Colloidal gold immunochromatography test strip for rapidly detecting fomesafen as well as preparation and detection methods of colloidal gold immunochromatography test strip

Abstract

The invention provides a colloidal gold immunochromatography test strip detection method for rapidly detecting fomesafen, which comprises the following steps of: preparing fomesafen hapten and complete antigen: introducing carboxyl at a sulfonylamino part in a fomesafen structure to prepare immune hapten; coupling the immune hapten with thyroglobulin through a carbodiimide method to prepare an immunogen; preparation of a monoclonal antibody: immunizing a mouse with the obtained immunogen and preparing the fomesafen monoclonal antibody through a hybridoma technology; and preparation of the colloidal gold immunochromatography test strip: spraying the prepared coating antigen on a nitrocellulose membrane to serve as a detection line, and spraying a goat-anti-mouse secondary antibody on the nitrocellulose membrane to serve as a quality control line. The elimination line value of the test strip for detecting fomesafen residues is 16ng / mL. According to the method, the fomesafen residue in the field sample can be rapidly detected, so that the method has important guiding significance on planting of next-stubble crops.

Description

TECHNICAL FIELD

[0001] The present application relates to a colloidal gold immunochromatographic test strip for rapid detection of fomesafen and a preparation and detection method thereof, in particular to a colloidal gold immunochromatographic test strip capable of detecting fomesafen residues in vegetables, cereals and soil. BACKGROUND

[0002] Fomesafen (Fomesafen) alias tiger Wei, molecular formula is C 15 H 10 ClF3N2O6S, its chemical name is 5-[2-chloro-4-(trifluoromethyl) phenoxy]-N-(methylsulfonyl)-2-nitrobenzene, which was introduced by British Syngenta in 1982. It is a diphenyl ether herbicide, mainly used for controlling annual and perennial broadleaf weeds in soybean and peanut fields. Its mode of action is a kind of selective herbicide of protoporphyrinogen oxidase (PPO) inhibitor, which mainly destroys the photosynthesis of weeds, so as to make the weeds yellow and wither and die. With the continuous expansion of soybean planting area in China, the use amount of fomesafen also increases year by year. However, due to the high residual amount and long residual period of the herbicide in soil, the large use of fomesafen will not only cause phytotoxicity to the succeeding crops, but also have a certain influence on soil enzyme activity and microbial community. In addition, fomesafen will flow into ponds and rivers through surface runoff with rainfall, thereby threatening the safety of aquatic organisms and causing groundwater pollution, which threatens human health. According to the maximum residue limit of pesticides in food published in 2021, the maximum residue limit of fomesafen in cereals, soybeans and peanut kernels is 0.05, 0.1 and 0.2 mg / kg respectively.

[0003] At present, the detection of benzenesulfonamide herbicide is mainly dependent on traditional large-scale instruments, including liquid chromatography-tandem mass spectrometry (LC-MS), ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) and solid-liquid extraction (SLE / LTP) coupled with high-performance diode array detector (HPLC / DAD). Although the instrument detection method has the characteristics of accuracy, sensitivity and good selectivity, it generally needs to be operated by professional personnel, and the sample pretreatment and purification steps are complicated, and cannot be used for on-site rapid detection. In recent years, the immunoassay method is widely used for the rapid detection of pesticides and their metabolites, veterinary drugs, food toxins and other pollutants due to its high efficiency, rapidness, high sensitivity and easy operation. The enzyme-linked immunoassay (ELISA) and colloidal gold immunochromatography test strip are widely used in the immunoassay method, and the main principle is based on the specific binding of antigen and antibody, so as to achieve the purpose of quantitative or qualitative detection. At present, there is only one literature report on the immunoassay method of benzenesulfonamide herbicide, which is the indirect competitive enzyme-linked immunoassay method based on the benzenesulfonamide herbicide polyclonal antibody published by the laboratory in the early stage. However, the ELISA experiment has many operation steps, and the detection result needs to be read by the aid of an enzyme-labeled instrument, so it is not suitable for on-site rapid detection. The colloidal gold immunochromatography test strip can be observed by naked eye, has the advantages of fast detection speed, low cost and no need for external instruments, and is suitable for on-site rapid detection. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a colloidal gold immunochromatography test strip for rapid detection of benzenesulfonamide herbicide and a preparation and detection method thereof. The semi-antigen of benzenesulfonamide herbicide is designed and synthesized, and then coupled with a carrier protein to obtain a complete antigen, and then the mice are immunized, and the high-sensitivity benzenesulfonamide herbicide monoclonal antibody is obtained by using the hybridoma technology. On this basis, the colloidal gold is prepared and coupled with the monoclonal antibody to obtain the gold-labeled antibody, so as to prepare the colloidal gold immunochromatography test strip for benzenesulfonamide herbicide residue detection and apply it.

[0005] The first object of the present application is to design and synthesize the semi-antigen and artificial antigen of benzenesulfonamide herbicide.

[0006] The second object of the present application is to prepare the high-sensitivity and high-specificity benzenesulfonamide herbicide monoclonal antibody.

[0007] The third object of the present application is to provide a colloidal gold label based on the benzenesulfonamide herbicide monoclonal antibody.

[0008] The fourth object of the present application is to prepare the benzenesulfonamide herbicide colloidal gold immunochromatography test strip.

[0009] The fifth object of the present application is to provide a colloidal gold immunochromatography test strip for detecting the benzenesulfonamide herbicide residues in soil, agricultural products and the like.

[0010] The above-mentioned object of the present application is achieved by the following technical solutions.

[0011] The present application provides preparation of sulfonamide herbicide hapten and artificial antigen

[0012] Design and synthesis of sulfonamide herbicide hapten: sulfonamide herbicide immune hapten is prepared by introducing carboxyl group to the sulfonamide group of sulfonamide herbicide for derivatization;

[0013] Preparation of artificial antigen: the above-mentioned sulfonamide herbicide immune hapten is coupled with thyroglobulin (Thy) to prepare immune antigen by carbodiimide method, and 5-(2-chloro-2,2,2-trifluoro-p-methyl phenoxy)-2-nitrobenzoic acid is coupled with bovine serum albumin (BSA) to prepare coating antigen by the same method.

[0014] In the above-mentioned scheme, the synthesis method of the hapten is specifically:

[0015] 1.828 mmol of 5-(2-chloro-2,2,2-trifluoro-p-methyl phenoxy)-2-nitrobenzoic acid is added to a 50 mL single-necked round-bottom flask, then an appropriate amount of SOCl2 is added, heated to reflux at 80℃ for 2 h, and then the solvent is removed to obtain compound I; the amount of compound I prepared and the volume of the reaction vessel are enlarged or reduced in proportion;

[0016] 1.306 mmol of 3-aminosulfonyl propionic acid is added to a 50 mL round-bottom flask, and 5 mL of methanol is added for stirring and dissolution, then SOCl2 is slowly added under ice bath conditions, after the addition is completed, stirring is carried out at room temperature for 15 minutes, then heating reflux reaction is carried out at 80℃. After the reaction is completed, the solvent is removed, an appropriate amount of water is added for dilution, NaHCO3 is added to adjust the pH to 7-8, then extraction is carried out with ethyl acetate, washing is carried out with saturated brine, drying is carried out with anhydrous sodium sulfate, vacuum filtration and concentration are carried out to remove excess solvent, and brown oil compound II is obtained.

[0017] 0.436 mmol of compound I is dissolved in 5 mL of dichloromethane, 0.5232 mmol of triethylamine is added for reaction, then 0.530 mmol of compound II is weighed and dissolved in 2 mL of dichloromethane, and then slowly added to the above-mentioned reaction system, after reaction at room temperature for 3 h, TCL is used to detect the reaction progress. After the reaction is completed, the crude product is purified by 100-200 mesh silica gel column chromatography, the eluent is petroleum ether: ethyl acetate with a boiling range of 60-90℃, the volume ratio is 1:1-3, and compound III is purified.

[0018] Take 0.04 mmol of compound III, and add 5 mL of methanol for dissolution, then add 0.08 mmol of solid NaOH, heat to reflux at 80°C for 2 h, then remove the methanol, then add water and ethyl acetate, take the aqueous phase and adjust the pH to 2-3 with hydrochloric acid, add ethyl acetate, collect the organic phase, dry, and rotary evaporate to obtain the florasulam hapten.

[0019] Preferably, the heating reflux time during the synthesis of compound II is 3-6 h.

[0020] Preferably, the NaHCO3 is a saturated solution, and the concentration of the hydrochloric acid is 1 M.

[0021] In the above scheme, the preparation of the complete antigen is to couple the obtained florasulam hapten with thyroglobulin by the carbodiimide method, and to prepare the coated antigen by coupling 5-(2-chloro-2,2,2-trifluoro-p-tolyloxy)-2-nitrobenzoic acid with bovine serum albumin by the same method. The specific steps are as follows:

[0022] Dissolve the florasulam hapten in dichloromethane, add N-hydroxysuccinimide (NHS) and stir at room temperature for 10 min, then add 1-ethylcarbodiimide hydrochloride (EDC), and react at room temperature for 3-4 h for activation. Dissolve the Thy protein in a borate buffer, and then slowly add the above activated hapten solution, which has been concentrated under reduced pressure and dissolved in a small amount of N,N-dimethylformamide, to the Thy protein solution. React at room temperature overnight, then dialyze in a phosphate buffer (PBS) for 72 h, and then store at -20°C for standby use.

[0023] Preparation of the coated antigen: Dissolve the intermediate 5-(2-chloro-2,2,2-trifluoro-p-tolyloxy)-2-nitrobenzoic acid of florasulam in dichloromethane, add N-hydroxysuccinimide (NHS) and stir at room temperature for 10 min, then add 1-ethylcarbodiimide hydrochloride (EDC), and react at room temperature for 3-4 h for activation. Slowly add the activated 5-(2-chloro-2,2,2-trifluoro-p-tolyloxy)-2-nitrobenzoic acid to bovine serum albumin (BSA) dissolved in a borate buffer, and react at room temperature overnight. Dialyze in a phosphate buffer (PBS) for 72 h, and then store at -20°C for standby use.

[0024] Preferably, in the activation process of the florasulam hapten and the intermediate 5-(2-chloro-2,2,2-trifluoro-p-tolyloxy)-2-nitrobenzoic acid, the molar ratio of the florasulam hapten or 5-(2-chloro-2,2,2-trifluoro-p-tolyloxy)-2-nitrobenzoic acid to N-hydroxysuccinimide (NHS) and 1-ethylcarbodiimide hydrochloride (EDC) is 1:1.0-1.2:1.2-1.5.

[0025] Preferably, during the synthesis of the immunogen and the coating antigen, the molar ratio of the fomesafen hapten and the thyroglobulin (Thy) is 1:400-600, and the molar ratio of the 5-(2-chloro-2,2,2-trifluoro-p-tolyloxy)-2-nitrobenzoic acid and the bovine serum albumin (BSA) is 1:200-300.

[0026] The present application provides preparation and purification of fomesafen monoclonal antibody.

[0027] Preparation of the monoclonal antibody: immunize mice with the immunogen, screen by hybridoma technology, and obtain a fomesafen monoclonal cell strain with good competitiveness by indirect competitive ELISA, and then purify the fomesafen monoclonal antibody by preparing ascites and using a Protein A column.

[0028] In the above scheme, the specific method for preparing the fomesafen monoclonal antibody is as follows: screen by hybridoma technology to obtain a high-quality monoclonal cell strain, then prepare ascites by injecting the cell strain into the abdominal cavity of a mouse, centrifuge the obtained ascites, filter the supernatant with a 0.22 μm filter membrane, and purify the ascites, the specific steps being as follows: first, wash the column with 10-20 mL of elution buffer to remove impurities in the column, then wash the column with 10-20 mL of binding buffer, dilute the ascites with the binding buffer at a ratio of 1:1-3, load the diluted ascites, collect the supernatant, repeat the loading 1-2 times, wash the column after loading with 20-30 mL of binding buffer, then elute the antibodies combined in the column with the elution buffer, neutralize the eluate with an appropriate amount of neutralization buffer, mix rapidly, and maintain the pH value in the centrifuge tube at 7. Equilibrate the column after elution with 10-20 mL of binding buffer, then wash the small column with 20% ethanol binding buffer, finally fill the small column with the solution, and store at 4°C. The purified antibody is dialyzed with normal saline, aliquoted, and stored at -20°C.

[0029] The elution buffer used for the above purification is prepared as follows: citric acid 4.2028 g, sodium citrate 1.4705 g, and ultrapure water to 250 mL; the binding buffer is prepared as follows: NaH2PO4·2H2O 0.5928 g, Na2HPO4·12H2O 2.2196 g, and ultrapure water to 1000 mL; and the neutralization buffer is prepared as follows: Tris 30.285 g, ultrapure water to 250 mL, and hydrochloric acid to adjust the pH to 9. The above buffers are all filtered with a 0.22 μm filter membrane after preparation, and then stored at 4°C.

[0030] Preferably, the centrifugation conditions for the ascites are 12000 rpm, 4°C, and 10 min.

[0031] Preferably, the amount of the neutralization buffer is 200-300 μL.

[0032] Preferably, the physiological saline concentration used in the dialysis process is 0.1% to 1.5%.

[0033] The present application provides preparation of colloidal gold marker of monoclonal antibody of sulfentrazone.

[0034] Preparation of colloidal gold solution: colloidal gold is prepared by trisodium citrate reduction method, specifically: take a certain amount of ultrapure water in a triangular flask, heat and stir, after the water boils, add 1% tetrachlorogold acid solution, boil for 10 min again, then add 1% trisodium citrate, the solution color changes from blue-violet-wine red, continue to boil for 10 min until the color changes to wine red, get colloidal gold solution, cool and store at 4℃ in the dark.

[0035] Preparation of colloidal gold marker: take 1-2 mL of colloidal gold buffer, adjust the pH value of the colloidal gold buffer to 8.2 with potassium carbonate, add the prepared sulfentrazone monoclonal antibody, react at room temperature for 30-60 min in the dark, then add 100 μL of BSA sodium borate buffer for blocking for 20-60 min, then centrifuge the labeled gold antibody at 12000 rpm at 4℃ for 15 min, discard the supernatant, resuspend with 1 / 10 volume of resuspension solution to obtain colloidal gold labeled antibody.

[0036] Preferably, the ratio of the amount of ultrapure water, tetrachlorogold acid and trisodium citrate is 100-200 mL: 1-3 mL: 2-5 mL.

[0037] Preferably, the ratio of the amount of colloidal gold solution, monoclonal antibody and blocking solution is 1-5 mL: 5-20 μg: 100-300 μL.

[0038] Preferably, the concentration of BSA in the blocking solution is 1%-15%.

[0039] Preferably, the resuspension solution is prepared as a sodium borate solution containing 2% BSA and 2% sucrose.

[0040] The present application provides assembly of sulfentrazone colloidal gold immunochromatography test strip.

[0041] The assembly of the colloidal gold immunochromatography test strip: the test strip is composed of PVC bottom plate, nitrocellulose (NC) membrane (quality control line T line sprayed with goat anti-mouse secondary antibody and detection line C line sprayed with coated antigen), sample pad, water absorption pad, etc. The goat anti-mouse secondary antibody is sprayed on the nitrocellulose (NC) membrane near the end of the water absorption pad as the quality control line (T line), and the coated antigen is diluted to a certain concentration and sprayed on the nitrocellulose (NC) membrane near the end of the sample pad as the detection line (C line).

[0042] Preferably, the specification of the PVC base plate is (1.5+2.5+2) cm, the nitrocellulose (NC) membrane is pasted in the middle of the PVC base plate, that is, 2.5 cm, the water absorption pad is pasted at 2.0 cm, and the sample pad is pasted at 1.5 cm, and when the water absorption pad and the sample pad are pasted, attention should be paid to cover the nitrocellulose (NC) membrane by 2-3 mm.

[0043] A method for using the colloidal gold immunochromatography test strip for rapidly detecting benoxacor: a sample to be detected is extracted with a sample extraction solution, a gold-labeled antibody is added to a micropore for incubation at room temperature for 5 min, the colloidal gold immunochromatography test strip for benoxacor is inserted into the micropore, and a detection result is read after 5-8 min.

[0044] Preferably, the amount of the antibody added in the preparation of the gold-labeled antibody is 2-10 muL, and the dilution multiple required by the sample diluent is 5-40 times.

[0045] The application provides the application of the colloidal gold immunochromatography test strip for benoxacor.

[0046] The application provides a method for detecting benoxacor pesticide residues in soil and agricultural products, adopts a test strip to detect a soil sample, and judges the residue of the benoxacor pesticide according to a color development result of the test strip.

[0047] Compared with the existing detection technology, the application has the following beneficial effects:

[0048] 1. The application derives the benoxacor hapten by introducing a carboxyl group on the basis of retaining the structure of benoxacor to the greatest extent, continues to couple the hapten with thyroglobulin to obtain an immunogen, couples 5-(2-chloro-2,2,2-trifluoro-p-tolyloxy)-2-nitrobenzoic acid, an intermediate compound in the synthesis process of benoxacor, with bovine serum albumin to obtain a coating antigen, immunizes mice with the prepared immunogen to obtain a benoxacor monoclonal antibody with high specificity through a hybridoma technology. Then, based on the obtained benoxacor monoclonal antibody, a benoxacor colloidal gold immunochromatography test strip with high sensitivity and specificity is constructed. The preparation of the test strip realizes rapid detection of benoxacor residues in the field, does not need to rely on instruments, and has the advantages of convenient and fast detection and low cost.

[0049] 2. The residues of benoxacor not only cause phytotoxicity to subsequent crops, but also threaten human health. The colloidal gold immunochromatography test strip prepared in the application can specifically detect benoxacor, and has important significance for the detection of benoxacor residues.

[0050] 3. The prepared fomesafen colloidal gold immunochromatography test strip has high sensitivity, the detection result can be observed by naked eyes, the detection cost is low, the detection speed is fast, the sample extract liquid is directly diluted, and then incubated with the fomesafen gold-labeled antibody in micropores for 5 minutes, the colloidal gold immunochromatography test strip is inserted, and the detection result can be determined after 5-8 minutes, and the fomesafen colloidal gold immunochromatography test strip is suitable for rapid detection of a large number of samples in the field.

[0051] 4. The prepared fomesafen colloidal gold immunochromatography test strip has low technical requirements for the operator, the detection result can be observed by naked eyes, the operation does not need to be assisted by instruments, the rapid detection demand of farmers in the field can be met, the preparation method is simple, and the fomesafen colloidal gold immunochromatography test strip has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 A synthetic route map is designed for the fomesafen immunohapten of the application;

[0053] Figure 2 The ultraviolet absorption peak and transmission electron microscope characterization diagram of the colloidal gold prepared in the application are shown;

[0054] Figure 3 The salt precipitation test curve of the colloidal gold-labeled fomesafen monoclonal antibody is shown;

[0055] Figure 4 The structure schematic diagram of the colloidal gold immunochromatography test strip provided by the embodiment of the application is shown (wherein, 1 is a PVC bottom plate, 2 is a sample pad, 3 is a nitrocellulose membrane, 4 is a water absorption pad, 5 is a T line of fomesafen coated antigen, and 6 is a goat anti-mouse secondary antibody as a C line);

[0056] Figure 5 The detection result judgment rule of the colloidal gold immunochromatography test strip of the application is shown;

[0057] Figure 6 The detection results of different concentrations of fomesafen standard solution under natural light are shown;

[0058] Figure 7 A is the detection result diagram of 0.5 times of the recommended dose of fomesafen in the field (the test strip detection results are control, 0d, 3d, 7d, 10d, 15d and 30d after spraying, respectively); Figure 7 B is the detection result diagram of 1.0 times of the recommended dose of fomesafen in the field (the test strip detection results are control, 0d, 3d, 7d, 10d, 15d and 30d after spraying, respectively); Figure 7 C is the detection result diagram of 2.0 times of the recommended dose of fomesafen in the field (the test strip detection results are control, 0d, 3d, 7d, 10d, 15d and 30d after spraying, respectively). DETAILED DESCRIPTION

[0059] In order to make the purposes, technical solutions and innovations of the present application more clear and explicit, the technical solutions of the present application are described in detail below in combination with the drawings and examples, and the examples do not limit the present application in any form. Although the present application is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the related art that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the present application, and all of them should be covered in the scope of the claims of the present application.

[0060] The preparation and application of a colloidal gold immunochromatography test strip based on monoclonal antibody of fomesafen, including the design and synthesis of hapten, the preparation of artificial antigen, the screening of monoclonal antibody, the labeling of gold-labeled antibody, and the preparation and application of fomesafen colloidal gold immunochromatography test strip. The colloidal gold immunochromatography test strip of the present application has high detection sensitivity, low cost, low technical requirements for operators, and does not require the aid of instruments. This method is suitable for rapid residue detection in the field.

[0061] Example 1 Preparation of fomesafen hapten and complete antigen

[0062] To prepare a colloidal gold immunochromatography test strip of fomesafen, it is necessary to obtain a fomesafen monoclonal antibody with good sensitivity first. The prerequisite for obtaining a monoclonal antibody is to design and synthesize a fomesafen hapten and an immunogen. The specific hapten and immunogen synthesis route is shown in Figure 1 The specific steps are as follows:

[0063] Preparation of hapten: by introducing a carboxyl group into the sulfonamide group of fomesafen for derivatization, fomesafen immunohapten is prepared;

[0064] 1.828 mmol of trifluorocarboxy ether was weighed into a 50 mL single-necked round-bottom flask, and an appropriate amount of SOCl2 was added. After heating at 80℃ for 2h, the solvent was removed to obtain compound I. The amount of compound I prepared and the volume of the reaction vessel were scaled up or down in proportion.

[0065] 1.306 mmol of 3-aminosulfonylpropionic acid was added to a 50 mL round-bottom flask, and methanol was added for stirring and dissolution. Then, SOCl2 was slowly added under ice bath conditions, and after the addition was completed, the solution was stirred at room temperature for 15 min, and then heated and refluxed at 80℃. After the reaction was completed, the solvent was removed, water was added for dilution, NaHCO3 was added to adjust the pH to 7-8, and then extracted with ethyl acetate, washed with saturated brine, dried with anhydrous sodium sulfate, concentrated by vacuum filtration, and the excess solvent was removed to obtain brown oil compound II.

[0066] Take 0.436 mmol of compound I, dissolve in 5 mL of dichloromethane, add 0.5232 mmol of triethylamine, and then take 0.530 mmol of compound II, dissolve in 2 mL of dichloromethane, and slowly add to the above reaction system. After reaction at room temperature for 3 h, the reaction progress is detected by TCL. After the reaction is completed, the crude product is purified by column chromatography on 100-200 mesh silica gel, and the eluent is petroleum ether: ethyl acetate with a boiling range of 60-90°C at a volume ratio of 1:1-3. Compound III is obtained after purification.

[0067] Take 0.04 mmol of compound III, add an appropriate amount of methanol to dissolve, and then add 0.08 mmol of solid NaOH. After heating and refluxing at 80°C for 2 h, the methanol is removed, and then water and ethyl acetate are added. The aqueous phase is taken and adjusted to pH 2-3 with hydrochloric acid. Ethyl acetate is added, the organic phase is collected, dried, and rotary evaporated to obtain the flumetralin hapten.

[0068] Preferably, the heating and refluxing time during the synthesis of compound II is 3-6 h.

[0069] Preferably, the NaHCO3 is a saturated solution, and the concentration of hydrochloric acid is 1 M.

[0070] Preparation of the complete antigen: The obtained hapten is coupled with thyroglobulin by the carbodiimide method to prepare an immunogen, and 5-(2-chloro-2,2,2-trifluoro-p-tolyloxy)-2-nitrobenzoic acid is coupled with bovine serum albumin by the same method to prepare a coating antigen. The specific steps are as follows:

[0071] Dissolve the flumetralin hapten in dichloromethane, add N-hydroxysuccinimide (NHS) and stir at room temperature for 10 min, then add 1-ethylcarbodiimide hydrochloride (EDC) and react at room temperature for 3-4 h to activate. Dissolve the thyroglobulin in a borate buffer, and then slowly add the activated hapten solution to the thyroglobulin solution after concentrating under reduced pressure and dissolving in a small amount of N,N-dimethylformamide solvent. After reaction at room temperature overnight, dialyze against a phosphate buffer (PBS) for 72 h, and then store at -20°C for later use.

[0072] Preparation of coated antigen: intermediate compound 5-(2-chloro-2,2,2-trifluoro-p- methoxyphenoxy)-2-nitrobenzoic acid of fomesafen was also dissolved in dichloromethane, N- hydroxysuccinimide (NHS) was added and stirred at room temperature for 10 min, then 1- ethylcarbodiimide hydrochloride (EDC) was added, and the reaction was carried out at room temperature for 3-4 h for activation. The activated 5-(2-chloro-2,2,2-trifluoro-p- methoxyphenoxy)-2-nitrobenzoic acid was added dropwise to bovine serum albumin (BSA) dissolved in borate buffer, and after overnight reaction, it was also dialyzed against phosphate buffer (PBS) for 72 h and stored at -20 °C for standby

[0073] Preferably, in the activation process of the fomesafen hapten and intermediate compound 5-(2-chloro-2,2,2-trifluoro-p-methoxyphenoxy)-2-nitrobenzoic acid, the molar ratio of the fomesafen hapten or 5-(2-chloro-2,2,2-trifluoro-p-methoxyphenoxy)-2-nitrobenzoic acid to N- hydroxysuccinimide (NHS) and 1-ethylcarbodiimide hydrochloride EDC is 1:1.2:1.5

[0074] Preferably, in the synthesis process of the immunogen and the coated antigen, the molar ratio of the fomesafen hapten and thyroglobulin is 1:400-600, and the molar ratio of 5-(2-chloro-2,2,2-trifluoro-p-methoxyphenoxy)-2-nitrobenzoic acid and bovine serum albumin is 1:200-300.

[0075] The bovine serum albumin and thyroglobulin were purchased from Solarbio Reagent Co., Ltd., the 5-(2-chloro-2,2,2-trifluoro-p-methoxyphenoxy)-2-nitrobenzoic acid was purchased from Anhui Zesheng Reagent Co., Ltd., and the rest of the drugs were purchased from Shanghai Macklin Reagent Co., Ltd. and Shanghai Bide Medicine.

[0076] Example 2 Preparation of fomesafen monoclonal antibody

[0077] (1) Mouse immunization

[0078] The mice were immunized by intraperitoneal injection. At the first immunization, 6-8 week old BALB / c female mice were selected, the prepared florasulam immunogen was diluted to 1 mg / mL with sterilized PBS (0.01M PBS), and then emulsified with the same volume of Freund's complete adjuvant (FCA). After complete emulsification, each mouse was injected with 100 μg (i.e. each mouse was injected with 200 μL of the emulsified immunogen), and each immunized mouse was labeled. After 21 days, the second immunization was performed on the mice with the same dose. Since the second immunization, Freund's complete adjuvant was replaced by Freund's incomplete adjuvant (FIA) in the emulsification process. Thereafter, the mice were immunized every two weeks. Starting from the third immunization, the tail blood of the mice was taken every time for immunization, and the tail blood titer was determined by indirect competitive enzyme-linked immunoassay. The tail blood of mice that had not been immunized was collected as a negative control. The obtained tail blood was incubated at 37°C for 30 min, and then centrifuged at 8000 rpm / min for 10 min at 25°C. The supernatant was collected. The supernatant was subjected to titer and competition determination by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to analyze the immunization effect, and the mice with good immunization effect and excellent competition were selected for booster immunization and cell fusion.

[0079] After the fifth immunization, cell fusion was performed, and the mice were given a booster immunization with the same dose of immunogen without any adjuvant three days before fusion. Three days later, the mice were sacrificed by cervical dislocation after blood sampling by removing the eyeball. The mice were soaked in 75% alcohol for 10 minutes, then quickly moved to a clean bench, and the mouse spleen cells were obtained by dissection. The spleen cells were filtered with a cell screen to prepare a cell suspension. The cell suspension and myeloma cells were mixed at a ratio of 5:1, centrifuged at 1000 rpm for 10 min, and then the supernatant was poured out. The cell pellet was knocked into a water-like state and placed in a 37°C water bath for warming. 1 mL of PEG1500 was drawn into a syringe and added dropwise into the centrifuge tube within 1 min. After the addition was completed, it was placed for 1 min, and then DMEM culture solution was added to terminate the PEG reaction, 3 s per drop at the first minute, 2 s per drop at the second minute, 1 s per drop at the third minute, and the DMEM culture solution was quickly added to 30 mL at the fourth minute. The fused cells were placed for 10 min, and then centrifuged at 800 rpm for 8 min. The centrifuged cells were transferred to a clean bench, the supernatant was poured out, 20 mL of 20% newborn calf serum HAT culture solution was added to resuspend the cells, and half of the cells were transferred to another centrifuge tube. Both tubes were diluted to 30 mL with 20% newborn calf serum HAT, and then 150 μL per well was added to a 96-well plate and placed in a cell culture incubator. After one week, the number of cell clusters in each well was counted under a microscope, the cell fusion rate was calculated, and when the cell clusters grew to a large area (usually 5-10 days after fusion), the cell culture solution in each well was removed, the supernatant was screened using indirect competitive enzyme-linked immunoassay, and the cell well with high titer and high inhibition rate was selected for subcloning. After three subcloning, the obtained cells were expanded and cultured to obtain the target hybridoma strain.

[0080] (2) Preparation and purification of monoclonal antibody of benzenesulfonamide herbicide

[0081] After the hybridoma cell line obtained above is cultured in large scale, a cell suspension is prepared, each mouse is injected with 500 μL of the cell suspension, and the mice are continuously raised for about one week. When the mice's abdomen is swollen, the ascites is obviously increased, and the mice have a phenomenon of hair explosion, the ascites is extracted with a sterilized needle. The ascites obtained is centrifuged, the supernatant is filtered with a 0.22 μm filter membrane, and the ascites obtained is purified according to the following steps: first, 10-20 mL of elution buffer is used to wash the column to remove the sundries in the column, then 10-20 mL of binding buffer is used to wash the column, the ascites is diluted according to 1:1-3 with the binding buffer, the diluted ascites is loaded, the supernatant is collected, the loading is repeated for 1-2 times, 20-30 mL of the binding buffer is used to elute the column after loading, then the antibody combined in the column is eluted with the elution buffer, the eluate is neutralized with an appropriate amount of neutralization buffer, and the mixture is quickly mixed to keep the pH value in the centrifuge tube at 7. The column after elution is equilibrated with 10-20 mL of the binding buffer, then the column is washed with 20% ethanol binding buffer, finally the column is filled with the solution, and the column is stored at 4°C. The antibody after purification is dialyzed with normal saline, and is stored at -20°C after being divided into small portions.

[0082] The buffer solutions used in the above purification, such as the elution buffer, are prepared as follows: 4.2028 g of citric acid and 1.4705 g of sodium citrate are dissolved in ultrapure water to make up to 250 mL; the binding buffer is prepared by dissolving 0.5928 g of NaH2PO4·2H2O and 2.2196 g of Na2HPO4·12H2O in ultrapure water to make up to 1000 mL; and the neutralization buffer is prepared by dissolving 30.285 g of Tris in ultrapure water to make up to 250 mL, and the pH value is adjusted to 9 with hydrochloric acid. The above buffer solutions are filtered with a 0.22 μm filter membrane after preparation, and are stored at 4°C.

[0083] Example 3 Preparation of a colloidal gold marker of a monoclone antibody of florasulam

[0084] (1) Preparation of a colloidal gold solution: the colloidal gold solution is prepared by the trisodium citrate reduction method, specifically, an appropriate amount of ultrapure water is taken in a flask, heated and stirred, 1% tetrachloroauric acid solution is added when the water is boiling, after boiling for 10 min, 1% trisodium citrate is added, the solution color changes from blue to purple to wine red, and the wine red color is maintained after boiling for another 10 min, and the colloidal gold solution is obtained, which is stored at 4°C in the dark after cooling. Figure 2 The transmission electron microscope characterization diagram of the colloidal gold prepared in the present application is shown in Fig. 1. Figure 2 The ultraviolet absorption spectrum and the transmission electron microscope (TEM) image after the colloidal gold solution is diluted 5 times with ultrapure water are shown in Fig. 2, the colloidal gold is observed to have good dispersibility under the TEM, the colloidal gold particle size is uniform, the particle size is about 20 nm, and there is no agglomeration. The colloidal gold can be used for the preparation of a colloidal gold immunochromatographic test strip.

[0085] (2) Gold antibody preparation: Take 1-2 mL colloidal gold buffer, adjust the pH value of the colloidal gold buffer to 8.2 with potassium carbonate, add the prepared flumetralin monoclonal antibody, react at room temperature for 30-60 min, then add 100 μL of BSA sodium borate buffer for blocking for 20-60 min, then centrifuge the labeled gold antibody at 12000 rpm at 4°C for 15 min, discard the supernatant, resuspend with 100 μL of resuspension solution, and then store at 4°C.

[0086] Preferably, the ratio of the amount of ultrapure water, tetrachloroauric acid and trisodium citrate is 100-200 mL: 1-3 mL: 2-5 mL.

[0087] Preferably, the ratio of the amount of colloidal gold solution, monoclonal antibody and blocking solution is 1-5 mL: 5-20 μg: 100-300 μL.

[0088] Preferably, after the colloidal gold adsorbs the antibody, the antibody can enhance the stability of the colloidal gold in high-concentration salt solution and reduce the aggregation of the colloidal gold. The optimal amount of monoclonal antibody is determined by measuring the ultraviolet absorption difference of the colloidal gold solution before and after adding salt. Figure 3 As shown in the salt precipitation test for antibody labeling, the concentration of the antibody used for labeling is determined by the concentration of the antibody used for labeling, and the absorbance difference at 525 nm before and after adding salt is determined. When the difference tends to decrease until the line is flat, the concentration is determined as the final selected concentration of the antibody used for labeling. At this time, the concentration of flumetralin monoclonal antibody is 62.5 μg / mL.

[0089] (3) Assembly of flumetralin colloidal gold immunochromatography test strip: as shown in Figure 4 The colloidal gold immunochromatography test strip for rapid detection of flumetralin according to the present application is composed of a PVC base plate, a nitrocellulose (NC) membrane (a quality control line T line sprayed with goat anti-mouse secondary antibody and a detection line C line sprayed with a coated antigen), a sample pad, a water absorption pad and the like. The goat anti-mouse secondary antibody is sprayed on the nitrocellulose (NC) membrane near the end of the water absorption pad as the quality control line (T line), and the coated antigen is diluted to a certain concentration and sprayed on the nitrocellulose (NC) membrane near the end of the sample pad as the detection line (C line). After the test strip is cut, it is placed in a 37°C oven for 1-2 h, and then cut into a test strip with a width of 4 mm using a strip cutter and stored in a self-sealing bag containing a drying agent.

[0090] Preferably, the specifications of the PVC base plate are (1.5+2.5+2) cm, the nitrocellulose (NC) membrane is attached to the middle of the PVC base plate, i.e. at 2.5 cm, the water absorption pad is attached at 2.0 cm, and the sample pad is attached at 1.5 cm. When attaching the water absorption pad and the sample pad, attention should be paid to covering the nitrocellulose (NC) membrane by 2-3 mm.

[0091] Performance evaluation of halosafen colloidal gold immunochromatographic test strip

[0092] (1) Determination of detection limit

[0093] A detection method of a colloidal gold immunochromatographic test strip for rapid detection of halosafen: dilute halosafen with 1% PBST buffer in a gradient, set the concentration of halosafen as 0, 1.25, 2.5, 5, 10, 20 ng / mL, add the colloidal gold labeled with halosafen monoclonal antibody to obtain a gold-labeled antibody, incubate at room temperature for 5 min, insert the halosafen colloidal gold immunochromatographic test strip into a micropore, and read the detection result after 5-8 min, and the result interpretation rule is as shown in Figure 5 .

[0094] Result interpretation:

[0095] Negative (-): The color of the T line (detection line, close to one end of the sample hole) is as deep as the color of the C line (quality control line), indicating that the residual content of halosafen in the sample is lower than the detection limit value or there is no pesticide residue;

[0096] Positive (+): The color of the T line is shallower than the color of the C line, or the T line is colorless, indicating that the sample contains halosafen pesticide, and the shallower the color of the T line compared with the color of the C line, the more halosafen residue;

[0097] Invalid result: The C line does not develop color, the T line develops color, or both the C line and the T line do not develop color, indicating that the detection result is invalid.

[0098] As shown in Figure 6 , the detection image of different concentrations of halosafen standard solution under natural light, the results show that the detection limit of the test strip for halosafen is 2.5 ng / mL, according to the maximum residue limit of pesticides in food issued in 2021, the maximum residue limit of halosafen in grains, soybeans and peanut kernels is 0.05, 0.1 and 0.2 mg / kg, and according to the above, the application reaches the national limit standard.

[0099] (2) Specificity test

[0100] Determination of antibody sensitivity and cross-reactivity rate: select compounds similar in structure to halosafen such as trifluron, flumetralin, fluroxypyr, bifenox, lactofen and dichlorprop, determine the inhibitory concentration (IC 50 ) of the structural analogues by indirect competitive enzyme-linked immunoassay, and then calculate the cross-reactivity of various structural analogues. The cross-reactivity formula is: cross-reactivity = IC 50 (halosafen) / IC 50(Other structural analogs) x 100%, the cross-reactivity of 6 fluorochloridone structural analogs is shown in Table 1. The results show that the monoclonal antibody of fluorochloridone has cross-reactivity with fluroxypyr and carfentrazone-ethyl.

[0101] Table 1 Determination of cross-reactivity of fluorochloridone antibody

[0102]

[0103]

[0104] Example 5 Application of fluorochloridone colloidal gold immunochromatography test strip

[0105] The soybean field is divided into three plots, and the corresponding plots are sprayed with 0.5 times, 1.0 times and 2.0 times of the field recommended dose of fluorochloridone (25% fluorochloridone aqueous agent, 160 mL / acre) respectively. Each plot is sampled by the four-point method, that is, four samples are collected from the four directions (northwest, northeast, southwest and southeast) of the diagonal line of the plot, and one sample is collected from the center of the plot. Five samples are mixed to obtain a mixed sample. Add the sample to 60% acetonitrile-PBS, vortex for 5 min, 8000 rpm; 4℃; centrifuge for 10 min. The supernatant is filtered through a 0.22 μm filter membrane. Since the soil matrix will affect the test results when testing soil, the dilution method is generally used to eliminate the matrix effect. The present application has determined that the supernatant needs to be diluted by 32 times to eliminate the matrix effect.

[0106] The prepared test strip in Example 3 is used for detection. The treated supernatant is diluted and added to the microwells, and the gold-labeled antibody is added. The test strip is placed in the microwell, and the liquid flow is timed. After 5-8 min of reaction, the result is determined. The detection results are shown in Table 2. Figure 7 As can be seen from the detection results, all the detection results are positive, except that the 30 d sampling result of 0.5 times of the field recommended dose of fluorochloridone is weakly positive, and the other detection results are strongly positive. It shows that the detection method of fluorochloridone of the present application can meet the requirements of the detection results of actual samples.

[0107] The above examples only describe the preferred mode of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application are made, which should fall within the protection scope determined by the claims of the present application.

Claims

1. A method for preparing a flusulfanilamide monoclonal antibody, characterized in that, Its preparation method specifically includes the following steps: The design and synthesis route of the flumetsulam hapten: The flumetsulam hapten was prepared by introducing a carboxyl group to derivatize the sulfonamide site of flumetsulam. The specific design and synthesis route of the hapten is as follows: The method for synthesizing the flusulfanilamide hapten according to claim 1 specifically comprises the following steps: A. Preparation method of compound I Weigh 1.828 mmol of trifluorocarboxymethyl ether into a 50 mL single-necked round-bottom flask, then add an appropriate amount of SOCl2, heat at 80 °C under reflux for 2 h to remove the solvent and obtain compound I. The amount of compound I prepared and the volume of the reaction vessel are increased or decreased proportionally. B. Preparation method of compound II 1.306 mmol of 3-aminosulfonylpropionic acid was added to methanol in a 50 mL round-bottom flask and stirred until homogeneous. SOCl2 was slowly added dropwise under ice bath conditions. After the addition was complete, the mixture was stirred at room temperature for 15 min, and then heated to reflux at 80 °C. The solvent was removed, the mixture was diluted with water, and NaHCO3 was added to adjust the pH to 7-8. After extraction with ethyl acetate, the organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The mixture was then concentrated by vacuum filtration to remove excess solvent, yielding a brown oily compound II. C. Preparation method of compound III 0.436 mmol of compound I was dissolved in 5 mL of dichloromethane, and 0.5232 mmol of triethylamine was added. Then, 0.530 mmol of compound II was weighed, dissolved in 2 mL of dichloromethane, and slowly added dropwise to the reaction system. The reaction was carried out at room temperature for 3 h. The reaction progress was monitored by TCL. After the reaction was completed, the residue was purified by silica gel column chromatography (100-200 mesh) using petroleum ether:ethyl acetate (boiling range 60-90 °C) in a volume ratio of 1:1-3 to obtain compound III. D. Methods for preparing haptens Take 0.04 mmol of compound III, dissolve it in an appropriate amount of methanol, then add 0.08 mmol of NaOH, heat under reflux at 80 °C for 2 h, concentrate under reduced pressure to remove methanol, add water and ethyl acetate, retain the aqueous phase, adjust the pH to 2-3 with hydrochloric acid, add ethyl acetate, collect the organic phase, dry, and evaporate to dryness. Preparation of flusulfanilamide immunoantigen: The flusulfanilamide hapten was conjugated with thyroglobulin using the carbodiimide method to prepare the immunoantigen. Specifically, the flusulfanilamide hapten was dissolved in dichloromethane, N-hydroxysuccinimide (NHS) was added, and the mixture was stirred at room temperature for 10 min. Then, 1-ethylcarbodiimide hydrochloride (EDC) was added, and the mixture was activated at room temperature for 3-4 h. Thyroglobulin was dissolved in borate buffer. The activated hapten solvent was concentrated under reduced pressure and then dissolved in a small amount of N,N-dimethylformamide. The solution was slowly added dropwise to the thyroglobulin solution, reacted overnight at room temperature, and then dialyzed against phosphate-buffered saline (PBS) for 72 h. The solution was then stored at -20°C for later use. Preparation of flusulfanilamide-coated antigen: Flusulfanilamide was dissolved in dichloromethane, N-hydroxysuccinimide (NHS) was added, and the mixture was stirred at room temperature for 10 min. Then, 1-ethylcarbodiimide hydrochloride (EDC) was added, and the mixture was activated by reacting at room temperature for 3-4 h. The activated flusulfanilamide was then added dropwise to bovine serum albumin dissolved in borate buffer, and the mixture was reacted overnight. After reacting, the mixture was dialyzed against phosphate-buffered saline (PBS) for 72 h and stored at -20°C for later use. Preparation of flusulfanilamide monoclonal antibody: The immunogen was diluted to the required concentration and adjuvant was added to immunize mice. After four immunizations, a cell fusion experiment was performed, and the flusulfanilamide monoclonal antibody was obtained by screening using an indirect competitive enzyme-linked immunosorbent assay.

2. A colloidal gold marker based on flusulfanilamide monoclonal antibody, characterized in that, The preparation method is as follows: the pH value of the colloidal gold solution is adjusted to 8.2, then 5-20 μg of flusulfanilamide monoclonal antibody is added, and the reaction is carried out at room temperature in the dark for 30-60 min. Then, 100 μL of 1%-15% BSA sodium borate buffer is added to block for 20-60 min. After centrifugation, the supernatant is discarded, and the precipitate is resuspended in colloidal gold reconstitution solution. The colloidal gold solution was prepared by the reduction method of trisodium citrate, wherein the mass fraction of tetrachloroauric acid and trisodium citrate was 1%, and the volume ratio used was 1-3 mL: 2-5 mL. The concentration of the blocking solution, BSA sodium borate buffer, is 1% to 15%. The colloidal gold complex solution was prepared as a sodium borate solution containing 2% BSA and 2% sucrose.

3. The colloidal gold label of flusulfanilamide monoclonal antibody according to claim 2, characterized in that, The pH of the colloidal gold solution was 8.2, the amount of flusulfanilamide monoclonal antibody added was 5–20 μg, and the concentration of the blocking buffer BSA sodium borate buffer was 1%–15%.

4. A colloidal gold immunochromatographic test strip for rapid detection of flusulfanilamide, characterized in that, The colloidal gold immunochromatographic test strip consists of a PVC base plate, an NC membrane (with a T-line coated with goat anti-mouse secondary antibody and a C-line coated with a coated antigen), a sample pad, and an absorbent pad.

5. The colloidal gold immunochromatographic test strip according to claim 4, characterized in its application in the rapid detection of flumetsulam residues in soil, involves incubating the extract of the soil sample to be tested with the colloidal gold marker according to claim 2, and then inserting it into the colloidal gold immunochromatographic test strip according to claim 4. The residue status of flumetsulam pesticide is determined based on the color development result of the test strip after 5-8 minutes.

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