A hapten, antigen, antibody, preparation method and photochemiluminescence quantitative detection kit for zearalenone (Z)

Abstract

The present invention relates to the field of immunoassay technology, and provides a zearalenone (Z) hapten, antigen, antibody, preparation method, and photochemiluminescence quantitative detection kit. The present invention uses a cyclic carboxylic acid anhydride as a reactant, and introduces an active group with a carbon chain of n1 (2≤n1≤22) carbon atoms between two hydroxyl groups on the benzene ring of the zearalenone (Z) molecule through a Friedel-Crafts acylation reaction to obtain a hapten. After the hapten is coupled to a carrier protein, the immunogenicity of the antigen can be enhanced, and antibodies with high titer and high sensitivity can be produced after immunization of the host animal. The results of the examples show that after the zearalenone (Z) antigen of the present invention is injected into mice for immunization, the antibody titer produced is 1:5000, and the IC 50 The zearalenone (Z) antigen and antibody of the present invention are applied to the rapid quantitative detection of zearalenone (Z) by photoluminescence, and the detection limit and false negative rate are all qualified, the steps are simple, and the industrial value is high.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a zearalenone (Z) hapten, antigen, antibody and preparation method thereof, and a photo-induced chemiluminescence quantitative detection kit. Background Art

[0002] Zearalenone (Z) is a mycotoxin produced by mold. It is a secondary metabolite produced by fungi. From corn, wheat, and peanuts to fruits, seasonings, traditional Chinese medicine, feed, and milk, mycotoxins are ubiquitous in our daily lives. Zearalenone (Z) is highly harmful to livestock and poultry breeding, often preventing a breeding base from recovering to its original level within a year and causing widespread quality degradation. It can be said that mycotoxin contamination in grain and oil products has become a major hidden danger to my country's food security and food safety, seriously endangering public health and economic development.

[0003] Current food safety rapid testing products are divided into two types according to their properties: qualitative and quantitative testing. Qualitative products are mainly colloidal gold test strips, and quantitative products mainly include ELISA kits, fluorescent quantitative test strips, PCR detection kits, conventional chemiluminescence kits, etc. Qualitative products can only perform qualitative analysis, and the products are greatly affected by different food matrices (color depth), and the qualitative test results have relatively little significance for food safety supervision; while quantitative detection products such as ELISA kits, PCR detection kits, and conventional chemiluminescence kits are relatively complex to operate, have long detection times, relatively high costs for instruments and consumables, and relatively high requirements for operators; fluorescent quantitative test strips are similar to colloidal gold qualitative detection test strips in that they are simple and fast to operate, but because of the principle of immunochromatography technology, the test results have poor parallelism and large errors; the currently used fluorescent quantitative detection test strips are mainly equipped with single-channel detection equipment, so the detection efficiency is low, which will affect the detection efficiency for large-scale sample detection scenarios; photochemiluminescence technology is a new fourth-generation chemiluminescence technology. Since it is a homogeneous reaction platform, no separation system is required during the detection process. It has the advantages of no wash, low background, high sensitivity, good repeatability, simple operation and short time consumption, and multi-channel simultaneous detection and analysis equipment is designed to improve sample detection efficiency.

[0004] However, the current ELISA kits and photochemiluminescence rapid quantitative detection kits for zearalenone (Z) generally have problems such as insufficient antibody detection sensitivity and low titer. Summary of the Invention

[0005] In view of this, the present invention provides a zearalenone (Z) hapten, antigen, antibody, preparation method, and photochemiluminescence quantitative detection kit. The antigen obtained by coupling the zearalenone (Z) hapten provided by the present invention with a carrier protein has high immunogenicity and can produce high-titer and high-sensitivity antibodies after immunization of a host animal, which is beneficial for improving the accuracy of immunoassay detection of zearalenone (Z).

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A zearalenone (Z) hapten having the structure shown in Formula I:

[0008]

[0009] In formula I: 0≤n2≤20.

[0010] The present invention provides a method for preparing the zearalenone (Z) hapten described in the above scheme, comprising the following steps:

[0011] Zearalenone (Z), a cyclic carboxylic acid anhydride and a catalyst are mixed to carry out a Friedel-Crafts acylation reaction to obtain a zearalenone (Z) hapten having the structure described in Formula I; the cyclic carboxylic acid anhydride is the anhydride of a straight-chain alkyl diacid, and the straight-chain alkyl diacid contains n1 carbon atoms, where n1 is 2 to 22.

[0012] Preferably, the temperature of the Friedel-Crafts acylation reaction is 80-85° C., and the reaction time is 2-3 hours; the molar ratio of zearalenone (Z), catalyst and glutaric anhydride is 1:2-2.5:1-1.2.

[0013] The present invention also provides a zearalenone (Z) antigen, comprising the zearalenone (Z) hapten described in the above scheme, and a carrier protein coupled to the zearalenone (Z) hapten.

[0014] Preferably, the carrier protein and the zearalenone (Z) hapten are coupled via amide bonds.

[0015] The present invention also provides a method for preparing zearalenone (Z) antigen, comprising the following steps:

[0016] The zearalenone (Z) hapten, coupling activator and polar solvent described in the above technical solution are mixed and activated to obtain an activated zearalenone (Z) hapten solution;

[0017] The activated zearalenone (Z) hapten solution and the carrier protein buffer solution are mixed to carry out coupling reaction to obtain the zearalenone (Z) antigen.

[0018] The present invention also provides a zearalenone (Z) antibody, which is obtained by immunizing a host animal with the zearalenone (Z) antigen described in the above technical solution or the zearalenone (Z) antigen prepared by the preparation method described in the above technical solution.

[0019] The present invention also provides a test paper or a kit for detecting zearalenone (Z), which contains the zearalenone (Z) antibody described in the above technical solution.

[0020] Preferably, the kit is a photochemiluminescence quantitative detection kit; the reagents in the photochemiluminescence rapid quantitative detection kit include chemiluminescence detection reagents; the chemiluminescence detection reagents include oxygen-accepting microspheres labeled with zearalenone (Z) antibodies, biotin labeled with zearalenone (Z) antigens, and oxygen-donating microspheres labeled with streptavidin.

[0021] The present invention also provides the use of the zearalenone (Z) antibody described in the above technical solution or the test paper or kit described in the above technical solution in detecting zearalenone (Z) for non-diagnostic purposes.

[0022] The present invention provides a zearalenone (Z) hapten, the structural formula of which is shown in Formula I. The quality of antibodies depends on the structural characteristics of the hapten, and therefore the design of the hapten is crucial. The present invention introduces an active group with a carbon chain consisting of n2+2 carbon atoms between the two hydroxyl groups on the benzene ring of the zearalenone (Z) molecule. The n2+2 carbon atom spacer arm enables the molecule to be fully exposed outside the protein after being coupled to a carrier protein, thereby enhancing the immunogenicity of the antigen. After immunization of a host animal, it can produce antibodies with high titer and sensitivity, thereby improving the accuracy of zearalenone (Z) analysis and detection.

[0023] The present invention also provides a method for preparing the zearalenone (Z) hapten described in the above scheme. The present invention introduces an active group with an n2+2 carbon atom carbon chain at the middle position between the two hydroxyl groups of the benzene ring on the zearalenone (Z) molecule through a Friedel-Crafts acylation reaction. The preparation method of the present invention can generate the zearalenone (Z) hapten in one step, with mild reaction conditions, simple operation, and easy post-processing.

[0024] The present invention also provides a zearalenone (Z) antigen, which is obtained by coupling the zearalenone (Z) hapten described in the above scheme with a carrier protein. After the zearalenone (Z) hapten provided by the present invention is coupled with a carrier protein, the resulting antigen has strong immunogenicity. The results of the examples show that after the zearalenone (Z) antigen of the present invention is injected into mice for immunization, the antibody titer generated is 1:5000, and the half inhibitory concentration (IC 50) is less than 10ppb / mL; when the zearalenone (Z) antigen and antibody obtained by the present invention are applied to the rapid quantitative detection of zearalenone (Z) by photoinduced chemiluminescence, the four dimensions of detection limit, false negative rate, false positive rate, and instrument method compliance rate are all qualified, and the detection steps are simple, which has good practical value and industrial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the product composition of the photochemiluminescence rapid quantitative detection kit of the present invention. Figure 1 Middle: 1-sample well, 2-foam packaging, 3-R1, 4-R2, 5-R3.

[0026] Figure 2 This is the H NMR spectrum of the zearalenone (Z) hapten prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0027] The present invention provides a zearalenone (Z) hapten having a structure shown in Formula I:

[0028]

[0029] In formula I: 0≤n2≤20.

[0030] In the present invention, n2 represents the number of methylene groups, specifically a positive integer of 0 to 20, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, more preferably 2 to 6, and even more preferably 3.

[0031] The present invention also provides a method for preparing the zearalenone (Z) hapten described in the above scheme, comprising the following steps:

[0032] Zearalenone (Z), a cyclic carboxylic acid anhydride and a catalyst are mixed to carry out a Friedel-Crafts acylation reaction to obtain a zearalenone (Z) hapten having the structure described in Formula I; the cyclic carboxylic acid anhydride is the anhydride of a straight-chain alkyl diacid, and the straight-chain alkyl diacid contains n1 carbon atoms, where n1 is 2 to 22.

[0033] In the present invention, the synthesis route of the compound having the structure represented by the zearalenone (Z) hapten is shown in Chemical Reaction Formula 1:

[0034]

[0035] In the present invention, the cyclic carboxylic acid anhydride is specifically the anhydride of a linear alkyl diacid, wherein the linear alkyl diacid contains n1 carbon atoms (including the carbon atoms in the carboxyl group), n1 is 2 to 22, and the relationship between n2 and n1 is: n2 = n1-2; specifically, the cyclic carboxylic acid anhydride is preferably oxalic anhydride, malonic anhydride, succinic anhydride, maleic anhydride, glutaric anhydride or adipic anhydride, and more preferably glutaric anhydride.

[0036] In the present invention, the temperature of the Friedel-Crafts acylation reaction is preferably 80-85°C; the reaction time is preferably 2-3 hours; the molar ratio of zearalenone (Z), catalyst, and cyclic carboxylic acid anhydride is preferably 1:2-2.5:1-1.2, more preferably 1:2-2.2:1-1.1. The catalyst is preferably aluminum trichloride, more preferably anhydrous aluminum trichloride. The reaction solvent of the Friedel-Crafts acylation reaction is preferably a chlorinated alkane, more preferably 1,2-dichloroethane.

[0037] The present invention preferably places the catalyst in a dry container and dissolves it in a solvent, then adds cyclic carboxylic acid anhydride to obtain a mixed solution of cyclic carboxylic acid anhydride and the catalyst, and then slowly drips the zearalenone (Z) solution into the mixed solution of cyclic carboxylic acid anhydride and the catalyst. After the dripping is completed, the Friedel-Crafts acylation reaction is carried out; during the reaction, TLC is preferably used to monitor the progress of the reaction, and the reaction is carried out until the raw material point disappears. In the present invention, the method for dissolving the catalyst is preferably: first place the catalyst in a dry container, and then add 1,2-dichloroethane dropwise at 0-5°C, stirring while adding dropwise. After the solvent is added, continue stirring for 10-15 minutes to obtain a clear anhydrous aluminum chloride solution; after adding the cyclic carboxylic acid anhydride, it is preferably stirred again for 30-40 minutes. The method for preparing the zearalenone (Z) solution is preferably to dissolve zearalenone (Z) in 1,2-dichloroethane.

[0038] After the Friedel-Crafts acylation reaction is completed, the present invention preferably performs post-treatment on the obtained reaction solution. The post-treatment preferably comprises: cooling the reaction solution obtained by the Friedel-Crafts acylation reaction to room temperature, and then mixing it with ice to obtain a mixed solution; extracting the mixed solution with ethyl acetate, washing the obtained organic phase, and then distilling it under reduced pressure to obtain a residue, dissolving the residue with ethyl acetate, mixing it with silica gel, and purifying it by dry column chromatography, collecting the main ultraviolet point component, and obtaining a light yellow oily liquid, which is the zearalenone (Z) hapten. In the present invention, the volume ratio of the ice cubes to the reaction liquid is preferably 1:1; the mixing of the ice cubes and the reaction liquid is preferably carried out under stirring conditions, and the stirring time is preferably 10 to 20 minutes; the number of extractions with ethyl acetate is preferably more than 1 time, more preferably 2 to 3 times. When the number of extractions is greater than 1 time, the organic phases obtained from multiple extractions are preferably combined; the volume of ethyl acetate used in each extraction is preferably 1 / 2 of the volume of the reaction liquid; the washing preferably includes washing with saturated brine and washing with deionized water, and the number of washings with saturated brine is preferably 3 to 4 times; when mixing the sample with silica gel, it is preferably mixed with silica gel with a mass twice that of the residue.

[0039] The present invention also provides a zearalenone (Z) antigen, comprising the zearalenone (Z) hapten described in the above scheme, and a carrier protein coupled to the zearalenone (Z) hapten; the carrier protein and the zearalenone (Z) hapten are preferably coupled by amide bond coupling; and 45 to 50 hapten molecules are preferably coupled to one carrier protein molecule.

[0040] In the present invention, the carrier protein is preferably ovalbumin (OVA); when the carrier protein is ovalbumin, the structure of the zearalenone (Z) antigen is shown in Formula II:

[0041]

[0042] In formula II: 0≤n2≤20.

[0043] In the present invention, when the carrier protein is preferably ovalbumin (OVA), the zearalenone (Z) antigen is specifically zearalenone (Z) coating.

[0044] In the present invention, the carrier protein is preferably bovine serum albumin (BSA); when the carrier protein is bovine serum albumin, the structure of the zearalenone (Z) antigen is shown in Formula III:

[0045]

[0046] In formula III: 0≤n2≤20.

[0047] In the present invention, when the carrier protein is preferably bovine serum albumin (BSA), the zearalenone (Z) antigen is specifically a zearalenone (Z) immunogen.

[0048] The present invention also provides a method for preparing the zearalenone (Z) antigen described in the above scheme, comprising the following steps:

[0049] The zearalenone (Z) hapten, coupling activator and polar solvent described in the above technical solution are mixed and activated to obtain an activated zearalenone (Z) hapten solution;

[0050] The activated zearalenone (Z) hapten solution and the carrier protein buffer solution are mixed to carry out coupling reaction to obtain the zearalenone (Z) antigen.

[0051] In the present invention, when the carrier protein is preferably ovalbumin (OVA), the synthesis route of the zearalenone (Z) antigen is as shown in Chemical Reaction Formula 2:

[0052]

[0053] When the carrier protein is preferably bovine serum albumin (BSA), the synthesis route of the zearalenone (Z) antigen is as shown in Chemical Reaction Formula 3:

[0054]

[0055] The present invention activates the zearalenone (Z) hapten described in the above embodiment by mixing a coupling activator and a polar solvent to obtain an activated zearalenone (Z) hapten solution. In the present invention, the polar solvent is preferably one or two of 1,4-dioxane, acetone, DMF, and DMSO. The coupling activator is preferably one or more of tri-n-butylamine, isobutyl chloroformate, dicyclohexylcarbodiimide (DCC), and N-hydroxysuccinimide (NHS).

[0056] In the present invention, when the carrier protein is preferably ovalbumin (OVA), the polar solvent is preferably 1,4-dioxane, and the ratio of the zearalenone (Z) hapten to the polar solvent is preferably 10 mg:1 mL; the coupling activator is preferably tri-n-butylamine and isobutyl chloroformate; the molar ratio of the zearalenone (Z) hapten, tri-n-butylamine, and isobutyl chloroformate is preferably 1:2 to 2.2:1 to 1.2; the activation temperature is preferably room temperature, and the activation time is preferably 12 to 16 hours. In a specific embodiment of the present invention, the hapten is preferably first dissolved in a polar solvent, followed by the addition of tri-n-butylamine, and then the addition of isobutyl chloroformate under ice bath conditions; after the addition of isobutyl chloroformate is completed, the reaction is preferably carried out under ice bath conditions for 0.5 to 1 hour, and then the activation reaction is carried out at room temperature.

[0057] In the present invention, when the carrier protein is preferably bovine serum albumin (BSA), the specific process conditions for the activation are the same as when the carrier protein is ovalbumin (OVA), and only OVA needs to be replaced with BSA, which will not be repeated here.

[0058] In the present invention, in the activated zearalenone (Z) hapten solution, the zearalenone (Z) hapten exists in the form of a zearalenone (Z) hapten activated ester; the hapten activated ester is a key intermediate in the synthesis of two antigens, and the structure of the zearalenone (Z) hapten activated ester is shown in Formula IV:

[0059]

[0060] After obtaining the activated zearalenone (Z) hapten solution, the present invention mixes the activated zearalenone (Z) hapten solution with a buffer solution of a carrier protein to perform a coupling reaction to obtain the zearalenone (Z) antigen. In the present invention, the concentration of the carrier protein in the buffer solution of the carrier protein is preferably 10 to 12 mg / mL; the buffer solution of the carrier protein is specifically obtained by dissolving the carrier protein in a buffer solution; the buffer solution is preferably a disodium hydrogen phosphate-sodium dihydrogen phosphate (PB) buffer solution, the concentration of the PB buffer solution is preferably 0.05 to 0.1 mol / L, and the pH value is preferably 8.0 to 8.5; the molar ratio of the carrier protein to the activated ester of the zearalenone (Z) hapten is preferably 1:45 to 50; in a specific embodiment of the present invention, the activated zearalenone (Z) hapten solution is preferably added dropwise to the buffer solution of the carrier protein; the temperature of the coupling reaction is preferably room temperature, and the time is preferably 6 to 8 hours.

[0061] After the coupling reaction is completed, the present invention preferably centrifuges the obtained reaction solution and dialyzes the obtained supernatant to obtain the zearalenone (Z) antigen. The dialysate used for the dialysis is preferably PB buffer, and the pH of the PB buffer is preferably 6.9 to 7.2; the molecular weight cutoff of the dialysis bag for dialysis is preferably 8000 to 14000 Da; the dialysis time is preferably 2 to 3 days, and when the carrier protein is BSA, the dialysis time is more preferably 3 days; the amount of dialysate used each time is preferably 500 to 800 mL; and the dialysate is preferably replaced 2 to 3 times a day. After the dialysis is completed, the solution in the dialysis bag is preferably collected and aliquoted and frozen for later use.

[0062] The present invention also provides a zearalenone (Z) antibody, which is obtained by immunizing a host animal with the zearalenone (Z) antigen described in the above technical solution or the zearalenone (Z) antigen prepared by the preparation method described in the above technical solution; the host animal is preferably a mouse; the present invention has no special requirements for the immunization method, and methods well known to those skilled in the art can be used; in a specific embodiment of the present invention, the zearalenone (Z) immunogen dilution is preferably emulsified with incomplete Freund's adjuvant before immunizing the host animal; the diluent used for the zearalenone (Z) immunogen dilution is preferably a sterilized 1wt% sodium chloride solution.

[0063] In a specific embodiment of the present invention, the process for preparing the zearalenone (Z) antibody is preferably as follows:

[0064] Immunizing a host animal with the emulsified zearalenone (Z) immunogen to obtain an immune animal, performing a serum test on the immune animal using an ELISA method, and preparing a monoclonal antibody according to a monoclonal antibody preparation method when the serum parameters meet the requirements;

[0065] extracting spleen cells from the immunized animal and fusing them with syngeneic myeloma cells to obtain fused cells;

[0066] performing hybridoma screening on the fusion cells;

[0067] The limiting dilution method is used to clone and culture hybridoma cells, and immunological methods are used to screen positive hybridoma cells that can produce the required monoclonal antibodies and then clone and expand them.

[0068] The present invention also provides a test paper or kit for detecting zearalenone (Z), comprising the zearalenone (Z) antibody described in the above scheme. In the present invention, the kit is preferably a photoluminescence rapid quantitative detection kit; the photoluminescence rapid quantitative detection kit comprises a chemiluminescence detection reagent; the chemiluminescence detection reagent preferably comprises oxygen-accepting microspheres labeled with the zearalenone (Z) antibody, biotin labeled with the zearalenone (Z) antigen, and oxygen-donating microspheres labeled with streptavidin. In the present invention, the oxygen acceptor microspheres labeled with the zearalenone (Z) antibody are obtained by labeling the zearalenone (Z) antibody described in the present invention with the oxygen acceptor microspheres. The present invention has no special requirements for the labeling method and the type of oxygen acceptor microspheres, and those familiar to those skilled in the art can be used; the biotin labeled with the zearalenone (Z) antigen is obtained by labeling the zearalenone (Z) antigen described in the present invention (in a specific embodiment of the present invention, preferably the zearalenone (Z) coating) with biotin. The present invention has no special requirements for the labeling method and biotin, and those familiar to those skilled in the art can be used; the oxygen supply microspheres labeled with streptavidin are obtained by labeling the oxygen supply microspheres with streptavidin, and the oxygen supply microspheres labeled with streptavidin are microspheres familiar to those skilled in the art. In a specific embodiment of the present invention, the oxygen acceptor microspheres labeled with the zearalenone (Z) antibody, the biotin labeled with the zearalenone (Z) antigen, and the oxygen supply microspheres labeled with streptavidin are respectively denoted as reagents R1, R2, and R3.

[0069] In the present invention, the photochemiluminescence rapid quantitative detection kit preferably also includes a sample plate and foam packaging; the sample plate is preferably provided with a sample well in the center, the shape of the sample plate is preferably square, the material is preferably PP material, and the color is preferably black; the shape of the sample well is preferably circular, and the material of the sample well is preferably PVC microporous plate; the foam packaging is preferably a foam pad, and the surface of the foam pad is die-cut with corresponding size holes according to the size of the reagent bottle for fixing the photochemiluminescence detection reagents R1, R2 and R3. In the present invention, the product composition of the photochemiluminescence rapid quantitative detection kit is as follows: Figure 1 As shown, Figure 1 Middle: 1-sample well, 2-foam packaging, 3-R1, 4-R2, 5-R3.

[0070] In the present invention, when using the photochemiluminescence rapid quantitative detection kit for detection, the specific detection method is preferably as follows: adding the sample solution to be tested, reagent R1, reagent R2, and reagent R3 to the sample wells respectively; placing the sample wells in the corresponding positions of the photochemiluminescence detector, detecting the light signal values, and calculating the content of zearalenone (Z) in the sample solution to be tested based on the corresponding relationship between the different light signal values ​​and the concentration of the standard. In a specific embodiment of the present invention, it is preferred to set the detection sample information on the instrument display of the photochemiluminescence detector, and wait for the instrument to automatically read the results.

[0071] In the present invention, the preparation method of the sample to be tested liquid is preferably: the sample is crushed and then sieved to obtain sample powder; the sample powder and the extractant are mixed and vortexed to obtain a sample extraction suspension; the sample extraction suspension is centrifuged or allowed to stand, and the supernatant is taken; the supernatant is mixed with a sample diluent to obtain the sample to be tested liquid; the crushing is preferably performed in a crusher, and the vortexing and centrifugation are preferably performed in a centrifuge tube; the extractant is preferably an ethanol-water solution; the volume ratio of ethanol and water in the ethanol-water solution is preferably 1:4, and the amount ratio of the sample powder and the extractant is preferably 1g:(5-10)mL; the sample diluent is preferably PB buffer, the pH of the PB buffer is preferably 7.2, and the concentration is preferably 0.05mol / L; the volume ratio of the supernatant and the sample diluent is preferably 1:2 to 1:5.

[0072] In the present invention, the photochemiluminescence rapid quantitative detection kit is preferably used to quantitatively detect the content of zearalenone (Z) in feed raw materials or finished feed; the feed raw materials preferably include corn, wheat, flour, wheat bran, soybean meal, peanut meal, rice bran meal, corn gluten meal, corn germ meal, puffed corn, DDGS, sprayed corn husks, waste flour or citric acid residue.

[0073] The present invention also provides the use of the zearalenone (Z) antibody or the test paper or kit described in the above scheme for non-diagnostic detection of zearalenone (Z); the method for detecting zearalenone (Z) is preferably an immunoassay, more preferably an ELLSA kit method or a photochemiluminescence assay. Immunizing mice with the zearalenone (Z) antigen of the present invention produces antibodies with high titer and sensitivity, and their application in the detection of zearalenone (Z) can improve the accuracy and sensitivity of the detection method.

[0074] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0075] Example 1

[0076] Synthesis of Zearalenone (Z) Hapten

[0077] 5.99 g of anhydrous aluminum chloride was added to a clean and dry 250 mL round-bottom flask, and 62.5 mL of 1,2-dichloroethane was added dropwise with stirring at 0°C. After the addition was complete, the mixture was stirred for 10 min. After the solution became clear and there was no white smoke, 2.64 g of glutaric anhydride was added and stirred for 30 min. After 6.13 g of zearalenone (Z) was fully dissolved in 32 mL of 1,2-dichloroethane, the mixture was slowly added dropwise to the reaction system. After the addition was complete, the mixture was refluxed at 80°C and monitored by TLC. After 2 h of reaction, the raw material spot disappeared. The reaction was stopped, the reaction solution was cooled to room temperature, poured into ice cubes made by freezing 94.5 mL of deionized water, stirred for 10 minutes, extracted twice with ethyl acetate, consuming 47 mL of ethyl acetate each time, and the organic phases were combined and washed three times with saturated brine, consuming 47 mL of saturated brine each time, and once with 94 mL of deionized water. The solvent was removed by distillation under reduced pressure, and the residue was dissolved in 94 mL of ethyl acetate. The sample was mixed with 24 g of silica gel and purified by dry column chromatography. The main ultraviolet point component was collected to obtain a light yellow oily liquid, which was the zearalenone (Z) hapten. After testing, the purity of the hapten was 96.7%. The product was subjected to nuclear magnetic resonance detection, and the results were as follows: Figure 2 As shown, according to Figure 2 It can be seen that the product has the target structure.

[0078] Synthesis of Zearalenone (Z) Antigen

[0079] 1: Synthesis of Zearalenone (Z) Coating

[0080] 10 mg of hapten was dissolved in 1 mL of 1,4-dioxane, 6.48 mg of tri-n-butylamine was added, and 3.14 mg of isobutyl chloroformate was added under ice bath. After addition, the reaction was allowed to proceed for 0.5 h, and then the reaction was allowed to proceed at room temperature for 12 h. 22.86 mg of OVA was weighed and dissolved in 1.2 mL of PB buffer (PB buffer concentration was 0.05 mol / L, pH = 8.0). After clarification, the prepared hapten activation solution was added dropwise. After addition, the reaction was allowed to proceed at room temperature for 6 h. The reaction solution was then centrifuged, the supernatant was aspirated, and dialyzed in a dialysis bag with a molecular weight cutoff of 8000-14000 for 2 days. The dialysate was 0.02 mol / L, pH = 6.9 PB buffer. The dialysate was changed twice a day, with 500 mL of dialysate used each time. After dialysis, the solution in the dialysis bag was aspirated and aliquoted and frozen for later use.

[0081] 2: Synthesis of Zearalenone (Z) Immunogen

[0082] The other conditions were the same as those for the synthesis of the zearalenone (Z) coating, except that 22.86 mg of OVA was replaced with 12.24 mg of BSA.

[0083] Example 2

[0084] Synthesis of Zearalenone (Z) Hapten

[0085] 1.77 g of anhydrous aluminum chloride was added to a clean and dry 50 mL round-bottom flask. 12.5 mL of 1,2-dichloroethane was added dropwise with stirring at 5 ° C. After the addition was complete, the mixture was stirred for 15 min. After the solution became clear and there was no white smoke, 0.73 g of glutaric anhydride was added and stirred for 35 min. 1.70 g of zearalenone (Z) was fully dissolved in 6.25 mL of 1,2-dichloroethane and then slowly added dropwise to the reaction system. After the addition was complete, the mixture was refluxed at 85 ° C and monitored by TLC. After 3 h of reaction, the raw material spot disappeared. The reaction was stopped, and the reaction solution was cooled to room temperature and poured into ice cubes made by freezing 18.75 mL of deionized water. The mixture was stirred for 20 min and extracted with ethyl acetate three times, consuming 9.38 mL of ethyl acetate each time. The organic phases were combined and washed with saturated brine four times, consuming 9.38 mL of saturated brine each time, and washed once with 18.75 mL of deionized water. The solvent was removed by distillation under reduced pressure, and the mixture was dissolved in 18.75 mL of ethyl acetate and mixed with 6 g of silica gel. The sample was purified by dry column chromatography, and the main ultraviolet point component was collected to obtain a light yellow oily liquid, which was the zearalenone (Z) hapten. The purity of the hapten was 90.2% after testing.

[0086] Synthesis of Zearalenone (Z) Antigen

[0087] 1: Synthesis of Zearalenone (Z) Coating

[0088] 15 mg of hapten was dissolved in 1.5 mL of 1,4-dioxane, 13.6 mg of tri-n-butylamine was added, and 5.25 mg of isobutyl chloroformate was added under ice bath. After addition, the reaction was allowed to proceed for 1 h, and then the reaction was allowed to proceed at room temperature for 15 h. 32.2 mg of OVA was weighed and dissolved in 3 mL of PB buffer (PB buffer concentration was 0.08 mol / L, pH = 8.3). After clarification, the prepared hapten activation solution was added dropwise. After addition, the reaction was allowed to proceed at room temperature for 7 h. The reaction solution was then centrifuged, the supernatant was aspirated, and dialyzed in a dialysis bag with a molecular weight cutoff of 8000-14000 for 3 days. The dialysate was 600 mL of 0.04 mol / L, pH = 7.2 PB buffer. The dialysate was changed 3 times a day. After dialysis, the solution in the dialysis bag was aspirated and frozen for later use.

[0089] 2: Synthesis of Zearalenone (Z) Immunogen

[0090] The other conditions were the same as those for the synthesis of the zearalenone (Z) coating, except that 22.86 mg OVA was replaced with 18.37 mg BSA.

[0091] Example 3

[0092] Synthesis of Zearalenone (Z) Hapten

[0093] 0.33 g of anhydrous aluminum chloride was added to a clean and dry 25 mL round-bottom flask. 6.25 mL of 1,2-dichloroethane was added dropwise with stirring at 3°C. After the addition was complete, the mixture was stirred for 12 min. When the solution became clear and there was no white smoke, 0.133 g of glutaric anhydride was added and stirred for 40 min. 0.31 g of zearalenone (Z) was fully dissolved in 3.125 mL of 1,2-dichloroethane and then slowly added dropwise to the reaction system. After the addition was complete, the mixture was refluxed at 85°C and monitored by TLC. After 3 h of reaction, the raw material spot disappeared. Stop the reaction, cool the reaction solution to room temperature, pour into ice cubes of the same volume as the reaction solution, stir for 15 minutes, extract with ethyl acetate three times, consuming 4.69 mL of ethyl acetate each time, combine the organic phases, wash with saturated brine three times, consuming 4.69 mL of saturated brine each time, and then wash once with 9.3 mL of deionized water. Remove the solvent by distillation under reduced pressure, dissolve in 9.3 mL of ethyl acetate, mix with 1.5 g of silica gel, purify by dry column chromatography, collect the main ultraviolet point component, and obtain a light yellow oily liquid, which is the zearalenone (Z) hapten.

[0094] Synthesis of Zearalenone (Z) Antigen

[0095] 1: Synthesis of Zearalenone (Z) Coating

[0096] 20 mg of hapten was dissolved in 2 mL of 1,4-dioxane, 16 mg of tri-n-butylamine was added, and 7.54 mg of isobutyl chloroformate was added under ice bath. After addition, the reaction was allowed to proceed for 40 min, and then the reaction was allowed to proceed at room temperature for 14 h. 41.16 mg of OVA was weighed and dissolved in 4 mL of PB buffer (PB buffer concentration was 0.1 mol / L, pH = 8.5). After clarification, the prepared hapten activation solution was added dropwise. After addition, the reaction was allowed to proceed at room temperature for 6.5 h. The reaction solution was then centrifuged, the supernatant was aspirated, and dialyzed in a dialysis bag with a molecular weight cutoff of 8000-14000 for 2.5 days. The dialysate was 800 mL of 0.03 mol / L, pH = 7.1 PB buffer. The dialysate was changed three times a day. After dialysis, the solution in the dialysis bag was aspirated and frozen for later use.

[0097] 2: Synthesis of Zearalenone (Z) Immunogen

[0098] The other conditions were the same as those for the synthesis of the zearalenone (Z) coating, except that 22.86 mg OVA was replaced with 24.49 mg BSA.

[0099] Test Example 1 ELISA test

[0100] Immunization of mice: Fifteen purebred BALB / c mice, aged 4-6 weeks, were selected and color-coded 1-5. Six 2mL syringes were used. Three were filled with 0.5mL of incomplete Freund's adjuvant, and the remaining three were filled with 0.5mL of zearalenone (Z) immunogen diluted with sterile 1% sodium chloride solution, respectively, at concentrations of 50μg, 100μg, and 200μg. After labeling, the syringes for the incomplete Freund's adjuvant and immunogen were connected with infusion tubing (the length should be long enough to align the two syringes). The syringes were then manually pushed back and forth until complete emulsification was achieved (emulsification was indicated by a white, milky liquid, slight resistance during pushing, and small, non-spreading droplets). The emulsification process was continued until complete, as this would prevent emulsification. Once emulsification was complete, multiple subcutaneous injections (on the back and abdomen) were performed, with approximately 0.2mL injected per mouse. The same method is repeated every 2 weeks, and blood sampling test is started after completing 3 times;

[0101] Blood collection method: Grab the mouse's scalp by the neck with the thumb, index finger, and middle finger of your left hand, and secure the tail with the pinky and ring fingers. Gently press the skin around the eye to be removed, causing the eyeball to become congested and protrude. Insert a glass capillary vertically through the mouse's eyeball, gently rotating it left and right until blood flows into the capillary (approximately 2 / 3 of the way into the capillary). Remove the blood from the capillary and blow it into a 0.5 mL centrifuge tube (store at low temperature).

[0102] After blood collection, the samples were placed in a 37°C oven for 30 min, and then centrifuged in a low-temperature high-speed centrifuge (10,000 rpm) for 10 min to separate the serum for ELISA testing.

[0103] Monoclonal antibody preparation process:

[0104] Cell fusion: Mice are killed using carbon dioxide gas, and the spleens are aseptically removed and crushed in a dish to prepare a splenocyte suspension. Prepared syngeneic myeloma cells are mixed with mouse spleen cells in a specific ratio, and the fusion promoter polyethylene glycol is added. Under the influence of polyethylene glycol, various lymphocytes can fuse with myeloma cells to form hybridoma cells.

[0105] Selective culture: The purpose of selective culture is to screen for fused hybridoma cells, typically using HAT selective medium. In HAT medium, unfused myeloma cells, lacking hypoxanthine-guanine phosphoribosyltransferase (HPT), cannot synthesize DNA using the salvage pathway and die. Although unfused lymphocytes possess HPT, they cannot survive long-term in vitro and gradually die. Only fused hybridoma cells, which inherit HPT from spleen cells and possess the indefinite proliferation characteristics of myeloma cells, can survive and proliferate in HAT medium.

[0106] Screening and cloning of positive hybridoma clones: Only a minority of hybridoma cells grown in HAT medium secrete the desired specific monoclonal antibody, so screening and cloning are essential. The limiting dilution method is typically used for hybridoma cell cloning. Using sensitive, rapid, and specific immunological methods, positive hybridoma cells that produce the desired monoclonal antibody are screened and cloned for expansion. After comprehensive characterization of the immunoglobulin type, subclass, specificity, affinity, antigen epitope recognition, and molecular weight of the secreted monoclonal antibody, the cells are promptly cryopreserved.

[0107] To prepare large quantities of monoclonal antibodies, Balb / c mice are pretreated by intraperitoneal injection of 0.5 ml of liquid paraffin or pristane. One to two weeks later, hybridoma cells are inoculated intraperitoneally. The hybridoma cells proliferate within the mouse peritoneal cavity and produce and secrete monoclonal antibodies. After approximately one to two weeks, the mouse abdomen will become visibly enlarged. Ascites fluid is then withdrawn with a syringe to obtain large quantities of monoclonal antibodies.

[0108] ELISA testing of newly synthesized antigens and preparation of antibodies

[0109] 1. The reagent formula used is as follows:

[0110] Blocking solution (1 L volume): 2.5 g skim milk powder, 8 g sodium chloride, 0.6 g potassium dihydrogen phosphate, 5.8 g disodium hydrogen phosphate, 50 g sucrose, 0.9 g potassium chloride, 0.5 mL preservative, 50 mL bovine serum, and the balance is water.

[0111] Enzyme dilute (16 L): sodium chloride 128 g, potassium dihydrogen phosphate 9.48 g, disodium hydrogen phosphate 92.8 g, glycerol 800 mL, bovine serum 3200 mL, preservative 12 mL, water 12 L.

[0112] Sample dilution (4 L): sodium chloride 23.38 g, potassium chloride 0.118 g, potassium dihydrogen phosphate 3.48 g, sodium dihydrogen phosphate 2.19 g, sodium hydrogen phosphate 20.64 g, Triton 40 mL, preservative 1.5 mL, and the balance is water.

[0113] Substrate A solution (100 mL): 0.48 g citric acid, 0.05 g urea peroxide, 90 mL glacial acetic acid, and the balance water.

[0114] Substrate B solution (400 mL): 0.164 g citric acid, 0.2 g TMB hydrochloride, 4 mL N,N-dimethylformamide, 14 mL methanol, 160 μL 1 mol / L hydrochloric acid, and the balance water.

[0115] 20× concentrated washing solution (40 L): 2560 g sodium chloride, 928 g disodium hydrogen phosphate, 80 g potassium dihydrogen phosphate, 1.44 g potassium chloride, 12 mL preservative, 800 mL Tween 20, and the balance water.

[0116] 2. The specific test methods are as follows:

[0117] (1) Antigen coating: dilute the zearalenone (Z) coating agent to a ratio of 1K:2W using CB buffer. After dilution, add 100 μL / well to the ELISA microplate, cover with a cover film, and incubate at 37°C for 2 h.

[0118] (2) Blocking: After coating, remove and discard the coating solution and use washing working solution (20× concentrated washing solution diluted to 1×) at 250 μL / well or wash once with a washing pot, soak for 30 seconds, discard the solution and pat dry with absorbent paper or towel, add blocking solution at 150 μL / well, place in a 37°C constant temperature incubator for reaction for 2 hours, remove and discard the blocking solution and pat dry for use.

[0119] (3) Dilution of different standard concentrations: Use 0.02 mol / L PBS1 buffer to dilute the directly purchased 100 μg / mL high-standard zearalenone (Z) to 0 ppb (directly PBS1 buffer), 1 ppb / mL, 10 ppb / mL, and 100 ppb / mL.

[0120] (4) Dilution of enzyme-labeled secondary antibody working solution: Dilute the directly purchased enzyme-labeled secondary antigen solution to a concentration of 1:1K with enzyme dilution buffer.

[0121] (5) Dilution of serum or monoclonal antibody working solution: dilute the serum or prepared monoclonal antibody to a concentration of 1:1000, 1:5000, 1:20000, or 1:100000 using sample dilution buffer.

[0122] (6) Testing steps: Test according to the chessboard method. The specific steps are as follows:

[0123] 1) Add standard / sample: Add 50 μL of standard / sample to the corresponding microwells.

[0124] 2) Serum or monoclonal antibody working solution: Add 50 μL of serum or monoclonal antibody working solution to each well, gently shake to mix, cover the plate with a cover film, and incubate at 37°C in a dark environment for 30 min.

[0125] 3) Wash the plate and add enzyme-labeled secondary antibody working solution: Carefully peel off the cover film, shake off the liquid in the wells, add 250μL / well of washing working solution, wash thoroughly 4-5 times, with 10 seconds between each wash, discard the washing solution in the wells, pat dry with absorbent paper (air bubbles that are not removed after patting dry can be punctured with an unused pipette tip), add 100μL / well of enzyme-labeled secondary antibody working solution, cover the plate with cover film, and incubate at 37℃ in a dark environment for 30 minutes.

[0126] 4) Washing: Carefully peel off the cover film, shake off the liquid in the wells, add 250 μL / well of washing solution, and wash thoroughly 4-5 times, with 10 seconds between each wash. Pour off the washing solution in the wells and pat dry with absorbent paper (air bubbles that are not removed after patting dry can be punctured with an unused pipette tip).

[0127] 5) Color development: Add 50 μL / well of substrate solution A and solution B, gently shake to mix, cover the plate with a cover film, and incubate at 37°C in a dark environment for 15 minutes.

[0128] 6) Determination: Add 50 μL / well of stop solution, gently shake to mix, set the microplate reader at 450 nm, and measure the OD value of each well.

[0129] When the relevant technical parameters are met and the OD value of the maximum antibody dilution is around 2.0, the maximum dilution factor of the antibody is the antibody titer.

[0130] The test results are shown in Tables 1 to 5.

[0131] Table 1 2 Serum test data after immunization

[0132]

[0133] Table 2 3 Serum test data after immunization

[0134]

[0135] Table 3 4 Serum test data after immunization

[0136]

[0137] Table 4 Whole serum test data

[0138]

[0139] Table 5 Preparation of monoclonal antibody test data

[0140]

[0141]

[0142] According to the data in Tables 1 to 3, it can be seen that the concentration of zearalenone (Z) antibodies in the serum gradually increases with the increase in the number of immunizations; according to the data in Table 4, it can be seen that the concentration of zearalenone (Z) in the serum has basically reached 1:20000, the antigen concentration is 1:1000 or the serum concentration is 1:1000, the antigen concentration is 1:20000, IC 50 According to the results in Table 5, the titer of the prepared monoclonal antibody was 1:5000, and the half-inhibitory concentration (IC 50 ) is less than 10ppb / mL.

[0143] Test Example 2

[0144] Photoluminescence rapid quantitative detection kit for zearalenone (Z) in cereal feed

[0145] A rapid quantitative detection kit for zearalenone (Z) in cereal feed, comprising: Figure 1 shown.

[0146] Its components and functions are shown in Table 6:

[0147] Table 6 Product composition and specifications

[0148]

[0149] Sample well: The outer square of the sample well is made of black PP material. Its main function is to fix the middle sample well and put it into the photochemiluminescence detection instrument for result testing. The middle circular hole is a PVC microplate, which is mainly used to hold the sample to be tested and as a reaction container.

[0150] Foam packaging: The main material is foam pad, and the corresponding size holes are die-cut according to the size of the reagent bottle to fix the photochemiluminescence detection reagent (R1, R2, R3);

[0151] Reagent R1: oxygen acceptor microspheres labeled with zearalenone (Z) antibody (the oxygen acceptor microspheres were purchased directly from Beijing Kemei Biotechnology Co., Ltd., and the zearalenone (Z) antibody was the antibody obtained in Test Example 1);

[0152] Reagent R2: biotin for labeling the zearalenone (Z) antigen (biotin was purchased directly from Sigma, and the zearalenone (Z) antigen was the zearalenone (Z) coating agent prepared in Example 1);

[0153] R3 reagent: oxygen-donating microspheres labeled with streptavidin (purchased directly from Beijing Kemei Biotechnology Co., Ltd.).

[0154] The relevant data of the zearalenone (Z) curve are shown in Table 7:

[0155] Table 7 Zearalenone curve

[0156] Standard concentration (ng / g) Absorbance value 0 79543 50 73538 150 65097 450 40056 900 11431

[0157] Calibration curve fitting equation: y = -75.12x + 77220, R 2 =0.99, where x is the concentration of mithralenone (Z) in ng / g, and y is the absorbance value.

[0158] Kit performance

[0159] 1. Quantitative detection range: 25~800μg / kg

[0160] 2. Accuracy: 80% to 120%

[0161] 3. Precision: CV ≤ 15%

[0162] 4. Detection time: 20 minutes (5 minutes pre-treatment + 15 minutes incubation)

[0163] 5. Test specifications: 48 samples

[0164] Required equipment and consumables

[0165] (1) Photochemiluminescence immunoassay

[0166] (2) Crusher

[0167] (3) Pipette (20-200 μL, 100-1000 μL)

[0168] (4) Centrifuge

[0169] (5) Balance: Sensitivity 0.01g

[0170] (6) Timer

[0171] (7) Crusher

[0172] (8) 50mL centrifuge tube

[0173] (9) 2mL polystyrene centrifuge tube

[0174] Preparation before testing

[0175] 1. Return samples, reagents, and sample diluents to room temperature before use (reagents take about 30 minutes to return to room temperature);

[0176] 2. Turn on the light-stimulated chemiluminescence detector and preheat it in advance in preparation for detection.

[0177] Sample preparation steps

[0178] 1. The sample should be returned to room temperature of 18-25°C before testing;

[0179] 2. Sample processing method:

[0180] (1) The sample was fully crushed using a crusher (passed through a 20-mesh sieve);

[0181] (2) Take 2.0 ± 0.05 g of the crushed sample and place it in a 50 mL centrifuge tube. Add 10 mL of sample extract (20% ethanol-water solution (v / v = 1:4)), vortex and shake for 1 min. Place in a centrifuge and centrifuge at 3000 rpm.

[0182] (3) Take 200 μL of supernatant and add it to 400 μL of sample diluent, shake and mix to obtain the sample solution for testing.

[0183] Detection steps

[0184] 1. Add 100 μL of sample solution, 70 μL each of reagent R1, reagent R2, and reagent R3 to the sample wells;

[0185] 2. Place the sample well in the corresponding position of the light-excited chemiluminescence detector, insert the pipette tip in the corresponding position, and cover the machine;

[0186] 3. Set the test sample information on the instrument display and wait for the instrument to automatically read the zearalenone (Z) test concentration value.

[0187] Precautions

[0188] 1. The test strip box should be refrigerated at 4°C away from light. Avoid strong light when taking reagents R1, R2, and R3 (the test strip box is the test kit);

[0189] 2. The operation should be carried out strictly according to the instructions. Do not mix different batches of reagent kits and sample diluents;

[0190] 3. The extracted sample solution should be tested within 4 hours. The diluted sample should only be tested once and should be diluted again when retesting is required.

[0191] 4. Consumables such as pipette tips and centrifuge tubes cannot be reused to avoid cross contamination.

[0192] Storage and shelf life

[0193] 1. Storage conditions: Store the kit in a cool, dark, and dry place at 4°C.

[0194] 2. Validity period: This product is valid for 6 months.

[0195] Test Example 3

[0196] Verification of technical parameters of the photoluminescence rapid quantitative detection kit for zearalenone (Z) in cereal feed

[0197] 1. Evaluation indicators

[0198] Evaluation indicators include four dimensions: detection limit, false negative rate, false positive rate, and instrument method compliance rate.

[0199] 2. Evaluation Method

[0200] (1) Detection limit

[0201] Take blank test samples from a batch of products and add 1 / 2 detection limit, 1 times detection limit, 2 times detection limit of the test drug standard, and blank samples, 10 copies each, and measure with the test products.

[0202] (2) False negative rate

[0203] Three batches of the product to be tested were used to test 40 positive samples (20 samples with the product to be tested added at the detection limit and 20 samples with the product to be tested added at a concentration twice the detection limit). The number of negative samples / total number of samples × 100% is the false negative rate (the average false negative rate of the three batches of products).

[0204] (3) False positive rate

[0205] Three batches of the test product were used to test 40 negative samples (20 negative blank samples of the test product and 20 samples with the test product added at a concentration of 1 / 2 the detection limit). The number of test-positive samples / total number of samples × 100% is the false positive rate.

[0206] (IV) Instrument and method compliance

[0207] One batch of products was used to measure 20 samples (10 negative and 10 positive) that had been tested using national standard instrument methods.

[0208] (5) Coefficient of variation

[0209] It includes at least 3 batches of product testing, and 10 positive samples (10 samples with the product to be tested added at the detection limit) are tested for each batch of product.

[0210] The detection method and sample pre-treatment method in the above evaluation were carried out in accordance with Test Example 2.

[0211] 3. Evaluation Results

[0212] (1) Detection limit

[0213] Table 8 Detection limit determination results

[0214]

[0215]

[0216] Note: The qualified range of recovery rate of spike concentration is 80%-120%.

[0217] (2) False negative rate

[0218] Table 9 False negative rate determination results

[0219]

[0220]

[0221] Note: The false negative rate must be less than 5%.

[0222] (3) False positive rate

[0223] Table 10 False positive rate determination results

[0224]

[0225] Note: The false positive rate must be less than 10%.

[0226] (IV) Instrument method compliance rate

[0227] Table 11 Instrument method compliance test results

[0228]

[0229] Note: A positive or negative conformity rate of not less than 90% is considered qualified. The national standard number of the instrument method is GB5009.209-2016 National Food Safety Standard Determination of Zearalenone in Food Method 1, and the rapid test product is the kit of the present invention.

[0230] (5) Coefficient of variation

[0231] Table 12 Coefficient of variation determination results

[0232]

[0233] Note: Coefficient of variation = standard deviation / mean value × 100%

[0234] According to the data in Tables 7 to 11, it can be seen that the photochemiluminescence rapid quantitative detection kit for zearalenone (Z) of the present invention has a low minimum detection limit for zearalenone (Z), high sensitivity, and the false negative rate, false positive rate, and instrument method dimensions are all qualified.

[0235] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A zearalenone (Z) hapten, characterized in that: It has the structure shown in formula I: Formula I; In formula I: n2 is 3.

2. The method for preparing the zearalenone (Z) hapten according to claim 1, characterized in that: The following steps are involved: Zearalenone (Z), a cyclic carboxylic acid anhydride and a catalyst are mixed to carry out a Friedel-Crafts acylation reaction to obtain a zearalenone (Z) hapten having a structure described in Formula I; the cyclic carboxylic acid anhydride is glutaric anhydride.

3. The preparation method according to claim 2, characterized in that The temperature of the Friedel-Crafts acylation reaction is 80-85°C, and the reaction time is 2-3 hours; The molar ratio of the zearalenone (Z), the catalyst and the glutaric anhydride is 1:2-2.5:1-1.

2.

4. A zearalenone (Z) antigen, characterized in that: The invention comprises the zearalenone (Z) hapten according to claim 1 and a carrier protein coupled to the zearalenone (Z) hapten.

5. The zearalenone (Z) antigen according to claim 4, characterized in that The coupling is an amide bond coupling.

6. The method for preparing the zearalenone (Z) antigen according to any one of claims 4 to 5, characterized in that: The following steps are involved: The zearalenone (Z) hapten according to claim 1, a coupling activator and a polar solvent are mixed and activated to obtain an activated zearalenone (Z) hapten solution; The activated zearalenone (Z) hapten solution and the carrier protein buffer solution are mixed to carry out coupling reaction to obtain the zearalenone (Z) antigen.

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