Halogen-modified haptens for enhancing the affinity of antibodies, and methods of making and using the same

Abstract

The present application relates to the technical field of bio-chemical engineering, and particularly relates to a halogen-modified hapten for enhancing antibody affinity, and a preparation method and application thereof. The present application provides a halogen-modified sulfonamide hapten for enhancing antibody affinity, which is obtained by adopting halogen-modified sulfonamides, and the antibody prepared by the halogen-modified sulfonamide hapten has relatively high titer and affinity. The present application provides a new and effective approach for preparing high-affinity sulfonamide antibodies.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, and in particular to a halogen-modified hapten that enhances antibody affinity, its preparation method, and its application. Background Technology

[0002] Sulfonamides (SAs) are a class of veterinary drugs commonly used in animal husbandry. Due to their broad antibacterial spectrum, stability, variety, ease of use, and low cost, they are widely used in livestock farming for the prevention and treatment of infectious diseases. However, improper use and failure to adhere to withdrawal periods during the farming process lead to sulfonamide residues in animal-derived foods, posing potential health risks to humans. Currently, China, the European Union, the United States, Japan, and other countries and regions have stipulated maximum residue limits (MRLs) for sulfonamides in animal-derived foods. Besides controlling the irrational use of sulfonamides at the source, detecting and monitoring drug residues in animal-derived foods is also a crucial measure to ensure food safety. Therefore, establishing rapid and sensitive methods for detecting sulfonamide residues is of great significance for ensuring food safety and human health.

[0003] Immunoassay methods based on antigen-antibody specific recognition have become the mainstream technology for rapid detection of veterinary drug residues due to their advantages such as low cost, simple operation, and large sample throughput. Immunoassay methods for detecting small molecule compounds are considered "reagent-limited" analyses, where antibody affinity plays a crucial role in detection sensitivity. It is known that the key to preparing high-affinity antibodies lies in the structure and properties of the hapten; however, current hapten designs are mostly based on retaining the common structure of the target molecule and directly introducing spacer arms such as succinic anhydride or phthalic anhydride. This strategy, based on the principle of "most similarity" and "trial and error," suffers from problems such as simple structure, poor immunogenicity, and insufficient diversity, making it difficult to prepare high-affinity antibodies and hindering the development of immunoassay methods.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a halogen-modified sulfonamide drug hapten that enhances antibody affinity, its preparation method, and its application.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a halogen-modified sulfonamide drug hapten, the general structural formula of which is shown in formula (I) or formula (II);

[0008] Formula (I), SBZ dist. Halogen-modified haptens:

[0009] ;

[0010] Formula (II), SBZ prox. Halogen-modified haptens:

[0011] ;

[0012] In formula (I), R1 is a benzene ring group containing halogen modification, and the halogen modification site is not limited;

[0013] In formula (II), R2 is a benzene ring group containing halogen modification, and the halogen modification site is located at the ortho or meta position of the benzene ring;

[0014] The halogen is selected from at least one of fluorine, chlorine, and bromine.

[0015] Halogen modification can lead to subtle differences in conformation, charge distribution, and physicochemical properties between haptens and target molecules. This invention utilizes halogen modification of sulfonamide drugs to obtain haptens, resulting in antibodies with relatively high titers and affinities. This may be related to the high electronegativity and hydrophobicity of halogens, which effectively increase electrostatic and hydrophobic interactions between the hapten and antibody, thereby enhancing antibody affinity. Furthermore, this invention provides an effective route for preparing high-affinity antibodies against sulfonamide drugs.

[0016] Preferably, R1 is selected from any one of the following groups:

[0017]

[0018] .

[0019] Preferably, R2 is selected from any one of the following groups:

[0020] .

[0021] Secondly, the present invention provides a method for preparing the halogen-modified sulfonamide drug hapten.

[0022] When the general structural formula of the halogen-modified sulfonamide drug hapten is as shown in formula (I), its preparation method includes the following steps: adding 4-acetaminophen-1-sulfonyl chloride to a pyridine solution containing halogen-modified aniline, and reacting to obtain compound 3; placing compound 3 in NaOH solution, and reacting to obtain compound 4; adding compound 5 to the pyridine solution of compound 4, and reacting to obtain compound 6; adding lithium hydroxide monohydrate solution to a mixed solution of THF and MeOH containing compound 6, and reacting to obtain the halogen-modified sulfonamide drug hapten shown in formula (I);

[0023] The general structural formula of compound 3 is shown in Formula 3; the general structural formula of compound 4 is shown in Formula 4; the general structural formula of compound 5 is shown in Formula 5; and the general structural formula of compound 6 is shown in Formula 6.

[0024] .

[0025] The synthetic route for the halogen-modified sulfonamide drug hapten shown in formula (I) is as follows:

[0026]

[0027] In a more preferred and specific embodiment of the present invention, the method for preparing the halogen-modified sulfonamide drug hapten of formula (I) includes the following steps:

[0028] (1) At 20 °C, 4-acetamidobenzene-1-sulfonyl chloride (compound 1, 15 mmol, 3.50 g) was added to a solution of pyridine (30 mL) containing halogen-modified aniline (compound 2, 15 mmol). The reaction mixture was then heated at 70 °C (oil bath) for 16 hours. The reaction mixture was then cooled to ambient temperature (20 °C). The pyridine was removed under vacuum using a rotary evaporator to obtain a viscous residue. The residue was suspended in 1 N HCl (100 mL) and stirred vigorously for 1 hour. A large amount of precipitate appeared in the mixture. The precipitate was collected by filtration, and the filter cake was washed with water (50 mL x 2). The solid was then dried under vacuum (60 °C) for 1 hour to obtain a gray to white solid. The solid was mixed with anhydrous ethanol (20 mL) and heated under reflux for 1 hour. The mixture was then cooled to ambient temperature. The precipitate in the mixture was collected by filtration and dried under vacuum (60 °C, 1 hour) to obtain compound 3;

[0029] (2) Compound 3 (3-4 mmol) was added to a 2M, 10 mL NaOH solution. The mixture became homogenized immediately and was then heated at 105 °C (oil bath) for 1 hour. The reaction mixture was cooled to room temperature and 3M HCl was added to adjust the pH to approximately 4. The reaction mixture was then stirred at room temperature for approximately 15 minutes. The white precipitate that formed from the mixture was then filtered under vacuum, washed with water (20 mL), and dried under vacuum (60 °C, 1 hour) to give a grayish-white to white solid of pure compound 4.

[0030] (3) Compound 5 was added to a solution of pyridine (10 mL) containing approximately 3 mmol of compound 4. The mixture immediately turned pale yellow and was stirred at room temperature for 10 minutes, then at 50°C for 16 hours. The mixture was then cooled to room temperature and concentrated under vacuum using a rotary evaporator to obtain a residue. EtOAc (100 mL) was added to the residue, and the mixture was washed with water (100 mL), 1N HCl (100 mL), water (50 mL), saturated NaHCO3 (50 mL), water (50 mL), and brine (50 mL), and dried over anhydrous sodium sulfate. The solvent was removed under vacuum to obtain a pale yellow solid. The solid was recrystallized from EtOAc (15 mL) to obtain a grayish-white powder or a white powder, which is the desired compound 6.

[0031] (4) A solution of lithium hydroxide monohydrate (2.10 g, 50 mmol) in water (20 mL) was added to a solution of compound 6 in THF (10 mL) and MeOH (10 mL). The reaction mixture became turbid after the addition. The reaction mixture was then stirred at room temperature for 16 hours. HCl solution (3 M, 20 mL) was then added to the reaction mixture to adjust the pH to approximately 1. The mixture was then stirred at room temperature for 1 hour. THF and MeOH were removed under vacuum using a rotary evaporator. The precipitate from the reaction mixture was then collected by filtration, washed with water (30 mL), and dried under vacuum (60 °C, 1 hour) to give compound (I).

[0032] When the general structural formula of the halogen-modified sulfonamide drug hapten is as shown in formula (II), its preparation method includes the following steps: dissolving halogen-modified 4-aminobenzoic acid in acetone, adding pyridine and 4-acetaminobenzenesulfonyl chloride in sequence, and reacting to obtain compound 10; dissolving compound 10 in THF, adding KOH aqueous solution, and reacting to obtain the halogen-modified sulfonamide drug hapten shown in formula (II);

[0033] The general structural formula of compound 10 is shown in Formula 10;

[0034] .

[0035] The synthetic route for the halogen-modified sulfonamide drug hapten shown in formula (II) is as follows:

[0036]

[0037] In a more preferred and specific embodiment of the present invention, the method for preparing the halogen-modified sulfonamide drug hapten of formula (II) includes the following steps:

[0038] (1) Dissolve 1.0 g of halogen-modified 4-aminobenzoic acid (compound 9, 1.0 eq.) in 15 ml of acetone, add 5 ml of pyridine, stir for 5 min, then add 4-acetamidobenzenesulfonyl chloride (compound 8, 1.2 eq.), and continue stirring for 3 h after the addition is complete. Filter, wash the filter cake with 10 mL of acetone, and dry to obtain compound 10.

[0039] (2) Dissolve 1.16-1.38 g of compound 10 (1.0 eq.) in 20 ml THF, add 10 ml of 10% KOH aqueous solution, and stir at 45 °C for 6 h. The reaction is monitored by TLC until it ends. Cool the reaction system to room temperature, adjust the pH of the system to neutral with 2N HCl (aq.), and then desolvate and concentrate. The concentrate is purified by silica gel column chromatography (DCM / MeOH=10 / 1) to obtain compound (II).

[0040] Thirdly, the present invention provides a halogen-modified sulfonamide drug artificial antigen, comprising the halogen-modified sulfonamide drug hapten and a carrier protein coupled to the halogen-modified sulfonamide drug hapten, obtained by coupling the halogen-modified sulfonamide drug hapten and the carrier protein. The halogen-modified sulfonamide drug artificial antigen can serve as either an immunogen or a coating antigen.

[0041] Preferably, the carrier protein is selected from bovine serum albumin and ovalbumin.

[0042] Preferably, the molar ratio of the compound shown in formula (Ⅰ) to the carrier protein (such as bovine serum albumin) is 3.3-10.0;

[0043] And / or, the molar ratio of the compound shown in formula (II) to the carrier protein (such as bovine serum albumin) is 17.0-29.4.

[0044] Fourthly, the present invention provides a method for preparing the halogen-modified sulfonamide drug artificial antigen, wherein a carrier protein is coupled to the carboxyl carbon of the halogen-modified sulfonamide drug hapten using an activated ester method.

[0045] Fifthly, the present invention provides the use of the halogen-modified sulfonamide drug hapten or the halogen-modified sulfonamide drug artificial antigen in the preparation or detection of anti-sulfonamide drug specific antibodies.

[0046] In a sixth aspect, the present invention provides specific antibodies prepared from said artificial antigens, including polyclonal antibodies and / or monoclonal antibodies.

[0047] In a seventh aspect, the present invention provides the application of the specific antibody in the preparation of sulfonamide drug detection reagents or kits.

[0048] Preferably, the reagent or kit is a fluorescence polarization detection kit for detecting sulfonamide drugs, an enzyme-linked immunosorbent assay (ELISA) kit for detecting sulfonamide drugs, or a lateral flow chromatography test strip for detecting sulfonamide drugs.

[0049] Preferably, the sulfonamide drugs include at least one of sulfanilamide, sulfacetyl, sulfaguanidine, sulfadiazine, phthalimidethiazole, sulfadimethoxazole, sulfathiazole, sulfamethoxypyrazine, sulfadiazine, sulfamethoxypyrazine, sulfasalazine, sulfanilide, sulfanilide, sulfadiazine, sulfachlorpyridazine, sulfaethoxypyridazine, sulfaquinoxaline, sulfachlorpyridazine, sulfamethoxypyrazine, sulfao-dimethoxypyrimidine, sulfa-m-dimethoxypyrimidine, sulfadimethylisopyrimidine, sulfa-m-dimethoxypyrimidine, sulfadiazine, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfamethoxypyrimidine, and sulfabromodimethoxypyrimidine.

[0050] Eighthly, the present invention also provides a sulfonamide drug detection reagent or kit prepared from the said specific antibody.

[0051] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0052] This invention discloses for the first time two sets of novel halogen-modified sulfonamide drug haptens, artificial antigens, and their preparation methods. Sulfabenzene (SBZ) in sulfonamide drugs is chemically modified structurally by coupling spacer arms at the amino and phenyl ends, respectively. Multiple halogen-modified haptens containing fluorine, chlorine, bromine, etc., are designed at positions far from and near the spacer arms, respectively, as shown in formula (I) SBZ. dist. Halogen-modified haptens and formula (II)SBZ prox. Halogen-modified haptens break through the traditional "most similarity" design principle. This study preliminarily explored the effects of halogen modification type, site, and quantity on antibody affinity using both polyclonal and monoclonal antibodies.

[0053] Subsequent experimental results showed that, compared with the unmodified hapten, formula (I)SBZ dist. Halogen-modified haptens (except for 5F modification) all enhanced the affinity of polyclonal antibodies for sulfanilide with low-level halogen modification, up to a maximum increase of 3.02-fold. Except for high-level modifications with 1F2Cl, 3Cl, and 5F, the monoclonal antibodies prepared with low-level halogen modification showed increased affinity for sulfanilide from 1.47 to 699.81-fold and for sulfanilide from 1.75 to 5747.13-fold. Formula (II) SBZ prox.The polyclonal antibodies prepared from halogen-modified haptens all showed increased affinity for total salicylic acid (SA) molecules, five-membered ring SA molecules, and six-membered ring SA molecules. The meta-fluorine-modified hapten (group 1F') showed the highest increase in affinity for five-membered ring SA molecules, six-membered ring SA molecules, and total SA molecules by 1.47-fold, 1.47-fold, and 1.40-fold, respectively; while the ortho-fluorine-modified hapten (group 1F) showed the highest increase in affinity for five-membered ring SA molecules, six-membered ring SA molecules, and total SA molecules by 1.69-fold, 1.49-fold, and 1.23-fold, respectively. Monoclonal antibody mAb 1F-15H3, prepared from an ortho-fluorine modified hapten, showed enhanced affinity for 14 sulfonamide drugs. The affinity for sulfadiazine was increased by 229.43 to 306.87 times, and the affinity for sulfathiazole, sulfamethoxypyrazine, sulfadimethoprim, sulfamethoxypyrimidine, and sulfadimethoxypyrimidine was increased by 18.64 to 78.88 times.

[0054] The halogen-modified hapten provided by this invention significantly enhances antibody affinity, offering a new approach and method for the rational design of small molecule compound haptens and the preparation of high-affinity antibodies.

[0055] In addition, the types, sites, and quantities of halogen-modified haptens provided by this invention can also serve as a reference for the design of other small molecule compound haptens. Attached Figure Description

[0056] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0057] Figure 1 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Flowchart for the preparation of halogen-modified haptens.

[0058] Figure 2 SBZ as shown in formula (II) in Embodiment 1 of the present invention prox. Flowchart for the preparation of halogen-modified haptens.

[0059] Figure 3 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -1) 1 H NMR).

[0060] Figure 4 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -1) 13 C NMR).

[0061] Figure 5 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -1F ( 1 H NMR).

[0062] Figure 6 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -1F ( 13 C NMR).

[0063] Figure 7 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -1Cl) ( 1 H NMR).

[0064] Figure 8 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -1Cl) 13 C NMR).

[0065] Figure 9 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -1Br) 1 H NMR).

[0066] Figure 10 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -1Br) 13 C NMR).

[0067] Figure 11 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -2F ( 1 H NMR).

[0068] Figure 12 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -2F ( 13 C NMR).

[0069] Figure 13 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -2Cl) ( 1 H NMR).

[0070] Figure 14 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -2Cl) ( 13 C NMR).

[0071] Figure 15 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of (-2Br) 1 H NMR).

[0072] Figure 16 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of (-2Br) 13 C NMR).

[0073] Figure 17 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -3F ( 1 H NMR).

[0074] Figure 18 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -3F ( 13 C NMR).

[0075] Figure 19 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -1F2Cl) ( 1 H NMR).

[0076] Figure 20 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -1F2Cl) ( 13 C NMR).

[0077] Figure 21 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -3Cl) ( 1 H NMR).

[0078] Figure 22 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. The NMR spectrum of -3Cl) ( 13 C NMR).

[0079] Figure 23 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. NMR spectrum of -5F ( 1 H NMR).

[0080] Figure 24 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Halogen-modified hapten (SBZ) dist. NMR spectrum of -5F ( 13 C NMR).

[0081] Figure 25 SBZ as shown in formula (II) in Embodiment 1 of the present invention prox. Halogen-modified hapten (SBZ) prox. The NMR spectrum of -1F ( 1 H NMR).

[0082] Figure 26 SBZ as shown in formula (II) in Embodiment 1 of the present invention prox. Halogen-modified hapten (SBZ) prox. The NMR spectrum of -1Cl) 1 H NMR).

[0083] Figure 27 SBZ as shown in formula (II) in Embodiment 1 of the present invention prox. Halogen-modified hapten (SBZ) prox. The NMR spectrum of -1Br) 1 H NMR).

[0084] Figure 28 SBZ as shown in formula (II) in Embodiment 1 of the present invention prox. Halogen-modified hapten (SBZ) prox. NMR spectrum of -Me ( 1 H NMR).

[0085] Figure 29 SBZ as shown in formula (II) in Embodiment 1 of the present invention prox. Halogen-modified hapten (SBZ) prox. The NMR spectrum of -MeO ( 1 H NMR).

[0086] Figure 30 SBZ as shown in formula (II) in Embodiment 1 of the present invention prox. Halogen-modified hapten (SBZ) prox. The NMR spectrum of -1F') 1 H NMR).

[0087] Figure 31 SBZ as shown in formula (I) in Embodiment 1 of the present invention dist. Molecular superposition of halogen-modified haptens (A), partial Mulliken charge (B), and distribution of molecular surface electrostatic potential (C).

[0088] Figure 32 SBZ as shown in formula (II) in Embodiment 1 of the present invention prox. Molecular superposition of halogen-modified haptens (A), distribution of molecular surface electrostatic potential (B), partial Mulliken charge (C), and surface integral distribution within different electrostatic potential ranges (D).

[0089] Figure 33 SBZ as shown in formula (I) in Embodiment 2 of the present invention dist. MALDI-TOF-MS image of artificial antigens prepared from halogen-modified haptens.

[0090] Figure 34 SBZ as shown in formula (II) in Embodiment 2 of the present invention prox. MALDI-TOF-MS image of artificial antigens prepared from halogen-modified haptens.

[0091] Figure 35 SBZ as shown in formula (I) in Embodiment 4 of the present invention dist. The inhibition rates of antibodies against (A) sulfanilide (5 μg / mL) and (B) the corresponding hapten (5 μg / mL) after secondary immunization with halogen-modified artificial antigens, and the inhibition rates of antibodies against (C) sulfanilide (5 μg / mL) and (D) the corresponding hapten (5 μg / mL) after tertiary immunization with halogen-modified artificial antigens.

[0092] Figure 36 SBZ as shown in formula (II) in Embodiment 4 of the present invention prox.The inhibition rates of antibodies against (A) a mixture of SAs (1400 μg / mL), (B) a mixture of five-membered ring SAs (250 μg / mL), and (C) a mixture of six-membered ring SAs (800 μg / mL) after secondary, tertiary, and quaternary immunizations with halogen-modified artificial antigens. Detailed Implementation

[0093] This invention discloses a halogen-modified sulfonamide drug hapten structure that enhances antibody affinity, its preparation method, and its application. The general structural formula of the halogen-modified sulfonamide drug hapten is shown in formula (I) or formula (II):

[0094] Formula (I): SBZ dist. Halogen-modified haptens

[0095] Formula (II): SBZ prox. Halogen-modified haptens

[0096] In formula (I), R1 is a benzene ring group containing halogen modification, wherein the halogen is selected from fluorine, chlorine or bromine, and R2 in formula (II) is a benzene ring group containing halogen modification, wherein the halogen is selected from fluorine, chlorine or bromine, and the modification site is located at the ortho or meta position of the benzene ring.

[0097] This invention chemically modifies the structure of sulfabenz (SBZ) in sulfonamide drugs by coupling spacer arms to the amino and phenyl ends, respectively, and designing sulfabenz haptens containing three typical halogens—fluorine, chlorine, and bromine—at positions far from and near the spacer arms, as shown in formulas (I) and (II), respectively, and naming them SBZ. dist. Halogen-modified haptens and SBZ prox. Halogen-modified haptens. R1 and R2 are benzene ring groups containing different types, sites, and numbers of halogen-modified groups. This invention utilizes quantum chemistry and molecular simulation techniques to characterize the above-mentioned halogen-modified haptens, clarifying the influence of halogen modification on the hapten's structure and properties. Through immunogen synthesis and animal immunization processes, antibodies prepared from the two groups of halogen-modified haptens effectively enhance affinity, providing a new approach and method for the rational design of small molecule compound haptens and the preparation of high-affinity antibodies.

[0098] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0099] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0100] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0101] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0102] Example 1: Preparation and characterization of halogen-modified sulfonamide drug haptens

[0103] I. Preparation and Characterization of Halogen-Modified Sulfonamide Haptens

[0104] 1. SBZ as shown in equation (I) dist. Preparation of halogen-modified haptens

[0105] (1) At 20 °C, 4-acetamidobenzene-1-sulfonyl chloride (1, 15 mmol, 3.50 g) was added to a solution of pyridine (30 mL) containing halogen-modified aniline (2, 15 mmol). The reaction mixture was then heated at 70 °C (oil bath) for 16 hours. The reaction mixture was then cooled to ambient temperature (20 °C). The pyridine was removed under vacuum using a rotary evaporator to obtain a viscous residue. The residue was suspended in 1 N HCl (100 mL) and stirred vigorously for 1 hour. A large amount of precipitate appeared in the mixture. The precipitate was collected by filtration, and the filter cake was washed with water (50 mL x 2). The solid was then dried under vacuum (60 °C) for 1 hour to obtain a gray to white solid. The solid was mixed with anhydrous ethanol (20 mL) and heated under reflux for 1 hour. The mixture was then cooled to ambient temperature. The precipitate in the mixture was collected by filtration and dried under vacuum (60 °C, 1 hour) to give compound 3.

[0106] (2) Compound 3 (3-4 mmol) was added to a 2M, 10 mL NaOH solution. The mixture became homogenized immediately and was then heated at 105 °C (oil bath) for 1 hour. The reaction mixture was cooled to room temperature and 3M HCl was added to adjust the pH to approximately 4. The reaction mixture was then stirred at room temperature for approximately 15 minutes. The white precipitate that formed from the mixture was then filtered under vacuum, washed with water (20 mL), and dried under vacuum (60 °C, 1 hour) to give a grayish-white to white solid of pure compound 4.

[0107] (3) Compound 5 was added to a solution of pyridine (10 mL) containing approximately 3 mmol of compound 4. The mixture immediately turned pale yellow and was stirred at room temperature for 10 minutes, then at 50°C for 16 hours. The mixture was then cooled to room temperature and concentrated under vacuum using a rotary evaporator to obtain a residue. EtOAc (100 mL) was added to the residue, and the mixture was washed with water (100 mL), 1N HCl (100 mL), water (50 mL), saturated NaHCO3 (50 mL), water (50 mL), and brine (50 mL), and dried over anhydrous sodium sulfate. The solvent was removed under vacuum to obtain a pale yellow solid. The solid was recrystallized from EtOAc (15 mL) to obtain a grayish-white powder or a white powder, which is the desired compound 6.

[0108] (4) A solution of lithium hydroxide monohydrate (2.10 g, 50 mmol) in water (20 mL) was added to a solution of compound 6 in THF (10 mL) and MeOH (10 mL). The reaction mixture became turbid after the addition. The reaction mixture was then stirred at room temperature for 16 hours. HCl solution (3 M, 20 mL) was then added to the reaction mixture to adjust the pH to approximately 1. The mixture was then stirred at room temperature for 1 hour. THF and MeOH were removed under vacuum using a rotary evaporator. The precipitate from the reaction mixture was then collected by filtration, washed with water (30 mL), and dried under vacuum (60 °C, 1 hour) to obtain compound (I). For the specific synthetic route, see [link to synthetic route]. Figure 1 Take the synthesized SBZ dist. Halogen-modified haptens were analyzed by proton NMR spectroscopy (NMR spectroscopy). 1 H-NMR and carbon spectroscopy (H-NMR) 13 ¹³C-NMR spectroscopy was used to determine the structure as follows: Figure 3-24 As shown.

[0109] 2. SBZ as shown in equation (II) prox. Preparation of halogen-modified haptens

[0110] (1) Dissolve 1.0 g of halogen-modified 4-aminobenzoic acid (9, 1.0 eq.) in 15 ml of acetone, add 5 ml of pyridine, stir for 5 min, then add 4-acetamidobenzenesulfonyl chloride (8, 1.2 eq.), and continue stirring for 3 h after the addition is complete. Filter, wash the filter cake with 10 mL of acetone, and dry to obtain compound 10.

[0111] (2) Dissolve 1.16–1.38 g of compound 10 (1.0 eq.) in 20 ml of THF, add 10 ml of 10% KOH aqueous solution, and stir at 45 °C for 6 h. The reaction is monitored by TLC until it is complete. Cool the reaction system to room temperature and react with 2N HCl. (aq.) The system pH was adjusted to neutral, then dissolved and concentrated. The concentrate was purified by silica gel column chromatography (DCM / MeOH = 10 / 1) to obtain compound (II). For the specific synthetic route, see [link to synthetic route]. Figure 2 Take the synthesized SBZ prox. Halogen-modified haptens were analyzed by proton NMR spectroscopy (NMR spectroscopy). 1 ¹H-NMR spectroscopy was used to determine the structure as follows: Figure 25-30 As shown.

[0112] 3. Characterization of halogen-modified haptens shown in formulas (I) and (II)

[0113] Based on M06-2X density functional theory and TZVP basis set, the geometry of halogen-modified haptens was optimized, and molecular superposition was performed using Discovery Studio software. SBZ as shown in equation (I) was extracted from the calculation results. dist. Halogen-modified haptens (Table 1) and SBZ as shown in formula (II) prox. Physicochemical parameters of halogen-modified haptens (Table 2), including molecular weight (MW), dipole moment (μ), molecular polarity index (MPI), lipid-water partition coefficient (cLogP), and molecular van der Waals volume (V). m The van der Waals surface area (SA), polar surface area (PSA), and solvent-accessible surface area (SASA) were calculated. The RMSD was calculated to measure the difference between the two structures when the halogen-modified hapten was geometrically optimized at the TVZP / M06-2X level, and the SBZ was calculated as shown in Equation (I). dist. Halogen-modified haptens ( Figure 31 ) and Equation (II) as shown in SBZ prox. Halogen-modified haptens ( Figure 32 The distribution of Mulliken charge and molecular surface electrostatic potential.

[0114] The results show that for equation (I)SBZ dist. Halogen modification of haptens affects various physicochemical properties, including Mullliken charge, polarity, hydrophobicity, and molecular surface electrostatic potential distribution. Except for 5F modification, which significantly alters the hapten conformation (RMSD 4.95), other low-quantity halogen modifications have minimal impact on hapten conformation (RMSD less than 0.40). For formula (II) SBZ... prox. Halogen modification of haptens significantly affects various physicochemical properties of haptens, including conformation (RMSD greater than 1.75), Mullliken charge, polarity, hydrophobicity, and distribution of electrostatic potential on the molecular surface.

[0115] Table 1 shows the SBZ shown in Equation (I). dist. Physicochemical parameters of halogen-modified haptens

[0116]

[0117] Note: 'a' represents the calculation of RMSD to measure SBZ at the TVZP / M06-2X level. dist. Differences between two structures when optimizing geometric superposition of halogen-modified haptens.

[0118] Table 2 shows the SBZ shown in formula (II). prox. Physicochemical parameters of halogen-modified haptens

[0119]

[0120] Note: 'a' represents the calculation of RMSD to measure SBZ at the TVZP / M06-2X level. prox. Differences between two structures when optimizing geometric superposition of halogen-modified haptens.

[0121] Example 2 Preparation and characterization of halogen-modified sulfonamide drug artificial antigens

[0122] The difference between the preparation methods of the immunogen and the coating agent lies in the type of carrier protein used. The immunogen carrier protein mainly uses BSA, while the coating agent carrier protein mainly uses OVA. The coupling method used is the active ester method.

[0123] I. Synthesis and Identification of Halogen-Modified Sulfonamide Drug Immunogens

[0124] 1. Preparation of halogen-modified sulfonamide drug immunogens

[0125] (1) Dissolve the compound (I) prepared in Example 1 with 0.091 mM in 1 mL DMF, add 0.136 mM NHS and 0.136 mM EDC, stir at room temperature for 24 h to obtain solution I.

[0126] (2) Add 40 mg BSA to 10 mL PBS buffer and dissolve it completely to obtain solution II.

[0127] (3) Slowly add solution I to solution II, stir slowly at 4°C for 24 h, then transfer to a dialysis bag and dialyze in PBS at 4°C for 72 h (with 6 water changes in between) to obtain the halogen-modified sulfonamide immunogen solution, which is stored at -20°C. The halogen-modified sulfonamide immunogen synthesized from the compound shown in formula (I) is abbreviated as SBZ. dist. -BSA.

[0128] (4) The halogen-modified sulfonamide immunogen synthesized by the compound shown in formula (II) was prepared using the same three steps as described above. This is abbreviated as SBZ. prox. -BSA.

[0129] 2. Identification of immunogens from halogen-modified sulfonamide drugs

[0130] SBZ was determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). dist. -BSA solution and SBZ prox. - The binding ratio of BSA to hapten in BSA solution. Results are shown in [link to results]. Figure 33, Figure 34 .

[0131] The binding ratios of the artificial antigens represented by compounds in formulas (I) and (II) are calculated using the following formulas (Tables 3 and 4):

[0132] Binding ratio = {M(conjugate) - M(protein)} / M(hapten)

[0133] II. Synthesis of Halogen-Modified Sulfonamide Drug Coating Agents

[0134] Using OVA instead of BSA, the preparation method and steps of the coating agent are the same as those for the immunogen. The compounds shown in formulas (I) and (II) are abbreviated as SBZ for the synthesis of halogen-modified sulfonamide drug coating agents. dist. -OVA and SBZ prox. -OVA.

[0135] Table 3 shows the SBZ shown in Equation (I). dist. Binding ratio of halogen-modified sulfonamide drug artificial antigens

[0136]

[0137] Table 4 shows the SBZ in formula (II). prox. The binding ratio of halogen-modified sulfonamide drug artificial antigens

[0138]

[0139] Example 3 Preparation of halogen-modified sulfonamide drug antibodies

[0140] The SBZ prepared in Example 2 dist. -BSA and SBZ prox. - 144 female BALB / c mice (SPF grade) aged 6-8 weeks were immunized with BSA and randomly divided into 18 groups of 8 mice each. The immunization program consisted of one basic immunization and several booster immunizations.

[0141] For the first immunization, 100 μg of immunogen was mixed with an equal volume of Freund's complete adjuvant, emulsified, and injected subcutaneously into the neck and back of mice at multiple sites, with 200 μL injected into each mouse for basic immunization.

[0142] 100 μg of immunogen was mixed with an equal volume of Freund's incomplete adjuvant and emulsified. Booster immunizations were administered every 3 weeks after the initial immunization, with a volume of 200 μL per animal.

[0143] Starting with the second immunization, blood was collected from the orbital rim 14 days after each immunization. Serum was separated, and the titer of serum antibodies and the inhibition rate of sulfonamide analogs were detected by indirect competitive ELISA. After blood collection, the blood was allowed to stand at 37 °C for 30 minutes, then centrifuged at 3000 rpm for 20 minutes. The supernatant was collected as polyclonal antibody and aliquoted and stored at -20 °C.

[0144] Example 4: Preparation and determination of halogen-modified polyclonal antibodies against sulfonamide drugs

[0145] I. The indirect competitive ELISA method was used to detect antibody affinity. The specific operating steps are as follows:

[0146] (1) Coating: The coating material was serially diluted with 0.05 M, pH 9.6 carbonate buffer, 100 μL / well, and reacted at 37 ℃ for 2 h.

[0147] (2) Washing: Pour out the solution in the plate, shake dry, and wash once with washing solution for 3 minutes each time.

[0148] (3) Sealing: After patting dry, add 150 μL / well sealing solution and react at 37 ℃ for 1 h. Wash and pat dry for later use.

[0149] (4) Prepare standard solution: Dilute sulfamethoxam standard to 5 μg / mL and use it for SBZ as shown in formula (I). dist.Detection of antibodies prepared from halogen-modified sulfonamide drug artificial antigens; 28 SAs (sulfanilide, sulfacetyl, sulfadidine, sulfadiazine, phthalimide, sulfadiazine, sulfadiazine, sulfathiazole, sulfadiazine, sulfamethoxazole, sulfadiazine, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfanilide, sulfadiazine, sulfapyridine, sulfachlorpyridazine, sulfamethoxypyridazine, sulfaquinoxaline, sulfachlorpyridazine, sulfamethoxypyridazine, sulfao-dimethoxypyrimidine, sulfamethoxypyrimidine, sulfadimethylisopyrimidine, sulfamethoxypyrimidine, sulfadiazine, sulfamethoxypyrimidine, sulfamethoxrimidine ... Pyrimidine, sulfadimethylpyrimidine, sulfabromide dimethylpyrimidine), 5 five-membered ring SAs (sulfadimethoxazole, sulfathiazole, sulfamethylisoxazole, sulfadimethoxazole, sulfamethoxypyrimidine), and 16 six-membered ring SAs (sulfapyridine, sulfachlorpyridazine, sulfamethoxypyridazine, sulfaethoxypyridazine, sulfaquinoxaline, sulfachlorpyridazine, sulfamethoxypyridazine, sulfao-dimethoxypyrimidine, sulfa-m-methoxypyrimidine, sulfadimethylisopyrimidine, sulfa-m-dimethoxypyrimidine, sulfadiazine, sulfa-p-methoxypyrimidine, sulfamethylpyrimidine, sulfabromide dimethylpyrimidine) were all prepared in a total quantity of 2. The mixed standard at mg / mL was then diluted with PBS to the required concentrations (the concentrations of the SAs mixed standard, the five-membered ring SAs mixed standard, and the six-membered ring SAs mixed standard were 1400 μg / mL, 250 μg / mL, and 800 μg / mL, respectively) for use in SBZ as shown in formula (II). prox. Detection of antibodies prepared from halogen-modified sulfonamide drug artificial antigens.

[0150] (5) Sample addition: Add 50 μL of diluted standard of each concentration to each well, and then add 50 μL of antibody at the optimal dilution factor per well. Incubate at 37 °C for 30 min. After thorough washing, add 100 μL of HRP-goat anti-mouse IgG diluted 1:5000 per well and incubate at 37 °C for 30 min.

[0151] (6) Colorimetric reaction: Take out the microplate, wash it thoroughly, add 100 μL of TMB colorimetric solution to each well, and react at 37 ℃ in the dark for 15 min.

[0152] (7) Termination and measurement: Add 100 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0153] (8) Data processing: Equation (I) SBZ dist. Halogen-modified artificial antigens of sulfonamide drugs (Table 5) Figure 35 ) and formula (II) SBZ prox. Halogen-modified artificial antigens for sulfonamide drugs (Tables 6, 7, and 8) Figure 36 The affinity of the antibody is calculated according to the following formula:

[0154] Inhibition rate (%) = (B0–B) / B0 × 100%, where B0 and B are the OD values ​​when no standard is present and when a standard is present, respectively.

[0155] The results showed that, compared with the unmodified hapten, formula (I)SBZ dist. Halogen-modified haptens (except for 5F modification) all exhibit increased antibody affinity for sulfanilamide under low-level halogen modification. In particular, haptens modified with one chlorine or bromine atom showed an almost three-fold increase in antibody affinity for sulfanilamide after two and three immunizations, a significant difference compared to the unmodified group. Furthermore, formula (I)SBZ dist. Halogen-modified haptens can increase the antibody affinity for the corresponding hapten by 1.73 to 5.85 times compared to unmodified haptens.

[0156] The results showed that, compared with the unmodified hapten, formula (II)SBZ prox. The antibodies prepared from halogen-modified haptens all showed increased affinity for total salicylic acid (SA) molecules, five-membered ring SA molecules, and six-membered ring SA molecules. The meta-fluorine-modified hapten (group 1F') showed the highest increase in affinity for five-membered ring SA molecules, six-membered ring SA molecules, and total SA molecules by 1.47-fold, 1.47-fold, and 1.40-fold, respectively; while the ortho-fluorine-modified hapten (group 1F) showed the highest increase in affinity for five-membered ring SA molecules, six-membered ring SA molecules, and total SA molecules by 1.69-fold, 1.49-fold, and 1.23-fold, respectively.

[0157] Table 5 Formula (I) SBZ dist. Affinity of halogen-modified sulfonamide drug artificial antigens for the preparation of polyclonal antibodies

[0158]

[0159] Note: a represents the optimal inhibition rate against SBZ (5 μg / mL) and the corresponding hapten (5 μg / mL) at different antibody dilutions. b represents the inhibition rate of SBZ compared to the unmodified group. dist. The fold difference in average inhibition rate among the halogen-modified groups.

[0160] Table 6 Formula (II) SBZ prox. Affinity of polyclonal antibodies prepared from halogen-modified sulfonamide drug artificial antigens to total SAs

[0161]

[0162] Table 7 Formula (II) SBZ prox. Affinity of polyclonal antibodies prepared from halogen-modified sulfonamide drug artificial antigens to five-membered ring SAs

[0163]

[0164] Table 8 Formula (II) SBZ prox. Affinity of polyclonal antibodies to six-membered ring SAs prepared from halogen-modified sulfonamide drug artificial antigens

[0165]

[0166] Example 5: Preparation and determination of halogen-modified monoclonal antibodies against sulfonamide drugs

[0167] I. Cell Fusion and Cloning

[0168] (1) Select BALB / c mice with the highest serum inhibition rate in each group for shock immunization and cell fusion experiments. The immunization method for shock immunization was intraperitoneal injection of 3 times the immunogen dose.

[0169] (2) Three days after the shock immunization, spleen cells were taken and fused with SP2 / 0 myeloma cells at a ratio of 5:1 (quantity ratio). The cell supernatant was measured by indirect competitive ELISA, and positive wells were screened.

[0170] (3) The positive wells were cloned using the limiting dilution method to obtain hybridoma cell lines that can secrete monoclonal antibodies against sulfonamide drugs.

[0171] II. Cell cryopreservation and thawing

[0172] Hybridoma cells were prepared into 1×10⁻⁶ cells using cryopreservation solution. 6 Cell suspensions of cells / mL were stored long-term in liquid nitrogen. Upon thawing, the cryovials were removed and immediately placed in a 37 °C water bath for rapid thawing. After centrifugation to remove the cryopreservation solution, the cells were transferred to culture flasks for incubation.

[0173] III. Preparation of Monoclonal Antibodies

[0174] BALB / c mice were intraperitoneally injected with sterile paraffin oil (0.5 mL / mouse). Seven days later, hybridoma cells prepared above were injected intraperitoneally (5 × 10⁻⁶ cells / mouse). 5 (each individual). Ascites fluid was collected 7 days later, which was the prepared monoclonal antibody and stored at -20 ℃.

[0175] IV. Identification of Monoclonal Antibodies

[0176] The monoclonal antibody solution obtained in step three was used to detect antibody affinity using an indirect competitive ELISA method. The specific steps are as follows:

[0177] (1) Coating: The coating material was serially diluted with 0.05 M, pH 9.6 carbonate buffer, 100 μL / well, and reacted at 37 ℃ for 2 h.

[0178] (2) Washing: Pour out the solution in the plate, shake dry, and wash once with washing solution for 3 minutes each time.

[0179] (3) Sealing: After patting dry, add 150 μL / well sealing solution and react at 37 ℃ for 1 h. Wash and pat dry for later use.

[0180] (4) Preparation of standard solutions: Sulfabenzamide and 29 SAs (sulfanilide, sulfacetyl, sulfaguanidine, sulfabenzylpyrazole, phthalimidethiazole, sulfadimethoxazole, sulfathiazole, sulfamethoxypyrazole, sulfamethoxazole, sulfamethoxypyrazole, sulfamethoxazole, sulfasalazine, sulfanilide, sulfanilide, sulfadiazine, sulfachlorpyridazine, sulfaethoxypyridazine, sulfaquinoxaline, sulfachlorpyridazine, sulfamethoxypyridazine, sulfao-dimethoxypyrimidine, sulfa-m-methoxypyrimidine, sulfa-m-dimethoxypyrimidine, sulfadiazine, sulfa-p-methoxypyrimidine, sulfamethylpyrimidine, sulfabromodimethoxypyrimidine) standard solutions were serially diluted 3-fold to obtain 8 concentrations. Each concentration was set up with 3 replicates for use in SBZ as shown in formula (I). dist. As shown in equation (II), SBZ prox. Determination of monoclonal antibodies prepared from halogen-modified sulfonamide drug artificial antigens.

[0181] (5) Sample addition: Add 50 μL of diluted standard of each concentration to each well, and then add 50 μL of antibody at the optimal dilution factor per well. Incubate at 37 °C for 30 min. After thorough washing, add 100 μL of HRP-goat anti-mouse IgG diluted 1:5000 per well and incubate at 37 °C for 30 min.

[0182] (6) Colorimetric reaction: Take out the microplate, wash it thoroughly, add 100 μL of TMB colorimetric solution to each well, and react at 37 ℃ in the dark for 15 min.

[0183] (7) Termination and measurement: Add 100 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0184] (8) Data processing: The standard concentration was used as the log value on the x-axis and the OD value on the y-axis. The four-parameter equation of Origin 8.5 was used for fitting to establish a standard curve and obtain the IC. 50 The values ​​are shown in Tables 9 and 10.

[0185] The results showed that, compared with the unmodified hapten, formula (I)SBZ dist.Halogen-modified haptens (excluding high-quantity modifications of 1F, 2Cl, 3Cl, and 5F) resulted in monoclonal antibodies with 1.47 to 699.81-fold increased affinity for sulfanilide and 1.75 to 5747.13-fold increased affinity for sulfanilide under low-quantity halogen modification.

[0186] In addition, equation (II)SBZ prox. The monoclonal antibody mAb 1F-14D4 / 15H3, prepared from a halogen-modified hapten, showed superior affinity for sulfacetyl, sulfaquinoxaline, sulfamethoxypyrazine, and sulfamethoxypyrimidine compared to mAb 1-17H1 / 18A10. In particular, mAb 1F-15H3 exhibited enhanced affinity for 14 sulfonamide drugs, including sulfacetyl, sulfaguanidine, phthalimidethiazole, sulfathiazole, sulfadimidine, sulfmethathiazole, sulfaethoxypyridazine, sulfaquinoxaline, sulfamethoxypyrazine, sulfadimethylisopyrimidine, sulfamethoxypyrimidine, sulfamethylpyrimidine, sulfamethoxypyrimidine, and sulfabromodimethylpyrimidine. Among them, the affinity for sulfadimidine increased by 229.43 to 306.87 times, and the affinity for sulfathiazole, sulfamethoxypyrazine, sulfadimethoprim, sulfamethoxypyrazine, and sulfadimethoprim increased by 18.64 to 78.88 times. Only the affinity for five sulfonamide drugs—sulfanilide, sulfadiazine, sulfadimidine, sulfasalazine, and sulfadiazine—decreased.

[0187] Table 9 Formula (I) SBZ dist. Affinity IC50 of halogen-modified sulfonamide drug artificial antigens for the preparation of monoclonal antibodies 50 (ng / mL)

[0188]

[0189] Note: 'a' represents the difference between SBZ and the unmodified group. dist. Monoclonal antibodies IC prepared by halogen modification group 50 The difference multiple.

[0190] Table 10 Formula (II) SBZ prox. Affinity IC50 of halogen-modified sulfonamide drug artificial antigens for the preparation of monoclonal antibodies 50 (ng / mL)

[0191]

[0192]

[0193] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of halogen-modified sulfonamide drug haptens in the preparation or detection of anti-sulfonamide drug specific antibodies, characterized in that, Its general structural formula is shown in formula (I) or formula (II); Formula (I), SBZ dist. Halogen-modified haptens: ； Formula (II), SBZ prox. Halogen-modified haptens: ； In formula (I), It contains a benzene ring group modified with halogen, and the halogen modification site is not limited; In formula (II), It is a benzene ring group containing halogen-modified groups, with the halogen modification site located at the ortho or meta position on the benzene ring; The halogen is selected from at least one of fluorine, chlorine, and bromine.

2. A halogen-modified artificial antigen for sulfonamide drugs, characterized in that, It includes the halogen-modified sulfonamide drug hapten of claim 1 and the carrier protein coupled to the halogen-modified sulfonamide drug hapten.

3. The artificial antigen according to claim 2, characterized in that, The carrier protein is selected from bovine serum albumin and ovalbumin.

4. The halogen-modified sulfonamide drug artificial antigen according to claim 3, characterized in that, The molar ratio of the compound shown in formula (Ⅰ) to the carrier protein is 3.3-10.0; And / or, the molar ratio of the compound shown in formula (II) to the carrier protein is 17.0-29.

4.

5. The use of the halogen-modified sulfonamide drug artificial antigen according to any one of claims 2-4 in the preparation or detection of anti-sulfonamide drug specific antibodies.

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