Triazolone haptens, artificial antigens and uses thereof

Abstract

The application discloses a triazolone hapten, an artificial antigen and application thereof. The triazolone hapten provided by the application not only completely retains all groups of the triazolone, but also directly connects with two conjugated double bonds through a coupling arm, so that the small molecule structure on the prepared triazolone artificial antigen is more prominent, and the structure characteristics of the triazolone are more conducive to being presented, and the immunogenicity of the antigen is improved. The triazolone artificial antigen and the monoclonal antibody provided by the application are used for ELISA detection, and the specificity is high, the IC 50 value is 4.62 ug / L. The colloidal gold immunochromatography technology established by using the triazolone artificial antigen and the monoclonal antibody provided by the application has high sensitivity, and the detection lower limit of a triazolone standard sample can reach 10 ug / kg, and the detection lower limit in a fruit and vegetable sample can reach 50 ug / kg, so that qualitative detection of the triazolone can be rapidly and conveniently realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological chemical industry, in particular to a triazolone hapten, artificial antigen and application thereof. BACKGROUND

[0002] Triazolone is a triazole fungicide with high efficiency, low toxicity, low residue, long persistence and strong systemicity. After being absorbed by various parts of plants, it can be conducted in the plant body and has the effects of prevention, eradication and treatment on rust and powdery mildew. It is effective on diseases of various crops such as corn blight, wheat cloud pattern disease, wheat leaf blight, pineapple black rot, corn silk head smut, etc. Due to its good fungicidal effect, triazolone is widely used in various fruits and vegetables. The maximum residue limit (MRL) of triazolone in various foods is stipulated in GB 2763-2016 Maximum Residue Limits of Pesticides in Foods, which is 0.2 mg / kg in cereals (except wheat) and dry grains (except corn), 1 mg / kg in solanaceous vegetables, and 1 mg / kg in citrus, apples and bananas.

[0003] In the prior art, the methods for detecting triazolone mainly include instrumental methods and immunological analysis detection techniques. However, instrumental methods are expensive, time-consuming and require professional operation, which cannot realize on-site detection and rapid inspection in a true sense, bringing great inconvenience to daily detection work. Moreover, the artificial antigens prepared directly based on triazolone have the defects of poor sensitivity and low cross-reactivity in the existing immunological analysis detection techniques, which cannot meet the actual use requirements of the market. Therefore, it is urgent to develop triazolone haptens or artificial antigens with high specificity and establish a rapid, high-sensitivity, simple and low-cost detection method for triazolone. SUMMARY

[0004] In order to solve the problem of lack of rapid, high-sensitivity, simple and low-cost triazolone immunodetection methods in the prior art, the present application provides a triazolone hapten, artificial antigen and application thereof.

[0005] The first object of the present application is to provide a triazolone hapten.

[0006] The second object of the present application is to provide a preparation method of a compound with a structural formula as shown in formula (I),

[0007]

[0008] The third object of the present application is to provide the application of a compound with a structural formula as shown in formula (I) in the preparation of a triazolone antigen,

[0009]

[0010] The fourth object of the present application is to provide a triazolinone artificial antigen.

[0011] The fifth object of the present application is to provide a preparation method of the above-mentioned triazolinone artificial antigen.

[0012] The sixth object of the present application is to provide an application of the compound with the structural formula as shown in formula (II) in the preparation of a triazolinone artificial antibody,

[0013]

[0014] In the formula, Protein is a carrier protein, and the carrier protein is bovine serum albumin or hemocyanin.

[0015] The seventh object of the present application is to provide a triazolinone artificial antigen combination.

[0016] The eighth object of the present application is to provide an application of the above-mentioned triazolinone artificial antigen combination in the preparation of a kit for detecting triazolinone.

[0017] The ninth object of the present application is to provide a kit for detecting triazolinone.

[0018] In order to achieve the above-mentioned objects, the present application is realized by the following scheme:

[0019] The detection effect of the immunoassay detection technology is determined by the performance of the antigen and the antibody, and the key of the antigen and the antibody is the hapten, so the structural design of the hapten is particularly important to obtain the antigen and the antibody with excellent performance. The prepared hapten of the present application not only retains all groups of triazolinone completely, but also the designed coupling arm is directly connected with two conjugated double bonds, so that the small molecule structure on the prepared artificial antigen is more prominent, which is more conducive to presenting the structural characteristics of triazolinone and improving the immunogenicity of the antigen.

[0020] A triazolinone hapten with a structural formula as shown in formula (I),

[0021]

[0022] The preparation method of the compound with the structural formula as shown in formula (I) is that 1,2,4-triazole-3-carboxylic acid methyl ester, chloro ether ketone, N,N-dimethyl formamide, K2CO3 and n-tetrabutylammonium iodide are fully reacted, and then an intermediate product is obtained through ethyl acetate extraction; the intermediate product is fully hydrolyzed under alkaline conditions, and the obtained hydrolysis product is extracted through dichloromethane, and then the pH is adjusted to be acidic, and then the compound with the structural formula as shown in formula (I) is obtained.

[0023]

[0024] The structural formula of the 1,2,4-triazole-3-carboxylic acid methyl ester is:

[0025] The structural formula of the chloro ether ketone is:

[0026] The structural formula of the intermediate product is:

[0027] Preferably, the molar ratio of the 1,2,4-triazole-3-carboxylic acid methyl ester, the chloro ether ketone and K2CO3 is 1:1.5-2.5:1.5-2.5.

[0028] More preferably, the molar ratio of the 1,2,4-triazole-3-carboxylic acid methyl ester, the chloro ether ketone and K2CO3 is 1:2:2.

[0029] The n-tetrabutylammonium iodide is a catalyst. Preferably, the amount of the n-tetrabutylammonium iodide is 6%-8% of the total mass of the reaction system.

[0030] More preferably, the amount of the n-tetrabutylammonium iodide is 7% of the total mass of the reaction system.

[0031] Specifically, the preparation method comprises the following steps:

[0032] Take 2.0g of 1,2,4-triazole-3-carboxylic acid methyl ester, (15.74mmol, CAS:4928-88-5) in a 100mL round-bottom flask, and then sequentially add 20mL of N,N-dimethylformamide and 8.22g of chloro ether ketone (31.47mmol, CAS:57000-78-9). After stirring and dissolving, sequentially add 4.35g of K2CO3 (31.47mmol) and 0.20g of n-tetrabutylammonium iodide. The obtained reaction mixture is reacted at room temperature for 48h-56h. After the reaction is completed, 100mL of purified water is added to the reaction product, extracted with ethyl acetate twice, and then the solvent is removed under reduced pressure to obtain the intermediate product crude product. The intermediate product crude product is dissolved in 5mL of methanol, and then 20mL of 6mol / L lithium hydroxide aqueous solution is added. The obtained reaction mixture is reacted at room temperature for 15-20h, 100mL of saturated sodium chloride aqueous solution is added, and then extracted with 30mL of dichloromethane twice. The aqueous phase is collected, and then the pH value is adjusted to 4-5 with 4M hydrochloric acid. The solid is precipitated, filtered, and dried to obtain 0.94g of triazole ketone hapten.

[0033] The use of the compound with the structural formula as shown in formula (I) in the preparation of a triazole ketone artificial antigen should also be within the protection scope of the present application,

[0034]

[0035] A triazolone artificial antigen, which is obtained by coupling a carrier protein to the triazolone hapten, and has a structural formula as shown in formula (II),

[0036]

[0037] In the formula, Protein is a carrier protein, and the carrier protein is bovine serum albumin or hemocyanin.

[0038] A preparation method of the triazolone artificial antigen, which comprises the following steps:

[0039] Preferably, the active ester method comprises the following steps:

[0040] The triazolone hapten, dimethylformamide, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide are fully reacted to obtain solution A; the carrier protein is dissolved in a phosphate buffer solution to obtain solution B; after solution A and solution B are fully reacted, dialysis is performed, and the triazolone artificial antigen is obtained.

[0041] Specifically, the preparation method of the triazolone artificial antigen with the carrier protein being bovine serum albumin is as follows:

[0042] 10 mg of the triazolone hapten is dissolved in 0.2 mL of dimethylformamide, and after being fully stirred, 5 mg of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide and 5 mg of N-hydroxysuccinimide are added, and stirring is performed at room temperature for 4 h to obtain hapten active ester 1; 45 mg of bovine serum albumin is fully dissolved in 4.5 mL of a 0.01 mol / L PBS solution to obtain carrier protein solution 1; the hapten active ester 1 obtained in the previous step is slowly added dropwise to the carrier protein solution 1 while stirring, and stirring is performed at room temperature for 16 h to 24 h; the reaction solution obtained in the previous step is dialyzed against a 0.01 mol / L PBS solution, and dialysis is performed at room temperature for 3 days, and the dialysis solution is replaced three times per day to remove unreacted small molecules, and the triazolone artificial antigen is obtained.

[0043] Specifically, the preparation method of the triazolone artificial antigen with the carrier protein being hemocyanin is as follows:

[0044] Take 10 mg of the above triazole ketone hapten, dissolve in 0.2 mL of dimethylformamide, stir thoroughly, then add 10 mg of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide and 10 mg of N-hydroxysuccinimide, stir at room temperature for 4 h to obtain hapten activated ester 2; take 45 mg of hemocyanin, dissolve it in 4.5 mL of 0.01 mol / L PBS solution to obtain carrier protein solution 2; slowly add the hapten activated ester 2 obtained in the previous step drop by drop to the carrier protein solution 2 while stirring, and stir at room temperature for 16-24 h; dialyze the reaction solution obtained in the previous step against 0.01 mol / L PBS, dialyze at room temperature for 3 days, and change the dialysate 3 times a day to remove unreacted small molecules, to obtain.

[0045] The use of the compound of formula (II) in the preparation of a triazole ketone antibody should also be within the scope of the present application

[0046]

[0047] Protein is a carrier protein, and the carrier protein is bovine serum albumin or hemocyanin.

[0048] A triazole ketone antibody is prepared by immunizing an animal with the above triazole ketone artificial antigen with hemocyanin as the carrier protein as the immunogen.

[0049] Preferably, the triazole ketone antibody is a monoclonal antibody, and a hybridoma cell is obtained by immunizing an animal with the above triazole ketone artificial antigen with hemocyanin as the carrier protein as the immunogen. The hybridoma cell is cultured and the cells are collected for animal immunization to obtain ascites, which is purified by identification.

[0050] A triazole ketone artificial antigen combination includes an immunogen and a coating agent, the immunogen is obtained by coupling the above triazole ketone hapten with hemocyanin; and the coating agent is obtained by coupling the above triazole ketone hapten with bovine serum albumin.

[0051] The use of the above triazole ketone artificial antigen combination in the preparation of a kit for detecting triazole ketone should also be within the scope of the present application.

[0052] An immunochromatographic test strip for detecting triazole ketone includes a base plate, a sample pad, a reaction membrane and a water absorption pad are sequentially arranged on the base plate, the reaction membrane is a nitrocellulose membrane provided with a detection zone and a quality control zone; the detection zone is coated with a coating agent obtained by coupling the above triazole ketone hapten with hemocyanin, and the quality control zone is coated with IgG. The detection zone is a T line, and the quality control zone is a C line.

[0053] Preferably, the sample pad is soaked in a sample processing solution, which is a 0.1M PB buffer containing 0.3% wt Tween 20, 1% wt sucrose, 0.5% wt BSA and 0.05% wt sodium azide.

[0054] Specifically, the preparation method of the immunochromatographic test strip is as follows:

[0055] Using bovine serum albumin as the carrier protein, a triazolone artificial antigen was prepared and its concentration was adjusted to 0.05 mg / mL to 0.2 mg / mL with coating buffer (0.01 M PBS buffer containing 1% wt sucrose and 0.05% wt sodium azide, pH = 7.6). Mouse IgG was also adjusted to 0.1 mg / mL to 0.5 mg / mL with coating buffer. Using a nitrocellulose membrane as the reaction membrane, the diluted antigen was sprayed onto the T line of the reaction membrane at a membrane volume of 0.8 μL / cm to 1.2 μL / cm, and the diluted mouse IgG was sprayed onto the C line of the reaction membrane. The T line and C line were spaced 2.5 mm apart. The membrane was then placed in an oven at 45°C for 12 h to 16 h to obtain the antigen-coated reaction membrane.

[0056] The cut 30cm×30cm blank sample pads were immersed in the sample preparation solution (0.1M PB buffer containing 0.3%wt Tween 20, 1%wt sucrose, 0.5%wt BSA and 0.05%wt sodium azide) for 5 minutes, then removed and dried at 37℃ for 16 hours to obtain the sample pads.

[0057] Using a PVC board as the base, the above-mentioned antigen-coated reaction membrane is pasted in the middle of the PVC board. The absorbent pad and the above-mentioned sample pad are pasted on both ends of the reaction membrane, with the absorbent pad near the C line and the sample pad near the T line, thus obtaining the test strip. The test strip is cut into test strips with a width of 3mm, and the test strips are loaded into the test strip card, with the sample pad near the sample application hole, thus obtaining the test strip.

[0058] A kit for detecting triazolone comprises an immunochromatographic test strip and an antibody for detecting triazolone; the immunochromatographic test strip includes a base plate, on which a sample pad, a reaction membrane, and an absorbent pad are sequentially overlapped; the reaction membrane is a nitrocellulose membrane having a detection zone and a control zone; the detection zone is coated with a coating antigen obtained by conjugating the above-mentioned triazolone hapten with hemocyanin, and the control zone is coated with IgG.

[0059] Preferably, the sample pad of the immunochromatographic test strip is soaked in a sample processing solution, which is a 0.1M PB buffer containing 0.3% wt Tween 20, 1% wt sucrose, 0.5% wt BSA and 0.05% wt sodium azide.

[0060] Specifically, the preparation method of the immunochromatography test strip is as follows:

[0061] The triazone artificial antigen with bovine serum albumin as the carrier protein is used as the antigen, and the concentration is adjusted to 0.05 mg / mL-0.2 mg / mL with a coating buffer (0.01M PBS buffer containing 1%wt sucrose and 0.05%wt sodium azide, pH=7.6); and the mouse IgG is also adjusted to a concentration of 0.1 mg / mL-0.5 mg / mL with the coating buffer; the nitrocellulose membrane is used as the reaction membrane, and the diluted antigen is sprayed on the T line of the reaction membrane at a membrane liquid amount of 0.8 μL / cm-1.2 μL / cm, and the diluted mouse IgG is sprayed on the C line of the reaction membrane, and the interval between the T line and the C line is 2.5 mm, and the reaction membrane coated with the antigen is placed in a 45°C oven for 12 h-16 h, to obtain the reaction membrane coated with the antigen.

[0062] The cut 30 cm x 30 cm blank sample pad is soaked in a sample processing solution (0.1M PB buffer containing 0.3%wt Tween 20, 1%wt sucrose, 0.5%wt BSA and 0.05%wt sodium azide), and after soaking for 5 min, it is taken out and dried at 37°C for 16 h, to obtain the sample pad.

[0063] The reaction membrane coated with the antigen is pasted in the middle of the PVC plate, and the water absorption pad and the sample pad are pasted at both ends of the reaction membrane, with the water absorption pad adjacent to the C line and the sample pad adjacent to the T line, to obtain the test paper plate; the test paper plate is cut into a test paper strip with a width of 3 mm, and the test paper strip is loaded into the test paper card with the sample pad adjacent to the sample addition hole.

[0064] Preferably, the antibody for detecting triazone is a colloidal gold-labeled antibody.

[0065] More preferably, the antibody is a monoclonal antibody, which is obtained by immunizing animals with the above-mentioned triazone artificial antigen with hemocyanin as the carrier protein as the immunogen, culturing the obtained hybridoma cells, collecting the cells for animal immunization to obtain ascites, and purifying and identifying the ascites.

[0066] Further preferably, the colloidal gold-labeled antibody is obtained by fully reacting the above-mentioned monoclonal antibody with colloidal gold at a mass-volume ratio of (3-10 μg):1 mL.

[0067] More preferably, the colloidal gold-labeled antibody is obtained by fully reacting the above-mentioned monoclonal antibody with colloidal gold at a mass-volume ratio of 5 μg:1 mL.

[0068] Specifically, the preparation method of the colloidal gold-labeled antibody is as follows:

[0069] Take 1 g of chloroauric acid, dissolve with pure water and ultrasonic, and then dilute to 100 mL to obtain a chloroauric acid solution, which is stored at 4°C in the dark. Take 1 mL of the colloidal gold solution and add an appropriate amount of 0.1 mol / L K2CO3 solution (the amount added is the minimum amount required for no discoloration during subsequent labeling). Add 5 μg of the monoclonal antibody to the colloidal gold solution, and react at room temperature for 5 minutes. Then add 10 μL of 10% wt bovine serum albumin for blocking, mix thoroughly, and centrifuge at 12,000 rpm for 10 minutes. Discard the supernatant to obtain the colloidal gold antibody.

[0070] Take 1 mL of the colloidal gold solution, add an appropriate amount of 0.1 mol / L K2CO3 solution (the amount added is the minimum amount required for no discoloration during subsequent labeling), and add 5 μg of the monoclonal antibody to the colloidal gold solution. React at room temperature for 5 minutes, then add 10 μL of 10% wt bovine serum albumin for blocking. Mix thoroughly, centrifuge at 12,000 rpm for 10 minutes, and discard the supernatant to obtain the colloidal gold antibody.

[0071] A method for detecting triazolone for non-disease treatment and diagnosis purposes, which uses any of the above-mentioned kits to detect the sample to be tested.

[0072] Preferably, the method comprises the following steps:

[0073] After the sample to be tested is fully reacted with the colloidal gold antibody, it is fully contacted with the sample pad of the immunochromatography test strip. The result is determined according to the color development of the immunochromatography test strip. The specific determination method is as follows:

[0074] If the C line does not develop color, the detection result is invalid. If the C line develops color, and the T line develops color more strongly than the C line or has no obvious difference from the C line, the detection result is negative (-), indicating that there is no triazolone in the sample to be tested. If the C line develops color, and the T line develops color significantly weaker than the C line or does not develop color, the detection result is positive (+), indicating that there is triazolone in the sample to be tested.

[0075] Preferably, the sample to be tested is obtained by crushing, extracting, and solid-liquid separation.

[0076] More preferably, the crushing method comprises cutting into fragments of 1 cm square.

[0077] More preferably, the extraction method comprises thoroughly shaking and mixing the crushed sample with 2.5 times the volume of diluent.

[0078] More preferably, the solid-liquid separation method comprises standing.

[0079] Further preferably, the solid-liquid separation method further comprises filtration or centrifugation.

[0080] Still further preferably, the centrifugation is 4000 revolutions for 3 minutes.

[0081] Still further preferably, the filtration is filter paper filtration.

[0082] Compared with the prior art, the present application has the following beneficial effects:

[0083] The triazolinone hapten provided by the present application not only retains all groups of triazolinone, but also has a coupling arm directly connected to two conjugated double bonds, so that the small molecule structure on the prepared triazolinone artificial antigen is more prominent, and the structure characteristics of triazolinone are more conducive to being presented, thereby improving the immunogenicity of the antigen. 50 The ELISA detection using the triazolinone artificial antigen and the monoclonal antibody provided by the present application has high specificity, and the IC50 value is 4.62 μg / L.The colloidal gold immunochromatography technology established using the triazolinone artificial antigen and the monoclonal antibody provided by the present application has high sensitivity, and the detection lower limit of the triazolinone standard sample is 10 μg / kg, and the detection lower limit in the fruit and vegetable sample is 50 μg / kg, so that the qualitative detection of triazolinone can be quickly and conveniently realized. BRIEF DESCRIPTION OF DRAWINGS

[0084] Figure 1 The synthesis route of the triazolinone hapten is shown in the following figure.

[0085] Figure 2 The mass spectrum result of the triazolinone hapten is shown in the following figure.

[0086] Figure 3 The synthesis route of the triazolinone artificial antigen is shown in the following figure.

[0087] Figure 4 The ultraviolet scanning identification result of the triazolinone artificial antigen is shown in the following figure.

[0088] Figure 5 The standard curve of the triazolinone standard sample solution is shown in the following figure.

[0089] Figure 6 The detection result interpretation diagram of the colloidal gold qualitative immunochromatography kit is shown in the following figure. DETAILED DESCRIPTION

[0090] The present application will be further described in detail below in combination with the drawings and specific examples in the specification. The examples are only used to explain the present application, and are not used to limit the scope of the present application. The test methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

[0091] Example 1: Synthesis and identification of triazolinone hapten

[0092] 1. Synthesis of triazolinone hapten

[0093] The synthesis route of the triazolinone hapten of the present application is shown in the following figure, and the specific steps are as follows: Figure 1

[0094] (1) Take 2.0 g of compound b (i.e. 1,2,4-triazole-3-carboxylic acid methyl ester, CAS: 4928-88-5, 15.74 mmol) in a 100 mL round-bottom flask, and then sequentially add 20 mL of N,N-dimethylformamide (DMF) and 8.22 g of compound a (i.e. chloro ether ketone, CAS: 57000-78-9, 31.47 mmol), stir to dissolve, and then sequentially add 4.35 g of K2CO3 (31.47 mmol) and 0.20 g of n-tetrabutylammonium iodide, and the obtained reaction mixture is reacted at room temperature for 48-56 h. After the reaction is completed, 100 mL of purified water is added to the reaction product, extracted twice with ethyl acetate, and then the solvent is removed under reduced pressure to obtain an intermediate product, i.e. a crude compound c.

[0095] (2) The crude compound c is dissolved in 5 mL of methanol, and then 20 mL of a 6 mol / L lithium hydroxide aqueous solution is added, and the obtained reaction mixture is reacted at room temperature for 15-20 h, 100 mL of a saturated sodium chloride aqueous solution is added, and then extracted twice with 30 mL of dichloromethane, the aqueous phase is collected, and then the pH value is adjusted to 4-5 with 4M hydrochloric acid, a solid is precipitated, filtered, and dried to obtain 0.94 g of a triazolone hapten.

[0096] The structural formula of the triazolone hapten is shown in formula (I):

[0097]

[0098] The mass spectrum result of the triazolone hapten is shown in Figure 2 , and specifically: ESI-MS: 336 [M-1].

[0099] Example 2: Synthesis and identification of a triazolone artificial antigen

[0100] 1. Synthesis of a triazolone artificial antigen

[0101] The structural formula of the triazolone artificial antigen of the present application is shown in formula (II),

[0102]

[0103] , wherein Protein is a carrier protein, and the carrier protein is bovine serum albumin (BSA) or hemocyanin (KLH).

[0104] The synthesis route of the triazolone artificial antigen is shown in Figure 3 , and the specific steps are as follows:

[0105] (1) Triazolone artificial antigen with bovine serum albumin as carrier protein

[0106] Take 10 mg of the triazolone hapten prepared in Example 1, dissolve it in 0.2 mL of dimethylformamide (DMF), after stirring sufficiently, add 5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 5 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h, to obtain hapten activated ester 1.

[0107] Take 45 mg of BSA, dissolve it in 4.5 mL of 0.01 mol / L PBS solution, to obtain carrier protein solution 1; slowly add the hapten activated ester 1 obtained in the previous step drop by drop to the carrier protein solution 1 while stirring, and stir at room temperature for 16-24 h.

[0108] Dialyze the reaction solution obtained in the previous step with 0.01 mol / L PBS, dialyze at room temperature for 3 days, change the dialysate 3 times a day, to remove the unreacted small molecule substances, and the dialysate obtained is the triazolone artificial antigen with BSA as the carrier protein, which is stored at 4°C after being divided into small portions.

[0109] (2) Triazolone artificial antigen with hemocyanin as carrier protein

[0110] Take 10 mg of the triazolone hapten prepared in Example 1, dissolve it in 0.2 mL of dimethylformamide (DMF), after stirring sufficiently, add 5 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 5 mg of N-hydroxysuccinimide (NHS), stir at room temperature for 4 h, to obtain hapten activated ester 1.

[0111] Take 45 mg of BSA, dissolve it in 4.5 mL of 0.01 mol / L PBS solution, to obtain carrier protein solution 1; slowly add the hapten activated ester 1 obtained in the previous step drop by drop to the carrier protein solution 1 while stirring, and stir at room temperature for 16-24 h.

[0112] Dialyze the reaction solution obtained in the previous step with 0.01 mol / L PBS, dialyze at room temperature for 3 days, change the dialysate 3 times a day, to remove the unreacted small molecule substances, and the dialysate obtained is the triazolone artificial antigen with BSA as the carrier protein, which is stored at 4°C after being divided into small portions.

[0113] 2. Identification of triazolone artificial antigen

[0114] Take the above BSA, KLH, triazolone hapten, triazolone artificial antigen with BSA as carrier protein, and triazolone artificial antigen with hemocyanin as carrier protein, and identify them by ultraviolet full wavelength method.

[0115] As Figure 4As shown in the ultraviolet scanning identification results of the triazolinone hapten (hapten), bovine serum albumin (BSA), hemocyanin (KLH), triazolinone hapten-bovine serum albumin (hapten-BSA) and triazolinone hapten-hemocyanin (hapten-KLH), bovine serum albumin (BSA) and hemocyanin (KLH) have typical absorption between 250 nm and 290 nm, and the absorption of triazolinone hapten, triazolinone hapten-bovine serum albumin and triazolinone hapten-hemocyanin conjugates has obvious deviation at 250 nm to 290 nm, which shows that the artificial antigen conjugate has different ultraviolet absorption characteristics from its precursor substance, indicating that the hapten is successfully coupled with the carrier protein.

[0116] Example 3 Preparation of triazolinone monoclonal antibody

[0117] 1. Animal immunization

[0118] The triazolinone artificial antigen prepared in Example 2 with hemocyanin as the carrier protein was used as the immunogen, and was emulsified with an equal volume of Freund's adjuvant to immunize BALB / C mice, with the immunization dose of each mouse being 50 μg to 100 μg. The immunization interval was 2 weeks, and after 3 times of immunization, the tail vein blood of the mice was taken to detect the serum titer. If the antibody titer does not meet the requirements, booster immunization is needed.

[0119] After the antibody titer no longer increases, 100 μg of the whole antigen was used for subcutaneous booster immunization.

[0120] 2. Preparation of hybridoma cells

[0121] Five days after the last booster immunization, the mouse spleen cells were taken and fused with human osteosarcoma SP20 cells. The fused cells were screened in HAT medium, and after 5 days, the complete medium was replaced with HAT medium for culture.

[0122] The cell supernatant was detected by ELISA, and the cells in the wells with strong positive detection results were subjected to limited dilution cloning culture. After 3 times of cloning culture detection, the cells in the wells with positive results were hybridoma cells secreting monoclonal antibodies.

[0123] 3. Preparation of monoclonal antibody

[0124] After the hybridoma cells were amplified and cultured, they were inoculated into the abdominal cavity of mice to produce antibody-containing ascites. The ascites was purified by caprylic acid-ammonium sulfate precipitation to obtain high-purity and high-specificity triazolinone monoclonal antibody.

[0125] Example 4 Performance evaluation of ELISA

[0126] 1. Experimental method

[0127] The performance of the artificial antigen and the monoclonal antibody prepared by the present application is evaluated by using the ELISA method, including the following steps:

[0128] (1) Antigen coating

[0129] The carrier protein of the triazolinone artificial antigen prepared in Example 2 is diluted to 0.2 μg / mL with a carbonate buffer solution at pH 9.6 as a coating diluent, and 100 μL / well is added to a polystyrene microplate, and coating is performed overnight at 4°C, and then the plate is spun dry, and washed with PBST three times.

[0130] (2) Blocking

[0131] 1%wt BSA-containing phosphate buffer solution is added at 280 μL / well, and blocking is performed at 37°C for 1 h, and then the plate is spun dry, and washed with PBST three times, and then dried and stored in a vacuum package.

[0132] (3) Primary antibody dilution

[0133] The triazolinone monoclonal antibody prepared in Example 3 is diluted to 0.04 μg / mL with a phosphate buffer solution (pH 7.4) containing 0.05%wt sodium azide, and stored at 4°C for standby use.

[0134] (4) Preparation of standard solution

[0135] The triazolinone standard is dissolved in 0.01M PBS to obtain triazolinone standard solutions with concentrations of 0 μg / L, 1 μg / L, 3 μg / L, 9 μg / L, 27 μg / L, and 81 μg / L.

[0136] (5) Sample addition and primary antibody incubation

[0137] The triazolinone standard solution is added to the micro-wells of the enzyme-labeled plate coated with the triazolinone artificial antigen at 100 μL / well, and the triazolinone monoclonal antibody solution is added at 20 μL / well, and then reaction is performed at 37°C for 0.5 h, and the plate is spun dry.

[0138] (6) Primary antibody washing

[0139] PBST is added at 280 μL / well, and the plate is washed three times and then spun dry.

[0140] (7) Secondary antibody incubation

[0141] HRP enzyme-labeled goat anti-mouse IgG enzyme-labeled secondary antibody is added at 100 μL / well, and reaction is performed at 37°C for 0.5 h.

[0142] (8) Secondary antibody washing

[0143] PBST is added at 280 μL / well, and the plate is washed three times again and then spun dry.

[0144] (9) Color development

[0145] Add 50 μL / well of color developing solution A and color developing solution B respectively, and react at 37°C for 15 min; add 50 μL / well of 2M sulfuric acid to terminate the color development.

[0146] (10) Absorbance measurement

[0147] Put the microwell enzyme-coated plate obtained in the previous step into an enzyme marker, and set the enzyme marker to measure the OD value of each well at 450 nm wavelength.

[0148] 2. Experimental results

[0149] Table 1. OD values of triazone standard solutions of different concentrations measured by ELISA

[0150]

[0151] The OD value measurement results of triazone standard solutions of different concentrations are shown in Table 1. Through the data shown in Table 1, a standard curve is drawn by using ELISA Calc software for four-parameter Logistic curve fitting, as shown in Figure 5 The linear equation is as follows:

[0152] y = (A-D) / [1 + (x / C) B] + D, r 2 = 0.9998; wherein A = 1.85574, B = 0.81651, C = 4.02831,

[0153] D = 0.09949, x represents the concentration of the measured substance, and y represents the OD value.

[0154] The IC 50 value is 4.62 μg / L, and it is linearly related at 1 μg / L to 81 μg / L.

[0155] Example 5. A colloidal gold qualitative immunochromatographic kit for detecting triazone and effect evaluation

[0156] I. Composition of colloidal gold qualitative immunochromatographic kit

[0157] 1. Immunochromatographic test card

[0158] The preparation method of the immunochromatographic test card is as follows:

[0159] (1) Preparation of reaction film coated with artificial antigen and goat anti-mouse IgG

[0160] The triazolinone artificial antigen prepared by using the carrier protein prepared in Example 2 as bovine serum albumin was used as an antigen, and was adjusted to a concentration of 0.05 mg / mL to 0.2 mg / mL with a coating buffer (0.01 M PBS buffer containing 1% wt sucrose and 0.05% wt sodium azide, pH = 7.6); and the mouse IgG was also adjusted to a concentration of 0.1 mg / mL to 0.5 mg / mL with the coating buffer. The nitrocellulose membrane (NC membrane) was used as a reaction membrane, and the diluted antigen was sprayed onto the detection zone (T line) on the reaction membrane at a membrane liquid amount of 0.8 μL / cm to 1.2 μL / cm, and the diluted mouse IgG was sprayed onto the control zone (C line) on the reaction membrane, and the interval between the detection zone and the control zone was 2.5 mm, and the reaction membrane was placed in a 45°C oven for 12 h to 16 h, and was placed in a constant temperature and humidity preservation box for standby.

[0161] (2) Preparation of sample pad

[0162] The cut 30 cm x 30 cm blank sample pad was soaked in a sample preparation solution (0.1 M PB buffer containing 0.3% wt Tween 20, 1% wt sucrose, 0.5% wt BSA and 0.05% wt sodium azide), and was taken out after soaking for 5 min, and was dried at 37°C for 16 h, and was placed in a constant temperature and humidity preservation box for standby.

[0163] (3) Assembly

[0164] The PVC plate was used as a base plate, the reaction membrane prepared in step 1 was pasted in the middle of the PVC plate, and the water absorption pad and the sample pad prepared in step 2 were pasted at both ends of the reaction membrane, and the water absorption pad was adjacent to the control zone (C line), and the sample pad was adjacent to the detection zone (T line), and thus a test strip was obtained. The test strip was cut into a test strip with a width of 3 mm. The test strip was loaded into a test strip card, and the sample pad was adjacent to the sample hole, and thus a colloidal gold qualitative immunochromatography test strip card was obtained.

[0165] 2. Gold-labeled micropore

[0166] The preparation method of the gold-labeled micropore is as follows:

[0167] (1) Preparation of colloidal gold solution

[0168] 1 g chloroauric acid was dissolved with pure water and ultrasonic to constant volume to 100 mL, and thus a chloroauric acid solution was obtained, which was stored at 4°C and protected from light for standby. 1 mL of the chloroauric acid solution was taken into 100 mL of pure water, heated to boiling, 0.5 mL of 0.06% wt sodium citrate solution was added, and heating was continued for 10 min. After cooling to room temperature, pure water was added to make up to 100 mL, and thus a colloidal gold solution was obtained, which was stored at room temperature and protected from light for standby. All glassware used needed to be soaked with a mixture of potassium permanganate and sulfuric acid overnight, and was cleaned and dried before use.

[0169] (2) Labeling of triazolone monoclonal antibodies

[0170] Take 1 mL of colloidal gold solution, add an appropriate amount of 0.1 mol / L K2CO3 solution (the amount added is the minimum amount required to prevent discoloration in the subsequent labeling process), add 5 μg of the monoclonal antibody to the colloidal gold solution, react at room temperature for 5 minutes, then add 10 μL of 10% wt bovine serum albumin for blocking, mix thoroughly, centrifuge at 12,000 rpm for 10 minutes, discard all the supernatant, and the resulting precipitate is the colloidal gold-labeled triazolone monoclonal antibody.

[0171] (3) Drying of the micropores of the gold label

[0172] The colloidal gold-labeled triazolone monoclonal antibody obtained in the previous step was reconstituted with 1 mL of gold diluent (an aqueous solution containing 2% wt Tris, 5% wt bovine serum albumin, 0.05% wt thimerosal, and 5% wt sucrose). The solution was then aliquoted into microwells at 10 μL / well and dried at 37°C for 16 hours to obtain gold-labeled microwells, which were then stored for later use.

[0173] II. Instructions for use of the colloidal gold qualitative immunochromatographic assay kit

[0174] (1) Sample processing

[0175] Take 2g of fruit and vegetable sample (4g of cross-sectional or epidermal sample for tubers), cut into approximately 1cm square pieces, place in a 50mL centrifuge tube, add 5mL of 0.01M PB, shake vigorously to mix for 2 minutes, let stand for 2 minutes, and collect the supernatant to obtain the sample solution. If turbidity or too many impurities occur after adding diluent, filter with filter paper or centrifuge at 4000 rpm for 3 minutes before collecting the supernatant. Before testing, dilute the sample solution with diluent in different proportions using centrifuge tubes to obtain the test solution.

[0176] (2) Detection

[0177] Add 100 μL of the test solution to the gold-labeled microwell, repeatedly pipet to reconstitute the solution, let it stand for 3 minutes, then transfer the solution from the gold-labeled microwell to the sample well of the immunochromatographic test strip. Start timing after adding the sample, and observe the results after standing for 5 to 8 minutes. Results exceeding 8 minutes are invalid. The test should be performed in triplicate.

[0178] (3) Result Interpretation

[0179] like Figure 6 As shown, the specific interpretation method is as follows:

[0180] When the C line does not develop color, it indicates that the detection result is invalid, the operation process is incorrect or the test strip has been invalid; when the C line develops color and the T line develops color stronger than the C line or has no obvious difference with the C line, the detection result is negative (-), indicating that there is no triazolone in the sample to be tested; when the C line develops color and the T line develops color obviously weaker than the C line or the T line does not develop color, the detection result is positive (+), indicating that there is triazolone in the sample to be tested.

[0181] III. Performance evaluation of colloidal gold qualitative immunochromatography kit

[0182] 1. Sensitivity

[0183] The triazolone standard was diluted to concentrations of 0 μg / L, 5 μg / L, 10 μg / L, 20 μg / L and 40 μg / L with 0.01M PBS buffer solution, and the colloidal gold qualitative immunochromatography kit of the embodiment was used for detection.

[0184] Table 2 Sensitivity determination results

[0185]

[0186] As shown in Table 2, the colloidal gold qualitative immunochromatography kit prepared by the application has high sensitivity in the detection of triazolone, and the lower limit of detection can reach 10 μg / L.

[0187] 2. Stability

[0188] The storage condition of the colloidal gold qualitative immunochromatography test paper card of the embodiment is room temperature. In order to determine the stability of the test strip, an accelerated destructive test was performed on the test strip, which was continuously placed at 45℃ for 60 days, and on the 0th day, the 5th day, the 10th day, the 20th day, the 30th day, the 40th day, the 50th day and the 60th day, the triazolone standard was diluted to concentrations of 0 μg / L, 5 μg / L, 10 μg / L and 20 μg / L with 0.01M PBS buffer solution, and the colloidal gold qualitative immunochromatography kit of the embodiment was used for detection, and 3 groups of repeats were set in the experiment.

[0189] Table 3 Stability determination results

[0190]

[0191] As shown in Table 3, after the colloidal gold qualitative immunochromatography test paper card stored at 45℃ was sealed and stored for 60 days, the color development depth judgment results of the T line and the C line of the test paper card had no obvious change, indicating that the colloidal gold qualitative immunochromatography test paper card could be stably stored for at least 60 days at 45℃ in the accelerated experiment. 37.5 days at 45℃ is equivalent to one year of storage at room temperature, therefore, the triazolone colloidal gold qualitative immunochromatography test paper card prepared by the application can be stably stored at room temperature for more than one year, which can fully meet the requirements of the market in the storage and transportation process.

[0192] 3. Detection limit of spiked fruit and vegetable samples

[0193] Cabbage, pea, Chinese cabbage, lettuce, flowering Chinese cabbage, cowpea, apple, pear, orange and litchi were used as the samples to be detected. The sample solutions of the samples to be detected were obtained according to the sample processing method in the example. The sample solutions of the samples to be detected were detected by gradient spiking with triazole ketone standard, with spiking gradients of 0 μg / kg, 25 μg / kg, 50 μg / kg, 100 μg / kg and 200 μg / kg. The colloidal gold qualitative immunochromatography kit of the example was used for detection.

[0194] Table 4. Results of detection limit determination of spiked fruit and vegetable samples

[0195]

[0196]

[0197] As shown in Table 4, the detection results of the colloidal gold qualitative immunochromatography kit prepared in the example in 10 samples were good in repeatability. When the content of triazole ketone in the samples was lower than 50 μg / kg, it was negative; when it was higher than 50 μg / kg, it was positive. Therefore, the detection limit of the triazole ketone colloidal gold immunochromatography reagent card prepared in the example in fruit and vegetable samples was 50 μg / kg.

[0198] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. For those skilled in the art, based on the above description and ideas, other different forms of changes or variations can be made, which do not need to be or cannot be exhaustively listed here. Any modification, equivalent replacement and improvement made within the spirit and principles of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. Use of a compound with structural formula as shown in formula (I) in the preparation of triazolinone artificial antigen, 2. A triazolinone artificial antigen, characterized in that, The triazolinone artificial antigen is obtained by coupling a carrier protein with a compound with structural formula as shown in formula (I), and has a structural formula as shown in formula (II). In the formula, Protein is a carrier protein, and the carrier protein is bovine serum albumin or hemocyanin.

3. A process for the preparation of the triazolone artificial antigen according to claim 2, characterized in that, The compound with structural formula as shown in formula (I) is coupled with a carrier protein by an active ester method.

4. Use of a compound with structural formula as shown in formula (II) in the preparation of triazolinone antibody, wherein Protein is a carrier protein, and the carrier protein is bovine serum albumin or hemocyanin.

5. A triazolinone artificial antigen combination, characterized in that, The immunogen is obtained by coupling hemocyanin with a compound with structural formula as shown in formula (I); and the coating antigen is obtained by coupling bovine serum albumin with a compound with structural formula as shown in formula (I).

6. Use of the triazolinone artificial antigen combination of claim 5 in the preparation of a kit for detecting triazolinone.

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