Diflubenzuron hapten, complete antigen, antibody as well as synthesis and application of diflubenzuron hapten, complete antigen and antibody

Abstract

The invention discloses a diflubenzuron hapten, a complete antigen, an antibody and synthesis and application of the diflubenzuron hapten, the complete antigen and the antibody. The diflubenzuron hapten, the complete antigen and the antibody have good specificity and sensitivity when applied to diflubenzuron detection, the IC50 value is as low as 0.2 mu g / L, the detection limit of diflubenzuron in a standard solution reaches 2 mu g / L, and the detection limit of a to-be-detected sample reaches 10 mu g / kg. By utilizing the diflubenzuron hapten, the complete antigen and the antibody, the purpose of rapidly and accurately detecting the diflubenzuron in a sample can be achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and more specifically to a diflubenzuron hapten, a complete antigen, an antibody, and the synthesis and application thereof. Background Art

[0002] Diflubenzuron is a benzoyl-specific, low-toxic insecticide and chitin synthase inhibitor. It is a third-generation pesticide developed after second-generation pesticides such as organophosphates and carbamates. It primarily acts as a stomach toxicant, with less contact activity and lacking systemic properties. It kills insects by inhibiting chitin biosynthesis. Its mechanism of action differs from other insecticides, resulting in high insecticidal activity, a broad spectrum of activity, low residual activity, and strong selectivity.

[0003] Because diflubenzuron is a highly effective, low-toxic, low-residue pesticide that is environmentally friendly to non-target organisms, its relatively low residue levels have led to insufficient attention regarding its pesticide residues. However, a lack of scientific knowledge and an excessive pursuit of control efficacy have led to excessive pesticide use, resulting in excessive residues at harvest time. Spraying also pollutes water, soil, and the atmosphere. Continued exposure can interfere with metabolism, damage the thyroid gland, and cause hepatotoxicity. Diflubenzuron hydrolyzes in the body to produce chloroaniline, which can cause methemoglobinemia. Long-term consumption of foods containing excessive levels of diflubenzuron may adversely affect human health.

[0004] Immunoassays have become an important technology for pesticide residue detection due to their simplicity, low cost, and high specificity. Their core approach is the preparation of specific antibodies, which in turn relies on the design of highly effective haptens. Currently, there are few reports on immunoassays for diflubenzuron, and their sensitivity is relatively poor.

[0005] Therefore, there is a current need to develop a new diflubenzuron hapten and, through optimizing the hapten design to prepare complete antigens and specific antibodies, improve the specificity and sensitivity of detection. Furthermore, the development of high-performance immunoassay devices based on this approach can enhance practicality, detection efficiency, and accuracy. Summary of the Invention

[0006] The purpose of the present invention is to overcome the defects and shortcomings of the prior art diflubenzuron hapten, complete antigen, antibody, and provide a diflubenzuron hapten, complete antigen, antibody and their synthesis and application.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention improves the immunogenicity of diflubenzuron hapten by optimizing the spatial configuration and chemical properties, and provides a diflubenzuron hapten, the structure of which is shown in formula (I):

[0009]

[0010] Specifically, the present invention connects one end of the urea group to a carbon atom of the 4-chlorophenyl group via a nitrogen atom, and the other end of the urea group is connected to the carbonyl carbon atom of the 2-fluorobenzoyl group via a nitrogen atom. -O(CH2)3COOH is introduced at the ortho position of the benzene ring as a coupling site. Its function is achieved through the following features:

[0011] (1) Ortho-position configuration optimization: The present invention provides a longer spacer arm through -O(CH2)3COOH to prevent the core structure of diflubenzuron from being shielded. The urea group and -O(CH2)3COOH of the present invention are in the ortho position on the benzene ring, which significantly exposes the core structure of diflubenzuron (urea group and its aromatic ring) while ensuring the stability of the diflubenzuron hapten structure. This design avoids the folding of the hapten molecule caused by the long spacer arm, causing the hapten molecule to be shielded close to the carrier protein surface, thereby improving the immune recognition efficiency of the antigen epitope.

[0012] (2) Spacer arm length and carboxyl modification: The present invention provides higher reactivity through -O(CH2)3COOH, more efficiently couples with carrier proteins, does not require an additional amidation step, and simplifies the synthesis route.

[0013] By optimizing the spatial configuration and chemical properties, the present invention exposes the core structure of diflubenzuron and enhances the efficiency of the immune response. This design not only simplifies the synthesis pathway for the hapten but also significantly improves the specificity and sensitivity of the antibody while maintaining chemical stability, laying the foundation for improved immunoassay performance.

[0014] Furthermore, the present invention provides a method for preparing the above-mentioned diflubenzuron hapten. Based on the structural design of diflubenzuron, the present invention sequentially performs substitution reaction, hydrolysis and amide reaction to prepare the diflubenzuron hapten having the structure shown in formula (I).

[0015] The above-mentioned diflubenzuron hapten can be prepared by the following steps:

[0016] S1. Aromatic amide compound a (5-fluoro-2-hydroxybenzamide, CAS: 56874-97-6) and halogenated ester compound b (ethyl 4-bromobutyrate, CAS: 2969-81-5) are subjected to a substitution reaction under alkaline conditions to obtain a first intermediate compound c;

[0017] S2. The first intermediate product compound c is hydrolyzed in an alkaline solution, and the pH is adjusted to precipitate a solid to obtain a second intermediate product compound d;

[0018] S3. The second intermediate compound d is subjected to an amide reaction with an aromatic isocyanate compound e (p-chlorophenyl isocyanate, CAS: 104-12-1) in an organic solvent to obtain a diflubenzuron hapten having a structure as shown in formula (I).

[0019] Preferably, the substitution reaction in step S1 is carried out in a polar aprotic solvent.

[0020] More preferably, the polar aprotic solvent is N,N-dimethylformamide (DMF).

[0021] Preferably, in step S1, the ratio of compound a to compound b is (650-870) mg: (1.2-1.6) mL.

[0022] More preferably, in step S1, the ratio of compound a to compound b is 790 mg:1.46 mL.

[0023] Preferably, the alkaline condition in step S1 is pH=8.5-10.5.

[0024] More preferably, the alkaline condition in step S1 is pH=9.6.

[0025] Optionally, potassium carbonate may be used to regulate the alkaline conditions in step S1.

[0026] Preferably, the alkaline solution in step S2 is a lithium hydroxide aqueous solution.

[0027] More preferably, the alkaline solution in step S2 is a 4-8 mol / L lithium hydroxide aqueous solution (most preferably 6 mol / L).

[0028] Preferably, in step S2, adjusting the pH to precipitate solids is to adjust the pH to 4-5 to precipitate solids.

[0029] Preferably, in step S3, the ratio of compound d to compound e is (120-200) mg:(220-360) mg.

[0030] More preferably, in step S3, the ratio of compound d to compound e is 160 mg:290 mg.

[0031] Preferably, the amide reaction in step S3 is carried out in a non-polar to medium-polar solvent environment.

[0032] More preferably, the non-polar to medium-polar solvent environment is provided by a mixed solvent of toluene and ethyl acetate.

[0033] As an optional embodiment, the method for preparing the above-mentioned diflubenzuron hapten comprises the following steps:

[0034] S1. Compound a was thoroughly stirred and mixed with N,N-dimethylformamide, potassium carbonate, and compound b, and the mixture was allowed to react at room temperature for 2 to 8 hours. After the reaction was complete, purified water was added, the aqueous phase was extracted twice with ethyl acetate, and the organic phase was decompressed to remove the solvent to obtain compound c.

[0035] S2. Dissolve compound c in methanol, add aqueous lithium hydroxide solution, react at room temperature for 10-12 hours, add purified water, and extract twice with ethyl acetate. Adjust the pH value of the aqueous phase to 4-5 with 4M hydrochloric acid. The solid precipitates, is filtered, and dried to obtain compound d.

[0036] S3. Dissolve compound d in toluene and ethyl acetate, add compound e, stir at 115°C, react for 4-8 hours, cool to room temperature, and filter to obtain diflubenzuron hapten.

[0037] Further preferably, in step S1, the ratio of compound a, N,N-dimethylformamide, potassium carbonate, and compound b is (650-870) mg:(4.1-5.5) mL:(1100-1550) mg:(1.2-1.6) mL.

[0038] Most preferably, in step S1, the ratio of compound a, N,N-dimethylformamide, potassium carbonate, and compound b is 790 mg:5 mL:1400 mg:1.46 mL.

[0039] Further preferably, in step S3, the ratio of toluene to ethyl acetate is (1-1.2):(1-1.5).

[0040] Most preferably, the ratio of toluene to ethyl acetate in step S3 is 1:1.

[0041] Furthermore, the present invention provides a complete diflubenzuron antigen, which is obtained by coupling the above-mentioned diflubenzuron hapten with a carrier protein.

[0042] Wherein, the carrier protein is selected from one of bovine serum albumin (BSA), lactoferrin (LF), ovalbumin (OVA), human serum albumin (HSA), and hemocyanin (KLH).

[0043] As an optional embodiment, the method for preparing the complete diflubenzuron antigen comprises the following steps:

[0044] S1. The above-mentioned diflubenzuron hapten was dissolved in dimethylformamide (DMF), and EDC and N-hydroxysuccinimide (NHS) were added to react to obtain a hapten-activated ester;

[0045] S2. adding the hapten activated ester to the carrier protein solution;

[0046] S3. The solution obtained in step S2 is dialyzed to obtain the complete diflubenzuron antigen.

[0047] Furthermore, the present invention provides a diflubenzuron antibody obtained by immunizing an animal with the above-mentioned diflubenzuron complete antigen.

[0048] Preferably, the diflubenzuron antibody is a diflubenzuron monoclonal antibody.

[0049] As an optional embodiment, the method for preparing the diflubenzuron monoclonal antibody comprises the following steps:

[0050] S1. Immunize mice with complete diflubenzuron antigen;

[0051] S2. Detect the antibody titer of mouse serum. If the titer is insufficient, perform a booster immunization.

[0052] S3. Mice were boosted with complete diflubenzuron antigen, and spleen cells were fused with mouse myeloma cells;

[0053] S4. Screen the fused cells using HAT medium;

[0054] S5. Hybridoma cells secreting diflubenzuron monoclonal antibodies were screened by ELISA and cloned by limiting dilution method;

[0055] S6. After the hybridoma cells are expanded and cultured, they are injected into the peritoneal cavity of mice to induce ascites, and the ascites is purified to obtain the above-mentioned diflubenzuron monoclonal antibody.

[0056] The performance of the diflubenzuron monoclonal antibody of the above alternative embodiment was evaluated by enzyme-linked immunosorbent assay (ELISA). 50 The value is 0.2μg / L, and it is linear in the range of 0.06 to 1.57μg / L, indicating that the antibody has strong specificity.

[0057] Furthermore, the present invention discloses the use of the above-mentioned diflubenzuron hapten, diflubenzuron complete antigen and diflubenzuron antibody in the detection of diflubenzuron.

[0058] Furthermore, the present invention discloses the use of the above-mentioned diflubenzuron hapten, diflubenzuron complete antigen and diflubenzuron antibody in the preparation of a diflubenzuron immunoassay device.

[0059] Furthermore, the present invention provides a diflubenzuron immunoassay device containing the above-mentioned diflubenzuron antibody.

[0060] The diflubenzuron immunoassay device includes but is not limited to one or more of enzyme immunoassay, immunochromatography, immunosensor, immunocolloidal gold, etc.

[0061] Preferably, the diflubenzuron immunoassay device is a diflubenzuron colloidal gold immunochromatographic assay device.

[0062] As an optional embodiment, the preparation method of the diflubenzuron colloidal gold immunochromatographic detection device comprises the following steps:

[0063] S1. Preparation of reaction membrane: spraying a solution containing the above-mentioned complete diflubenzuron antigen in the detection area (T line) of a nitrocellulose membrane (NC membrane), and spraying mouse immunoglobulin (mouse IgG) in the control area (C line);

[0064] S2. Preparation of colloidal gold labeled micropores: colloidal gold was prepared by sodium citrate reduction method, the above-mentioned diflubenzuron monoclonal antibody was combined with colloidal gold, bovine serum albumin was added for blocking, the precipitate was re-dissolved after centrifugation, and aliquoted into micropores;

[0065] S3. Prepare the sample pad: soak the blank sample pad in buffer and dry it for later use;

[0066] S4. Assemble the test card: stack the sample pad obtained in step S3, the reaction membrane obtained in step S1, and the absorbent pad on a PVC board, cut into test strips, and load them into the test card housing.

[0067] Preferably, a 0.2-0.6 mg / mL diflubenzuron complete antigen solution is sprayed in the detection area (T line), and a 0.05-0.1 mg / mL mouse immunoglobulin (mouse IgG) solution is sprayed in the control area (C line).

[0068] More preferably, a 0.3 mg / mL diflubenzuron complete antigen solution is sprayed on the detection area (T line), and a 0.08 mg / mL mouse immunoglobulin (mouse IgG) solution is sprayed on the control area (C line).

[0069] The method for using the diflubenzuron colloidal gold immunochromatographic detection device of the above-mentioned optional embodiment comprises the following steps:

[0070] S1. Preparation of the test solution: The test sample was added to a phosphate buffer solution and mixed, and the mixture was allowed to stand. The supernatant was mixed with a phosphate buffer solution to prepare the test solution.

[0071] S2. Detection: Add the test solution to the colloidal gold-labeled microwells of the diflubenzuron colloidal gold immunochromatographic detection device, mix to re-dissolve the colloidal gold-labeled antibody, let it stand for 3-7 minutes, and then transfer the solution in the colloidal gold-labeled microwells to the sample wells of the test card of the diflubenzuron colloidal gold immunochromatographic detection device. Start the timer after adding the sample, observe the results within 5-8 minutes, and determine the presence of diflubenzuron residue based on the color development results. After 8 minutes, the test is deemed invalid. The results are determined as follows:

[0072] (1) Negative (-): The color of the T line is stronger than that of the C line or there is no significant difference in color with the C line;

[0073] (2) Positive (+): The color of the T line is significantly weaker than that of the C line or the T line does not show any color;

[0074] (3) Invalid: Line C does not appear, the operation process is incorrect or the test strip has expired.

[0075] Sensitivity tests using a standard solution of diflubenzuron and a sample showed that the diflubenzuron colloidal gold immunochromatographic detection device of the aforementioned alternative embodiment had a detection limit of 2 μg / L for diflubenzuron in the standard solution and a detection limit of 10 μg / kg for diflubenzuron in the sample. The diflubenzuron colloidal gold immunochromatographic detection device of the present invention has a low detection limit for diflubenzuron in samples, meeting the requirements for trace detection and providing excellent detection results.

[0076] Through stability testing, the above-mentioned optional implementation scheme of the diflubenzuron colloidal gold immunochromatographic detection device has good stability under storage conditions, can still accurately detect diflubenzuron residues after storage, can be stably stored at room temperature for more than one year, and can meet the market requirements during storage and transportation.

[0077] Compared with the prior art, the present invention has the following significant beneficial effects:

[0078] (1) Strong specificity. The present invention prepares complete antigens and antibodies based on the novel diflubenzuron hapten, and the antibody has high affinity and its IC 50 The value is as low as 0.2μg / L, which is better than the existing technology. The constructed diflubenzuron immunoassay device can reduce non-specific reactions and improve detection accuracy.

[0079] (2) High sensitivity. The diflubenzuron immunoassay device prepared based on the novel diflubenzuron hapten of the present invention has a detection limit of 2 μg / L for diflubenzuron in standard solution and a detection limit of 10 μg / kg for the sample to be tested. The sensitivity is greatly improved, and the detection capability of low-concentration diflubenzuron residues is significantly enhanced.

[0080] (3) Strong anti-interference ability. The present invention optimizes the design of the diflubenzuron hapten structure and enhances the specificity of the antibody, so that the diflubenzuron immunoassay device can maintain stable performance in the environment of the sample to be tested. The present invention significantly reduces the influence of the matrix effect, and the detection limit of the sample to be tested reaches 10μg / kg, ensuring the reliability of practical application.

[0081] (4) Good stability. The diflubenzuron immunoassay device prepared based on the novel diflubenzuron hapten of the present invention can be stably stored at room temperature for more than one year and can still accurately detect diflubenzuron residues after storage.

[0082] (5) Rapid and portable. The diflubenzuron immunoassay device prepared based on the novel diflubenzuron hapten of the present invention takes only 5 to 8 minutes to detect, does not require laboratory equipment, and is easy to operate.

[0083] In summary, the present invention significantly improves the immune efficiency of antibodies and the performance of immunoassay devices by designing a novel diflubenzuron hapten structure with optimized spatial configuration and chemical properties. The resulting diflubenzuron immunoassay device combines high sensitivity, strong specificity, anti-interference capabilities, and rapid portability, overcoming the shortcomings of existing technologies and providing an efficient and reliable solution for the rapid screening of diflubenzuron in samples. This represents a significant technological advancement and holds broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 Mass spectrum of the diflubenzuron hapten of the present invention.

[0085] Figure 2 The synthesis route of the complete diflubenzuron antigen of the present invention.

[0086] Figure 3 A standard curve of the diflubenzuron monoclonal antibody ELISA according to one embodiment of the present invention.

[0087] Figure 4 Schematic diagram of the result judgment criteria of the diflubenzuron colloidal gold immunochromatographic detection device of the present invention. DETAILED DESCRIPTION

[0088] The present invention will be further described below with reference to the accompanying drawings and specific examples. The examples are only used to explain the present invention, but the examples do not limit the present invention in any form.

[0089] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0090] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0091] Example 1 Preparation and Identification of Diflubenzuron Hapten

[0092] 1. Preparation of Diflubenzuron Hapten

[0093] S1. Take 790 mg of compound a (5.09 mmol, 5-fluoro-2-hydroxybenzamide, CAS: 56874-97-6) and place it in a 50 mL round-bottom flask. Then, add 5 mL of N,N-dimethylformamide, 1.4 g of potassium carbonate (10.13 mmol), and 1.46 mL of compound b (10.19 mmol, ethyl 4-bromobutyrate, CAS: 2969-81-5) in this order. After thorough stirring and mixing, react at room temperature for 2 h to 8 h. After the reaction is complete, add 50 mL of purified water, extract the aqueous phase twice with ethyl acetate, and remove the solvent from the organic phase under reduced pressure to obtain compound c.

[0094] S2. Dissolve 1.63 g of compound c in 20 mL of methanol, add 20 mL of 6 mol / L aqueous lithium hydroxide solution, react at room temperature for 10-12 h, add 70 mL of purified water, extract twice with 30 mL of ethyl acetate, adjust the pH of the aqueous phase to 4-5 with 4 M hydrochloric acid, precipitate a solid, filter, and dry to obtain compound d.

[0095] S3.160 mg of compound d was dissolved in 15 mL of a mixed solution of toluene and ethyl acetate (1:1), and 290 mg of compound e (1.93 mmol, p-chlorophenyl isocyanate, CAS: 104-12-1) was added and stirred at 115°C for 4-8 h. The mixture was cooled to room temperature and filtered to obtain the diflubenzuron hapten.

[0096] 2. Identification of Diflubenzuron Hapten

[0097] The prepared diflubenzuron hapten was identified by mass spectrometry, and the mass spectrum detection results were as follows: Figure 1 As shown. Figure 1 The molecular ion peak of diflubenzuron hapten in ESI negative ion mode was [MH]-, m / z 392.9 (the theoretical molecular weight of diflubenzuron hapten was 394.07 Da), proving that the diflubenzuron hapten represented by formula (I) was synthesized.

[0098] Example 2 Preparation of complete diflubenzuron antigen

[0099] The synthesis process of the complete diflubenzuron antigen of the present invention is as follows ( Figure 2 Synthetic route shown):

[0100] S1. Take 5 mg of the diflubenzuron hapten prepared in Example 1 and dissolve it in 0.1 mL of dimethylformamide (DMF). After stirring thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS) and stir at room temperature for 4 h to obtain an activated hapten ester.

[0101] S2. Weigh 30 mg of lactoferrin (LF) and dissolve it in 5 mL of PBS solution with a concentration of 0.01 mol / L to form a lactoferrin carrier solution. The hapten-activated ester obtained in step S1 was slowly added dropwise to the lactoferrin carrier solution under stirring and stirred at room temperature for 16 to 24 h.

[0102] S3. The solution obtained in step S2 was dialyzed against 0.01 mol / L PBS solution at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules to obtain the complete diflubenzuron antigen (i.e., diflubenzuron hapten-LF conjugate), which was then aliquoted and stored at 4°C for later use.

[0103] Example 3 Preparation of complete diflubenzuron antigen

[0104] The synthesis process of the complete diflubenzuron antigen of the present invention is as follows ( Figure 2 Synthetic route shown):

[0105] S1. Take 10 mg of the diflubenzuron hapten prepared in Example 1 and dissolve it in 0.2 mL of dimethylformamide (DMF). After stirring thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS) and stir at room temperature for 4 h to obtain an activated hapten ester.

[0106] S2. Weigh 40 mg of bovine serum albumin (BSA) and dissolve it in 4 mL of 0.01 mol / L PBS solution to form a bovine serum albumin carrier solution. The hapten-activated ester obtained in step S1 was slowly added dropwise to the bovine serum albumin carrier solution under stirring and stirred at room temperature for 16 to 24 h.

[0107] S3. The solution obtained in step S2 was dialyzed against 0.01 mol / L PBS solution at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules to obtain the complete diflubenzuron antigen (i.e., diflubenzuron hapten-BSA conjugate), which was then aliquoted and stored at 4°C for later use.

[0108] Example 4 Preparation of complete diflubenzuron antigen

[0109] The synthesis process of the complete diflubenzuron antigen of the present invention is as follows ( Figure 2 Synthetic route shown):

[0110] S1. Take 5 mg of the diflubenzuron hapten prepared in Example 1 and dissolve it in 0.3 mL of dimethylformamide (DMF). After stirring thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS) and stir at room temperature for 4 h to obtain an activated hapten ester.

[0111] S2. Weigh 50 mg of ovalbumin (OVA) and dissolve it in 0.3 mL of 0.01 mol / L PBS to form an ovalbumin carrier solution. Slowly add the hapten-activated ester obtained in step S1 dropwise to the ovalbumin carrier solution under stirring and stir at room temperature for 16 to 24 hours.

[0112] S3. The solution obtained in step S2 was dialyzed against 0.01 mol / L PBS solution at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules to obtain the complete diflubenzuron antigen (i.e., diflubenzuron hapten-OVA conjugate), which was then aliquoted and stored at 4°C for later use.

[0113] Example 5 Preparation of complete diflubenzuron antigen

[0114] The synthesis process of the complete diflubenzuron antigen of the present invention is as follows ( Figure 2 Synthetic route shown):

[0115] S1. Take 15 mg of the diflubenzuron hapten prepared in Example 1 and dissolve it in 0.3 mL of dimethylformamide (DMF). After stirring thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS) and stir at room temperature for 4 h to obtain an activated hapten ester.

[0116] S2. Weigh 80 mg of human serum albumin (HSA) and dissolve it in 0.3 mL of 0.01 mol / L PBS solution to form a human serum albumin carrier solution. The hapten-activated ester obtained in step S1 was slowly added dropwise to the human serum albumin carrier solution under stirring and stirred at room temperature for 16 to 24 hours.

[0117] S3. The solution obtained in step S2 was dialyzed against 0.01 mol / L PBS solution at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules to obtain the complete diflubenzuron antigen (i.e., diflubenzuron hapten-HSA conjugate), which was aliquoted and stored at 4°C for later use.

[0118] Example 6 Preparation of complete diflubenzuron antigen

[0119] The synthesis process of the complete diflubenzuron antigen of the present invention is as follows ( Figure 2 Synthetic route shown):

[0120] S1. Take 15 mg of the diflubenzuron hapten prepared in Example 1 and dissolve it in 0.3 mL of dimethylformamide (DMF). After stirring thoroughly, add 5 mg of EDC and 5 mg of N-hydroxysuccinimide (NHS) and stir at room temperature for 4 h to obtain an activated hapten ester.

[0121] S2. Weigh 80 mg of hemocyanin (KLH) and dissolve it in 0.3 mL of 0.01 mol / L PBS solution to form a hemocyanin (KLH) carrier solution. Slowly add the hapten-activated ester obtained in step S1 dropwise to the hemocyanin (KLH) carrier solution under stirring and stir at room temperature for 16 to 24 hours.

[0122] S3. The solution obtained in step S2 was dialyzed against 0.01 mol / L PBS solution at room temperature for 3 days, with the dialysate changed 3 times a day to remove unreacted small molecules to obtain the complete diflubenzuron antigen (i.e., diflubenzuron hapten-KLH conjugate), which was aliquoted and stored at 4°C for later use.

[0123] In the following Examples 7 and 8, the complete diflubenzuron antigen (diflubenzuron hapten-LF conjugate) prepared in Example 2 was used as the immunization antigen to prepare diflubenzuron monoclonal antibodies.

[0124] Example 7 Preparation of Diflubenzuron Monoclonal Antibody

[0125] S1. Complete diflubenzuron antigen was emulsified with an equal volume of Freund's adjuvant and subcutaneously immunized BALB / C mice. Each mouse received a 100 μg dose, with an interval of 2 weeks between immunizations for a total of 3 immunizations.

[0126] S2. After the third immunization, blood was collected from the tail vein of the mice to test the serum antibody titer. If the antibody titer was lower than 1:8000, booster immunizations were performed with 100 μg of the vaccine every 7 days until the titer stabilized at or above 1:32000.

[0127] S3. Subcutaneous booster immunization was performed with 100 μg of diflubenzuron complete antigen in 0.01 M PBS solution. Five days later, mouse spleen cells were harvested and fused with SP20 mouse myeloma cells.

[0128] S4. The fused cells were screened in HAT medium and then cultured in complete medium (fetal bovine serum, basal medium, streptomycin-penicillin) after 5 days.

[0129] S5. Detect cell supernatant by ELISA and screen OD 450 Cells in the strongly positive wells with a score >1.0 were cloned and cultured three times using the limiting dilution method to confirm that the positive cells were hybridoma cells secreting the diflubenzuron monoclonal antibody;

[0130] S6. After hybridoma cells were expanded and cultured, 1×10 6 The cells were injected into the peritoneal cavity of mice to induce ascites, and the ascites was purified by caprylic acid-ammonium sulfate precipitation to obtain the diflubenzuron monoclonal antibody.

[0131] Example 8 Preparation of Diflubenzuron Monoclonal Antibody

[0132] S1. Complete diflubenzuron antigen was emulsified with an equal volume of Freund's adjuvant and subcutaneously immunized BALB / C mice. Each mouse received a 50 μg dose, with an interval of 2 weeks between immunizations for a total of 3 immunizations.

[0133] S2. After the third immunization, blood was collected from the tail vein of the mice to test the serum antibody titer. If the antibody titer was lower than 1:8000, 50 μg of the antibody was given as a booster immunization every 7 days until the titer stabilized at or above 1:32000.

[0134] S3. Subcutaneous booster immunization was performed with 50 μg of diflubenzuron complete antigen in 0.01 M PBS solution. Five days later, mouse spleen cells were harvested and fused with SP20 mouse myeloma cells.

[0135] S4. The fused cells were screened in HAT medium and then cultured in complete medium (fetal bovine serum, basal medium, streptomycin-penicillin) after 5 days.

[0136] S5. Detect cell supernatant by ELISA and screen OD 450 Cells in the strongly positive wells with a score >1.0 were cloned and cultured three times using the limiting dilution method to confirm that the positive cells were hybridoma cells secreting the diflubenzuron monoclonal antibody;

[0137] S6. After hybridoma cells were expanded and cultured, 1×10 6 The cells were injected into the peritoneal cavity of mice to induce ascites, and the ascites was purified by caprylic acid-ammonium sulfate precipitation to obtain the diflubenzuron monoclonal antibody.

[0138] In the following Example 9, the complete diflubenzuron antigen (diflubenzuron hapten-BSA conjugate) prepared in Example 3 was used as the coating antigen, and the diflubenzuron monoclonal antibody prepared in Example 7 was used as an example to test the performance of the diflubenzuron monoclonal antibody.

[0139] Example 9 ELISA Performance Evaluation of Diflubenzuron Monoclonal Antibody

[0140] The performance of the diflubenzuron monoclonal antibody was evaluated by enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows:

[0141] S1. Using carbonate buffer (pH 9.6) as the coating diluent, dilute the complete diflubenzuron antigen to 0.1 μg / mL and add 100 μL / well to a polystyrene microplate. Coat overnight at 4°C, spin dry, then add 280 μL / well of 1% BSA solution. Block the plate in phosphate buffer containing 1% BSA at 37°C for 1 hour, spin dry, and vacuum-pack for storage.

[0142] S2. Dilute the diflubenzuron monoclonal antibody to 0.1 μg / mL in phosphate buffer (pH 7.4) containing 0.05% sodium azide and store at 4°C until use.

[0143] S3. Add 50 μL / well of the diflubenzuron standard solution (0-1.6 μg / L) to the microwell ELISA plate coated with the complete diflubenzuron antigen in step S1, and then add 50 μL / well of the diflubenzuron monoclonal antibody solution in step S2, react at 37°C for 0.5 h, and spin dry;

[0144] S4. Add 280 μL / well of wash buffer, wash three times, pat dry, then add 100 μL / well of enzyme-labeled secondary antibody and incubate at 37°C for 0.5 h.

[0145] S5. Wash again three times and pat dry. Add chromogen A and chromogen B (Aladdin TMB chromogen, chromogen A product number T117927, chromogen B product number T117928) at 50 μL / well, respectively. Incubate at 37°C for 15 min. Terminate the reaction by adding 1 M sulfuric acid solution at 50 μL / well. Set the microplate reader to 450 nm to measure the OD value of each well. The results are shown in Table 1.

[0146] Table 1 Concentration and OD value (450nm) of diflubenzuron standard solution

[0147]

[0148] According to the data in Table 1, ELISACalc software was used to fit the four-parameter logistic curve to draw the standard curve ( Figure 3 ), the linear equation of the standard curve is: y = B + (AB) / (1 + (x / C) ^ D), r 2 =0.997578, A=0.87431±0.03856, B=0.03404±0.00299, C=0.31251±0.06846, D=0.8578±0.068, x represents the concentration of the test substance, y represents the OD value, and the IC value of the diflubenzuron monoclonal antibody was obtained by calculation. 50 The value was 0.2 μg / L, and a linear relationship was observed between 0.06 and 1.57 μg / L, demonstrating that the diflubenzuron monoclonal antibody prepared in this invention has high affinity and specificity, significantly improving the detection performance of diflubenzuron. This performance is derived from the optimized design of the novel hapten structure and provides a basis for the high sensitivity of the diflubenzuron immunoassay device.

[0149] In the following Examples 10 to 15, the complete diflubenzuron antigen (diflubenzuron hapten-BSA conjugate) prepared in Example 3 was used as the coating antigen, and the diflubenzuron monoclonal antibody prepared in Example 7 was used as an example to prepare diflubenzuron colloidal gold immunochromatographic detection devices.

[0150] Example 10 Preparation of Diflubenzuron Colloidal Gold Immunochromatographic Detection Device

[0151] S1. Preparation of reaction membrane coated with complete diflubenzuron antigen and mouse IgG

[0152] Using nitrocellulose membrane (NC membrane) as the substrate, a 0.3 mg / mL diflubenzuron complete antigen solution was sprayed on the test area (T line), and a 0.08 mg / mL mouse immunoglobulin (mouse IgG) solution was sprayed on the control area (C line). Both solutions were prepared with coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS buffer, pH = 7.6) at a spray volume of 1.0 μL / cm. The test and control areas were separated by 2.5 mm. The membranes were oven-dried at 45°C for 12 hours and stored at 20-25°C and ≤35% humidity.

[0153] S2. Preparation of Microwells Containing Colloidal Gold-Labeled Diflubenzuron Monoclonal Antibodies

[0154] S2.1 Preparation of colloidal gold

[0155] Take 1g of chloroauric acid, dissolve it with pure water by ultrasonication, and then dilute to 100mL; take 4mL of the above solution and add it to 100mL of pure water, heat it to boiling, then add 1.2mL of 0.06% sodium citrate solution to reduce it, and continue heating for 10 minutes; after heating, cool it to 25°C and make up to the original volume with pure water to obtain a colloidal gold solution, which should be stored in the dark; all glassware used should be soaked in a mixture of potassium permanganate and sulfuric acid overnight, and then washed and dried before use;

[0156] S2.2 Labeling of Diflubenzuron Monoclonal Antibody

[0157] The colloidal gold solution was dispensed into 1 mL / vial containers and the pH was adjusted dropwise to various values ​​within the range of 7.0 to 9.0 using 0.1 mol / L K2CO3 solution to optimize the binding of the antibody to the colloidal gold. 5 μg of diflubenzuron monoclonal antibody was added to the colloidal gold solution at various pH values ​​and allowed to react at 25°C for 5 minutes. The color of the solution was observed, and the lowest pH at which red color remained was selected as the optimal pH. The solution was blocked with 10 μL of 10% bovine serum albumin (BSA) solution and centrifuged at 4°C, 12,000 rpm, for 10 minutes. The supernatant was discarded and the precipitate was collected.

[0158] S2.3 Preparation of colloidal gold-labeled microwells

[0159] Redissolve the precipitate (i.e., labeled antibody) in colloidal gold diluent containing 0.02 M Tris, 2% bovine serum albumin, 0.05% thimerosal, and 5% sucrose, dispense into microwells at 12 μL / well, dry at 37°C for 16 hours, and store until ready for use.

[0160] S3. Preparation of sample pad

[0161] Soak the cut 30×30 cm blank sample pad in 0.05 M PBS buffer containing 0.05% Tween 20, 1% sucrose, 0.5% PVP40, and 0.05% sodium azide for 5 minutes, then take it out and dry it at 37°C for 16 hours, and store it at a temperature of 20-25°C and a humidity of ≤35%.

[0162] S4. Assembling colloidal gold chromatography detection card

[0163] The reaction membrane prepared in step S1 is stacked in the middle of the PVC board backing; the sample pad prepared in step S3 is stacked on the side of the reaction membrane close to the detection area, and the absorbent pad is stacked on the side close to the control area; the sample pad and the absorbent pad are connected to the two ends of the reaction membrane, respectively, with the connection parts of the sample pad, reaction membrane and absorbent pad overlapping by 1 to 2 mm, and then attached to the PVC board; the bottom plate is cut into 3 mm wide test strips using a strip cutter and loaded into a detection cartridge with a sample addition hole, with the sample addition hole aligned with the sample pad area of ​​the test strip.

[0164] Example 11 Preparation of Diflubenzuron Colloidal Gold Immunochromatographic Detection Device

[0165] S1. Preparation of reaction membrane

[0166] Using nitrocellulose membrane (NC membrane) as the substrate, a 0.2 mg / mL diflubenzuron complete antigen solution was sprayed on the test area (T line), and a 0.05 mg / mL mouse immunoglobulin (mouse IgG) solution was sprayed on the control area (C line). Both solutions were prepared in coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS, pH 7.6) at a spray volume of 0.8 μL / cm. The test and control areas were separated by a 2.5 mm gap. The membranes were oven-dried at 45°C for 12 hours and stored under constant temperature and humidity conditions.

[0167] The other steps (S2 to S4) are the same as S2 to S4 of Example 10.

[0168] Example 12 Preparation of Diflubenzuron Colloidal Gold Immunochromatographic Detection Device

[0169] S1. Preparation of reaction membrane

[0170] Using nitrocellulose membrane (NC membrane) as the substrate, a 0.4 mg / mL diflubenzuron complete antigen solution was sprayed on the test area (T line), and a 0.08 mg / mL mouse immunoglobulin (mouse IgG) solution was sprayed on the control area (C line). Both solutions were prepared in coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS, pH 7.6) at a spray volume of 1.0 μL / cm. The test and control areas were separated by a 2.5 mm gap. The membranes were oven-dried at 45°C for 12 hours and stored under constant temperature and humidity conditions.

[0171] The other steps (S2 to S4) are the same as S2 to S4 of Example 10.

[0172] Example 13 Preparation of Diflubenzuron Colloidal Gold Immunochromatographic Detection Device

[0173] S1. Preparation of reaction membrane

[0174] Using nitrocellulose membrane (NC membrane) as the substrate, a 0.6 mg / mL diflubenzuron complete antigen solution was sprayed on the test area (T line), and a 0.1 mg / mL mouse immunoglobulin (mouse IgG) solution was sprayed on the control area (C line). Both solutions were prepared in coating buffer (0.01 M PBS buffer, pH 7.6, containing 1% sucrose, 0.05% sodium azide), with a spray volume of 1.2 μL / cm. The test and control areas were separated by a 2.5 mm gap. The membranes were oven-dried at 45°C for 12 hours and stored under constant temperature and humidity conditions.

[0175] The other steps (S2 to S4) are the same as S2 to S4 of Example 10.

[0176] Example 14 Preparation of Diflubenzuron Colloidal Gold Immunochromatographic Detection Device

[0177] S1. Preparation of reaction membrane

[0178] Using nitrocellulose membrane (NC membrane) as the substrate, a 0.3 mg / mL diflubenzuron complete antigen solution was sprayed on the test area (T line), and a 0.07 mg / mL mouse immunoglobulin (mouse IgG) solution was sprayed on the control area (C line). Both solutions were prepared in coating buffer (0.01 M PBS buffer, pH 7.6, containing 1% sucrose, 0.05% sodium azide), with a spray volume of 1.1 μL / cm. The test and control areas were separated by a 2.5 mm gap. The membranes were oven-dried at 45°C for 12 hours and stored under constant temperature and humidity conditions.

[0179] The other steps (S2 to S4) are the same as S2 to S4 of Example 10.

[0180] Example 15 Preparation of Diflubenzuron Colloidal Gold Immunochromatographic Detection Device

[0181] S1. Preparation of reaction membrane

[0182] Using nitrocellulose membrane (NC membrane) as the substrate, a 0.5 mg / mL diflubenzuron complete antigen solution was sprayed on the test area (T line), and a 0.06 mg / mL mouse immunoglobulin (mouse IgG) solution was sprayed on the control area (C line). Both solutions were prepared in coating buffer (containing 1% sucrose, 0.05% sodium azide, and 0.01 M PBS, pH 7.6) at a spray volume of 0.9 μL / cm. The test and control areas were separated by a 2.5 mm gap. The membranes were oven-dried at 45°C for 12 hours and stored under constant temperature and humidity conditions.

[0183] The other steps (S2 to S4) are the same as S2 to S4 of Example 10.

[0184] The following Examples 16 to 17 take the diflubenzuron colloidal gold immunochromatographic detection device prepared in Example 10 as an example to illustrate its use and test its sensitivity and stability.

[0185] Example 16 Detection Method and Sensitivity Determination of Diflubenzuron Colloidal Gold Immunochromatographic Detection Device

[0186] 1. Testing of Diflubenzuron Standard Solution Samples

[0187] A series of different concentrations of diflubenzuron standard solutions of 0, 0.5, 1, 2, and 4 μg / L were prepared with 0.01M PBS buffer. The different concentrations of diflubenzuron standard solutions of 100 μL were then taken and added to the colloidal gold labeled micropores of the diflubenzuron colloidal gold immunochromatographic detection device. The solution was repeatedly pipetted and redissolved uniformly. After standing for 5 min, the solution in the colloidal gold labeled micropores was transferred to the sample addition hole of the diflubenzuron colloidal gold immunochromatographic detection device test card of the present invention. After adding the sample, the timing was started, and the results were observed for 5 to 8 min (the signal intensity was balanced during this time period, and the specific reading time could be adjusted according to the ambient temperature, such as reading the result at 6 minutes). After 8 min, the reading was invalid. The detection experiment was repeated for 3 groups.

[0188] The result judgment criteria are as follows Figure 4 As shown, specifically:

[0189] (1) Negative (-): The color of the T line is stronger than that of the C line or there is no significant difference in color with the C line;

[0190] (2) Positive (+): The color of the T line is significantly weaker than that of the C line or the T line does not show any color;

[0191] (3) Invalid: Line C does not appear, the operation process is incorrect or the test strip has expired.

[0192] The test results are shown in Table 2. Table 2 shows that the sensitivity of the diflubenzuron colloidal gold immunochromatographic detection device prepared by the present invention to diflubenzuron is 2 μg / L.

[0193] Table 2 Determination results of diflubenzuron standard solutions at different concentrations

[0194]

[0195] Note: “-” indicates a negative test result, and “+” indicates a positive test result.

[0196] 2. Detection of Diflubenzuron in the Test Samples

[0197] Lettuce, cowpea, cucumber, lettuce, Chinese cabbage, leek, rapeseed, spinach, celery, green pepper, and sugar orange were selected as blank samples. After sampling and processing according to GB 23200.45, GB / T 5009.147-2003, and NY / T 1720, the samples were placed in a 50mL centrifuge tube, 4mL of 0.01M PB buffer was added, and the mixture was vigorously shaken for 2 minutes. After the centrifuge tube was allowed to stand for 2 minutes, 100μL of the supernatant was added to 400μL of 0.1M PB buffer and shaken to prepare the test solution. 100 μL of the test solution was added to the colloidal gold-labeled microwells of the diflubenzuron colloidal gold immunochromatographic detection device of the present invention. The solution was repeatedly pipetted to uniformly redissolve the solution. After standing for 5 minutes, the solution in the colloidal gold-labeled microwells was transferred to the sample wells of the diflubenzuron colloidal gold immunochromatographic detection device of the present invention. The timer was started after the sample was added, and after 5 to 8 minutes, the results were observed according to the judgment criteria. A gradient spike test was performed on blank samples of 11 kinds of fruits and vegetables using a standard solution of diflubenzuron, with the spike gradients being 0, 5, 10, 20, and 40 μg / kg, respectively. The test was repeated three times.

[0198] The result judgment criteria are as follows Figure 4 As shown, specifically:

[0199] (1) Negative (-): The color of the T line is stronger than that of the C line or there is no significant difference in color with the C line;

[0200] (2) Positive (+): The color of the T line is significantly weaker than that of the C line or the T line does not show any color;

[0201] (3) Invalid: Line C does not appear, the operation process is incorrect or the test strip has expired.

[0202] The test results are shown in Table 3. Table 3 shows that a negative result is obtained when the diflubenzuron content in the test sample is less than 10 μg / kg; a positive result is obtained when the content is greater than or equal to 10 μg / kg. Therefore, the detection limit of the diflubenzuron colloidal gold immunochromatographic detection device prepared in the present invention for diflubenzuron in the test sample is 10 μg / kg.

[0203] Table 3 Detection limits of spiked samples

[0204]

[0205] Note: “-” indicates a negative test result, and “+” indicates a positive test result.

[0206] Example 17 Stability Test of Diflubenzuron Colloidal Gold Immunochromatographic Detection Device

[0207] In order to evaluate the stability of the diflubenzuron colloidal gold immunochromatographic detection device of the present invention under storage conditions and determine the shelf life of the detection device, an accelerated destructive test was performed on the detection device.

[0208] The test was carried out at 45°C for 60 consecutive days to simulate one year of long-term storage, and the color changes of negative and diflubenzuron standard solutions were detected on the 0th day, the 5th day, the 10th day, the 20th day, the 30th day, the 40th day, the 50th day and the 60th day, respectively, with three replicates.

[0209] The test results are shown in Table 4. Table 4 shows that after the diflubenzuron colloidal gold immunochromatographic detection device of the present invention was sealed and stored at 45°C for 60 days, the T / C color depth reading of the test card did not change significantly. These results demonstrate that the diflubenzuron colloidal gold immunochromatographic detection device prepared by the present invention exhibits excellent stability, can still accurately detect diflubenzuron residues after storage, and can be stably stored at room temperature for over one year, meeting market requirements for storage and transportation.

[0210] Table 4 Stability test results of diflubenzuron colloidal gold immunochromatographic detection device

[0211]

[0212] Note: “-” indicates a negative test result, and “+” indicates a positive test result.

[0213] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A diflubenzuron hapten, characterized in that Its structure is shown in formula (I):

2. A method for preparing the diflubenzuron hapten according to claim 1, characterized in that: The following steps are involved: S1. The aromatic amide compound a and the halogenated ester compound b are subjected to a substitution reaction under alkaline conditions to obtain a first intermediate compound c, as shown in the reaction formula (II): S2. The first intermediate product compound c is hydrolyzed in an alkaline solution, and the pH is adjusted to precipitate a solid to obtain a second intermediate product compound d. The reaction formula is shown in formula (III): S3. The second intermediate compound d is subjected to an amide reaction with an aromatic isocyanate compound e in an organic solvent to obtain the diflubenzuron hapten according to claim 1, wherein the reaction formula is as shown in formula (IV):

3. A complete diflubenzuron antigen, characterized in that: The diflubenzuron hapten according to claim 1 is coupled with a carrier protein, and its structure is shown in formula (V):

4. The complete diflubenzuron antigen according to claim 3, characterized in that The carrier protein is selected from the group consisting of bovine serum albumin, lactoferrin, ovalbumin, human serum albumin, and hemocyanin.

5. A diflubenzuron antibody, characterized in that The method is prepared by immunizing an animal with the complete diflubenzuron antigen according to claim 3 or 4.

6. The diflubenzuron antibody according to claim 5, characterized in that The diflubenzuron antibody is a diflubenzuron monoclonal antibody.

7. Use of any one of the diflubenzuron hapten according to claim 1, the diflubenzuron complete antigen according to claim 3 or 4, and the diflubenzuron antibody according to claim 5 or 6, or a combination thereof, in the detection of diflubenzuron.

8. Use of any one of the diflubenzuron hapten according to claim 1, the diflubenzuron complete antigen according to claim 3 or 4, and the diflubenzuron antibody according to claim 5 or 6, or a combination thereof, in the preparation of a diflubenzuron immunoassay device.

9. A diflubenzuron immunoassay device, characterized in that: Containing the diflubenzuron antibody according to claim 5 or 6.

10. The diflubenzuron immunoassay device according to claim 9, characterized in that: The diflubenzuron immunoassay device is a diflubenzuron colloidal gold immunochromatographic assay device.

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