Preparation and application of cyromazine artificial hapten, antigen and antibody

Abstract

The invention discloses design and synthesis of a hapten of cyromazine, development of a cyromazine artificial antigen and a cyromazine antibody, and application of the cyromazine artificial antigen and the cyromazine antibody in the field of food safety rapid detection. The aminocarboxylic acid modified cyromazine hapten product is prepared through the steps of cyromazine hapten design, connecting arm introduction, synthesis, purification and the like. The cyromazine hapten with the structure is coupled with keyhole limpet hemocyanin to form an artificial immune antigen with immunogenicity, and is coupled with chicken ovalbumin to form an artificial competitive antigen. Tests show that the artificial immune antigen has high immunocompetence and can stimulate an animal immune system to generate an anti-cyromazine antibody, and the titer of an immune Blab / c mouse serum antibody is 16000. The IC50 of an enzyme-linked immunosorbent assay (ELISA) detection method established by the cyromazine artificial competitive antigen and the anti-cyromazine antibody is 5.3 [mu] g / L, and the detection sensitivity of an established gold-labeled immunochromatography detection method reaches 50 [mu] g / L.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay detection of small molecule compounds, specifically involving techniques such as organic synthesis, immunochemistry, biochemistry, and physicochemical determination. In particular, it relates to the design and synthesis of haptens and antigens of the insecticide cyromazine, which has a functional carboxylic acid group; mouse immunization; hybridoma cell screening; preparation of specific monoclonal antibodies; and the establishment of immunoassay methods. Specifically, this is reflected in the application of ELISA detection methods and rapid detection methods using gold-labeled immunochromatographic strips, and its application in the field of agricultural product testing. Background Technology

[0002] Cyromazine (also known as cyromazine) is a low-toxicity insecticide belonging to the insect growth regulator class. It has very strong selectivity, primarily active against Diptera insects. Its mechanism of action involves causing morphological abnormalities in Diptera larvae and pupae, resulting in incomplete or inhibited adult emergence. This pesticide has contact and stomach poison effects, strong systemic conductivity, and a long residual effect, but its rate of action is relatively slow. Cyromazine has no toxic side effects on humans or livestock and is environmentally safe. Cyromazine is suitable for use on various fruits and vegetables, primarily exhibiting good insecticidal activity against fly pests. Currently, in fruit and vegetable production, it is mainly used to control various leafminers such as the American leafminer, South American leafminer, bean stalk leafminer, onion leafminer, and trifoliate leafminer on various fruits, solanaceous vegetables, beans, and various leafy vegetables, as well as root maggots (leek maggots) on leeks, onions, and garlic. A comprehensive evaluation of cyromazine's toxicity classifies it as a low-toxicity pesticide, with an acceptable daily intake (ADI) of 0.06 mg / kg bw. Maximum residue levels in food are as follows: cereals 3 mg / kg, vegetables 0.1-20 mg / kg, fruits 0.5 mg / kg, edible fungi 1-7 mg / kg, seasonings 10 mg / kg, meat 0.1-0.3 mg / kg, eggs 0.3 mg / kg, and raw milk 0.01 mg / kg. Therefore, national food safety regulations impose certain requirements on the cyromazine content in food. We need to correctly analyze the advantages and disadvantages of cyromazine, rationally manage its use in non-point source pollution control, and strengthen on-site monitoring and supervision.

[0003] This invention provides technical support for on-site monitoring and detection of cyromazine by developing a rapid detection method for cyromazine. Currently, the main quantitative detection methods for cyromazine residues are instrumental analysis methods such as high-performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS). These methods offer high sensitivity and accuracy, but require sophisticated equipment, complex sample preparation, and highly skilled analysts, making them unsuitable for rapid on-site detection of large batches of samples. This invention develops an immunochemical analysis technique based on cyromazine antigen and antibody, overcoming the aforementioned shortcomings and offering advantages such as high sensitivity, high specificity, rapid and convenient use, and environmental friendliness. To date, there are no publicly reported domestic studies on monoclonal antibody hybridoma cell technology for cyromazine, nor on the application of the specific antibodies generated from it for the rapid detection and analysis of cyromazine. Summary of the Invention

[0004] This invention discloses the design and synthesis of cyromazine hapten, the preparation of cyromazine artificial antigen, and the preparation of its monoclonal antibody. The purpose is to provide methods for preparing cyromazine hapten and corresponding artificial antigens and antibodies, and to apply them to the rapid immunoassay analysis of cyromazine in samples.

[0005] The immunogenicity of an antigen is a crucial factor determining whether an animal's immune system can produce antibodies. However, cyromazine, as a small molecule compound, lacks natural immunogenicity, meaning it cannot stimulate the animal's immune system to produce antibodies. Therefore, this invention selects cyromazine-based compounds, represented by cyromazine, and designs and modifies their structure to obtain cyromazine hapten molecules containing amide carboxylic acid groups. A certain number of these hapten molecules are then condensed and coupled with protein macromolecules to obtain immunogens and coating antigens.

[0006] The unique feature of this invention lies in overcoming the difficulties in the chemical synthesis of cyromazine hapten, thus establishing a complete technical route for the synthesis of cyromazine hapten. When used to immunize animals, it can produce highly specific antibodies with high affinity, and based on this, an ELISA method (enzyme-linked immunosorbent assay) and a gold-labeled immunochromatographic test strip method have been established, which can accurately detect the content of cyromazine.

[0007] Another unique feature of this invention is the use of keyhole hemocyanin-conjugated small molecules as immunogens, which increases the conjugation ratio of hapten molecules to protein molecules in the conjugation reaction and enhances their immunogenicity.

[0008] The structure of cyromazine is shown below:

[0009]

[0010] To solve the above-mentioned technical problems, the technical solution adopted in this invention is as follows: the artificial hapten of cyromazine is an aminocarboxylic acid group substituted at the R site of one amino group on the melamine ring. R can be one or more of aminopropionic acid, aminobutyric acid, aminovaleric acid, and aminohexanoic acid derivative groups. The molecular structural formula is as follows:

[0011]

[0012] Furthermore, the cyromazine hapten was prepared by substitution and hydrolysis of 2-aminomelamine. It was obtained by introducing aminocarboxylic acid into 2-aminomelamine under strong alkaline catalysis. The molecular structure is as follows:

[0013]

[0014] This invention also discloses a cyromazine immunogen (MYA-KLH), which is synthesized by coupling a cyromazine artificial hapten with a modified R site structure with keyhole hemocyanin (KLH). The molecular structure of MYA-KLH is as follows:

[0015]

[0016] The artificially coated cyromazine antigen (MYA-OVA) is synthesized by coupling the cyromazine artificial hapten with the modified R site structure of cyromazine with chicken ovalbumin (OVA). The molecular structure of MYA-OVA is as follows:

[0017]

[0018] The present invention also discloses a cyromazine antibody, which is a monoclonal antibody capable of generating a specific immune response with cyromazine immunogen or cyromazine artificially coated antigen.

[0019] Anti-cyromazine monoclonal antibodies are monoclonal antibodies that can produce specific immune responses to MYA-KLH or MYA-OVA, as well as cyromazine compounds.

[0020] An application of cyromazine antibody: This antibody can be used for the rapid and sensitive detection of cyromazine content in agricultural products, specifically in ELISA immunoassay and rapid detection methods using gold nanoparticle-based immunochromatographic strips. Application areas include agricultural products such as vegetables, fruits, eggs, dairy products, and meat.

[0021] The cyromazine was prepared via a series of reactions including substitution and hydrolysis. The synthetic route is as follows:

[0022]

[0023] The preparation method of cyromazine hapten includes the following steps:

[0024] (1) Dissolve 2-aminomelamine in KOH alkaline solution, add 1-2 molar equivalents of methyl bromide, stir the reaction and heat to 60-80℃, and the substitution reaction yields melamine amino methyl bromide.

[0025] (2) Under alkaline conditions of NaOH, melamine methyl ester is hydrolyzed into melamine amino acid, cooled to 5-10℃, filtered, and the solid sample obtained by filtration is recrystallized with 1,4-dioxane and dried to obtain cyromazine hapten.

[0026] The preparation method of MYA-KLH is the carbodiimide method, which includes the following steps:

[0027] (1) Dissolve 0.005 mmol / L of cyromazine hapten in 0.2 mL of DMF, add 0.01-0.02 mmol / L of N-hydroxysuccinimide (NHS), stir for 15 min, then add 0.01-0.02 mmol / L of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and stir overnight at room temperature. The reaction solution is the cyromazine hapten activation solution.

[0028] (2) Prepare a 5-10 mg / mL KLH or OVA solution using 0.01 M carbonate buffer (pH 9.0). Add the above activation solution dropwise under stirring at room temperature, and then slowly stir magnetically for 3 hours. Transfer the reaction solution into a pretreated dialysis bag and dialyze three times with 100 times the volume of 0.01 M phosphate buffer (PBS), changing the solution every 6 hours.

[0029] (3) After dialysis, take out the dialysate, mix it well and dispense it into low-adsorption centrifuge tubes to obtain MYA-KLH or MYA-BSA, and store it at -20℃.

[0030] The preparation method of MYA-OVA is the mixed anhydride method, which includes the following steps:

[0031] (1) Dissolve 0.01 mmol / L of cyromazine hapten in 0.2 mL of DMF. Add 0.02-0.04 mmol / L of tri-n-butylamine and 0.02-0.04 mmol / L of isobutyl chloroformate while stirring. Stir at room temperature for 1 h to obtain cyromazine hapten activated solution.

[0032] (2) Prepare a 10-20 mg / mL OVA solution with 0.01 M carbonate buffer (pH 9.0), add the above cyromazine hapten activation solution dropwise under stirring at room temperature, continue stirring for 3 h, put the reaction solution into a pretreated dialysis bag, dialyze with 100 times the volume of 0.01 M PBS 3 times, and change the solution every 6 h.

[0033] (3) After dialysis, take out the dialysate, mix it well and dispense it into low-adsorption centrifuge tubes to obtain MYA-OVA solution, and store it at -20℃.

[0034] The preparation method of anti-cyromazine monoclonal antibody includes animal immunization, hybridoma cell screening, and antibody preparation, comprising the following steps:

[0035] (1) Immunization: Six-week-old female BALB / c mice were used for immunization. The immunization methods were subcutaneous multi-point and intraperitoneal Freund's adjuvant injection. A booster immunization was performed 21 days after the initial immunization, and a booster immunization was performed every 14 days, for a total of 3 booster immunizations and 1 final immunization. The specific experimental methods are as follows:

[0036] Primary immunization: Dissolve 0.1-0.2 mg of MYA-KLH in 0.9% physiological saline. Take an equal volume of Freund's complete adjuvant and load it into syringes. Use an emulsifying tube to connect the syringes and thoroughly emulsify the immunogen MYA-KLH. Administer subcutaneous injections to mice at seven points: neck, waist, limbs, and intraperitoneal injection.

[0037] Boosting immunization: Dissolve 0.05-0.1 mg of MYA-KLH in 0.9% physiological saline. Take an equal volume of Freund's complete adjuvant and load it into syringes. Use an emulsification tube to connect the syringes and thoroughly emulsify the immunogen MYA-KLH or MYA-BSA. Administer intraperitoneal injection to mice for immunization.

[0038] Last immunization: Dissolve 0.05-0.1 mg of MYA-KLH in 0.9% saline and administer via intraperitoneal injection.

[0039] (2) Hybridoma cell screening: Mouse antibody titers were monitored regularly. After two booster immunizations, mouse tail blood titers were measured every week, using absorbance values ​​(OD). 450nm A value ≥1 indicates a positive result. Using MYA-OVA as the coating agent, mice were immunized for the last time when their tail blood titer reached 1:1000–1:4000. Three days after the last immunization, the spleen was harvested by cervical dislocation for hybridoma cell fusion, and monoclonal hybridoma cells were screened. A homologous indirect competitive ELISA method was used, with 5–10 μg / mL MYA-OVA coated and 100–1000 ng / mL cyromazine standard solution used as the competitive agent. Strongly positive OD values ​​were selected. 450nm Cell lines with ≥0.5 and inhibition rate ≥50% were subcloned 2-3 times consecutively to obtain stable monoclonal hybridoma cell lines.

[0040] (3) Antibody preparation: Seven-week-old F1 mice were selected and injected intraperitoneally with 500 μL of phytidine per mouse. Seven days later, the stable monoclonal hybridoma cell lines obtained in step (2) (cell density approximately 2 × 10⁻⁶) were used. 6500 μL / mouse was injected into the peritoneal cavity of mice. After 7–10 days, once the peritoneal cavity had expanded, ascites fluid was collected and purified using the octanoic acid-ammonium sulfate precipitation method. The purified ascites fluid was coated with MYA-OVA, and the antibody titer and inhibition rate against cyromazine were determined indirectly.

[0041] Steps for ELISA detection and analysis of cyromazine:

[0042] The monoclonal antibody against cyromazine is used for the rapid detection of cyromazine content in a sample, and the immunoassay is an enzyme-linked immunosorbent assay (ELISA). The application of the cyromazine antibody for the immunoassay of cyromazine content includes the following steps:

[0043] (1) Coating: Dissolve MYA-OVA in 0.01mol / L carbonate buffer (pH 9.0), coat 100μl / well onto the microplate, and refrigerate overnight at 4℃. After removal, wash 3 times with 0.01M PBST.

[0044] (2) Blocking: After coating, 2% skim milk powder was prepared with 0.01mol / L PBS (pH 7.4), and 300μl / well was used to block the microplate. The plate was then incubated at 37℃ for 30 minutes. After removal, the plate was washed twice with 0.01mol / L PBST.

[0045] (3) Competitive reaction: Dissolve the sample to be tested in 0.01 mol / L PBS solution of 30% methanol, add 50 μl / well to the microplate, add 50 μl / well of cyromazine antibody solution, incubate at 37℃ for 0.5 hours, and wash the plate 3 times after taking it out.

[0046] (4) Enzyme-labeled secondary antibody reaction: Dilute rabbit anti-mouse enzyme-labeled secondary antibody 20,000 times with 0.01 mol / L PBS (pH 7.4), add 100 μl / well, and incubate at 37℃ for 0.5 hours. After incubation, wash the plate 4 times.

[0047] (5) Color development and termination: The substrate for color development is tetramethylbenzidine (TMB) solution. Add 100 μl / well during color development and add 50 μl / well of 2 mol / L H2SO4 after 15 minutes to terminate the color development.

[0048] (6) After the color development is complete, place the ELISA plate on the microplate reader and read the OD value. 450nm Numerical analysis, calculation, and data analysis.

[0049] Rapid detection method steps for cyromazine immunochromatographic test strips:

[0050] The aforementioned cyromazine-coated antigen and cyromazine monoclonal antibody are used for the rapid detection of cyromazine content in agricultural products and environmental samples. The immunoassay is performed using a colloidal gold immunochromatographic test strip. The application of cyromazine antibody in the rapid detection method using the immunochromatographic test strip includes the following steps:

[0051] (1) The coated antigen MYA-OVA was streaked on the chromatographic membrane at a concentration of 0.05-0.5 mg / mL as the detection line (T line), and the goat anti-mouse secondary antibody was streaked on the chromatographic membrane at a concentration of 0.05-0.4 mg / mL as the quality control line (C line). The membrane was then dried in a forced-air drying oven.

[0052] (2) Take the above-mentioned chromatography membrane, and assemble it with the sample pad and absorbent pad to make chromatography test paper, and cut it into 3-4 mm test paper strips.

[0053] (3) The monoclonal antibody against cyromazine was labeled onto 30 nm colloidal gold particles at a concentration of 2-10 μg / mL to prepare gold-labeled cyromazine antibody. The prepared gold-labeled antibody was added to 10-50 μL in a 96-well microplate and lyophilized or dried by forced air drying.

[0054] (4) Prepare a standard solution of cyromazine with a concentration range of 0.01-10 mg / L. Take the test strip from step 2 and the gold microwell from step 3 for detection. The sample volume is 100-200 μL.

[0055] (5) When the concentration of cyromazine colloidal gold immunochromatographic test strip is 0.05 mg / L or above, the test strip shows a positive result, with no T line and a C line showing color; when the concentration is below 0.05 mg / L, the test strip shows a negative result, with both the T line and the C line showing color.

[0056] This invention has the following advantages and effects compared to existing analytical techniques:

[0057] (1) The designed and synthesized cyromazine hapten has high similarity to the target analyte and retains the characteristic structure of cyromazine intact, laying the foundation for the preparation of cyromazine antibodies with good specificity;

[0058] (2) Experimental verification shows that the above hapten has a simple synthesis method, high synthesis efficiency, and few reaction steps. The cyromazine hapten only requires 4 reaction steps to synthesize, thereby improving the controllability and timeliness of the reaction.

[0059] (3) The cyromazine antibody exhibits good detection sensitivity. The MYA-OVA coating concentration was 1 μg / mL, the antibody concentration was 1 μg / mL, and the cyromazine standard solution was prepared using 0.01 mol / L phosphate buffer with 10% methanol. An ELISA standard curve was established, and the linear equation was obtained as: y = 15.612ln(x) + 24.104, R0 2 =0.9962, estimated IC50 It reached 5.3 μg / mL.

[0060] (4) This invention is the first to apply cyromazine monoclonal antibody to gold-labeled immunochromatographic test strips for rapid sample detection. The operation is simple and convenient, and the detection can be completed in 15 minutes. The detection limit is 50 μg / mL, which can meet the needs of rapid detection.

[0061] Therefore, the method for synthesizing haptens in this invention is easier to promote and popularize compared with other methods. The antibody involved in this invention has high activity and has been successfully applied to the ELISA detection method. Its operation is simple and rapid, requiring only 1 hour and 15 minutes, and the detection accuracy can reach over 95%. The gold-labeled immunochromatographic test strip developed in this invention can be used for rapid food safety testing. Therefore, this invention not only demonstrates excellent detection results in a laboratory environment but also lays the foundation for developing low-cost, high-efficiency, and fast-operation rapid immunoassay tools, suitable for on-site rapid testing needs, and has good application prospects, offering both economic and social benefits. Attached Figure Description

[0062] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0063] Figure 1 For cyromazine hapten 1 H NMR spectrum.

[0064] Figure 2 The image shows the HPLC-MS spectrum of the cyromazine hapten.

[0065] Figure 3 The standard curve diagram established for the ELISA analysis method of cyromazine of the present invention.

[0066] Figure 4 This is the colorimetric image of the gold-labeled immunochromatographic test strip for cyromazine of the present invention. Detailed Implementation

[0067] The following embodiments of the present invention are merely illustrative of the invention and should not be construed as limiting the scope or content of the invention. The present invention will now be further illustrated through these embodiments.

[0068] Example 1

[0069] (1) Synthesis of cyromazine hapten

[0070] Synthesis method of cyromazine hapten: 2g of 2-aminomelamine was dissolved in KOH alkaline solution, and 1.5 molar equivalents of methyl bromide butyrate were added. The mixture was heated to 80℃ under stirring to obtain methyl melamine-aminobutyrate. Under NaOH alkaline conditions, methyl melamine-aminobutyrate was hydrolyzed to melamine-aminobutyric acid. The solution was cooled to 10℃, and the obtained solid sample was recrystallized from 1,4-dioxane. After drying, 0.636g of cyromazine hapten solid was obtained with a purity of 97.0%. Characterization was performed as follows: 1 H NMR (400MHz, CD3OD) δ1.39-1.43(m,2H),1.55-1.59(m,2H),1.61-1.67(m,2H),2.32(t,J=7.2Hz,2H),2.75-2.86(m,2H).HRMS(ESI-TOF)m / z:[M+H] + calcd.forC9H 18 N7O2256.1516, found 256.1522. The synthetic route of the cyromazine hapten is as follows:

[0071]

[0072] (2) Preparation of artificial antigens MYA-KLH and MYA-OVA for cyromazine

[0073] Dissolve 0.005 mmol / L of cyromazine hapten in 0.2 mL of DMF, add 0.01 mmol / L of NHS, stir for 15 min, then add 0.001 mmol / L of EDC, and stir overnight at room temperature. The reaction solution is the cyromazine hapten activation solution. Prepare a 5 mg / mL KLH solution using 0.01 M carbonate buffer (pH 9.0), and add the above hapten activation solution dropwise under stirring at room temperature, then slowly stir magnetically for 3 h. Transfer the reaction solution to a pretreated dialysis bag and dialyze three times with 100 times the volume of 0.01 M phosphate buffer (PBS), changing the buffer every 6 h. After dialysis, remove the dialysate, mix well, and aliquot into low-adsorption centrifuge tubes to obtain cyromazine immunogen MYA-KLH, and store at -20℃. The molecular structure of MYA-KLH is:

[0074]

[0075] 0.01 mmol / L of cyromazine hapten was dissolved in 0.2 mL of DMF. 0.02 mmol / L of tri-n-butylamine and 0.02 mmol / L of isobutyl chloroformate were added with stirring, and the mixture was stirred at room temperature for 1 h to obtain the activated cyromazine hapten solution. A 10 mg / mL OVA solution was prepared using 0.01 M carbonate buffer (pH 9.0), and the above activated hapten solution was added dropwise with stirring at room temperature. The reaction was continued for 3 h. The reaction solution was then transferred to a pretreated dialysis bag and dialyzed three times with 100 volumes of 0.01 M PBS, changing the solution every 6 h. After dialysis, the dialysate was collected, mixed, and aliquoted into low-adsorption centrifuge tubes to obtain the original MYA-OVA solution coated with cyromazine, which was stored at -20 °C. The molecular structure of MYA-OVA is:

[0076]

[0077] (3) Preparation of hybridoma cell lines of cyromazine monoclonal antibody

[0078] Six-week-old female BALB / c mice were used for immunization. The immunization method was subcutaneous multi-site Freund's adjuvant injection. Booster immunizations were administered 21 days after the initial immunization, with booster immunizations every 14 days, for a total of three booster immunizations and one final immunization. For the initial immunization, 0.2 mg of MYA-KLH was dissolved in 0.9% saline, and an equal volume of Freund's complete adjuvant was added to syringes. The MYA-KLH immunogen was thoroughly emulsified using an emulsion tube connected to the syringe, and injected subcutaneously at seven points: neck, back, limbs, and abdomen. For the booster immunization, 0.1 mg of MYA-KLH was dissolved in 0.9% saline, and an equal volume of Freund's complete adjuvant was added to syringes. The MYA-KLH immunogen was thoroughly emulsified using an emulsion tube connected to the syringe, and injected intraperitoneally. For the final immunization, 0.1 mg of MYA-KLH was dissolved in 0.9% saline and injected directly into the peritoneum.

[0079] Antibody titers were monitored regularly during mouse immunization. After two booster immunizations, tail blood titers were measured every week, expressed as absorbance values ​​(OD). 450nm A value ≥1 indicates a positive result. Using MYA-OVA as the coating agent, mouse tail blood titers reaching 1:16000 were tested for a final immunization. Three days after the final immunization, the spleen was harvested via cervical dislocation for hybridoma cell fusion, and monoclonal hybridoma cells were screened. A homologous indirect competitive ELISA method was used, with 5 μg / mL MYA-OVA coated and 100 ng / mL cyromazine standard solution used as the competing drug. Strongly positive OD values ​​were selected. 450nm Cell lines with ≥0.5 and inhibition rate ≥50% were subcloned twice to obtain stable monoclonal hybridoma cell lines.

[0080] (4) Preparation of cyromazine monoclonal antibody

[0081] Six-week-old F1 mice were selected and injected intraperitoneally with 500 μL of berberine per mouse. Seven days later, the selected stable monoclonal hybridoma cell lines of cyromazine (cell density approximately 2 × 10⁻⁶) were used. 5 The ascites fluid was injected into the peritoneal cavity of mice at a dose of 500 μL / mouse. One week later, the peritoneal cavity of the mice began to expand. The ascites fluid was collected and purified by the octanoic acid-ammonium sulfate precipitation method to obtain the cyromazine monoclonal antibody.

[0082] Example 2

[0083] A rapid ELISA detection method was established using the cyromazine monoclonal antibody obtained in Example 1.

[0084] MYA-OVA was dissolved in 0.01 mol / L carbonate buffer (pH 9.0) and coated onto a microplate at 100 μl / well. The plate was incubated overnight at 4°C. After coating, the plate was washed three times with 0.01 M PBST. Following coating, 2% skim milk powder was prepared in 0.01 mol / L PBS (pH 7.4) and 300 μl / well was used to block the microplate. The plate was incubated at 37°C for 30 minutes and then washed twice with 0.01 mol / L PBST. The sample to be tested was dissolved in 0.01 mol / L PBS solution (30% methanol) and added to the microplate at 50 μl / well. Then, 50 μl of cyromazine antibody solution was added to each well. The plate was incubated at 37°C for 0.5 hours and then washed three times. Rabbit anti-mouse enzyme-labeled secondary antibody was diluted 20,000 times with 0.01 mol / L PBS (pH 7.4), and 100 μl was added to each well. The plate was incubated at 37°C for 0.5 hours, and then washed four times. The chromogenic substrate was tetramethylbenzidine (TMB) solution, 100 μl of which was added to each well. After 15 minutes of development, 50 μl of 2 mol / L H₂SO₄ was added to terminate the reaction. After the reaction was terminated, the plate was read using a microplate reader to determine the OD value. 450nm Numerical analysis, calculation, and data analysis.

[0085] The inhibition rate of the target analyte concentration on the antibody is calculated as I = ((Amax - Amin) - (Ai - Amin) / (Amax - Amin)) × 100, and the binding rate of the antibody to the antigen is calculated as B / B0 = (Ai - Amin) / (Amax - Amin), where Amax is the average absorbance value of the blank wells, Amin is the average absorbance value of the control wells containing pre-immunized BLAB / c mouse serum, and Ai is the average absorbance value of the sample wells. A standard curve is plotted with the inhibition rate I or binding rate B / B0 as the ordinate and the analyte concentration C as the abscissa, as shown in the attached figure. Figure 3 IC detection 50It can stably reach 5.3 μg / L. Using the target analyte concentrations corresponding to 5% and 95% inhibition rates as the minimum and maximum detection concentrations, respectively, the detection range is 0.3–93.8 μg / L.

[0086] GB2763-2021, the National Food Safety Standard for Maximum Residue Limits of Pesticides in Food, stipulates that the maximum residue limit for cyromazine in vegetables, fruits, and eggs is 0.1-10 mg / kg. This method has a sensitivity of 0.3 mg / kg, which meets the detection requirements of most maximum residue limits (MRLs) in food. Actual samples tested included broccoli (MRL = 1 mg / kg), celery (MRL = 4 mg / kg), cowpeas (MRL = 0.5 mg / kg), mango (MRL = 0.5 mg / kg), melon (MRL = 0.5 mg / kg), eggs (MRL = 0.3 mg / kg), water, and soil. Negative samples and cyromazine-positive additives were set up at concentrations of 0.03 mg / kg, 0.3 mg / kg, and 3 mg / kg, respectively. Samples were extracted using an extraction buffer, and the final test samples were diluted 6 times. The concentrations of cyromazine in the blank and additive samples were calculated based on the ELISA standard curve, and the results were consistent with the additive concentrations. Specific data are shown in Table 1.

[0087] Table 1. ELISA detection results of cyromazine in actual samples.

[0088]

[0089] Example 3

[0090] An immunochromatographic rapid test strip was prepared using the cyromazine-coated antigen and cyromazine antibody obtained in Example 1. The detection limit of the rapid test strip method for cyromazine was 0.3 mg / kg, and this method was applied to the detection of actual samples.

[0091] Actual sample testing included broccoli, celery, cowpeas, mango, melon, and eggs. Blank samples and positive additives for cyromazine were prepared, and samples were extracted using an extraction buffer. Blank samples and additives with concentrations below the detection limit of cyromazine all showed negative results on the test strips; additives with concentrations at or above the detection limit of cyromazine all showed positive results. The test results indicate that the rapid test strip for cyromazine has high accuracy. Specific results are shown in Table 2.

[0092] Table 2. Detection results of cyromazine test strips in actual samples.

[0093]

[0094] Finally, it should be stated that this invention is not limited to the above three embodiments, and may have other derivative uses. Any other variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. The artificial hapten of cyromazine is an aminocarboxylic acid group substituted at one amino R site on the melamine ring, wherein R can be one or more of aminopropionic acid, aminobutyric acid, aminovaleric acid, and aminohexanoic acid derivative groups, characterized in that... The molecular structural formula is:

2. The artificial hapten of cyromazine according to claim 1, characterized in that: The cyromazine hapten was prepared by substitution and hydrolysis of 2-aminomelamine. It was obtained by introducing aminocarboxylic acid into 2-aminomelamine under strong alkaline catalysis. The molecular structure is as follows:

3. A cyromazine immunogen, characterized in that: The artificial hapten of cyromazine, modified from the R site structure of cyromazine as described in claim 1 or 2, is coupled with keyhole hemocyanin (KLH), and its molecular structure is as follows:

4. The artificially coated antigen of cyromazine, characterized in that: The artificial hapten of cyromazine, modified from the R site structure of cyromazine as described in claim 1 or 2, is coupled with ovalbumin (OVA), and the molecular structure of the artificial cyromazine-coated antigen is as follows:

5. A cyromazine antibody, characterized in that: A monoclonal antibody that can generate a specific immune response with the cyromazine immunogen of claim 3 or the cyromazine artificially coated antigen of claim 4.

6. The application of the cyromazine antibody as described in claim 5, characterized in that: Cyromazine antibodies can be used for rapid and sensitive detection of cyromazine content in samples, specifically in the application of ELISA immunoassay methods and rapid detection methods using gold-labeled immunochromatographic strips.

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