Preparation method and application of thiazinone hapten, complete antigen and antibody

Abstract

The present invention provides, for the first time, methods for preparing and applying thiazinone haptens, complete antigens, and antibodies, belonging to the field of immunoassay technology. The structural design and synthesis of thiazinone haptens, antibody preparation, and the establishment of immunoassay methods are all still under research at home and abroad. The thiazinone hapten provided by the present invention can fully expose the thiazinone antigenic determinants to the host animal's immune system. The complete antigen provided by the present invention is a compound having the structure represented by Formula I coupled with a carrier protein to obtain a thiazinone complete antigen. After the complete antigen prepared by the present invention is used to immunize a host animal, it can stimulate the body to produce highly sensitive antiserum / antibodies, and the prepared antiserum / antibodies can specifically recognize thiazinone. The antiserum / antibody provided by the present invention can be used to establish thiazinone immunological analysis methods or detection reagents. The present invention will provide strong technical support for protecting thiazinone residues in food and agricultural products in my country and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a preparation method and application of thiazinone hapten, complete antigen and antibody. Background Art

[0002] Buprofezin, also known as Buprofezin, is an insect growth regulator insecticide. Its chemical formula is 2-tert-butylimino-3-isopropyl-5-phenyl-3,4,5,6-tetrahydro-2H-1,3,5-thiadiazin-4-one, CAS registration number: 69327-76-0, and its molecular formula is: C 16 H 23 N3SO4 has a relative molecular weight of 305.4. The pure product is white crystals with a melting point of 104.5°C to 105.5°C and a relative density of 1.18 (25°C). It is stable to acids, bases, light, and heat. The molecular structure of thiazinone is shown in the figure below.

[0003]

[0004] Buprofezin belongs to the heterocyclic thiadiazine class of insect growth regulators. Its mechanism of action is to inhibit chitin synthesis and interfere with insect metabolism. Its strong contact effect can cause nymphs to molt or develop wing deformities, leading to slow death. It is effective against planthoppers, leafhoppers, whiteflies, and scale insects. It is primarily used for pest control in crops such as rice, fruit trees, tea trees, and vegetables, and exhibits persistent larvicidal activity against Coleoptera, some Homoptera, and Acarina.

[0005] As one of the most successful insect growth regulators (IGRs) globally, thiazolinone is an effective control agent for Homoptera pests (such as rice planthoppers, whiteflies, and scale insects) and is widely used in over 80 countries and regions. It is a chitin synthesis inhibitor. Due to its unique mechanism of action, strong selectivity, and safety to beneficial organisms at recommended doses, it is often used as an essential agent in rotation programs and is widely used in integrated pest management (IPM). At a time when highly toxic pesticides are banned and resistance to conventional pesticides is developing, thiazolinone is experiencing a new round of development opportunities.

[0006] Currently, there are still gaps in research on the design and synthesis of thiazolinone's hapten structure, antibody preparation, and the establishment of immunoassay methods, both domestically and internationally. Pesticide residue detection technology has long been of great interest, and the development of sensitive, accurate, and efficient pesticide residue detection methods has long been a goal pursued by researchers. The long-lasting effect of thiazolinone is a double-edged sword that can cause a series of environmental problems. The residues and accumulation of thiazolinone pesticides caused by improper use and ecological recycling seriously affect the environment and ecological safety, threatening human health. Excessive pesticide residues remain a major factor affecting the quality and safety of my country's food and agricultural products. The haptens, complete antigens, and anti-thiazolinone antibodies prepared in the present invention are the basis for the development of immunoassay methods. The highly sensitive anti-thiazolinone antibodies prepared in the present invention can be used to establish immunoassay methods that specifically recognize thiazolinone, which will provide strong technical support for protecting my country's food and agricultural products from thiazolinone residues. Summary of the Invention

[0007] In view of this, the present invention aims to provide, for the first time, methods for preparing and using thiazinone haptens, complete antigens, and antibodies. The complete antigen prepared from the thiazinone haptens of the present invention is used to immunize a host animal, thereby obtaining antiserum or antibodies that are highly sensitive and can specifically recognize thiazinone.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a thiazinone hapten having a structure shown in Formula I:

[0010]

[0011] In formula I, R1 is -(CH2)n-COOH, -(CH2)n-OH, -(CH2)n-NH2, -(CH2)n-COOR2, wherein m and n are independently integers from 0 to 6;

[0012] R2 is

[0013] The present invention provides a method for preparing the above-mentioned thiazinone hapten, comprising the following steps:

[0014] Mixing 5-methyl-2-pyrrolidone, hydrochloric acid and water to carry out a ring-opening reaction to obtain a compound having a structure shown in formula a;

[0015]

[0016] Mixing the compound having the structure shown in formula a, methanol and an esterification reaction catalyst to carry out an esterification reaction to obtain a compound having the structure shown in formula b;

[0017]

[0018] Mixing the compound having the structure shown in formula b, tert-butyl isosulfate, an inorganic base catalyst, and a first organic solvent, and performing a substitution reaction to obtain a compound having the structure shown in formula c;

[0019]

[0020] Mixing the compound having the structure shown in formula c, an inorganic strong base aqueous solution and a second organic solvent, and performing a hydrolysis reaction to obtain a compound having the structure shown in formula d;

[0021]

[0022] Mixing N-methylformanilide, sulfonyl chloride, an azo initiator, and a third organic solvent to carry out an acyl chlorination reaction to obtain a compound having a structure shown in formula e;

[0023]

[0024] The compound having the structure shown in formula d, the compound having the structure shown in formula e, an inorganic strong base aqueous solution, a phase transfer catalyst and a third organic solvent are mixed to perform a cyclization reaction to obtain a thiazinone hapten having the structure shown in formula I-1;

[0025]

[0026] Mixing the thiazinone hapten having the structure shown in Formula I-1, N-hydroxysuccinimide, carbodiimide hydrochloride, and a fourth organic solvent to perform a carboxyl activation reaction to obtain a thiazinone hapten having the structure shown in Formula I-2;

[0027]

[0028] Preferably, the ring-opening reaction temperature is 95-100°C and the time is 7.5-8h;

[0029] The esterification reaction temperature is 75-80°C and the time is 5.5-6h;

[0030] The temperature of the substitution reaction is 20-25°C and the time is 9.5-10 hours;

[0031] The temperature of the hydrolysis reaction is 20-25° C., and the time is 5-5.5 hours.

[0032] Preferably, the temperature of the acyl chlorination reaction is 70°C to reflux temperature, and the time is 2.5 to 3 hours;

[0033] The temperature of the cyclization reaction is 20-25° C., and the time is 8-8.5 hours.

[0034] The present invention provides a complete thiazinone antigen, comprising the above-mentioned thiazinone hapten and a carrier protein coupled with the thiazinone hapten.

[0035] The present invention provides the use of the above-mentioned complete thiazinone antigen in preparing thiazinone antiserum or thiazinone antibody.

[0036] The present invention provides anti-thiazinone polyclonal antibodies, anti-thiazinone monoclonal antibodies or anti-thiazinone nano antibodies, which are obtained by emulsifying the above-mentioned thiazinone complete antigen and then immunizing a host animal.

[0037] The present invention provides a method for preparing the above-mentioned anti-thiazinone polyclonal antibody, anti-thiazinone monoclonal antibody or anti-thiazinone nanobody, comprising the following steps:

[0038] The host animal is immunized with the complete thiazide antigen emulsified;

[0039] After boosting the immunized host animal, whole blood of the immunized animal is collected, and the serum is separated and purified to obtain anti-buprofen polyclonal antibodies;

[0040] Alternatively, spleen cells are isolated from the host animal, and after the spleen cells are fused with SP2 / 0 tumor cells in vitro, a monoclonal hybridoma cell line capable of secreting anti-thiazinone antibodies is screened, and an anti-thiazinone monoclonal antibody is prepared based on the monoclonal hybridoma cell line;

[0041] Alternatively, mRNA is extracted from IgG of B lymphocytes in the peripheral blood of the host animal for the construction of a nanoantibody library, nanoantibodies are screened in the constructed nanoantibody library, and the nanoantibodies obtained by the screening are induced to express and purified to obtain anti-thiazinone nanoantibodies.

[0042] The present invention provides a kit for detecting thiazinone, comprising the above-mentioned anti-thiazinone polyclonal antibody, anti-thiazinone monoclonal antibody or anti-thiazinone nanoantibody or the anti-thiazinone polyclonal antibody, anti-thiazinone monoclonal antibody or anti-thiazinone nanoantibody prepared by the above-mentioned preparation method.

[0043] Application of anti-thiazinone polyclonal antibody, anti-thiazinone monoclonal antibody or anti-thiazinone nano antibody or kit for detecting thiazinone in detecting thiazinone.

[0044] The present invention provides a thiazinone hapten having the structure shown in Formula I. Using the antigenic determinant of the thiazinone structure as a matrix, a carbon linker arm and an active group capable of coupling to a carrier protein, namely, an R1 linker group, are introduced to obtain a compound having the structure shown in Formula I. This compound can fully expose the thiazinone antigenic determinant to the host animal's immune system. Furthermore, the thiazinone hapten provided by the present invention has good stability, requires few synthetic steps, is low in cost, and employs simple reaction conditions. The resulting hapten is high in purity, and its solubility and stability meet the requirements for coupling to a carrier protein.

[0045] The complete thiazinone antigen provided by the present invention is obtained by coupling a compound having the structure represented by Formula I with a carrier protein. The complete antigen prepared by the present invention, when used to immunize a host animal, can stimulate the body to produce highly sensitive anti-thiazinone serum and / or antibodies. The prepared anti-thiazinone antiserum and / or antibodies can specifically recognize thiazinone. The anti-thiazinone serum or anti-thiazinone antibodies provided by the present invention can be used to establish thiazinone immunological analysis methods or detection reagents. This invention will provide strong technical support for ensuring the quality and safety of food and agricultural products in my country and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a synthetic route for the thiazinone hapten;

[0047] Figure 2 is a TLC spectrum of a thiazinone hapten having the structure shown in Formula I-1;

[0048] Figure 3 is the HRMS spectrum of the thiazinone hapten having the structure shown in Formula I-1;

[0049] Figure 4 A thiazinone hapten having the structure shown in formula I-1 1 H NMR spectrum;

[0050] Figure 5 A thiazinone hapten having the structure shown in formula I-1 13 C NMR spectrum;

[0051] Figure 6 This is the MALDI-TOF-MS detection spectrum of OVA standard;

[0052] Figure 7 This is the MALDI-TOF-MS detection spectrum of the complete thiazinone antigen II-1;

[0053] Figure 8 This is the MALDI-TOF-MS detection spectrum of BSA standard;

[0054] Figure 9This is the MALDI-TOF-MS detection spectrum of the complete thiazide antigen II-2. DETAILED DESCRIPTION

[0055] The present invention provides a thiazinone hapten having a structure shown in Formula I:

[0056]

[0057] In formula I, R1 is -(CH2)n-COOH, -(CH2)n-OH, -(CH2)n-NH2, -(CH2)n-COOR2, wherein m and n are independently integers of 0 to 6, preferably 1 to 5, more preferably 2 to 4;

[0058] R2 is

[0059] In the present invention, "*" represents a linking site.

[0060] In the present invention, the thiazinone hapten preferably has a structure shown in Formula I-1 or Formula I-2:

[0061]

[0062] The present invention provides a method for preparing the above-mentioned thiazinone hapten, comprising the following steps:

[0063] Mixing 5-methyl-2-pyrrolidone, hydrochloric acid and water to carry out a ring-opening reaction to obtain a compound having a structure shown in formula a;

[0064]

[0065] Mixing the compound having the structure shown in formula a, methanol and an esterification reaction catalyst to carry out an esterification reaction to obtain a compound having the structure shown in formula b;

[0066]

[0067] Mixing the compound having the structure shown in formula b, tert-butyl isosulfate, an inorganic base catalyst and a first organic solvent, and performing a substitution reaction to obtain a compound having the structure shown in formula c;

[0068]

[0069] Mixing the compound having the structure shown in formula c, an inorganic strong base aqueous solution and a second organic solvent, and performing a hydrolysis reaction to obtain a compound having the structure shown in formula d;

[0070]

[0071] Mixing N-methylformanilide, sulfonyl chloride, an azo initiator, and a third organic solvent to carry out an acyl chlorination reaction to obtain a compound having a structure shown in formula e;

[0072]

[0073] The compound having the structure shown in formula d, the compound having the structure shown in formula e, an inorganic strong base aqueous solution, a phase transfer catalyst and a third organic solvent are mixed to perform a cyclization reaction to obtain a thiazinone hapten having the structure shown in formula I-1;

[0074] The thiazinone hapten having the structure shown in formula I-1, N-hydroxysuccinimide, carbodiimide hydrochloride and a fourth organic solvent are mixed to perform a carboxyl activation reaction to obtain a thiazinone hapten having the structure shown in formula I-2.

[0075] The present invention comprises mixing 5-methyl-2-pyrrolidone, hydrochloric acid, and water to perform a ring-opening reaction to obtain a compound having the structure represented by Formula (a). In the present invention, the hydrochloric acid and water are preferably added in the form of an aqueous hydrochloric acid solution. In the present invention, the concentration of the aqueous hydrochloric acid solution is preferably 5.5 to 6 mol / L, more preferably 6 mol / L.

[0076] In the present invention, the temperature of the ring-opening reaction is preferably 95-100° C., more preferably 100° C.; the time is preferably 8-8.5 h, more preferably 8 h.

[0077] After the ring-opening reaction, the present invention preferably performs post-treatment on the obtained ring-opening reaction solution, and the post-treatment preferably comprises the following steps:

[0078] The obtained ring-opening reaction liquid is sequentially cooled and concentrated for the first time, and the obtained concentrated liquid is dissolved in methanol and concentrated for the second time.

[0079] In the present invention, the cooling is preferably to 20-25° C., more preferably to room temperature. The present invention has no special requirements for the specific operation methods of the first concentration and the second concentration, and concentration methods well known to those skilled in the art can be used.

[0080] In the present invention, the compound having the structure represented by Formula a, methanol, and an esterification reaction catalyst are mixed to carry out an esterification reaction to obtain a compound having the structure represented by Formula b. In the present invention, the esterification reaction catalyst is preferably thionyl chloride. In the present invention, the molar ratio of the compound having the structure represented by Formula a to the esterification reaction catalyst is preferably 1:2 to 2.5, more preferably 1:2.

[0081] In the present invention, the temperature of the esterification reaction is preferably 80-85°C, more preferably 80°C; the time is preferably 6-6.5 hours, more preferably 6 hours. In the present invention, the esterification reaction is preferably carried out in nitrogen atmosphere.

[0082] After the esterification reaction, the present invention preferably performs post-treatment on the obtained esterification reaction liquid, and the post-treatment preferably includes the following steps:

[0083] The esterification reaction liquid is sequentially cooled and concentrated for the first time, and the obtained concentrated liquid is dissolved in methanol and concentrated for the second time.

[0084] In the present invention, the cooling is preferably to 20-25° C., more preferably to room temperature. The present invention has no special requirements for the specific operation methods of the first concentration and the second concentration, and concentration methods well known to those skilled in the art can be used.

[0085] In the present invention, the compound having the structure represented by Formula B, tert-butyl isosulfate, an inorganic base catalyst, and a first organic solvent are mixed and subjected to a substitution reaction to obtain a compound having the structure represented by Formula C. In the present invention, the inorganic base catalyst is preferably sodium bicarbonate; and the first organic solvent is preferably dichloromethane.

[0086] In the present invention, the molar ratio of the compound having the structure represented by formula b to tert-butyl isosulfate is preferably 1:1 to 1.2, more preferably 1:1.2; the molar ratio of the compound having the structure represented by formula b to the inorganic base catalyst is preferably 1:1.5 to 2, more preferably 1:2.

[0087] In the present invention, the temperature of the substitution reaction is preferably 20-25° C., more preferably room temperature; and the time is preferably 10-10.5 h, more preferably 10 h.

[0088] In the present invention, after the substitution reaction, the present invention preferably performs post-treatment on the obtained substitution reaction liquid, and the post-treatment preferably includes the following steps:

[0089] The substitution reaction liquid is subjected to solid-liquid separation, and the obtained liquid is concentrated and purified by column chromatography to obtain a pure compound having the structure shown in formula C.

[0090] In the present invention, the solid-liquid separation method is preferably filtration; the concentration method is preferably concentration under reduced pressure. In the present invention, the mobile phase for column chromatography purification is preferably petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is preferably from 20:1 to 5:1. In the present invention, the column chromatography purification is preferably performed using a silica gel column.

[0091] In the present invention, the compound having the structure represented by formula c, an aqueous solution of an inorganic strong base, and a second organic solvent are mixed and hydrolyzed to obtain a compound having the structure represented by formula d. In the present invention, the inorganic strong base is preferably sodium hydroxide; and the second organic solvent is preferably tetrahydrofuran.

[0092] In the present invention, the molar ratio of the compound having the structure represented by formula C to the inorganic strong base is preferably 1:1.5 to 2, more preferably 1:2.

[0093] In the present invention, the temperature of the hydrolysis reaction is preferably 20-25° C., more preferably room temperature; and the time is preferably 5-5.5 h, more preferably 5 h.

[0094] After the hydrolysis reaction, the present invention preferably performs post-treatment on the obtained hydrolysis reaction solution, and the post-treatment preferably includes the following steps:

[0095] The organic solvent of the hydrolysis reaction solution is removed, and the pH value of the remaining liquid is adjusted to 3-5 with hydrochloric acid. Extraction, concentration, and column chromatography purification are performed in sequence to obtain a pure compound having the structure shown in Formula d.

[0096] In the present invention, the method for removing the organic solvent is preferably concentration under reduced pressure. In the present invention, the extraction agent used in the extraction is preferably ethyl acetate; the number of extractions is preferably 3 times; after the extraction, the present invention preferably combines the organic phases.

[0097] In the present invention, the concentration method is preferably reduced pressure concentration. In the present invention, the mobile phase for the column chromatography purification is preferably petroleum ether, ethyl acetate and formic acid, and the volume ratio of petroleum ether, ethyl acetate and formic acid is preferably from 2:1:0.05 to 1:1:0.05. In the present invention, the column chromatography purification is preferably performed using a silica gel column.

[0098] The present invention comprises mixing N-methylformanilide, sulfonyl chloride, an azo initiator, and a third organic solvent, and performing an acyl chlorination reaction to obtain a compound having a structure represented by Formula e. In the present invention, the azo initiator is preferably azobisisobutyronitrile. In the present invention, the third organic solvent is preferably carbon tetrachloride.

[0099] In the present invention, the molar ratio of N-methylformanilide to sulfonyl chloride is preferably 1:2-3, more preferably 1:3; the molar ratio of N-methylformanilide to azo initiator is preferably 1:1-1.6, more preferably 1:1.6.

[0100] In the present invention, the temperature of the acyl chlorination reaction is preferably 85 to 90° C., more preferably 90° C.; the time is preferably 3 to 3.5 h, more preferably 3 h.

[0101] After the acyl chlorination reaction, the present invention preferably removes the organic solvent from the obtained acyl chlorination reaction solution and directly proceeds to the next reaction.

[0102] In the present invention, the compound having the structure represented by Formula d, the compound having the structure represented by Formula e, an aqueous solution of a strong inorganic base, a phase transfer catalyst, and a third organic solvent are mixed and subjected to a cyclization reaction to obtain a thiazinone hapten having the structure represented by Formula I-1. In the present invention, the strong inorganic base is preferably sodium hydroxide; the phase transfer catalyst is preferably tetrabutylammonium bromide; and the third organic solvent is preferably 1,4-dioxetane.

[0103] In the present invention, the molar ratio of the compound having the structure represented by formula d to the compound having the structure represented by formula e is preferably 1:1-1.5, more preferably 1:1.5; the molar ratio of the compound having the structure represented by formula d to the inorganic strong base is preferably 1:2.5-3, more preferably 1:3; the molar ratio of the compound having the structure represented by formula d to the phase transfer catalyst is preferably 1:0.1-0.2, more preferably 1:0.2.

[0104] In the present invention, the temperature of the cyclization reaction is preferably 20-25° C., more preferably room temperature; and the time is preferably 8-8.5 h, more preferably 8 h.

[0105] After the cyclization reaction, the present invention preferably performs post-treatment on the obtained cyclization reaction solution, and the post-treatment preferably includes the following steps:

[0106] The organic solvent in the cyclization reaction solution is removed, and the pH value of the remaining liquid is adjusted to 3-5 with hydrochloric acid. Extraction, concentration, and column chromatography purification are performed in sequence to obtain a pure thiazinone hapten having the structure shown in Formula I-1.

[0107] In the present invention, the method for removing the organic solvent is preferably concentration under reduced pressure. In the present invention, the extraction agent used in the extraction is preferably ethyl acetate; the number of extractions is preferably 3 times; after the extraction, the present invention preferably combines the organic phases.

[0108] In the present invention, the concentration method is preferably reduced pressure concentration. In the present invention, the mobile phase of the column chromatography purification is preferably petroleum ether, ethyl acetate and triethylamine, and the volume ratio of the petroleum ether, ethyl acetate and triethylamine is preferably from 2:1:0.05 to 1:1:0.05. In the present invention, the column chromatography purification is preferably performed using a silica gel column.

[0109] In the present invention, the thiazinone hapten having the structure represented by Formula I-1, N-hydroxysuccinimide, carbodiimide hydrochloride, and a fourth organic solvent are mixed and a carboxyl group activation reaction is performed to obtain the thiazinone hapten having the structure represented by Formula I-2. In the present invention, the fourth organic solvent is preferably N,N-dimethylformamide.

[0110] In the present invention, the molar ratio of the thiazinone hapten having the structure represented by Formula I-1, N-hydroxysuccinimide and carbodiimide hydrochloride is preferably 1:2:2.

[0111] In the present invention, the temperature for the carboxyl activation is preferably 4-8°C, more preferably 4°C; the time is preferably 10-10.5 hours, more preferably 10 hours. In the present invention, the carboxyl activation reaction is preferably carried out under stirring.

[0112] In the present invention, after the carboxyl activation reaction, the obtained carboxyl activation solution can be directly used for subsequent coupling with a carrier protein.

[0113] In the present invention, the synthesis route of the thiazinone hapten is preferably as follows: Figure 1 shown.

[0114] The present invention provides a complete thiazinone antigen, comprising the above-mentioned thiazinone hapten and a carrier protein coupled to the thiazinone hapten. In the present invention, the carrier protein is preferably one or more of bovine serum albumin, ovalbumin, and keyhole limpet hemocyanin.

[0115] In the present invention, the schematic structure of the complete thiazinone antigen is preferably as shown in Formula II:

[0116]

[0117] Preferably, the schematic structure of the complete thiazinone antigen is shown in Formula II-1 or Formula II-2:

[0118]

[0119] The present invention provides a method for preparing the above-mentioned complete thiazinone antigen, comprising the following steps:

[0120] The thiazinone hapten, carrier protein and buffer solution are mixed and coupled to obtain the thiazinone complete antigen.

[0121] In the present invention, the buffer solution is preferably one or more of carbonate buffer, phosphate buffer, borate buffer and 4-hydroxyethylpiperazineethanesulfonic acid buffer; the pH value of the buffer solution is preferably 5 to 9, more preferably 7.4.

[0122] In the present invention, the molar ratio of the thiazinone hapten to the carrier protein is preferably 30-60:1, more preferably 40-50:1; the temperature of the coupling reaction is preferably 0-50°C, more preferably 4-10°C; and the time is preferably 8-36h, more preferably 12-24h.

[0123] In the present invention, after the coupling reaction, the obtained coupling reaction liquid is preferably dialyzed. In the present invention, the dialysate used for the dialysis is preferably a PBS solution; in the present invention, the pH value of the dialysate is preferably 7 to 10, more preferably 7.4; and the concentration is preferably 0.01 to 0.2 mol / L, more preferably 0.01 mol / L.

[0124] The present invention provides the use of the above complete thiazinone antigen in preparing anti-thiazinone antibodies. In the present invention, the anti-thiazinone antibodies are preferably anti-thiazinone serum.

[0125] The present invention provides anti-thiazinone polyclonal antibodies, anti-thiazinone monoclonal antibodies or anti-thiazinone nano antibodies, which are obtained by emulsifying the above-mentioned thiazinone complete antigen and then immunizing a host animal.

[0126] The present invention provides a method for preparing an anti-thiazinone polyclonal antibody, an anti-thiazinone monoclonal antibody or an anti-thiazinone nanobody, comprising the following steps:

[0127] emulsifying the above complete thiazinone antigen and immunizing the host animal;

[0128] After boosting the immunization of the immunized host animal, the whole blood of the immunized animal is collected, and the serum is separated and purified to obtain anti-thiazinone polyclonal antibodies.

[0129] Alternatively, spleen cells are isolated from the host animal, and after the spleen cells are fused with SP2 / 0 tumor cells in vitro, a monoclonal hybridoma cell line capable of secreting anti-thiazinone antibodies is screened, and an anti-thiazinone monoclonal antibody is prepared based on the monoclonal hybridoma cell line;

[0130] Alternatively, mRNA is extracted from IgG of B lymphocytes in the peripheral blood of the host animal for the construction of a nanoantibody library, nanoantibodies are screened in the constructed nanoantibody library, and the nanoantibodies obtained by the screening are induced to express and purified to obtain anti-thiazinone nanoantibodies.

[0131] In the present invention, the host animal is preferably one or more of Balb / c mice, New Zealand white rabbits, camelids, cartilaginous fish, goats and chickens, the camelids are preferably alpacas and / or camels, and the cartilaginous fish are preferably crocodiles.

[0132] The present invention has no special requirements for the immunization host animal and the method for screening monoclonal hybridoma cell lines, and the above methods well known to those skilled in the art can be used.

[0133] The present invention provides a kit for detecting thiazinone, comprising the above-mentioned anti-thiazinone polyclonal antibody or anti-thiazinone monoclonal antibody or anti-thiazinone nanoantibody or the anti-thiazinone polyclonal antibody, anti-thiazinone monoclonal antibody or anti-thiazinone nanoantibody prepared by the preparation method according to claim 8.

[0134] The present invention provides use of the anti-thiazinone polyclonal antibody, anti-thiazinone monoclonal antibody or anti-thiazinone nanoantibody or the kit for detecting thiazinone according to claim 9 in detecting thiazinone.

[0135] The preparation methods and applications of the thiazinone hapten, complete antigen and antibody provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0136] The sources of some materials in the examples: 5-methyl-2-pyrrolidone, dichlorothionyl, tert-butyl isothioate, silica gel column (200-300 mesh), tetrahydrofuran, ethyl acetate, N-methylformanilide, carbon tetrachloride, sulfonyl chloride, tetrabutylammonium bromide, 1,4-dioxane, petroleum ether, ethyl acetate, and triethylamine were all purchased from Shanghai Anaiji Chemical; 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.

[0137] Example 1

[0138] Preparation of thiazinone hapten having the structure shown in formula I-1:

[0139] (1) Preparation of a compound having a structure shown in formula a:

[0140] To a 100 mL round-bottom flask at room temperature, 5-methyl-2-pyrrolidone (9.9 g, 100 mmol, 1.0 equiv) and 50 mL of 6 mol / L aqueous hydrochloric acid were added. A condenser was then added and the mixture was refluxed in an oil bath at 100°C for 8 h. TLC monitoring showed that the reaction of the starting material was complete. Post-treatment: Cool to room temperature, concentrate under vacuum, dissolve in methanol, and re-concentrate under vacuum three times. The obtained product was pure enough to be used directly in the next step.

[0141] (2) Preparation of the compound having the structure shown in formula b

[0142] Dissolve the crude product of Formula (a) directly in 100 mL of dry methanol at room temperature. Then, slowly add thionyl chloride (14.5 mL, 200 mmol, 2.0 equiv) dropwise under nitrogen. Reflux at 80°C for 6 h. If TLC monitoring indicates complete reaction, proceed with post-processing by cooling to room temperature, concentrating under vacuum, then dissolving in methanol and concentrating under vacuum three times. The resulting product is sufficiently pure for direct use in the next step.

[0143] (3) Preparation of the compound having the structure shown in formula c

[0144] The crude product of formula (b) was dissolved in 100 mL of dry dichloromethane at room temperature. Sodium bicarbonate (16.8 g, 200 mmol, 2.0 equiv) was added under nitrogen and stirred until homogeneous. Tert-butyl isosulfate (15.2 mL, 120 mmol, 1.2 equiv) was slowly added dropwise under nitrogen. The reaction was continued at room temperature for 10 h under nitrogen. TLC monitoring indicated complete reaction. Post-treatment: The sodium bicarbonate solid was removed by filtration. The filtrate was concentrated under reduced pressure and purified on a silica gel column using a mobile phase of petroleum ether / ethyl acetate (from 20 / 1 to 5 / 1). This afforded 16.8 g of pure A-3, a 68.3% yield for the three-step process from A-1 to A-3.

[0145] (4) Preparation of the compound having the structure shown in formula d

[0146] At room temperature, pure product of formula c (16.8 g, 68.3 mmol, 1.0 equiv) was dissolved in 70 mL of tetrahydrofuran. Separately, sodium hydroxide (5.46 g, 136.5 mmol, 2.0 equiv) was dissolved in 70 mL of water and the resulting aqueous sodium hydroxide solution was slowly added dropwise to the A-3 solution. The reaction was allowed to proceed at room temperature for 5 h. TLC monitoring indicated complete reaction. Post-treatment: Concentrate under reduced pressure to remove the tetrahydrofuran. Adjust the pH to 3-5 with 2 mol / L hydrochloric acid (HCl). The aqueous phase was then extracted three times with ethyl acetate (100 mL x 3). The organic phases were combined, dried, filtered, and concentrated under reduced pressure to yield the crude product. Purification was then performed on a silica gel column using a mobile phase consisting of petroleum ether / ethyl acetate / formic acid (2:1:0.05 to 1:1:0.05). This afforded 12.7 g of pure A-4 in an 80.1% yield.

[0147] (5) Preparation of the compound having the structure shown in Formula e

[0148] At room temperature, under nitrogen, N-methylformanilide (2.46 mL, 20 mmol, 1.0 equiv), AIBN (263 mg, 1.6 mmol, 0.08 equiv), and 20 mL of carbon tetrachloride were added to the reaction flask. The mixture was then heated to 70°C and sulfonyl chloride (4.86 mL, 60 mmol, 3.0 equiv) was slowly added dropwise. After the addition, the temperature was raised to 90°C and the reaction was continued for 3 h. TLC monitoring showed that the raw material had reacted completely. Post-treatment: the solvent was removed under reduced pressure and the crude product was used directly in the next reaction.

[0149] (6) Preparation of Thiazinon Hapten with the Structure of Formula I-1

[0150] Formula d (232 mg, 1.0 mmol, 1.0 equiv), tetrabutylammonium bromide (64 mg, 0.2 mmol, 0.2 equiv), and 8 mL of 1,4-dioxane were added to a reaction tube at room temperature and stirred uniformly. 1.2 mL (3.0 equiv) of 10% aqueous sodium hydroxide solution was then added. A 2 mL 1,4-dioxane solution of formula e (305 mg, 1.5 mmol, 1.5 equiv) was then slowly added dropwise to the above system. The reaction was stirred at room temperature for 8 h. TLC monitoring showed that the reaction of the starting material was complete. Post-treatment: 1,4-dioxane was removed by concentration under reduced pressure, and the pH value was adjusted to 3-5 with 2 mol / L hydrochloric acid (HCl). The aqueous phase was extracted with ethyl acetate three times (100 mL×3). The organic phases were combined, dried, filtered, and concentrated under reduced pressure to obtain the crude product, which was then purified on a silica gel column with a mobile phase of petroleum ether / ethyl acetate / triethylamine (2:1:0.05 to 1:1:0.05). Then 52 g of pure product was obtained with a yield of 14.3%.

[0151] The TLC spectrum of the thiazinone hapten having the structure shown in formula I-1 is as follows Figure 2 As shown; HRMS spectrum as Figure 3 As shown; 1 H NMR spectrum Figure 4 As shown; 13 C NMR spectrum Figure 5 The theoretical molecular weight of thiazolinone hapten is 363.16.

[0152] The structural characterization data are as follows:

[0153] 1 H-NMR (400M, CDCl3): δ1.33(s,9,tBu),1.49(d,3,J=8.0Hz,CH3),2.02-2.11(m,1,H 4-1 ),2.40(t,2,J=8.0Hz,H3),2.55-2.48(m,1,H 4-1),4.61-4.55(m,1,H5),4.76(d,1,J=12.0Hz,H 2-1 ),4.82(d,1,J=12.0Hz,H 2-1 ),7.22-7.40(m,5,Ph).

[0154] 13 C NMR(101MHz,DMSO)δ174.33,154.49,142.61,141.42,128.79,125.81,124.84,53.84,53.80,47 .11,45.46,40.12,39.91,39.70,39.50,39.29,39.08,38.87,31.63,29.29,28.96,18.36,9.99.

[0155] HRMS called for C9H 14 ClN5O2:[M+H + ]363.16,found 364.1712.

[0156] Example 2

[0157] Preparation of thiazinone hapten having the structure shown in formula I-2:

[0158] The molar ratio of thiazinone hapten to carrier protein was 50:1. 19.63 mg (0.027 mmol) of thiazinone hapten I-1, 6.21 mg (0.054 mmol) of N-hydroxysuccinimide (NHS), and 10.35 mg (0.054 mmol) of dicarbodiimide hydrochloride (EDC) were weighed and dissolved in 1 mL of anhydrous N,N-dimethylformamide (DMF). The mixture was stirred magnetically in a refrigerator at 4°C overnight (10 h). After completion of the reaction, a reaction product containing thiazinone hapten B was obtained, which could be directly used for subsequent carrier protein coupling.

[0159] Example 3

[0160] Synthetic complete thiazinone antigen II-1:

[0161]

[0162] 429 μL of the supernatant containing the thiazinone hapten obtained in Example 2 was slowly added dropwise to a carrier protein OVA (ovalbumin) solution (the carrier protein solution was prepared by dissolving 10 mg of OVA in 1 mL of phosphate buffer (0.01 mol / L PBS, pH = 7.4) and mixing). The reaction was stirred at 25°C for 4 h. The resulting reaction solution was dialyzed six times against 0.01 mol / L PBS (pH = 7.4). The dialyzed reaction product solution was quickly frozen with liquid nitrogen and stored at -20°C until use. This yielded the compound represented by Formula II-1, a conjugate of the thiazinone hapten and OVA.

[0163] The MALDI-TOF-MS detection spectrum of OVA standard is as follows Figure 6 As shown, the MALDI-TOF-MS detection spectrum of thiazinone complete antigen II-1 is as follows Figure 7 As shown. Figure 6 、 7 As can be seen, the single-charged ion peaks obtained for OVA and complete antigen II-1 are 44586.160 and 46129.253, respectively. The carrier protein to hapten coupling ratio = (complete antigen molecular weight - carrier protein molecular weight) / hapten molecular weight. The coupling ratio for complete antigen II-1 was calculated using the formula: 1:4 (rounded to the nearest integer).

[0164] Example 4

[0165] Synthetic complete thiazinone antigen II-2:

[0166]

[0167] 571 μL of the supernatant containing the thiazinone hapten obtained in Example 2 was slowly added dropwise to a carrier protein BSA (bovine serum albumin) solution (the carrier protein solution was prepared by dissolving 20 mg of BSA in 2 mL of phosphate buffer (0.01 mol / L PBS, pH = 7.4) and mixing thoroughly). The reaction was stirred at 25°C for 4 h. The resulting reaction solution was dialyzed six times against 0.01 mol / L PBS (pH = 7.4), quickly frozen in liquid nitrogen, and stored at -20°C until use. This yielded the compound represented by Formula II-2, a conjugate of the thiazinone hapten and BSA.

[0168] The MALDI-TOF-MS detection spectrum of BSA standard is as follows Figure 8 As shown, the MALDI-TOF-MS detection spectrum of thiazinone complete antigen II-2 is as follows Figure 9 As shown. Figure 8 、 9As can be seen, the single-charged ion peaks obtained for BSA and complete antigen II-2 are 67334.423 and 72971.617, respectively. The carrier protein to hapten coupling ratio = (complete antigen molecular weight - carrier protein molecular weight) / hapten molecular weight. The coupling ratio of complete antigen II-2 was calculated to be 1:15 (rounded to the nearest whole number).

[0169] Example 5

[0170] 1. Preparation of polyclonal antibodies using the complete thiazide antigen II-2 of Example 4

[0171] 1.1 Preparation of polyclonal antibodies using the complete thiazide antigen II-2 of Example 4

[0172] (1) 7- to 8-week-old Balb / c mice were selected as experimental animals (7- to 8-week-old Balb / c mice weigh approximately 23-25 ​​g).

[0173] (2) Primary Immunization: 1 mL of the complete buprofen II-2 antigen solution (concentration: 1 mg / mL) obtained in Example 4 was sterile filtered and then added with an equal volume of Freund's complete adjuvant. The mixture was stirred thoroughly until emulsified and did not diffuse when dropped into water. The emulsified complete antigen was injected into the peritoneal cavity and subcutaneously on the back of mice, with a total injection dose of 0.1 mg of emulsified antigen per mouse.

[0174] (3) Booster immunization: 2 weeks after the initial immunization, take 1 mL of the above-mentioned thiazinone complete antigen II-2 antigen solution (concentration is 1 mg / mL), then add 1 mL of Freund's incomplete adjuvant, stir thoroughly to emulsify, until it does not spread when dropped into water. The emulsified complete antigen is injected into the mouse's abdominal cavity and back subcutaneously, with a total injection dose of 0.1 mg of emulsified antigen per mouse. Booster immunization is performed every 14 days. Starting from the third booster immunization, blood is collected from the mouse's eye sockets 3 to 5 days after each immunization, and the titer and inhibition rate of anti-thiazinone serum / antibody after each immunization are determined by ic-ELISA. The coating antigen is thiazinone complete antigen II-1 (1 mg / mL) diluted in 4 gradients, namely 0.5×10 3 , 1×10 3 , 2×10 3 and 4×10 3 . Waiting for titer ≥6.4×10 4 After that (the titer is defined as the dilution multiple of serum when the color value of the control well is about 1.0), the eyeballs are enucleated and blood is collected. The blood is placed in a constant temperature incubator at 37°C for 30 minutes, and then placed in a refrigerator at 4°C for 2 hours. Then, the blood is centrifuged at 4°C and 10,000 r / min for 5 minutes to separate the antiserum, thus obtaining the anti-thiazinone serum / antibody. The titer and inhibition rate of the serum / antibody checkerboard of the anti-thiazinone fusion mouse are determined by ic-ELISA.

[0175] 1.2 Determination of titer and inhibition rate of anti-thiazide serum / antibody

[0176] The various buffers used in the following experiments are as follows:

[0177] (1) Coating buffer (CBS, pH 9.6, 0.05 M carbonate buffer): Weigh 1.5 g of Na2CO3 and 2.94 g of NaHCO3, and dilute to 1 L with ultrapure water;

[0178] (2) Phosphate buffer (0.01 M PBS, pH = 7.4): Weigh 0.2 g of KH2PO4, 8 g of NaCl, and 2.92 g of NaH2PO4·12H2O, and dilute to 1 L with ultrapure water;

[0179] (3) Washing buffer (PBST): Add 0.1% Tween-20 by volume to the prepared 0.01 M PBS solution;

[0180] (4) Sample diluent (PBSTG): Add 1% by volume of Tween-20 and 1 g of gelatin (melted in a microwave oven) to the prepared phosphate buffer solution;

[0181] (5) Chromogenic substrate buffer: Weigh 0.1 g potassium sorbate and 46.04 g potassium dihydrogen citrate hydrate, and dilute to 1 L with ultrapure water;

[0182] (6) Stop solution (1 mol / L HCl): Measure 440 mL of distilled water, add 40 mL of concentrated hydrochloric acid (w / v = 98%) dropwise and stir.

[0183] 1.3 Determination of titer and inhibition rate of anti-thiazide serum / antibody

[0184] 1.3.1. Preparation of Buprofezin Complete Antigen II-1 Coating Antigen Solution

[0185] The complete thiazinone antigen II-1 prepared in Example 3 was diluted with coating buffer (CBS) as needed to obtain coating antigen solutions of different concentrations of the complete thiazinone antigen II-1.

[0186] 1.3.2. Preparation of Buprofezin Standard Solution

[0187] (1) Weigh 10 mg of thiazinone standard sample and dissolve it in 10 mL of acetonitrile to obtain a 1 mg / mL thiazinone standard solution.

[0188] (2) The 1 mg / mL thiazinone standard solution in (1) was prepared with sample diluent (PBSTG) to a final concentration of 1000 ng / mL thiazinone standard solution.

[0189] 1.3.3. Preparation of anti-buprofen serum / antibody dilutions

[0190] The thiazinone antiserum / antibody prepared in the above steps was gradiently diluted with PBSTG according to experimental requirements to obtain anti-thiazinone serum / antibody dilution solution.

[0191] 1.3.4. ic-ELISA Assay Procedure

[0192] (1) Coating: Add 100 μL of the complete thiazide antigen II-1 prepared in step 1 (after dilution with CBS solution, the original concentration is 1 mg / mL) to each well of a 96-well transparent ELISA plate, incubate in a 37°C constant temperature incubator for 3 h, wash three times with PBST, and spin dry.

[0193] (2) Competition: Add 50 μL of PBSTG to each control well; add 50 μL of the thiazinone standard solution prepared in step 2 to each inhibition well. Add the anti-thiazinone antiserum / antibody dilution obtained in step 3 to the ELISA plate (50 μL / well), incubate in a 37°C incubator for 30 min, wash the plate three times with PBST, and spin dry.

[0194] (3) Add enzyme-labeled secondary antibody: Dilute goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP) with PBSTG to the working concentration of 100 μL per well, incubate in a 37°C constant temperature incubator for 30 min, wash the plate 3 times with PBST, and spin dry.

[0195] (4) Color development: The color development solution for each ELISA plate is: add 200 μL of TMB storage color development solution to 11 mL of color development substrate buffer, and add 3.4 μL of hydrogen peroxide (w:v = 30%). Add 100 μL of prepared TMB color development solution to each well. Prepare the color development solution immediately before use and develop the color at room temperature in the dark for 15 minutes.

[0196] (5) Stop the reaction: Add 50 μL of 1M HCL stop buffer to each well. After the reaction is terminated, measure the OD value of each well of the plate using a microplate reader. 450nm value.

[0197] 1.4 Results of serum / antibody titer and inhibition rate determination in anti-thiazinone fusion mice

[0198] The serum titer of fusion mice was determined by IC-ELISA.

[0199] The inhibition rate was calculated as follows:

[0200] I=(CI) / C×100%

[0201] "C" is the OD of the control well 450nm Value; "I" is the inhibition hole OD450nm “IR” is the inhibition rate.

[0202] After the fourth and fifth immunizations of anti-thiazinone fusion mice, the titers and inhibition rates of anti-thiazinone serum / antibodies (TMB color development for 15 min, inhibition by 1000 ng / mL thiazinone standard) are shown in Table 1. 4 After (titer is defined as OD 450 When the nm value is approximately 1.0, the dilution factor of the antiserum / antibody is calculated. Eyeballs are enucleated and blood is collected. The blood is placed in a 37°C incubator for 30 minutes, then in a 4°C refrigerator for 2 hours. The blood is then centrifuged at 4°C, 10,000 rpm, and 5 minutes. The serum is then separated to obtain the anti-buprofen serum / antibody, which is used in the following experiments.

[0203] Table 1 Results of the fourth and fifth serum / antibody titer determinations of anti-thiazinone fusion mice

[0204]

[0205] In Table 1, "I" represents the inhibition well in the ELISA plate, "C" represents the control well in the ELISA plate, and "IR" represents the inhibition rate. The inhibition concentration of the thiazolinone standard is 1000 ng / mL.

[0206] The results in Table 1 show that after the fifth immunization, when the coating antigen was diluted to 0.5×10 3 and anti-buprofen serum diluted 1:6.4×10 4 At this time, the OD value of the antiserum is about 1.0, which indicates that the complete thiazinone antigen II-2 prepared in Example 4 can be used as an immunogen to prepare anti-thiazinone serum / antibody.

[0207] The results in Table 1 demonstrate that the thiazinone hapten designed and synthesized by the present invention can fully expose the antigenic determinant to the host animal and exhibit antigenicity. The complete antigen prepared by the present invention, when used to immunize the host animal (Balb / c mouse), can stimulate the production of highly sensitive anti-thiazinone serum / antibodies. The resulting antiserum / antibody can specifically recognize thiazinone.

[0208] The results of the antiserum / antibody checkerboard assay against thiazinone-fused mice (TMB color development for 15 min, inhibition by 1000 ng / mL thiazinone standard) are shown in Table 2 .

[0209] Table 2 Results of titer determination of checkerboard antiserum / antibody against thiazinone fusion mice

[0210]

[0211]

[0212] In Table 2, "I" represents the inhibition well in the ELISA plate, "C" represents the control well in the ELISA plate, and "IR" represents the inhibition rate. The inhibition concentration of the thiazolinone standard is 1000 ng / mL.

[0213] The results in Table 2 show that when the coating antigen was diluted to 4×10 3 times, the anti-buprofen serum / antibody dilution was 6.4×10 4 times, at this time OD 450nm The value of is around 1.0, and the inhibition rate is 82.35%, which is a good inhibitory effect. This shows that after immunizing Balb / c mice with the complete thiazinone antigen II-2 prepared in Example 5, the anti-thiazinone serum / antibody titer of the screened fusion mice is high, and the inhibition rate is good.

[0214] Example 6

[0215] 2. Preparation of Nanobodies Using the Thiazide Complete Antigen II-2 of Example 4

[0216] 2.1 Immunizing Alpacas

[0217] Three-year-old male alpacas were selected as host animals, with an immunization dose of 200 μg. The immunization strategy was similar to that used for mouse immunization: Freund's complete adjuvant (primary immunization) or Freund's incomplete adjuvant (boosting immunization) was fully emulsified with complete antigen II-2 at a 1:1 ratio. The emulsified immunogen (complete antigen II-2) was injected subcutaneously on the neck and back of the alpaca at multiple points. The immunization strategy was to have two weeks between each booster immunization, for a total of six immunizations. Starting after the third immunization, blood was collected using a blood collection tube 7-10 days after each booster immunization to measure the serum / antibody titer and inhibition rate after each booster immunization. One week after the fifth immunization, 10-15 mL of peripheral blood was collected from the alpaca using a blood collection tube, with 1 mL reserved for serum titer measurement. The remaining blood was then used to extract total RNA and lyse red blood cells using the Trizol method. The remaining white blood cells were dissolved in Trizol and stored at -80°C.

[0218] 2.2 Determination steps and results of alpaca serum titer and inhibition rate

[0219] 2.2.1 Determination of alpaca serum titer and specificity

[0220] ① Coating: Dilute 1 mg / mL buprofezin-OVA to 1:1×10 3 , 1:2×10 3 , 1:4×10 3 , 1:8×10 3 , 100 μL was added to each well of the ELISA plate, incubated at 37°C for 3 h, CBS was discarded, and the plate was washed 4 times with PBST;

[0221] ② Blocking: Add 200 μL of blocking solution to each well, incubate at 37°C for 1 hour, discard the blocking solution, and wash the plate 4 times with PBST;

[0222] ③Add standard and serum: dilute serum with PBSTG to 1:1×10 3 , 1:2×10 3 , 1:4×10 3 , 1:8×10 3 , 50 μL of buprofen standard (1000 ng / mL and 0 ng / mL) was first added to each well, followed by 50 μL of diluted serum / antibody, incubated at 37°C for 30 min, and the plate was washed four times;

[0223] ④Add secondary antibody: HRP-labeled anti-His tag mouse monoclonal antibody diluted to 1:10000 with PBSTG, add 100 μL to each well, incubate at 37°C for 30 min, and wash the plate 4 times;

[0224] ⑤ Color development: Add 100 μL TMB single-component color development solution to each well and develop the color for 15 min at 20-25°C in the dark.

[0225] ⑥Termination and detection: Add 50 μL of stop solution to each well to terminate the reaction and use a microplate reader to measure OD 450 Values ​​at nm;

[0226] ⑦ Titer determination: The maximum dilution factor of the serum when the absorbance is 1.0 is the serum titer

[0227] ⑧Specificity determination: The specificity of the serum is determined based on the inhibition rate. The inhibition rate calculation formula is the same as 1.4.

[0228] 2.2.2 Results of titer and specificity determination of alpaca serum

[0229] The serum titer and inhibition rate were determined by IC-ELISA. The results of alpaca negative serum determination showed that the immunized alpaca was a blank alpaca. The results of alpaca serum titer and inhibition rate determination after the third, fourth and fifth immunizations are shown in Table 3. Table 3 Results of alpaca serum titer and inhibition rate determination

[0230]

[0231]

[0232] In Table 3, "I" indicates the inhibition well in the ELISA plate, "C" indicates the control well in the ELISA plate, and "IR" indicates the inhibition rate. The inhibition concentration of the thiazolinone standard is 1000 ng / mL.

[0233] The results in Table 3 show that after the fifth immunization, the coating original (complete antigen II-1) was diluted 1×10 3times, dilute alpaca serum to 1×10 3 When the serum titer and inhibition rate are slightly different from the results after the fourth immunization, the alpaca peripheral blood can be collected for the construction of the nanoantibody library.

[0234] In summary, the antigenic determinants of the thiazinone hapten structure provided by the present invention are fully exposed to the host animal. Immunization of the host animal with the complete antigen provided by the present invention can stimulate the production of highly sensitive anti-thiazinone serum / antibodies that specifically recognize thiazinone. The anti-thiazinone serum / antibodies provided by the present invention can be used to establish thiazinone immunological analysis methods or detection reagents.

[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 thiazinone hapten having the structure shown in Formula I-1 or Formula I-2:

2. The method for preparing the thiazinone hapten according to claim 1, comprising the following steps: Mixing 5-methyl-2-pyrrolidone, hydrochloric acid and water to carry out a ring-opening reaction to obtain a compound having a structure shown in formula a; Mixing the compound having the structure shown in formula a, methanol and an esterification reaction catalyst to carry out an esterification reaction to obtain a compound having the structure shown in formula b; Mixing the compound having the structure shown in formula b, tert-butyl isosulfate, an inorganic base catalyst and a first organic solvent, and performing a substitution reaction to obtain a compound having the structure shown in formula c; Mixing the compound having the structure shown in formula c, an inorganic strong base aqueous solution and a second organic solvent, and performing a hydrolysis reaction to obtain a compound having the structure shown in formula d; Mixing N-methylformanilide, sulfonyl chloride, an azo initiator, and a third organic solvent to carry out an acyl chlorination reaction to obtain a compound having a structure shown in formula e; The compound having the structure shown in formula d, the compound having the structure shown in formula e, an inorganic strong base aqueous solution, a phase transfer catalyst and a third organic solvent are mixed to perform a cyclization reaction to obtain a thiazinone hapten having the structure shown in formula I-1; Mixing the thiazinone hapten having the structure shown in Formula I-1, N-hydroxysuccinimide, carbodiimide hydrochloride, and a fourth organic solvent to perform a carboxyl activation reaction to obtain a thiazinone hapten having the structure shown in Formula I-2; 3. The preparation method according to claim 2, characterized in that The ring-opening reaction temperature is 95-100°C and the time is 7.5-8h; The esterification reaction temperature is 75-80°C and the time is 5.5-6h; The temperature of the substitution reaction is 20-25°C and the time is 9.5-10 hours; The temperature of the hydrolysis reaction is 20-25° C., and the time is 5-5.5 hours.

4. The preparation method according to claim 2, characterized in that The temperature of the acyl chlorination reaction is 70°C to reflux temperature, and the time is 2.5 to 3 hours; The temperature of the cyclization reaction is 20-25° C., and the time is 8-8.5 hours.

5. A complete thiazinone antigen, comprising the thiazinone hapten according to claim 1 and a carrier protein coupled to the thiazinone hapten.

6. Use of the complete thiazinone antigen according to claim 5 in the preparation of thiazinone antiserum or thiazinone antibody.

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