CN114276292B - Quinclorac hapten and preparation method and application thereof - Google Patents

Abstract

The invention discloses a quinclorac hapten, a preparation method and application thereof. The quinclorac hapten is synthesized by adopting a secondary chemistry, so that the characteristic structure of quinclorac is reserved to the greatest extent, and a proper connecting arm is introduced to expose the characteristic structure of quinclorac as far as possible. Coupling quinclorac hapten with carrier protein to prepare quinclorac artificial antigen. The monoclonal antibody of quinclorac is obtained by utilizing the artificial antigen, and has high sensitivity and strong specificity. Sensitivity (IC) of quinclorac monoclonal antibodies as determined by indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) ₅₀ ) 1.63ng/mL, linear range (IC ₁₀ ‑IC ₉₀ ) 0.28-10.38 ng/mL, and has no cross reaction with the quinclorac structural analogues pesticide (CR < 0.1%). The colloidal gold test strip prepared based on the quinclorac monoclonal antibody has high sensitivity and strong specificity, has the detection limit of 8ng/mL, and can be used for rapidly detecting quinclorac residues in agricultural products and environmental samples.

Description

Quinclorac hapten and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and in particular relates to a quinclorac hapten, and a preparation method and application thereof

Background

The quinclorac belongs to hormone type quinoline carboxylic acid herbicide, is systemic conduction type selective post-emergence herbicide, and is mainly used for preventing and controlling barnyard grass in paddy fields. The quinclorac is transferred to other parts after being absorbed by roots and leaves of weeds to induce the weeds to synthesize substances such as ethylene, cyanide, abscisic acid and the like, so that the weeds are killed. The quinclorac has long pot life, small dosage and special effect on barnyard grass, and is one of main rice field herbicide varieties in China. The quinclorac is slowly degraded in soil, and is easy to produce phytotoxicity to the sensitive crop of the aftercrop, such as solanaceae, leguminous and umbelliferone crops. In addition, in paddy rice and tobacco rotation areas, the low concentration of quinclorac in the soil can cause tobacco to generate phytotoxicity, thereby bringing great economic loss to farmers. According to national standard GB2763-2021 maximum residue limit of pesticides in food of 9 months and 3 days of 2021 in China, the allowable daily intake of quinclorac is 0.4mg/kg bw, and the maximum residue limits of quinclorac in brown rice, sorghum and cowberry fruit are 1mg/kg, 0.1mg/kg and 1.5mg/kg respectively. Therefore, it is necessary to enhance the detection of quinclorac residue.

The quinclorac detection method mainly comprises an instrument detection method, including gas chromatography, liquid chromatography, gas chromatography and mass spectrometry, and liquid chromatography and mass spectrometry. The instrument detection method has the advantages of high accuracy and good repeatability, but the instrument analysis pretreatment is complex, the organic solvent consumption is large, expensive instrument equipment and professional operators are needed, and the on-site detection of the sample cannot be met. The immunoassay is an analytical method based on antigen-antibody specific reaction, and can realize qualitative and quantitative detection of target analytes. The immunoassay has the advantages of high sensitivity, strong specificity, rapidness, convenience and low price, and can realize the on-site detection of the sample and the instrument detection as a complementary detection method. Antibodies are the core reagents for immunoassays, while obtaining high quality antibodies is largely dependent on rational hapten design. Therefore, reasonably designing and synthesizing the quinclorac hapten is a key point for establishing a quick, simple, sensitive and accurate immunoassay method.

Currently, quinclorac immunoassay methods have been reported. Zhu Guonian (patent No. CN 200410018337.1) reacting quinclorac with thionyl chloride to prepare quinclorac acylate, and reacting the acylate with aminobutyric acid and aminocaproic acid to obtain two quinclorac hapten, respectively, to prepare quinclorac polyclonal antibody, the Inhibitory Concentration (IC) of which in enzyme-linked immunosorbent assay (ELISA) ₅₀ ) 2.7 μg/mL; chen Li (patent number: CN 201610151290.9) reacting quinclorac with bromoacetyl chloride to obtain quinclorac hapten, and preparing quinclorac monoThe monoclonal antibody is used for developing a quinclorac colloidal gold test strip, and the detection limit is 0.8mg/kg; chen Li (patent number: CN 201811104625.7) reacting quinclorac with (1, 3-dioxacyclohexylethyl) magnesium bromide to give propiolactone and reacting with trifluoroacetic acid to give hapten, to give quinclorac monoclonal antibody, which is used as IC in ELISA ₅₀ The range is 2.3-5.8 ng/mL, but is not used in the test strip. Therefore, there is a need to further improve the structure of quinclorac hapten and establish a sensitive and rapid immunoassay method.

Disclosure of Invention

The invention provides a quinclorac hapten structure and preparation and application thereof.

In the first aspect, the quinclorac hapten structure designed by the invention is as follows.

In a second aspect, the present invention provides a method for preparing the hapten

The hapten preparation steps are as follows:

step 1) adding 2.74g of 2-amino-6-chlorobenzoic acid into a reaction bottle, sequentially adding 1mL of water and 6mL of concentrated sulfuric acid, heating to 140 ℃, blackening the solution, adding 0.23g of sodium iodide, dropwise adding 1.5mL of glycerol, and reacting for 6.5h under heat preservation and stirring. The reaction solution is cooled to room temperature, slowly transferred into 50mL of ice water, the pH value is adjusted to 5 by using 1M sodium hydroxide solution, and the product 1 is obtained after suction filtration and concentration.

Step 2) 2.76g of the product 1, 3.72g of potassium carbonate and 15mL of N, N-dimethylformamide are added into a reaction bottle in sequence, 1.3mL of methyl iodide is added dropwise under stirring at room temperature, and the mixture is introduced into a drying tube and stirred at room temperature for reaction for 12h. After the reaction, the solid residue is removed by suction filtration, the filtrate is poured into a separating funnel, 250mL of saturated sodium bicarbonate solution is added for shaking, standing and layering are carried out, the aqueous phase is extracted 3 times by 20mL of ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate, and the crude product is obtained by concentration. Dissolving the crude product with a small amount of dichloromethane, loading the solution, purifying the solution by a silica gel column, eluting and separating the solution by petroleum ether and ethyl acetate in a volume ratio of 5:1, and concentrating the solution to obtain a product 2.

Step 3) 4.12g of product 2 and 30mL of acetic acid were added to the reaction flask, 7.51g of N-iodosuccinimide was slowly added with stirring at room temperature, the temperature was raised to 50℃and the reaction was stirred for 12 hours. After the reaction, concentrating to remove acetic acid to obtain solid. 10mL of ethyl acetate was added for ultrasonic dissolution, 250mL of saturated sodium bicarbonate was added for stirring, a large amount of bubbles were generated, and after the bubbles were dissipated, the mixture was transferred to a separating funnel and allowed to stand for delamination. The aqueous phase was extracted 3 times with 20mL ethyl acetate and the organic phases were combined. 25mL of a saturated sodium thiosulfate solution was added to the organic phase, the solution turned from mauve to pale yellow, the organic phase was washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated to give a crude product. Dissolving the crude product with a small amount of dichloromethane, loading the solution, purifying the solution by a silica gel column, eluting and separating the solution by petroleum ether and ethyl acetate in a volume ratio of 10:1 to 5:1, and concentrating the solution to obtain a product 3.

Step 4) adding 1.18g of the product 3 into a reaction bottle, adding 20mL of dichloromethane for dissolution, adding 1mL of triethylamine and 0.6mL of 3-butyn-1-ol, rapidly adding 124mg of bis (triphenylphosphine) palladium dichloride and 67mg of cuprous iodide, continuously introducing nitrogen for 3 times, and stirring at 25 ℃ for reaction for 8 hours. After the reaction, the reaction solution was poured into a separating funnel, 250mL of saturated sodium bicarbonate was added, the mixture was shaken, left to stand and separate layers, the aqueous phase was extracted 3 times with 20mL of dichloromethane, the organic phases were combined, the organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. Dissolving the crude product with a small amount of dichloromethane, loading the solution, purifying the solution by a silica gel column, eluting and separating the solution by petroleum ether and ethyl acetate in a volume ratio of 1:1, and concentrating the solution to obtain a product 4.

Step 5) adding 58mg of the product 4, 22mg of Pd/C (water content 55%, pd content 10%) and 2mL of methanol into a reaction bottle respectively, purging with nitrogen for 3 times, introducing hydrogen under the protection of nitrogen, and stirring at 25 ℃ for reaction for 10 hours. After the reaction is finished, the reaction solution is filtered, and the filtrate is concentrated to obtain a crude product. Dissolving the crude product with a small amount of dichloromethane, loading the solution, purifying the solution by a silica gel column, eluting and separating the solution by using dichloromethane and ethyl acetate with the volume ratio of 1:1, and concentrating the solution to obtain a product 5.

Step 6) 25mg of the product 5, 0.5mL of methanol, 0.5mL of 1M sodium hydroxide solution were added to each reaction flask, and the reaction was stirred at 60℃for 12 hours. After the reaction is finished, the reaction solution is concentrated to obtain a crude product. Dissolving the crude product with a small amount of dichloromethane, loading the solution, purifying the solution by a silica gel column, eluting and separating the solution by using dichloromethane and methanol with the volume ratio of 20:1, and concentrating the solution to obtain the quinclorac hapten.

In a third aspect, the invention provides an artificial antigen of the quinclorac hapten, wherein the artificial antigen is obtained by coupling hapten and carrier protein through activated hydroxyl. The quinclorac artificial antigen is divided into an immune antigen and a coating antigen, wherein one quinclorac immune antigen is obtained by coupling quinclorac hapten with Bovine Serum Albumin (BSA), and one quinclorac coating antigen is obtained by coupling quinclorac hapten with chicken Ovalbumin (OVA) respectively.

Step 1) 10mg of quinclorac hapten, 54mg of N, N' -disuccinimide carbonate and 26mg of 4-dimethylaminopyridine are dissolved in 2mM N, N-dimethylformamide and stirred at room temperature for reaction for 6h in the absence of light.

Step 2) centrifuging the reaction solution obtained in the step 1) at 8000rpm for 10min, taking half of the supernatant, slowly dripping the half of the supernatant into 4mL of phosphate buffer solution (0.14M, pH =7.4) containing 40mg of Bovine Serum Albumin (BSA), and stirring and reacting at room temperature for 12h in a dark place to prepare the quinclorac immune antigen; the remaining supernatant was slowly added dropwise to 3mL of a phosphate buffer solution (0.14M, pH =7.4) containing 30mg of chicken Ovalbumin (OVA), and the reaction was stirred at room temperature for 12 hours under light-shielding conditions to prepare a quinclorac-coated antigen.

And 3) respectively filling the quinclorac immune antigen and the coating antigen obtained in the step 2) into a dialysis bag, dialyzing and removing impurities by using a phosphate buffer solution (0.14M, pH =7.4), replacing the dialysate every 6 hours, taking out after 3 days of dialysis, and sub-packaging and storing at the temperature of-20 ℃.

In a fourth aspect, the invention provides a quinclorac monoclonal antibody, which is prepared from the quinclorac artificial antigen and can specifically recognize quinclorac or quinclorac hapten and artificial antigen.

In a fifth aspect, the invention provides a colloidal gold test strip for detecting quinclorac residues in agricultural products and environmental samples.

The invention has the following beneficial effects:

the quinclorac hapten provided by the invention not only maintains the characteristic structure of quinclorac to the greatest extent, but also introduces a connecting arm with a proper structure, so that the quinclorac characteristic structure is fully exposed to the organism as much as possible, and the immune effect is enhanced; the quinclorac hapten provided by the invention has hydroxyl at the connecting arm, which is beneficial to preparing quinclorac artificial antigen; the quinclorac artificial antigen provided by the invention has strong immunogenicity, and is favorable for stimulating an organism to complete immune response, so that a high-quality monoclonal antibody is obtained, and a core reagent is provided for establishing a quinclorac immune analysis method.

The quinclorac monoclonal antibody obtained by adopting the quinclorac artificial antigen provided by the invention has better potency, specificity and affinity. mid-Inhibitory Concentration (IC) of quinclorac monoclonal antibodies as determined by competitive enzyme-linked immunosorbent assay (IC-ELISA) ₅₀ ) 1.63ng/mL, linear range (IC ₁₀ -IC ₉₀ ) The cross reaction with the quinclorac structural analogue is less than 0.1% at 0.28-10.38 ng/mL, and the sensitivity and the specificity of the antibody are superior to those of the reported researches. The minimum detection limit of the colloidal gold test strip obtained by adopting the quinclorac monoclonal antibody provided by the research is 8ng/mL, and compared with the reported minimum detection limit, the colloidal gold test strip is improved by 100 times. Therefore, the quinclorac hapten and the artificial antigen provided by the invention are more beneficial to obtaining the quinclorac monoclonal antibody with high sensitivity and strong specificity, and the ELISA method and the colloidal gold test strip developed based on the monoclonal antibody also have better sensitivity.

Drawings

FIG. 1 shows the route of quinclorac hapten synthesis.

FIG. 2 is a standard curve of quinclorac monoclonal antibody ic-ELISA.

FIG. 3 is a top view of a quinclorac colloidal gold test strip.

FIG. 4 is a side view of a quinclorac colloidal gold test strip; in the figure, 1: lining board, 2: sample pad, 3: gold mark binding pad, 4: cellulose film, 5: invisible detection line, 6: stealth control line, 7: absorbent pad, 8-1: sample immersion end protection film, 8-2: handle end protection film, 9: and (5) identifying lines.

FIG. 5 is a graph showing the judgment of the results of a quinclorac colloidal gold test strip; in the figure, A, B indicates that the detection result is negative, C, D indicates that the detection result is positive, and E, F indicates that the detection result is ineffective.

Detailed Description

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention, in conjunction with the accompanying drawings. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1: synthesis of quinclorac hapten is shown in figure 1.

Step 1) adding 2.74g of 2-amino-6-chlorobenzoic acid into a reaction bottle, sequentially adding 1mL of water and 6mL of concentrated sulfuric acid, heating to 140 ℃, blackening the solution, adding 0.23g of sodium iodide, dropwise adding 1.5mL of glycerol, and reacting for 6.5h under heat preservation and stirring. The reaction solution is cooled to room temperature, slowly transferred into 50mL of ice water, the pH value is adjusted to 5 by using 1M sodium hydroxide solution, and the product 1 is obtained after suction filtration and concentration.

Step 2) 2.76g of the product 1, 3.72g of potassium carbonate and 15mL of N, N-dimethylformamide are added into a reaction bottle in sequence, 1.3mL of methyl iodide is added dropwise under stirring at room temperature, and the mixture is introduced into a drying tube and stirred at room temperature for reaction for 12h. After the reaction, the solid residue is removed by suction filtration, the filtrate is poured into a separating funnel, 250mL of saturated sodium bicarbonate solution is added for shaking, standing and layering are carried out, the aqueous phase is extracted 3 times by 20mL of ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate, and the crude product is obtained by concentration. Dissolving the crude product with a small amount of dichloromethane, loading the solution, purifying the solution by a silica gel column, eluting and separating the solution by petroleum ether and ethyl acetate in a volume ratio of 5:1, and concentrating the solution to obtain a product 2.

Step 3) 4.12g of product 2 and 30mL of acetic acid were added to the reaction flask, 7.51g N-iodosuccinimide was slowly added with stirring at room temperature, the temperature was raised to 50℃and the reaction was stirred for 12 hours. After the reaction, concentrating to remove acetic acid to obtain solid. 10mL of ethyl acetate was added for ultrasonic dissolution, 250mL of saturated sodium bicarbonate was added for stirring, a large amount of bubbles were generated, and after the bubbles were dissipated, the mixture was transferred to a separating funnel and allowed to stand for delamination. The aqueous phase was extracted 3 times with 20mL ethyl acetate and the organic phases were combined. 25mL of a saturated sodium thiosulfate solution was added to the organic phase, the solution turned from mauve to pale yellow, the organic phase was washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated to give a crude product. Dissolving the crude product with a small amount of dichloromethane, loading the solution, purifying the solution by a silica gel column, eluting and separating the solution by petroleum ether and ethyl acetate in a volume ratio of 10:1 to 5:1, and concentrating the solution to obtain a product 3.

Step 4) adding 1.18g of the product 3 into a reaction bottle, adding 20mL of dichloromethane for dissolution, adding 1mL of triethylamine and 0.6mL of 3-butyn-1-ol, rapidly adding 124mg of bis (triphenylphosphine) palladium dichloride and 67mg of cuprous iodide, continuously introducing nitrogen for 3 times, and stirring at 25 ℃ for reaction for 8 hours. After the reaction, the reaction solution was poured into a separating funnel, 250mL of saturated sodium bicarbonate was added, the mixture was shaken, left to stand and separate layers, the aqueous phase was extracted 3 times with 20mL of dichloromethane, the organic phases were combined, the organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. Dissolving the crude product with a small amount of dichloromethane, loading the solution, purifying the solution by a silica gel column, eluting and separating the solution by petroleum ether and ethyl acetate in a volume ratio of 1:1, and concentrating the solution to obtain a product 4.

Step 5) adding 58mg of the product 4, 22mg of Pd/C (water content 55%, pd content 10%) and 2mL of methanol into a reaction bottle respectively, purging with nitrogen for 3 times, introducing hydrogen under the protection of nitrogen, and stirring at 25 ℃ for reaction for 10 hours. After the reaction is finished, the reaction solution is filtered, and the filtrate is concentrated to obtain a crude product. Dissolving the crude product with a small amount of dichloromethane, loading the solution, purifying the solution by a silica gel column, eluting and separating the solution by using dichloromethane and ethyl acetate with the volume ratio of 1:1, and concentrating the solution to obtain a product 5.

Step 6) 25mg of the product 5, 0.5mL of methanol, 0.5mL of 1M sodium hydroxide solution were added to each reaction flask, and the reaction was stirred at 60℃for 12 hours. After the reaction is finished, the reaction solution is concentrated to obtain a crude product. Dissolving the crude product with a small amount of dichloromethane, loading the solution, purifying the solution by a silica gel column, eluting and separating the solution by using dichloromethane and methanol with the volume ratio of 20:1, and concentrating the solution to obtain the quinclorac hapten.

Example 2: identification of quinclorac hapten

Results of the quinclorac hapten nuclear magnetic identification: 1H NMR (500 MHz, DMSO-d 6) delta 8.87 (d, J=2.2 Hz, 1H), 8.25 (d, J=2.2 Hz, 1H), 8.02 (d, J=8.9 Hz, 1H), 7.67 (d, J=8.8 Hz, 1H), 4.41 (s, 1H), 3.43 (t, J=6.1 Hz, 2H), 2.81 (t, J=7.6 Hz, 2H), 1.71 (p, J=7.6 Hz, 2H), 1.47 (dt, J=13.7, 6.5Hz, 2H).

Example 3: preparation of quinclorac artificial antigen

Step 1) 10mg of quinclorac hapten, 54mg of N, N' -disuccinimide carbonate and 26mg of 4-dimethylaminopyridine are dissolved in 2mM N, N-dimethylformamide and stirred at room temperature for reaction for 6h in the absence of light.

Step 2) centrifuging the reaction solution obtained in the step 1) at 8000rpm for 10min, taking half of the supernatant, slowly dripping the half of the supernatant into 4mL of phosphate buffer solution (0.14M, pH =7.4) containing 40mg of Bovine Serum Albumin (BSA), and stirring and reacting at room temperature for 12h in a dark place to prepare the quinclorac immune antigen; the remaining supernatant was slowly added dropwise to 3mL of a phosphate buffer solution (0.14M, pH =7.4) containing 30mg of chicken Ovalbumin (OVA), and the reaction was stirred at room temperature for 12 hours under light-shielding conditions to prepare a quinclorac-coated antigen.

And 3) respectively filling the quinclorac immune antigen and the coating antigen obtained in the step 2) into a dialysis bag, dialyzing and removing impurities by using a phosphate buffer solution (0.14M, pH =7.4), replacing the dialysate every 6 hours, taking out after 3 days of dialysis, and sub-packaging and storing at the temperature of-20 ℃.

Example 4: quinclorac artificial antigen immune mouse

Five 6-8 week old BALB/c females were immunized with quinclorac immunization antigen. Fully emulsifying quinclorac immune antigen and an adjuvant according to the volume dosage of 1:1, and performing immunization by adopting an intraperitoneal injection mode, wherein the injection dosage is 100 mug/dose, and the immunization is performed for five times at two-week intervals. First immunization was with Freund's complete adjuvant, and the remaining four times with Freund's incomplete adjuvant. Three days prior to cell fusion, five immunized mice were subjected to sprint immunization without adjuvant at an injection dose of 100 μg/mouse.

Example 5: cell fusion

Step 1) obtaining mouse myeloma cells: three bottles of mouse myeloma cells (SP 2/0) in logarithmic growth phase were selected, SP2/0 was blown down with DMEM medium preheated at 37℃and transferred to a 50mL centrifuge tube to a volume of 35mL.

Step 2) obtaining mouse spleen cells: three days in advance, the eyeballs of the mice after sprinting and immunization are removed, the necks of the mice are killed, the spleens of the mice are removed by medical ophthalmic forceps and scissors, fat and connective tissues are removed, the mice are transferred into a 70 mu m cell screen, the spleens are broken by a sterile syringe needle, spleen cells are released into DMEM solution, and the spleen cell solution is transferred into a 50mL centrifuge tube to be fixed to 35mL.

Step 3) cell fusion: SP2/0 was centrifuged (1000 rpm,10 min) with mouse spleen cells, the supernatant was discarded, resuspended in DMEM medium and counted. Mixing SP2/0 and spleen cells at a ratio of 1:10, centrifuging (1000 rpm,10 min), discarding supernatant, loosening cell pellet, and placing in a constant temperature water bath at 37deg.C. 1mL PEG1500 to 1mL sterile syringe was pipetted into the centrifuge tube at a slow-first-fast speed over 1min, allowed to stand for 1min, 30mL DMEM broth was added to the centrifuge tube at a slow-first-fast speed, PEG action stopped, allowed to stand for 10min, centrifuged (800 rpm,8 min), and the supernatant discarded. The bottom cells were gently resuspended with the appropriate amount of HAT medium, added with the appropriate amount of interleukins and antibiotics, mixed well and added drop-wise to 96 well cell culture plates and placed in carbon dioxide incubator for culture.

Example 6: cell strain selection

When the cell mass grows to more than one fourth of the cell hole area, taking a proper amount of cell supernatant, measuring the titer and the inhibition rate of the cell supernatant by adopting an ELISA method, selecting a cell hole with high inhibition rate, and subcloning cells in the hole by adopting a limiting dilution method until a monoclonal cell strain capable of stably secreting the quinclorac monoclonal antibody can be obtained.

Example 7: antibody preparation

Monoclonal antibodies are prepared by culturing target hybridoma cells in vivo. Sterile paraffin oil was intraperitoneally injected into mice at 500 μl/mouse. Proper amount of (2X 10) is added within 7-14 days after injecting paraffin oil ⁶ Individual cells/mouse) hybridoma cells were injected into mice, cultured for about 7 days, and a significant increase in the abdomen was observed, and ascites was extracted using a sterilized 16-gauge needle. After the ascites was centrifuged (8000 rpm,10 min), the supernatant was collected, and the collected supernatant was purified by using a Protein A immunochromatographic column to obtain a quinclorac monoclonal antibody.

Example 8: antibody specificity evaluation

Determination of quinclorac by indirect competition ELISA methodIC of pesticide similar to its structure ₅₀ According to formula CR (cross-reactivity) =ic ₅₀ (quinclorac)/IC ₅₀ (its structural analogue pesticide). Times.100% calculate the cross-reactivity. The results show that the cross reaction rate of the quinclorac monoclonal antibody to the structural analogue pesticide is less than 0.1%. Therefore, the quinclorac monoclonal antibody has strong specificity.

Example 9: preparation of quinclorac ELISA standard curve

(1) Coating: diluting the coated antigen to a target concentration with carbonate buffer CBS (0. M, pH =9.6), adding to 96-well elisa plate, 100 μl/well, coating overnight at 4 ℃;

(2) Washing the plate: the liquid in the well was dried by beating, and washed five times with pH7.4 containing 0.05% Tween20 and 0.14M Phosphate Buffer Solution (PBST), and the absorbent paper was dried by beating;

(3) Closing: blocking was performed using 1% gelatin in pH7.4, 0.14M Phosphate Buffered Saline (PBS), 200. Mu.L/well, and incubated at 37℃for 2h;

(4) Washing the plate: and (2);

(5) Adding primary antibody: diluting a quinclorac standard substance to a series of concentrations of 50 mu L/hole, diluting an antibody to a certain concentration of 50 mu L/hole by using PBS, uniformly mixing, adding 100 mu LPBS as a negative control, adding 50 mu L of diluted antibody and 50 mu L of PBS as a positive control, and incubating for 1h at 37 ℃;

(6) Washing the plate: and (2);

(7) Adding a secondary antibody: diluting horseradish peroxidase-goat anti-mouse IgG with PBS 20000 times, 100 μL/well, and incubating at 37deg.C for 1h;

(8) Washing the plate: and (2);

(9) Color development: preparation of a color developing solution (25 mL of a pH 5.50.1M citric acid buffer solution was added with 0.4mL of a 6mg/mL tetramethylbenzidine DMSO solution, 0.1mL of 1% H) ₂ O ₂ ) 100. Mu.L/well, incubation at 37℃for 15min;

(10) And (3) terminating: 50 mu L of stop solution (2M sulfuric acid solution) is added into each hole, and an enzyme-labeled instrument is adopted to read OD ₄₅₀ A value;

(11) And (3) drawing an ELISA standard curve: the concentration of the quinclorac standard solution is taken as the horizontal directionCoordinates, in terms of binding ratio B/B0 (B is OD of corresponding wells of standard solutions of different concentrations) ₄₅₀ Value, B0 is OD of positive control well ₄₅₀ Values) establish a standard curve for the ordinate, as shown in fig. 2. The results indicate that the inhibitory concentration of the monoclonal antibody (IC ₅₀ ) 1.63ng/mL, linear range (IC ₁₀ -IC ₉₀ ) 0.28-10.38 ng/mL.

Example 10: preparation of quinclorac rapid detection test strip

1. Preparation of gold-labeled antibody

Step 1) preparing colloidal gold by adopting a sodium citrate reduction method: 100mL of ultrapure water was taken into a 250mL Erlenmeyer flask, heated to boiling, and 1mL of 1% HAuCl was added ₄ After boiling, 1.8mL of 1% aqueous sodium citrate solution is added rapidly, and when the solution turns red, stirring and heating are continued for 5min, and after cooling, the solution is stored at 4 ℃ in a dark place.

Step 2) labeling quinclorac monoclonal antibody by colloidal gold: the optimal amount of antibody was determined by salt precipitation experiments. With 0.1. 0.1M K ₂ CO ₃ 1mL of the colloidal gold solution was adjusted to pH 8.2, 0.75. Mu.g of antibody was added, the mixture was shaken at room temperature for 20 minutes, 100. Mu.L of 10% BSA solution was added for blocking, the mixture was shaken at room temperature for 10 minutes, centrifuged at 8000rpm for 10 minutes, the supernatant was discarded, and 100. Mu.L of the resuspension (0.01M borate buffer solution containing 3% sucrose, 2% BSA) was used for resuspension, and the mixture was stored at 4℃in the dark.

2. Coating antigen and sheep anti-mouse IgG coated cellulose membrane

And (5) scribing the film by using an XYZ-3000 three-dimensional film spraying instrument. The coated antigen is coated on a detection line (T line), the concentration of the coated antigen is 0.3mg/mL, the goat anti-mouse IgG is coated on a quality control line (C line), the concentration of the goat anti-mouse IgG is 0.2mg/mL, the distance between the two lines is 5mm, and the coated goat anti-mouse IgG is dried in an oven at 37 ℃ for 1h after the film is drawn.

3. Preparation of gold mark binding pad

The conjugate pad was soaked with 4% BSA in PBS for 1h and then dried in an oven at 37℃for 2h. Uniformly spraying the quinclorac gold-labeled antibody on the pretreated bonding pad by using an XYZ-3000 three-dimensional film spraying instrument according to the concentration of 12.5 mu L per cm, and then drying the bonding pad in a drying oven at 37 ℃ for 2 hours for standby.

4. Assembly of test strips

The dried cellulose film is stuck in the middle of the PVC bottom plate, the water absorption pad is stuck at the upper end of the cellulose film and is overlapped with the cellulose film by 1mm, the bonding pad is stuck at the lower end of the cellulose film and is overlapped with the cellulose film by 1mm, and the sample pad is stuck at the lower end of the bonding pad and is overlapped with the bonding pad by 2mm. After assembly, the test plate was cut into 4mm wide strips using a slitter as shown in FIGS. 3 and 4.

Example 11: test of quinclorac rapid detection test strip

1. Sensitivity test of test strip

A series of standard solutions of quinclorac at concentrations (128, 64, 32, 16, 8, 4, 2, 0 ng/mL) were prepared with PBS containing 10% acetonitrile, 100. Mu.L each was added dropwise to the test strip sample pad, and the color development was observed over 8-10 min. The result determination is based on the graph shown in fig. 5. The results show that the standard solutions of quinclorac are positive when the concentrations of the standard solutions of quinclorac are 128, 64, 32, 16 and 8ng/mL respectively, and negative when the concentrations of the standard solutions of quinclorac are 4, 2 and 0ng/mL respectively. Thus, the sensitivity of the quinclorac test strip was 8ng/mL.

2. Specificity test of test strip

The quinclorac structural analogues of pesticides (imidazoquinolinate, ethoxyquin, quizalofop-p-ethyl, bensulfuron-methyl, pendimethalin and atrazine) are diluted into 10000ng/mL standard solution by using PBS containing 10% acetonitrile, 100 mu L of each standard solution is dripped to a test strip sample pad, and the color development result is observed within 8-10 min. The results showed that all test strips were negative. Therefore, the quinclorac test strip and the pesticide with the structural analogue thereof have no cross reaction and strong specificity.

3. Test paper strip for detecting soil sample

Weighing 5g of a soil blank sample into a 50mL centrifuge tube, adding a quinclorac standard substance into the soil to enable the final concentration of the quinclorac in the soil to be 0.07mg/kg, and standing for 2h. 10mL of 50% acetonitrile-0.05% formic acid aqueous solution is added, the mixture is manually shaken up and down for 1min, then 1mL of soil solution is taken, diluted by 2.5 times by PBS, the mixture is uniformly shaken, 100 mu L of the mixture is taken and dripped to a sample pad of a test strip, and a color development result is observed within 8-10 min. The result shows that the test strip is positive in color development, and the rapid detection of quinclorac residue in a soil sample can be satisfied.

4. Test strip stability test

The test strip is placed in an aluminum foil bag and stored at room temperature in vacuum. And taking out the test strip after three months, and measuring the sensitivity of the test strip, wherein the result shows that the sensitivity of the test strip is 8ng/mL, the color development is uniform, the strip is clear, and the stability of the quinclorac test strip is good.

Claims (3)

1. A quinclorac hapten has a chemical structure as follows:

2. the method for preparing the hapten of quinclorac according to claim 1, wherein:

step 1) adding 2.74g of 2-amino-6-chlorobenzoic acid into a reaction bottle, sequentially adding 1mL of water and 6mL of concentrated sulfuric acid, heating to 140 ℃, blackening the solution, adding 0.23g of sodium iodide, dropwise adding 1.5mL of glycerol, carrying out heat preservation and stirring reaction for 6h, cooling the reaction solution to room temperature, slowly transferring to 50mL of ice water, regulating the pH value to 5 by using 1M sodium hydroxide solution, carrying out suction filtration and concentration to obtain a product 1,

step 2) adding 2.76g of product 1, 3.72g of potassium carbonate 15mL of N, N-dimethylformamide into a reaction bottle in sequence, dropwise adding 1.3mL of methyl iodide under stirring at 25 ℃, connecting into a drying pipe, stirring at 25 ℃ for reaction for 12 hours, after the reaction is finished, filtering out the reaction liquid to remove solid residues, pouring filtrate into a separating funnel, adding 250mL of saturated sodium bicarbonate solution for shaking, standing for layering, extracting the water phase with 20mL of ethyl acetate for 3 times, merging the organic phases, washing the organic phases with saturated saline, drying the organic phases with anhydrous sodium sulfate, concentrating to obtain a crude product, dissolving the crude product with a small amount of dichloromethane, loading the crude product into a silica gel column for purification, eluting and separating by adopting petroleum ether and ethyl acetate solution with the volume ratio of 5:1, concentrating to obtain the product 2,

step 3) adding 4.12g of 2 and 30mL of acetic acid into a reaction bottle, slowly adding 7.51-g N-iodosuccinimide under stirring at room temperature, heating to 50 ℃, stirring for reaction for 12 hours, concentrating to remove acetic acid to obtain a solid, adding 10mL of ethyl acetate for ultrasonic dissolution, adding 250mL of saturated sodium bicarbonate for stirring, generating a large amount of bubbles, transferring to a separating funnel after the bubbles dissipate, standing for layering, extracting an aqueous phase with 20mL of ethyl acetate for 3 times, combining an organic phase, adding 25mL of saturated sodium thiosulfate solution into the organic phase, changing the solution from mauve to light yellow, washing the organic phase with saturated saline water, drying anhydrous sodium sulfate, concentrating to obtain a crude product, dissolving the crude product with a small amount of dichloromethane, loading the crude product, purifying by a silica gel column, eluting and separating by using petroleum ether and ethyl acetate solution with the volume ratio of 10:1 to 5:1, concentrating to obtain a product 3,

step 4) adding 1.18g of a product 3 into a reaction bottle, adding 20mL of dichloromethane for dissolution, adding 1mL of triethylamine and 0.6mL of 3-butyn-1-ol, then rapidly adding 124mg of bis (triphenylphosphine) palladium dichloride and 67mg of cuprous iodide, continuously introducing nitrogen for 3 times, stirring at 25 ℃ for reaction for 8 hours, pouring the reaction solution into a separating funnel after the reaction is finished, adding 250mL of saturated sodium bicarbonate for shaking, standing for layering, extracting an aqueous phase with 20mL of dichloromethane for 3 times, merging organic phases, washing the organic phases with saturated saline water, drying with anhydrous sodium sulfate, concentrating to obtain a crude product, dissolving the crude product with a small amount of dichloromethane, loading the crude product, purifying by a silica gel column, eluting and separating by adopting petroleum ether and ethyl acetate solution with the volume ratio of 1:1, concentrating to obtain a product 4,

step 5) adding 58mg of a product 4, 22mg of Pd/C with water content of 55% and Pd content of 10% and 2mL of methanol into a reaction bottle respectively, purging with nitrogen for 3 times, introducing hydrogen under the protection of nitrogen, stirring at 25 ℃ for reaction for 10 hours, filtering the reaction liquid after the reaction is finished, concentrating the filtrate to obtain a crude product, dissolving the crude product with a small amount of dichloromethane, loading the crude product into a silica gel column for purification, eluting and separating with a dichloromethane/ethyl acetate solution with volume ratio of 1:1, concentrating to obtain a product 5,

step 6) respectively adding 25mg of product 5, 0.5mL of methanol and 0.5mL of 1M sodium hydroxide solution into a reaction bottle, stirring at 60 ℃ for reaction for 12 hours, concentrating the reaction solution after the reaction is finished to obtain a crude product, dissolving the crude product with a small amount of dichloromethane, loading the solution into a silica gel column for purification, eluting and separating the solution by adopting dichloromethane and methanol solution with the volume ratio of 20:1, concentrating the solution to obtain the quinclorac hapten,

3. a quinclorac artificial antigen obtained by coupling a quinclorac hapten with a carrier protein according to claim 1.

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