CN113896743B - Dipterex hapten, preparation method thereof, antigen, antibody and application thereof - Google Patents
Dipterex hapten, preparation method thereof, antigen, antibody and application thereof Download PDFInfo
- Publication number
- CN113896743B CN113896743B CN202111299579.2A CN202111299579A CN113896743B CN 113896743 B CN113896743 B CN 113896743B CN 202111299579 A CN202111299579 A CN 202111299579A CN 113896743 B CN113896743 B CN 113896743B
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- Prior art keywords
- trichlorfon
- hapten
- dipterex
- antibody
- antigen
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/28—Phosphorus compounds with one or more P—C bonds
- C07F9/38—Phosphonic acids RP(=O)(OH)2; Thiophosphonic acids, i.e. RP(=X)(XH)2 (X = S, Se)
- C07F9/40—Esters thereof
- C07F9/4003—Esters thereof the acid moiety containing a substituent or a structure which is considered as characteristic
- C07F9/4006—Esters of acyclic acids which can have further substituents on alkyl
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- 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
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- 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
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- 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
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- 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
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/548—Carbohydrates, e.g. dextran
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/10—Insecticides
Abstract
The invention relates to the technical field of drug immunodetection synthesis, and in particular discloses a trichlorfon hapten, a trichlorfon hapten intermediate, a synthesis method thereof, a trichlorfon antigen based on the trichlorfon hapten, a trichlorfon antibody specific to the trichlorfon antigen, application thereof and the like. When the dipterex antigen and the dipterex antibody are applied to the dipterex colloidal gold chromatography detection device, the sensitivity can reach 100 mug/kg.
Description
Technical Field
The invention relates to the technical field of drug immunodetection synthesis, in particular to a high-purity trichlorfon hapten as well as a synthesis method and application thereof.
Background
Trichlorfon is an organophosphorus insecticide, belongs to phosphate, is white crystalline powder, and has low volatility. In 1952, trichlorfon was synthesized by Bayer in Germany, and has been used in China for more than 50 years, and has wide application range in agriculture, and is mainly used for preventing and controlling cabbage caterpillar, sang Ye silkworm, weevil, fruit fly and other various pests. However, due to its toxicity, trichlorfon was listed as a class 3 carcinogen by the world health organization international cancer research institute, tanglong et al, at day 27 of 10 in 2017, which showed that trichlorfon had reproductive toxicity to male animals.
The dipterex can be detected and analyzed by catalytic photometry, gas chromatography mass spectrometry, liquid chromatography mass spectrometry, etc.
The catalytic photometry needs to be heated in a water bath, and the color development is easily interfered by metal ions; the gas chromatography needs a plurality of links such as conversion, extraction and the like, is complex to operate and is easy to introduce errors; the gas chromatography mass spectrometry needs to perform water sample pretreatment, and is used for measuring after the trichlorfon is converted into dichlorvos, so that the operation is complicated, or the decomposition products of the trichlorfon are measured, but the deviation and the qualitative errors of the measurement result can be caused; fan Jun adopts liquid chromatography mass spectrometry of sample injection after extraction, which takes a long time and pollutes the environment by the reagents used for extraction and purification. The sensitivity of chromatography is higher than other methods, which can solve part of the practical problems with higher accuracy. However, in general, the sample pretreatment process of these physicochemical analysis methods is quite complex, the required instruments are also relatively expensive, the efficiency is relatively low, the analysis cost is relatively high, and the limitation on the physicochemical properties of the object to be measured is strict. These methods do not respond quickly to the problem of trichlorfon residue in animal edible tissues, nor are they suitable as a primary screening method for trichlorfon residue in edible animal tissues.
Aiming at the trend of stricter and stricter food safety requirements in China at present, a method for measuring the residual quantity of trichlorfon in animal-derived food, which has high sensitivity, strong specificity, simplicity and easiness, is established, and has great significance for strengthening the trade import and export of food. The immunoassay method overcomes the defects of a physicochemical detection method, and has the most outstanding advantages of small sampling amount, simple pretreatment, large capacity, low instrumentation degree, high sensitivity, and the analysis efficiency which can reach tens of times of that of a chromatographic analysis method, thereby being applicable to rapid screening detection of large-scale sample trichlorfon residues. The method can be used for simultaneously measuring a large number of samples and is used for primary screening of large-batch detection; the investment is low, the operation is simple and convenient, and the method is suitable for basic supervision departments.
When an immunological detection method is established and the detection method is applied to detect the residual quantity of the trichlorfon, the key technology is that the antibody with strong specificity and high sensitivity can be obtained, and the aim is to be realized if the proper trichlorfon hapten is prepared. However, in this respect, the reports on the correlation are very limited. Therefore, there is a need to further explore new synthetic methods for dipterex haptens and the new dipterex haptens thus obtained, to facilitate rapid mass screening of dipterex residues.
Disclosure of Invention
In order to overcome the defects in the prior art, the inventor develops a method for synthesizing the trichlorfon hapten aiming at the structural characteristics of trichlorfon, and obtains a novel trichlorfon hapten, antigen and antibody thereof. By utilizing the trichlorfon hapten, the trichlorfon antigen and the trichlorfon antibody, trichlorfon in a sample can be effectively detected in an immunodetection mode.
Accordingly, in a first aspect of the present invention there is provided a trichlorfon hapten of formula (I):
wherein the R is 1 Selected from C 0 -C 6 An alkylene group; r is R 2 Selected from C 1 -C 6 An alkylene group.
In a second aspect of the invention, there is provided an intermediate for use in the manufacture of the dipterex hapten of the first aspect of the invention having the structural formula (II):
wherein R is 1 Selected from C 0 -C 6 An alkylene group.
In a third aspect of the invention there is provided a method of preparing a trichlorfon hapten according to the first aspect of the invention, the method comprising the steps of:
step 1: at low temperature, phosphorus trichloride and the structural formula are shown as followsThe compound 1 of (2) is subjected to reduced pressure distillation to obtain an intermediate 1:
wherein R is 1 Selected from C 0 -C 6 An alkylene group;
step 2: reacting said intermediate 1 with anhydrous methanol and pyridine under basic conditions at low temperature, to give intermediate 2:
step 3: reacting the intermediate 2 with chloral under the condition of an acid binding agent to obtain an intermediate 3:
step 4: bringing said intermediate 3 into contact with a structural formula as shown inThe compound 2 of (2) is reacted in the presence of an initiator to obtain the trichlorfon hapten shown in the formula (I):
wherein R is 2 Selected from C 1 -C 6 An alkylene group.
In a fourth aspect of the invention there is provided a trichlorfon antigen, the immunizing antigen comprising: the dipterex hapten of the first aspect of the invention and a carrier protein coupled to the dipterex hapten.
In a fifth aspect of the invention there is provided a trichlorfon antibody specific for the trichlorfon antigen of the fourth aspect of the invention.
In a sixth aspect of the invention there is provided the use of a trichlorfon hapten according to the first aspect of the invention, a trichlorfon antigen according to the fourth aspect of the invention, a trichlorfon antibody according to the fifth aspect of the invention in a trichlorfon immunoassay.
In a seventh aspect of the present invention, there is provided a dipterex colloidal gold chromatography detection device, comprising a test strip and a reaction cup, wherein the test strip comprises a reaction membrane, the reaction membrane is provided with a detection line and a quality control line, the detection line is coated with the dipterex antigen of the fourth aspect of the present invention, and the reaction cup contains the dipterex antibody of the fifth aspect of the present invention labeled with colloidal gold.
In an eighth aspect of the invention, there is provided a method of detecting trichlorfon in a sample, the method comprising: the colloidal gold chromatography detection apparatus of the seventh aspect of the invention is brought into contact with a sample.
The invention has the following beneficial effects:
according to the invention, on the basis of not changing the structure of trichlorfon, trichlorfon derivative with olefin is synthesized, and trichlorfon hapten with carboxyl connecting arm is derived through click reaction. The synthesized trichlorfon hapten not only maintains the characteristic structure of trichlorfon to the greatest extent, but also has carboxyl which can be coupled with carrier protein, so that trichlorfon antigen with immunogenicity can be obtained after the trichlorfon hapten is coupled with the carrier protein.
The dipterex antigen provided by the invention can stimulate animals to carry out immune response so as to generate antibodies with stronger specificity, higher sensitivity and long shelf life.
The synthesis method of the trichlorfon hapten has the advantages of easily available raw materials, simple reaction operation and easily controlled reaction conditions.
The method for synthesizing the dipterex hapten has high purity and yield of the dipterex hapten, the yield of the synthesized hapten can reach more than 50 percent, and the overall synthesis cost is more advantageous.
Drawings
FIG. 1 is a mass spectrum of the dipterex hapten of the present invention.
Fig. 2 is a graph of the uv absorption profile of the trichlorfon antigen of the present invention.
Fig. 3 is a schematic diagram of a colloidal gold detection apparatus prepared in example 4.
Fig. 4 is a schematic diagram of a result determination according to an embodiment of the present invention.
Detailed Description
The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments that can be obtained by a person skilled in the art based on the embodiments of the present invention are within the scope of the present invention.
The immunological detection analysis method is an analysis method for detecting various substances (drugs, hormones, proteins, microorganisms, etc.) by utilizing the specific binding of antigen and antibody, and the key to establishing an immunological detection analysis method for a small molecular compound is to be able to produce an antibody having high affinity and high specificity for the small molecular compound, while the key to producing such an antibody is the design and synthesis of an artificial antigen or an artificial hapten. In particular to the invention, the key step of establishing an immunological detection and analysis method of the trichlorfon is to design and synthesize a proper trichlorfon hapten.
Accordingly, in a first aspect, the present invention provides a trichlorfon hapten having the structural formula shown in formula (I):
wherein the R is 1 Selected from C 0 -C 6 An alkylene group; the R is 2 Selected from C 1 -C 6 An alkylene group.
Specifically, the R 1 May not be present (i.e. when R 1 Is C 0 When) is either-CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -、-(CH 2 ) 4 -、-(CH 2 ) 5 -or- (CH) 2 ) 6 -；R 2 Can be-CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -、-(CH 2 ) 4 -、-(CH 2 ) 5 -or- (CH) 2 ) 6 -。
In a preferred embodiment, R 1 Is- (CH) 2 ) 2 -，R 2 Is- (CH) 2 ) 2 -at this point, the trichlorfon hapten has the structural formula (I-1) shown below:
as understood by those skilled in the art, by hapten is meant a class of small molecule substances: it alone does not induce an immune response, i.e. is not immunogenic, but it is immunogenic when crosslinked or conjugated to a carrier such as a macromolecular protein or non-antigenic polylysine, thereby inducing an immune response. Such small molecule substances may bind to the response-effect product and be antigenic, i.e. immunoreactive, but not immunogenic.
The molecular structural formula of trichlorfon is shown as follows:
trichlorfon itself does not possess antigenic or hapten properties. Thus, if antibodies to trichlorfon are to be produced, trichlorfon molecules need to be engineered first. In the present invention, the inventor makes a methoxy group on the trichlorfon molecule on the basis of not changing trichlorfon structureRadical (CH) 3 -O-) reacts with enols to synthesize dipterex derivatives with olefins, which are then rendered hapten by click reaction with carboxyl linker arms. Experiments show that the dipterex hapten and the carrier protein are coupled to obtain the dipterex antigen, and the dipterex hapten and the carrier protein can stimulate animals to carry out immune response after being immunized, and generate antibodies with stronger specificity and higher sensitivity, thereby providing a foundation for the subsequent establishment of various immune analysis methods of dipterex.
The dipterex hapten disclosed by the invention is simple and convenient in synthesis method and higher in purity, can be applied to synthesis of an antigen system suitable for animal immunity, fills up the blank in the technical field of a domestic dipterex immunology detection method, and lays a foundation for further development of the dipterex immunodetection method.
In a second aspect, the present invention provides an intermediate for the preparation of the dipterex hapten of the first aspect of the invention having the structural formula (II):
wherein R is 1 Selected from C 0 -C 6 An alkylene group.
Specifically, the R 1 May not be present (i.e. when R 1 Is C 0 When) is selected from-CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -、-(CH 2 ) 4 -、-(CH 2 ) 5 -or- (CH) 2 ) 6 -。
In a preferred embodiment, R 1 Is- (CH) 2 ) 2 In this case, the intermediate of the trichlorfon hapten has the following structural formula (II-1):
from the process according to the third aspect of the invention, which is described in detail below, it is known that the final product (I), the trichlorfon hapten according to the invention, can be prepared directly from this intermediate.
In a third aspect, the present invention also provides a method of preparing the dipterex hapten of the first aspect of the invention comprising the steps of:
step 1: at low temperature, phosphorus trichloride and the structural formula areThe compound 1 of (2) is subjected to reduced pressure distillation to obtain an intermediate 1:
wherein R is 1 Selected from C 0 -C 6 An alkylene group;
step 2: reacting the intermediate 1 with anhydrous methanol and pyridine under alkaline conditions at low temperature to obtain an intermediate 2:
step 3: reacting the intermediate 2 with chloral under the condition of an acid binding agent to obtain an intermediate 3:
step 4: bringing said intermediate 3 into contact with a structural formula as shown inThe compound 2 of (2) is reacted in the presence of an initiator to obtain the trichlorfon hapten shown in the formula (I):
wherein R is 2 Selected from C 1 -C 6 An alkylene group.
The radicals R of this aspect of the invention 1 And R is 2 The radicals R as referred to in the first aspect of the invention 1 And R is 2 The same shall be said, and therefore the definition thereof will be specifically referred to the relevant description of the first aspect of the present invention, and will not be repeated here.
The specific reaction process of the method is as follows:
in a preferred embodiment, in step 1, the molar ratio of phosphorus trichloride to compound 1 is 1 (1-1.1).
In a preferred embodiment, in step 1, the reaction system is filled with nitrogen, and the dropwise addition of compound 1 is started after the phosphorus trichloride has been cooled to a temperature of from-75℃to-85℃such as-78 ℃; continuously blowing out generated hydrogen chloride by nitrogen in the dropping process, wherein the dropping time is more than 5 hours; stopping refrigeration after the dripping is completed, keeping stirring under the nitrogen atmosphere, slowly heating and reacting overnight; after completion of the reaction, distillation under reduced pressure is carried out, and a fraction of 0.1 to 0.3kPa, for example, 0.2kPa, 45℃to 50℃is collected, namely, intermediate 1.
In a preferred embodiment, in step 2, the molar ratio of intermediate 1, anhydrous methanol and pyridine is 1:1 (2-5).
In a preferred embodiment, in step 2, the intermediate 1 is dissolved in tetrahydrofuran and cooled to-75 ℃ to-85 ℃, for example-78 ℃; anhydrous methanol and a part of pyridine are dissolved in tetrahydrofuran and slowly added dropwise to the reaction system at-75 ℃ to-85 ℃, for example-78 ℃ for 0.8-1.2h, for example 1h. After the completion of the dropwise addition, reacting at-75 ℃ to-85 ℃, for example-78 ℃, then heating to room temperature and stirring; after the reaction is finished, cooling to-1 ℃ to 1 ℃, for example, 0 ℃, dissolving water and the other part of pyridine in tetrahydrofuran, dripping into a reaction system, and continuing the reaction after the dripping is finished; after the reaction is completed, filtering is carried out, and the filtrate is dried by spin and then is filtered through a column to obtain an intermediate 2.
In a preferred embodiment, in step 3, the molar ratio of intermediate 2, chloral and acid-binding agent is 1 (1-1.2): 1-3.
In a preferred embodiment, in step 3, intermediate 2 is dissolved in dichloromethane, acid binding agent and chloral are added, stirring is carried out at room temperature overnight, and after the reaction is completed, the mixture is dried by spin-drying and then passed through a column to obtain intermediate 3.
In a further preferred embodiment, the acid-binding agent is selected from the group consisting of organic bases selected from at least one of triethylamine, diethylamine, N-diisopropylethylamine, 4-dimethylaminopyridine, 1, 8-diazabicycloundec-7-ene, tetramethylethylenediamine and pyridine.
In a preferred embodiment, in step 4, the molar ratio of intermediate 3, compound 2 and initiator is 1 (1-2): 0.1-0.3.
In a preferred embodiment, the initiator comprises benzoin dimethyl ether, and/or azobisisobutyronitrile.
In a preferred embodiment, in step 4, intermediate 3, compound 2 and initiator are dissolved by adding methylene chloride and reacted at room temperature under co-irradiation of 350nm to 380nm and 250nm to 280nm ultraviolet lamps. After the reaction is completed, spin-drying and column passing are carried out.
In a preferred embodiment of the invention, the method comprises the steps of:
step 1: reacting phosphorus trichloride with 3-butene-1-ol at low temperature, and distilling under reduced pressure to obtain an intermediate 1':
step 2: reacting the intermediate 1 'with anhydrous methanol and pyridine under alkaline conditions at low temperature to obtain an intermediate 2':
step 3: reacting the intermediate 2 'with chloral under the condition of an acid binding agent to obtain an intermediate 3':
step 4: reacting the intermediate 3' with 3-mercaptopropionic acid to obtain the trichlorfon hapten shown in formula (I-1):
the specific reaction process of the above-described method of the present invention is as follows:
in a preferred embodiment, in step 1, the molar ratio of phosphorus trichloride to 3-buten-1-ol is 1 (1-1.1).
In a preferred embodiment, in step 1, phosphorus trichloride is placed in a three-necked flask and 3-buten-1-ol is placed in a constant pressure dropping funnel; filling the whole system with nitrogen, cooling phosphorus trichloride in the three-neck flask to-78 ℃, and then beginning to dropwise add 3-butene-1-ol; continuously blowing out generated hydrogen chloride by nitrogen in the dropping process, wherein the dropping time is more than 5 hours; stopping refrigerating after the dripping is completed, and keeping stirring under the nitrogen atmosphere to slowly raise the temperature for reaction overnight; after the reaction is completed, adding a thorn type fractionating tower, carrying out warm water bath reduced pressure distillation, and collecting fractions of 0.2kPa at 45-50 ℃ which are intermediates 1'.
In a preferred embodiment, in step 2, the molar ratio of intermediate 1', anhydrous methanol and pyridine is 1:1 (2-5).
In a preferred embodiment, in step 2, said intermediate 1' is dissolved in tetrahydrofuran and placed in a round bottom flask cooled to-78 ℃; weighing anhydrous methanol and partial pyridine, dissolving the anhydrous methanol and partial pyridine in tetrahydrofuran, and slowly dripping the solution into a reaction system at the temperature of minus 78 ℃ for about 1h; after the dripping is completed, reacting at-78 ℃, then heating to room temperature and stirring; after the reaction is finished, cooling to 0 ℃, taking water and the other part of pyridine to dissolve in tetrahydrofuran, dripping into a reaction system, and continuing the reaction after the dripping is finished; after the reaction is completed, filtering is carried out, a filter cake is washed by dichloromethane, and after the filtrate is dried in a spinning manner, the filtrate is filtered by dichloromethane and then is subjected to column chromatography to obtain an intermediate 2'.
In a preferred embodiment, in step 3, the molar ratio of the intermediate 2', chloral and acid-binding agent is 1 (1-1.2): 1-3.
In a preferred embodiment, in step 3, intermediate 2' is dissolved in dichloromethane and triethylamine and chloral are added; stirring overnight at room temperature, and directly spin-drying the mixture after the reaction is completed to obtain an intermediate 3'.
In a preferred embodiment, in step 4, the reaction system is an intermediate 3', 3-mercaptopropionic acid, and an initiator, wherein the mass ratio of the intermediate 3', 3-mercaptopropionic acid to the initiator is 1 (1-2): (0.1-0.3), and the initiator comprises benzoin dimethyl ether and azobisisobutyronitrile.
In a preferred embodiment, in step 4, intermediate 3', 3-mercaptopropionic acid and benzoin dimethyl ether are dissolved in methylene chloride and reacted at room temperature under co-irradiation of 365nm and 275nm ultraviolet lamps; after the reaction is completed, spin-drying and column passing are carried out.
According to the structural characteristics of the trichlorfon, the synthesis method of the trichlorfon hapten is reasonably designed, the raw materials used are easy to obtain, the reaction operation is simple, and the reaction conditions are easy to control. By adopting the method for synthesizing the dipterex hapten, the purity and the yield of the obtained dipterex hapten are high, the yield of the synthesized hapten can reach more than 50%, and the overall synthesis cost is more advantageous.
It is to be noted that the present invention is not particularly limited to the above reaction time, and specifically, the reaction time is limited to the time of complete reaction, and the above reaction time is merely an example and is not limited thereto.
As mentioned above, the dipterex hapten is only immunoreactive and not immunogenic and does not alone stimulate the production of the corresponding antibodies by the animal. Therefore, in order to render the dipterex hapten immunogenic, it is necessary to couple, bind or crosslink the dipterex hapten with a carrier such as a macromolecular protein, thereby producing a dipterex-coupled antigen that is both immunoreactive and immunogenic. Methods of coupling, binding or cross-linking between hapten and carrier molecules are known in the art.
Thus, according to a fourth aspect of the present invention there is provided a trichlorfon antigen comprising the trichlorfon hapten of the first aspect of the present invention and a carrier protein coupled to the trichlorfon hapten.
The term "carrier protein" as referred to herein is any substance capable of coupling, binding or cross-linking with a hapten and thereby producing a substance that is both immunogenic and immunoreactive, including, for example, macromolecular proteins or non-antigenic polylysines, and the like. As examples, carrier proteins that may be used include, but are not limited to, macromolecular proteins such as Bovine Serum Albumin (BSA), human Serum Albumin (HSA), chicken Ovalbumin (OVA), hemocyanin (KLH).
After entering the body, the antigen stimulates B cells, induces proliferation and differentiation of the cells, and then generates specific antibodies. In particular, the dipterex hapten and carrier protein coupling of the dipterex hapten are utilized to immunize animals, so that the animals can be stimulated to carry out immune response, and antibodies with strong specificity, high sensitivity and long quality guarantee period can be generated.
Thus, according to a fifth aspect of the present invention there is provided a trichlorfon antibody which is an antibody specific for the trichlorfon antigen of the fourth aspect of the present invention.
The dipterex antibody may be a monoclonal antibody or a polyclonal antibody. Alternatively, antibodies to trichlorfon may be prepared using methods known to those of ordinary skill in the art. For example, in the case where the trichlorfon antibody is a polyclonal antibody, it can be obtained by immunizing a mammal such as a mouse, rat, rabbit, goat, sheep, primate (excluding human) or the like with trichlorfon antigen, followed by isolation of serum. In the case where the trichlorfon antibody is a monoclonal antibody, the monoclonal antibody can be obtained by producing and culturing hybridoma cells and collecting the culture medium, or the hybridoma cells thus produced can be inoculated into a body of a mammal such as a mouse, a rat, a rabbit, a goat, a sheep, a primate (excluding a human), or the like by intraperitoneal injection, and ascites is collected when the abdomen of the inoculated animal is significantly enlarged, thereby obtaining the monoclonal antibody.
As will be appreciated by those skilled in the art, there is no particular limitation on the source of the trichlorfon antibody, which may be derived from any mammal, including, for example, mice, rats, rabbits, goats, sheep, primates (excluding humans), and the like, but is not limited thereto. In a specific embodiment, the trichlorfon antibody is a polyclonal or monoclonal antibody derived from mice, rats, rabbits, goats, sheep, primates (excluding humans).
Based on the need of immunological detection, the inventor applies the trichlorfon hapten of the first aspect of the invention, the trichlorfon antigen of the fourth aspect of the invention and the trichlorfon antibody of the fifth aspect of the invention to immunological detection to detect trichlorfon pesticide residue.
Thus, according to a sixth aspect of the present invention there is provided the use of a trichlorfon hapten according to the first aspect of the present invention, a trichlorfon antigen according to the fourth aspect of the present invention, and/or a trichlorfon antibody according to the fifth aspect of the present invention in an immunological assay.
According to a seventh aspect of the present invention, there is provided a dipterex colloidal gold chromatography detection device comprising a test strip and a reaction cup, wherein the test strip comprises a reaction membrane on which a detection line and a quality control line are provided, and wherein the detection line is coated with the dipterex antigen of the fourth aspect of the present invention, and wherein the reaction cup contains a dipterex antibody of the fifth aspect of the present invention (abbreviated as "gold labeled antibody") labeled with colloidal gold.
According to some embodiments of the invention, the test strip may also include other components such as a bottom plate, a sample absorbing pad, and a water absorbing pad. In this case, a sample absorbing pad, a reaction membrane, and a water absorbing pad are sequentially attached to the base plate. In the present invention, the sample absorbing pad may be a glass fiber cotton, a nylon film, a polyvinylidene fluoride film or a polyvinyl acetate film; the reaction membrane can be a nitrocellulose membrane, a pure cellulose membrane or a carboxylated cellulose membrane; the water absorption pad can be water absorption filter paper or oil filtering paper; the base plate may be a non-absorbent flexible material such as a rigid plastic strip such as a PVC base plate or a non-absorbent rigid paper strip or other rigid non-absorbent material.
In the invention, the reaction film comprises a detection line and a quality control line. Typically, the detection line is disposed on the side close to the sample absorbing pad. The detection line is prepared by linear spotting of trichlorfon antigen (i.e. trichlorfon hapten-carrier protein conjugate in the present invention) on a reaction membrane. The quality control line can be obtained by linearly spotting an antigen or an antibody on a reaction membrane.
In one embodiment of the invention, the quality control line is obtained by spotting a second antibody of the dipterex antibody provided by the invention. When the dipterex antibody marked by the colloidal gold moves to the quality control line, the dipterex antibody can generate a combination reaction with a second antibody forming the quality control line, thereby developing the color.
If the quality control line develops color, the detection system is indicated to be established, and the detection result is available. In contrast, if the quality control line does not develop, the detection system is indicated to be not established, and the detection result is not available.
As described above, the reaction cuvette contains a colloidal gold-labeled trichlorfon antibody of the fifth aspect of the present invention, which is specific for the trichlorfon antigen of the fourth aspect of the present invention.
The dipterex antibody is only required to be capable of antigen-antibody binding reaction with dipterex antigen, regardless of whether it is a monoclonal antibody or a polyclonal antibody. However, as will be appreciated by those skilled in the art, monoclonal antibodies are more suitable from the standpoint of requiring greater specificity. Thus in the present invention, the trichlorfon antibody is preferably a monoclonal antibody.
In the invention, the sample application preparation of the quality control line can be performed according to the type of the dipterex antibody marked by the colloidal gold. Specifically, if the dipterex antibody marked by the colloidal gold is a dipterex monoclonal antibody, the quality control line can be prepared by adopting a goat anti-mouse antibody to perform linear sample application, and if the dipterex antibody marked by the colloidal gold is a dipterex polyclonal antibody, the quality control line can be prepared by adopting a goat anti-rabbit antibody to perform linear sample application.
In one embodiment of the invention, the invention provides a dipterex colloidal gold chromatography detection device, which comprises a test strip and a reaction cup. As shown in fig. 3, the test strip comprises a bottom plate, and a sample absorption pad, a reaction membrane and absorbent paper which are sequentially paved on the bottom plate, wherein the reaction membrane comprises a detection line and a quality control line in the direction from the sample absorption pad to the absorbent paper, and the detection line is prepared from trichlorfon antigen; as shown in fig. 3, the cuvette contains a colloidal gold-labeled trichlorfon antibody, which is a murine monoclonal antibody specific to trichlorfon antigen, and the quality control line is a goat anti-mouse antibody to trichlorfon antibody.
In one embodiment of the present invention, the dipterex colloidal gold chromatography detection device of the present invention can be prepared by the following preparation method: preparing a reaction membrane, preparing a detection line by adopting the trichlorfon antigen of the invention to carry out linear sample application on the reaction membrane, and preparing a quality control line by adopting a goat anti-mouse antibody aiming at trichlorfon antibody through linear sample application; sequentially splicing and adhering a sample absorption pad, a reaction film and water absorption paper on the bottom plate along the same direction, thereby assembling a test strip; adding the dipterex antibody marked by the colloidal gold into a micropore reaction cup, freeze-drying, and adding a micropore plug into the micropore reaction cup. The components or assemblies used in the preparation method are as described above for the dipterex colloidal gold chromatography detection device of the invention.
According to an eighth aspect of the present invention there is provided a method of detecting trichlorfon in a sample, the method comprising: the dipterex colloidal gold chromatography detection device provided in the seventh aspect of the invention is brought into contact with a sample.
In the present invention, the sample may be any sample suspected of exceeding the residual amount of trichlorfon, which may be vegetables, fruits, edible animal tissue, water or soil.
According to some embodiments of the present invention, prior to detecting trichlorfon in a sample using the method of the present invention, trichlorfon may be subjected to pretreatment according to the sample, which is a general method known in the art for pretreatment of a sample for detection, and is not particularly limited herein.
In a further embodiment, after the sample is pretreated, the sample is dripped into a micropore reaction cup, after being uniformly mixed, a test strip is inserted into the micropore reaction cup, the sample solution to be detected and the gold-labeled antibody in the micropore are combined and then are diffused to a reaction membrane together, and if the quality control line is observed to show a mauve strip, the detection system is indicated to be established and available. Fig. 4 illustrates a determination result of detecting trichlorfon using the method provided by the present invention according to some embodiments of the present invention, the determination method is as follows:
(1) If the detection line (T line) does not develop color or develops color more shallowly than the quality control line (C line), the sample is indicated to contain trichlorfon. When the sample liquid to be detected contains trichlorfon, trichlorfon in the sample liquid to be detected can be combined with the gold-labeled antibody in the diffusion process, so that the antigen binding point of trichlorfon on the gold-labeled antibody is completely closed, the combination of the gold-labeled antibody and trichlorfon antigen on the reaction film is prevented, the T line does not develop color or the T line is lighter than the C line, and the anti-antibody can be combined with the gold-labeled antibody, and the C line develops color.
(2) If the detection line shows a purple red stripe like the quality control line and the color depth of the detection line is equal to or deeper than the color depth of the quality control line, the sample is indicated to contain no trichlorfon. Because the sample liquid to be detected does not contain trichlorfon, the antigen binding site on the gold-labeled antibody cannot be blocked, and then the gold-labeled antibody can be coupled and combined with trichlorfon antigen on the reaction film, the T line is developed, meanwhile, the anti-antibody can be combined with the gold-labeled antibody, and the C line is developed, and at the moment, the color of the T line is darker than or the color of the C line is the same.
(3) If neither the T line nor the C line on the reaction membrane develops color, the test strip fails.
The present invention will be described in detail with reference to the following examples, which are only preferred embodiments of the present invention and are not limiting thereof.
EXAMPLE 1 Synthesis and identification of dipterex hapten
The synthesis method of the trichlorfon hapten comprises the following steps:
step 1: phosphorus trichloride (190.4 g,1.387 mol) was weighed into a three-necked flask, and 3-buten-1-ol (100.61 g,1.387 mol) was placed in a constant pressure dropping funnel. The whole system is filled with nitrogen, the temperature of the phosphorus trichloride in the three-neck flask is reduced to-78 ℃, and then 3-butene-1-ol is added dropwise. In the dropping process, the generated hydrogen chloride is continuously blown out by nitrogen, and the dropping time is more than 5 hours. After the completion of the dropwise addition, the cooling was stopped, stirring was maintained under nitrogen atmosphere, and the temperature was slowly raised and the reaction was allowed to proceed overnight. After the reaction is completed, adding a thorn-type fractionating tower, carrying out warm water bath reduced pressure distillation, and collecting fractions of 0.2kPa, 45-50 ℃ which are intermediates
Step 2: dipterex hapten intermediate 1' (8.16 g,47.2 mmol) was weighed out, dissolved in 160mL of tetrahydrofuran and placed in a round bottom flask cooled to-78 ℃. Anhydrous methanol (1.521 g,47.2 mmol) and pyridine (3.284 g,47.2mmol,3.80 mL) were weighed, dissolved in 20mL tetrahydrofuran, and slowly added dropwise to the reaction system at-78℃for 1h. After completion of the dropwise addition, the reaction was carried out at-78℃for 2 hours, and then the temperature was raised to room temperature and stirred for 2 hours. After the completion of the reaction, the temperature was lowered to 0℃and water (0.9 mL) and pyridine (4.11 g,52mmol,18 mL) were taken and dissolved in 20mL of tetrahydrofuran, and the reaction system was dropped, followed by continuing the reaction for 1.5 hours after the completion of the dropping. Filtering after the reaction is finished, washing a filter cake by using dichloromethane, spin-drying the filtrate, and passing the filtrate through a column by using dichloromethane to obtain colorless oily substance, namely an intermediate 2'
Step 3: the trichlorfon hapten intermediate 2' (6.7 g, 44.264 mmol) was weighed out and dissolved in 103mL of dichloromethane, and triethylamine (836 mg,8.266mmol,1.15 mL) and chloral (6.09 g,41.328 mmol) were added. Stirring overnight at room temperature, and finishing the reactionDirectly spin-drying and passing through a column to obtain colorless oily substance, namely an intermediate
Step 4: the trichlorfon hapten intermediate 3' (1 g,3.361 mmol), 3-mercaptopropionic acid (317 mg,3.361 mmol) and benzoin dimethyl ether (258 mg,1.008 mmol) were weighed out and dissolved in 40mL of absolute dry dichloromethane and reacted for 2h at room temperature under the common irradiation of 365nm and 275nm ultraviolet lamps. After the reaction is completed, spin-drying and column passing are carried out to obtain colorless oily matter, namely trichlorfon hapten
And (3) identification: mass spectrometry EI-MS (negative) was used to identify the dipterex hapten, and the mass spectrum was shown in figure 1. From the mass spectrum, the molecular ion peak of hapten is m/z:401[ M-H ]] - And is the highest peak, which is consistent with the molecular weight (402) of the trichlorfon hapten, indicating that the trichlorfon hapten represented by formula (I-1) was successfully synthesized.
Example 2 preparation and identification of dipterex antigens
Preparation: 0.1mmol of the trichlorfon hapten prepared in example 1 was dissolved in 2mL of Dimethylformamide (DMF), and 41.3mg of Dicyclohexylcarbodiimide (DCC) and 17.3mg of N-hydroxysuccinimide (NHS) were added with stirring. The reaction was magnetically stirred at 4 ℃ overnight, the supernatant was retained after centrifugation and labeled as solution a. Bovine Serum Albumin (BSA) and Ovalbumin (OVA) were weighed out at 140mg each, and dissolved in 10mL of PBS (pH 8.0) at a concentration of 0.1mol/L, followed by adding 1mL of DMF thereto, and stirring and dissolving, thereby obtaining solution B. Under magnetic stirring, the solution A was gradually dropped into the solution B, and reacted at 4℃for 12 hours. After centrifugation, the supernatant was taken and dialyzed with physiological saline at 4℃for 3 days, and the dialysate was changed 3 times per day, whereby dipterex conjugated antigens conjugated with Bovine Serum Albumin (BSA) and Ovalbumin (OVA), respectively, were obtained. The obtained trichlorfon conjugated antigen is split into 0.5mL centrifuge tubes at a concentration of 1mg/mL and frozen in a refrigerator at-20 ℃.
And (3) identification: and (3) respectively scanning full wavelength of 200-600 nm on the trichlorfon Hapten (Hapten), BSA and the Hapten-BSA, OVA, hapten-OVA by adopting an ultraviolet scanner so as to observe whether the conjugate and the carrier protein are different or not, thereby identifying whether the trichlorfon Hapten, the BSA and the OVA are successfully coupled or not. As can be seen from FIG. 2, the absorption curves of the immunogens (Hapten-BSA and Hapten-OVA) are significantly different from the dipterex Hapten (Hapten), BSA, OVA, exhibiting cumulative absorption characteristics of BSA or OVA with the dipterex Hapten, indicating successful coupling of the dipterex Hapten with the carrier proteins BSA, OVA.
EXAMPLE 3 preparation of dipterex monoclonal antibodies
A trichlorfon monoclonal antibody was prepared using trichlorfon immune antigen (Hapten-BSA) prepared in example 2. Specifically, 4 BALB/C mice with the age of 6 weeks are immunized by using the identified trichlorfon immune antigen, blood is taken for measuring the titer after three times of boosting, the mice are immunized by using twice doses of antigen without adding an adjuvant after the serum titer is not increased, spleen cells are prepared by taking spleen after three days, spleen cells are prepared by taking spleen under the aseptic condition, the spleen cells and the bone marrow cells of the vigorous mice are mixed in a 50mL centrifuge tube according to the proportion of 8:1, 30mL serum-free IPMI1640 culture medium is added, the supernatant is discarded after centrifugation for 5min at 1100r/min, and the cell mass is gently vibrated loose and placed in a water bath at 37 ℃. Slowly adding 1mL of 50% PEG-4000 into cells, dripping over 1min while gently stirring the bottom sediment, standing for 1min, slowly adding 1mL of serum-free medium along the tube wall at constant speed for the first 30s, adding 2mL for the last 30s, rapidly adding 27mL to terminate fusion process, centrifuging at 1100r/min for 5min, discarding supernatant, re-suspending with HAT selective medium, adding into 96-well cell culture plate with feeder cells, adding CO with volume fraction of 5% at 37deg.C 2 Culturing under the condition. And after 7 days, changing into HT culture solution, screening by using an indirect ELISA method when the number of hybrid cells in the holes reaches more than 300, selecting holes with strong positivity, good inhibition effect and vigorous cell growth for limited dilution cloning, carrying out cloning culture and detection for more than 3 times, wherein the cells in the holes which are positive are hybridoma cells secreting monoclonal antibodies, and carrying out expanded culture on the hybridoma cells for preparing the monoclonal antibodies.
Production of trichlorfon-resistant single by in-vivo induced ascites methodCloning the antibody. Selecting 4 produced Kunming mice, injecting liquid paraffin oil into abdominal cavity 0.5 mL/mouse, and injecting the hybridoma cells into abdominal cavity 3-5×10 after 7 days 6 After 10 days, ascites was collected when the abdomen of the mice had significantly distended. Purifying ascites by an n-octanoic acid-ammonium sulfate precipitation method, and measuring the content of the anti-trichlorfon monoclonal antibody by ultraviolet.
Example 4 dipterex colloidal gold detection device
4.1 preparation of colloidal gold
1mL of 1% chloroauric acid solution is taken, 99mL of ultrapure water is added into the chloroauric acid solution with the final concentration of 0.01%, after heating and boiling, 1.6mL of 1% trisodium citrate is taken and rapidly added into the boiled chloroauric acid solution, the solution is continuously heated until the solution turns from light yellow to blue black and finally turns into bright red, after the color is stable, the solution is continuously heated for 5min, and the solution is cooled at room temperature and is supplemented with water loss to the original volume.
4.2 preparation of colloidal gold-labeled dipterex monoclonal antibody
Adjusting the pH value of the colloidal gold solution to 8.0, uniformly stirring by a constant-speed stirrer, simultaneously dropwise adding trichlorfon monoclonal antibody, adding polyethylene glycol (PEG) with equivalent antibody amount after 1h, fully reacting for 30min, adding Bovine Serum Albumin (BSA) with equivalent antibody amount, and continuously stirring for 30min after the addition. Centrifuging at 9000rpm for 30min to obtain homogeneous gold-labeled antibody precipitate, and adding p-nitrophenyl butyrate (PNPB) for resuspension.
4.3 preparation of colloidal gold detection device
As shown in fig. 3, on the base plate, a sample pad, a nitrocellulose membrane sprayed with trichlorfon Hapten-BSA (detection line) and goat anti-mouse IgG (quality control line) and a water absorbing paper were sequentially bonded in the same direction.
Adding colloidal gold labeled monoclonal antibody into the reaction cup, and freeze-drying.
Example 5 colloidal gold rapid detection of trichlorfon
5.1 pretreatment of samples: 5g of chopped fruit and vegetable samples are taken and added into an extraction bottle containing 5mL of phosphate buffer solution with pH7.4, the bottle cap is screwed down and shaken vigorously for about 30 seconds, and the liquid to be detected is obtained after uniform mixing.
5.2, detection: unscrewing the upper cover of the reagent bottle, vertically dripping 9-10 drops (about 200 mu L) of the liquid to be detected into the reaction cup, and pumping up and down for 10 times to mix uniformly. Starting the first reaction at 20-40 ℃ and timing for 3 minutes; inserting the test strip into a reaction cup, starting a second reaction step at 20-40 ℃ and timing for 3 minutes; the test strip was removed from the well, the sample pad at the lower end of the test strip was gently scraped off, and the results were interpreted and shown in Table 1.
5.3 interpretation of results
TABLE 1
The results are shown in Table 2, in comparison with the results of other verification methods.
TABLE 2
Example 6 sensitivity of dipterex colloidal gold detection device
And (3) adopting a gradient experiment, adding standard substances with different gradient concentrations of 25 mug/kg, 50 mug/kg, 100 mug/kg, 150 mug/kg, 200 mug/kg and the like into a reaction cup, and detecting by using a test strip, wherein the concentration of the detection line T when the strip is just eliminated is the sensitivity of the colloidal gold detection device. Experiments show that the T line disappears when the standard substance with the concentration of 100 mug/kg is added, and the sensitivity of the dipterex colloidal gold detection device is shown as follows: 100 mug/kg of trichlorfon.
Example 7 specificity experiment of dipterex colloidal gold detection device
To the negative sample, 100. Mu.g/kg of trichlorfon, 10mg/kg of dichlorvos, 10mg/kg of methamidophos, 10mg/kg of ethidium, 10mg/kg of dimethoate, 10mg/kg of omethoate, 10mg/kg of chlorpyrifos and 10mg/kg of phoxim were added. The experimental result shows that only the samples added with the trichlorfon can be detected, but the samples added with the trichlorfon, methamidophos, ethiprole, dimethoate, omethoate, chlorpyrifos and phoxim can not be detected, which proves that the detection device has better specificity on the trichlorfon.
Example 8 shelf-life experiment of dipterex colloidal gold detection device
And (3) respectively carrying out shelf life experiments on three batches of conventionally produced products, placing the products in an indoor room temperature environment for maintenance, taking 12 devices every 1 month, detecting by using quality control samples, respectively carrying out negative and standard adding experiments of 50 mug/kg, 100 mug/kg and 150 mug/kg, repeating the experiments for three times, observing data change, and inspecting shelf life time. The negative development decreased from 13 months and the product quality did not change significantly over 1 year, thus confirming a shelf life of 1 year.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (18)
1. The trichlorfon hapten is characterized in that the structural formula of the trichlorfon hapten is shown as a formula (I):
wherein the R is 1 Selected from C 0 -C 6 An alkylene group; the R is 2 Selected from C 1 -C 6 An alkylene group.
2. The trichlorfon hapten of claim 1, wherein the trichlorfon hapten has a structure as shown in formula (I-1):
3. an intermediate for preparing the trichlorfon hapten as set forth in claim 1 or 2, which has a structural formula as shown in formula (II):
wherein the R is 1 Selected from C 0 -C 6 An alkylene group.
4. An intermediate for a trichlorfon hapten according to claim 3, characterized in that the intermediate has a structure as shown in formula (II-1):
5. a method of preparing the trichlorfon hapten of claim 1 or 2, comprising the steps of:
step 1: at low temperature, phosphorus trichloride and the structural formula areThe compound 1 of (2) is subjected to reduced pressure distillation to obtain an intermediate 1:
wherein R is 1 Selected from C 0 -C 6 An alkylene group;
step 2: reacting said intermediate 1 with anhydrous methanol and pyridine under basic conditions at low temperature, to give intermediate 2:
step 3: reacting the intermediate 2 with chloral under the condition of an acid binding agent to obtain an intermediate 3:
step 4: the intermediate 3 and the structural formula are as followsThe compound 2 of (2) is reacted in the presence of an initiator to obtain the trichlorfon hapten shown in the formula (I):
wherein R is 2 Selected from C 1 -C 6 An alkylene group.
6. The method according to claim 5, wherein the molar ratio of phosphorus trichloride to said compound 1 is 1 (1-1.1).
7. The process according to claim 5 or 6, wherein the molar ratio of intermediate 1, anhydrous methanol and pyridine is 1:1 (2-5).
8. The method according to claim 5 or 6, wherein the molar ratio of the intermediate 2, chloral and acid-binding agent is 1 (1-1.2): 1-3.
9. The method according to claim 5 or 6, wherein the acid-binding agent is selected from an organic base selected from at least one of triethylamine, diethylamine, N-diisopropylethylamine, 4-dimethylaminopyridine, 1, 8-diazabicycloundec-7-ene, tetramethyl ethylenediamine and pyridine.
10. The process according to claim 5 or 6, wherein the molar ratio of intermediate 3, compound 2 and initiator is 1 (1-2): 0.1-0.3.
11. The method according to claim 10, wherein the initiator is benzoin dimethyl ether, and/or azobisisobutyronitrile.
12. A trichlorfon antigen, the trichlorfon immunizing antigen comprising: the trichlorfon hapten of claim 1 or 2, and a carrier protein coupled to the trichlorfon hapten.
13. The trichlorfon antigen of claim 12, wherein the carrier protein comprises bovine serum albumin, human serum albumin, chicken ovalbumin or hemocyanin.
14. A trichlorfon antibody, characterized in that it is an antibody specific for a trichlorfon antigen according to claim 12 or 13.
15. The dipterex antibody of claim 14, wherein the dipterex antibody is a dipterex monoclonal antibody or a dipterex polyclonal antibody.
16. Use of the trichlorfon hapten as claimed in claim 1 or 2, the trichlorfon antigen as claimed in claim 12 or 13, and the trichlorfon antibody as claimed in claim 14 or 15 in trichlorfon immunological detection.
17. The dipterex colloidal gold chromatography detection device is characterized by comprising a test strip and a reaction cup, wherein the test strip comprises a reaction membrane, a detection line and a quality control line are arranged on the reaction membrane, the detection line is coated with the dipterex antigen according to claim 12 or 13, and the reaction cup contains the dipterex antibody according to claim 14 or 15 marked by colloidal gold.
18. A method of detecting trichlorfon in a sample, the method comprising: the colloidal gold chromatography detection apparatus according to claim 17 is contacted with a sample, wherein the sample is a vegetable, fruit, edible animal tissue, water or soil.
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| CN102002103A (en) * | 2009-09-03 | 2011-04-06 | 天津科技大学 | Pesticide trichlorfon-resistant specific antibody |
| CN108469517A (en) * | 2018-03-16 | 2018-08-31 | 北方工业大学 | Time-resolved fluorescent test strip for detecting trichlorfon and application thereof |
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