CN115947688B - Pyrazolonamine hapten, antigen, antibody, detection device and preparation and application thereof - Google Patents
Pyrazolonamine hapten, antigen, antibody, detection device and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a isopyrazam hapten, an antigen, an antibody, a detection device and preparation and application thereof, and relates to a isopyrazam hapten, an antigen, an antibody, a colloidal gold chromatography detection device and preparation and application thereof in detecting isopyrazam residues in foods. The invention provides an artificial antigen prepared from a isopyrazam hapten coupled carrier protein, and an antibody which is specifically aimed at isopyrazam and is produced by a body of the animal after the animal is immunized, wherein the titer is high, and the IC50 value is 22.3ng/mL. The invention utilizes the specific immunological reaction of antigen and antibody and the easily identified immune mark amplification technology, can qualitatively and semi-quantitatively detect the residual isopyrazam in food, and has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, on-site rapid detection and the like.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a isopyrazam hapten, an antigen, an antibody, a detection device and preparation and application thereof.
Background
Isopyrazam (Isopyrazam) is an active ingredient of a bactericide, is a new generation succinate dehydrogenase inhibitor (SDHI), has both prevention and treatment effects, mainly prevents and treats cereal leaf diseases, and also has yield increasing effect. Can be used for fruit vegetables, brassica, cucurbits, bananas, rapes, pomes and ornamental plants to prevent and treat fungal diseases. Strict limits are imposed on the residue of isopyrazam in agricultural products in the United states, japan and other countries. The development of the method for rapidly detecting the isopyrazam residue has important significance for guaranteeing food safety and ensuring smooth export of agricultural products in China.
Common detection methods of the isopyrazam comprise gas chromatography, and the like, and the detection methods have higher accuracy, but rely on large-scale detection equipment, have complex operation and high detection cost, and are not suitable for large-scale popularization and application; and the results cannot be immediately displayed, and the method is not suitable for departments such as basic management and the like to rapidly detect and monitor the suspected objects on line. The immunological detection 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 a plurality of advantages compared with methods such as instrument detection and the like.
When an immunological detection method is established and the detection method is applied to detect the residual quantity of the isopyrazam, the key technology is that the antibody with strong specificity and high sensitivity can be obtained, and the aim is to be fulfilled if a proper isopyrazam hapten is required to be synthesized and prepared. However, no related report on the isopyrazam hapten is available in China.
Disclosure of Invention
The invention aims to provide a isopyrazam hapten, an antigen, an antibody, a detection device and a preparation and detection method thereof, and aims to detect residual isopyrazam in food.
According to one aspect of the present invention, there is provided a isopyrazam hapten having the structure shown in formula (I):
according to another aspect of the present invention, there is provided a method for preparing a isopyrazam hapten, comprising the steps of:
s1, adding 4, 4-difluoro acetoacetic acid ethyl ester, triethyl orthoformate and acetic anhydride into a single-neck flask, carrying out reflux stirring reaction, and carrying out reduced pressure distillation after the reaction is finished to obtain an intermediate 1, wherein the reaction formula in the step is shown as a formula (II):
s2, dissolving the intermediate 1 by using ethanol, adding hydrazine hydrate, refluxing and stirring for reaction, removing ethanol by rotary evaporation under reduced pressure after the reaction is finished, adding a proper amount of water, extracting for 2-3 times by using ethyl acetate, merging organic phases, evaporating to dryness, and purifying by using a column to obtain an intermediate 2, wherein the reaction formula in the step is shown as a formula (III):
s3, adding the intermediate 2, anhydrous potassium carbonate and DMF into a 100mL single-neck flask, stirring uniformly, adding 6-bromo-1-hexene, stirring at 85 ℃ for reaction, cooling to room temperature after the reaction is finished, performing oil pump reduced pressure rotary evaporation, distilling off solvent DMF, adding a proper amount of water, extracting for 2-3 times by using ethyl acetate, merging organic phases, evaporating to dryness, and purifying by a column to obtain an intermediate 3, wherein the reaction formula in the step is shown as a formula (IV):
s4, dissolving the intermediate 3 by using methanol, adding a sodium hydroxide solution, refluxing and stirring for reaction overnight, adjusting a system to be slightly acidic by using hydrochloric acid after the reaction is finished, extracting for 2-3 times by using ethyl acetate, combining organic phases, washing by using water, drying by using anhydrous sodium sulfate, and evaporating to dryness to obtain an intermediate 4, wherein the reaction formula of the step is shown as a formula (V):
s5, dissolving the intermediate 4 by using dichloromethane, adding thionyl chloride, carrying out reflux stirring reaction, evaporating the solvent after the reaction is finished to obtain an intermediate 5, wherein the reaction formula in the step is shown as a formula (VI):
S6, adding the isopyrazam into a single-neck flask, adding methanol to dissolve the isopyrazam completely, adding sodium hydroxide solution, refluxing and stirring for reaction, cooling to room temperature after the reaction is finished, removing the methanol by reduced pressure rotary evaporation, adding hydrochloric acid solution to adjust the pH of the solution to about 8, extracting for 2-3 times by using ethyl acetate, merging organic phases, washing by water, evaporating to dryness, and purifying by a column to obtain an intermediate 6, wherein the reaction formula of the step is shown as a formula (VII):
s7, dissolving the intermediate 6 by using anhydrous pyridine, dropwise adding the intermediate 5 under stirring at room temperature after dissolving, stirring at room temperature for reaction, and carrying out reduced pressure rotary evaporation by an oil pump after the reaction is finished, and purifying by passing the distilled solvent pyridine through a column to obtain an intermediate 7, wherein the reaction formula in the step is shown as a formula (VIII):
s8, putting the intermediate 7 into a single-neck flask, adding acetonitrile and water, then adding sodium periodate, then adding ruthenium trichloride, finally adding carbon tetrachloride, heating a mixed system to reflux for reaction at 60 ℃, filtering the system to remove insoluble substances after the reaction is finished, concentrating, adding water for dilution, extracting for 2-3 times by using ethyl acetate, merging organic phases, drying by using anhydrous sodium sulfate, evaporating, and purifying by a column to obtain the isopyrazam hapten, wherein the reaction formula of the step is shown as a formula (IX):
In some embodiments, the mass volume fraction of sodium hydroxide solution in step S4 is 10%.
In some embodiments, the molar concentration of the hydrochloric acid solution is 6mol/L.
In some embodiments, the mass volume fraction of sodium hydroxide solution in step S6 is 25%.
According to a further aspect of the invention, the isopyrazam antigen is a conjugate of isopyrazam hapten and a carrier protein, the carrier protein being one of bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin.
According to a fourth aspect of the present invention, the isopyrazam antibody is prepared from isopyrazam antigen by animal immunization, and the isopyrazam antibody is isopyrazam monoclonal antibody.
According to a fifth aspect of the invention, there is provided the use of isopyrazam hapten, isopyrazam antigen in immunological detection of isopyrazam.
According to a sixth aspect of the present invention there is provided the use of a isopyrazam antibody in the immunological detection of isopyrazam.
According to a seventh aspect of the present invention, there is provided a rapid detection method of isopyrazam residue in a food, the method comprising detecting isopyrazam residue in a food using isopyrazam antibody.
The invention has the beneficial effects that: the invention provides an artificial antigen prepared from a isopyrazam hapten coupled carrier protein, and a high-specificity antibody specific to isopyrazam generated by a body of the animal after an experimental animal is immunized, wherein the titer is high, and the IC50 value is 22.3ng/mL. The invention can qualitatively and semi-quantitatively detect the residual isopyrazam in food by utilizing the specific immunological reaction of antigen and antibody and the easily identified immune labeling amplification technology. The method has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, capability of realizing on-site rapid detection and the like.
Drawings
FIG. 1 is a mass spectrum of a isopyrazam hapten according to one embodiment of the invention.
FIG. 2 is a standard curve of an indirect competition ELISA established based on a isopyrazam monoclonal antibody according to one embodiment of the invention.
Detailed Description
The invention will be described in further detail with reference to specific embodiments thereof, it being understood that these embodiments are for purposes of illustration only and not for purposes of limiting the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the invention, and are defined in the claims appended hereto. Unless otherwise specified, all materials and reagents of the invention are those commercially available in the conventional market.
Example 1 in this example, pure chromatographically isopyrazam supplied by isopyrazam select Cook Chemicals, 97% bromopropyne supplied by 3-bromopropyne select Cook Chemicals, 60% sodium hydride supplied by sodium hydride select Cook Chemicals, analytically pure ethyl acetate supplied by ethyl acetate select Cook Chemicals, 97% 4-azidobutyric acid supplied by 4-azidobutyric acid select Zhengzhou alpha Chemicals, 99% L-ascorbic acid supplied by L-ascorbic acid select Cook Chemicals, the following examples 2-6 all employ the reagents of example 1.
Example 2 a method for preparing a isopyrazam hapten comprising the steps of:
s1, adding 9.0g of 4, 4-difluoro acetoacetic acid ethyl ester, 10.9g of triethyl orthoformate and 50mL of acetic anhydride into a single-neck flask, carrying out reflux stirring reaction for 5h, and carrying out reduced pressure distillation after the reaction is finished to obtain an intermediate 1;
s2, dissolving about 10g of the intermediate 1 in the previous step by using 50mL of ethanol, adding 2.5mL of hydrazine hydrate, refluxing and stirring for reaction for 1.5h, removing ethanol by rotary evaporation under reduced pressure after the reaction is finished, adding a proper amount of water, extracting for 2-3 times by using ethyl acetate, merging organic phases, evaporating to dryness, and purifying by a column to obtain an intermediate 2;
s3, weighing 4.0g of the intermediate 2, adding 5.0g of anhydrous potassium carbonate and 30mL of DMF into a 100mL single-neck flask, stirring uniformly, adding 4.0g of 6-bromo-1-hexene, stirring at 85 ℃ for reaction for 3 hours, cooling to room temperature after the reaction is finished, performing rotary evaporation under reduced pressure by an oil pump, distilling off the solvent DMF, and adding a proper amount of water. Extracting with ethyl acetate for 2-3 times, mixing organic phases, evaporating to dryness, and purifying by column to obtain intermediate 3;
s4, dissolving about 5g of the intermediate 3 in 10mL of methanol, adding 15mL of 10% sodium hydroxide solution, refluxing and stirring for reaction overnight, regulating a system to be slightly acidic by using 6mol/L hydrochloric acid solution after the reaction is finished, extracting for 2-3 times by using ethyl acetate, merging organic phases, washing by using water, drying by using anhydrous sodium sulfate, and evaporating to obtain an intermediate 4;
s5, dissolving about 4g of the intermediate 4 obtained in the previous step by using 10mL of dichloromethane, adding 5mL of thionyl chloride, carrying out reflux stirring reaction for 4 hours, and evaporating the solvent after the reaction is finished to obtain an intermediate 5;
s6, adding 10.0g of isopyrazam into a single-neck flask, adding methanol to dissolve completely, adding 100mL of 25% sodium hydroxide solution with mass and volume fraction, refluxing and stirring for reaction for 20h, cooling to room temperature after the reaction is finished, removing the methanol by rotary evaporation under reduced pressure, adding 6mol/L hydrochloric acid solution to adjust the pH of the system to about 8, extracting for 2-3 times by using ethyl acetate, merging organic phases, washing by water, evaporating to dryness, and purifying by a column to obtain an intermediate 6;
s7, weighing 3.7g of intermediate 6, dissolving the intermediate 6 with 20mL of anhydrous pyridine, dropwise adding the intermediate 5 obtained in the step 5 under stirring at room temperature after dissolving, stirring at room temperature for reaction for 5 hours after the dropwise adding is finished, and performing rotary evaporation under reduced pressure by an oil pump after the reaction is finished, and evaporating a solvent pyridine, and purifying by a column to obtain an intermediate 7;
s8, putting 2.8g of the intermediate 7 obtained in the previous step into a single-neck flask, adding 20mL of acetonitrile and 30mL of water, then adding 5.2g of sodium periodate, then adding 0.3g of ruthenium trichloride, finally adding 20mL of carbon tetrachloride, heating a mixed system to reflux for 2h at 60 ℃, filtering the system to remove insoluble substances after the reaction is finished, concentrating, adding water for dilution, extracting for 2-3 times by using ethyl acetate, merging organic phases, drying by using anhydrous sodium sulfate, evaporating, purifying by using a column, and obtaining the isopyrazam hapten, wherein the structure of the isopyrazam hapten is shown as a formula (I).
The mass spectrometry is adopted to identify the isopyrazam hapten, and the obtained mass spectrum is shown in the attached figure 1 of the specification. As can be seen from a mass spectrum, the molecular ion peak of the isopyrazam hapten is M/z 443.7 [ M-H ]] - And the highest peak is consistent with the molecular weight 445.22 of the isopyrazam hapten, which shows that the isopyrazam hapten shown in the formula (I) is successfully synthesized.
EXAMPLE 3 preparation of antigen for immunization and antigen for coating of isopyrazam
Preparation of antigen for immunization with isopyrazam: weighing 25mg of isopyrazam hapten, dissolving in 2ml of N, N-Dimethylformamide (DMF), adding 20mg of N-hydroxysuccinimide (NHS) and 30mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl), and reacting for 6 hours at room temperature to obtain an activated liquid; 60mg of Bovine Serum Albumin (BSA) is dissolved in 2ml of boric acid buffer solution with the pH value of 0.05M and 9.0, 1ml of DMF and 0.5ml of the activating solution are added for reaction for 4 hours at room temperature, PBS (0.02 mol/L, phosphate buffer solution with the pH value of 7.4) is used for dialysis, each 4 times of h is used for changing the solution for 1 time, 7-8 times of changing the solution is used for dialysis, centrifugation is carried out for 5 minutes at 4000 revolutions per minute, supernatant is taken, and a isopyrazam hapten-BSA conjugate is obtained, namely the isopyrazam antigen for immunization, split charging is carried out, and the isopyrazam antigen is stored at the temperature of minus 20 ℃.
Preparation of antigen for coating isopyrazam: weighing 12 mg of isopyrazam hapten, dissolving in 1ml of anhydrous N, N-Dimethylformamide (DMF), sequentially adding 4 mu l of diisopropylethylamine and 5 mu l of isobutyl chloroformate, and reacting at 0 ℃ for 30min to obtain solution A; dissolving 70 mg chicken Ovalbumin (OVA) with 0.1M boric acid buffer solution 7ml to obtain solution B; dropwise adding the solution A into the solution B, reacting for 4 hours at room temperature, dialyzing with PBS (0.02 mol/L, phosphate buffer solution with pH=7.4), changing the solution for 1 time every 4h times, changing the solution for 7-8 times, centrifuging for 5 minutes at 4000 revolutions/min after dialyzing, taking the supernatant, and obtaining the isopyrazam hapten-OVA conjugate, namely the isopyrazam coating antigen, packaging, and storing at-20 ℃.
EXAMPLE 4 preparation, purification and establishment of an Indirect competitive ELISA Standard Curve of a Pyrazolidinium monoclonal antibody
The preparation method of the isopyrazam monoclonal antibody comprises the following steps:
after the antigen for immunization of the isopyrazam obtained in example 3 was identified, 10 8-week-old BALB/C mice were immunized, three times of booster immunization were performed, the titers were measured by blood sampling, and after the serum titers were no longer increased, the mice were immunized with twice the amount of the antigen for immunization without adjuvant, and after three days, the mice were cervical-removed and killed. Spleen cells were prepared under aseptic conditions, mixed with growing mouse myeloma cells in a 50mL centrifuge tube at a ratio of 8:1, added with 30mL serum-free IPMI1640 medium, centrifuged at 1100r/min for 5min to discard supernatant, and the cell mass was gently shaken loose and placed in a 37 ℃ water bath. Slowly adding 1mL 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, stopping 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 spread thereon, adding CO with volume fraction of 5% at 37deg.C, and concentrating under reduced pressure 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.
The monoclonal antibody of the anti-isopyrazam is produced by adopting an in-vivo induced ascites method. Selecting 4 produced Kunming mice, injecting liquid paraffin oil into abdominal cavity 0.5 mL/mouse, and injecting 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 isopyrazam monoclonal antibody by ultraviolet. A standard curve is established according to indirect ELISA, see figure 2 of the specification. The IC50 of the curve is 22.3ng/mL, the detection limit (the mass concentration of parathion at 90% of the binding rate) is 0.890ng/mL, the detection range (the mass concentration of parathion at 20% -80% of the binding rate) is 3.4-550.1 ng/mL, and the detection requirement of the GB2763-2021 on the residue of the isopyrazam in fruits and vegetables can be met.
Example 5 preparation of a Pyrazolonamine colloidal gold chromatography detection device
5.1 Preparation of colloidal gold solution
Diluting 1% chloroauric acid solution into 0.01% (mass fraction) by using double distilled deionized water, placing 100ml of 0.01% chloroauric acid solution into a conical flask, heating to boiling by using a constant-temperature electromagnetic stirrer, rapidly adding 1.6 ml of 1% trisodium citrate solution at one time, continuously stirring at a constant speed, heating until the solution is transparent red, stopping stirring, continuously heating for 5min after color stabilization, cooling to room temperature, recovering to original volume by using deionized water, obtaining colloidal gold solution, and preserving at 4 ℃;
5.2 Preparation of isopyrazam monoclonal antibody-colloidal gold marker
Taking 100ml of prepared 0.01% colloidal gold solution, adjusting the pH value of the colloidal gold solution to about 8.5 by using 0.1mol/L potassium carbonate under magnetic stirring, adding 30-40 mug of the isopyrazam monoclonal antibody into the colloidal gold solution according to the standard of adding the isopyrazam monoclonal antibody into each ml of the colloidal gold solution, adding the isopyrazam monoclonal antibody prepared in the example 4 into the colloidal gold solution, and standing for 20min at the room temperature of 25 ℃ after uniformly stirring. Adding 1ml of 10% Bovine Serum Albumin (BSA) solution, stirring by using a magnetic stirrer (rotating speed 150e/min,30 min), standing for 20min, centrifuging at 12000rpm for 40min at 4 ℃, discarding supernatant, re-suspending the rest precipitate by using 20ml of colloid Jin Chong suspension (2% sucrose, 1% BSA and 0.3% Tween 20 and prepared by 0.01mol/L phosphate buffer solution with pH of 7.4), and standing at 4 ℃ for later use to prepare a isopyrazam monoclonal antibody-colloidal gold marker;
5.3 preparation of Pyrazolonamine colloidal gold detection device
Spraying a isopyrazam monoclonal antibody-colloidal gold marker on a gold-labeled pad according to the amount of 5 mu l/cm by a film spraying instrument, drying for 2 hours at 37 ℃, coating a detection area (T line) and a quality control area (C line) on an NC film, coating the detection area (T line) with the isopyrazam coating antigen prepared in the embodiment 3, sequentially adhering the NC film, the gold-labeled pad, a sample pad and a water-absorbing pad on a PVC bottom plate, cutting into test strips with the width of 4mm after the adhesion, placing in a special plastic card, adding a drying agent, sealing, and obtaining the isopyrazam colloidal gold chromatography detection card, and preserving at room temperature.
Example 6 Rapid detection method of isopyrazam residue in food
6.1 sample pretreatment: 1.0 g+/-0.01 g of crushed food sample to be detected is weighed into a 50mL polystyrene centrifuge tube, tris-PBS buffer solution with pH of 7.2 is added for dilution by 10 times, a cover is covered, a vortex mixer is evenly mixed or manually oscillated up and down for evenly mixing 30s, and the mixture is kept stand for 5min, and supernatant fluid is sucked to be detected.
6.2, detection: 3-5 drops are added into the sample adding hole of the isopyrazam colloidal gold chromatography detection card prepared in the example 5, and after 3-5min, the result is judged.
Result determination
The result determination is performed by comparing the color shades of the control line (C line) and the detection line (T line).
Positive: when the quality control area (C line) and the detection area (T line) both show strips, judging that the strips are positive, namely that the food has the residue of the isopyrazam, and the residue is expressed by "+";
negative: when the quality control area (C line) shows a strip, the detection area (T line) does not develop color, and the detection area is judged as negative, namely, the food has no residual isopyrazam or the residual isopyrazam content in the food is lower than the detection limit, and the mark is indicated by "-;
invalidation: when the quality control area (C line) does not show the strip, the test paper fails.
The foregoing is merely illustrative of some embodiments of the invention, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the inventive concept.
Claims (4)
1. A method for preparing a isopyrazam hapten, comprising the steps of:
s1, adding 4, 4-difluoro acetoacetic acid ethyl ester, triethyl orthoformate and acetic anhydride into a single-neck flask, carrying out reflux stirring reaction, and carrying out reduced pressure distillation after the reaction is finished to obtain an intermediate 1, wherein the reaction in the step is shown as the following formula (II):
s2, dissolving the intermediate 1 by using ethanol, adding hydrazine hydrate, refluxing and stirring for reaction, removing ethanol by rotary evaporation under reduced pressure after the reaction is finished, adding a proper amount of water, extracting for 2-3 times by using ethyl acetate, merging organic phases, evaporating to dryness, and purifying by a column to obtain an intermediate 2, wherein the reaction in the step is shown as the following formula (III):
s3, adding the intermediate 2, anhydrous potassium carbonate and DMF into a 100mL single-neck flask, stirring uniformly, adding 6-bromo-1-hexene, stirring at 85 ℃ for reaction, cooling to room temperature after the reaction is finished, performing oil pump decompression rotary evaporation, distilling off solvent DMF, adding a proper amount of water, extracting for 2-3 times by using ethyl acetate, merging organic phases, evaporating to dryness, and performing column purification to obtain an intermediate 3, wherein the reaction in the step is as shown in the following formula (IV):
s4, dissolving the intermediate 3 by using methanol, adding a sodium hydroxide solution, refluxing and stirring for reaction overnight, adjusting a system to be slightly acidic by using a hydrochloric acid solution after the reaction is finished, extracting for 2-3 times by using ethyl acetate, merging organic phases, washing by using water, drying by using anhydrous sodium sulfate, and evaporating to dryness to obtain an intermediate 4, wherein the reaction in the step is shown in the following formula (V):
s5, dissolving the intermediate 4 by using dichloromethane, adding thionyl chloride, carrying out reflux stirring reaction, evaporating the solvent after the reaction is finished to obtain an intermediate 5, wherein the reaction in the step is shown as the following formula (VI)
s6, adding the isopyrazam into a single-neck flask, adding methanol to dissolve the isopyrazam completely, adding sodium hydroxide solution, refluxing and stirring for reaction, cooling to room temperature after the reaction is finished, removing the methanol by rotary evaporation under reduced pressure, adding hydrochloric acid solution to adjust the pH of the system to about 8, extracting for 2-3 times by using ethyl acetate, merging organic phases, washing with water, evaporating to dryness, and purifying by a column to obtain an intermediate 6, wherein the reaction in the step is shown as the following formula (VII):
s7, dissolving the intermediate 6 by using anhydrous pyridine, dropwise adding the intermediate 5 under stirring at room temperature after dissolving, stirring at room temperature for reaction after the dropwise adding is finished, and performing oil pump reduced pressure rotary evaporation after the reaction is finished, distilling out the solvent pyridine and purifying by using a column to obtain the intermediate 7, wherein the reaction formula in the step is shown as a formula (VIII):
s8, putting the intermediate 7 into a single-neck flask, adding acetonitrile and water, then adding sodium periodate, then adding ruthenium trichloride, finally adding carbon tetrachloride, heating a mixed system to perform reflux reaction at 60 ℃, filtering a system to remove insoluble substances after the reaction is finished, concentrating, adding water for dilution, extracting for 2-3 times by using ethyl acetate, merging organic phases, drying by using anhydrous sodium sulfate, evaporating, and purifying by a column to obtain the isopyrazam hapten, wherein the reaction formula of the step is shown as a formula (IX):
2. The method according to claim 1, wherein the sodium hydroxide solution in step S4 has a mass volume fraction of 10%.
3. The method according to claim 1, wherein the molar concentration of the hydrochloric acid solution is 6mol/L.
4. The method according to claim 1, wherein the mass volume fraction of sodium hydroxide solution in step S6 is 25%.
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