm extension site, and reacts with succinic anhydride to generate amide, so that the obtained hapten and a skeleton structure of the saccharin sodium to be detected have high overlapping degree, the characteristic structure of the saccharin sodium is exposed, and a foundation is laid for generating a high-specificity antibody;
the overlapping degree of the skeleton structure of the saccharin sodium hapten Ri and the saccharin sodium of a detection object is high, so that the immunogenicity of the saccharin sodium hapten Ri is effectively improved; the skeleton structure of saccharin sodium of the detection object is shown as follows:
example 2 preparation of sodium saccharin Artificial antigen
1. Experimental methods
A method for preparing saccharin sodium artificial antigen comprises the following steps:
coupling the saccharin sodium hapten Ri obtained in the example 1 with egg white albumin OVA (albumin) and lactoferrin LF (lactoferrin) by an active ester method, dissolving the saccharin sodium hapten Ri in DMF, adding 1.5eq NHS and EDC into the solution, and reacting at 4 ℃ overnight to obtain solution A; dropwise adding the solution A into a PBS buffer solution of carrier protein, and continuously reacting for 8 hours at 4 ℃; dialyzing the reaction solution at 4 deg.C for 3 days, changing the dialysate twice per day, dialyzing for 6 times, and collecting the solution in dialysis bag to obtain saccharin sodium artificial antigen (saccharin sodium complete antigen Ri-LF and saccharin sodium complete antigen Ri-OVA). The structural formula of the saccharin sodium artificial antigen is shown as the formula (II):
Example 3 identification of sodium saccharin artificial antigen.
(1) Animal immunization
Healthy 6-week-old Balb/c female mice were used as experimental animals and the complete antigen Ri-LF as immunogen, and were injected subcutaneously into the neck, back and abdominal cavity of the mice at a dose of 0.5mL (containing 0.5mg of immunogen) per immunization. The first immunization is carried out by emulsifying 0.5mL of complete Freund's adjuvant with antigen and then using the emulsified complete Freund's adjuvant for immunization, after 4 weeks, 0.5mL of incomplete Freund's adjuvant is used for enhancing immunization after being emulsified with antigen and then is immunized once every 2 weeks, a small amount of blood is taken from tail vein during the immunization for antibody quality identification, after the antibody is stabilized, the mouse with the best performance is selected for cell fusion, and the mouse with 0.5mg of immunogen is directly injected for one-time additional immunization in abdominal cavity 3 days before the cell fusion.
(2) Evaluation of antiserum Effect
The saccharin sodium complete antigen Ri-OVA prepared in example 2 was used as a coating antigen, the collected mouse serum was used as a detection antibody, the antiserum titer and inhibition rate of the mouse serum were measured by an indirect competitive ELISA method, and the titer and inhibition rate of each antiserum were comprehensively considered and evaluated. The specific operation steps are as follows:
wrapping a board: the saccharin sodium complete antigen Ri-OVA was diluted to 1000ng/mL with 0.05M carbonate buffer (pH9.6) and coated overnight at 4 ℃ in 100. mu.L/well; discarding the coating solution, washing with PBST for 2 times, adding 120 μ L of blocking solution (5% skimmed milk) into each well, and blocking at 37 deg.C for 3 hr; removing the sealing liquid, drying at 37 ℃ for 60min, and packaging with a sealing bag at 4 ℃ for later use to obtain the wrapped ELISA plate.
Serum titer and inhibition detection: the titer of the ELISA plate coated in the step 1) is listed as follows: add 50. mu.L PBS and 50. mu.L serum diluted by gradient multiple (1K, 2K, 4K, 8K, 16K, 32K, 64K) separately to each well; inhibition column: each well was added 50. mu.L of diluted 1000ng/mL drug (saccharin sodium) and 50. mu.L of serum diluted in gradient multiples (1K, 2K, 4K, 8K, 16K, 32K, 64K) to make 2 replicates. Incubating for 40min at 37 ℃, washing for five times by PBST, patting dry the liquid in the hole, adding enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) diluted by 1:5000, incubating for 30min at 37 ℃, washing for five times by PBST, patting dry the liquid in the hole, adding 100 mu L of TMB substrate liquid, and developing for 10min at 37 ℃ in a dark place; add 50. mu.L of stop solution (2M H)2SO4) Terminating the reaction; the absorbance at 450nm was read with a microplate reader.
(3) Results of the experiment
The inhibition detection results of antiserum obtained by immunizing Balb/c female mice are shown in Table 1, and it can be seen that all the immunized mice generate mouse polyclonal antibody, the obtained antiserum has an inhibition effect on saccharin sodium serving as a target analyte, and the inhibition effect of No. 4 mice is most obvious. The hapten prepared by the invention can be used for the subsequent preparation of the saccharin sodium monoclonal antibody and the establishment of an immunodetection method.
TABLE 1 potency and inhibition of antisera
EXAMPLE 4 preparation of monoclonal antibody to sodium saccharin
(1) Cell fusion
Spleen cells of an immunized No. 4 mouse are mixed with mouse myeloma cells (SP2/0) in a logarithmic growth phase, then preheated fusion agent (PEG4000) is slowly added within 45s for fusion, HAT culture medium is used for uniform suspension, a proper amount of feeder cells are added, the mixture is cultured in a 96-well culture plate and cultured in a 5% CO2 incubator at 37 ℃ for 5 days, half liquid change is carried out by using HT culture medium, and full liquid change is carried out after 9 days.
(2) Screening for Positive hybridomas
After the cells are fused, when the cells grow to 1/4 of the area of the culture hole, screening the hybridoma cells by adopting a step screening method; primarily selecting an indirect ELISA method, coating an ELISA plate with a coating antigen (the optimal coating concentration and the positive serum dilution are titrated conventionally by a square matrix method in advance), adding a culture supernatant of a detected hole, incubating, washing, adding goat anti-mouse IgG-HRP, and adding a TMB substrate solution for color reaction; screening the screened positive hole by using an indirect competitive ELISA method, mixing cell supernatant and 1000ng/mL saccharin sodium in equal volume, performing water bath at 37 ℃ for 30min, and adding the mixture into a coated ELISA plate; meanwhile, PBS is used for replacing saccharin sodium as a contrast, and the other steps are the same as the above; OD after blocking by saccharin sodium450nmThe value is reduced to be below 50 percent of the control hole, the control hole is judged to be positive, the control hole is detected to be positive for 2 times and 3 times, and the limiting dilution method is immediately used for subcloning;
(3) expanded culture of hybridoma cells
Carrying out expanded culture on the hybridoma after 2-3 times of subcloning and strain establishment, collecting supernatant, measuring titer by using indirect ELISA, and freezing and storing; injecting 0.5 mL/mouse of liquid paraffin into the abdominal cavity of a Balb/c mouse aged 8-10 weeks, injecting 1-2 × 10 hybridoma cells into the abdominal cavity after 7-10 days6And extracting ascites from the mice 7-10 days later, centrifuging to obtain supernatant, measuring titer, and freezing for later use.
EXAMPLE 5 establishment of an Indirect competitive ELISA Standard Curve based on monoclonal antibodies
1. Coating and sealing
Ri-OVA coating stock was diluted to 500ng/mL with coating solution and coated overnight at 37 ℃. Washing with PBST (0.01M PBS, 0.06% Tween-20(v/v)) twice the next day, adding 2% skimmed milk powder, sealing at 37 deg.C for 3 hr, discarding the sealing solution, oven drying at 37 deg.C for 60min, and packaging with sealing bag at 4 deg.C.
2. Establishment of a Standard Curve
(1) Experimental methods
Adding 50 mu L of saccharin sodium resistant monoclonal antibody with the concentration of 3.5ug/mL and a series of saccharin sodium standard substances with different concentrations into each well of the coated ELISA plate, incubating for 40min at 37 ℃, washing for five times by PBST, patting the liquid in the wells, adding 1:5000 diluted ELISA secondary antibody (goat anti-mouse IgG-HRP), incubating for 40min at 37 ℃, washing for five times by PBST, patting the liquid in the wells, adding 100 mu LTMB substrate liquid, and developing for 10min at 37 ℃ in a dark place; add 50. mu.L of stop solution (2M H)2SO4) Terminating the reaction; the absorbance at 450nm was read with a microplate reader. The concentration of saccharin sodium standard substance is used for the abscissa, B/B0(OD of wells to which sodium saccharin was added450OD450 of wells without saccharin sodium) as ordinate, an indirect competition standard curve was established.
(2) Results of the experiment
An indirect competition ELISA standard curve graph established based on the monoclonal antibody is shown in figure 2, and can be seen that the standard curve is S-shaped, the linear correlation is better, the lowest detection limit of saccharin sodium is 30.48ng/mL, and IC (integrated Circuit)50489.68ng/mL, a linear range of 103.26-2322.01 ng/mL, high sensitivity and wide linear range.
The hapten Ri of the saccharin sodium takes an amino functional group on a benzene ring of the saccharin sodium analog 6-saccharin as an arm extension site, and reacts with succinic anhydride to generate amide, so that the obtained hapten and a skeleton structure of the saccharin sodium to be detected have high overlapping degree, the characteristic structure of the saccharin sodium is exposed, and a foundation is laid for generating a high-specificity antibody; further using the artificial antigen obtained by coupling the carboxyl terminal of the saccharin sodium hapten Ri and lactoferrin as an immunogen to immunize animals, preparing hybridoma monoclonal antibody cell strains through cell fusion, and obtaining saccharinA sodium high specificity monoclonal antibody. An analysis method for detecting the saccharin sodium based on the monoclonal antibody is established by using the antibody, and the minimum detection limit of the saccharin sodium is 30.48ng/mL and IC50489.68ng/mL, and the linear range is 103.26-2322.01 ng/mL; and the preparation method of the saccharin sodium hapten Ri, the artificial antigen and the antibody is simple and easy to implement, low in cost and wide in application prospect.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A saccharin sodium hapten Ri having a structural formula as shown in formula (I):
2. a process for the preparation of the sodium saccharin hapten Ri as claimed in claim 1, which is obtained by reacting the amino group on the benzene ring in the sodium saccharin analog 6-aminosaccharin with succinic anhydride.
3. Use of the saccharin sodium hapten Ri of claim 1 in the preparation of a saccharin sodium artificial antigen.
4. A sodium saccharin artificial antigen based on the sodium saccharin hapten Ri of claim 1, having a structural formula according to formula (II):
5. A process for preparing the artificial saccharin sodium antigen as claimed in claim 4, which comprises coupling the carboxy terminus of the saccharin sodium hapten with carrier protein by active ester method to obtain the artificial saccharin sodium antigen; the method specifically comprises the following steps:
(1) dissolving the saccharin sodium hapten in N, N-Dimethylformamide (DMF), adding 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), and stirring at 4 ℃ to react;
(2) weighing carrier protein, dissolving the carrier protein in a phosphate buffer solution, dropwise adding the reaction solution obtained in the step (1) into the carrier protein phosphate buffer solution, and continuing the reaction at 4 ℃.
(3) Dialyzing the reaction solution at 4 deg.C for 3 days, and changing the dialyzate for 2 times per day to obtain saccharin sodium artificial antigen.
6. The combination of an immunogen and a coating antigen for the enzyme-linked immunosorbent assay (ELISA) of the saccharin sodium hapten Ri according to claim 1, wherein the immunogen is a conjugate of the saccharin sodium hapten Ri and lactoferrin, and the coating antigen is a conjugate of the saccharin sodium hapten Ri and egg albumin.
7. The use of the saccharin sodium hapten Ri of claim 1, the saccharin sodium artificial antigen of claim 4 or the combination of the immunogen and the coating antigen of claim 6 in the detection of saccharin sodium or in the preparation of a saccharin sodium detection kit.
8. A saccharin sodium antibody according to claim 1, which is prepared by using a complete antigen obtained by coupling the saccharin sodium hapten Ri to lactoferrin as an immunogen.
9. A method for detecting saccharin sodium based on claim 1 or based on a saccharin sodium antibody, characterized in that the detection is carried out based on an indirect ELISA method, using a complete antigen obtained by coupling the hapten Ri with chicken ovalbumin as a coating antigen, and using the saccharin sodium antibody as a detection antibody.
10. A saccharin sodium assay kit according to claim 6, comprising a combination of the immunogen and a coating source.
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| B. UNTERHALT ET AL.: "Immunchemosche Bestimmung von Saccharin", 《PHARNAZIE》 * |
| 刘丽 主编, 河南科学技术出版社 * |
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| CN113930398A (en) * | 2021-10-19 | 2022-01-14 | 江南大学 | Hybridoma cell strain secreting anti-saccharin sodium monoclonal antibody and application thereof |
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