CN117362240A - Pyrimethanil hapten and artificial antigen as well as preparation methods and application thereof - Google Patents

Pyrimethanil hapten and artificial antigen as well as preparation methods and application thereof Download PDF

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CN117362240A
CN117362240A CN202311307816.4A CN202311307816A CN117362240A CN 117362240 A CN117362240 A CN 117362240A CN 202311307816 A CN202311307816 A CN 202311307816A CN 117362240 A CN117362240 A CN 117362240A
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pyrimethanil
hapten
artificial antigen
compound
antigen
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刘波
袁利鹏
华彦涛
梁铃渝
文锦谦
伍惠冰
姚楷淳
吴芷茗
戴蕤
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GUANGDONG AIB POLYTECHNIC COLLEGE
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Abstract

The invention disclosesA pyrimethanil hapten and an artificial antigen as well as a preparation method and application thereof are provided. The structural formula of the pyrimethanil hapten isThe arm introduced by the pyrimethanil hapten has active groups, the benzene ring and the dimethylpyrimidine ring structure of the object to be detected are completely reserved, and recognition sites are enhanced. The pyrimethanil artificial antigen and monoclonal antibody of the invention are used for ELISA detection with strong specificity and IC 50 The value is 1.97 mug/L, which is better than the prior monoclonal antibody. The colloidal gold immunochromatographic test strip prepared by the invention has the sensitivity of 10 mug/L for detecting the pyrimethanil.

Description

Pyrimethanil hapten and artificial antigen as well as preparation methods and application thereof
Technical Field
The invention relates to the field of biochemical engineering, in particular to a pyrimethanil hapten and an artificial antigen as well as a preparation method and application thereof.
Background
The pyrimethanil is a high-efficiency and low-toxicity bactericide for preventing and treating gray mold, has systemic conduction and fumigation effects, can be quickly transferred to various parts in a plant body after being applied, and effectively inhibits the generation of pathogenic bacteria infection enzyme, so that pathogenic bacteria infection is prevented, and pathogenic bacteria are thoroughly killed, and the special action surface is widely used. The toxicity of pyrimethanil in mammals is low, but studies have shown that pyrimethanil has potential oncogenic toxicity in mice, rats, dogs and aquatic organisms.
The conventional instrument detection method has the advantages that equipment and instruments are expensive, the detection time is long, professional operation is needed, and the on-site rapid spot check detection cannot be realized; the other detection method is an immunological detection technology, has the characteristics of high specificity and high selectivity, and is very suitable for detecting trace components of complex matrixes. The performance of the immunodetection product is determined by the performance of the antigen and the antibody, and the key of the antigen and the antibody is the hapten, so that the structural design of the hapten is important for obtaining the antigen and the antibody with excellent performance. Therefore, the development of the pyrimethanil hapten or the artificial antigen with high specificity has important significance for a quick, high-sensitivity and low-cost detection method of pyrimethanil.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art and provides a pyrimethanil hapten, an artificial antigen, a preparation method and application thereof.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided:
the structure of the pyrimethanil hapten is shown as a formula I:
in a second aspect of the invention, there is provided:
the preparation method of the pyrimethanil hapten comprises the following steps:
1) The compound A and the compound B undergo substitution reaction under alkaline conditions to obtain a compound C;
2) Hydrolyzing the compound C to obtain pyrimethanil hapten;
wherein the structural formula of the compound A isThe structural formula of the compound B is
The structural formula of the compound C is
In a third aspect of the invention, there is provided:
an artificial antigen of pyrimethanil, which is obtained by coupling the pyrimethanil hapten and a protein carrier according to the first aspect of the invention.
In some examples of the pyrimethanil artificial antigen, the pyrimethanil artificial antigen has a structural formula as shown in formula II:
wherein protein is a protein carrier.
In some examples of the pyrimethanil artificial antigen, the protein carrier is at least one of bovine serum albumin, ovalbumin, human serum albumin, or hemocyanin.
In a fourth aspect of the invention, there is provided:
the preparation method of the pyrimethanil artificial antigen is characterized by comprising the following steps of:
1) Mixing hapten, carbodiimide and N-hydroxysuccinimide shown in formula I, and performing an activation reaction to obtain hapten activated ester;
2) Mixing carrier protein with hapten activated ester, and carrying out amidation reaction to obtain the artificial antigen.
In some examples of the preparation method, the activation reaction time is 3 to 5 hours, and the amidation reaction time is 16 to 24 hours.
In a fifth aspect of the invention, there is provided:
the invention relates to an application of an artificial antigen of pyrimethanil in preparation of ELISA detection of pyrimethanil.
In a sixth aspect of the invention, there is provided:
a test strip or test reagent for detecting pyrimethanil, wherein the test strip or test reagent comprises the pyrimethanil artificial antigen according to the third aspect of the invention.
In a seventh aspect of the invention, there is provided:
the pyrimethanil monoclonal antibody is prepared by immunizing an animal with the pyrimethanil artificial antigen as an immunogen.
The beneficial effects of the invention are as follows:
1. the arm introduced by the pyrimethanil hapten has active groups, the benzene ring and the dimethylpyrimidine ring structure of the object to be detected are completely reserved, and recognition sites are enhanced.
2. The pyrimethanil artificial antigen and monoclonal antibody of the invention are used for ELISA detection with strong specificity and IC 50 The value is 1.97 mug/L, which is better than the prior monoclonal antibody.
3. The pyrimethanil artificial antigen and the monoclonal antibody are used for a colloidal gold immunochromatography technology, and can rapidly and conveniently detect pyrimethanil, and the colloidal gold immunochromatography test strip prepared by the invention has the detection sensitivity of 10 mug/L for pyrimethanil.
Drawings
FIG. 1 is a synthetic route for pyrimethanil hapten.
FIG. 2 is a mass spectrum of pyrimethanil hapten.
FIG. 3 is a synthetic route pattern for pyrimethanil artificial antigen.
Fig. 4 is a standard graph of pyrimethanil.
Detailed Description
The following disclosure provides many different embodiments, or examples, for implementing different aspects of the invention.
Preparation of pyrimethanil hapten
As shown in FIG. 1, the pyrimethanil hapten of the invention is prepared by the following method, and the steps are as follows:
1) 3.00g (15.06 mmol) of Compound A was taken in a 100ml round bottom flask and 20ml DMF, 3.12. 3.12g K were added sequentially 2 CO 3 (22.59 mmol), 4.41g (22.59 mmol) of Compound B and a catalytic amount of tetrabutylammonium bromide (200 mg) were reacted at room temperature for more than 48 h. After the reaction, 90ml of purified water was added thereto, and both were extracted with ethyl acetateThen, the solvent was removed under reduced pressure, and the mixture was purified by column chromatography to obtain 1.75g of Compound C.
2) 1.75g (5.58 mmol) of Compound 3 was dissolved in 10ml of methanol, and then 10ml of 4mol/L aqueous lithium hydroxide solution was added, the reaction mixture was reacted at RT for 8 hours or more, 80ml of aqueous sodium chloride solution was added, and extraction was performed twice with 30ml of methylene chloride, the pH of the aqueous phase was adjusted to 4 to 5 with 4M dilute hydrochloric acid, filtration and drying were performed to obtain 1.07g of hapten, and the mass spectrum of the hapten was shown in FIG. 2.
Example 1
As shown in the figure, the synthesis method of the pyrimethanil artificial antigen with the carrier protein being bovine serum albumin comprises the following steps:
1) Dissolving 10mg of the prepared pyrimethanil hapten in 0.2mL of Dimethylformamide (DMF), stirring fully, adding 10mg of carbodiimide (EDC) and 10mg of N-hydroxysuccinimide (NHS), and stirring at room temperature for 4 hours to obtain hapten activated ester;
2) Weighing 30mg of Bovine Serum Albumin (BSA), fully dissolving the BSA in 3mL of 0.1mol/L CB buffer solution to form a carrier protein solution, dropwise and slowly dropwise adding the hapten-activated ester into the carrier protein solution under stirring, and stirring at room temperature for 16-24 hours;
3) Dialyzing the solution prepared in the step 2) with 0.01mol/L PBS at room temperature for 3 days, and changing the dialysate 3 times per day to remove unreacted micromolecular substances;
4) Subpackaging, and storing at 4deg.C for use.
Example 2
As shown in fig. 3, the synthesis method of the pyrimethanil artificial antigen with the carrier protein being hemocyanin comprises the following steps:
1) Dissolving 5mg of the prepared pyrimethanil hapten in 0.2mL of Dimethylformamide (DMF), stirring fully, adding 10mg of EDC and 10mg of N-hydroxysuccinimide (NHS), and stirring at room temperature for 4 hours to obtain hapten activated ester;
2) 15mg of hemocyanin (KLH) is weighed and fully dissolved in 3mL of 0.1mol/L CB buffer solution to form a carrier protein solution, the hapten-activated ester is slowly dripped into the carrier protein solution dropwise under stirring, and the carrier protein solution is stirred at room temperature for 16-24 hours;
3) Dialyzing the solution prepared in the step 2) with 0.01mol/L PBS at room temperature for 3 days, and changing the dialysate 3 times per day to remove unreacted micromolecular substances;
4) Subpackaging, and storing at 4deg.C for use.
Application of pyrimethanil artificial antigen in preparation of pyrimethanil-resistant monoclonal antibody
The preparation method of the pyrimethanil-resistant monoclonal antibody comprises the following steps:
BALB/C mice were immunized by emulsifying the pyrimethanil artificial antigen of example 2 with an equal volume of Freund's adjuvant. The immunization dose of each mouse is 100 mug, the immunization interval is 2 weeks, and the serum titer is detected by taking the tail venous blood of the mouse after 3 times of immunization. If the antibody titer is not required, the immunization needs to be enhanced, after the antibody titer is no longer increased, 100 mug of whole antigen is used for subcutaneous enhanced immunization, and after 5 days, the spleen cells of the mice are taken and fused with SP20 cells (myeloma cells of the mice). The fused cells were selected in HAT medium and cultured after 5 days by replacing the HAT medium with complete medium. And (3) detecting cell supernatants by ELISA, and carrying out cloning culture on the cells in the holes with strong positive detection results by a limiting dilution method, wherein the cells in the holes which are positive after 3 times of cloning culture detection are hybridoma cells secreting monoclonal antibodies. After the hybridoma cells are cultured in an enlarged manner, the hybridoma cells are inoculated into the abdominal cavity of a mouse, and ascites containing the antibody is produced. Purifying ascites by caprylic acid-ammonium sulfate precipitation method, and freeze drying to obtain monoclonal antibody with high purity and high specificity.
Application of pyrimethanil artificial antigen in ELISA and effect evaluation
The pyrimethanil artificial antigen in the example 1 is diluted to 0.05 mug/mL by using a carbonate buffer solution with pH of 9.6 as a coating diluent, added into a polystyrene micro-pore plate according to 100 mug/hole, coated overnight at 4 ℃, dried, added with 1% BSA according to 280 mug/hole, blocked for 1h at 37 ℃ in a phosphate buffer solution, dried, and vacuum packaged for storage.
Adding 100 mu L/hole of a standard solution of pyrimethanil (subjected to gradient dilution by phosphate buffer solution with pH 7.4) into a microporous ELISA plate coated with the artificial antigen of pyrimethanil, then correspondingly adding 20 mu L/hole of a monoclonal antibody solution of pyrimethanil (subjected to dilution to 0.08 mu g/mL by phosphate buffer solution with 0.05% sodium azide and pH 7.4) and reacting at 37 ℃ for 0.5h; after spin-drying, 250 mu L/hole of washing liquid is added, and the mixture is washed for 3 times and then is patted dry; then adding 100 mu L/hole of enzyme-labeled secondary antibody, and reacting for 0.5h at 37 ℃; washing for 3 times again, then beating to dry, adding 100 mu L/Kong Xianse liquid, and reacting for 15min at 37 ℃; add 0.5M sulfuric acid 50. Mu.L/Kong Zhongzhi, set the microplate reader at 450nm wavelength to determine the OD per well. The results are shown in table 1 below:
TABLE 1 ELISA testing of OD values of standard solutions of pyrimethanil at different concentrations
By the data in table 1, four-parameter Logistic curve fitting is performed by using ELISA Calc software to draw a standard curve, and the obtained standard curve is shown in fig. 4, and the linear equation of pyrimethanil is as follows: y= (A-D)/[ 1+ (x/C)/(B)]+d, r2=0.999, a=2.53610, b=0.84793, c=1.72817, d=0.12939, x represents the concentration of the analyte, y represents the OD value, and IC is calculated 50 The value is 1.97 mug/L, and the linear relation is between 0.5 and 40.5 mug/L.
Application of pyrimethanil artificial antigen in preparation of colloidal gold immunochromatography test strip for detection
The preparation method of the pyrimethanil colloidal gold qualitative immunochromatographic reagent strip comprises the following steps:
1. preparing a reaction membrane coated with artificial antigen and sheep anti-mouse IgG:
the nitrocellulose membrane (NC membrane) is used as a reaction membrane, the concentration of the artificial antigen with carrier protein of bovine serum albumin is regulated to be 0.2-0.5 mg/mL by using a coating buffer solution, and the concentration of the goat anti-mouse IgG is also regulated to be 0.1-0.4 mg/mL by using the coating buffer solution. Spraying antigen and goat anti-mouse IgG to a detection area (T line) and a control area (C line) corresponding to the reaction membrane according to the membrane liquid amount of 0.8-1.2 mu L/cm, wherein the interval between the detection area and the control area is 2.5mm, placing in a 37 ℃ oven for treatment for 16-24 h, and placing in a constant temperature and constant humidity preservation box for standby; the coating buffer was a 0.01M PBS buffer with pH7.4 containing 2% sucrose, 0.05% sodium azide.
2. Preparing gold pads of nano gold-labeled pyrimethanil monoclonal antibodies:
a) Preparation of nano gold: preparation of a nano gold solution: 1g of chloroauric acid is taken, dissolved by pure water and ultrasound, and then fixed to 100ml, and stored at 4 ℃ in a dark place for standby. Taking 1ml of the solution to 100ml of pure water, heating to boil, adding 0.5ml of 1% sodium citrate solution, continuously heating for 10 minutes, cooling to room temperature, recovering to original volume (100 ml) by using pure water, and standing at normal temperature in dark place for standby. All glassware is soaked in a mixed solution of potassium permanganate and sulfuric acid overnight, washed and dried.
b) Preparation of nano gold labeled pyrimethanil monoclonal antibody: 8. Mu.g of pyrimethanil monoclonal antibody was added to 1ml of 0.01% gold nanoparticle solution, reacted at room temperature for 10 minutes, blocked by adding 10. Mu.l of 10% bovine serum albumin, centrifuged at 12000rpm for 10 minutes, and the whole supernatant was discarded.
c) Preparation of gold pads: adding 1ml of gold seed diluent containing 2% of Tris, 5% of bovine serum albumin, 0.05% of merthiolate and 5% of sucrose, re-dissolving, uniformly coating on glass fiber with the area of 5cm by 5cm, drying at 37 ℃ for 16-24 hours, and preserving for later use.
3. Preparation of sample pad:
soaking a cut blank sample pad with the thickness of 30cm in sample pad treatment liquid for 5min, taking out, drying at 37 ℃ for 16-24 h, and placing in a constant temperature and humidity preservation box for standby; the sample pad treatment was a 0.1M PB buffer containing 0.3% Tween 20, 1% sucrose, 0.5% BSA, 0.05% sodium azide.
4. Assembling a colloidal gold qualitative immunochromatography reagent strip:
and (3) superposing the reaction film prepared in the step (1) in the middle of the back lining of the PVC plate, respectively superposing the gold pad and the water absorption pad in the step (2) at two ends, wherein the reaction film is connected with the gold pad and the water absorption pad, the gold pad is connected with the sample pad, the detection area is close to the sample pad, the control area is close to the water absorption pad, a test paper board is obtained, the test paper board is cut into a 3mm test paper strip, the test paper strip is loaded in the test paper card, and the colloidal gold qualitative immunochromatography test paper card is obtained, and then the pyrimethanil colloidal gold immunochromatography test paper card prepared by the method is further detected.
Test for testing detection limit of pyrimethanil colloidal gold immunochromatographic test paper card
A series of standard solutions of pyrimethanil with different concentrations are prepared by using a 0.01M PBS buffer solution, then 100 mu l of the standard solution is added into a sample adding hole of the pyrimethanil colloidal gold immunochromatography test paper card, timing is started after sample adding, the result can be observed after 5-8 min, and interpretation is invalid after 8 min.
The naked eye interpretation method comprises the following steps: the color development of the T line is stronger than that of the C line or has no obvious difference with that of the C line, and the negative (-) detection result is indicated. The color development of the T line is obviously weaker than that of the C line or the T line does not develop, which indicates that the detection result is positive (+). Invalidation: the absence of line C indicates incorrect operation or failure of the test strip. The test set up was repeated for 3 groups. The rapid qualitative detection of the colloidal gold immunochromatography test paper card can be realized.
The measurement results were as follows:
table 2 determination results of colloidal gold qualitative immunochromatography test paper card for detecting pyrimethanil standard solution with different concentrations
The test strip provided by the invention can detect the pyrimethanil with different concentrations (table 2) by detecting the concentration value of the pyrimethanil series standard solution, and the result is shown in table 2, so that the colloidal gold qualitative immunochromatography test strip prepared by the invention has high sensitivity to pyrimethanil detection.
Stability test of colloidal gold qualitative immunochromatography test paper card
The condition for preserving the colloidal gold qualitative immunochromatography test paper card is room temperature, and in order to ensure the stability of the test paper, an accelerated destructive experiment is carried out on the test paper. The test was continued for 60 days at 45℃and negative and sulfamethazine standard solutions were tested for color development on day 0, day 5, day 10, day 20, day 30, day 40, day 50 and day 60, respectively, and the test set was repeated for 3 groups, with the results shown in Table 3: ("+" represents positive and "-" represents negative)
Table 3 stability test
As shown in Table 3, after the colloidal gold qualitative immunochromatographic test strip is hermetically stored at 45 ℃ for 60 days, the T/C color depth interpretation results of the test strip are not obviously changed, which indicates that the colloidal gold qualitative immunochromatographic test strip can be stably stored for at least 60 days at 45 ℃ in an accelerated experiment. Therefore, the pyrimethanil colloidal gold qualitative immunochromatographic test strip prepared by the invention can be stably stored for more than one year at room temperature, and can completely meet the requirements of the market in the storage and transportation processes.
The above description of the present invention is further illustrated in detail and should not be taken as limiting the practice of the present invention. It is within the scope of the present invention for those skilled in the art to make simple deductions or substitutions without departing from the concept of the present invention.

Claims (10)

1. The pyrimethanil hapten is characterized in that the structure of the pyrimethanil hapten is shown as a formula I:
2. the method for preparing pyrimethanil hapten according to claim 1, which comprises the following steps:
1) The compound A and the compound B undergo substitution reaction under alkaline conditions to obtain a compound C;
2) Hydrolyzing the compound C to obtain pyrimethanil hapten;
wherein the structural formula of the compound A isThe structural formula of the compound B isThe structural formula of the compound C is +.>
3. An artificial pyrimethanil antigen, which is obtained by coupling the hapten of pyrimethanil and a protein carrier according to claim 1.
4. A pyrimethanil artificial antigen according to claim 3, wherein the pyrimethanil artificial antigen has the structural formula shown in formula II:
wherein protein is a protein carrier.
5. The pyrimethanil artificial antigen according to claim 3, wherein the protein carrier is at least one of bovine serum albumin, ovalbumin, human serum albumin or hemocyanin.
6. The method for preparing the pyrimethanil artificial antigen according to any one of claims 3 to 5, comprising the following steps:
1) Mixing hapten, carbodiimide and N-hydroxysuccinimide shown in formula I, and performing an activation reaction to obtain hapten activated ester;
2) Mixing carrier protein with hapten activated ester, and carrying out amidation reaction to obtain the artificial antigen.
7. The method according to claim 6, wherein the activation reaction time is 3 to 5 hours, and the amidation reaction time is 16 to 24 hours.
8. Use of an artificial antigen of pyrimethanil according to any one of claims 3 to 5 for the preparation of an ELISA assay of pyrimethanil.
9. A test strip or test reagent for the detection of pyrimethanil, characterized in that the test strip or test reagent comprises the artificial antigen of pyrimethanil according to any one of claims 3 to 5.
10. A pyrimethanil monoclonal antibody, which is prepared by immunizing an animal with the pyrimethanil artificial antigen as claimed in any one of claims 3 to 5 as an immunogen.
CN202311307816.4A 2023-10-10 2023-10-10 Pyrimethanil hapten and artificial antigen as well as preparation methods and application thereof Pending CN117362240A (en)

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