Flusilazole hapten, complete antigen, antibody and preparation method and application thereof

Abstract

The present invention belongs to the technical field of immunoassay, and particularly relates to a flusilazole hapten, a complete antigen, an antibody, and a preparation method and application thereof. The present invention provides a flusilazole hapten having a structure shown in Formula I: R1 in Formula I is a linking arm capable of conjugating with a carrier protein in the flusilazole hapten structure. On the basis of retaining the basic structure of flusilazole, the flusilazole hapten provided by the present invention connects a linking arm and an active reaction group structure to the 5C atom in the "1,2,4-triazole" group structure of flusilazole. This not only effectively avoids non-specific binding caused by the linking arm, but also enables the characteristic structure of the flusilazole main body molecule to be fully exposed after the introduced linking arm and active reaction group are conjugated with the carrier protein. The prepared flusilazole complete antigen has immunogenicity, and the prepared anti-flusilazole antibody can be used to establish a flusilazole immunological detection method or detection reagent, having broad application prospects. #imgabs0#

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay, and specifically relates to flusilazole hapten, complete antigen, antibody, and preparation method and application thereof. Background Art

[0002] Flusilazole is called Flusilazole in English, its chemical name is: bis(4-fluorophenyl)methyl(1H-1,2,4-oxazol-1-ylmethylene)silane, its CAS registration number is: 85509-19-9, and its molecular formula is: C 16 H 15 F 2 N 3 Si, molecular weight: 315.4, Chinese aliases: Fuxing, Kejunxing, trade names: Nustar, Olymp, Punch, Fuxing, Kejunxing, Qiufu.

[0003] Flusilazole is a triazole systemic fungicide that has both protective and therapeutic effects and strong permeability. It can prevent and treat diseases caused by ascomycetes, basidiomycetes and some imperfect fungi. However, unreasonable flusilazole application methods and dosages will cause environmental pollution and pesticide residue problems. Residual flusilazole can be continuously transferred and migrated in the food chain through enrichment, thus causing adverse effects on the quality and safety of agricultural products and the ecological environment, and thus endangering human health. Therefore, it is necessary to monitor the residual level of flusilazole.

[0004] The detection methods for flusilazole in the national standard GB 2763-2021 are all instrumental analysis methods. Instrumental analysis methods have sufficient accuracy and high sensitivity, such as liquid chromatography (LC), LC-tandem mass spectrometry (LC-MS / MS), gas chromatography (GC) and GC-tandem mass spectrometry (GC-MS / MS), which are commonly used detection methods. However, these instrumental analysis methods usually require well-trained personnel, expensive instruments, complex sample pretreatment and time-consuming analysis, which are not suitable for on-site flusilazole residue detection. Immunoassay methods are due to their specific recognition of antigens and antibodies.

[0005] The immunoassay method has the advantages of being rapid, simple, real-time, easy to conduct on-site detection, simple sample pretreatment, high sensitivity, strong selectivity, and suitable for high-throughput analysis. It can also significantly reduce the cost of detection. Therefore, the preparation of flusilazole antibodies is of great significance for the establishment of flusilazole immunoassay methods. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a flusilazole hapten, a complete antigen, an antibody preparation method and application. The flusilazole hapten provided by the present invention can fully highlight the flusilazole antigenic determinant cluster after coupling, and the prepared flusilazole complete antigen has a higher specific recognition of the characteristic three-dimensional structure and immunoactive chemical groups of flusilazole, so that the obtained flusilazole antibody has good specificity and high sensitivity, and can be used for immunoassay detection of flusilazole.

[0007] The present invention provides a flusilazole hapten having a structure shown in Formula I:

[0008]

[0009] In the formula I, R 1 It is the connecting arm and active reaction group in the flusilazole hapten structure that can be coupled with the carrier protein.

[0010] Preferably, the R 1 Includes-(CH 2 ) n -COOH, -(CH 2 ) n -NH 2 、-(CH 2 ) n -OH, -(CH 2 ) n -COOR 2 , n is 0 to 6, the -(CH 2 ) n -COOR 2 R 2 include

[0011] Preferably, the flusilazole hapten has a structure shown in Formula I-1 or Formula I-2:

[0012]

[0013] The present invention provides a method for preparing the flusilazole hapten described in the above technical solution, comprising the following steps:

[0014] In the compound structure shown in Formula II, R 3 The group is replaced by R through chemical reaction 1 Group, to obtain the flusilazole hapten represented by formula I;

[0015]

[0016] In the formula II, R 3 H or

[0017] Preferably, the method for preparing the flusilazole hapten shown in formula I-1 comprises the following steps:

[0018] The compound represented by Formula 1, benzyl alcohol and p-toluenesulfonic acid are dissolved in an organic solvent, and a first nucleophilic substitution reaction is performed to obtain a compound represented by Formula 2;

[0019]

[0020] Dissolving the compound represented by Formula 2 and triphenylphosphine in an organic solvent, and performing a first addition reaction to obtain a compound represented by Formula 3;

[0021]

[0022] The compound of Formula 4 and isopropylmagnesium bromide are dissolved in an organic solvent to carry out a nucleophilic addition reaction to obtain a nucleophilic addition reaction solution, and the nucleophilic addition reaction solution and the organic solvent are mixed to continue the reaction to obtain a compound of Formula 5;

[0023]

[0024] In a protective gas atmosphere, dissolving the compound represented by Formula 5, the compound represented by Formula 3 and sodium hydride in an organic solvent for a second addition reaction to obtain a compound represented by Formula 6;

[0025] The compound represented by Formula 6 and a palladium-carbon catalyst are dissolved in an organic solvent, and a reduction reaction is carried out in a reducing gas atmosphere to obtain a flusilazole hapten having a structure represented by Formula I-1;

[0026]

[0027] The present invention provides a complete flusilazole antigen, which is obtained by coupling the flusilazole hapten described in the above technical solution or the flusilazole hapten prepared by the preparation method described in the above technical solution with a carrier protein.

[0028] The present invention provides a flusilazole antibody, which is obtained by emulsifying the flusilazole complete antigen described in the above technical solution and then immunizing a host animal.

[0029] The present invention provides a method for preparing the flusilazole antibody described in the above technical solution, comprising the following steps:

[0030] emulsifying the flusilazole complete antigen described in the above technical scheme and immunizing the host animal;

[0031] The preparation method of flusilazole polyclonal antibody comprises: after boosting immunization of an immune host animal, collecting blood of the immune animal and separating the blood to obtain the flusilazole polyclonal antibody;

[0032] The method for preparing flusilazole monoclonal antibody comprises: extracting spleen cells from an immunized host animal, fusing the spleen cells with SP2 / 0 tumor cells, screening monoclonal hybridoma cell lines capable of secreting flusilazole antibodies, and preparing the flusilazole monoclonal antibody based on the monoclonal hybridoma cell lines;

[0033] The preparation method of flusilazole nanoantibody is as follows: extract mRNA transcribed from B lymphocytes in the blood of immunized host animals, reverse transcribe it into cDNA, use cDNA as substrate for PCR amplification to obtain diversified nanoantibody gene fragments, and then construct a phage or yeast antibody expression library with the nanoantibody gene fragments, and obtain flusilazole nanoantibody after screening.

[0034] The present invention provides a reagent or a kit for detecting flusilazole, which contains the flusilazole antibody described in the above technical solution or the flusilazole antibody prepared by the preparation method described in the above technical solution.

[0035] The present invention provides use of the flusilazole antibody described in the above technical solution or the flusilazole antibody prepared by the preparation method described in the above technical solution or the reagent or kit described in the above technical solution in detecting flusilazole.

[0036] The present invention provides a flusilazole hapten having a structure shown in Formula I:

[0037]

[0038] In the formula I, R 1 It is the connecting arm and active reaction group in the flusilazole hapten structure that can be coupled with the carrier protein.

[0039] The flusilazole hapten provided by the present invention connects a connecting arm and an active reaction group to the 5C atom in the "1,2,4-triazole" group structure of flusilazole on the basis of retaining the basic structure of flusilazole, which can not only effectively avoid the nonspecific binding caused by the connecting arm, but also the introduced connecting arm and active reaction group can fully expose the characteristic structure of the flusilazole main molecule after coupling with the carrier protein, and the prepared flusilazole complete antigen has higher specificity.

[0040] The present invention provides a flusilazole complete antigen, wherein the flusilazole hapten is prepared by the preparation method described in the above technical scheme, and the active group of the flusilazole hapten is activated and coupled with a carrier protein to obtain the flusilazole hapten. The anti-flusilazole serum or anti-flusilazole antibody obtained by immunization with the flusilazole complete antigen provided by the present invention can efficiently recognize and specifically bind to flusilazole.

[0041] The present invention provides an anti-flusilazole antibody, which can stimulate the host animal body to produce anti-flusilazole antibodies with good sensitivity and specificity after the flusilazole complete antigen described in the above technical solution is emulsified and immunized to the host animal, and can provide a technical basis for establishing the immune application of flusilazole. The flusilazole antibody provided by the present invention can achieve specific recognition and binding to flusilazole, has good specificity for flusilazole and a low minimum detection limit, can be used to detect flusilazole, and provides technical support for the rapid detection of flusilazole residues in fruits, vegetables, and grains. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The synthetic route of the flusilazole hapten shown in Formula I-1 in Example 1;

[0043] Figure 2 The synthetic route of the flusilazole hapten shown in formula I-2 in Example 2;

[0044] Figure 3 The synthetic route of the flusilazole complete antigen represented by Formula III of the present invention;

[0045] Figure 4 is a liquid chromatogram of the flusilazole hapten represented by formula I-1 in Example 1;

[0046] Figure 5 is the flusilazole hapten of formula I-1 in Example 1 1 H NMR spectrum;

[0047] Figure 6 This is the MALDI-TOF-MS spectrum of the OVA standard in Example 3;

[0048] Figure 7 This is the MADLI-TOF-MS spectrum of the complete flusilazole antigen represented by formula III-1 in Example 3;

[0049] Figure 8 This is the MADLI-TOF-MS spectrum of the BSA standard in Example 4;

[0050] Fig. 9 This is the MALDI-TOF-MS spectrum of the complete flusilazole antigen represented by formula III-2 in Example 4. DETAILED DESCRIPTION

[0051] The present invention provides a flusilazole hapten having a structure shown in Formula I:

[0052]

[0053] In the formula I, R 1 It is the connecting arm and active reaction group in the flusilazole hapten structure that can be coupled with the carrier protein.

[0054] In the present invention, the R 1 Preferably, it includes -(CH 2 ) n -COOH, -(CH 2 ) n -NH 2 、-(CH 2 ) n -OH, -(CH 2 ) n -COOR 2 , n is 0 to 6, the -(CH 2 ) n -COOR 2 R 2 include

[0055] In the present invention, the flusilazole hapten preferably has a structure shown in Formula I-1 or Formula I-2:

[0056]

[0057] The present invention provides a method for preparing the flusilazole hapten described in the above technical solution, comprising the following steps:

[0058] In the compound structure shown in Formula II, R 3 The group is replaced by R through chemical reaction 1 Group, to obtain the flusilazole hapten represented by formula I;

[0059]

[0060] In the formula II, R 3 H or

[0061] In the present invention, the method for preparing the flusilazole hapten represented by formula I-1 preferably comprises the following steps:

[0062] The compound represented by Formula 1, benzyl alcohol and p-toluenesulfonic acid are dissolved in an organic solvent (hereinafter referred to as the first organic solvent), and a first nucleophilic substitution reaction is performed to obtain a compound represented by Formula 2;

[0063]

[0064] The compound represented by Formula 2 and triphenylphosphine are dissolved in an organic solvent (hereinafter referred to as the second organic solvent) to perform a first addition reaction to obtain a compound represented by Formula 3;

[0065]

[0066] The compound of Formula 4 and isopropyl magnesium bromide are dissolved in an organic solvent (hereinafter referred to as the third organic solvent) to carry out a nucleophilic addition reaction to obtain a nucleophilic addition reaction solution, and the nucleophilic addition reaction solution and an organic solvent (hereinafter referred to as the fourth organic solvent) are mixed and reacted to obtain a compound of Formula 5;

[0067]

[0068] In a protective gas atmosphere, the compound represented by Formula 5, the compound represented by Formula 3, and sodium hydride are dissolved in an organic solvent (hereinafter referred to as the fifth organic solvent) to carry out a second addition reaction to obtain a compound represented by Formula 6;

[0069] The compound represented by Formula 6 and the palladium-carbon catalyst are dissolved in an organic solvent (hereinafter referred to as the sixth organic solvent), and a reduction reaction is carried out in a reducing gas atmosphere to obtain a flusilazole hapten having a structure represented by Formula I-1;

[0070]

[0071] The present invention dissolves the compound shown in Formula 1, benzyl alcohol and p-toluenesulfonic acid in a first organic solvent, and performs a first nucleophilic substitution reaction to obtain the compound shown in Formula 2.

[0072] In the present invention, the second organic solvent is preferably cyclohexane.

[0073] In the present invention, the molar ratio of the compound represented by Formula 1 to benzyl alcohol is preferably 51:66.

[0074] In the present invention, the molar ratio of the compound represented by Formula 1 to p-toluenesulfonic acid is preferably 51:5.1.

[0075] In the present invention, the ratio of the amount of the compound represented by Formula 1 to the volume of the first organic solvent is preferably 51 mmol:70 mL.

[0076] In the present invention, the first nucleophilic substitution reaction is preferably carried out under heating reflux conditions.

[0077] In the present invention, the insulation time of the first nucleophilic substitution reaction is preferably 16 hours.

[0078] In the present invention, the water generated by the first nucleophilic substitution reaction is preferably separated.

[0079] In the present invention, after the first nucleophilic substitution reaction, a first nucleophilic substitution reaction liquid is obtained. In the present invention, the first nucleophilic substitution reaction liquid is preferably post-treated to obtain a compound shown in Formula 2. In the present invention, the post-treatment preferably includes: washing, drying and solvent removal in sequence. In the present invention, the washing is preferably washed with a saturated sodium bicarbonate aqueous solution. In the present invention, the drying is preferably dried with anhydrous sodium sulfate. In the present invention, the specific embodiment of the solvent removal is preferably distillation under reduced pressure.

[0080] In the present invention, the compound represented by formula 2 is specifically 4-bromobutyric acid benzyl ester.

[0081] After obtaining the compound represented by Formula 2, the present invention dissolves the compound represented by Formula 2 and triphenylphosphine in a second organic solvent, and performs a first addition reaction to obtain the compound represented by Formula 3.

[0082] In the present invention, the second organic solvent is preferably toluene.

[0083] In the present invention, the molar ratio of the compound represented by Formula 2 to triphenylphosphine is preferably 49.9:50.

[0084] In the present invention, the ratio of the amount of the compound represented by Formula 2 to the volume of the second organic solvent is preferably 49.9 mmol:200 mL.

[0085] In the present invention, the first addition reaction is preferably carried out under heating reflux conditions.

[0086] In the present invention, the insulation time of the first addition reaction is preferably 16 hours.

[0087] In the present invention, after the first addition reaction, a first addition reaction liquid is obtained. In the present invention, the first addition reaction liquid is preferably post-treated to obtain a compound shown in Formula 3. In the present invention, the post-treatment preferably includes: performing, recrystallization and drying in sequence. In the present invention, the specific implementation of the solvent removal is preferably reduced pressure distillation. In the present invention, the recrystallization is preferably: dissolving the oily substance obtained by the solvent removal in methanol to obtain a methanol solution, and mixing the methanol solution, tetrahydrofuran and cyclohexane to precipitate a solid product; in the present invention, the volume ratio of methanol, tetrahydrofuran and cyclohexane is preferably 1:1:1. The present invention preferably dries the solid product obtained by the recrystallization to obtain a compound shown in Formula 3.

[0088] In the present invention, the compound of formula 4 and isopropyl magnesium bromide are dissolved in a third organic solvent to carry out a nucleophilic addition reaction, and after obtaining a nucleophilic addition reaction liquid, the nucleophilic addition reaction liquid is mixed with a fourth organic solvent to continue the reaction, so as to obtain a compound of formula 5.

[0089] In the present invention, the third organic solvent is preferably anhydrous tetrahydrofuran.

[0090] In the present invention, the molar ratio of the compound of formula 4 to isopropylmagnesium bromide is preferably 50:52.

[0091] In the present invention, the specific implementation process of dissolving the compound of formula 4 and isopropyl magnesium bromide in the third organic solvent is preferably: dissolving the compound shown in formula 4 in part of the fourth organic solvent to obtain a solution of the compound shown in formula 4; dissolving the isopropyl magnesium bromide in the remaining third organic solvent to obtain an isopropyl magnesium bromide solution. In the present invention, the molar concentration of the isopropyl magnesium bromide solution is preferably 2 mol / L; mixing the solution of the compound shown in formula 4 and the isopropyl magnesium bromide solution. In the present invention, the temperature at which the solution of the compound shown in formula 4 and the isopropyl magnesium bromide solution are mixed is preferably 0°C.

[0092] In the present invention, the temperature of the nucleophilic addition reaction is preferably 0°C.

[0093] In the present invention, the holding time of the nucleophilic addition reaction is preferably 1 h.

[0094] In the present invention, the nucleophilic addition reaction is preferably carried out in a protective gas atmosphere. In the present invention, the protective gas is preferably nitrogen.

[0095] In the present invention, the nucleophilic addition reaction is preferably carried out under stirring conditions, and the present invention has no special requirements for the specific implementation process of the stirring.

[0096] In the present invention, the fourth organic solvent is preferably N,N-dimethylformamide (DMF).

[0097] In the present invention, the ratio of the amount of the compound represented by Formula 4 to the volume of the fourth organic solvent is preferably 50 mmol:5 mL.

[0098] In the present invention, the temperature for mixing the nucleophilic addition reaction solution and the fourth organic solvent and continuing the reaction is preferably room temperature.

[0099] In the present invention, the holding time for mixing the nucleophilic addition reaction solution and the fourth organic solvent and continuing the reaction is preferably 12 hours.

[0100] In the present invention, the mixing of the nucleophilic addition reaction liquid and the fourth organic solvent for continued reaction is preferably carried out under stirring. The present invention has no special requirements for the specific implementation process of the stirring.

[0101] The present invention mixes the nucleophilic addition reaction liquid and the fourth organic solvent and continues to react to obtain a nucleophilic addition reaction liquid. The present invention performs post-treatment on the nucleophilic addition reaction liquid to obtain a compound shown in Formula 5. In the present invention, the post-treatment preferably includes: adding a saturated ammonium chloride solution for stratification, aqueous phase extraction, washing the organic phase after merging the extracted organic phase, drying, concentration and column chromatography purification. In the present invention, the volume of the saturated ammonium chloride solution and the amount of the compound described in Formula 4 are preferably 100mL: 50mmol. In the present invention, the extractant for aqueous phase extraction is preferably ethyl acetate. In the present invention, the number of extractions is preferably 2 times. In each extraction, the volume of the extractant and the amount of the compound described in Formula 4 are preferably 100mL: 50mmol. In the present invention, after extraction, the present invention combines the organic phases and washes the organic phase. In the present invention, the detergent used when washing the extracted organic phase is preferably a saturated NaCl solution. In the present invention, the volume of the saturated NaCl solution and the amount of the compound described in Formula 4 are preferably 100mL: 50mmol. In the present invention, the drying is preferably carried out by anhydrous sodium sulfate. In the present invention, the concentration is preferably rotary distillation. In the present invention, the column chromatography purification is preferably silica gel column chromatography purification. In the present invention, the eluent used in the column chromatography purification is preferably a mixed solvent of petroleum ether and ethyl acetate. In the present invention, the volume ratio of petroleum ether to ethyl acetate is preferably 10:1.

[0102] After obtaining the compound represented by Formula 5 and the compound represented by Formula 3, the present invention dissolves the compound represented by Formula 5, the compound represented by Formula 3 and sodium hydride in a fifth organic solvent in a protective gas atmosphere to carry out a second addition reaction to obtain a compound represented by Formula 6.

[0103] In the present invention, the protective gas is preferably nitrogen.

[0104] In the present invention, the fifth organic solvent is preferably tetrahydrofuran.

[0105] In the present invention, the molar ratio of the compound represented by Formula 5 to the compound represented by Formula 3 is preferably 20:22.

[0106] In the present invention, the molar ratio of the compound represented by Formula 3 to sodium hydride is preferably 22:24.

[0107] In the present invention, the ratio of the amount of the compound represented by Formula 3 to the volume of the fifth organic solvent is preferably 22 mmol:120 mL.

[0108] In the present invention, the specific implementation process of dissolving the compound of Formula 5, the compound of Formula 3 and sodium hydride in the fifth organic solvent is preferably: suspending the compound of Formula 3 in the fifth organic solvent to obtain a solution of the compound of Formula 3, adding the sodium hydride to the solution of the compound of Formula 3 for first mixing to obtain a first mixed solution; adding the compound of Formula 5 to the first mixed solution to carry out the second addition reaction. In the present invention, the temperature when the compound of Formula 3 is suspended in the fifth organic solvent is preferably 0°C, in the present invention, the temperature of the first mixing is preferably room temperature, the insulation time of the first mixing is preferably 1h, the first mixing is preferably carried out under stirring conditions, and the present invention has no special requirements for the specific implementation process of the stirring. In the present invention, the temperature of the second addition reaction is preferably room temperature, the insulation time of the second addition reaction is preferably 16h, the second addition reaction is preferably carried out under stirring conditions, and the present invention has no special requirements for the specific implementation process of the stirring.

[0109] In the present invention, the second addition reaction obtains a second addition reaction liquid, and the present invention preferably performs post-treatment on the second addition reaction liquid to obtain the compound shown in Formula 6. In the present invention, the post-treatment preferably includes: adding saturated ammonium chloride for stratification, aqueous phase extraction, washing the extracted organic phase, drying, concentration and column chromatography separation in sequence. In the present invention, the volume of the saturated ammonium chloride solution and the amount of the compound described in Formula 4 are preferably 200mL:22mmol. In the present invention, the extractant for aqueous phase extraction is preferably ethyl acetate. In the present invention, the number of extractions is preferably 1, and the volume of the extractant and the amount of the compound described in Formula 3 are preferably 100mL:22mmol. In the present invention, after extraction, the present invention combines the organic phases and washes the organic phases. In the present invention, the detergent used for washing the extracted organic phase is preferably a saturated NaCl solution. In the present invention, the volume of the saturated NaCl solution and the amount of the compound described in Formula 3 are preferably 100mL:22mmol. In the present invention, the drying is preferably carried out using anhydrous sodium sulfate. In the present invention, the concentration is specifically preferably rotary distillation. In the present invention, the column chromatography purification is preferably silica gel column chromatography purification. In the present invention, the eluent used in the column chromatography purification is preferably a mixed solvent of petroleum ether and ethyl acetate. In the present invention, the volume ratio of petroleum ether to ethyl acetate is preferably 10:1.

[0110] After obtaining the compound of Formula 6, the present invention dissolves the compound of Formula 6 and a palladium-carbon catalyst in a sixth organic solvent, and performs a reduction reaction in a reducing gas atmosphere to obtain a flusilazole hapten having a structure of Formula I-1;

[0111]

[0112] In the present invention, the sixth organic solvent is preferably ethyl acetate.

[0113] In the present invention, the mass ratio of the compound represented by Formula 6 to the palladium-carbon catalyst is preferably 3.41:0.3.

[0114] In the present invention, the ratio of the amount of the compound represented by Formula 6 to the volume of the sixth organic solvent is preferably 6.7 mmol:30 mL.

[0115] In the present invention, the dissolution order of the compound represented by Formula 6 and the palladium-carbon catalyst in the sixth organic solvent is preferably: dissolving the compound represented by Formula 6 in the sixth organic solvent to obtain a solution of the compound represented by Formula 6, and adding the palladium-carbon catalyst to the solution of the compound represented by Formula 6.

[0116] In the present invention, the reducing gas is preferably hydrogen.

[0117] In the present invention, the temperature of the reduction reaction is preferably room temperature.

[0118] In the present invention, the insulation time of the reduction reaction is preferably 16 hours.

[0119] In the present invention, the reduction reaction obtains a reduction reaction liquid. The present invention preferably performs post-treatment on the reduction reaction liquid to obtain the flusilazole hapten shown in formula I-1. In the present invention, the post-treatment preferably includes: sequentially performing solid-liquid separation, concentration and column chromatography separation. In the present invention, the solid-liquid separation is specifically preferably filtration. In the present invention, the column chromatography purification is preferably silica gel column chromatography purification. In the present invention, the eluent used for the column chromatography purification is preferably a mixed solvent of petroleum ether and ethyl acetate. In the present invention, the volume ratio of petroleum ether and ethyl acetate is preferably 10:1, v:v, gradient elution, wherein the concentration of ethyl acetate is from 0% to 100%, and the concentration of petroleum ether is from 100% to 0%.

[0120] In the present invention, the method for preparing the flusilazole hapten represented by formula I-2 preferably comprises the following steps:

[0121] The flusilazole hapten shown in formula I-1, N-hydroxysuccinimide (NHS), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) are dissolved in N,N-dimethylformamide (DMF) for substitution reaction to obtain the flusilazole hapten shown in formula I-2.

[0122] In the present invention, the molar ratio of the flusilazole hapten represented by formula I-1 to NHS is preferably 0.0265:0.053.

[0123] In the present invention, the molar ratio of the flusilazole hapten represented by formula I-1 to EDC is preferably 0.0265:0.053.

[0124] In the present invention, the ratio of the amount of the flusilazole hapten represented by formula I-1 to the volume of the DMF is preferably 0.0265 mmol:0.5 mL.

[0125] In the present invention, the temperature of the substitution reaction is preferably 4°C.

[0126] In the present invention, the insulation time of the substitution reaction is preferably 10 h.

[0127] In the present invention, the substitution reaction is preferably carried out under stirring conditions, and the stirring is preferably magnetic stirring.

[0128] The present invention provides a complete flusilazole antigen, which is obtained by coupling the flusilazole hapten described in the above technical solution or the flusilazole hapten prepared by the preparation method described in the above technical solution with a carrier protein.

[0129] In the present invention, the carrier protein preferably includes bovine serum albumin (BSA), ovalbumin (OVA) or keyhole limpet hemocyanin.

[0130] In the present invention, the structural formula of the flusilazole complete antigen is shown in Formula III:

[0131]

[0132] In a specific embodiment of the present invention, the structural formula of the flusilazole complete antigen is preferably as shown in Formula III-1 or Formula III-2:

[0133]

[0134] The present invention provides a method for preparing the flusilazole complete antigen described in the above technical solution, preferably comprising the following steps:

[0135] The flusilazole hapten and the buffer solution of the carrier protein are mixed to carry out coupling reaction to obtain the flusilazole complete antigen.

[0136] In the present invention, when the flusilazole hapten is preferably a flusilazole hapten of the structure shown in formula I-1, the present invention preferably prepares the flusilazole hapten shown in formula I-2 according to the preparation method of the flusilazole hapten shown in formula I-2 and then conducts a coupling reaction with a carrier protein.

[0137] In the present invention, the flusilazole hapten is preferably coupled with the carrier protein in the form of an organic solution of the flusilazole hapten.

[0138] In a specific embodiment of the present invention, the flusilazole hapten is preferably prepared by using the substitution reaction supernatant obtained by the preparation method of the flusilazole hapten shown in formula I-2 described in the above technical scheme as a raw material for coupling reaction with the carrier protein.

[0139] In the present invention, in the reaction system of the coupling reaction, the molar ratio of the flusilazole hapten to the carrier protein is preferably (10-50):1, more preferably 50:1.

[0140] In the present invention, the reaction temperature of the coupling reaction is preferably 0-50°C, more preferably 4°C; the time of the coupling reaction is preferably 8-36h, more preferably 12h; the reaction pH of the coupling reaction is preferably 5-9, more preferably 7.4.

[0141] In the present invention, the buffer solution of the carrier protein is preferably at least one of carbonate buffer (CBS), phosphate buffer (PBS), borate buffer and 4-hydroxyethylpiperazineethanesulfonic acid buffer of the carrier protein. In the present invention, the pH value of the buffer solution of the carrier protein is preferably 5 to 9, more preferably 7.4.

[0142] In the present invention, after the coupling reaction, the present invention preferably further comprises dialyzing the reaction system of the coupling reaction, and the dialysate used for the dialysis is preferably a PBS solution; the pH of the PBS solution is preferably 7-10, more preferably 7.4; the concentration of the PBS solution is preferably 0.01-0.2 mol / L, more preferably 0.01 mol / L.

[0143] The flusilazole hapten of the invention has good stability, few synthesis steps, low synthesis cost, simple reaction conditions, high purity of the synthesized hapten, and solubility and stability that can meet the requirements of coupling carrier protein.

[0144] The present invention also provides the use of the hapten described in the above scheme or the complete flusilazole antigen in the preparation of flusilazole antibodies.

[0145] In the present invention, the flusilazole antibody is preferably anti-flusilazole serum.

[0146] The present invention provides a flusilazole antibody, which is obtained by emulsifying the flusilazole complete antigen described in the above technical solution and then immunizing a host animal.

[0147] The present invention provides a method for preparing the flusilazole antibody described in the above technical solution, comprising the following steps:

[0148] emulsifying the flusilazole complete antigen described in the above technical scheme and immunizing the host animal;

[0149] The preparation method of flusilazole polyclonal antibody comprises: after boosting immunization of an immune host animal, collecting blood of the immune animal and separating the blood to obtain the flusilazole polyclonal antibody;

[0150] The method for preparing flusilazole monoclonal antibody comprises: extracting spleen cells from an immunized host animal, fusing the spleen cells with SP2 / 0 tumor cells, screening monoclonal hybridoma cell lines capable of secreting flusilazole antibodies, and preparing the flusilazole monoclonal antibody based on the monoclonal hybridoma cell lines;

[0151] The preparation method of flusilazole nanoantibody is as follows: extracting mRNA transcribed from B lymphocytes in the blood of immunized host animals, reverse transcribing it into cDNA, using the cDNA as a substrate for PCR amplification to obtain diversified nanoantibody gene fragments, constructing a phage or yeast antibody expression library with the nanoantibody gene fragments, and obtaining flusilazole nanoantibody through screening.

[0152] In the present invention, when preparing flusilazole nanobodies, the host animal is preferably a camel or a cartilaginous fish.

[0153] In the present invention, monoclonal hybridoma cell lines are easy to prepare for those skilled in the art. For example, spleen cells are obtained after immunizing a host animal with the above-mentioned complete antigen, and the spleen cells are fused with SP2 / 0 tumor cells in vitro. Single hybridomas that can secrete flusilazole antibodies are screened through selective culture medium to obtain hybridoma cell lines. Flusilazole nanoantibodies can also be obtained by genetic engineering technology and other means. These technologies are easy to implement for those skilled in the art. Based on this, no matter what method is used to prepare flusilazole monoclonal antibodies or flusilazole nanoantibodies, as long as the hapten or complete antigen provided by the present invention is used, it belongs to the protection scope of the present invention.

[0154] Based on the hapten or complete antigen disclosed in the present invention, it is easy for those skilled in the art to prepare anti-flusilazole antibodies by using a suitable antibody preparation method, no matter what kind of animal is used for immunization, no matter how the immunization conditions or parameters are set or changed, as long as the hapten or complete antigen provided by the present invention is used, it falls within the protection scope of the present invention.

[0155] The antibody or antiserum prepared by immunizing a host animal with the complete flusilazole antigen provided by the present invention can specifically bind to flusilazole and has a good immune effect.

[0156] The present invention provides a reagent or a kit for detecting flusilazole, which contains the flusilazole antibody described in the above technical solution or the flusilazole antibody prepared by the preparation method described in the above technical solution.

[0157] The present invention provides use of the flusilazole antibody described in the above technical solution or the flusilazole antibody prepared by the preparation method described in the above technical solution or the reagent or kit described in the above technical solution in detecting flusilazole.

[0158] The synthesis route of the hapten provided by the present invention is relatively simple, the synthesis cost is low, and its solubility and stability are good; and the antiserum obtained by immunizing the host animal with the complete antigen of the present invention has a good titer, and the spleen cells obtained by immunizing the mouse with the complete antigen can be effectively used for the fusion of hybridoma cells and the preparation of anti-flusilazole monoclonal antibodies. The complete antigen is used to immunize alpacas, and the peripheral blood of the alpacas is collected, and its B lymphocytes are collected. By constructing a phage or yeast nanoantibody expression library, a nanoantibody that specifically recognizes flusilazole can be obtained through multiple rounds of solid phase competition panning. The flusilazole antibody prepared by the flusilazole complete antigen provided by the present invention has good specificity and a low minimum detection limit, can achieve specific recognition of flusilazole, can be used to establish a flusilazole immunological detection method or detection reagent, and has broad application prospects.

[0159] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0160] Sources of some materials in the examples:

[0161] Sodium bicarbonate, ethanol, hydrochloric acid, sodium dihydrogen phosphate dodecahydrate, sodium chloride, gelatin, citric acid monohydrate, and Tween-20 were all purchased from Sinopharm Chemical Reagent Co., Ltd.; anhydrous N,N-dimethylformamide (DMF) was purchased from Aladdin; 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Freund's complete adjuvant, Freund's incomplete adjuvant, bovine serum albumin (BSA), and ovalbumin (OVA) were all purchased from Sigma; goat anti-mouse IgG-HRP was purchased from Jackson.

[0162] Example 1

[0163] The flusilazole hapten shown in the synthetic formula I-1 has the following structure:

[0164]

[0165] Synthesis route see Figure 1 .

[0166] 4-bromobutyric acid (8.51 g, 51 mmol, 1 equiv.) was dissolved in cyclohexane (70 mL) to obtain a 4-bromobutyric acid solution. Benzyl alcohol (6.8 mL, 66 mmol, 1.3 equiv.) and p-toluenesulfonic acid (0.97 g, 5.1 mmol, 0.1 equiv.) were added to the 4-bromobutyric acid solution to form a mixed solution. The mixed solution was heated to reflux for 16 h, and the water generated during the reflux process was separated. After the reaction was completed, the reaction system was washed with a saturated sodium bicarbonate aqueous solution, dried over anhydrous sodium sulfate, and the residue after the solvent was recovered by rotary evaporation was subjected to reduced pressure distillation to obtain the product of the compound shown in formula 2, which was a colorless oil (12.84 g, 98%).

[0167] Triphenylphosphine (13.10 g, 50 mmol) was added to a toluene (200 mL) solution of the compound shown in Formula 2 (12.84 g, 49.9 mmol), and the reaction system formed by the compound shown in Formula 2 and triphenylphosphine was heated to reflux for 16 h. After the reaction was completed, the system was concentrated to obtain a viscous oily product. The viscous oily product was dissolved in methanol (50 mL) to obtain a viscous oily product methanol solution. Tetrahydrofuran (50 mL) and cyclohexane (50 mL) were added to the viscous oily product methanol solution, and stirred to precipitate a solid product. The solid product was collected by filtration and dried to obtain a compound shown in Formula 3 (20.76 g, 80%).

[0168] Under nitrogen protection, the compound shown in formula 4 (flusilazole, 15.50 g, 50 mmol) was dissolved in anhydrous THF, and a THF solution of isopropyl magnesium bromide (2M, 26 mL, 52 mmol) was added by injection at 0°C. The reaction system of flusilazole and isopropyl magnesium bromide was stirred for reaction at 0°C for 1 h, and then DMF (5 mL) was added by injection. The mixture was stirred at room temperature overnight, and a saturated ammonium chloride solution (100 mL) was added to the reaction system. The mixture was then extracted twice with ethyl acetate (each time with a volume of 100 mL of ethyl acetate). The combined organic phase was washed with saturated NaCl (100 mL), dried over anhydrous sodium sulfate, and concentrated to obtain a preliminary product (16.30 g). The mixture was eluted by silica gel column chromatography (petroleum ether:ethyl acetate=10:1, v:v) to obtain a compound shown in formula 5 (8.58 g, 50%).

[0169] Under nitrogen protection, the compound shown in formula 3 (11.42g, 22mmol) was suspended in anhydrous THF (120mL), cooled to 0°C in an ice bath, sodium hydride (960mg, 24mmol) was added to the reaction system, and then the compound shown in formula 3 and the sodium hydride reaction system were stirred at room temperature for 1h, and the compound shown in formula 5 (6.86g, 20mmol) was added to the reaction system, and the reaction system was stirred at room temperature for 16h. Saturated ammonium chloride (200mL) was added to separate the layers, and the aqueous layer was extracted with ethyl acetate (100mL). The organic phase was washed with saturated NaCl (100mL), dried over anhydrous sodium sulfate, and concentrated to obtain an oily substance (15g) as a preliminary product, and the preliminary product was eluted by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1, v:v) to obtain the compound shown in formula 6 (5.10g, 50%).

[0170] Palladium carbon (10%, 300 mg) was added to a solution of the compound (3.41 g, 6.7 mmol) in ethyl acetate (30 mL) of Formula 6 to obtain a mixed solution, the atmosphere of the mixed solution was replaced with nitrogen, and then hydrogenated under a hydrogen atmosphere, and stirred at room temperature for 16 h. The reaction was completed. The reaction mixture was filtered to remove palladium carbon, and the filtered reaction liquid was concentrated to an oily product, and the oily product was separated and purified by a silica gel column chromatography (petroleum ether: ethyl acetate = 10: 1, v: v, gradient elution, wherein the concentration of ethyl acetate was from 0% to 100%, and the concentration of petroleum ether was from 100% to 0%) to obtain the flusilazole hapten (1.65 g, 59%) shown in Formula I-1.

[0171] Figure 4 is a liquid chromatogram of the flusilazole hapten represented by formula I-1 in Example 1; liquid chromatogram ( Figure 4 ) shows only one sample peak, and the molecular formula of flusilazole hapten is C 21 H 23 F 2 N 3 O 2 Si, the exact molecular weight theoretical value is 415.51.

[0172] The product prepared in Example 1 was subjected to liquid chromatography, 1 H NMR ( Figure 5 ) confirmed that it was the flusilazole hapten represented by formula I-1: 3-(4-chloro-6-(ethylamino)-1,3,5-triazine-2-ylamino)butyric acid.

[0173] 1 H NMR (400 MHz, CDCl 3)δ0.76(3,3H),1.61-1.68(m,4H),2.33(t,J=6.4Hz,2H),2.56(t,J=6.4 Hz,2H),4.02(s,2H),7.08-7.12(m,4H).7.46-7.49(m,4H),7.85(s,1H).

[0174] Example 2

[0175] The flusilazole hapten shown in the synthetic formula I-2 has the following structure:

[0176]

[0177] Synthesis route see Figure 2 .

[0178] 11.02 mg (0.0265 mmol) of the flusilazole immune antigen of formula I-2 prepared in Example 1, 6.10 mg (0.053 mmol) of NHS and 10.16 mg (0.053 mmol) of EDC were fully dissolved in 0.5 mL of anhydrous DMF, and reacted in a refrigerator at 4°C with magnetic stirring for 10 h. After the reaction was completed, a reaction solution containing the flusilazole hapten of formula I-2 was obtained. The reaction solution containing the flusilazole hapten of formula I-2 prepared in Example 2 can be directly used for coupling with the buffer solution of the carrier protein.

[0179] Example 3

[0180] The complete flusilazole antigen shown in the synthetic formula III-1 is:

[0181]

[0182] Synthesis route see Figure 3 .

[0183] Synthesis method:

[0184] The supernatant of the reaction solution containing the flusilazole hapten shown in formula I-2 obtained in Example 2 was slowly added dropwise to the PBS buffer solution of OVA, wherein the PBS buffer solution of OVA was obtained by dissolving 10 mg of OVA in 1 mL of PBS buffer solution with a pH value of 7.4 and mixing evenly, and the molar ratio of the flusilazole hapten shown in formula I-2 to OVA was 50:1, and the reaction was stirred at 25°C for 4 hours, and the obtained reaction solution was dialyzed six times in a PBS buffer solution with a pH value of 7.4 and a concentration of 0.01 mol / L, and the dialyzed reaction product solution was diluted to a 1 mg / mL solution. The role of dialysis is to remove the flusilazole hapten shown in formula I-1 or other small molecules in the unreacted solution, and obtain the flusilazole complete antigen shown in formula III-1, that is, the conjugate of the flusilazole hapten described in formula I-1 and OVA.

[0185] OVA detected by MALDI-TOF-MS Figure 6 ) and the flusilazole complete antigen represented by formula III-1 ( Figure 7 ) The single-charged ion peaks are 44557.794 and 47163.915. The calculation formula for the coupling ratio of the carrier protein OVA to the flusilazole hapten described in Formula I-1 is: coupling ratio = (complete antigen molecular weight - carrier protein molecular weight) / hapten molecular weight. The coupling ratio of the flusilazole complete antigen shown in Formula III-1 calculated by the coupling ratio formula is 1:6.

[0186] Example 4

[0187] The complete flusilazole antigen shown in the synthetic formula III-2 is:

[0188]

[0189] The supernatant of the reaction solution containing the flusilazole hapten shown in formula I-2 obtained in Example 2 was slowly added dropwise to the PBS buffer solution of BSA, wherein the PBS buffer solution of BSA was obtained by dissolving 20 mg BSA in 5 mL phosphate buffer (PBS) with a pH value of 7.4 and mixing, and the molar ratio of the flusilazole hapten shown in formula I-2 to BSA was 60:1, and the reaction was stirred at 25°C for 4 hours, and the obtained reaction solution was dialyzed six times in a PBS buffer solution with a pH value of 7.4 and a concentration of 0.01 mol / L, and the dialyzed reaction product solution was diluted to a 1 mg / mL solution, and after quick freezing with liquid nitrogen, it was placed at -20°C for standby use. The flusilazole complete antigen shown in formula III-2 was obtained, that is, the conjugate of the flusilazole hapten described in formula I-1 and BSA.

[0190] BSA standard obtained by MALDI-TOF-MS detection ( Figure 8 ) and the flusilazole complete antigen represented by formula III-2 ( Fig. 9 ) single-charged ion peaks 67860.266 and 81119.433. The coupling ratio of the flusilazole complete antigen shown in Formula III-2 was calculated to be 1:31 by the coupling ratio formula.

[0191] Example 5

[0192] Application of flusilazole complete antigen

[0193] 1. Preparation of Flusilazole Antibody Using the Flusilazole Complete Antigen of Formula III-2 Prepared in Example 4

[0194] (1) 6- to 8-week-old Balb / c mice were used as experimental animals (8-week-old Balb / c mice weigh approximately 23 to 25 g).

[0195] (2) Primary immunization: The diluted flusilazole complete antigen solution (concentration of 1 mg / mL) of formula III-2 obtained in Example 4 was filtered through a sterile filter and added with an equal volume of Freund's complete adjuvant. The mixture was stirred and emulsified until it did not diffuse when dropped into water. The emulsified flusilazole complete antigen of formula III-2 was injected into the abdominal cavity and back subcutaneously of mice, with a total injection dose of 0.1 mg of emulsified antigen per mouse.

[0196] (3) Reinforced immunization: 2 weeks after the initial immunization, take 1 mL of the diluted flusilazole complete antigen solution of formula III-2, add 1 mL of Freund's incomplete adjuvant, and stir thoroughly to emulsify until it does not spread when dropped into water. The emulsified flusilazole complete antigen of formula III-2 is injected intraperitoneally and subcutaneously at multiple points on the back of mice, with a total injection dose of 0.1 mg of emulsified antigen per mouse. The booster immunization was performed every 14 days. Starting from the third booster immunization, blood was collected from the mouse eye sockets 3 days after each immunization to determine the flusilazole antibody titer in the serum. The test method was ELISA. The coated antigen was 1 mg / mL of the flusilazole complete antigen shown in formula III-1. The serum was diluted to the required gradient, which was 1000 times, 4000 times, 8000 times and 16000 times respectively. After the titer was ≥1:64000 (the titer was defined as the dilution multiple of the serum when the zero-hole color value was about 1.0), the eyeball was enucleated and blood was collected. The blood was placed in a 37°C constant temperature incubator for 30 minutes, then placed in a 4°C refrigerator for 2 hours, and then centrifuged in a centrifuge at 4°C and 10000r / min for 5 minutes to separate the serum to obtain flusilazole antiserum. It was used in the following experiments.

[0197] 2. Detection of Flusilazole Antibody Titer in Flusilazole Antiserum

[0198] The various buffers used in the following experiments are as follows:

[0199] (1) Coating buffer (CBS, pH 9.6, 0.05 M carbonate buffer): weigh Na 2 CO 3 1.5 g NaHCO 3 2.94 g, dilute to 1000 mL with ultrapure water;

[0200] (2) Phosphate buffer (0.01 M PBS, pH 7.4): weigh KH 2 PO 4 0.2 g, NaCl 8 g, NaH 2 PO 4 12H 2 O 2.92g, ultrapure water to 1L;

[0201] (3) Washing buffer (PBST): Add 0.1% Tween-20 by volume to the prepared 0.01 M PBS;

[0202] (4) Sample diluent (PBSTG): Add 1% Tween-20 and 1 g gelatin (melted in a microwave oven) at a volume ratio to the prepared 0.01 M PBS;

[0203] (5) Substrate buffer: pH 5.5: weigh Na 2 HPO 3 12H 2 O 9.22g, citric acid monohydrate 2.55g, measure 0.5mL of Tween-20, and make up to 1L with ultrapure water;

[0204] (6) Stop solution (1 M HCl): Measure 440 mL of distilled water, add 40 mL of 98% (volume / volume) concentrated hydrochloric acid dropwise and stir.

[0205] (I) Determination of the titer and inhibition rate of flusilazole antibodies in flusilazole antiserum

[0206] 1. Preparation of Flusilazole Complete Antigen Coated Antigen Solution Shown in Formula III-1

[0207] The complete flusilazole antigen represented by formula III-1 prepared in the above Example 3 was gradiently diluted with CBS as needed to obtain coated antigen solutions of the complete flusilazole antigen represented by formula III-1 with different concentrations.

[0208] 2. Preparation of Flusilazole Standard Solution

[0209] (1) Weigh 10 mg of flusilazole standard sample and dissolve it in 10 mL of methanol to obtain a 1 mg / mL flusilazole standard solution.

[0210] (2) The 1 mg / mL flusilazole standard solution in (1) was prepared with PBSTG to a final concentration of 1000 ng / mL flusilazole standard solution.

[0211] 3. Preparation of Flusilazole Antiserum Dilution

[0212] The flusilazole antiserum prepared in the above step was gradiently diluted with PBSTG to obtain a flusilazole antiserum dilution solution.

[0213] 4. Determination of Flusilazole Antiserum Titer and Inhibition Rate

[0214] ① Coating: Add 100 μL of CBS containing the complete flusilazole antigen of formula III-1 prepared in step 1 to each well of a 96-well ELISA plate, incubate at 37°C for 3 h, wash three times with PBST, and spin dry.

[0215] ② Competition: Add 50 μL PBSTG to each well of the zero well; add 50 μL flusilazole standard solution prepared in step 2 to each well of the inhibition well.

[0216] ③ Add the flusilazole antiserum dilution to the ELISA plate (50 μL / well), incubate at 37°C for 30 min, wash the plate three times with PBST, and spin dry.

[0217] ④ Add enzyme-labeled secondary antibody: dilute goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson) 1000 times with PBS (0.1M, pH=9.6), add 100 μL to each well, incubate at 37°C for 30 min, wash the plate three times with PBST, and spin dry.

[0218] ⑤ Color development: Prepare the color development substrate immediately before use, add 100 μL TMB single-component color development solution to each well, and develop the color for 15 minutes at room temperature in the dark.

[0219] ⑥ Termination and detection: Add 50 μL 1M HCl to each well to terminate the reaction, and use an enzyme reader to detect the absorbance value (OD value) of each well at 450 nm.

[0220] ⑦ Titer determination: When the OD value is about 1.0, the maximum dilution multiple of the flusilazole antiserum is the titer of the flusilazole antiserum.

[0221] ⑧Specificity determination: The specificity of serum is determined based on the inhibition rate.

[0222] The calculation formula of inhibition rate is: inhibition rate = [(OD value of control well - OD value of inhibition well) / OD value of control well] × 100%. After the fourth immunization, the titer results of flusilazole antiserum are shown in Table 1.

[0223] Table 1 Flusilazole antiserum titer and inhibition rate (TMB color development 15min, 1000ng / mL standard sample inhibition)

[0224]

[0225] Note: I represents the inhibition well in the ELISA plate, C represents the control well in the ELISA plate, IR represents the inhibition rate, and K represents 1000 times.

[0226] The results in Table 1 show that the titer and inhibition rate of flusilazole antiserum after four immunizations are very high, indicating that the flusilazole complete antigen represented by formula III-2 prepared in Example 4 above can be used as an immunogen to prepare flusilazole antibodies.

[0227] The results in Table 1 show that after four immunizations, when the coating antigen dilution was 1:8000 and the flusilazole antiserum dilution was 1:128000, the inhibition rate was 62.6%, and the inhibition effect was better at this time. This shows that the flusilazole complete antigen shown in Formula III-2 prepared in Example 4 above can be used as an immunogen to prepare antibodies for detecting flusilazole.

[0228] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention rather than all the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A flusilazole hapten having a structure shown in Formula I: The R 1 For -(CH 2 ) 4 -COOH or -(CH 2 ) 4 -COOR 2 , the R 2 for 2. The method for preparing the flusilazole hapten according to claim 1, The following steps are involved: In the compound structure shown in Formula II, R 3 The group is replaced by R through chemical reaction 1 Group, to obtain the flusilazole hapten represented by formula I; In the formula II, R 3 H or 3. The preparation method according to claim 2, It is characterized in that R 1 For -(CH 2 ) 4 The preparation method of the flusilazole hapten of -COOH comprises the following steps: The compound represented by Formula 1, benzyl alcohol and p-toluenesulfonic acid are dissolved in an organic solvent, and a first nucleophilic substitution reaction is performed to obtain a compound represented by Formula 2; Dissolving the compound represented by Formula 2 and triphenylphosphine in an organic solvent, and performing a first addition reaction to obtain a compound represented by Formula 3; The compound of Formula 4 and isopropylmagnesium bromide are dissolved in an organic solvent to carry out a nucleophilic addition reaction to obtain a nucleophilic addition reaction solution, and the nucleophilic addition reaction solution and the organic solvent are mixed to continue the reaction to obtain a compound of Formula 5; In a protective gas atmosphere, dissolving the compound represented by Formula 5, the compound represented by Formula 3 and sodium hydride in an organic solvent for a second addition reaction to obtain a compound represented by Formula 6; The compound represented by Formula 6 and the palladium carbon catalyst are dissolved in an organic solvent and subjected to reduction reaction in a reducing gas atmosphere to obtain R 1 For -(CH 2 ) 4 -COOH of the flusilazole hapten;

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