CN111690007A - Diazinon hapten and artificial antigen as well as preparation method and application thereof - Google Patents
Diazinon hapten and artificial antigen as well as preparation method and application thereof Download PDFInfo
- Publication number
- CN111690007A CN111690007A CN202010697067.0A CN202010697067A CN111690007A CN 111690007 A CN111690007 A CN 111690007A CN 202010697067 A CN202010697067 A CN 202010697067A CN 111690007 A CN111690007 A CN 111690007A
- Authority
- CN
- China
- Prior art keywords
- diazinon
- hapten
- artificial antigen
- specific antibody
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
- C07F9/6509—Six-membered rings
- C07F9/6512—Six-membered rings having the nitrogen atoms in positions 1 and 3
-
- 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
- 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
-
- 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
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to diazinon hapten and artificial antigen as well as a preparation method and application thereof. The invention takes trichloro-sulfur phosphorus as an initiator, reacts with ethanol in an alkaline environment, and then reacts with (2-isopropyl-6-oxo-1, 6-dihydro-4-pyrimidyl) acetic acid to obtain a compound containing carboxyl groups, namely diazinon hapten; then coupling the hapten with carrier protein to obtain the artificial antigen. The artificial antigen can be used for immunizing animals to generate specific antibodies aiming at diazinon, and can be used for establishing an enzyme-linked immunosorbent assay method of the diazinon and a colloidal gold test strip rapid detection method, so that the artificial antigen can be used for rapidly detecting the residue of the diazinon in vegetables and fruits.
Description
Technical Field
The invention belongs to the field of food safety detection. More specifically, the invention relates to diazinon hapten and artificial antigen, and a preparation method and application thereof.
Background
Diazinon (diazinon), also known as diazinon, is an efficient and moderately toxic organophosphorus insecticide and acaricide, has the effects of contact poisoning, stomach poisoning, fumigation and systemic absorption, has a good control effect on lepidoptera, homoptera and other pests, and can be used for preventing and controlling grubs, mole crickets, wireworms and other underground pests by dressing or mixing with soil. As organophosphorus insecticides, diazinon exerts an effect on the nervous system of humans and, upon excessive exposure, causes symptoms of nausea, headache, vomiting, diarrhea, general weakness, etc. The national standard GB 2763 and 2019 maximum limit of pesticide residue in food safety national standard food stipulate that the residue limit of diazinon in vegetables and fruits is 0.01-1 mg/kg.
At present, the analysis and detection methods for diazinon residue at home and abroad mainly comprise liquid chromatography, gas chromatography, chromatography-mass spectrometry combined technology and the like, but have the defects of complicated sample pretreatment, long detection time, expensive instruments and the like, cannot be widely applied in China, and do 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 the residue of the diazinon.
When an immunological detection method is established and the detection method is applied to detecting the residual amount of diazinon, the key technology is that an antibody with strong specificity and high sensitivity can be obtained, and the aim is to realize the aim under the precondition that a proper diazinon hapten is synthesized and prepared.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a hapten which can furthest reserve the characteristic structure of diazinon 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.
The invention firstly takes trichloro-sulfur phosphorus as an initiator to react with ethanol in an alkaline environment, and then the trichloro-sulfur phosphorus reacts with (2-isopropyl-6-oxo-1, 6-dihydro-4-pyrimidyl) acetic acid to obtain a compound containing carboxyl groups, namely diazinon hapten; then coupling with carrier protein to obtain its artificial antigen.
The diazinon hapten provided by the invention takes trichlorothion as an initiator, reacts with ethanol in an alkaline environment, and then reacts with (2-isopropyl-6-oxo-1, 6-dihydro-4-pyrimidyl) acetic acid to obtain a compound containing a carboxyl group, namely the diazinon hapten.
Preferably, the structure of the diazinon hapten is shown as the formula (I):
the diazinon hapten with the structure of the formula (I) can be prepared by the following method: taking trichloro sulfur phosphorus as an initiator, reacting with ethanol in an alkaline environment to obtain 2-ethoxy-chloro-sulfur phosphorus, and reacting the 2-ethoxy-chloro-sulfur phosphorus with (2-isopropyl-6-oxo-1, 6-dihydro-4-pyrimidyl) acetic acid to obtain diazinon hapten.
The preparation method comprises the following steps:
dissolving 1.69g of trichloro sulfur phosphorus by adding 30mL of absolute ethyl alcohol, cooling to 0-5 ℃ in an ice bath, adding 1.38g of anhydrous potassium carbonate, continuing stirring for 3 hours, stopping the reaction, adding 200mL of 0-5 ℃ cold water, adding 100mL of trichloromethane, extracting, separating out a water phase, drying organic phase anhydrous sodium sulfate, and evaporating to dryness to obtain 2-ethoxy-chloro-sulfur phosphorus; then, dissolving 2-ethoxy-chloro-sulfur-phosphorus with 20mL of pyridine, adding (2-isopropyl-6-oxo-1, 6-dihydro-4-pyrimidyl) acetic acid 1.99g and KOH 1.22g, stirring at room temperature for 2h, adding 100mL of water, adjusting the pH value to 6 with 6mol/L hydrochloric acid, adding 1, 2-dichloroethane 200mL for extraction, separating the water phase, drying with anhydrous sodium sulfate, evaporating to dryness, purifying with a silica gel column, eluting and separating with a methanol-dichloromethane mixed solvent with the volume ratio of 10:1 to obtain the target compound.
Compared with diazinon, a spacer arm containing two carbon atoms is added in a target compound molecule, the structure of the diazinon is completely reserved, and the target compound can be used as a diazinon hapten.
The diazinon artificial antigen provided by the invention is obtained by coupling the diazinon hapten and carrier protein.
The carrier protein of the invention is selected from BSA, OVA and the like.
The diazinon artificial antigen can be prepared by the following method: and synthesizing the diazinon artificial antigen by adopting an active ester method.
The invention also provides a specific antibody prepared from the diazinon artificial antigen, wherein the antibody is a polyclonal antibody or a monoclonal antibody.
The invention also provides a diazinon detection reagent or kit prepared from the diazinon hapten, the diazinon artificial antigen or the specific antibody.
The invention also provides the application of the diazinon hapten, the diazinon artificial antigen, the specific antibody or the detection reagent or the kit in ELISA immunoassay detection of the diazinon.
The invention also provides the application of the diazinon hapten, the diazinon artificial antigen, the specific antibody or the detection reagent in preparing a colloidal gold detection test strip for diazinon.
After a mouse is immunized by the conjugate of the hapten and the carrier protein BSA provided by the invention, the titer of the antiserum of the mouse can reach 100000, and the sensitivity is 0.025 mu g/L. Experimental results show that the diazinon hapten and artificial antigen synthesized by the invention can generate high-sensitivity and high-specificity antibodies. The antigen and the antibody can be used for establishing immunoassay methods such as enzyme-linked immunosorbent assay and the like, and lay a foundation for detecting residue of diazinon in vegetables and fruits.
The invention modifies the structure of diazinon to obtain hapten, prepares the artificial antigen of diazinon, and the artificial antigen can be used for immunizing animals to generate specific antibody aiming at the diazinon, can be used for establishing an enzyme-linked immunosorbent assay method of the diazinon and a colloidal gold test strip rapid detection method, thereby being used for rapidly detecting the residue of the diazinon in food and having wide application prospect.
Drawings
FIG. 1 is a scheme showing the synthesis of diazinon hapten with the structure of formula (I) according to the present 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 Synthesis of diazinon haptens
Dissolving 1.69g of trichloro sulfur phosphorus by adding 30mL of absolute ethyl alcohol, cooling to 0-5 ℃ in an ice bath, adding 1.38g of anhydrous potassium carbonate, continuing stirring for 3 hours, stopping the reaction, adding 200mL of 0-5 ℃ cold water, adding 100mL of trichloromethane, extracting, separating out a water phase, drying organic phase anhydrous sodium sulfate, and evaporating to dryness to obtain 2-ethoxy-chloro-sulfur phosphorus; then, dissolving 2-ethoxy-chloro-sulfur-phosphorus with 20mL of pyridine, adding (2-isopropyl-6-oxo-1, 6-dihydro-4-pyrimidyl) acetic acid 1.99g and KOH 1.22g, stirring at room temperature for 2h, adding 100mL of water, adjusting the pH value to 6 with 6mol/L hydrochloric acid, adding 1, 2-dichloroethane 200mL for extraction, separating the water phase, drying with anhydrous sodium sulfate, evaporating to dryness, purifying with a silica gel column, eluting and separating with a methanol-dichloromethane mixed solvent with the volume ratio of 10:1 to obtain 0.67g of diazinon hapten.
EXAMPLE 2 preparation of Artificial antigen of diazinon
Conjugates of haptens to BSA were used as immunogens and conjugates to OVA as coaters.
1. Preparation of diazinon immunogens
Taking 26mg of diazinon hapten, adding 2mL of DMF (dimethyl formamide) for dissolving, adding 17mg of NHS (N-hydroxysuccinimide) and 20mg of EDC (EDC), and reacting at room temperature for 3 hours to obtain a hapten solution A; taking 100mg of BSA, and adding 0.05mol/L of CB buffer solution for dissolving to obtain solution B; dripping the A solution into the B solution, reacting for 12h at 4 ℃, dialyzing and purifying for 3 days by using 0.02mol/L PBS, changing the solution three times every day to obtain the diazinon phosphohapten-BSA conjugate which is the immunogen, and storing at-20 ℃ for later use.
2. Preparation of diazinon-coated antigen
Taking 15mg of diazinon hapten, adding 2mL of dioxane for dissolving, adding 8.9mg of NHS and 13.1mg of EDC, and reacting at room temperature for 3h to obtain a hapten solution A; dissolving OVA 100mg in CB buffer solution of 0.05mol/L to obtain solution B; dripping the A solution into the B solution, reacting for 12h at 4 ℃, dialyzing and purifying for 3 days by using 0.02mol/L PBS, and changing the solution three times every day to obtain the diazinon phosphorus hapten-OVA conjugate which is the coating antigen, and storing at-20 ℃ for later use.
EXAMPLE 3 preparation of diazinon monoclonal antibodies
1. Animal immunization
10 healthy female Balb/c mice (divided into two groups A and B, and 5 mice in each group) with the age of 6-8 weeks are taken, emulsified by Freund's complete adjuvant for primary immunization, and injected subcutaneously at multiple points on the back of the neck, wherein the immunization dose of each mouse is 200 mu g of diazinon immunogen in the embodiment 2; 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 the diazinon coated antigen in example 2 by 1:1000 with 0.05mol/L of carbonate buffer solution with pH9.6, coating an enzyme label plate by 100 mu L per well, incubating at 37 ℃ for 2h, throwing off the 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 intervals 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, diazinon is selected as a standard substance, and inhibition effect determination is carried out on positive cells by indirect competition ELISA. And selecting a well with better inhibition on the diazinon standard substance, carrying out subcloning by adopting a limiting dilution method, and carrying out detection by adopting the same method. Repeating the steps for three times to obtain the cell strain capable of stably secreting the diazinon 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 and a microscope at a cell concentration of1.0×106～1.5×106In 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 titer determination
And (3) measuring the antibody titer by adopting an indirect ELISA method, and referring to step 1, measuring the serum titer of the animal immunity. The result shows that the titer of the diazinon 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 diazinon monoclonal antibody to the diazinon and other organophosphorus pesticides is as follows: 100 percent of diazinon, and less than 1 percent of pyrimidylphosphine, pirimiphos-methyl, fenitrothion, fenthion, parathion-methyl, dichlorvos, carboxim, chlorpyrifos, simazine and atrazine. 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 (8)
1. The diazinon hapten is characterized by having a structure shown as a formula (I):
2. a method of producing the diazinon hapten of claim 1, wherein the method comprises the steps of: dissolving 1.69g of trichloro sulfur phosphorus by adding 30mL of absolute ethyl alcohol, cooling to 0-5 ℃ in an ice bath, adding 1.38g of anhydrous potassium carbonate, continuing stirring for 3 hours, stopping the reaction, adding 200mL of 0-5 ℃ cold water, adding 100mL of trichloromethane, extracting, separating out a water phase, drying organic phase anhydrous sodium sulfate, and evaporating to dryness to obtain 2-ethoxy-chloro-sulfur phosphorus; then, dissolving 2-ethoxy-chloro-sulfur-phosphorus with 20mL of pyridine, adding (2-isopropyl-6-oxo-1, 6-dihydro-4-pyrimidyl) acetic acid 1.99g and KOH 1.22g, stirring at room temperature for 2h, adding 100mL of water, adjusting the pH value to 6 with 6mol/L hydrochloric acid, adding 1, 2-dichloroethane 200mL for extraction, separating the water phase, drying with anhydrous sodium sulfate, evaporating to dryness, purifying with a silica gel column, eluting and separating with a methanol-dichloromethane mixed solvent with the volume ratio of 10:1 to obtain the target compound.
3. The diazinon artificial antigen is characterized by comprising the following components in parts by weight: obtained by coupling the diazinon hapten as described in claim 1 with a carrier protein; wherein, the carrier protein is selected from BSA and OVA.
4. A method for producing the diazinon artificial antigen according to claim 3, wherein the method comprises the steps of: and synthesizing the diazinon artificial antigen by adopting an active ester method.
5. A specific antibody prepared from the diazinon artificial antigen of claim 3.
6. A diazinon detection reagent or kit prepared from the diazinon hapten as defined in claim 1, the diazinon artificial antigen as defined in claim 3 or the specific antibody as defined in claim 5.
7. Use of the diazinon hapten of claim 1, the diazinon artificial antigen of claim 3, the specific antibody of claim 5 or the detection reagent or kit of claim 6 in ELISA immunoassay detection of diazinon.
8. Use of the diazinon hapten of claim 1, the diazinon artificial antigen of claim 3, the specific antibody of claim 5 or the detection reagent of claim 6 in preparing a colloidal gold test strip for diazinon.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010697067.0A CN111690007B (en) | 2020-07-20 | 2020-07-20 | Diazinon hapten and artificial antigen as well as preparation methods and applications thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010697067.0A CN111690007B (en) | 2020-07-20 | 2020-07-20 | Diazinon hapten and artificial antigen as well as preparation methods and applications thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111690007A true CN111690007A (en) | 2020-09-22 |
| CN111690007B CN111690007B (en) | 2023-10-20 |
Family
ID=72486298
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010697067.0A Active CN111690007B (en) | 2020-07-20 | 2020-07-20 | Diazinon hapten and artificial antigen as well as preparation methods and applications thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111690007B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1431213A (en) * | 2003-01-13 | 2003-07-23 | 浙江大学 | Method for preparing artificial hapten, artificial antigen and specific antibody of chlorpyrifos and its usage |
| KR20050026153A (en) * | 2003-09-09 | 2005-03-15 | 삼성에버랜드 주식회사 | Antibody for sulfamethazine and analysis method of sulfamethazine |
| CN101013129A (en) * | 2007-02-13 | 2007-08-08 | 北京望尔生物技术有限公司 | Enzyme linked immunosorbent reagent casing for detecting furacilin metabolite and uses thereof |
| CN102372737A (en) * | 2010-08-09 | 2012-03-14 | 贾明宏 | Preparation method and application of parathion-methyl artificial hapten, artificial antigen and specific antibody |
| CN110642793A (en) * | 2019-09-24 | 2020-01-03 | 北京勤邦生物技术有限公司 | Azoxystrobin hapten, artificial antigen and antibody, and preparation method and application thereof |
| CN111269262A (en) * | 2020-03-03 | 2020-06-12 | 北京勤邦生物技术有限公司 | Profenofos hapten, artificial antigen and antibody, and preparation method and application thereof |
-
2020
- 2020-07-20 CN CN202010697067.0A patent/CN111690007B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1431213A (en) * | 2003-01-13 | 2003-07-23 | 浙江大学 | Method for preparing artificial hapten, artificial antigen and specific antibody of chlorpyrifos and its usage |
| KR20050026153A (en) * | 2003-09-09 | 2005-03-15 | 삼성에버랜드 주식회사 | Antibody for sulfamethazine and analysis method of sulfamethazine |
| CN101013129A (en) * | 2007-02-13 | 2007-08-08 | 北京望尔生物技术有限公司 | Enzyme linked immunosorbent reagent casing for detecting furacilin metabolite and uses thereof |
| CN102372737A (en) * | 2010-08-09 | 2012-03-14 | 贾明宏 | Preparation method and application of parathion-methyl artificial hapten, artificial antigen and specific antibody |
| CN110642793A (en) * | 2019-09-24 | 2020-01-03 | 北京勤邦生物技术有限公司 | Azoxystrobin hapten, artificial antigen and antibody, and preparation method and application thereof |
| CN111269262A (en) * | 2020-03-03 | 2020-06-12 | 北京勤邦生物技术有限公司 | Profenofos hapten, artificial antigen and antibody, and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111690007B (en) | 2023-10-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111978304B (en) | Difenoconazole hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN110498766B (en) | Fluazinam hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN110845444B (en) | Dimethomorph hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN101413955B (en) | ELISA test box for detecting zearalenone and preparing and detecting method thereof | |
| CN111333570B (en) | Haloxyfop hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN114315722B (en) | Preparation and application of tolfenpyrad artificial hapten and antibody thereof | |
| CN110642793A (en) | Azoxystrobin hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN111269262B (en) | Profenofos hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN110845429A (en) | Tebuconazole hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN111943882B (en) | Pythium hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN110627726B (en) | Prochloraz hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN101100456A (en) | Fipronil artificial hapten and synthesis method, antigen and antibody and application | |
| CN113264834A (en) | Abienol hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN103288963B (en) | Monoclonal antibody for kitasamycin residual detection and preparation method and application thereof | |
| CN112480167B (en) | Isocarbophos hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN110590561B (en) | Herbicidal ether hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN113024415B (en) | Cyhalothrin hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN111690007A (en) | Diazinon hapten and artificial antigen as well as preparation method and application thereof | |
| CN111825562B (en) | Chlordan hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN111875652A (en) | Pleocidin hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN113896758A (en) | Solanum nigrum semiantigen, artificial antigen and antibody, and preparation method and application thereof | |
| CN113527390A (en) | Tulathromycin hapten, artificial antigen and antibody, and preparation method and application thereof | |
| CN109608479A (en) | Preparation method and application of a tetramine hapten and monoclonal antibody | |
| CN111943881B (en) | Chlorfenapyr hapten, artificial antigen and antibody as well as preparation methods and applications thereof | |
| CN111961002A (en) | Pyraclostrobin hapten, artificial antigen and antibody as well as preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: No.8, Gaoxin 4th Street, Huilongguan international information industry base, Changping District, Beijing Applicant after: Beijing Qinbang Technology Co.,Ltd. Address before: No.8, Gaoxin 4th Street, Huilongguan international information industry base, Changping District, Beijing Applicant before: BEIJING KWINBON BIOTECHNOLOGY Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |