WO2020248317A1 - 毒黄素半抗原、人工抗原、抗体及其合成方法和应用 - Google Patents
毒黄素半抗原、人工抗原、抗体及其合成方法和应用 Download PDFInfo
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- WO2020248317A1 WO2020248317A1 PCT/CN2019/095190 CN2019095190W WO2020248317A1 WO 2020248317 A1 WO2020248317 A1 WO 2020248317A1 CN 2019095190 W CN2019095190 W CN 2019095190W WO 2020248317 A1 WO2020248317 A1 WO 2020248317A1
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- WIPO (PCT)
- Prior art keywords
- toxoflavin
- compound
- hapten
- antigen
- toxin
- Prior art date
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- SLGRAIAQIAUZAQ-UHFFFAOYSA-N toxoflavin Chemical compound CN1N=CN=C2C1=NC(=O)N(C)C2=O SLGRAIAQIAUZAQ-UHFFFAOYSA-N 0.000 title claims abstract description 150
- 239000000427 antigen Substances 0.000 title claims abstract description 74
- 108091007433 antigens Proteins 0.000 title claims abstract description 74
- 102000036639 antigens Human genes 0.000 title claims abstract description 74
- 238000010189 synthetic method Methods 0.000 title description 2
- 238000000034 method Methods 0.000 claims abstract description 70
- 241001465754 Metazoa Species 0.000 claims abstract description 12
- 239000003053 toxin Substances 0.000 claims description 42
- 231100000765 toxin Toxicity 0.000 claims description 42
- 108700012359 toxins Proteins 0.000 claims description 42
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 claims description 32
- 230000001900 immune effect Effects 0.000 claims description 24
- 102000014914 Carrier Proteins Human genes 0.000 claims description 19
- 108010078791 Carrier Proteins Proteins 0.000 claims description 19
- 150000001875 compounds Chemical class 0.000 claims description 17
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 15
- 230000002194 synthesizing effect Effects 0.000 claims description 14
- 229940125904 compound 1 Drugs 0.000 claims description 13
- 229940125782 compound 2 Drugs 0.000 claims description 13
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- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 12
- 229940126062 Compound A Drugs 0.000 claims description 12
- NLDMNSXOCDLTTB-UHFFFAOYSA-N Heterophylliin A Natural products O1C2COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC2C(OC(=O)C=2C=C(O)C(O)=C(O)C=2)C(O)C1OC(=O)C1=CC(O)=C(O)C(O)=C1 NLDMNSXOCDLTTB-UHFFFAOYSA-N 0.000 claims description 12
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- 238000000576 coating method Methods 0.000 claims description 11
- 229940126214 compound 3 Drugs 0.000 claims description 9
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 claims description 8
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims description 8
- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 claims description 8
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 8
- 238000010168 coupling process Methods 0.000 claims description 6
- 230000002378 acidificating effect Effects 0.000 claims description 5
- 230000008878 coupling Effects 0.000 claims description 5
- 238000005859 coupling reaction Methods 0.000 claims description 5
- 238000006460 hydrolysis reaction Methods 0.000 claims description 5
- HDZGCSFEDULWCS-UHFFFAOYSA-N monomethylhydrazine Chemical compound CNN HDZGCSFEDULWCS-UHFFFAOYSA-N 0.000 claims description 5
- 125000003754 ethoxycarbonyl group Chemical group C(=O)(OCC)* 0.000 claims description 4
- 230000003053 immunization Effects 0.000 claims description 4
- NIZHERJWXFHGGU-UHFFFAOYSA-N isocyanato(trimethyl)silane Chemical compound C[Si](C)(C)N=C=O NIZHERJWXFHGGU-UHFFFAOYSA-N 0.000 claims description 4
- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 claims description 4
- 235000010288 sodium nitrite Nutrition 0.000 claims description 4
- 238000011896 sensitive detection Methods 0.000 abstract 1
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- 241000209094 Oryza Species 0.000 description 11
- 235000007164 Oryza sativa Nutrition 0.000 description 11
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- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- 108010058846 Ovalbumin Proteins 0.000 description 8
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- 229940092253 ovalbumin Drugs 0.000 description 8
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
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- 238000001308 synthesis method Methods 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 108091006905 Human Serum Albumin Proteins 0.000 description 4
- 102000008100 Human Serum Albumin Human genes 0.000 description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
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- 150000003384 small molecules Chemical class 0.000 description 3
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- 108060003552 hemocyanin Proteins 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- RGOJCHYYBKMRLL-UHFFFAOYSA-N 4-(trifluoromethoxy)benzenesulfonamide Chemical compound NS(=O)(=O)C1=CC=C(OC(F)(F)F)C=C1 RGOJCHYYBKMRLL-UHFFFAOYSA-N 0.000 description 1
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- 208000019331 Foodborne disease Diseases 0.000 description 1
- OWIKHYCFFJSOEH-UHFFFAOYSA-N Isocyanic acid Chemical compound N=C=O OWIKHYCFFJSOEH-UHFFFAOYSA-N 0.000 description 1
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- 229920002534 Polyethylene Glycol 1450 Polymers 0.000 description 1
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- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 1
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- 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 1
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- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
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- SHCXABJSXUACKU-XTXDISFPSA-N isobongkrekic acid Natural products COC(CC=C/C=C/CCC=CCC(C)C=CC(=C/C(=O)O)CC(=O)O)C(=C/C=C(C)/C(=O)O)C SHCXABJSXUACKU-XTXDISFPSA-N 0.000 description 1
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- 229940124547 specific antidotes Drugs 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
Definitions
- the present invention relates to the technical field of immunochemical analysis, in particular to toxoflavin haptens, artificial antigens, antibodies, and synthetic methods and applications thereof.
- Fermented rice noodle poisoning is a bacterial food poisoning with a very high fatality rate in the vast rural areas of our country.
- Pseudomonas cocotoxin fermented rice noodle subspecies is the cause of fermented rice noodle food poisoning and spoiled white fungus poisoning.
- the pathogenic substances of this bacteria are two toxins produced during growth and reproduction: bongkrekic acid. acid, BA) and toxoflavin (TF). Both toxins are small molecules of fatty acids, which are highly toxic, heat-resistant, and highly toxic, and cannot be destroyed by general cooking methods. Therefore, food is easily poisoned after being contaminated. There is currently no specific antidote. The production of fermented acid is much higher than toxoflavin.
- the methods used to detect rice yeast acid mainly include traditional detection methods, such as spectrophotometry, high performance liquid chromatography and their combined technology.
- traditional detection methods such as spectrophotometry, high performance liquid chromatography and their combined technology.
- the equipment is expensive and the detection time is long. , And require professional operation, so it is impossible to achieve real on-site detection.
- the immunological detection and analysis technology has been widely used in the field of drug residue detection due to its high sensitivity, high specificity, rapidity, and easy operation. It has many advantages compared with inspection methods such as instruments. Therefore, the immunoassay provides a new analysis and detection method for the study of the residues of rice yeast acid.
- the key technology is to be able to obtain antibodies with strong specificity and high sensitivity.
- the prerequisite is to synthesize and prepare Appropriate hapten of rice yeast acid.
- the rice yeast acid has a chain structure, and it is difficult for immunized animals to obtain high-quality antibodies.
- Toxaxanthin is a three-dimensional molecule composed of two parallel rings. Compared with orysonic acid, immunized animals are more likely to obtain highly sensitive and specific antibodies.
- the immunodetection of toxoflavin can indirectly detect orycholic acid, which provides a simple and reliable detection scheme for the poisoning of orycholic acid.
- the present invention provides toxoflavin haptens, artificial antigens, antibodies and their synthesis methods and applications.
- Toxoflavin hapten which has the following structural formula:
- n is the number of -CH 2 groups and n is an integer of 1-7.
- the present invention also provides a method for synthesizing the above-mentioned toxoflavin hapten, which includes the following steps:
- the compound 2 has the structural formula
- the compound 4 undergoes a hydrolysis reaction to obtain the toxoflavin hapten.
- the method for synthesizing the compound 1 is: (1) providing compound A, the molecular formula of the compound A is
- the present invention also provides toxaxanthin artificial antigens, which include toxaxanthin immune antigens and toxaxanthin coating antigens, which are obtained by coupling the toxin haptens and carrier proteins.
- the carrier protein is any one of bovine serum albumin (BSA), ovalbumin (OVA), human serum albumin (HAS) or hemocyanin (KLH).
- BSA bovine serum albumin
- OVA ovalbumin
- HAS human serum albumin
- KLH hemocyanin
- the structure of the toxin hapten 2 ⁇ n ⁇ 7; when preparing the toxin-coated antigen, the structure of the toxin hapten, 1 ⁇ n ⁇ 6.
- n is 4 in the structure of the toxin hapten; when preparing the toxin hapten coating antigen, n is 4 in the structure of the toxin hapten.
- the present invention also provides a method for synthesizing the toxaxanthin artificial antigen, which is prepared by coupling the toxin hapten with a carrier protein by a mixed acid anhydride method.
- the present invention also provides a toxaxanthin antibody, which is obtained by immunizing animals with the above-mentioned toxlavin immune antigen.
- sample to be tested is treated with methanol and hydroxylamine hydrochloride, wherein the concentration of the hydroxylamine hydrochloride is 0.2M-0.8M.
- the immunological method is any one of immunological analysis methods including immunochromatography, enzyme-linked immunosorbent assay, time-resolved fluorescence immunoassay, and chemiluminescence immunoassay.
- Et is ethyl
- Ac is acetyl
- Me is methyl
- the fatty chain linking arm is derived from the nitrogen atom at the 6 position of toxaxanthin, and the end of the derived fatty chain linking arm has a carboxyl group.
- the toxin hapten of the present invention retains the characteristics of toxin to the greatest extent
- the structure can better expose the antigenic determinants of the hapten, so that the immunogenicity of the toxoflavin hapten is significantly enhanced, and it has a carboxyl group that can be coupled to the carrier protein; the toxoflavin hapten is coupled to the carrier protein
- the obtained toxaxanthin immune antigen to immunize the animal is more conducive to stimulating the animal immune response to produce more specific and sensitive antibodies.
- the immunological method using this antibody can detect 0.03ng/ml toxin contamination , It provides a simple and quick solution for the rice yeast acidosis. The entire detection process only takes more than ten minutes, which can fully meet the actual detection needs.
- the method for synthesizing the toxicoflavin hapten of the present invention has readily available raw materials, simple reaction operation, and easy control of reaction conditions.
- the method for synthesizing the toxin hapten of the present invention has a simple synthesis route, the purity and yield of the toxin hapten is high, and the overall synthesis cost is more advantageous.
- Figure 1 shows the synthetic route of the toxoflavin hapten of the present invention.
- the present invention provides toxoflavin hapten, which has the following structural formula:
- n is the number of -CH 2 groups and n is an integer of 1-7.
- Toxaxanthin is not immunogenic due to its small molecular weight (molecular weight is less than 1000) and cannot produce antibodies in animals. It must be coupled with macromolecular carrier proteins to prepare artificial antigens to induce the production of specific antibodies. Since there is no active group on the toxaxanthin molecule that can be directly coupled to the carrier protein, it is necessary to introduce a suitable linking arm and an active group coupled to the carrier protein in the appropriate position on the toxoflavin molecule through the derivatization process. .
- the fatty chain linking arm is derived from the nitrogen atom at the 6 position of toxaxanthin, and the end of the derived fatty chain linking arm has a carboxyl group.
- the toxin hapten of the present invention retains the characteristics of toxin to the greatest extent
- the structure can better expose the antigenic determinants of the hapten, so that the immunogenicity of the toxoflavin hapten is significantly enhanced, and it has a carboxyl group that can be coupled to the carrier protein; the toxoflavin hapten is coupled to the carrier protein
- the obtained toxaxanthin immune antigen to immunize the animal is more conducive to stimulating the animal immune response to produce more specific and sensitive antibodies.
- the immunological method using this antibody can detect 0.03ng/ml toxin contamination , It provides a simple and quick solution for the rice yeast acidosis. The entire detection process only takes more than ten minutes, which can fully meet the actual detection needs.
- the toxin hapten of the present invention is not only simple in synthesis method and high in purity, but also can be applied to synthesize an antigen system suitable for animal immunity, which makes up for the blank in the technical field of domestic toxin immunology methods, and is a toxin immunity The further development of detection methods laid the foundation.
- the present invention provides a method for synthesizing the above-mentioned toxoflavin hapten.
- the method for synthesizing the toxin hapten of the above structure includes the following steps:
- the compound 2 has the structural formula
- the substitution reaction between methylhydrazine and compound 1 is based on the principle that those skilled in the art can know it through technical manuals or through conventional experimental methods.
- the present invention does not specifically limit the specific operation of the reaction. Those skilled in the art Routine adjustments can be made according to actual needs.
- compound 2 and formaldehyde undergo a carbonyl amine condensation reaction under acidic conditions to form a carbon-nitrogen double bond.
- the reaction principles of the carbonylamine condensation reaction are all known to those skilled in the art through technical manuals or through conventional experimental methods.
- the present invention does not specifically limit the specific operation of the reaction, and those skilled in the art can make routine adjustments according to actual needs.
- compound 3 undergoes a ring-forming reaction.
- the reaction principles are all known to the skilled person through technical manuals or through conventional experimental methods.
- the present invention does not specifically limit the specific operation of the reaction, and those skilled in the art can follow the actual conditions. Regular adjustments are required.
- the compound 4 undergoes a hydrolysis reaction to obtain the toxoflavin hapten.
- compound 4 undergoes a hydrolysis reaction.
- the reaction principles are all known to the skilled person through technical manuals or through conventional experimental methods.
- the present invention does not specifically limit the specific operation of the reaction, and those skilled in the art can according to actual needs. Make regular adjustments.
- the synthesis method of the compound 1 is: (1) providing compound A, the molecular formula of the compound A is
- the invention rationally designs the synthesis method of the toxin hapten, the synthesis route is simple, the raw materials used are easily available, the reaction operation is relatively simple, and the reaction conditions are easy to control.
- the purity and yield of the toxin hapten are high, and the overall synthesis cost is more advantageous.
- the present invention provides toxaxanthin artificial antigens, which include toxaxanthin immune antigens and toxaxanthin coating antigens, which are obtained by coupling the toxin haptens described above with carrier proteins .
- the carrier protein is any one of bovine serum albumin (BSA), ovalbumin (OVA), human serum albumin (HAS) or hemocyanin (KLH), but is not limited to the materials listed above It may also be other materials that are not listed in this embodiment but are well known to those skilled in the art.
- BSA bovine serum albumin
- OVA ovalbumin
- HAS human serum albumin
- KLH hemocyanin
- n is 4 in the structure of the toxin hapten; when preparing the toxin hapten coating antigen, n is 4 in the structure of the toxin hapten.
- the inventors found that the toxaxanthin-coated antigen is synthesized by the mixed acid anhydride method.
- the toxoflavin-coated antigen n is 4, and the toxoflavin immune antigen is n is 4, the established immunological method has higher Compared with the EDC method to synthesize toxoflavin-coated antigen and other combinations, the detection limit is improved by nearly an order of magnitude, and an unexpected technical effect has been achieved.
- the present invention provides a method for synthesizing toxoflavin artificial antigen, which is prepared by coupling the toxoflavin hapten with a carrier protein by a mixed acid anhydride method, and the steps are as follows:
- the present invention also provides a toxoflavin antibody, which is obtained by immunizing an animal with the above-mentioned toxoflavin immune antigen.
- the present invention provides an immunological method for detecting toxoflavin, which uses the above-mentioned toxoflavin antibody and toxaxanthin to coat the antigen.
- the immunological method is any immunological analysis method including immunochromatography, enzyme-linked immunosorbent assay, time-resolved fluorescence immunoassay, and chemiluminescence immunoassay.
- the treatment liquid is methanol and hydroxylamine hydrochloride, where the concentration of the hydroxylamine hydrochloride is 0.2M-0.8M, which can improve the sensitivity of detection.
- the compound 2 has the structural formula
- the compound 4 undergoes a hydrolysis reaction to obtain the toxoflavin hapten.
- the synthesis method of the compound 1 is: (1) providing compound A, the molecular formula of the compound A is
- Example 2 Mix and emulsify the toxoflavin immune antigen synthesized in Example 2 with an equal volume of Freund’s adjuvant (Freund's complete adjuvant for the first time, and Freund's incomplete adjuvant for the second, third, and fourth time) to immunize Bal B/ In mouse C, ELISA was used to evaluate the immune effect of different toxoflavin immune antigens, and to prepare monoclonal antibodies to toxaxanthin. Table 1 shows the evaluation of different toxaxanthin immune antigen immune serum.
- Example 5 Immunological method for detecting toxoflavin
- nanoparticle-labeled toxoflavin antibody Prepare 40nm colloidal gold particles according to the Frens method (1973); adjust the pH according to the amount of 3 ⁇ L 0.2M potassium carbonate per ml of colloidal gold particles; add 3 ⁇ g toxin per ml of colloidal gold particles Flavin monoclonal antibody, react at room temperature for 15 minutes; add bovine serum albumin (BSA) to a final concentration of 1%, and let stand at room temperature for 15 minutes; centrifuge at 10000 rpm for 10 minutes; discard the supernatant and add 1/10 colloidal gold volume to the resuspension (10mM Tris(PH8.0), 0.5% Tween20, 0.4% casein, 2% sucrose) resuspend the particles;
- BSA bovine serum albumin
- binding pad spray the nanoparticle-labeled toxiflavin antibody on the Osron 8964 glass cellulose membrane according to 5 ⁇ L/cm, dry it at 37°C, and cut it to a width of 0.4cm for use;
- sample pad soak the polyester cellulose film with [100mM Tris(PH8.0), 0.5% Tween20], dry at 37°C, and cut it to 1.2cm width for use;
- test strip Lap and paste the sample pad, bonding pad, chromatography membrane, and absorbent pad onto the PVC bottom plate in turn, cut them into 3.5mm wide test strips, dry and store them for later use;
- test strips (1) Sample processing: Take 10g of sample (fungus, patient vomit, fermented rice noodle products, etc.), add 20ml of sample treatment solution, shake for 1min; centrifuge at 10000rpm for 1min, take the supernatant for testing; 2) Test sample: Insert one end of the sample pad of the test strip into the supernatant to be tested. Note that the binding pad should be on the top of the page and let it stand for 10 minutes to read the result. When the test line does not show color, the result is positive; when the test line goes out, the result is negative.
- Table 2 shows the comparison results of the lower detection limit of the test strip when the samples are processed by different treatment solutions. From the results, the combined application of methanol and hydroxylamine hydrochloride can improve the sensitivity of detection. When the concentration of hydroxylamine hydrochloride is between 0.2M and 0.8M, the lower limit of detection is the best. The application of hydroxylamine hydrochloride (0.4M) alone cannot improve the sensitivity of detection.
- Treatment liquid formula Lower limit of detection 1 20% methanol 25ng/ml 2 20% methanol, 0.1M hydroxylamine hydrochloride 15ng/ml 3 20% methanol, 0.2M hydroxylamine hydrochloride 5ng/ml 4 20% methanol, 0.4M hydroxylamine hydrochloride 0.25ng/ml 6 20% methanol, 0.6M hydroxylamine hydrochloride 1.25ng/ml 7 20% methanol, 0.8M hydroxylamine hydrochloride 15ng/ml 8 20% methanol, 1M hydroxylamine hydrochloride 15ng/ml 9 0.4M Hydroxylamine Hydrochloride 30ng/ml
- Example 6 The influence of using mixed acid anhydride method and EDC method to synthesize toxoflavin coated antigen
- Example 3 The mixed acid anhydride method in Example 3 was used to prepare different toxoflavin-coated antigens, and the immunological method for detecting toxoflavin was established according to the method of Example 5.
- the different toxin-coated antigens prepared by the mixed acid anhydride method See Table 4 for the lower detection limit of immunological methods.
- EDC method to prepare different toxoflavin coating antigens take 20 mg of carrier protein (ovalbumin, OVA) and dissolve in 4ml phosphate buffer (pH 7.4), add 5 mg toxoflavin hapten, mix well, and add 10mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), react at room temperature (25°C) for 2 hours, dialysis to remove small molecules not bound to the carrier protein, and collect The protein after dialysis is the toxoflavin-coated antigen; the immunological method for detecting toxoflavin was established according to the method in Example 5. For the lower detection limit of the immunological method of different toxoflavin-coated antigens prepared by the EDC method, see table 3.
- Example 3 The mixed acid anhydride method in Example 3 was used to prepare the toxoflavin coating antigen (n is 4), the method of Example 4 was used to prepare the toxoflavin antibody (n is 4), and the method of Example 5 was used to establish the detection of toxoflavin.
- a total of 118 samples (45 from patient’s vomitus, 11 from fungus samples, and 62 from fermented rice noodle products) were tested using immunological methods to evaluate the specificity of the method. No false positive results were found, indicating the method Has good specificity.
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Abstract
公开了毒黄素半抗原及毒黄素人工抗原的制备方法,用所制备的人工抗原免疫动物可以获得高特异、高灵敏的抗体。用该方法制备的抗原、抗体,以及样品处理,可为毒黄素的高灵敏检测提供简便快捷的技术途径。
Description
本发明涉及了免疫化学分析技术领域,特别是涉及了毒黄素半抗原、人工抗原、抗体及其合成方法和应用。
酵米面中毒是我国广大农村致死率极高的一种细菌性食物中毒。椰毒假单胞菌酵米面亚种是引起酵米面食物中毒和变质银耳中毒的原因,研究证明该菌的致病物质是其在生长繁殖过程中产生的两种毒素:米酵菌酸(bongkrekic acid,BA)和毒黄素(toxoflavin,TF)。两种毒素均为脂肪酸类的小分子物质,具有剧毒、耐热、毒性强,不能被一般的烹调方法所破坏,故食品被污染后极易引起中毒,目前尚无特效解毒药物,且米酵菌酸的产量远高于毒黄素。
目前用于检测米酵菌酸的方法主要有传统的检测方法,如分光光度法、高效液相色谱法及其联用技术等,以上方法虽然可以精确定量,但由于设备仪器昂贵,检测时间长,且需专业人员操作,因此无法实现真正意义上的现场检测。而免疫学检测分析技术以其高灵敏、特异性高、快速、操作简便等优点在药物残留检测领域已被广泛应用,比起仪器等检验方法有很多优势。所以免疫分析为米酵菌酸残留研究提供了一条新的分析检测方法。
在建立免疫学检测方法并应用该检测方法检测米酵菌酸残留量时,关键技术在于能够获取到特异性强、灵敏度高的抗体,而要实现这一目标,前提条件就是得合成、制备出合适的米酵菌酸半抗原。但是米酵菌酸为链状结构,免疫动物难以获得高质量的抗体。而毒黄素则是由两个并环组成的立体状的分子,相对于米酵菌酸,免疫动物更容易获得高灵敏、高特异的抗体。通过免疫检测毒黄素可以间接检测米酵菌酸,对米酵菌酸的中毒检测提供一种简便可靠的检测方案。但是,目前,国内还没有针对毒黄素半抗原的相关报道。
发明概述
为了弥补已有技术的缺陷,本发明提供毒黄素半抗原、人工抗原、抗体及其合成方法和应用。
问题的解决方案
本发明所要解决的技术问题通过以下技术方案予以实现:
毒黄素半抗原,其具有如下结构式:
其中,n为-CH
2基团数目,n为1-7的整数。
本发明还提供上述毒黄素半抗原的合成方法,包括如下步骤:
S1.甲基肼与化合物1进行反应,得到化合物2,所述化合物1具有结构式
;所述化合物2具有结构式
S2.在酸性条件下,所述化合物2与甲醛进行反应,得到化合物3,所述化合物3 具有结构式
S3.在亚硝酸钠和乙酸存在下,所述化合物3进行反应,得到化合物4,所述化合物4具有结构式
S4.所述化合物4进行水解反应,得到所述毒黄素半抗原。
进一步地,所述化合物1的合成方法为:(1)提供化合物A,所述化合物A的分子式为
NH
2(CH
2)
nCOOEt
;(2)所述化合物A与三甲基硅基异氰酸酯进行反应,得到化合物B,所述化合物B具有结构式
;(3)所述化合物B与丙二酸、乙酸酐进行反应,得到化合物C,所述化合物 C具有结构式
;(4)所述化合物C与三氯氧磷进行反应,得到所述化合物1。
本发明还提供毒黄素人工抗原,所述毒黄素人工抗原包括毒黄素免疫抗原和毒黄素包被抗原,其是由上述毒黄素半抗原与载体蛋白偶联得到的。
进一步地,所述载体蛋白为牛血清白蛋白(BSA),卵清蛋白(OVA),人血清白蛋白(HAS)或血蓝蛋白(KLH)中的任意一种。
进一步地,制备毒黄素免疫抗原时,所述毒黄素半抗原结构中,2≤n≤7;制备毒黄素包被抗原时,所述毒黄素半抗原结构中,1≤n≤6。
进一步地,制备毒黄素免疫抗原时,所述毒黄素半抗原结构中,n为4;制备毒黄素包被抗原时,所述毒黄素半抗原结构中,n为4。
本发明还提供毒黄素人工抗原的合成方法,其是采用混合酸酐法将所述毒黄素半抗原与载体蛋白偶联制备得到。
本发明还提供一种毒黄素抗体,它是由上述毒黄素免疫抗原经动物免疫得到。
一种检测毒黄素的免疫学方法,其采用上述毒黄素抗体。
进一步地,待测样品经甲醇和盐酸羟胺处理,其中,所述盐酸羟胺的浓度为0.2M-0.8M。
进一步地,所述免疫学方法为免疫层析法、酶联免疫吸附试验、时间分辨荧光免疫分析、化学发光免疫分析在内的任意一种免疫学分析方法。
特别说明,本申请文件中化学结构式中Et为乙基;Ac为乙酰基;Me为甲基。
发明的有益效果
本发明具有如下有益效果:
本发明中,在毒黄素6位上的氮原子上衍生脂肪链连接臂,衍生的脂肪链连接臂末段带有羧基,本发明的毒黄素半抗原最大程度保留了毒黄素的特征结构,能更好地暴露半抗原的抗原决定簇,使得毒黄素半抗原的免疫原性明显增强,又具有可以与载体蛋白发生偶联的羧基;该毒黄素半抗原与载体蛋白偶联后得到的毒黄素免疫抗原去免疫动物,更有利于刺激动物免疫应答产生特异性更强、灵敏度更高的抗体,采用该抗体的免疫学方法可以检测出0.03ng/ml的毒黄素污染,为米酵菌酸中毒提供了简便快捷的解决方案,整个检测流程只需要十多分钟,完全能够满足实际检测需求。
本发明中毒黄素半抗原的合成方法,使用的原料易得,反应操作较为简单,反应条件易于控制。本发明的毒黄素半抗原合成方法,合成路线简单,毒黄素半抗原的纯度和收率高,整体的合成成本更具优势。
对附图的简要说明
图1为本发明毒黄素半抗原的合成路线。
发明实施例
第一方面,本发明提供毒黄素半抗原,其具有如下结构式:
其中,n为-CH
2基团数目,n为1-7的整数。
人工抗原的合成是免疫检测中抗体制备和建立免疫分析方法最关键的步骤,半抗原的合成是人工抗原合成的关键。毒黄素因分子量小而不具有免疫原性(分子量小于1000),不能在动物体内产生抗体,必须与大分子载体蛋白偶联制备人工抗原,才能诱导特异性抗体的产生。由于毒黄素分子上没有可以直接与载 体蛋白偶联的活性基团,因此需先通过衍生过程在毒黄素小分子上适当的位置引入合适的连接臂和与载体蛋白偶联的活性基团。不同结构的连接臂、不同的偶联方法或连接臂的引入位置,都影响后续建立的免疫学方法检测的灵敏度和特异性。目前,以毒黄素衍生物为半抗原制备合成抗体和人工抗原进行检测毒黄素的免疫学方法鲜有报道。
本发明中,在毒黄素6位上的氮原子上衍生脂肪链连接臂,衍生的脂肪链连接臂末段带有羧基,本发明的毒黄素半抗原最大程度保留了毒黄素的特征结构,能更好地暴露半抗原的抗原决定簇,使得毒黄素半抗原的免疫原性明显增强,又具有可以与载体蛋白发生偶联的羧基;该毒黄素半抗原与载体蛋白偶联后得到的毒黄素免疫抗原去免疫动物,更有利于刺激动物免疫应答产生特异性更强、灵敏度更高的抗体,采用该抗体的免疫学方法可以检测出0.03ng/ml的毒黄素污染,为米酵菌酸中毒提供了简便快捷的解决方案,整个检测流程只需要十多分钟,完全能够满足实际检测需求。
本发明的毒黄素半抗原,不仅合成方法简便、纯度较高,而且能应用于合成适于动物免疫的抗原体系,弥补了国内毒黄素免疫学方法技术领域的空白,为毒黄素免疫检测方法的进一步发展奠定了基础。
第二方面,本发明提供上述毒黄素半抗原的合成方法。
具体地,上述结构的毒黄素半抗原的合成方法包括如下步骤:
S1.甲基肼与化合物1进行反应,得到化合物2,所述化合物1具有结构式
;所述化合物2具有结构式
此步骤中,甲基肼与化合物1进行取代反应,其原理都为本技术人员均可通过技术手册得知或通过常规实验方法获知,本发明对反应的具体操作不作具体限定,本领域技术人员可以根据实际需要进行常规调整。
S2.在酸性条件下,所述化合物2与甲醛进行反应,得到化合物3,所述化合物3具有结构式
此步骤中,化合物2和甲醛在酸性条件下进行羰胺缩合反应,形成碳氮双键。羰胺缩合反应的反应原理都为本技术人员均可通过技术手册得知或通过常规实验方法获知,本发明对反应的具体操作不作具体限定,本领域技术人员可以根据实际需要进行常规调整。
S3.在亚硝酸钠和乙酸存在下,所述化合物3进行反应,得到化合物4,所述化合物4具有结构式
此步骤中,化合物3进行成环反应,反应原理都为本技术人员均可通过技术手册得知或通过常规实验方法获知,本发明对反应的具体操作不作具体限定,本领域技术人员可以根据实际需要进行常规调整。
S4.所述化合物4进行水解反应,得到所述毒黄素半抗原。
此步骤中,化合物4进行水解反应,反应原理都为本技术人员均可通过技术手册得知或通过常规实验方法获知,本发明对反应的具体操作不作具体限定,本领域技术人员可以根据实际需要进行常规调整。
其中,所述化合物1的合成方法为:(1)提供化合物A,所述化合物A的分子式为
NH
2(CH
2)
nCOOEt
;(2)所述化合物A与三甲基硅基异氰酸酯进行反应,得到化合物B,所述化合物B具有结构式
;(3)所述化合物B与丙二酸、乙酸酐进行反应,得到化合物C,所述化合物C具有结构式
;(4)所述化合物C与三氯氧磷进行反应,得到所述化合物1。
本发明根据毒黄素的结构特点,合理设计毒黄素半抗原的合成方法,合成路线 简单,使用的原料易得,反应操作较为简单,反应条件易于控制。本发明的毒黄素半抗原合成方法,毒黄素半抗原的纯度和收率高,整体的合成成本更具优势。
第三方面,本发明提供毒黄素人工抗原,所述毒黄素人工抗原包括毒黄素免疫抗原和毒黄素包被抗原,其是由上述毒黄素半抗原与载体蛋白偶联得到的。
所述载体蛋白为牛血清白蛋白(BSA),卵清蛋白(OVA),人血清白蛋白(HAS)或血蓝蛋白(KLH)中的任意一种,但不限于前面所列举的几种材料,也可以是其他未列举在本实施例中的但被本领域技术人员所熟知的其他材料。
其中,制备毒黄素免疫抗原时,所述毒黄素半抗原结构中,2≤n≤7;制备毒黄素包被抗原时,所述毒黄素半抗原结构中,1≤n≤6。
更优选地,制备毒黄素免疫抗原时,所述毒黄素半抗原结构中,n为4;制备毒黄素包被抗原时,所述毒黄素半抗原结构中,n为4。发明人发现,采用混合酸酐法合成毒黄素包被抗原,当毒黄素包被抗原中,n为4,而毒黄素免疫抗原中n为4时,所建立的免疫学方法具有更高的灵敏度,较之EDC法合成毒黄素包被抗原和其它组合,检测下限提高了近一个数量级,取得了预料不到的技术效果。
第四方面,本发明提供毒黄素人工抗原的合成方法,其是采用混合酸酐法将所述毒黄素半抗原与载体蛋白偶联制备得到,步骤如下:
称取5mg毒黄素半抗原,溶于1ml DMF中,置于4℃下10min,加入8.5μl三乙胺,再加入6.3ul氯甲酸异丁酯,4℃搅拌反应30min,得甲液;
取20mg OVA溶于1.8ml 50%DMF中,预冷至4℃,置于冰浴不断搅拌,用1M NaOH调节PH至8.0,得毒黄素人工抗原。
发明人发现,采用混合酸酐法合成毒黄素包被抗原,相较于EDC法合成毒黄素包被抗原,所建立的免疫学方法具有更高的灵敏度。
第五方面,本发明还提供一种毒黄素抗体,它是由上述毒黄素免疫抗原经动物免疫得到。
第六方面,本发明提供一种检测毒黄素的免疫学方法,其采用上述毒黄素抗体和毒黄素包被抗原。
所述免疫学方法为免疫层析法、酶联免疫吸附试验、时间分辨荧光免疫分析、 化学发光免疫分析在内的任意一种免疫学分析方法。
检测毒黄素的免疫学方法中,需要采用处理液对待测样品进行处理。发明人在实践中发现,处理液为甲醇和盐酸羟胺,其中,所述盐酸羟胺的浓度为0.2M-0.8M时,可以提高检测的灵敏性。
下面结合实施例对本发明进行详细的说明,实施例仅是本发明的优选实施方式,不是对本发明的限定。
实施例1:毒黄素半抗原的合成
S1.甲基肼与化合物1进行反应,得到化合物2,所述化合物1具有结构式
;所述化合物2具有结构式
S2.在酸性条件下,所述化合物2与甲醛进行反应,得到化合物3,所述化合物3具有结构式
S3.在亚硝酸钠和乙酸存在下,所述化合物3进行反应,得到化合物4,所述化合物4具有结构式
S4.所述化合物4进行水解反应,得到所述毒黄素半抗原。
其中,所述化合物1的合成方法为:(1)提供化合物A,所述化合物A的分子式为
NH
2(CH
2)
nCOOEt
;(2)所述化合物A与三甲基硅基异氰酸酯进行反应,得到化合物B,所述化合物B具有结构式
;(3)所述化合物B与丙二酸、乙酸酐进行反应,得到化合物C,所述化合物C具有结构式
实施例2:毒黄素免疫抗原的合成
取载体蛋白(牛血清白蛋白,BSA)20mg溶于4ml磷酸盐缓冲液(pH7.4)中,加入5mg毒黄素半抗原,混匀,加入10mg EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐),室温(25℃)反应2小时,透析除去未结合到载体蛋白上的小分子,收集透析后的蛋白质即为毒黄素免疫抗原。
实施例3:毒黄素包被抗原的合成(混合酸酐法)
称取5mg毒黄素半抗原,溶于1ml DMF中,置于4℃下10min,加入8.5μl三乙胺,再加入6.3ul氯甲酸异丁酯,4℃搅拌反应30min,得甲液;
取20mg OVA溶于1.8ml 50%DMF中,预冷至4℃,置于冰浴不断搅拌,用1M NaOH调节PH至8.0,得毒黄素包被抗原。
实施例4:毒黄素抗体
将实施例2合成的毒黄素免疫抗原与等体积弗氏佐剂(首次用弗氏完全佐剂,第二、三、四次用弗氏不完全佐剂)混合、乳化,免疫Bal B/C鼠,采用ELISA评价不同毒黄素免疫抗原的免疫效果,并制备毒黄素单克隆抗体。表1为不同毒黄素免疫抗原免疫血清评价情况。
其中,毒黄素单克隆抗体的制备流程为:
(1)融合前3天,用50μg抗原经腹腔加强免疫一次;
(2)融合前一天,制备饲养细胞,按照100μL/孔铺到96孔板中;
(3)摘眼球取血,脱颈处死,于75%酒精中浸泡5min;
(4)取出脾脏,于纱网上碾磨,显微镜下计数;
(5)1200rpm,5min,弃掉上清;
(6)按照1个骨髓瘤细胞对应5个脾细胞的比例加入骨髓瘤细胞,补加1640培养基至30mL,充分混匀;
(7)1200rpm,10min,弃上清;
(8)轻弹管底,充分重悬细胞;
(9)于37℃下,1min内沿管壁加入50%PEG1450 1mL;
(10)缓慢加入25mL 1640培养基;
(11)900rpm,7min,弃上清;
(12)120mL HAT培养液重悬细胞,按照100μL/孔铺到准备好的饲养细胞板中;于37℃ 5%二氧化碳培养箱中培养;
(13)第8天用HT培养基换液;
(14)第10天ELISA检测;
(15)用完全培养基稀释ELISA检测呈强阳性的孔中的细胞,使细胞浓度为5cells/mL;
(16)将细胞悬液按照200μL/孔分装到96孔板中,置于37℃ 5%二氧化碳培养箱中培养;
(17)待微孔中的细胞长到约100cells/孔时,进行ELISA检测;
(18)再次进行克隆筛选,直至所有有细胞的孔均呈阳性反应;
(19)随后转入24孔细胞培养板进行培养,随后用细胞培养瓶进行培养;
(20)按照100万个细胞/只小鼠的剂量腹腔注射BalB/C小鼠;
(21)收集腹水,纯化其中的抗体。
表1用不同免疫抗原的免疫学清与不同包被抗原的反应情况
实施例5:检测毒黄素的免疫学方法
1.纳米颗粒标记毒黄素抗体的制备:按照Frens法(1973)制备40nm胶体金颗粒;按照每毫升胶体金颗粒加入3μL 0.2M碳酸钾的量调节PH;按照每毫升胶体金颗粒加入3μg毒黄素单克隆抗体,室温反应15min;加入牛血清白蛋白(BSA),使其终浓度为1%,室温静置15min;10000rpm离心10min;弃掉上清,加入1/10胶体金体积的重悬液(10mM Tris(PH8.0),0.5%Tween20、0.4%酪蛋白、2%蔗糖)重悬颗粒;
2.结合垫的制备:按照5μL/cm喷纳米颗粒标记毒黄素抗体于奥斯龙8964玻璃纤维素膜上,置于37℃烘干,裁成0.4cm宽,备用;
3.层析膜的制备:按照1μL/cm喷涂浓度为1mg/mL的毒黄素包被抗原于硝酸纤维素膜(Millipore 135)上,作为检测线;按照1μL/cm喷涂浓度为0.2mg/mL的羊抗鼠IgG作为质控线,置于室温晾干,备用;
4.样品垫的制备:用[100mM Tris(PH8.0),0.5%Tween20]浸泡聚酯纤维素膜,置于37℃烘干,裁成1.2cm宽,备用;
5.试纸条的组装:将样品垫、结合垫、层析膜、吸水垫依次搭接粘贴到PVC底板上,裁成3.5mm宽的试纸条,干燥保存,备用;
6.试纸条的使用:(1)样品处理:取样品(木耳、病人呕吐物、发酵米面制品等)10g,加入20ml样品处理液,振摇1min;10000rpm离心1min,取上清检测;(2)检测样品:将试纸条样品垫一端插入待检上清中,注意结合垫应在页面上方,静置10min,判读结果。当检测线不显色时,结果为阳性;当检测线出线时,结果为阴性。
表2为不同处理液处理样品时,试纸条的检测下限比对结果。从结果来看,联合应用甲醇和盐酸羟胺可以提高检测的灵敏性,其中盐酸羟胺的浓度在0.2M-0.8M之间时,检测下限最佳。而单独应用盐酸羟胺(0.4M),则不能改善检测的灵敏性。
表2不同处理液对于检测下限的影响
处理液 | 配方 | 检测下限(LOD) |
1 | 20%甲醇 | 25ng/ml |
2 | 20%甲醇,0.1M盐酸羟胺 | 15ng/ml |
3 | 20%甲醇,0.2M盐酸羟胺 | 5ng/ml |
4 | 20%甲醇,0.4M盐酸羟胺 | 0.25ng/ml |
6 | 20%甲醇,0.6M盐酸羟胺 | 1.25ng/ml |
7 | 20%甲醇,0.8M盐酸羟胺 | 15ng/ml |
8 | 20%甲醇,1M盐酸羟胺 | 15ng/ml |
9 | 0.4M盐酸羟胺 | 30ng/ml |
实施例6:采用混合酸酐法和EDC法合成毒黄素包被抗原的影响
采用实施例3中的混合酸酐法制备不同的毒黄素包被抗原,按照实施例5的方法建立检测毒黄素的免疫学方法,采用混合酸酐法制备的不同的毒黄素包被抗原的免疫学方法的检测下限参见表4。
采用EDC法制备不同的毒黄素包被抗原:取载体蛋白(卵清蛋白,OVA)20mg溶于4ml磷酸盐缓冲液(pH7.4)中,加入5mg毒黄素半抗原,混匀,加入10mg EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐),室温(25℃)反应2小时,透析除去未结合到载体蛋白上的小分子,收集透析后的蛋白质即为毒黄素包被抗 原;按照实施例5的方法建立检测毒黄素的免疫学方法,采用EDC法制备的不同的毒黄素包被抗原的免疫学方法的检测下限参见表3。
我们发现,采用混合酸酐法制备的包被抗原与某些抗体组合可以建立更为灵敏的免疫层析法,与表3结果相比,当包被抗原(n=4)、免疫抗原(n=4)时,所建立的方法具有很高的灵敏性。
表3包被抗原、抗体对于检测检测下限(LOD)的影响(单位:ng/ml)
表4混合酸酐法制备的包被抗原与不同抗体组合的免疫层析法检测下限(LOD)(单位:ng/ml)
实施例7:特异性评价
采用实施例3中的混合酸酐法制备毒黄素包被抗原(n为4),采用实施例4的方法制备毒黄素抗体(n为4),按照实施例5的方法建立检测毒黄素的免疫学方法检测阴样本,评价该方法的特异性,一共检测了118份样本(其中病人呕吐物45份、木耳样品11份、发酵米面制品62份),没有假阳性结果出现,说明该方法具有良好的特异性。
以上所述实施例仅表达了本发明的实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制,但凡采用等同替换或等效变换的形式所获得的技术方案,均应落在本发明的保护范围之内。
Claims (10)
- 毒黄素人工抗原,其特征在于,所述毒黄素人工抗原包括毒黄素免疫抗原和毒黄素包被抗原,其是由权利要求1所述的毒黄素半抗原与载体蛋白偶联得到的。
- 如权利要求4所述的毒黄素人工抗原,其特征在于,制备毒黄素免疫抗原时,所述毒黄素半抗原结构中,2≤n≤7;制备毒黄素包被抗原时,所述毒黄素半抗原结构中,1≤n≤6。
- 如权利要求4所述的毒黄素人工抗原,其特征在于,制备毒黄素免疫抗原时,所述毒黄素半抗原结构中,n为4;制备毒黄素包被抗原时,所述毒黄素半抗原结构中,n为4。
- 如权利要求4所述的毒黄素人工抗原的合成方法,其是采用混合酸酐法将所述毒黄素半抗原与载体蛋白偶联制备得到。
- 一种毒黄素抗体,其特征在于,其是由权利要求4所述的毒黄素免疫抗原经动物免疫得到。
- 一种检测毒黄素的免疫学方法,其特征在于,其采用权利要求8所述的毒黄素抗体。
- 如权利要求9所述的免疫学方法,其特征在于,待测样品经甲醇和盐酸羟胺处理,其中,所述盐酸羟胺的浓度为0.2M-0.8M。
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