CN112480167B - Isocarbophos hapten, artificial antigen and antibody as well as preparation method and application thereof - Google Patents
Isocarbophos hapten, artificial antigen and antibody as well as preparation method and application thereof Download PDFInfo
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- CN112480167B CN112480167B CN202011285140.XA CN202011285140A CN112480167B CN 112480167 B CN112480167 B CN 112480167B CN 202011285140 A CN202011285140 A CN 202011285140A CN 112480167 B CN112480167 B CN 112480167B
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- YFVOXLJXJBQDEF-UHFFFAOYSA-N isocarbophos Chemical compound COP(N)(=S)OC1=CC=CC=C1C(=O)OC(C)C YFVOXLJXJBQDEF-UHFFFAOYSA-N 0.000 title claims abstract description 105
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- 238000002360 preparation method Methods 0.000 title abstract description 14
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- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 11
- 241001465754 Metazoa Species 0.000 claims abstract description 11
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- 238000010168 coupling process Methods 0.000 claims abstract description 4
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- SCYULBFZEHDVBN-UHFFFAOYSA-N 1,1-Dichloroethane Chemical compound CC(Cl)Cl SCYULBFZEHDVBN-UHFFFAOYSA-N 0.000 claims description 3
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- 241000283707 Capra Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
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- PNVJTZOFSHSLTO-UHFFFAOYSA-N Fenthion Chemical compound COP(=S)(OC)OC1=CC=C(SC)C(C)=C1 PNVJTZOFSHSLTO-UHFFFAOYSA-N 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
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- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- MUUGCDGGIVCSDT-UHFFFAOYSA-N [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O Chemical compound [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O MUUGCDGGIVCSDT-UHFFFAOYSA-N 0.000 description 1
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- HRBKVYFZANMGRE-UHFFFAOYSA-N chlorpyrifos-methyl Chemical compound COP(=S)(OC)OC1=NC(Cl)=C(Cl)C=C1Cl HRBKVYFZANMGRE-UHFFFAOYSA-N 0.000 description 1
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- ZNOLGFHPUIJIMJ-UHFFFAOYSA-N fenitrothion Chemical compound COP(=S)(OC)OC1=CC=C([N+]([O-])=O)C(C)=C1 ZNOLGFHPUIJIMJ-UHFFFAOYSA-N 0.000 description 1
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- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
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- ATROHALUCMTWTB-OWBHPGMISA-N phoxim Chemical compound CCOP(=S)(OCC)O\N=C(\C#N)C1=CC=CC=C1 ATROHALUCMTWTB-OWBHPGMISA-N 0.000 description 1
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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 Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/22—Amides of acids of phosphorus
- C07F9/24—Esteramides
- C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
- C07F9/242—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyaryl compounds
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
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- Immunology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
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- Biophysics (AREA)
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- Urology & Nephrology (AREA)
- Gastroenterology & Hepatology (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The isocarbophos hapten, the artificial antigen and the antibody as well as the preparation method and the application thereof are disclosed, the isocarbophos hapten provided by the invention not only furthest reserves the characteristic structure of isocarbophos, so that the immunogenicity of the isocarbophos hapten is obviously enhanced, but also has amino which can be coupled with carrier protein; the isocarbophos artificial antigen obtained by coupling isocarbophos hapten and carrier protein is used for immunizing animals, so that the animal immune response can be stimulated to generate antibodies with stronger specificity and higher sensitivity, the sensitivity of the isocarbophos antibody can reach 0.03 mu g/L through detection, the cross reaction rate with other organophosphorus insecticides is low, and a foundation is provided for the subsequent establishment of various immunoassay methods of isocarbophos.
Description
Technical Field
The invention belongs to the field of food safety detection. More particularly, the invention relates to isocarbophos hapten, artificial antigen and antibody, and preparation methods and applications thereof.
Background
Isocarbophos is a quick-acting broad-spectrum organophosphorus insecticide, is mainly used for controlling pests of fruit trees, rice and cotton, and once becomes one of the main insecticides in agricultural production due to the advantages of high insecticidal efficiency, easy decomposition under alkaline conditions and the like. However, abuse of isocarbophos can not only cause pesticide residue in the environment, but also poison animals and people through the enrichment function of a food chain, and further influence the metabolic balance of a human body, especially more serious influence on the normal development of the nervous system of children. The national standard GB 2763 of China stipulates that the maximum residual limit of isocarbophos in vegetables and partial fruits is 0.05mg/kg, so that the establishment of a isocarbophos residual rapid detection technology has important significance for guaranteeing food safety and human health and reducing environmental pollution.
At present, the isocarbophos detection at home and abroad mainly adopts analysis methods such as a gas chromatography, a gas chromatography-mass spectrometry, a liquid chromatography tandem mass spectrometry and the like, and has the defects of complicated sample pretreatment, long detection time, expensive instruments and the like, so the isocarbophos detection method cannot be widely applied in China and does not meet the requirements of on-site detection on accurate detection and screening of a large number of samples at low cost in a short time. The immunological detection and analysis technology has the advantages of high sensitivity, high specificity, rapidness, simple and convenient operation and the like, is widely applied to the field of drug residue detection, and has many advantages compared with detection methods such as instruments and the like. Therefore, the immunoassay provides a new analysis and detection method for the research of isocarbophos residue.
When an immunological detection method is established and the detection method is applied to detect the residual quantity of isocarbophos, the key technology is that an antibody with strong specificity and high sensitivity can be obtained, and the goal is to realize the key technology, and the precondition is to synthesize and prepare a proper isocarbophos hapten.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a hapten which can furthest reserve the characteristic structure of isocarbophos and has a connecting arm with a certain length and a preparation method of the hapten; the artificial antigen prepared by the hapten and the antibody with high detection sensitivity and strong specificity; and the use of such haptens.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a isocarbophos hapten which has the following structural formula:
the isocarbophos hapten provided by the invention introduces an amino active group on the molecular structure of isocarbophos, so that the isocarbophos hapten can be coupled with carrier protein to obtain an artificial antigen for immunization; the isocarbophos hapten reserves all characteristic groups of isocarbophos, changes the original structural characteristics of isocarbophos to the minimum, and is coupled with carrier protein to highlight the unique structure of isocarbophos, thereby laying a foundation for generating an antibody with stronger specificity and higher sensitivity for subsequent stimulation of animal immune response.
In a second aspect, the invention provides a preparation method of the isocarbophos hapten, which comprises the following steps:
1) 4-nitro salicylic acid and isopropanol are subjected to esterification reaction in the presence of a catalyst, and the 4-nitro isopropyl salicylate is obtained after purification;
2) under the existence of a catalyst and under the alkaline condition, carrying out condensation reaction on 4-nitro isopropyl salicylate and O-methyl thiophosphoryl dichloride, and purifying to obtain 4-nitro isopropyl salicylate thiophosphoryl;
3) carrying out ammoniation reaction on 4-nitro isopropyl salicylate sulfur phosphorus and ammonia gas, and purifying to obtain nitro isocarbophos;
4) in the presence of a catalyst, nitro isocarbophos and hydrogen are subjected to reduction reaction, and isocarbophos hapten is obtained after purification.
Further, the step 1) includes the steps of: taking 1.83g of 4-nitro salicylic acid, adding 20mL of isopropanol to dissolve, fully stirring, adding 0.5mL of concentrated sulfuric acid, stirring at room temperature for 3h, stopping reaction, adding 200mL of water, adding 100mL of ethyl acetate to extract, adding 100mL of water to the organic phase, shaking, washing, concentrating and evaporating to dryness to obtain the 4-nitro isopropyl salicylate.
Further, the step 2) includes the following steps: directly adding 80mL of dichloromethane into 4-nitro isopropyl salicylate to dissolve, adding 1.64g O-methyl thiophosphoryl dichloride, fully stirring, adding 3mL of triethylamine, stirring for 4 hours at room temperature, stopping reaction, adding 100mL of water, supplementing 100mL of dichloromethane for extraction, adding 60mL of saturated saline solution into an organic phase, shaking, washing, concentrating and drying by distillation, applying to a silica gel column, and eluting and separating by using a mixed solvent of dichloromethane and methanol with the volume ratio of 10:1 to obtain 4-nitro isopropyl salicylate thiophosphate.
Further, the step 3) includes the following steps: adding 100mL of dichloroethane directly into 4-nitro salicylic acid isopropyl thiophosphate for dissolving, introducing ammonia gas under stirring, reacting at room temperature for 2h, stopping the reaction, adding 50mL of multiplied by 3, washing for 3 times, concentrating and evaporating an organic phase to dryness to obtain nitro hydrosulfur.
Further, the step 4) includes the following steps: and (2) directly adding 100mL of methanol into the nitro isocarbophos to dissolve, adding 0.5g of wet palladium-carbon, exhausting air, introducing hydrogen, stirring at room temperature for 2h, stopping reaction, performing suction filtration, drying by distillation, applying to a silica gel column, and eluting and separating by using a mixed solvent of dichloromethane and methanol in a volume ratio of 5:1 to obtain isocarbophos hapten.
According to the structural characteristics of isocarbophos, 4-nitro salicylic acid is used as an initial raw material, and a series of reactions such as carboxyl esterification, condensation with O-methyl thiophosphoryl dichloride, ammoniation, reduction and the like are carried out to synthesize the spacer arm with the amino group.
In a third aspect, the invention provides a isocarbophos artificial antigen which is a conjugate obtained by coupling a carrier protein and the isocarbophos hapten. The artificial antigen of isocarbophos can be used as immunogen and also can be used as coating antigen.
Further, the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin; bovine serum albumin and ovalbumin are preferred.
More specifically, the following are: an immunogen formed by isocarbophos hapten-Bovine Serum Albumin (BSA); isocarbophos hapten-Ovalbumin (OVA).
The isocarbophos hapten molecules are only immunoreactive and not immunogenic. Therefore, in order to confer immunogenicity on the isocarbophos hapten molecules, it is also necessary to couple and bind the isocarbophos hapten molecules to suitable carrier protein molecules, thereby producing artificial antigens of isocarbophos that are both immunoreactive and immunogenic.
In a fourth aspect, the invention provides a isocarbophos antibody, which is obtained by immunizing animals with the isocarbophos artificial antigen and can perform specific immune reaction with isocarbophos.
Further, the isocarbophos antibody is a monoclonal antibody or a polyclonal antibody. In addition, the isocarbophos antibody can be prepared by a method conventional in the art.
In a specific embodiment, the isocarbophos antibody is a murine monoclonal antibody specific for an artificial antigen of isocarbophos that is a hapten for isocarbophos as described above.
The isocarbophos antibody obtained by adopting the isocarbophos artificial antigen has better titer, specificity and affinity, and has low cross reaction rate with other organophosphorus insecticides.
In a fifth aspect, the invention provides an application of the isocarbophos antibody in detecting isocarbophos residues.
The invention induces immune animals to generate antibodies through the isocarbophos artificial antigen, thereby being used in isocarbophos immunodetection and analysis.
The isocarbophos immunoassay comprises but is not limited to an isocarbophos ELISA kit, an isocarbophos colloidal gold test strip and an isocarbophos time-resolved fluorescence test strip.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the isocarbophos hapten provided by the invention not only retains the characteristic structure of isocarbophos to the greatest extent, so that the immunogenicity of the isocarbophos hapten is obviously enhanced, but also has amino which can be coupled with carrier protein; the isocarbophos artificial antigen obtained by coupling isocarbophos hapten and carrier protein is used for immunizing animals, so that the method is more favorable for stimulating the immune response of the animals to generate antibodies with stronger specificity and higher sensitivity, and provides a foundation for subsequently establishing various immunoassay methods of isocarbophos.
The preparation method of the isocarbophos hapten has the advantages of easily available raw materials, simple reaction operation, easily controlled reaction conditions and high purity and yield of the prepared isocarbophos hapten.
The isocarbophos antibody obtained by adopting the isocarbophos artificial antigen has better titer, specificity and affinity, the sensitivity can reach 0.03 mu g/L, and the cross reaction rate with other organophosphorus insecticides is low.
Drawings
FIG. 1 is a synthetic route of isocarbophos hapten of the invention
Detailed Description
The present invention will be described in further detail with reference to specific examples, which are only preferred embodiments of the present invention and are not intended to limit the present invention.
Example 1
A preparation method of isocarbophos hapten comprises the following steps:
1) dissolving 1.83g of 4-nitro salicylic acid in 20mL of isopropanol, fully stirring, adding 0.5mL of concentrated sulfuric acid, stirring at room temperature for 3 hours, stopping reaction, adding 200mL of water, adding 100mL of ethyl acetate for extraction, adding 100mL of water in an organic phase, shaking, washing, concentrating and evaporating to dryness to obtain 4-nitro isopropyl salicylate;
2) directly adding 80mL of dichloromethane into the 4-nitro isopropyl salicylate to dissolve, adding 1.64g O-methyl thiophosphoryl dichloride, fully stirring, adding 3mL of triethylamine, stirring for 4 hours at room temperature, stopping reaction, adding 100mL of water, supplementing 100mL of dichloromethane for extraction, adding 60mL of saturated saline solution into an organic phase, shaking, washing, concentrating and evaporating to dryness, loading on a silica gel column, eluting and separating by using a mixed solvent with the volume ratio of dichloromethane to methanol being 10:1 to obtain 4-nitro isopropyl salicylate thiophosphate;
3) directly adding 100mL of dichloroethane to the 4-nitro salicylic acid isopropyl thiophosphate for dissolving, introducing ammonia gas under stirring, reacting at room temperature for 2 hours, stopping the reaction, adding 50mL of multiplied by 3 water, washing for 3 times, concentrating and evaporating an organic phase to dryness to obtain nitro isocarbophos;
4) and (2) directly adding 100mL of methanol into the nitro isocarbophos to dissolve, adding 0.5g of wet palladium-carbon, exhausting air, introducing hydrogen, stirring at room temperature for 2 hours, stopping reaction, performing suction filtration, drying by distillation, applying to a silica gel column, and eluting and separating by using a mixed solvent of dichloromethane and methanol in a volume ratio of 5:1 to obtain isocarbophos hapten.
Example 2
A preparation method of artificial isocarbophos antigen comprises the following steps:
taking 113mg of isocarbophos hapten prepared in example 1, adding 1mL of ethanol for dissolving, adding 2mL of water and 1mol/L of 1.2mL of HCl for fully dissolving, cooling to 0-5 ℃, adding 1mL of aqueous solution containing 25mg of sodium nitrite, and continuously stirring for 1h to obtain hapten activating solution A; taking 500mg of Bovine Serum Albumin (BSA), adding 7mL of 0.1mol/L CB buffer solution for dissolving, cooling to 0-5 ℃, dropwise adding all A solution to immediately turn into red solution, continuing to react for 2h, dialyzing and purifying for 3 days by using 0.02mol/L PB buffer solution, changing the solution for 3 times every day, centrifuging, subpackaging to obtain artificial antigen of isocarbophos coupled with the BSA, and subpackaging at-20 ℃ for storage.
Example 3
A preparation method of artificial isocarbophos antigen comprises the following steps:
taking 67mg of isocarbophos hapten prepared in example 1, adding 1mL of ethanol for dissolving, adding 2mL of water and 0.87mL of 1mol/L HCl for fully dissolving, cooling to 0-5 ℃, adding 1mL of aqueous solution containing 15.2mg of sodium nitrite, and continuously stirring for 1h to obtain hapten activating solution A; dissolving Ovalbumin (OVA)500mg in CB buffer solution 8mL of 0.1mol/L, cooling to 0-5 ℃, dropwise adding all A solution to immediately become red solution, continuously reacting for 2h, dialyzing and purifying for 3 days by PB buffer solution of 0.02mol/L, changing solution 3 times every day, centrifuging, subpackaging to obtain artificial antigen of isocarbophos coupled with ovalbumin, and subpackaging for preservation at-20 ℃.
Example 4
The preparation method of the isocarbophos antibody comprises the following steps:
1. animal immunization
10 healthy female Balb/c mice (divided into two groups of A and B, and 5 mice in each group) with 6-8 weeks are taken, primary immunization is emulsified by Freund complete adjuvant, and then subcutaneous multi-point injection is carried out on the back and the neck, wherein the immunization dose of each mouse is 200 mu g of isocarbophos artificial antigen coupled with bovine serum albumin; then boosting immunity and injecting subcutaneous multi-point on the back of the neck once every two weeks, and emulsifying by Freund incomplete adjuvant; the last immunization uses normal saline to replace Freund's incomplete adjuvant, and is injected in the abdominal cavity, and the injection dosage is the same as the previous times. The specific immunization procedure is shown in table 1.
Table 1 mouse immunization procedure
And (3) for the third time, the fourth time and 7d after the boosting immunization, the tail of the mouse is cut off, blood is taken, and the serum titer of the mouse is measured by an ELISA method, wherein the specific steps are as follows:
(1) diluting isocarbophos artificial antigen coupled with ovalbumin by 0.05mol/L of carbonate buffer solution with pH of 9.6 at a ratio of 1:1000, coating an enzyme label plate by 100 mu L of each hole, incubating at 37 ℃ for 2h, throwing off coating solution, washing for 1 time by PBST, and patting to dry;
(2) adding 150 mu L of sealing liquid into each hole, reacting at 37 ℃ for 2h, then pouring off the sealing liquid, and patting to dry;
(3) adding 50 mu L of antiserum diluted by PBS in each hole, reacting at 25 ℃ for 30min, then removing the reaction liquid, washing 3-5 times by PBST, and patting dry at intervals of 30s each time;
(4) adding 100 mu L/hole of horseradish peroxidase-labeled goat anti-mouse anti-antibody (1:1000) diluted by PBS, reacting for 30min at 25 ℃, washing for 3-5 times by PBST (PBST), and patting dry at an interval of 30s each time;
(5) adding 50 μ L of substrate developing solution A and B into each well, reacting at 25 deg.C in dark for 15min, adding 50 μ L of 2mol/L H into each well2SO4Terminating the reaction by the solution;
(6) measuring OD value with wavelength of 450nm by enzyme-labeling instrument, and determining OD of sample hole450The titer of positive sera was determined as a dilution factor close to 1.
2. Cell fusion
(1) Preparing feeder cells: the Balb/c mice of 8-10 weeks old are killed after neck breakage, soaked in 75% alcohol for 5min, immediately placed in an ultra-clean bench with the abdomen facing upwards in a plate or fixed on a dissecting plate. The skin of the abdomen of the mouse is clamped by an ophthalmic forceps, a small opening is cut by scissors, and the peritoneum is not cut to avoid the outflow and pollution of the abdominal cavity fluid. Then blunt dissection was performed up and down with scissors to fully expose the peritoneum. Wiping peritoneum with alcohol cotton ball for sterilization. 5mL of RPMI-1640 basic culture solution was aspirated by a syringe, injected into the abdominal cavity of the mouse, the syringe was gently withdrawn, and the leg and tail of the mouse were shaken several times. The liquid in the abdominal cavity is pumped back by the original syringe and is injected into the centrifuge tube. The operation is repeated for 3-4 times. Centrifuging at 1000r/min for 10min, and discarding the supernatant. Resuspending the cells with 20-50 mL of complete culture medium, adding 100 μ L/well dropwise to the culture plate, and placing in an incubator for later use.
(2) Preparation of splenocytes: 3d after enhancing the immunity, taking an immune Balb/c mouse, collecting blood from an orbit, dislocating and killing the mouse, disinfecting the mouse in 75% alcohol, taking the spleen, removing connective tissues, preparing a spleen cell suspension, transferring the spleen cell suspension into a 50mL centrifuge tube, adding RPMI-1640 to 30mL, centrifuging the spleen cell suspension at 1500-2000 r/min for 5min, removing a supernatant, adding RPMI-1640 to 30mL, and counting the number for later use.
(3) Myeloma cell preparation: taking 3 bottles of myeloma cells with good growth state (the number of living cells is more than 95 percent), completely blowing down the myeloma cells, transferring the myeloma cells into a 50mL centrifuge tube, adding RPMI-1640 to 30mL, centrifuging at 1500-2000 r/min for 5min, discarding the supernatant, adding RPMI-1640 to 30mL, and counting for later use.
(4) Cell mixing: spleen cells and myeloma cells are mixed and centrifuged at 1500-2000 r/min for 5min, wherein the ratio of the spleen cells to the myeloma cells is 8: 1.
(5) Cell fusion: centrifuging the mixed cells, pouring out the supernatant, making the precipitated cell mass into paste, placing the paste in a water bath at 37 ℃, adding 1mL of fusion agent which is polyethylene glycol (PEG)4000 within 1min, acting for 2min, slightly stirring the cells, adding 20mL of serum-free PEG nutrient solution within 4min, centrifuging at 1000r/min for 10min, and discarding the supernatant. The cells were resuspended in 20-50 mL of complete medium, plated on 96-well feeder cells-containing cell culture plates at 100. mu.L per well, and placed in an incubator.
3. Cell line selection
And (3) when the cells grow to 1/3-1/2 of the bottom of the hole, carrying out antibody detection. Screening culture wells with hybridoma cell growth by adopting an ELISA method, wherein the screening comprises two steps: in the first step, positive cell holes are screened by indirect ELISA, in the second step, isocarbophos is selected as a standard substance, and the inhibition effect of positive cells is measured by indirect competitive ELISA. And (4) selecting a hole with better inhibition on the isocarbophos standard substance, carrying out subcloning by adopting a limiting dilution method, and carrying out detection by using the same method. Repeating the steps for three times to obtain the cell strain capable of stably secreting the isocarbophos monoclonal antibody.
4. Preparation of ascites
Liquid paraffin was injected into Balb/c mice for 6-8 weeks at 500. mu.L/mouse. 10 days later, hybridoma cells in logarithmic growth phase were collected in RPMI-1640 basic medium, counted in a hemocytometer at a cell concentration of 1.0X 10 and a microscope6～1.5×106In the size per mL range. Each mouse was injected with 0.5mL hybridoma cells into the abdominal cavity. Note that after one week the abdomen of the mouse was enlarged, ascites was collected in the abdomen of the mouse with a sterile syringe once every one to two days, and this was repeated until the mouse died naturally. Centrifuging at 4 deg.C for 5min at 5000r/min, collecting supernatant, and removing fat and protein membrane floating on the upper layer of abdominal water.
5. Antibody purification
The monoclonal antibody is purified by an octanoic acid-ammonium sulfate method.
6. Antibody potency assay
And (3) measuring the antibody titer by adopting an indirect ELISA method, and referring to the step 1, measuring the serum titer of animal immunity. The result shows that the titer of the isocarbophos monoclonal antibody is more than or equal to 100000.
7. Antibody cross-reactivity assay
The indirect competitive ELISA method is adopted for determination, and the result shows that the cross reaction rate of the isocarbophos monoclonal antibody to isocarbophos and other organophosphorus insecticides is as follows: 100 percent of isocarbophos, and less than 1 percent of phorate, parathion, methyl parathion, fenitrothion, phoxim, chlorpyrifos, methyl chlorpyrifos, fenthion and profenofos. Therefore, the prepared antibody has better specificity.
The above-mentioned embodiments only express the embodiments of the present invention, and the description is more specific and detailed, but not understood as the limitation of the patent scope of the present invention, but all the technical solutions obtained by using the equivalent substitution or the equivalent transformation should fall within the protection scope of the present invention.
Claims (10)
1. The isocarbophos hapten is characterized by having the following structural formula:
2. the method for preparing isocarbophos hapten as claimed in claim 1, comprising the steps of:
1) carrying out esterification reaction on 4-nitro salicylic acid and isopropanol in the presence of a catalyst, and purifying to obtain 4-nitro isopropyl salicylate;
2) in the presence of a catalyst and under an alkaline condition, carrying out condensation reaction on 4-nitro isopropyl salicylate and O-methyl thiophosphoryl dichloride, and purifying to obtain 4-nitro isopropyl salicylate thiophosphate;
3) carrying out ammoniation reaction on 4-nitro isopropyl salicylate sulfur phosphorus and ammonia gas, and purifying to obtain nitro isocarbophos;
4) in the presence of a catalyst, nitro isocarbophos and hydrogen are subjected to reduction reaction, and isocarbophos hapten is obtained after purification.
3. The method for preparing isocarbophos hapten as claimed in claim 2, wherein the step 1) comprises the following steps: taking 1.83g of 4-nitro salicylic acid, adding 20mL of isopropanol to dissolve, fully stirring, adding 0.5mL of concentrated sulfuric acid, stirring at room temperature for 3h, stopping reaction, adding 200mL of water, adding 100mL of ethyl acetate to extract, adding 100mL of water to the organic phase, shaking, washing, concentrating and evaporating to dryness to obtain the 4-nitro isopropyl salicylate.
4. The method for preparing isocarbophos hapten as claimed in claim 2, wherein the step 2) comprises the steps of: directly adding 80mL of dichloromethane into 4-nitro isopropyl salicylate to dissolve, adding 1.64g O-methyl thiophosphoryl dichloride, fully stirring, adding 3mL of triethylamine, stirring for 4 hours at room temperature, stopping reaction, adding 100mL of water, supplementing 100mL of dichloromethane for extraction, adding 60mL of saturated saline solution into an organic phase, shaking, washing, concentrating and drying by distillation, applying to a silica gel column, and eluting and separating by using a mixed solvent of dichloromethane and methanol with the volume ratio of 10:1 to obtain 4-nitro isopropyl salicylate thiophosphate.
5. The method for preparing isocarbophos hapten as claimed in claim 2, wherein the step 3) comprises the following steps: adding 100mL of dichloroethane directly into 4-nitro salicylic acid isopropyl thiophosphate for dissolving, introducing ammonia gas under stirring, reacting at room temperature for 2h, stopping the reaction, adding 50mL of multiplied by 3, washing for 3 times, concentrating and evaporating an organic phase to dryness to obtain nitro isocarbophos.
6. The method for preparing isocarbophos hapten as claimed in claim 2, wherein the step 4) comprises the following steps: directly adding 100mL of methanol into nitroisocarbophos for dissolving, adding 0.5g of wet palladium-carbon, exhausting air, introducing hydrogen, stirring at room temperature for 2 hours, stopping reaction, performing suction filtration, drying by distillation, putting on a silica gel column, and performing elution separation by using a mixed solvent of dichloromethane and methanol in a volume ratio of 5:1 to obtain isocarbophos hapten.
7. An artificial antigen of isocarbophos, which is a conjugate obtained by coupling a carrier protein and the isocarbophos hapten as claimed in claim 1.
8. The artificial antigen of isocarbophos according to claim 7, wherein the carrier protein is bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.
9. An antibody against isocarbophos, which is obtained by immunizing an animal with the artificial antigen against isocarbophos according to claim 7, and which is capable of specifically immunoreacting with isocarbophos.
10. Use of the isocarbophos antibody of claim 9 for detecting isocarbophos residues.
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