Ethylmerphosal hapten, complete antigen, antibodies and methods of preparation and use

Abstract

This invention belongs to the field of immunoassay technology, specifically relating to acetylpyridinium hapten, complete antigen, antibody, preparation methods, and applications. The acetylpyridinium hapten provided by this invention has the structure shown in Formula I, where R1 is an active group capable of direct coupling with a carrier protein. The acetylpyridinium hapten provided by this invention retains the basic structure of acetylpyridinium, using the original -CH2-CH2-CH2-CH2- structure of acetylpyridinium as a linker arm. An active group R1 capable of direct coupling with a carrier protein is introduced into the linker arm at a site far from the characteristic structure of acetylpyridinium, effectively avoiding non-specific binding caused by the external linker arm. After coupling with the carrier protein, the introduced active group can fully expose the characteristic structure of the acetylpyridinium bulk molecule. The prepared acetylpyridinium complete antigen is immunogenic, and the prepared anti-acetylpyridinium antibody has good specificity and high sensitivity. It can be used to establish immunological detection methods or reagents for acetylpyridinium, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to ethirimol hapten, complete antigen, antibody, preparation method and application. Background Technology

[0002] Acetaminophen's English name is prometryn, its chemical name is 5-butyl-2-ethylamino-4-hydroxy-6-methylpyrimidine, its CAS registry number is 23947-60-6, and its molecular formula is C 11 H 19 N3O, with a molecular weight of 209.3, is also known in Chinese as ethirimol, acetaminophen, acetaminophen, 5-butyl-2-ethylamino-4-hydroxy-6-methylpyrimidine, ethirimol, or acetaminophen.

[0003] Ethirimol exhibits higher efficacy and safety against powdery mildew in many crops, including strawberries, watermelons, cucumbers, and grapes, compared to other imported or domestically produced products. However, improper application methods and dosages of ethirimol can lead to environmental pollution and pesticide residues. Residual ethirimol can accumulate and migrate throughout the food chain, negatively impacting the quality and safety of agricultural products, the ecological environment, and ultimately, human health. Therefore, monitoring ethirimol residue levels is necessary.

[0004] Many methods have been established for detecting acetaminophen residues in environmental samples, food, and agricultural products. Instrumental analytical methods, such as liquid chromatography (LC), LC-tandem mass spectrometry (LC-MS / MS), gas chromatography (GC), and GC-tandem mass spectrometry (GC-MS / MS), offer sufficient accuracy and high sensitivity and are commonly used. However, these instrumental analytical methods typically require highly trained personnel, expensive equipment, complex sample pretreatment, and time-consuming analytical procedures, making them unsuitable for on-site detection of acetaminophen residues.

[0005] Immunoassay methods offer advantages such as speed, simplicity, real-time performance, ease of on-site testing, simple sample pretreatment, high sensitivity, strong selectivity, and suitability for high-throughput analysis due to their specific recognition of antigens and antibodies. They can also significantly reduce testing costs. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a method for preparing acetaminophen hapten, complete antigen, and antibody, as well as their applications. The acetaminophen hapten provided by this invention, after conjugation, can fully highlight the acetaminophen antigenic determinant. The prepared acetaminophen complete antigen is immunogenic and can specifically recognize acetaminophen. The resulting anti-acetaminophen antibody has good specificity and high sensitivity, and can be used for the development of immunoassay methods for acetaminophen.

[0007] This invention provides an acetaminophen hapten having the structure shown in Formula I:

[0008]

[0009] In Formula I, R1 is an active group that can be directly coupled to the carrier protein.

[0010] Preferably, R1 includes -(CH2). n -COOH, -(CH2) n -NH2、-(CH2) n -OH, -(CH2) n -COOR2、 n is an integer from 0 to 3, m is an integer from 0 to 4, and -(CH2) n -COOR2 R2 in the middle includes

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

[0012]

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

[0014] By replacing the R3 group in the compound structure shown in Formula II with the R1 group through a chemical reaction, the ethirimol hapten with the structure shown in Formula I is obtained.

[0015]

[0016] R3 in Formula II is -CH3 or -COO-CH2-CH3.

[0017] Preferably, the method for preparing the ethirimol hapten with the structure shown in Formula I-1 includes the following steps:

[0018] The compound with the structure shown in Formula 5 and an inorganic strong base were dissolved in a mixed solution of organic solvent and water to carry out a hydrolysis reaction, yielding the ethirimol hapten with the structure shown in Formula I-1.

[0019]

[0020] This invention provides a complete acetaminophen antigen, which is obtained by conjugating the acetaminophen hapten or the acetaminophen hapten prepared by the preparation method described in the above technical solution with a carrier protein.

[0021] This invention provides an acetaminophen antibody, which is obtained by emulsifying the acetaminophen complete antigen described in the above technical solution and then immunizing a host animal.

[0022] This invention provides a method for preparing the acetaminophen antibody described in the above technical solution, comprising the following steps:

[0023] The host animal is immunized after emulsifying the ethirimol complete antigen described in the above technical solution.

[0024] The preparation method of acetaminophen polyclonal antibody is as follows: after boosting the immunization of the host animal, the blood of the immunized animal is collected and separated to obtain the acetaminophen polyclonal antibody;

[0025] The method for preparing ethirimol monoclonal antibody is as follows: extract spleen cells from immunized host animals, fuse the spleen cells with SP2 / 0 tumor cells, screen monoclonal hybridoma cell lines that can secrete ethirimol antibody, and prepare the ethirimol monoclonal antibody based on the monoclonal hybridoma cell lines.

[0026] The preparation method of ethirimol nanobodies is as follows: mRNA transcribed from B lymphocytes in the blood of immunized host animals is extracted, reverse transcribed into cDNA, and the cDNA is used as a substrate for PCR amplification to obtain diverse nanobody gene fragments. The diverse nanobody gene fragments are used to construct a phage or yeast antibody expression library, and ethirimol nanobodies are obtained after screening.

[0027] This invention provides a reagent or kit for detecting acetaminophen, containing the acetaminophen antibody described in the above technical solution or the acetaminophen antibody prepared by the preparation method described in the above technical solution.

[0028] This invention provides the application of the acetaminophen antibody described in the above-described technical solution, or the acetaminophen antibody prepared by the preparation method described in the above-described technical solution, or the reagent or kit described in the above-described technical solution, in the detection of acetaminophen.

[0029] This invention provides an acetaminophen hapten having the structure shown in Formula I:

[0030]

[0031] In Formula I, R1 is an active group that can be directly coupled to the carrier protein.

[0032] The acetylpyridinium hapten provided by this invention retains the basic structure of acetylpyridinium while using the original -CH2-CH2-CH2-CH2- structure of acetylpyridinium as the linker arm for all or part of the linker. An active group R1, capable of direct coupling with a carrier protein, is introduced at a site on the -CH2-CH2-CH2-CH2- structure far from the characteristic structure of acetylpyridinium. This not only effectively avoids non-specific binding caused by the external linker arm, but also fully exposes the characteristic structure of the acetylpyridinium molecule after coupling with the carrier protein. The resulting complete acetylpyridinium antigen is immunogenic. After immunizing host animals with the complete acetylpyridinium antigen, the prepared anti-acetylpyridinium serum or anti-acetylpyridinium antibody can be used to establish acetylpyridinium immunological detection methods or reagents, showing broad application prospects.

[0033] This invention provides a complete acetaminophen antigen, obtained by conjugating an acetaminophen hapten prepared by the method described in the above-described technical solution with a carrier protein. The anti-acetaminophen serum or anti-acetaminophen antibody obtained by immunization with the complete acetaminophen antigen provided by this invention can efficiently recognize and specifically bind to acetaminophen, providing a technical basis for the immunization application of acetaminophen.

[0034] This invention provides an acetaminophen antibody, obtained by emulsifying the complete acetaminophen antigen described in the above-mentioned technical solution and then immunizing a host animal. The anti-acetaminophen serum or anti-acetaminophen antibody obtained by immunizing with the complete acetaminophen antigen provided by this invention can efficiently recognize and specifically bind to acetaminophen.

[0035] In summary, this invention is the first to design and synthesize an acetylene hapten, and the complete acetylene antigen prepared by conjugating the acetylene hapten with a carrier protein possesses immunogenicity. This invention allows for the immunization of host animals with the complete acetylene antigen, resulting in the preparation of highly sensitive and specific anti-acetylene antibodies, providing a technical foundation for establishing immunoassay methods for anti-acetylene antibodies. Furthermore, the immunoassay method developed based on the anti-acetylene antibodies prepared in this invention can meet the need for rapid on-site detection of acetylene and has broad application prospects. Attached Figure Description

[0036] Figure 1 The synthetic route for the ethirimol hapten shown in Formula I-1 in Example 1 is as follows;

[0037] Figure 2 The synthetic route for the ethirimol hapten shown in Formula I-2 in Example 2 is as follows;

[0038] Figure 3 This is the synthetic route for the ethirimol complete antigen shown in Formula III of this invention;

[0039] Figure 4The liquid chromatogram of the ethirimol hapten shown in Formula I-1 in Example 1;

[0040] Figure 5 The mass spectrum of the acetaminophen hapten shown in Formula I-1 in Example 1 is obtained by LCMS.

[0041] Figure 6 The ethirimol hapten shown in Formula I-1 in Example 1 1 H NMR spectrum;

[0042] Figure 7 The MALDI-TOF-MS spectrum of the OVA standard in Example 3;

[0043] Figure 8 The MADLI-TOF-MS spectrum of the ethirimol complete antigen shown in Formula III-1 in Example 3;

[0044] Figure 9 The MADLI-TOF-MS spectrum of the BSA standard in Example 4;

[0045] Figure 10 The MALDI-TOF-MS spectrum of the ethirimol complete antigen shown in Formula III-2 in Example 4 is shown. Detailed Implementation

[0046] This invention provides an acetaminophen hapten having the structure shown in Formula I:

[0047]

[0048] In Formula I, R1 is an active group that can be directly coupled to the carrier protein.

[0049] In this invention, R1 preferably includes -(CH2). n -COOH, -(CH2) n -NH2、-(CH2) n -OH, -(CH2) n -COOR2、 n is an integer from 0 to 3, m is an integer from 0 to 4, and -(CH2) n -COOR2 R2 in the middle includes

[0050] In this invention, n is preferably an integer from 0 to 3, and more preferably 0.

[0051] In this invention, R1 is preferably -OH, -NH2, -COOH or -COOR2.

[0052] In this invention, the -(CH2) n The R2 in -COOR2 is specifically preferred.

[0053] In a specific embodiment of the present invention, the ethirimol hapten preferably has the structure shown in Formula I-1 or Formula I-2:

[0054]

[0055] This invention provides a method for preparing the ethirimol hapten described in the above technical solution, comprising the following steps:

[0056] By replacing the R3 group in the compound structure shown in Formula II with the R1 group through a chemical reaction, the ethirimol hapten shown in Formula I is obtained.

[0057]

[0058] R3 in Formula II is -CH3 or -COO-CH2-CH3.

[0059] In this invention, the method for preparing the ethirimol hapten with the structure shown in Formula I-1 preferably includes the following steps:

[0060] The compound with the structure shown in Formula 5 and an inorganic strong base were dissolved in a mixed solution of an organic solvent (hereinafter referred to as the first organic solvent) and water to carry out a hydrolysis reaction, so as to obtain the ethirimol hapten with the structure shown in Formula I-1.

[0061]

[0062] In this invention, the method for preparing the compound with the structure shown in Formula 5 preferably includes the following steps:

[0063] In a protective gas atmosphere, the compound with the structure shown in Formula 1, the compound with the structure shown in Formula 2, and sodium hydride (NaH) are dissolved in an organic solvent (hereinafter referred to as the second organic solvent) to carry out an alkylation reaction to obtain the compound with the structure shown in Formula 3.

[0064]

[0065] The compound with the structure shown in Formula 3, the compound with the structure shown in Formula 4, and sodium methoxide (CH3ONa) are dissolved in an organic solvent (hereinafter referred to as the third organic solvent) and subjected to a condensation reaction to obtain the compound with the structure shown in Formula 5.

[0066]

[0067] In a specific embodiment of the present invention, the compound with the structure shown in Formula 4 is specifically ethyl guanidine sulfate.

[0068] In this invention, under a protective gas atmosphere, the compound with the structure shown in Formula 1, the compound with the structure shown in Formula 2, and sodium hydride (NaH) are dissolved in tetrahydrofuran (THF) to carry out an alkylation reaction to obtain the compound shown in Formula 3.

[0069] In this invention, the molar ratio of the compound with the structure shown in Formula 1 to the compound with the structure shown in Formula 2 is preferably 3:2.

[0070] In this invention, the molar ratio of the compound with the structure shown in Formula 1 to NaH is preferably 1:1.

[0071] In this invention, the second polar organic solvent is preferably tetrahydrofuran (THF).

[0072] The present invention does not have any special requirements on the amount of THF used; it is sufficient to completely dissolve the compound with the structure shown in Formula 1, the compound with the structure shown in Formula 2, and NaH.

[0073] In this invention, the alkylation reaction is preferably carried out under reflux conditions.

[0074] In this invention, the alkylation reaction is preferably carried out for 12 hours.

[0075] In this invention, the protective gas atmosphere is preferably a nitrogen atmosphere.

[0076] In this invention, the alkylation reaction yields an alkylation reaction solution. Preferably, the alkylation reaction solution is post-treated to obtain a compound with the structure shown in Formula 3. The post-treatment preferably includes: sequentially adding a saturated ammonium chloride aqueous solution, adding water, extracting with ethyl acetate, combining the organic phases, washing the organic phases with a saturated NaCl aqueous solution, drying, concentrating, and purifying by column chromatography. In this invention, the volume ratio of the saturated ammonium chloride aqueous solution to the THF is preferably 1:3; the volume ratio of water to the saturated ammonium chloride aqueous solution is preferably 5:1; the ethyl acetate extraction is preferably performed twice, and the volume ratio of ethyl acetate to water used in each extraction is preferably 1:1; the drying is preferably performed using anhydrous sodium sulfate; this invention does not have special requirements for the specific implementation process of the concentration; in this invention, the column chromatography is preferably silica gel column chromatography, and the eluent used in the column chromatography is preferably petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate preferably 10:1.

[0077] In this invention, the compound with the structure shown in Formula 3 is a light yellow oily substance.

[0078] After obtaining the compound shown in Formula 3, the present invention dissolves the compound with the structure shown in Formula 3, the compound with the structure shown in Formula 4 (ethylguanidine sulfate), and sodium methoxide in a third organic solvent to carry out a condensation reaction to obtain the compound with the structure shown in Formula 5.

[0079] In this invention, the molar ratio of the compound shown in Formula 3 to the compound shown in Formula 4 is preferably 2:1.

[0080] In this invention, the molar ratio of the compound shown in Formula 3 to sodium methoxide is preferably 1:1.

[0081] In this invention, the third organic solvent is preferably methanol.

[0082] The present invention does not have any special requirements on the amount of methanol used; it is sufficient to completely dissolve the compound shown in Formula 3, the compound shown in Formula 4, and sodium methoxide.

[0083] In this invention, the temperature of the condensation reaction is preferably 70°C.

[0084] In this invention, the condensation reaction time is preferably 1 hour.

[0085] In this invention, the condensation reaction yields a condensation reaction solution. Preferably, the condensation reaction solution is post-treated to obtain a compound with the structure shown in Formula 5. The post-treatment preferably includes: sequentially removing the ethanol solvent, adding a saturated ammonium chloride aqueous solution, extracting with ethyl acetate, washing the organic phase with water, washing with a saturated NaCl solution, drying the washed organic phase, concentrating, and purifying by column chromatography. In this invention, the volume ratio of the saturated ammonium chloride aqueous solution to ethanol is preferably 4:3; the volume ratio of ethyl acetate to the saturated ammonium chloride aqueous solution is preferably 1:2; the organic phase is preferably dried and washed with anhydrous sodium sulfate; the specific implementation process of the concentration is not particularly required; the column chromatography is preferably silica gel column chromatography, and the eluent used in the column chromatography is preferably dichloromethane and methanol, with a volume ratio of dichloromethane to methanol preferably 50:1.

[0086] In this invention, the inorganic strong base is preferably NaOH.

[0087] In this invention, the first organic solvent is preferably ethanol.

[0088] In this invention, the preferred mass ratio of the compound with the structure shown in Formula 5 to the inorganic strong base is 11.56:8.

[0089] In this invention, the volume ratio of ethanol to water is preferably 1:1.

[0090] In this invention, the hydrolysis reaction is preferably carried out at room temperature.

[0091] In this invention, the hydrolysis reaction time is preferably 16 hours.

[0092] In this invention, the hydrolysis reaction yields a hydrolysis reaction solution. Preferably, the hydrolysis reaction solution undergoes post-treatment to obtain the ethirimol hapten with the structure described in Formula I-1. The post-treatment preferably includes the following steps: concentration, adjusting the pH of the concentrate to 4 with acetic acid to precipitate solids, solid-liquid separation, collecting the solid product and washing it with water, methanol slurrying, and drying. This invention does not have specific requirements for the specific implementation process of the concentration; the volume ratio of the water used for washing to the volume of methanol used in the methanol slurrying is preferably 1:2; this invention does not have specific requirements for the specific implementation process of the drying.

[0093] In this invention, the method for preparing the ethirimol hapten with the structure shown in Formula I-2 preferably includes the following steps:

[0094] The ethirimol hapten with the structure shown in Formula I-1, N-hydroxysuccinimide (NHS), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were dissolved in N,N-dimethylformamide (DMF) and a substitution reaction was carried out to obtain the ethirimol hapten with the structure shown in Formula I-2.

[0095] In this invention, the molar ratio of ethirimol hapten and NHS with the structure shown in Formula I-1 is preferably 0.021:0.042.

[0096] In this invention, the molar ratio of ethirimol hapten and EDC with the structure shown in Formula I-1 is preferably 0.021:0.042.

[0097] In this invention, the ethirimol hapten, NHS, and EDC with the structure shown in Formula I-1 are dissolved in the reaction solution formed by DMF, and the molar concentration of the ethirimol hapten with the structure shown in Formula I-1 is preferably 0.042 mol / L.

[0098] In this invention, the temperature of the substitution reaction is preferably 4°C.

[0099] In this invention, the holding time for the substitution reaction is preferably 10 hours.

[0100] In this invention, the substitution reaction is preferably carried out under stirring conditions, and the stirring is preferably magnetic stirring.

[0101] This invention provides a complete acetaminophen antigen, which is obtained by conjugating the acetaminophen hapten or the acetaminophen hapten prepared by the preparation method described in the above technical solution with a carrier protein.

[0102] In this invention, the carrier protein preferably includes bovine serum albumin (BSA), ovalbumin (OVA), or keyhole hemocyanin (KLH).

[0103] In this invention, the structural formula of the ethirimol complete antigen is preferably as shown in Formula III:

[0104]

[0105] In specific embodiments of the present invention, the structural formula of the ethirimol complete antigen is preferably shown as shown in Formula III-1 or Formula III-2:

[0106]

[0107] This invention provides a method for preparing the ethirimol complete antigen described in the above technical solution, preferably comprising the following steps:

[0108] The ethirimol hapten was coupled with a buffer solution of the carrier protein to obtain the complete ethirimol antigen.

[0109] In this invention, when the acetaminophen hapten is preferably the acetaminophen hapten with the structure shown in Formula I-1, it is preferable to prepare the acetaminophen hapten with the structure shown in Formula I-1 according to the preparation method of the acetaminophen hapten shown in Formula I-2 above, and then perform the coupling reaction with the carrier protein.

[0110] In this invention, the ethirimol hapten is preferably coupled to the carrier protein in the form of an organic solution of the ethirimol hapten.

[0111] In a specific embodiment of the present invention, the acetaminophen hapten is preferably prepared by the substitution reaction supernatant obtained by the preparation method of the acetaminophen hapten shown in Formula I-2 of the above technical solution as a raw material for coupling reaction with the carrier protein.

[0112] In this invention, in the reaction system of the coupling reaction, the molar ratio of the ethirimol hapten to the carrier protein is preferably (10-60):1, more preferably 60:1.

[0113] In this invention, the reaction temperature of the coupling reaction is preferably 0-50°C, more preferably 4°C; the reaction time is preferably 8-36 h, more preferably 12 h; and the reaction pH value of the coupling reaction is preferably 5-9, more preferably 7.4.

[0114] In this invention, the buffer solution for the carrier protein is preferably at least one selected from carbonate buffer (CBS), phosphate buffer (PBS), borate buffer, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer. In this invention, the pH of the buffer solution for the carrier protein is preferably 5 to 9, more preferably 7.4.

[0115] In this invention, after the coupling reaction, the invention preferably further includes dialysis of the reaction system of the coupling reaction, wherein the dialysis solution used for dialysis is preferably a PBS solution; the pH of the PBS solution is preferably 7-10, more preferably 7.4; and the concentration of the PBS solution is preferably 0.01-0.2 mol / L, more preferably 0.01 mol / L.

[0116] The ethirimol hapten of the present invention has good stability, requires few synthesis steps, has low synthesis cost, simple reaction conditions, high purity, and its solubility and stability can meet the requirements of coupling carrier proteins.

[0117] The present invention also provides the use of the hapten or the ethirimol complete antigen described in the above scheme in the preparation of ethirimol antibodies.

[0118] In this invention, the acetaminophen antibody is preferably anti-acetaminophen serum.

[0119] This invention provides an acetaminophen antibody, which is obtained by emulsifying the acetaminophen complete antigen described in the above technical solution and then immunizing a host animal.

[0120] This invention provides a method for preparing the acetaminophen antibody described in the above technical solution, comprising the following steps:

[0121] The host animal is immunized after emulsifying the ethirimol complete antigen described in the above technical solution.

[0122] The preparation method of acetaminophen polyclonal antibody is as follows: after boosting the immunization of the host animal, the blood of the immunized animal is collected and separated to obtain the acetaminophen polyclonal antibody;

[0123] The method for preparing ethirimol monoclonal antibody is as follows: extract spleen cells from immunized host animals, fuse the spleen cells with SP2 / 0 tumor cells, screen monoclonal hybridoma cell lines that can secrete ethirimol antibody, and prepare the ethirimol monoclonal antibody based on the monoclonal hybridoma cell lines.

[0124] The preparation method of ethirimol nanobodies is as follows: mRNA transcribed from B lymphocytes in the blood of immunized host animals is extracted, reverse transcribed into cDNA, and the cDNA is used as a substrate for PCR amplification to obtain diverse nanobody gene fragments. The diverse nanobody gene fragments are used to construct a phage or yeast antibody expression library, and ethirimol nanobodies are obtained after screening.

[0125] In this invention, when preparing ethirimol nanobodies, the host animal is preferably a camel or a cartilaginous fish.

[0126] In this invention, monoclonal hybridoma cell lines are easily prepared by those skilled in the art. For example, spleen cells can be obtained by immunizing host animals with the aforementioned complete antigen, and then fused with SP2 / 0 tumor cells in vitro. Monoclonal hybridoma cells that can secrete anti-ethirimol can be screened using selective culture media to obtain monoclonal hybridoma cell lines. Ethirimol nanobodies can also be obtained through genetic engineering techniques, which are easily implemented by those skilled in the art. Therefore, regardless of the method used to prepare ethirimol monoclonal antibodies, polyclonal antibodies, or nanobodies, as long as they use the hapten or complete antigen provided by this invention, they fall within the scope of protection of this invention.

[0127] Based on the hapten or complete antigen disclosed in this invention, those skilled in the art will readily conceive of using suitable antibody preparation methods to prepare antibodies against ethirimol. Regardless of the type of animal used for immunization or how the immunization conditions or parameters are set or changed, as long as the hapten or complete antigen provided by this invention is used, it falls within the protection scope of this invention.

[0128] Antibodies or antiserum prepared by immunizing host animals with the ethirimol complete antigen provided by this invention can specifically bind to ethirimol and have a good immunizing effect.

[0129] This invention provides a reagent or kit for detecting acetaminophen, containing the acetaminophen antibody described in the above technical solution or the anti-acetaminophen antibody prepared by the preparation method described in the above technical solution.

[0130] This invention provides the application of the acetaminophen antibody described in the above-described technical solution, or the anti-acetaminophen antibody prepared by the preparation method described in the above-described technical solution, or the reagent or kit described in the above-described technical solution, in the detection of acetaminophen.

[0131] The acetylpyridinium hapten provided by this invention has a simple synthetic route, low synthesis cost, and good solubility and stability. Furthermore, the antiserum obtained by immunizing host animals with the complete antigen of this invention has high titer and good properties. Spleen cells obtained by immunizing mice with this complete antigen can be effectively used for hybridoma cell fusion and the preparation of anti-acetylpyridinium monoclonal antibodies. Alpaca were immunized with this complete antigen, peripheral blood was collected, and B lymphocytes were harvested. A phage display nanobody expression library was constructed using phage display technology, and nanobodies specifically recognizing acetylpyridinium were obtained through a four-round solid-phase competitive screening method. The acetylpyridinium monoclonal antibodies, polyclonal antibodies, or nanobodies prepared from the acetylpyridinium complete antigen provided by this invention have good specificity and low detection limits, enabling specific recognition of acetylpyridinium. They can be used to establish acetylpyridinium immunological detection methods or reagents and have broad application prospects.

[0132] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0133] Sources of some materials in the example:

[0134] 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-Aldrich; goat anti-mouse IgG-HRP was purchased from Jackson Laboratories.

[0135] Example 1

[0136] The ethirimol hapten with the structure shown in Formula I-1 has the following structure:

[0137]

[0138] See the synthetic route. Figure 1 .

[0139] Under nitrogen protection, NaH (60%, 1.20 g, 30 mmol) was added to dry THF (30 mL), and the resulting mixture was cooled to 0 °C. Ethyl acetoacetate was then added to the mixture while stirring. Figure 11, 3.90 g, 30 mmol), then stirred at room temperature for 30 min, and then ethyl 5-bromo-valerate was added to the system. Figure 1 2, 4.18 g, 20 mmol); the reaction mixture was heated under reflux for 12 h. After cooling to room temperature, a saturated ammonium chloride solution (10 mL) was added, followed by water (50 mL); the mixture was extracted with ethyl acetate (50 mL × 2); the resulting organic phases were combined and washed with saturated brine (50 mL); dried over sodium sulfate, and concentrated to obtain an oil; the oil was separated by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1, v:v, elution) to give a pale yellow oil (3.28 g, yield 60%) of the compound with the structure shown in Formula 3. Figure 1 (3)

[0140] The compound with the structure shown in Formula 4 (ethyl guanidine sulfate, 8.5 g, 31 mmol) was dissolved in methanol (150 mL), cooled to 0 °C in an ice bath, and a methanol solution of 30 wt% sodium methoxide (11.2 g, 62 mmol) was added. The mixture was stirred for 30 min while maintaining the same temperature. Then, the compound with the structure shown in Formula 3 (16.0 g, 62 mmol) was added to the system, and the mixture was heated to 70 °C and stirred for 1 h. The resulting reaction solution was concentrated to remove ethanol. A saturated ammonium chloride solution (200 mL) was added to the residue. The residue was extracted with ethyl acetate (100 mL). The organic phase was washed with water (200 mL), then with saturated brine (100 mL), and dried over sodium sulfate. The concentrated residue was separated by silica gel column chromatography (dichloromethane:methanol = 50:1, v:v), and the compound with the structure shown in Formula 5 (12.4 g, yield 71%) was obtained.

[0141] The compound with the structure shown in Formula 5 (11.56 g, 40 mmol) was dissolved in ethanol (100 mL), and sodium hydroxide solution (NaOH 8.0 g, 100 mL water) was added. The resulting reaction solution was stirred at room temperature for 16 h. The reaction solution was concentrated, and the pH was adjusted to 4 with acetic acid. After stirring for 10 min, a solid precipitated out. The solid product was collected by filtration, washed with water (50 mL), and then slurried with methanol (100 mL). After drying, a white solid was obtained, which was the ethirimol hapten with the structure shown in Formula I-1.

[0142] Figure 4 The liquid chromatogram of the ethirimol hapten shown in Formula I-1 of Example 1; liquid chromatogram ( Figure 4 The result showed only one sample peak; the molecular formula of the ethirimol hapten is C. 12 H 19 N3O3, 254.15, with a theoretical molecular weight of 253.30.

[0143] The product prepared in Example 1 was subjected to liquid chromatography and LC-MS (… Figure 5 ), 1 H NMR ( Figure 6 The acetaminophen hapten shown in Formula I-1 is confirmed to be 5-(2-(ethylamino)-4-hydroxy-6-methylpyrimidin-5-yl)pentanoic acid, with the English name 5-(2-(ethylamino)-4-hydroxy-6-methylpyrimidin-5-yl)pentanoic acid.

[0144] 1 H NMR(400MHz,DMSO-d6)δ1.07(t,J=6.4Hz,3H),1.29-1.37(m,2H),1.44-1.52(m,2H),2.0 5(s,3H),2.19-2.28(m,4H),3.18-3.25(m,2H),6.12(s,1H),10.66(s,1H),11.91(s,1H).

[0145] Example 2

[0146] The ethirimol hapten shown in formula I-2 has the following structure:

[0147]

[0148] See the synthetic route. Figure 2 .

[0149] Weigh 5.37 mg (0.021 mmol) of ethirimol immunogen (Formula I-2) prepared in Example 1, 4.88 mg (0.042 mmol) of NHS, and 8.13 mg (0.042 mmol) of EDC, and dissolve them thoroughly in 0.5 mL of anhydrous DMF. The mixture is then magnetically stirred at 4°C for 10 h. After the reaction is complete, a reaction solution containing the ethirimol hapten (Formula I-2) is obtained. The reaction solution containing the ethirimol hapten (Formula I-2) prepared in Example 2 can be directly used for coupling with a buffer solution of a carrier protein.

[0150] Example 3

[0151] The complete ethirimol antigen with the structure shown in Formula III-1:

[0152]

[0153] See the synthetic route. Figure 3 .

[0154] Synthesis method:

[0155] The supernatant of the reaction solution containing the ethirimol hapten of Formula I-2 obtained in Example 2 was slowly added dropwise to a PBS buffer solution of OVA. The PBS buffer solution of OVA was obtained by dissolving 10 mg of OVA in 1 mL of a PBS buffer solution with a pH of 7.4 and mixing thoroughly. The molar ratio of the ethirimol hapten of Formula I-2 to OVA was 40:1. The reaction was stirred at 25°C for 4 hours. The resulting reaction solution was dialyzed six times with a PBS buffer solution with a pH of 7.4 and a concentration of 0.01 mol / L. The completely dialyzed reaction product solution was diluted to a concentration of 1 mg / mL. The purpose of dialysis is to remove the ethirimol hapten of Formula I-2 or other small molecules from the unreacted solution to obtain the complete ethirimol antigen with the structure shown in Formula III-1, i.e., the conjugate of the ethirimol hapten of Formula I-1 and OVA.

[0156] OVA obtained by MALDI-TOF-MS detection Figure 7 ) and the ethirimol complete antigen with the structure shown in Formula III-1 ( Figure 8 The single-charge ion peaks are 44721.036 and 47354.014. The coupling ratio of the carrier protein OVA to the ethirimol hapten described in Formula I-1 is calculated as follows: Coupling ratio = (Molecular weight of complete antigen - molecular weight of carrier protein) / molecular weight of hapten. The coupling ratio of the ethirimol complete antigen with the structure shown in Formula III-1 is calculated to be 1:10 using this formula.

[0157] Example 4

[0158] The complete ethirimol antigen with the structure shown in Formula III-2:

[0159]

[0160] The supernatant of the reaction solution containing the ethirimol hapten shown in Formula I-2 obtained in Example 2 was slowly added dropwise to a PBS buffer solution of BSA. The PBS buffer solution of BSA was obtained by dissolving 20 mg of BSA in 5 mL of phosphate-buffered saline (PBS) with a pH of 7.4. The molar ratio of the ethirimol hapten shown in Formula I-2 to BSA was 40:1. The reaction was stirred at 25°C for 4 h. The resulting reaction solution was dialyzed six times with a PBS buffer solution with a pH of 7.4 and a concentration of 0.01 mol / L. The completely dialyzed reaction product solution was diluted to a concentration of 1 mg / mL, flash-frozen in liquid nitrogen, and stored at -20°C for later use. The complete ethirimol antigen with the structure shown in Formula III-2 was obtained, i.e., the conjugate of the ethirimol hapten and BSA described in Formula I-1.

[0161] BSA standard obtained by MALDI-TOF-MS detection ( Figure 9 ) and the ethirimol complete antigen with the structure shown in Formula III-2 ( Figure 10 The single-charged ion peaks are 67510.359 and 75544.340. The coupling ratio of the ethirimol complete antigen with the structure shown in Formula III-2 is calculated to be 1:31 using the coupling ratio formula.

[0162] Example 5

[0163] Application of ethirimol complete antigen

[0164] I. Preparation of acetaminophen antibodies using the acetaminophen complete antigen with the structure shown in Formula III-2 obtained in Example 4.

[0165] (1) Balb / c mice aged 6 to 8 weeks were used as experimental animals (8-week-old Balb / c mice weighed about 23 to 25g).

[0166] (2) Primary immunization: The diluted ethirimol complete antigen solution (concentration of 1 mg / mL) of the structure shown in Formula III-2 obtained in Example 4 was filtered through a sterile filter and an equal volume of Freund's complete adjuvant was added. The mixture was stirred thoroughly to emulsify until it did not diffuse when dropped into water. The emulsified ethirimol complete antigen of the structure shown in Formula III-2 was injected into the peritoneum and subcutaneously on the back of mice. The total injection dose was 0.1 mg of emulsified antigen per mouse.

[0167] (3) Booster Immunization: Two weeks after the initial immunization, take 1 mL of the diluted ethirimol complete antigen solution (structure shown in Formula III-2) and add 1 mL of Freund's incomplete adjuvant. Emulsify thoroughly until it does not diffuse when dropped into water. Administer the emulsified ethirimol complete antigen (structure shown in Formula III-2) to mice via intraperitoneal injection and multiple subcutaneous injections on the back. The total injection dose is 0.1 mg of emulsified antigen per mouse. Booster immunizations are given every 14 days. Starting from the third booster immunization, blood is collected from the orbital sinus of mice 3 days after each immunization to determine the serum ethirimol antibody titer using intracranial ELISA. The acetaminophen complete antigen, with a structure shown in Formula III-1, was coated at a concentration of 1 mg / mL. Serum was diluted to the desired gradient until the titer reached ≥1:64000 (titer is defined as the dilution factor of serum when the zero-well colorimetric value is approximately 1.0). Blood was collected by enucleation. The blood was incubated at 37°C for 30 min, then incubated at 4°C for 2 h. Finally, the blood was centrifuged at 4°C and 10000 rpm for 5 min to separate the serum, yielding acetaminophen antiserum. This antiserum was used in the following experiments.

[0168] II. Detection of Anti-acetaminophen Serum Antibody Titer

[0169] The buffer solutions used in the following experiments are as follows:

[0170] (1) Coating buffer (CBS, pH 9.6, 0.05M carbonate buffer): Weigh 1.5g Na2CO3 and 2.94g NaHCO3, and bring the volume to 1000mL with ultrapure water;

[0171] (2) Phosphate buffer (0.01M PBS, pH 7.4): Weigh 0.2 g of KH2PO4, 8 g of NaCl, and 2.92 g of NaH2PO4·12H2O, and bring the volume to 1 L with ultrapure water;

[0172] (3) Washing buffer (PBST): Add 0.1% Tween-20 by volume to the prepared phosphate buffer solution;

[0173] (4) Sample dilution buffer (PBSTG): Add 1% Tween-20 and 1g gelatin (melted by microwave heating) to the prepared phosphate buffer solution;

[0174] (5) Substrate buffer (pH 5.5): Weigh 9.22g of Na2HPO3·12H2O, 2.55g of citric acid monohydrate, measure 0.5mL of Tween-20, and bring the volume up to 1L with ultrapure water;

[0175] (6) Termination solution (1M HCl): Measure 440mL of distilled water, add 40mL of 98% (volume / volume) concentrated hydrochloric acid dropwise and stir.

[0176] (I) Determination of the titer and inhibition rate of anti-ethirimol serum antibodies

[0177] 1. Preparation of ethirimol-coated antigen solution with the structure shown in Formula III-1

[0178] The ethirimol complete antigen with the structure shown in Formula III-1 prepared in Example 3 above was serially diluted with CBS at 1:1000, 1:2000, 1:4000 and 1:8000 to obtain coated antigen solutions of ethirimol complete antigen with the structure shown in Formula III-1 at different concentrations.

[0179] 2. Preparation of ethirimol standard solution

[0180] (1) Weigh 10 mg of ethirimol standard and dissolve it completely in 10 mL of methanol to obtain a 1 mg / mL ethirimol standard solution.

[0181] (2) Prepare a 1 mg / mL ethirimol standard solution with a final concentration of 1000 ng / mL using PBSTG.

[0182] 3. Preparation of ethirimol antiserum diluent

[0183] The ethirimol antiserum prepared in the above steps was serially diluted with PBSTG as needed to obtain ethirimol antiserum diluent.

[0184] 4. Determination of ethirimol antiserum titer and inhibition rate

[0185] ① Coating: Add 100 μL of the coating antigen solution of the ethirimol complete antigen with the structure shown in Formula III-1 prepared in step 1 to each well of a 96-well microplate, incubate at 37°C for 3 h, wash 3 times with PBST, and spin dry.

[0186] ② Competition: Add 50 μL of PBSTG to each well in the zero well; add 50 μL of the ethirimol standard solution prepared in step 2 to each well in the inhibition well.

[0187] ③ Add the diluent of ethirimol antiserum to the microplate (50 μL / well), incubate at 37°C for 30 min, wash the plate 3 times with PBST, and spin dry.

[0188] ④ Add enzyme-labeled secondary antibody: Dilute goat anti-mouse enzyme-labeled secondary antibody (IgG-HRP, Jackson) to working concentration with PBS (0.1M, pH=9.6), add 100μL to each well, incubate at 37℃ for 30min, wash the plate 3 times with PBST, and spin dry.

[0189] ⑤ Color development: Prepare the color development substrate fresh for use. Add 100 μL TMB single-component color development solution to each well and develop the color for 15 min at room temperature in the dark.

[0190] ⑥ Termination and detection: Add 50 μL of 1M HCl stop solution to each well to terminate the reaction, and use an ELISA reader to detect the absorbance value (OD value) of each well at 450 nm.

[0191] ⑦ Potency determination: When the OD value is around 1.0, the maximum dilution factor of the ethirimol antiserum is the potency of the ethirimol antiserum.

[0192] ⑧ Specificity determination: The specificity of the serum is determined based on the inhibition rate.

[0193] The inhibition rate was calculated using the formula: Inhibition rate = [(OD value of control well - OD value of inhibition well) / OD value of control well] × 100%. The titers of ethirimol antiserum after the third and fourth immunizations are shown in Table 1.

[0194] Table 1. Serum titer and inhibition rate of anti-etirifen (TMB color development for 15 min, inhibition of 1000 ng / mL standard sample)

[0195]

[0196] 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-fold.

[0197] Table 2. Serum titers and inhibition rates of ethirimol-fused mice (TMB staining for 15 min, inhibition by 1000 ng / mL standard sample).

[0198]

[0199]

[0200] 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-fold.

[0201] Table 1 shows that after the fourth immunization, when the coating antigen (ethirimol hapten-OVA) was diluted 8000 times, the serum titer against ethirimol was 256000, with an inhibition rate of 76.2%. This indicates that the ethirimol complete antigen with the structure shown in Formula III-2 prepared in Example 4 above has immunogenicity and good specificity, and can be used to prepare antibodies against ethirimol.

[0202] Table 2 shows that the serum titers of the fusion mice were high, and the inhibition rate was over 80%. This indicates that the spleen cells of the acetaminophen fusion mice with the structure shown in Formula III-2 prepared in Example 5 can be used for fusion experiments to prepare anti-acetaminophen antibodies.

[0203] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An ethirimol hapten having the structure shown in Formula I: Formula I; R1in said formula I is selected from -COOH or -COOR2, R2being .

2. The method for preparing the ethirimol hapten according to claim 1, comprising the following steps: By replacing the R3 group in the compound structure shown in Formula II with the R1 group through a chemical reaction, the ethirimol hapten with the structure shown in Formula I is obtained. Formula II, R3 in Formula II is -CH3 or -COO-CH2-CH3.

3. The preparation method according to claim 2, characterized in that, The preparation method of the acetaminophen hapten, R1, selected from -COOH, includes the following steps: The compound with the structure shown in Formula 5 and an inorganic strong base were dissolved in a mixed solution of organic solvent and water and hydrolyzed to obtain R1, which is an ethirimol hapten selected from -COOH. Formula 5.

4. An acetaminophen complete antigen, obtained by conjugating an acetaminophen hapten prepared by the method described in claim 1 or claim 2 or 3 with a carrier protein.

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