Thiazide hapten, artificial antigen and their preparation and application

Abstract

The present application relates to the field of food safety technology, and more particularly to a thiazinone hapten, an artificial antigen, and their preparation and application. The structural formula of the thiazinone hapten is as follows: This application designs a hapten based on a thiazinone compound as a raw material. The synthesized hapten can highly mimic the structural integrity of the target analyte, and the introduced active group of succinic anhydride, when coupled with a macromolecular protein to prepare an artificial antigen, can better induce an immune response and produce high-performance antibodies.

Description

Technical Field

[0001] The present application relates to the technical field of food safety, and in particular to a thiazinone hapten, an artificial antigen, and the preparation and application thereof. Background Art

[0002] Buprofezin, also known as buprofezin and rice thief, is a new type of selective insecticide that inhibits the growth and development of insects. It has strong contact and stomach toxicity, and is characterized by high activity, high selectivity, and a long residual effect. It can effectively control planthoppers and leafhoppers on rice, leafhoppers on tea and potatoes, whiteflies on citrus and vegetables, shield scales and mealybugs on citrus, and can also control scale insects on fruit trees and tea trees. It is often used for pest control of rice and vegetables. However, improper use can lead to excessive levels of buprofezin in foods such as vegetables, fruits, and tea, which can cause harm to people's health. As people pay more and more attention to food safety, the limit requirements for drug residues in vegetables and fruits are also becoming increasingly stringent. To protect consumer interests and food safety, my country's national standard GB 2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food" stipulates the maximum residue limits of buprofezin in food.

[0003] Currently, the main methods for detecting thiazolinone pesticide residues include gas chromatography (GC), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC / MS), liquid chromatography-mass spectrometry (LC / MS), and enzyme-linked immunosorbent assay (ELISA). These methods all require laboratory conditions, tedious and time-consuming sample pretreatment, and expensive instrumentation. These methods are costly, time-consuming, and complex to perform, resulting in significant limitations in practical applications and difficulty meeting the needs of rapid testing of large numbers of samples or on-site samples. Among them, an ELISA kit involves binding a known antibody or antigen to a solid-phase support while maintaining its immunological activity. During the assay, the sample to be tested and the enzyme-labeled antigen or antibody are reacted with the antibody or antigen adsorbed on the solid-phase support in various steps. Washing is then used to separate the antigen-antibody complex from the free component. Finally, an enzyme substrate is added to catalyze color development. A standard curve is generated based on the known residual substance and the inhibition rate of the test sample, followed by a semi-logarithmic relationship between the inhibition rate and the residual substance concentration, from which the concentration of the analyte can be inferred. For enzyme-linked immunosorbent assay (ELISA), each target protein requires a primary antibody that can specifically bind to it, which results in low experimental flexibility. The detection accuracy is not high, and the test results are affected by many factors, which may result in false positive or false negative results. The experimental operation is complex and the operation process needs to be strictly controlled. It is easy to be affected by the external environment and make errors. In addition, supporting instruments are required for data analysis, and there are differences between different instruments.

[0004] Compared with the above methods, immunochemical detection has the advantages of being rapid, specific, sensitive, accurate, capable of batch monitoring, simple sample processing, and capable of automated operation for drug detection. The prerequisite for immunochemical detection of thiazinone is the need for antibodies against thiazinone, and obtaining high-performance antibodies depends on the structural design of the hapten. Therefore, the molecular design and synthesis of the immune hapten is the most basic and critical step in producing high-performance antibodies and establishing a rapid detection technology for small molecule pesticide residues. The current preparation of thiazinone hapten (patent application number: CN202211388260.1) is not carried out using thiazinone compounds as raw materials, but is synthesized step by step through a series of compounds, and the integrity of the original structure cannot be guaranteed.

[0005] In view of this, this application is hereby filed. Summary of the Invention

[0006] One or more embodiments of the present application provide a thiazinone hapten, an artificial antigen, and their preparation and use, including the following technical solutions:

[0007] One or more embodiments of the present application provide a thiazinone hapten, the structural formula of which is as follows:

[0008]

[0009] One or more embodiments of the present application provide a method for preparing the thiazinone hapten, wherein the method comprises introducing compound I into The steps for preparing thiazinone hapten are described.

[0010] One or more embodiments of the present application provide an artificial antigen, comprising a carrier protein and the thiazinone hapten coupled to the carrier protein;

[0011] Optionally, the carrier protein is bovine serum albumin and / or ovalbumin.

[0012] One or more embodiments of the present application provide a method for preparing the artificial antigen, which comprises: mixing the carrier protein and the thiazinone hapten to perform a coupling reaction to prepare the artificial antigen.

[0013] One or more embodiments of the present application provide a method for preparing a thiazinone polyclonal antibody, the method comprising the steps of immunizing a mammal with an immunogen and isolating the thiazinone polyclonal antibody from the serum of the immunized mammal;

[0014] Wherein, the immunogen is the artificial antigen;

[0015] Optionally, the mammal comprises a lagomorph.

[0016] One or more embodiments of the present application provide a thiazinone polyclonal antibody, which is prepared by the preparation method.

[0017] One or more embodiments of the present application provide a thiazinone immunoassay test strip, which comprises one or more of the thiazinone polyclonal antibody and the artificial antigen.

[0018] In some embodiments of the present application, the thiazinone immunoassay test strip comprises the thiazinone polyclonal antibody and the artificial antigen.

[0019] In some embodiments of the present application, the thiazide immunoassay test strip comprises a sample pad, a conjugate gold pad, a reaction membrane, and a water-absorbing pad sequentially assembled on a bottom plate along a chromatography direction, wherein a detection line and a quality control line are provided on the reaction membrane;

[0020] The conjugate gold pad is coated with a conjugate, which includes the coupled thiazinone polyclonal antibody and colloidal gold; the quality control line is coated with a quality control protein, which specifically binds to the thiazinone polyclonal antibody; and the detection line is coated with the artificial antigen.

[0021] One or more embodiments of the present application provide a method for preparing the thiazinone immunoassay test strip, which includes the steps of preparing the thiazinone immunoassay test strip using one or more of the thiazinone polyclonal antibody and the artificial antigen.

[0022] One or more embodiments of the present application provide a method for detecting thiazinone, which includes the step of using the thiazinone polyclonal antibody or the thiazinone immunoassay test strip to detect thiazinone in a sample to be tested.

[0023] In some embodiments of the present application, the sample to be tested is a vegetable sample, a fruit sample, or a grain sample.

[0024] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the application will become apparent from the description and from the claims thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0026] Figure 1 A synthetic route for the thiazinone hapten;

[0027] Figure 2 Schematic diagram of the cross-sectional structure of the test strip;

[0028] Figure 3 This is a diagram showing the test strip test results. DETAILED DESCRIPTION

[0029] Below in conjunction with accompanying drawing, embodiment and example, the application is described in further detail.It should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms and is not limited to the embodiment and example described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the application.It should be understood that the application can be implemented without one or more of these details.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only and are not intended to limit this application.

[0031] the term

[0032] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0033] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0034] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0035] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0036] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0037] Herein, "preferred", "better", "more preferred" and "suitable" are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0038] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0039] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0040] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Furthermore, "first," "second," "third," "fourth," etc. serve only as non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0041] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0042] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0043] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0044] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0045] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0046] Immunoassays are gaining increasing attention in the field of pesticide residue detection due to their high specificity, ease of operation, and ability to rapidly detect large numbers of samples. Therefore, the development of efficient immunoassays is essential. However, these methods are determined by the properties of both the antigen and the antibody, and the structural design of the hapten is crucial for obtaining high-performance antibodies.

[0047] In a first aspect of the embodiments of the present application, a thiazinone hapten is provided, and the structural formula of the thiazinone hapten is as follows:

[0048]

[0049] The embodiment of the present application provides a novel thiazinone hapten, which is coupled to a carrier protein. The prepared complete antigen can produce highly efficient antibodies, and utilizes the specific immunological reaction of the antigen and antibody and the amplification effect of the easily detectable and identifiable marker to detect ultra-trace small molecule targets in the sample, which can be used for sample determination. The key to this application is the molecular design and synthesis of the hapten and the preparation of the complete antigen and antibody.

[0050] This application designs haptens based on thiazinone compounds as raw materials, retaining the chemical structure of thiazinone to the greatest extent possible. The synthesized haptens can highly simulate the integrity of the target analyte structure, and the introduced active group of succinic anhydride can be coupled with macromolecular proteins to prepare artificial antigens, which can better induce immune responses, specifically recognize thiazinone, and produce high-performance antibodies. Overall, the hapten improves the specificity of the hapten and adds reaction sites to the antigen preparation. The corresponding antibodies are applied to colloidal gold immunochromatographic rapid detection strips, which are time-saving, simple to operate, and low-cost, and are suitable for large-scale sample testing at grassroots units.

[0051] In a second aspect of the present invention, a method for preparing the thiazinone hapten is provided, wherein the method comprises introducing the thiazinone hapten into the compound I. The steps for preparing thiazinone hapten are described.

[0052] The present application does not specifically limit the specific steps of introducing succinic anhydride, including but not limited to Figure 1 The synthetic route shown and the specific steps described under item 1.1 of Example 1.

[0053] In a third aspect of the embodiments of the present application, an artificial antigen is provided, which includes a carrier protein and the thiazinone hapten coupled to the carrier protein.

[0054] In some embodiments, the carrier protein is bovine serum albumin and / or ovalbumin. In some embodiments, the carrier protein is bovine serum albumin, and the corresponding artificial antigen can be used to immunize animals to produce corresponding antibodies. In some embodiments, the carrier protein is ovalbumin, and the corresponding artificial antigen can be used as a coating agent to be coated on the test line of an immunoassay test strip to compete with the antigen in the sample for antibodies.

[0055] In a fourth aspect of the embodiments of the present application, a method for preparing the artificial antigen is provided, wherein the method comprises: mixing the carrier protein and the thiazinone hapten to perform a coupling reaction to prepare the artificial antigen.

[0056] The present application does not particularly limit the preparation method of the artificial antigen, including but not limited to the preparation steps under Item 1.2 and Item 1.3 in Example 1.

[0057] In a fifth aspect of the embodiments of the present application, there is provided a method for preparing a polyclonal antibody against thiazolinone, the method comprising the steps of immunizing a mammal with an immunogen and isolating the polyclonal antibody against thiazolinone from the serum of the immunized mammal;

[0058] Wherein, the immunogen is the artificial antigen;

[0059] Optionally, the mammal comprises a lagomorph.

[0060] In a sixth aspect of the embodiments of the present application, a thiazinone polyclonal antibody is provided, wherein the thiazinone polyclonal antibody is prepared by the preparation method.

[0061] In a seventh aspect of the embodiments of the present application, a thiazinone immunoassay test strip is provided, wherein the thiazinone immunoassay test strip comprises one or more of the thiazinone polyclonal antibody and the artificial antigen.

[0062] Optionally, the thiazinone immunoassay test strip comprises the thiazinone polyclonal antibody and the artificial antigen.

[0063] In some embodiments, the thiazide immunoassay test strip comprises a sample pad, a conjugate gold pad, a reaction membrane, and a water-absorbing pad assembled on a bottom plate in sequence along the chromatography direction, wherein the reaction membrane is provided with a detection line and a quality control line;

[0064] The conjugate gold pad is coated with conjugate 1, which includes the coupled thiazinone polyclonal antibody and colloidal gold; the quality control line is coated with quality control protein, which specifically binds to the thiazinone polyclonal antibody; and the detection line is coated with the artificial antigen.

[0065] The thiazinone rapid test strips of the present invention utilize a highly specific antibody-antigen reaction and competitive inhibition immunochromatographic analysis technique. A thiazinone polyclonal antibody-colloidal gold conjugate is immobilized on a gold conjugate pad. During flow, the thiazinone in the sample specifically binds to the thiazinone polyclonal antibody-colloidal gold conjugate on the gold conjugate pad, forming a thiazinone drug-antibody-colloidal gold complex. The thiazinone drug in the sample competes with the thiazinone hapten-carrier protein conjugate on the test line of the reaction membrane for binding to the thiazinone polyclonal antibody-colloidal gold conjugate. The presence or color depth of a red strip on the test line is used to determine whether the sample solution contains thiazinone residues.

[0066] During the test, the sample is treated and then dropped onto the sample pad of the test strip. When the concentration of thiazinone in the sample is lower than the detection limit (including zero), the thiazinone polyclonal antibody-colloidal gold conjugate will specifically bind to the thiazinone hapten-carrier protein conjugate fixed on the reaction membrane during the chromatography process, and a red strip will appear on the test line (T) and the quality control line (C). If the concentration of thiazinone in the sample is equal to or higher than the detection limit, the thiazinone polyclonal antibody-colloidal gold conjugate will completely bind to thiazinone, so that no red strip will appear at the T line due to the competitive reaction because it will not bind to the thiazinone hapten-carrier protein conjugate.

[0067] The test strips of the present application have the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitability for use in various units, simple storage, and long shelf life. The method of detecting thiazolinone residues using the test strips of the present invention is simple, rapid, intuitive, accurate, widely applicable, low cost, and easy to promote and use.

[0068] The embodiments of the present application overcome the characteristics of existing methods for detecting thiazolinone, such as high dependence on detection equipment and inability to achieve rapid detection of large batches of samples. The embodiments of the present application have high sensitivity and can achieve rapid detection and on-site monitoring of large batches of thiazolinone samples.

[0069] In the present application, the second quality control protein includes but is not limited to goat anti-rabbit IgG.

[0070] In an eighth aspect of the embodiments of the present application, a method for preparing the thiazinone immunoassay test strip is provided, which comprises the steps of preparing the thiazinone immunoassay test strip using one or more of the thiazinone polyclonal antibody and the artificial antigen.

[0071] In a ninth aspect of the embodiments of the present application, a method for detecting thiazin is provided, which comprises the steps of using the thiazin polyclonal antibody and the thiazin immunoassay test strip to detect thiazin in a sample to be tested.

[0072] Optionally, the sample to be tested is a vegetable sample, a fruit sample or a grain sample.

[0073] The concentration of concentrated sulfuric acid is typically 98.3%. This concentration refers to the mass fraction of sulfuric acid in a solution, that is, the proportion of pure sulfuric acid in the solution. Concentrated sulfuric acid is a highly corrosive mineral acid with chemical properties including dehydration, low volatility, acidity, and hygroscopicity. In practical applications, the concentration of concentrated sulfuric acid is often adjusted to around 98%. This is because sulfuric acid solutions at this concentration are highly corrosive and oxidizing, making them suitable for many chemical reactions and laboratory operations.

[0074] Concentrated hydrochloric acid is a highly corrosive chemical. Its concentration is typically expressed as a mass fraction, with commercially available concentrations ranging from 36% to 38%. The concentration of concentrated hydrochloric acid used in experiments also falls within this range, though the specific value may vary slightly depending on the production batch and intended use.

[0075] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0076] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0077] Example 1

[0078] 1. Preparation of Buprofezin Test Strips

[0079] The preparation method of the test strip mainly comprises the following steps:

[0080] (1) preparing a gold pad sprayed with a thiazinone antibody-colloidal gold label conjugate;

[0081] (2) preparing a reaction membrane having a detection line coated with a thiazinone hapten-BSA conjugate and a quality control line coated with a goat anti-rabbit IgG;

[0082] (3) Assemble the gold pad, reaction membrane, sample absorption pad, water absorption pad and bottom plate prepared in step (1) and step (2) into a test strip.

[0083] The following is a detailed step-by-step description:

[0084] 1.1 Synthesis of thiazinone hapten (synthesis route see attached Figure 1 )

[0085] 3 g of compound I was slowly added to 10 mL of concentrated sulfuric acid cooled to 0°C and completely dissolved. 1 g of KNO3 was then slowly added. The reaction was allowed to proceed at low temperature (0°C) for 3 hours. The pH was adjusted to 5-7 with NaHCO3, and the mixture was extracted three times with dichloromethane. The aqueous phase was removed, and the organic phase was evaporated to dryness. The mixture was recrystallized from petroleum ether and ethyl acetate to obtain 1.4 g of compound II.

[0086] To the total compound II (1.4 g), add SnCl2·2H2O (6.8 g) and concentrated hydrochloric acid (20 mL) in sequence, react at 60°C for 6 h, adjust the pH to 8-9 with NaHCO3, extract three times with dichloromethane, remove the aqueous phase, and evaporate the organic phase to dryness to obtain compound III (1 g).

[0087] To 1 g of the total compound III, 70 mL of dilute sulfuric acid (4 mol / L) was added, the mixture was cooled to 0°C, and 0.17 g of sodium nitrite solution (2.5 mmol) was slowly added. The mixture was reacted at 0°C for 3 h. 150 mL of dilute sulfuric acid (4 mol / L) was added and the mixture was reacted at 45°C for 3 h. The mixture was cooled to room temperature, the pH was adjusted to 6-7 with NaHCO3, and the mixture was extracted three times with dichloromethane. The aqueous phase was removed, and the organic phase was evaporated to dryness to obtain 0.6 g of compound IV.

[0088] 0.2 g of compound IV and 0.23 g of succinic anhydride were dissolved in pyridine, 300 μL of triethylamine and 0.05 g of 4-dimethylaminopyridine (DMAP) were added, and the reaction was refluxed overnight. The reaction solution was separated and purified by column chromatography, and then the separated and purified reactant was dissolved in methanol. The molar ratio of the separated and purified reactant to methanol was 1:5. Then, 1 mol / L aqueous sodium hydroxide solution was added and stirred at room temperature for 3 to 5 hours. After the reaction, the pH was adjusted to 6 to 7 with 1 mol / L hydrochloric acid. The resulting precipitate was the hapten product compound V.

[0089] 1.2 Preparation of immunogen

[0090] Dissolve 5 mg of compound V, 3 mg of NHS (activating reagent N-hydroxysuccinimide), and 10 mg of EDC (dehydrating agent carbodiimide) in 50-100 μL DMF (N,N-dimethylformamide) and stir in the dark at room temperature for 2-4 h to obtain thiazinone hapten activation solution A.

[0091] Weigh 20 mg of bovine serum albumin (BSA) and dissolve it in 1 mL of PBS buffer (0.01 mol / L, pH = 7.4) to obtain solution B; slowly add solution A dropwise to solution B, stir at 4°C for 12 h, stop the reaction, dialyze and purify in 0.01 mol / L PBS for 3 days, change the solution 3 times a day, and centrifuge and aliquot to obtain a thiazinone hapten-BSA conjugate, which is the immunogen.

[0092] 1.3 Preparation of coating

[0093] The same method as the immunogen preparation method was used, 5 mg of compound V was coupled with 10 mg of ovalbumin (OVA) in a PBS buffer solution at pH 7.4, and the thiazinone hapten-OVA conjugate was obtained after purification by dialysis to obtain the coating source.

[0094] 1.4 Preparation of Buprofezin Antibodies

[0095] The immunogen prepared in step 1.2 (immunogen concentration of 5 mg / mL) was emulsified evenly with the immune adjuvant (incomplete Freund's adjuvant for the first immunization and incomplete Freund's adjuvant for subsequent booster immunizations) in a volume ratio of 1:1 and immunized New Zealand white rabbits. The New Zealand white rabbits weighed 2.5-3 kg and were injected subcutaneously at multiple points in the neck and back. The second immunization was performed 4 weeks later, and booster immunizations were performed every 3 weeks thereafter, with each injection dose of 1 mg. Blood was collected from the ear vein one week after the third booster immunization, and the serum titer was determined using an indirect competitive ELISA. When the titer no longer increased, a booster immunization was performed using the ear vein. One week later, blood was collected from the heart, and the collected blood was used to obtain serum by incubating at 37°C for 0.5-1 hour, then standing at 4°C overnight, and then aspirating the precipitated serum with a pipette. Then, the supernatant was collected by centrifugation at 3000-5000 rpm at 4°C for 10 minutes. The antiserum was purified by ammonium sulfate precipitation to obtain polyclonal antibodies against buprofen and stored at -20°C until use.

[0096] 1.5 Preparation of thiazinone antibody-colloidal gold conjugate

[0097] (1) Preparation of colloidal gold

[0098] Dilute 1% chloroauric acid to 0.01% (mass fraction) with double-distilled deionized water. Place 100 mL in a conical flask and heat to boiling using a thermostatic electromagnetic stirrer. Add 125 mL of 1% trisodium citrate while continuing to stir at high temperature. Continue stirring at a constant speed until the solution turns a translucent red. Cool to room temperature and store at room temperature. The prepared colloidal gold should be pure, translucent, and free of precipitates and floating matter.

[0099] (2) Preparation of thiazinone antibody-colloidal gold conjugate and conjugate gold pad

[0100] The colloidal gold solution was adjusted to pH 7.0 with 0.1 mol / L potassium carbonate solution and stirred for 15 minutes. Thiazide polyclonal antibody was added to the colloidal gold solution at a standard of 5 μg of antibody per milliliter of colloidal gold solution (colloidal gold concentration of 0.0001 mol / L). Stirring was continued for 20 minutes. Then, 20% BSA was added to a final concentration of 1% (volume fraction) in the final product. Stirring was continued for 25 minutes. Centrifugation was performed at 10,000 rpm / min and 4°C for 20 minutes. The supernatant was discarded and the solution was reconstituted with reconstitution buffer at a volume of 1 / 10 of the initial colloidal gold solution. The solution was sprayed onto the conjugate pad at a spray rate of 2.0 μL / cm.

[0101] 1.6 Preparation of reaction membrane and sample pad

[0102] Take 0.2 mg / mL coating agent and 0.1 mg / mL goat anti-rabbit IgG, and spray them on nitrocellulose membrane 4 at a spray volume of 1.0 μL / cm using a film scribing machine to prepare a test line 5 and a quality control line 6, respectively. Place the sprayed nitrocellulose membrane 4 in a 37°C vacuum drying oven and dry it for 24 hours. The dilution coating solution of 0.2 mg / mL coating agent and 0.1 mg / mL goat anti-rabbit IgG is PBS buffer with a pH value of 7.4 containing 1% trehalose by mass.

[0103] 1.7 Preparation of sample pad

[0104] The sample absorption pad was pre-moistened with the sample treatment solution and placed in a drying oven at 37°C to dry for 24 h.

[0105] 1.8 Assembly of test strips

[0106] According to the attached Figure 2 The cross-sectional structure of the test strip is shown. The sample absorption pad 2, the conjugate gold pad 3, the reaction membrane 4, and the water absorbent pad 7 are pasted on the bottom plate 1 in order. The sample absorption pad covers the conjugate gold pad. Finally, it is cut into small strips with a width of 4 mm, placed in a special plastic card shell, and sealed with an aluminum foil bag. It can be stored for 12 months at 4-30°C.

[0107] 2. Detection of Buprofezin Residues in Samples

[0108] 2.1 Sample pretreatment

[0109] Cut the sample to be tested into pieces smaller than 1 cm square. Weigh 1 ± 0.1 g of sample into a 15 mL polystyrene centrifuge tube, add 3 mL of sample extract, cover, and vortex or manually shake for 1 minute. Let stand for 2 minutes. The supernatant is the sample solution. For melons and vegetables, pipette 100 μL of sample solution and add 900 μL of sample extract. Vortex and mix thoroughly to obtain the test solution.

[0110] The sample extract is a PB buffer solution with a concentration of 0.02M and a pH value of 7.4, wherein the PB buffer solution contains 0.5% by volume of Triton X-405 and 0.01% by mass of PC300.

[0111] 2.2 Testing with test strips

[0112] Use a pipette to draw up the sample solution to be tested and vertically drip 3 to 4 drops into the sample well. Start timing when the liquid flows, and let it react for 5 to 10 minutes before determining the result.

[0113] 2.3 Analysis of test results

[0114] If the test line (T) is darker than or the same color as the control line (C), the result is negative; if the test line (T) is lighter than the C line, or if the test line (T) does not show any color, the result is positive; if the control line (C) does not show any color, it may be due to improper operation or invalid test strip card. It is recommended to retest with a new test strip card. Figure 3 .

[0115] 3. Sample testing examples

[0116] 3.1 Detection Limit Example

[0117] Take blank cucumber, pumpkin and wax gourd samples, add thiamethoxam to them to the final concentration of 0.35 mg / kg, 0.7 mg / kg and 1.4 mg / kg respectively; take test strips for testing, and repeat the measurement 3 times for each sample.

[0118] When the test strips were used to test cucumber, pumpkin and winter melon samples, when there was no thiazolinone and its added concentration was 0.35 mg / kg, the test strips showed that the T line was darker than the C line or the color was the same as the C line, which was negative; when the thiazolinone added concentration was 0.7 mg / kg and 1.4 mg / kg, the test strips showed that the T line was lighter than the C line or had no color with the T line, which was positive, indicating that the detection limit of this test strip for thiazolinone in melons is 0.7 mg / kg.

[0119] 3.2 Sensitivity, false positive rate, and false negative rate test

[0120] Fifty blank cucumber, pumpkin, and wax gourd samples and 50 positive cucumber, pumpkin, and wax gourd samples spiked with buprofezin to a final concentration of 0.7 mg / kg and 1.4 mg / kg were tested using three batches of test strips. The results are shown in Table 1.

[0121] Table 1. Experimental results of performance index determination

[0122]

[0123]

[0124] The results show that the performance indicators of the test strip of the present application are sensitivity ≥ 99% and false negative rate ≤ 1%, indicating that the test strip for detecting thiazolinone of the present application can quickly detect thiazolinone residues in melon samples.

[0125] 3.3 Specificity test

[0126] The specificity of the test strip was determined by testing thiamethoxam, triazophos, isofenphos-methyl, methomyl, and clothianidin at 100 μg / L concentrations. The results were repeated three times to determine the strip's specificity. The results showed that the strip was negative for all of the above drugs, indicating no cross-contamination with the above drugs, indicating that the test strip had good specificity.

[0127] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0128] The above-described embodiments only express several implementation methods of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that, for those skilled in the art, several variations and improvements can be made without departing from the concept of the present application, and these all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A thiazinone hapten, characterized in that The structural formula of the thiazinone hapten is shown below:

2. The method for preparing the thiazinone hapten according to claim 1, characterized in that: The preparation method comprises introducing compound I into The following steps are used to prepare the thiazinone hapten:

3. An artificial antigen, characterized in that The artificial antigen comprises a carrier protein and the thiazinone hapten according to claim 1 coupled to the carrier protein.

4. The artificial antigen according to claim 3, characterized in that The carrier protein is bovine serum albumin and / or ovalbumin.

5. The method for preparing an artificial antigen according to any one of claims 3 to 4, characterized in that: The preparation method comprises: mixing the carrier protein and the thiazinone hapten to carry out a coupling reaction to prepare an artificial antigen.

6. A method for preparing a thiazolinone polyclonal antibody, characterized in that: The preparation method comprises the steps of immunizing a mammal with an immunogen and isolating thiazolinone polyclonal antibodies from the serum of the immunized mammal; Wherein, the immunogen is the artificial antigen according to any one of claims 3 to 4.

7. The method for preparing a thiazolinone polyclonal antibody according to claim 6, characterized in that: Said mammals include lagomorphs.

8. A thiazolinone polyclonal antibody, characterized in that The thiazinone polyclonal antibody is prepared by the preparation method according to any one of claims 6 to 7.

9. A thiazolinone immunoassay test strip, characterized in that: The thiazinone immunoassay test strip comprises the thiazinone polyclonal antibody according to claim 8 and one or more of the artificial antigens according to any one of claims 3 to 4.

10. The buprofezin immunoassay test strip according to claim 9, characterized in that: The thiazinone immunoassay test strip comprises the thiazinone polyclonal antibody and the artificial antigen.

11. The oxazinone immunoassay test strip according to any one of claims 9 to 10, characterized in that: The thiazide immunoassay test strip comprises a sample pad, a conjugate gold pad, a reaction membrane and a water-absorbing pad which are sequentially assembled on a bottom plate along a chromatography direction, wherein a detection line and a quality control line are provided on the reaction membrane; The conjugate gold pad is coated with a conjugate, which includes the coupled thiazinone polyclonal antibody and colloidal gold; the quality control line is coated with a quality control protein, which specifically binds to the thiazinone polyclonal antibody; and the detection line is coated with the artificial antigen.

12. The method for preparing the oxazinone immunoassay test strip according to any one of claims 9 to 11, characterized in that: The preparation method comprises the steps of preparing a buprofen immunoassay test strip using the buprofen polyclonal antibody according to claim 8 and one or more of the artificial antigens according to any one of claims 3 to 4.

13. A method for detecting thiazolinone, characterized in that: The detection method comprises the steps of using the thiazinone polyclonal antibody according to claim 8 or the thiazinone immunoassay test strip according to any one of claims 9 to 11 to detect thiazinone in the sample to be tested.

14. The method for detecting buprofezin according to claim 13, wherein: The sample to be tested is a vegetable sample, a fruit sample or a grain sample.

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