Hapten and artificial antigen for sulfonamide drug detection, construction method and application thereof

Abstract

The invention belongs to the technical field of immunodetection, and discloses a hapten and an artificial antigen for sulfonamide drug detection, a construction method and an application thereof, the structure of the hapten and the artificial antigen is shown as a formula (1): R1 =-COOH, R2 = R3 = H; r2 =-COOH, R1 = R3 = H; r3 is-COOH, and R1 is equal to R2 and H is equal to H. The invention discloses a novel sulfonamide immune hapten and a construction method of an artificial antigen of the novel sulfonamide immune hapten for the first time, and successfully obtains a specific antibody with high titer, high sensitivity and broad-spectrum recognition capability on the basis of immune animals of the artificial antigen.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay, and in particular to a hapten and an artificial antigen for detecting sulfonamide drugs, a construction method and applications thereof. Background Art

[0002] Sulfonamides (SAs) are widely used antibacterial drugs worldwide. Since their antibacterial activity was first discovered by G. Domagk in 1935, they have become an important treatment for infectious diseases. Their primary mechanism of action is based on the structural similarity between sulfonamide and para-aminobenzoic acid (PABA), which interferes with bacterial folate synthesis. Studies have shown that the sulfonamide group (SO2NH) is a key structure in their antibacterial activity. By introducing different functional groups at the R position, derivatives with diverse pharmacological activities can be synthesized, thereby expanding the antibacterial spectrum and enhancing efficacy.

[0003] However, the misuse or inappropriate use of sulfonamides can lead to residual contamination, posing a threat to environmental ecosystems and human health. For example, their residues can disrupt soil microbial communities or enter the human body through animal-derived foods, inducing allergic reactions, imbalances in the intestinal flora, inhibiting enzyme activity, and even promoting the development of drug-resistant pathogens. Furthermore, some metabolites are more toxic than the original drug, making long-term low-dose exposure more likely to induce drug resistance, posing a serious threat to public health.

[0004] To strengthen food safety management, my country has established strict limits for sulfonamide residues in its "Maximum Residue Limits of Veterinary Drugs in Food" (GB 31650-2019). The total residue limit is 100 μg / kg, and some drugs, such as sulfamethazine, have an even lower limit of 25 μg / kg in milk. Internationally, the EU and US also set limits of 100 μg / kg, while Japan is even stricter at 20 μg / kg. Therefore, there is an urgent need to develop an efficient, sensitive, and rapid detection method to monitor these residues.

[0005] Enzyme-linked immunosorbent assay (ELISA) has been widely used in drug residue detection due to its ease of use, low cost, and high sensitivity. Among these, indirect competitive ELISA (ic-ELISA) achieves quantitative analysis by competing for binding between antigens and antibodies. It offers the advantages of high specificity, a low limit of detection, and suitability for high-throughput screening.

[0006] The core of immunoassays lies in the quality of antibodies and the rational design of haptens. Although various synthetic strategies for sulfonamide haptens have been developed, the resulting antibodies often suffer from insufficient sensitivity and broad spectrum, limiting their application in general detection. First, relatively direct methods for synthesizing artificial sulfonamide antigens involve the diazotization of arylamino groups with the introduction of spacers such as glutaraldehyde and succinic anhydride. While these methods offer relatively simple synthetic routes, the resulting antibodies lack broad specificity and sensitivity, making them unsuitable for preparing general-purpose sulfonamide antibodies.

[0007] Some studies have used polyclonal antibodies or direct ELISA. While simple to perform, these methods lack specificity, sensitivity, and broad spectrum, making them difficult to meet the needs of complex sample testing. At the end of the 20th century, a study proposed using sulfathiazole derivatives as haptens and generated polyclonal antibodies. The resulting antibodies showed some inhibitory activity against nine different sulfonamide structures. Although subsequent studies have improved the purification of these polyclonal antibodies, they have yet to achieve a significant improvement in specificity and broad spectrum. In the early 21st century, researchers attempted to direct the immune response toward the common structure of sulfonamides by alternately immunizing animals with multiple immunogens. Mouse polyclonal antibodies do have broader specificity, but they have not improved the specificity of monoclonal antibodies. Similar studies have demonstrated that polyclonal antibodies exhibit high cross-reactivity in complex samples, potentially limiting the sensitivity, specificity, and robustness of analytical methods.

[0008] In the application of ELISA methods, direct enzyme-linked immunosorbent assay (Direct ELISA) has also demonstrated its limitations. Due to the lack of a signal amplification mechanism, its sensitivity and detection range are far inferior to those of indirect ELISA. After the hapten 5-[6-(4-amino-phenylsulfonylamino)-pyridin-3-yl]-2-methyl-pentanoic acid was designed and synthesized, a direct ELISA method was developed for the detection of sulfonamides in milk samples. This method was only able to identify four sulfonamides that met the EU maximum residue limits.

[0009] In contrast, monoclonal antibodies have the advantages of a single source and strong specificity, making them the key to building an efficient detection system. Studies have used specific structural sites to design haptens and obtained monoclonal antibodies that can recognize a variety of sulfonamides through ic-ELISA screening. Some of them can detect up to 30 types of SAs, showing good prospects. However, there is still room for improvement in their broad spectrum and sensitivity. In recent years, many studies have been devoted to optimizing hapten design and antibody screening strategies. For example, using a common skeleton structure to guide immune response, introducing diverse functional groups to enhance recognition ability, and combining new detection platforms (such as gold immunosensors and chemiluminescence) have all made progress in improving detection performance. However, no matter what strategy is adopted, the final effect is still highly dependent on the hapten structure and antibody characteristics.

[0010] In summary, despite significant progress in the detection of sulfonamide residues, challenges remain in terms of broad-spectrum detection, sensitivity, specificity, and stability. Therefore, the present invention aims to design and synthesize novel haptens, prepare monoclonal antibodies with broad spectrum and high sensitivity, and establish an optimized ic-ELISA detection system based on these haptens, providing effective technical support for the rapid screening of various SAs. Summary of the Invention

[0011] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a hapten and artificial antigen for the detection of sulfonamide drugs, a construction method and applications thereof.

[0012] The technical solution adopted by the present invention to solve its technical problem is:

[0013] A hapten for detecting sulfonamide drugs, the structure of which is shown in formula (1):

[0014]

[0015] Wherein, R1=-COOH, R2=R3=H; R2=-COOH, R1=R3=H; R3=-COOH, R1=R2=H;

[0016] Furthermore, the immune hapten is a compound represented by formula (I):

[0017]

[0018] Alternatively, the coated hapten is a compound represented by formula (II):

[0019]

[0020] Alternatively, the immune hapten is a compound represented by formula (III):

[0021]

[0022] The method for preparing the sulfonamide hapten as described above comprises the following steps:

[0023] The method for preparing an immune hapten comprises the following steps:

[0024] Step 1, N-acetylaminobenzenesulfonyl chloride and methyl m-aminobenzoate to obtain the intermediate of formula (2);

[0025] Step 2: The intermediate represented by formula (2) undergoes hydrolysis reaction to obtain the compound represented by formula (I);

[0026]

[0027] Alternatively, a method for preparing a coated hapten comprises the following steps:

[0028] Step 1: N-acetylaminobenzenesulfonyl chloride and methyl p-aminobenzoate are reacted to obtain an intermediate represented by formula (3); Step 2: The intermediate represented by formula (3) is hydrolyzed to obtain a compound represented by formula (II);

[0029]

[0030] Alternatively, a method for preparing an immune hapten comprises the following steps:

[0031] Step 1: N-acetylaminobenzenesulfonyl chloride and methyl p-aminobenzoate are reacted to obtain an intermediate of formula (4); Step 2: The intermediate of formula (4) is hydrolyzed to obtain a compound of formula (III);

[0032]

[0033] The artificial antigen of sulfonamide drugs is prepared using the sulfonamide drug hapten as described above.

[0034] The method for preparing the artificial antigen of sulfonamide drugs as described above is to couple the sulfonamide drug hapten with a carrier protein by the N-hydroxysuccinimide NHS active ester method; wherein the carrier protein is bovine serum albumin BSA.

[0035] Use of the hapten and / or artificial antigen and / or method as described above in any of the following applications:

[0036] 1) Preparation of anti-sulfonamide drug-specific antibodies;

[0037] 2) Detection of anti-sulfonamide drug-specific antibodies.

[0038] A specific antibody prepared by induction of the artificial antigen described above, wherein the specific antibody is a monoclonal antibody.

[0039] A sulfonamide drug detection reagent or kit comprising the specific antibody as described above.

[0040] Use of the specific antibodies described above in any of the following applications:

[0041] 1) Used to establish a method based on indirect competitive enzyme-linked immunosorbent assay (IC-ELISA);

[0042] 2) Used for sensitivity detection of sulfonamides;

[0043] 3) Used for the specific detection of sulfonamides;

[0044] 4) Used for testing the recovery rate of sulfonamide drugs.

[0045] Furthermore, the optimization of the ic-ELISA detection system includes screening of pH value and ionic strength;

[0046] Alternatively, the sulfonamide drugs include: sulfamethazine, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfaquinoxaline, sulfamethoxazole, sulfamethoxazole, sulfachloropyrazine sodium, sulfamethazine, sulfamethoxazole, sulfaphenazole, sulfachloropyrazine, sulfadiazine, sulfamethoxazole, sulfamethoxazole, sulfaphenazole, sulfachloropyrazine, sulfadiazine, sulfamethoxazole, sulfadoxine, and sulfathiazole.

[0047] The advantages and positive effects achieved by the present invention are:

[0048] 1. The present invention discloses for the first time a new type of sulfonamide drug immune hapten and a method for constructing an artificial antigen thereof, and successfully obtains specific antibodies with high titer, high sensitivity and broad-spectrum recognition ability by immunizing animals with the artificial antigen.

[0049] 2. The immune hapten provided by the present invention can be used to efficiently prepare anti-sulfonamide drug antibodies after coupling with a carrier protein. The preparation process is simple and low-cost. The obtained antibodies show advantages in sensitivity, recognition spectrum range and practicality, and have good industrial application prospects.

[0050] 3. Based on this, the present invention systematically optimizes and validates the ic-ELISA method to establish a high-performance immunoassay system. This system has a detection sensitivity of ng / mL and a broad-spectrum recognition capability, which can meet the needs of simultaneous detection of multiple sulfonamides.

[0051] Furthermore, a rapid and stable immunoassay method has been developed based on the specific antibodies constructed in this invention. This method demonstrates good spike recovery in complex matrices such as milk, fully demonstrating its reliability and practicality in real-world sample testing. This method provides practical technical support for rapid on-site screening of sulfonamide residues and has significant application and promotion value.

[0052] 4. The present invention relates to a method for synthesizing a sulfonamide drug hapten and an artificial antigen thereof, the optimization of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) detection conditions, and the application of the prepared monoclonal antibody in immunoassay. The structure of the hapten is shown in the figure, wherein R1 = -COOH, R2 = R3 = H; R2 = -COOH, R1 = R3 = H; R3 = -COOH, R1 = R2 = H. The artificial antigen is prepared by coupling the above hapten with a carrier protein. By immunizing animals with the immune hapten, specific monoclonal antibodies with high titer, high sensitivity and good broad spectrum can be obtained. The ic-ELISA detection system established based on the antibody has the optimal detection conditions of pH 7.5 and ionic strength 10mM. In this system, the half inhibitory concentration (IC50) of sulfamethazine is 2.5, and the half inhibitory concentration (IC60) of sulfamethazine is 1.5, and the half inhibitory concentration (IC6 ... 50 ) was 0.8334 ng / mL, and sulfamethoxazole was 4.7595 ng / mL. It showed good cross-reactivity to 19 sulfonamides, of which 7 compounds had a cross-reactivity rate exceeding 100%. When 17 sulfonamides were detected in milk samples, good recovery rates were obtained, verifying the practical applicability of this method. The immune hapten and antibody provided by the present invention, combined with coating material selection and ic-ELISA condition optimization, provide an effective technical means for constructing a rapid, simple, sensitive and broad-spectrum sulfonamide detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a synthetic route for the sulfonamide immune hapten (I) in Example 1 of the present invention;

[0054] Figure 2 The sulfonamide immune hapten (I) prepared in Example 1 of the present invention 1 H NMR spectrum (400 MHz, DMSO);

[0055] Figure 3 This is a synthetic route for the sulfonamide-coated hapten (II) in Example 2 of the present invention;

[0056] Figure 4 The sulfonamide-coated hapten (II) prepared in Example 2 of the present invention 1 H NMR spectrum (400 MHz, DMSO);

[0057] Figure 5 This is a UV-visible absorption spectrum of the sulfonamide immune hapten (I) coupled with the carrier protein in Example 4 of the present invention;

[0058] Figure 6 This is a UV-visible absorption spectrum of the sulfonamide-coated hapten (II) coupled with the carrier protein in Example 5 of the present invention;

[0059] Figure 7 This is a pH condition screening diagram for the sulfonamide enzyme-linked immunosorbent assay method in Example 8 of the present invention;

[0060] Figure 8 This is a diagram showing the ion strength condition screening method for the sulfonamide enzyme-linked immunosorbent assay in Example 8 of the present invention;

[0061] Figure 9 This is a standard curve diagram of the sulfamethazine enzyme-linked immunosorbent assay in Example 9 of the present invention;

[0062] Figure 10 This is the standard curve of sulfamethoxazole enzyme-linked immunosorbent assay in Example 9 of the present invention. DETAILED DESCRIPTION

[0063] The present invention will be further described below with reference to the following examples. The following examples are descriptive rather than restrictive, and the scope of protection of the present invention cannot be limited by the following examples.

[0064] The various experimental operations involved in the specific embodiments are all routine techniques in the field. For parts not specifically annotated in this document, ordinary technicians in this field can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of this invention to implement them.

[0065] The sulfonamide drug hapten provided by the present invention is 2-((4-aminophenyl)sulfonamido)benzoic acid, 3-((4-aminophenyl)sulfonamido)benzoic acid, or 4-((4-aminophenyl)sulfonamido)benzoic acid, and has a structure as shown in formula (1):

[0066]

[0067] Wherein, R1=-COOH, R2=R3=H; R2=-COOH, R1=R3=H; R3=-COOH, R1=R2=H;

[0068] Specifically, the sulfonamide immune hapten represented by formula (1) is a compound represented by formula (I):

[0069]

[0070] The sulfonamide drug-coated hapten represented by formula (1) is a compound represented by formula (II):

[0071]

[0072] The sulfonamide drug-coated hapten represented by formula (1) is a compound represented by formula (III):

[0073]

[0074] The present invention also provides a method for preparing the above-mentioned hapten, and the method for preparing the compound represented by formula (I) comprises the following steps:

[0075] 1) N-acetylaminobenzenesulfonyl chloride and methyl m-aminobenzoate to obtain the intermediate of formula (2);

[0076] 2) The intermediate represented by formula (2) undergoes hydrolysis reaction to obtain the compound represented by formula (I);

[0077] Wherein, the structure of the compound in step 1)-2) is as follows:

[0078]

[0079] Specifically, the preparation method of the compound represented by formula (I) comprises the following steps:

[0080] 1) Mix N-acetylaminobenzenesulfonyl chloride and methyl m-aminobenzoate, and dissolve them in an appropriate amount of 1,4-dioxane under inert gas protection;

[0081] 2) Add appropriate amount of anhydrous Et3N, heat and stir;

[0082] 3) After the reaction is complete, remove the solvent by rotary evaporation under reduced pressure to obtain the intermediate of formula (2);

[0083] 4) Dissolve the intermediate of formula (2) in an appropriate amount of NaOH, heat and stir, and monitor on a TLC plate;

[0084] 5) After the reaction is complete, stop the reaction, adjust the pH with HCl until a precipitate is produced, and filter to obtain formula (I).

[0085] In one embodiment of the present invention, the hapten is prepared as follows (synthetic route see Figure 1 ).

[0086] 1) Weigh 200 mg of N-acetylaminobenzenesulfonyl chloride and 107.82 mg of methyl m-aminobenzoate into a 100 mL round-bottom flask under inert gas protection and dissolve in 5 mL of 1,4-dioxane;

[0087] 2) Add 198 μl of Et3N, heat and stir at 100°C for 3 h, and monitor on a TLC plate (EA);

[0088] 3) After the reaction is complete, remove the solvent by rotary evaporation under reduced pressure to obtain the intermediate of formula (2);

[0089] 4) The intermediate of formula (2) was dissolved in 5 mL of 2 M NaOH, stirred under reflux at 90°C, and monitored by TLC plate (EA);

[0090] 5) After the reaction is complete, stop the reaction, adjust the pH to 3 with 4M HCl, and produce a large amount of yellow precipitate, which is filtered to obtain a yellow solid product of formula (I) (207.5 mg, yield 50%). 1 H-NMR) determination, such as Figure 2 shown.

[0091] The present invention also provides a method for preparing the above-mentioned hapten, and the method for preparing the compound represented by formula (II) comprises the following steps:

[0092] 1) N-acetylaminobenzenesulfonyl chloride and methyl p-aminobenzoate to obtain the intermediate of formula (3);

[0093] 2) The intermediate represented by formula (3) undergoes hydrolysis reaction to obtain the compound represented by formula (II);

[0094] Wherein, the structure of the compound in step 1)-2) is as follows:

[0095]

[0096] Specifically, the preparation method of the compound represented by formula (II) comprises the following steps:

[0097] 1) Mix N-acetylaminobenzenesulfonyl chloride and methyl p-aminobenzoate, and dissolve them in an appropriate amount of 1,4-dioxane under inert gas protection;

[0098] 2) Add appropriate amount of anhydrous Et3N, heat and stir, then transfer and stir at room temperature;

[0099] 3) After the reaction is complete, remove the solvent by rotary evaporation under reduced pressure;

[0100] 4) adding ethyl acetate and saturated NaOH aqueous solution to extract the organic phase, drying, filtering and rotary evaporation to obtain the intermediate of formula (3);

[0101] 5) Dissolve the intermediate of formula (3) in an appropriate amount of NaOH, heat and stir, and monitor on a TLC plate;

[0102] 6) After the reaction is complete, stop the reaction, adjust the pH with HCl until a precipitate is produced, and filter to obtain formula (II).

[0103] In one embodiment of the present invention, the hapten is prepared as follows (synthetic route see Figure 3 ).

[0104] 1) Weigh 300 mg of N-acetylaminobenzenesulfonyl chloride and 194.1 mg of methyl para-aminobenzoate into a 100 mL round-bottom flask under inert gas protection and dissolve in 5 mL of 1,4-dioxane;

[0105] 2) Add 357 μl of Et3N, heat and stir at 100°C for 2 h, then transfer to room temperature for 12 h, and monitor by TLC (EA); after stopping the reaction, remove the solvent by rotary evaporation under reduced pressure;

[0106] 3) adding ethyl acetate and saturated NaOH aqueous solution to extract three times, drying over anhydrous magnesium sulfate, filtering to obtain a filtrate, and drying under reduced pressure to obtain an intermediate of formula (3);

[0107] 4) The intermediate of formula (3) was dissolved in 5 mL of 2 M NaOH, stirred under reflux at 90°C, and monitored by TLC plate (EA);

[0108] 5) After the reaction is complete, stop the reaction, adjust the pH to 3 with 4M HCl, and produce a yellow precipitate, which is filtered to obtain a yellow solid product of formula (II) (58.5 mg, yield 15.6%). The synthesized sulfonamide drug hapten (II) was analyzed by H NMR ( 1 H-NMR) determination, such as Figure 4 shown.

[0109] The present invention also provides a method for preparing the above-mentioned hapten, and the method for preparing the compound represented by formula (III) comprises the following steps:

[0110] 1) N-acetylaminobenzenesulfonyl chloride and methyl anthranilate to obtain an intermediate of formula (4);

[0111] 2) The intermediate represented by formula (4) undergoes hydrolysis reaction to obtain the compound represented by formula (III);

[0112] Wherein, the structure of the compound in step 1)-2) is as follows:

[0113]

[0114] Specifically, the preparation method of the compound represented by formula (III) comprises the following steps:

[0115] 1) Mix N-acetylaminobenzenesulfonyl chloride and methyl anthranilate, and dissolve them in an appropriate amount of 1,4-dioxane under inert gas protection;

[0116] 2) Add appropriate amount of anhydrous Et3N, heat and stir, then transfer and stir at room temperature;

[0117] 3) After the reaction is complete, remove the solvent by rotary evaporation under reduced pressure;

[0118] 4) adding ethyl acetate and saturated NaOH aqueous solution to extract the organic phase, drying, filtering and rotary evaporation to obtain the intermediate of formula (4);

[0119] 5) Dissolve the intermediate of formula (4) in an appropriate amount of NaOH, heat and stir, and monitor on a TLC plate;

[0120] 6) After the reaction is complete, stop the reaction, adjust the pH with HCl until a precipitate is generated, and filter to obtain formula (III).

[0121] In one embodiment of the present invention, the hapten is prepared as follows:

[0122] 1) Weigh 200 mg of N-acetylaminobenzenesulfonyl chloride and 107.82 mg of methyl p-aminobenzoate into a 100 mL round-bottom flask under inert gas protection and dissolve in 5 mL of 1,4-dioxane;

[0123] 2) Add 198 μl of Et3N, heat and stir at 100°C for 3 h, then transfer to room temperature for 12 h, and monitor by TLC (EA); after stopping the reaction, remove the solvent by rotary evaporation under reduced pressure;

[0124] 3) adding ethyl acetate and saturated NaOH aqueous solution to extract three times, drying over anhydrous magnesium sulfate, filtering to obtain a filtrate, and drying under reduced pressure to obtain an intermediate of formula (4);

[0125] 4) The intermediate of formula (4) was dissolved in 5 mL of 2 M NaOH, stirred under reflux at 90°C, and monitored by TLC plate (EA);

[0126] 5) After the reaction was complete, the reaction was stopped and the pH was adjusted to 3 with 4 M HCl to produce a yellow precipitate, which was filtered to obtain a yellow solid product of formula (III) (100.07 mg, 40.0% yield).

[0127] Another object of the present invention is to provide an artificial antigen of sulfonamide drugs, which is obtained by coupling the sulfonamide drug immune and coated hapten with a carrier protein.

[0128] Wherein, the carrier protein is selected from bovine serum albumin (BSA);

[0129] The present invention also provides a method for preparing the artificial antigen, that is, coupling the sulfonamide drug hapten with a carrier protein through the N-hydroxysuccinimide active ester method to prepare the artificial antigen.

[0130] Specifically, the carboxyl groups on the drug molecules are converted into N-hydroxysuccinimide (NHS) active esters and then cross-linked with proteins.

[0131] More specifically, with the assistance of dicyclohexylcarbodiimide (DCC) or carbodiimide (EDC), a carboxyl-containing hapten or drug reacts with NHS to form an active ester derivative, which reacts with the amino group on the carrier protein to form a coupling product connected via an amide bond. The synthesized sulfonamide drug artificial antigen is scanned at 200–500 nm using a UV-visible spectrophotometer (UV2600), and the effect of the coupling reaction is judged by the changes in the characteristic peaks. Figure 5 、 6 shown.

[0132] Another object of the present invention is to provide specific antibodies produced by the above-mentioned sulfonamide immune haptens and artificial antigens prepared therefrom.

[0133] The specific antibody is a monoclonal antibody.

[0134] The present invention provides any of the following applications of the sulfonamide drug hapten or the sulfonamide drug artificial antigen:

[0135] 1) Preparation of anti-sulfonamide drug-specific antibodies;

[0136] 2) Detection of anti-sulfonamide drug-specific antibodies.

[0137] Another object of the present invention is to provide anti-sulfonamide monoclonal antibodies, which are obtained by immunizing experimental animals with the above-mentioned artificial antigens.

[0138] Specifically, the artificial antigen is obtained by coupling the sulfonamide immune hapten with bovine serum albumin.

[0139] The monoclonal antibody provided by the present invention is a sensitive, broad-spectrum recognition antibody that can simultaneously detect multiple sulfonamides.

[0140] The present invention provides any of the following applications of the specific antibody:

[0141] 1) Used for the detection of sulfonamides;

[0142] 2) To establish an enzyme-linked immunosorbent assay (ELISA) for sulfonamides based on an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA);

[0143] 3) Used for testing the recovery rate of sulfonamide drugs.

[0144] In the above application, the sulfonamide drugs include: sulfamethazine, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfaquinoxaline, sulfamethoxazole, sulfamethoxazole, sulfachloropyrazine sodium, sulfamethazine, sulfamethoxazole, sulfaphenazole, sulfachloropyrazine, sulfadiazine, sulfamethoxazole, sulfamethoxazole, sulfaphenazole, sulfachloropyrazine, sulfadiazine, sulfamethoxazole, sulfadoxine, and sulfathiazole.

[0145] The present invention also provides a rapid, sensitive and broad-spectrum enzyme-linked immunosorbent assay method for sulfonamides based on the monoclonal antibody and the coated antigen.

[0146] Specifically, the relevant preparation and testing are as follows:

[0147] Example 1 Preparation and Identification of Sulfonamide Immune Hapten I (3-((4-aminophenyl)sulfonamido)benzoic acid)

[0148] 1) Weigh 200 mg of N-acetylaminobenzenesulfonyl chloride and 107.82 mg of methyl m-aminobenzoate into a 100 mL round-bottom flask under inert gas protection and dissolve in 5 mL of 1,4-dioxane;

[0149] 2) Add 198 μl of Et3N, heat and stir at 100°C for 3 h, and monitor on a TLC plate (EA);

[0150] 3) After the reaction is complete, remove the solvent by rotary evaporation under reduced pressure to obtain the intermediate of formula (2);

[0151] 4) The intermediate of formula (2) was dissolved in 5 mL of 2 M NaOH, stirred under reflux at 90°C, and monitored by TLC plate (EA);

[0152] 5) After the reaction is complete, stop the reaction, adjust the pH to 3 with 4M HCl, and produce a large amount of yellow precipitate, which is filtered to obtain a yellow solid product of formula (I) (207.5 mg, yield 50%). 1 H-NMR) determination, such as Figure 2 Yellow solid product (207.5 mg, yield = 50%) 1 H NMR (400MHz, DMSO-d6) δ12.96(s,1H),10.15(s,1H),7.66(d,J=2.1Hz,1H),7.54(dt,J=7.1 ,1.8Hz,1H),7.38(d,J=8.7Hz,2H),7.36–7.27(m,2H),6.52(d,J=8.7Hz,2H),6.01(s,2H).

[0153]

[0154] Example 2 Preparation and Identification of Sulfonamide-Coated Hapten II (4-((4-aminophenyl)sulfonamido)benzoic acid).

[0155] 1) Weigh 300 mg of N-acetylaminobenzenesulfonyl chloride and 194.1 mg of methyl para-aminobenzoate into a 100 mL round-bottom flask under inert gas protection and dissolve in 5 mL of 1,4-dioxane;

[0156] 2) Add 357 μl of Et3N, heat and stir at 100°C for 2 h, and then react at room temperature for 12 h, monitored by TLC plate (EA);

[0157] 3) After stopping the reaction, remove the solvent by rotary evaporation under reduced pressure;

[0158] 4) adding ethyl acetate and saturated NaOH aqueous solution to extract three times, drying over anhydrous magnesium sulfate, filtering to obtain a filtrate, and drying under reduced pressure to obtain the intermediate of formula (3);

[0159] 5) The intermediate of formula (3) was dissolved in 5 mL of 2 M NaOH, stirred under reflux at 90°C, and monitored by TLC plate (EA);

[0160] 6) After the reaction is complete, stop the reaction, adjust the pH to 3 with 4M HCl, and produce a yellow precipitate, which is filtered to obtain a yellow solid product of formula (II) (58.5 mg, yield 15.6%). The synthesized sulfonamide drug hapten (II) was analyzed by H NMR ( 1 H-NMR) determination, such as Figure 4 Yellow solid product (58.5 mg, yield = 15.6%) 1 H NMR (400MHz, DMSO-d6) δ12.70 (s, 1H), 10.44 (s, 1H), 7.78 (d, J = 8.7Hz, 2H), 7. 45(d,J=8.8Hz,2H),7.15(d,J=8.7Hz,2H),6.54(d,J=8.8Hz,2H),6.06(s,2H).

[0161]

[0162] Example 3 Preparation of sulfonamide immune hapten III (2-((4-aminophenyl)sulfonamido)benzoic acid).

[0163] 1) Weigh 200 mg of N-acetylaminobenzenesulfonyl chloride and 107.82 mg of methyl anthranilate into a 100 mL round-bottom flask under inert gas protection and dissolve in 5 mL of 1,4-dioxane;

[0164] 2) Add 198 μl of Et3N, heat and stir at 100°C for 2 h, and then react at room temperature for 12 h, monitored by TLC plate (EA);

[0165] 3) After stopping the reaction, remove the solvent by rotary evaporation under reduced pressure;

[0166] 4) adding ethyl acetate and saturated NaOH aqueous solution to extract three times, drying over anhydrous magnesium sulfate, filtering to obtain a filtrate, and drying under reduced pressure to obtain an intermediate of formula (4);

[0167] 5) The intermediate of formula (4) was dissolved in 5 mL of 2 M NaOH, stirred under reflux at 90°C, and monitored by TLC plate (EA);

[0168] 6) After the reaction was complete, the reaction was stopped and the pH was adjusted to 3 with 4M HCl to produce a yellow precipitate, which was filtered to obtain a yellow solid product of formula (III) (107 mg, 40.0% yield).

[0169]

[0170] Example 4 Preparation and Identification of Sulfonamide Immune Hapten I (3-((4-aminophenyl)sulfonamido)benzoic acid)-Bovine Serum Albumin

[0171] 1) Dissolve 0.1 mmol of sulfonamide immune hapten I (3-((4-aminophenyl)sulfonamido)benzoic acid), 0.15 mmol of N-hydroxysuccinimide (NHS), and 0.15 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in 1.0 mL of N,N-dimethylformamide (DMF) and stir at room temperature for 2 h.

[0172] 2) Dissolve 250 μl of the system in 5 M bovine serum albumin (BSA) solution and gently stir at 4°C for 12 h.

[0173] 3) The supernatant was dialyzed against 0.1 M PBS buffer at 4°C for 3 days and then stored at -20°C to obtain the immune hapten I-BSA.

[0174] 4) Weigh a small amount of sulfonamide immune hapten I (3-((4-aminophenyl)sulfonamido)benzoic acid), bovine serum albumin, and immune hapten I-BSA, respectively, and use 0.1M PBS buffer to prepare the same concentration of hapten solution, carrier protein solution, and immune hapten I-BSA solution. Use a UV-visible spectrophotometer (UV2600) to scan at 200–500 nm, and judge the effect of the coupling reaction by the change of characteristic peaks, such as Figure 5As shown in the figure, UV-visible spectroscopy analysis revealed that immune hapten I (Hapten) exhibited a characteristic absorption peak at 260 nm, bovine serum albumin (BSA) exhibited an absorption peak at 278 nm, and the synthesized immune hapten I-BSA solution (Hapten-BSA, abbreviated as H-BSA) exhibited a new absorption peak at 266 nm. The significant shift in the maximum absorption wavelengths of the three solutions indicated that immune hapten I had been successfully coupled to BSA.

[0175] Example 5 Preparation and Identification of Sulfonamide-Coated Hapten II (4-((4-aminophenyl)sulfonamido)benzoic acid)-Bovine Serum Albumin

[0176] 1) Dissolve 0.1 mmol of sulfonamide-coated hapten II (4-((4-aminophenyl)sulfonamido)benzoic acid), 0.15 mmol of N-hydroxysuccinimide (NHS), and 0.15 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in 1.0 mL of N,N-dimethylformamide (DMF) and stir at room temperature for 2 h.

[0177] 2) Dissolve 250 μl of the system in 5 M BSA solution and stir gently at 4°C for 12 h.

[0178] 3) The supernatant was dialyzed against 0.1 M PBS buffer at 4°C for 3 days and then stored at -20°C.

[0179] 4) Weigh a small amount of sulfonamide-coated hapten II (4-((4-aminophenyl)sulfonamido)benzoic acid), bovine serum albumin, and coated hapten II-BSA, respectively, and use 0.1M PBS buffer to prepare the same concentration of hapten solution, carrier protein solution, and coated hapten II-BSA solution. Use a UV-visible spectrophotometer (UV2600) to scan at 200–500 nm. The effect of the coupling reaction can be judged by the change of the characteristic peak, such as Figure 6 UV-visible spectroscopy analysis revealed that coated hapten II (Hapten) exhibited a characteristic absorption peak at 260 nm, bovine serum albumin (BSA) exhibited an absorption peak at 278 nm, and the synthesized coated hapten II-BSA solution (Hapten-BSA, abbreviated as H-BSA) exhibited a new absorption peak at 267 nm. The significant shift in the maximum absorption wavelengths of the three solutions indicated that coated hapten II had been successfully coupled to BSA.

[0180] Example 6 Preparation of Sulfonamide Immune Hapten III (2-((4-aminophenyl)sulfonamido)benzoic acid)-Bovine Serum Albumin

[0181] 1) Dissolve 0.1 mmol of sulfonamide immune hapten III (2-((4-aminophenyl)sulfonamido)benzoic acid), 0.15 mmol of N-hydroxysuccinimide (NHS), and 0.15 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) in 1.0 mL of N,N-dimethylformamide (DMF) and stir at room temperature for 2 h.

[0182] 2) Dissolve 250 μl of the system in 5 M BSA solution and stir gently at 4°C for 12 h.

[0183] 3) The supernatant was dialyzed against 0.1 M PBS buffer at 4°C for 3 days and then stored at -20°C.

[0184] Example 7 Preparation of Monoclonal Antibodies for Detection of Sulfonamides

[0185] 1) Three 6-8 week old female BALB / c mice were immunized with the immune hapten I-BSA synthesized in Example 4 and the immune hapten III-BSA synthesized in Example 6, respectively. The immunization program included one primary immunization and multiple booster immunizations.

[0186] 2) For primary immunization, 100 μg of immunogen was emulsified with an equal volume of Freund's complete adjuvant (FCA) and injected subcutaneously at multiple sites in the back, neck, inner thigh, and abdomen of mice, with an injection volume of 200 μL per mouse.

[0187] 3) Four weeks after the initial immunization, the first booster immunization was performed using 50 μg of the immunogen emulsified with an equal volume of Freund's incomplete adjuvant (FIA). Thereafter, booster immunizations were performed every three weeks until the third booster immunization was completed, with an injection volume of 200 μL per animal.

[0188] 4) Starting from the second immunization, tail vein blood was collected on the 8th day after each immunization, serum was separated, and the titer, recognition spectrum and half inhibitory concentration (IC) of the antibodies in the serum were detected by indirect competitive ELISA. 50 ), to evaluate the quality of the immune response;

[0189] 5) Select antibodies with high titer and IC 50 Mice with low values ​​were given the fourth and fifth booster immunizations;

[0190] 6) After the booster immunization, the mice were anesthetized with ether and euthanized by cervical dislocation. The spleens were removed under sterile conditions for cell fusion.

[0191] 7) Mix the isolated splenocytes with SP2 / 0 myeloma cells at a ratio of 1:8 (cell ratio), slowly add 0.8 mL of 50% polyethylene glycol PEG1450 preheated to 37°C, and gently stir for 1 minute to promote cell fusion;

[0192] 8) After fusion is complete, gradually dilute to remove PEG and wash the cells several times with serum-free medium;

[0193] 9) The fused cells were resuspended in RPMI-1640 complete medium containing 1% HAT, seeded in 96-well plates, and cultured at 37°C and 5% CO2;

[0194] 10) After 7 days of culture, hybridoma cells in positive wells were screened by indirect competitive ELISA;

[0195] 11) Perform at least 5 rounds of subcloning screening to ultimately obtain a monoclonal cell line that stably secretes specific antibodies.

[0196] 12) After the obtained cell line is expanded and cultured, it is injected into the peritoneal cavity of mice that have been pre-injected with liquid paraffin to induce the formation of ascites, which is used for the subsequent preparation of high-titer monoclonal antibodies.

[0197] Example 8 Establishment of sulfonamide enzyme-linked immunosorbent assay method

[0198] 1. Reagent Preparation

[0199] 1) Dilution buffer (0.01 M PBS): Prepare by dissolving 1.15 g of anhydrous disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, 0.2 g of potassium chloride, and 8.0 g of sodium chloride in 1000 mL of water.

[0200] 2) Blocking buffer: 1 g of bovine serum albumin was added to 100 ml of 0.01 M PBS solution and 0.05% (volume concentration) Tween-20 was added.

[0201] 3) Washing buffer (PBST): Prepared by adding 2 mL of Tween-20 to 2 L of 0.01 M PBS.

[0202] 4) Substrate solution: Sodium acetate 13.6 g, citric acid 1.6 g, 3% H2O2, add distilled water to 500 mL, shake well, and store at 4°C in the dark.

[0203] 5) TMB storage solution: Dissolve 0.16 g of TMB in 10 mL of DMF and add 0.95 g of citric acid in 500 mL of distilled water. Store at 4°C in the dark.

[0204] 6) Stop solution: Take 22.2 mL of 98% concentrated sulfuric acid, add 177.8 mL of distilled water, and mix well.

[0205] 2. Coating concentration screening

[0206] 1) Use the matrix titration method to screen for the optimal coating concentration. Prepare 0.02μg / ml, 0.03μg / ml, 0.04μg / ml, 0.05μg / ml, 0.06μg / ml, and 0.07μg / ml of coating agent in dilution buffer. Add them vertically to a 96-well microtiter plate from columns 1 to 12 for coating. Set up one replicate for each dilution. Incubate at 4°C overnight.

[0207] 2) Subsequently, the plate was washed three times, 100 μl of blocking buffer was added to each well, and the plate was blocked at 37°C for 2 h;

[0208] 3) After blocking, discard the blocking buffer and add 50 μl of 0.01 M PBS buffer to columns 1, 3, 5, 7, 9, and 11, and 50 μl of 10 ng / mL (10 ppb) sulfamethazine to columns 2, 4, 6, 8, 10, and 12 for a competitive reaction.

[0209] 4) Dilute the monoclonal antibody 270,000-fold using dilution buffer and add it to a 96-well ELISA plate from row 1 to row 8, incubating at 37°C for 60 minutes.

[0210] 5) After washing the plate three times, add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (1:1000 dilution) to each well and continue incubation at 37°C for 40 minutes.

[0211] 6) After washing the plate three times, add 100 μL of substrate solution and TMB storage solution and develop color for 20 min (1:1, volume ratio).

[0212] 7) Add 100 μL of stop solution to terminate the enzymatic reaction. Finally, use a microplate reader to measure the absorbance of each well at a wavelength of 450 nm (OD 450 ). The monoclonal antibody prepared with immune hapten I-BSA was used to perform matrix titration on different concentrations of coated hapten II-BSA. The titration results are shown in Table 1. The preliminary screening criteria are: OD value close to 1.5, significant OD value variation between adjacent wells, and coating concentration with strong competitive ability. As can be seen from the data in Table 1, when the OD value is about 1.5, the OD value of the experimental group with a coating concentration of 0.02μg / mL is 0.01μg / mL when the antibody dilution factor is 256000. 450 Therefore, 0.02 μg / mL was determined to be the optimal coating antigen concentration for subsequent experiments.

[0213] Table 1 Matrix titration of I-BSA monoclonal antibody and II-BSA at different concentrations

[0214]

[0215] 3. Buffer pH and ionic strength screening

[0216] 1) Use the matrix method to screen the optimal pH and ionic strength of the buffer. Prepare a PBS dilution buffer with an ionic strength of 0.1M, and adjust the pH to 4.5, 6.0, 7.5, and 9.0 respectively. Subsequently, each group of buffers with different pH values ​​are configured with different gradients of ionic strength, namely: 0.0mM, 10.0mM, 20.0mM, 40.0mM, 60.0mM, and 80.0mM. Use the corresponding PBS buffer to prepare 0.02μg / mL of coating antigen. Add the coating to a 96-well ELISA plate vertically from the 1st to the 12th columns for coating, set one replicate for each dilution, and let it stand at 4°C for 12 hours;

[0217] 2) Wash the plate three times, add 100 μl of blocking buffer to each well, and block at 37°C for 2 h;

[0218] 3) After blocking, discard the blocking buffer and add 50 μl of 0.01 M PBS buffer to columns 1, 3, 5, 7, 9, and 11, and 50 μl of 10 ng / mL (10 ppb) sulfamethazine to columns 2, 4, 6, 8, 10, and 12 for a competitive reaction.

[0219] 4) Dilute the monoclonal antibody 270,000-fold with dilution buffer and add it to a 96-well ELISA plate from row 1 to row 8, incubating at 37°C for 60 minutes.

[0220] 5) After washing the plate three times, add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody (1:1000 dilution) to each well and incubate at 37°C for another 40 minutes.

[0221] 6) After washing the plate three times, add 100 μL of substrate solution and TMB storage solution and develop color for 20 min (1:1, volume ratio).

[0222] 7) Add 100 μL of stop solution to terminate the enzymatic reaction. Finally, use a microplate reader to measure the absorbance of each well at a wavelength of 450 nm (OD 450 ). The results of the matrix titration for buffer pH and ionic strength screening are shown in Figure 7 、 Figure 8 , preliminary estimate IC 50 , select OD max / IC 50 The highest value, better competition for pH and ionic strength. Figure 7 It can be seen that under different pH conditions, IC 50 The values ​​showed different differences. At pH 7.5, IC 50 The value reached the lowest point, OD max / IC 50 The pH value of 7.5 was the highest, showing the best competitive effect and sensitivity. This indicates that the buffer at pH 7.5 can provide the best antigen-antibody binding conditions, thereby improving the sensitivity and specificity of ic-ELISA detection. Therefore, pH 7.5 was determined to be the most suitable buffer pH value in this study, and this condition will be used in subsequent experiments to further optimize the detection performance. Figure 8 It can be seen that with the increase of ionic strength, IC 50 The values ​​showed significant changes. At 0mM, IC 50 The value is the highest and the detection sensitivity is the worst, which shows that the ionic strength in the buffer has a significant effect on the competitive recognition ability of antigen-antibody. Especially when the ionic strength is 10mM, IC 50 The lowest value, the highest sensitivity, OD max / IC 50 The IC value also reached the best (0.88), indicating that the competition effect between antigen and antibody was the most significant at this ionic strength. However, as the ionic strength increased further (such as 40.0mM, 60.0mM and 80.0mM), the IC 50 The value gradually increased and the detection sensitivity gradually decreased, which indicates that higher ionic strength may interfere with the specific binding of antigen and antibody, thereby affecting the detection effect. Therefore, this study determined 10mM as the optimal ionic strength condition and applied it to subsequent antibody identification experiments.

[0223] Example 9 Identification of Monoclonal Antibodies to Immune Hapten I-BSA

[0224] 1. Establishment of the standard curve

[0225] 1) Prepare 7 concentration gradients of sulfonamide drug standards including sulfamethazine, sulfamethoxazole, sulfamethoxazole, sulfaquinoxaline, sulfamethoxazole, sulfachloropyrazine sodium, sulfamethoxazole, sulfamethoxazole, sulfaphenazole, sulfachloropyrazine, sulfadiazine, sulfamethoxazole, sulfamethoxazole, sulfaquinoxaline, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfaquinoxaline, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxine, and sulfathiazole for indirect competitive ELISA.

[0226] 2) Use the checkerboard method to establish a standard curve. Coat 100 μL of the coating antigen (II-BSA) at a coating concentration of 0.02 μg / mL on a 96-well ELISA plate and incubate overnight at 4°C.

[0227] 3) Wash the wells three times with PBST and add 180 μL of blocking buffer for blocking. Incubate the plate at 37°C for 120 minutes.

[0228] 4) After blocking, each row of the 96-well plate (8 wells total) served as a sample group. 50 μL of a sulfonamide standard solution diluted in seven gradients was added to each well. Then, 50 μL of antibody dilution was added to the last row of wells, setting up a blank control. The concentration of the SAs standard solution was set to 0, and the cells were incubated at 37°C for 60 minutes.

[0229] 5) Wash the wells three times with PBST, add 100 μL of HRP-IgG (1:1000 dilution), and continue incubating at 37°C for 40 minutes.

[0230] 6) After incubation, wash again with PBST three times, add 100 μL substrate solution and TMB storage solution for color development for 20 min (1:1, volume ratio).

[0231] 7) Add 100 μL of stop solution to terminate the enzymatic reaction. After the reaction is completed, the absorbance value is measured at a wavelength of 450 nm using a microplate reader. Using Origin2024 software, the inhibition curve of competitive ELISA is drawn based on the relationship between SAs concentration and B / B0 value, and the half-maximal inhibitory concentration (IC) of SAs is calculated using the linear equation of the curve. 50 Among them, sulfamethazine showed the best sensitivity reaction, such as Figure 9 As shown, its IC 50 The value was 0.8833ng / mL, indicating that the monoclonal antibody produced had the strongest recognition ability for sulfadimethoxine. At the same time, sulfadimethoxine also showed good recognition effect, IC 50 The value is 4.7595ng / mL (see Figure 10 The standard curves of the remaining 17 sulfonamides also showed varying degrees of sensitivity, with IC values ​​of 14 drugs being 50 The values ​​were all less than 10 ng / mL, and the IC values ​​of 19 sulfonamides were 50 The values ​​were all less than 70 ng / mL. The relevant test results are shown in Table 2.

[0232] 2. Determination of cross-reactivity rate

[0233] In order to further evaluate the specificity and broad spectrum of monoclonal antibodies against sulfonamides, the present invention uses cross-reactivity (CR) to quantify the mutual recognition ability between different sulfonamides. Based on the IC-ELISA test data in Example 9, the competitive inhibition curve was drawn using Origin2024 software to calculate the half-maximal inhibitory concentration (IC) of various competitors for polyclonal antibodies. 50 ) and detection range (IC 20 -IC 80 ).

[0234] To further optimize the data, we used the IC 50 As a reference indicator, the cross-reaction rate (CR) was further calculated: that is, the IC 50 With sulfamethoxazole IC 50 The percentage value of IC 50 (SAs):IC 50 The results are shown in Table 2.

[0235] The results showed that all 19 sulfonamides exhibited varying degrees of cross-reactivity. Specifically, the cross-reactivity rate for eight sulfonamides was greater than or equal to 100%, while the cross-reactivity rate for another five sulfonamides exceeded 50%, demonstrating good broad-spectrum detection. This demonstrates that the monoclonal antibody produced not only has strong specificity for sulfamethazine but also has broad recognition capabilities for other sulfonamides, demonstrating good detection capabilities.

[0236] Table 2 ic-ELISA method IC 50 Value and antibody cross-reactivity rate

[0237]

[0238]

[0239] 3. Test of recovery rate of sulfonamides

[0240] The enzyme-linked immunosorbent assay (ELISA) optimized in Example 8 was used, with 100 μL of hapten II-BSA coated at a coating concentration of 0.02 μg / mL and sulfamethazine (50 ng / mL) as the standard. The enzyme-linked immunosorbent assay (ELISA) conditions were set to a dilution buffer with a pH of 7.5 and an ionic strength of 10 mM. This patent employed a matrix method to test negative sample matrices. The OD value trends of the milk sample and the blank control (buffer group) were essentially the same when the antibody was diluted at the same multiple, and both showed a negative OD value at a dilution multiple of 2.6*10 ^5 There was a significant difference when the two samples were tested, indicating that the titer and specificity were basically consistent. The test results showed that the milk sample was a negative sample and could be used for recovery rate testing.

[0241] Table 3 Addition recovery test

[0242] Antibody dilution factor milk Milk (50 ppb) PBS PBS (50 ppb) <![CDATA[1:10 ^3 ]]> 3.045 2.714 3.046 3.025 <![CDATA[1:4*10 ^3 ]]> 3.012 2.447 3.019 2.721 <![CDATA[1:1.6*10 ^4 ]]> 3.044 1.468 3.044 1.904 <![CDATA[1:6.4*10 ^4 ]]> 2.719 0.64 2.852 0.741 <![CDATA[1:2.6*10 ^5 ]]> 1.743 0.302 1.629 0.28 <![CDATA[1:1.0*10 ^6 ]]> 0.757 0.123 0.54 0.15 <![CDATA[1:4.1*10 ^6 ]]> 0.321 0.06 0.08 0.08 <![CDATA[1:1.6*10 ^7 ]]> 0.053 0.053 0.056 0.056

[0243] In order to achieve the convenience of the detection method and retain the rapidity and practicality of the method to the greatest extent, the present invention uses direct dilution of negative milk samples as a pretreatment strategy, that is, the milk samples are diluted with PBS buffer at a volume ratio of 1:1 (finally a 2-fold dilution).

[0244] The optimized ic-ELISA detection system described in Example 8 was used, with 100 μL of hapten II-BSA coated at a coating concentration of 0.02 μg / mL. An enzyme-linked immunosorbent assay (ELISA) was performed using a dilution buffer with a pH of 7.5 and an ionic strength of 10 mM. Standard curves for 17 sulfonamides were plotted using the checkerboard method in the standard buffer system to serve as reference standards for recovery assessment. The experimental procedure was as follows: Two wells were set up in the ELISA plate for each sulfonamide. A diluted negative sample matrix was added to one well, and a target sulfonamide standard at a specified concentration was added to the other well. A parallel control was set up for each group.

[0245] During the detection process, the absorbance value (OD 450 ) and average the data for each group. Then, the average OD 450 Substitute the values ​​into the corresponding standard curve fitting equation to calculate the corresponding theoretical concentration. The spike recovery (%) is calculated by multiplying the ratio of the theoretical concentration to the actual spike concentration by 100%. Table 4 shows the recovery results of spiked milk samples for 17 sulfonamides in a 2-fold dilution of the milk sample. The study found that the recoveries of the 17 sulfonamides ranged from 80% to 120%, meeting the basic recovery requirements for immunoassays in the food safety field and demonstrating excellent recovery capabilities.

[0246] Table 4 Recovery rate test of milk matrix addition

[0247] Drug name Abbreviation Recovery(%) Sulfadoxine SMD' 116.97 Sulfamethoxazole SMZ 115.85 Sulfadiazine SMPZ 109.64 Sulfadiazine SM2 105.96 Sulfisoxazole SIZ 103.62 Sulfadiazol SBA 101.59 Sulfadiazine SEP 99.61 Sulfadiazine SMD 93.42 Sulfathiazole ST 93.40 Sulfadiazine SCP 89.93 Sulfadiazine SMR 89.54 Sulfadiazine SDZ 89.30 Sulfadiazine SIM 89.21 Sulfadiazine sodium SPZ 86.17 Sulfamethoxazole SXL 83.98 Sulfadiazine SMP 83.05 Sulfadiazine SMM 81.39

[0248] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A hapten for detecting sulfonamides, characterized in that: Its structure is shown in formula (1): Among them, R1=-COOH, R2=R3=H; R2=-COOH, R1=R3=H; R3=-COOH, R1=R2=H.

2. The hapten according to claim 1, characterized in that: The immune hapten is a compound represented by formula (I): Alternatively, the coated hapten is a compound represented by formula (II): Alternatively, the immune hapten is a compound represented by formula (III):

3. The method for preparing a hapten according to claim 1 or 2, wherein: The steps include: The method for preparing an immune hapten comprises the following steps: Step 1, N-acetylaminobenzenesulfonyl chloride and methyl m-aminobenzoate to obtain the intermediate of formula (2); Step 2: The intermediate represented by formula (2) undergoes hydrolysis reaction to obtain the compound represented by formula (I); Alternatively, a method for preparing a coated hapten comprises the following steps: Step 1: N-acetylaminobenzenesulfonyl chloride and methyl p-aminobenzoate are reacted to obtain the intermediate represented by formula (3); Step 2: The intermediate represented by formula (3) undergoes hydrolysis reaction to obtain the compound represented by formula (II); Alternatively, a method for preparing an immune hapten comprises the following steps: Step 1: N-acetylaminobenzenesulfonyl chloride and methyl anthranilate are reacted to obtain an intermediate represented by formula (4); Step 2: The intermediate represented by formula (4) undergoes hydrolysis reaction to obtain a compound represented by formula (III); 4. An artificial antigen of sulfonamide drugs prepared using the hapten as claimed in claim 1 or 2.

5. The method for preparing the artificial antigen of sulfonamide drugs according to claim 4, wherein: The sulfonamide drug hapten is coupled to a carrier protein through the N-hydroxysuccinimide NHS active ester method; wherein the carrier protein is bovine serum albumin BSA.

6. Use of the hapten and / or artificial antigen and / or method according to any one of claims 1 to 5 in any of the following: 1) Preparation of anti-sulfonamide drug-specific antibodies; 2) Detection of anti-sulfonamide drug-specific antibodies.

7. A specific antibody prepared by induction using the artificial antigen according to claim 4, characterized in that: The specific antibody is a monoclonal antibody.

8. A sulfonamide drug detection reagent or kit comprising the specific antibody according to claim 7.

9. Use of the specific antibody according to claim 7 in any of the following: 1) Used to establish a method based on indirect competitive enzyme-linked immunosorbent assay (IC-ELISA); 2) Used for sensitivity detection of sulfonamides; 3) Used for the specific detection of sulfonamides; 4) Used for testing the recovery rate of sulfonamide drugs.

10. The use according to claim 9, characterized in that: The optimization of the ic-ELISA detection system includes screening of pH value and ionic strength; Alternatively, the sulfonamide drugs include sulfamethazine, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfaquinoxaline, sulfamethoxazole, sulfamethoxazole, sulfachloropyrazine sodium, sulfamethazine, sulfamethoxazole, sulfaphenazole, sulfachloropyrazine, sulfadiazine, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfaphenazole, sulfachloropyrazine, sulfadiazine, sulfamethoxazole, sulfadoxine, and sulfathiazole.

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