A pyrimethanil hapten, an artificial antigen, its preparation method and application

Abstract

This invention discloses a pyrimethanil hapten, an artificial antigen, and their preparation methods and applications. The structural formula of the pyrimethanil hapten is shown below. The arm introduced in this invention not only possesses an active group but also fully retains the benzene ring and dimethylpyrimidine ring structure of the target analyte, enhancing the recognition site. The pyrimethanil artificial antigen and monoclonal antibody of this invention exhibit high specificity for ELISA detection, with an IC50 rating of [missing information]. 50 The value is 1.97 μg / L, which is better than existing monoclonal antibodies. It can be used in colloidal gold immunochromatography to rapidly and conveniently detect pyrimethanil. The colloidal gold immunochromatographic test strip prepared in this invention has a detection sensitivity of 10 μg / L for pyrimethanil.

Description

Technical Field

[0001] This invention relates to the field of biochemical engineering, specifically to a pyrimethanil hapten, an artificial antigen, and their preparation methods and applications. Background Technology

[0002] Pyrimethanil is a highly effective, low-toxicity fungicide for controlling gray mold. It has systemic and fumigant action, allowing it to rapidly reach all parts of the plant after application. It effectively inhibits the production of pathogenic enzymes, thus preventing infection and completely killing the pathogen. Due to its unique action, it is widely used. While pyrimethanil has low toxicity in mammals, studies have shown it to have potential carcinogenic toxicity in mice, rats, dogs, and aquatic organisms.

[0003] Currently, conventional instrumental detection methods are not only expensive and time-consuming, but also require professional operation, making rapid on-site sampling impossible. Another detection method is immunological detection, which features high specificity and selectivity, making it highly suitable for detecting trace components in complex matrices. The performance of immunoassay products is determined by the performance of the antigen and antibody, and the key to both is the hapten. Therefore, the structural design of the hapten is crucial for obtaining high-performance antigens and antibodies. Thus, developing highly specific pyrimethanil haptens or artificial antigens is of paramount importance for rapid, highly sensitive, and low-cost detection methods for pyrimethanil. Summary of the Invention

[0004] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a pyrimethanil hapten, an artificial antigen, and its preparation method and application.

[0005] The technical solution adopted in this invention is:

[0006] The first aspect of the present invention provides:

[0007] A pyrimethanil hapten, the structure of which is shown in Formula I:

[0008]

[0009] A second aspect of the present invention provides:

[0010] The method for preparing the pyrimethanil hapten according to the first aspect of the present invention includes the following steps:

[0011] 1) Compound A and compound B undergo a substitution reaction under alkaline conditions to give compound C;

[0012] 2) Hydrolysis of compound C yields pyrimethanil hapten;

[0013] The structural formula of compound A is as follows: The structural formula of compound B is

[0014] The structural formula of compound C is

[0015] A third aspect of the present invention provides:

[0016] A pyrimethanil artificial antigen, wherein the pyrimethanil artificial antigen is obtained by conjugating the pyrimethanil hapten described in the first aspect of the present invention with a protein carrier.

[0017] In some examples of pyrimethanil artificial antigens, the structural formula of the pyrimethanil artificial antigen is shown in Formula II:

[0018]

[0019] Among them, protein is the protein carrier.

[0020] In some examples of pyrimethanil artificial antigens, the protein carrier is at least one of bovine serum albumin, ovalbumin, human serum albumin, or hemocyanin.

[0021] A fourth aspect of the present invention provides:

[0022] The method for preparing the pyrimethanil artificial antigen according to the third aspect of the present invention is characterized by comprising the following steps:

[0023] 1) Mix the hapten, carbodiimide and N-hydroxysuccinimide shown in Formula I, and activate them to obtain the activated hapten ester;

[0024] 2) The carrier protein is mixed with the hapten activated ester, and the artificial antigen is obtained by amidation reaction.

[0025] In some examples of preparation methods, the activation reaction time is 3-5 h, and the amidation reaction time is 16-24 h.

[0026] A fifth aspect of the present invention provides:

[0027] The application of the pyrimethanil artificial antigen described in the third aspect of this invention in the preparation of pyrimethanil ELISA detection.

[0028] A sixth aspect of the present invention provides:

[0029] A test strip or reagent for detecting pyrimethanil, said test strip or reagent containing the pyrimethanil artificial antigen as described in the third aspect of the present invention.

[0030] The seventh aspect of the present invention provides:

[0031] A pyrimethanil monoclonal antibody, wherein the pyrimethanil monoclonal antibody is prepared by immunizing animals with the pyrimethanil artificial antigen described in the third aspect of the present invention as an immunogen.

[0032] The beneficial effects of this invention are:

[0033] 1. The arm introduced by the pyrimethanil hapten of the present invention not only has an active group, but also completely retains the benzene ring and dimethylpyrimidine ring structure of the target analyte, thereby enhancing the recognition site.

[0034] 2. The pyrimethanil artificial antigen and monoclonal antibody of the present invention have high specificity for ELISA detection, IC50... 50 The value was 1.97 μg / L, which is better than existing monoclonal antibodies.

[0035] 3. The artificial antigen and monoclonal antibody of pyrimethanil of the present invention are used in colloidal gold immunochromatography, which can quickly and conveniently detect pyrimethanil. The colloidal gold immunochromatographic test strip prepared in the present invention has a detection sensitivity of 10 μg / L for pyrimethanil. Attached Figure Description

[0036] Figure 1 This is a synthetic route diagram for the pyrimethanil hapten.

[0037] Figure 2 This is the mass spectrum of the pyrimethanil hapten.

[0038] Figure 3 This is a synthetic route diagram for pyrimethanil artificial antigen.

[0039] Figure 4 This is the standard curve of pyrimethanil. Detailed Implementation

[0040] The following disclosure provides many different implementations or examples for different ways of implementing the present invention.

[0041] Preparation of pyrimethanil hapten

[0042] like Figure 1 As shown, the pyrimethanil hapten of the present invention is prepared by the following method, with the following steps:

[0043] 1) 3.00 g (15.06 mmol) of compound A was placed in a 100 mL round-bottom flask, followed by the addition of 20 mL of DMF, 3.12 g (22.59 mmol) of K₂CO₃, 4.41 g (22.59 mmol) of compound B, and a catalytic amount of tetrabutylammonium bromide (200 mg). The reaction was carried out at room temperature for at least 48 h. After the reaction was completed, 90 mL of purified water was added, and the mixture was extracted twice with ethyl acetate. The solvent was then removed under reduced pressure, and the mixture was purified by column chromatography to obtain 1.75 g of compound C.

[0044] 2) Dissolve 1.75 g (5.58 mmol) of compound 3 in 10 ml of methanol, then add 10 ml of 4 mol / L lithium hydroxide aqueous solution. React the reaction mixture at RT for at least 8 h. Add 80 ml of sodium chloride aqueous solution and extract twice with 30 ml of dichloromethane. Adjust the pH of the aqueous phase to 4–5 with 4 M dilute hydrochloric acid. Filter and dry to obtain 1.07 g of hapten. The mass spectrum of this hapten is shown below. Figure 2 As shown.

[0045] Example 1

[0046] As shown in the figure, the method for synthesizing the pyrimethanil artificial antigen with bovine serum albumin as the carrier protein is as follows:

[0047] 1) Take 10 mg of the prepared pyrimethanil hapten, dissolve it in 0.2 mL of dimethylformamide (DMF), stir thoroughly, add 10 mg of carbodiimide (EDC) and 10 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain the hapten activated ester;

[0048] 2) Weigh 30 mg of bovine serum albumin (BSA) and dissolve it completely in 3 mL of 0.1 mol / L CB buffer solution to form a carrier protein solution. Add the hapten activated ester dropwise slowly to the carrier protein solution while stirring, and stir at room temperature for 16–24 h.

[0049] 3) The solution obtained in step 2) is dialyzed with 0.01 mol / L PBS at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules.

[0050] 4) Dispense into smaller portions and store at 4°C for later use.

[0051] Example 2

[0052] like Figure 3 As shown, the method for synthesizing the pyrimethanil artificial antigen with hemocyanin as the carrier protein is as follows:

[0053] 1) Take 5 mg of the prepared pyrimethanil hapten, dissolve it in 0.2 mL of dimethylformamide (DMF), stir thoroughly, add 10 mg of EDC and 10 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h to obtain the hapten activated ester;

[0054] 2) Weigh 15 mg of hemocyanin (KLH) and dissolve it completely in 3 mL of 0.1 mol / L CB buffer solution to form a carrier protein solution. Add the hapten activated ester dropwise slowly to the carrier protein solution while stirring, and stir at room temperature for 16-24 h.

[0055] 3) The solution obtained in step 2) is dialyzed with 0.01 mol / L PBS at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules.

[0056] 4) Dispense into smaller portions and store at 4°C for later use.

[0057] Application of pyrimethanil artificial antigen in the preparation of anti-pyrimethanil monoclonal antibodies

[0058] The preparation method of anti-pyrimethanil monoclonal antibody is as follows:

[0059] Using the pyrimethanil artificial antigen from Example 2 as the immunogen, BALB / c mice were immunized with an equal volume of Freund's adjuvant. Each mouse received 100 μg of the immunogen, with immunizations spaced 2 weeks apart. After three immunizations, serum titers were measured using tail vein blood. If the antibody titer was insufficient, booster immunizations were administered. Once the antibody titer ceased to rise, a subcutaneous booster immunization with 100 μg of the complete antigen was performed. Five days later, mouse spleen cells were fused with SP20 cells (mouse myeloma cells). The fused cells were screened in HAT medium, and after 5 days, the medium was replaced with complete medium for further culturing. The cell supernatant was analyzed using ELISA. Cells showing strong positive results were subjected to limiting dilution clonal culture. Cells showing positive results after three clonal culture tests were identified as hybridoma cells secreting monoclonal antibodies. After amplification and culture of the hybridoma cells, they were inoculated into the peritoneal cavity of mice, producing antibody-containing ascites. Ascites fluid can be purified by octanoic acid-ammonium sulfate precipitation and then freeze-dried to obtain high-purity, high-specificity monoclonal antibodies.

[0060] Application and efficacy evaluation of pyrimethanil artificial antigen in ELISA

[0061] Using carbonate buffer at pH 9.6 as the coating diluent, the pyrimethanil artificial antigen in Example 1 was diluted to 0.05 μg / mL and added to polystyrene microplates at 100 μL / well. The plates were coated overnight at 4°C, dried, and then 1% BSA was added at 280 μL / well. The plates were blocked in phosphate buffer at 37°C for 1 h, dried, and then vacuum-packed for storage.

[0062] Add 100 μL of pyrimethanil standard solution per well (serially diluted with phosphate buffer at pH 7.4) to the microplate coated with pyrimethanil artificial antigen, followed by 20 μL of pyrimethanil monoclonal antibody solution per well (diluted to 0.08 μg / mL with phosphate buffer containing 0.05% sodium azide at pH 7.4, freshly prepared and used immediately). Incubate at 37°C for 0.5 h. After drying, add 250 μL of washing buffer per well, wash three times, and blot dry. Add 100 μL of enzyme-labeled secondary antibody per well, and incubate at 37°C for 0.5 h. Wash three times again, blot dry, add 100 μL of chromogenic solution per well, and incubate at 37°C for 15 min. Stop incubation with 50 μL of 0.5 M sulfuric acid per well. Set the microplate reader to 450 nm and measure the OD value of each well. The results are shown in Table 1 below.

[0063] Table 1. OD values ​​of pyrimethanil standard solutions at different concentrations as determined by ELISA

[0064]

[0065] Using the data in Table 1, a standard curve was plotted using ELISA Calc software to fit a four-parameter logistic curve. The resulting standard curve is shown below. Figure 4 As shown, the linear equation for pyrimethanil is: y=(AD) / [1+(x / C)^B]+D, r2=0.999, A=2.53610, B=0.84793, C=1.72817, D=0.12939, where x represents the concentration of the analyte and y represents the OD value. The IC50 value was calculated. 50 The value was 1.97 μg / L, and it showed a linear relationship in the range of 0.5–40.5 μg / L.

[0066] Application of pyrimethanil artificial antigen in the preparation of colloidal gold immunochromatographic test strips for detection

[0067] The preparation method of pyrimethanil colloidal gold qualitative immunochromatographic reagent strips is as follows:

[0068] 1. Preparation of reaction membranes coated with artificial antigens and goat anti-mouse IgG:

[0069] Using nitrocellulose membranes (NC membranes) as the reaction membrane, the concentration of the artificial antigen, with bovine serum albumin as the carrier protein, was adjusted to 0.2–0.5 mg / mL with coating buffer, and the concentration of goat anti-mouse IgG was also adjusted to 0.1–0.4 mg / mL with coating buffer. The antigen and goat anti-mouse IgG were sprayed onto the corresponding detection area (T line) and control area (C line) of the reaction membrane at a membrane volume of 0.8–1.2 μL / cm, with a 2.5 mm gap between the detection and control areas. The membranes were then placed in a 37°C oven for 16–24 hours and stored in a constant temperature and humidity incubator for later use. The coating buffer was 0.01 M PBS buffer with pH 7.4 containing 2% sucrose and 0.05% sodium azide.

[0070] 2. Preparation of a gold pad for gold nanoparticle-labeled pyrimethanil monoclonal antibody:

[0071] a) Preparation of gold nanoparticles: Preparation of gold nanoparticle solution: Take 1g of chloroauric acid, dissolve it in pure water and sonicate, then bring the volume to 100ml and store at 4℃ protected from light. Take 1ml of the above solution into 100ml of pure water, heat to boiling, then add 0.5ml of 1% sodium citrate solution, continue heating for 10 minutes, cool to room temperature, and restore the volume to the original volume (100ml) with pure water. Store at room temperature protected from light. All glassware used must be soaked overnight in a mixture of potassium permanganate and sulfuric acid, washed and dried before use.

[0072] b) Preparation of pyrimethanil monoclonal antibody labeled with gold nanoparticles: 8 μg of pyrimethanil monoclonal antibody was added to 1 ml of 0.01% gold nanoparticle solution and reacted at room temperature for 10 minutes. 10 μl of 10% bovine serum albumin was added for blocking. The mixture was centrifuged at 12000 rpm for 10 minutes and all supernatant was discarded.

[0073] c) Preparation of gold pad: Add 1 ml of gold diluent containing 2% Tris, 5% bovine serum albumin, 0.05% thimerosal and 5% sucrose to redissolve the gold, spread it evenly on a glass fiber with an area of ​​5cm*5cm, dry it at 37℃ for 16-24h and store it for later use.

[0074] 3. Preparation of the sample pad:

[0075] Immerse the cut 30*30cm blank sample pads in the sample pad treatment solution for 5 minutes, then remove and dry at 37℃ for 16-24 hours. Store in a constant temperature and humidity incubator for later use. The sample pad treatment solution is a 0.1M PB buffer solution containing 0.3% Tween 20, 1% sucrose, 0.5% BSA, and 0.05% sodium azide.

[0076] 4. Assembly of colloidal gold qualitative immunochromatographic reagent strips:

[0077] The reaction membrane prepared in step (1) is stacked in the middle of the PVC board backing, and the gold pad and absorbent pad from step (2) are stacked at both ends respectively. The reaction membrane, the gold pad, and the absorbent pad are connected together, and the gold pad is connected to the sample pad. The detection area is close to the sample pad, and the control area is close to the absorbent pad to obtain the test paper. The test paper is cut into 3mm test strips, and the test strips are loaded into the test paper card to obtain the colloidal gold qualitative immunochromatographic test paper card. Then, the pyrimethanil colloidal gold immunochromatographic test paper card prepared in this invention is further tested.

[0078] Detection limit test of pyrimethanil colloidal gold immunochromatographic test strip

[0079] Prepare a series of standard solutions of pyrimethanil with different concentrations using 0.01M PBS buffer. Then, add 100 μl of the standard solution to the sample well of the pyrimethanil colloidal gold immunochromatographic test strip card of this invention. Start timing after adding the sample, and observe the results in 5-8 minutes. Readings after 8 minutes are invalid.

[0080] Visual interpretation method: A stronger T-line than the C-line, or no significant difference in color between the two, indicates a negative result (-). A significantly weaker T-line than the C-line, or no T-line, indicates a positive result (+). Invalid: No C-line appears, indicating incorrect operation or an expired test strip. The test should be performed in triplicate. This allows for rapid qualitative detection using colloidal gold immunochromatographic test strips.

[0081] The measurement results are as follows:

[0082] Table 2. Results of the determination of different concentrations of pyrimethanil standard solution using colloidal gold qualitative immunochromatographic test strips.

[0083]

[0084] The concentration values ​​of the pyrimethanil series standard solutions were tested, and the results are shown in Table 2. The test strip of the present invention can detect different concentrations of pyrimethanil (Table 2). Therefore, the colloidal gold qualitative immunochromatographic test strip prepared in the present invention has high sensitivity for the detection of pyrimethanil.

[0085] Stability test of colloidal gold qualitative immunochromatographic test strips

[0086] The colloidal gold qualitative immunochromatographic test strips were stored at room temperature. To ensure the stability of the test strips, an accelerated destructive experiment was performed. The strips were continuously placed at 45℃ for 60 days, and negative results and color changes of sulfadiazine standard solution were detected on days 0, 5, 10, 20, 30, 40, 50, and 60, respectively. The experiment was conducted in triplicate, and the results are shown in Table 3: ("+" represents positive, "-" represents negative).

[0087] Table 3 Stability Test

[0088]

[0089] As shown in Table 3, after 60 days of sealed storage at 45℃, the T / C colorimetric depth of the colloidal gold qualitative immunochromatographic test strips showed no significant change, indicating that the colloidal gold qualitative immunochromatographic test strips can be stably stored for at least 60 days at 45℃ in accelerated experiments. Therefore, the pyrimethanil colloidal gold qualitative immunochromatographic test strips prepared in this invention can be stably stored at room temperature for more than one year, fully meeting the requirements of the market in terms of storage and transportation.

[0090] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. The application of the compound in the preparation of pyrimethanil hapten, characterized in that, The structure of the compound is shown in Formula I: Formula I.

2. The application according to claim 1, characterized in that, The structural formula of the pyrimethanil artificial antigen prepared from the pyrimethanil hapten is shown in Formula II: Formula II Among them, protein is the protein carrier.

3. The application according to claim 2, characterized in that, The protein carrier is at least one of bovine serum albumin, ovalbumin, human serum albumin, or hemocyanin.

4. The application according to claim 2, characterized in that, The preparation method of the pyrimethanil artificial antigen includes the following steps: 1) Mix the pyrimethanil hapten, carbodiimide and N-hydroxysuccinimide shown in Formula I, and activate them to obtain the hapten activated ester; 2) The protein carrier is mixed with the activated ester of pyrimethanil hapten and amidation reaction is carried out to obtain the artificial antigen.

5. The application according to claim 4, characterized in that, The activation reaction time is 3-5 h, and the amidation reaction time is 16-24 h.

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