Adamantylamine hydrophobic haptens, methods of synthesis and use thereof

Abstract

The present application relates to the technical field of biochemistry, and particularly relates to a kind of adamantylamine hydrophobic hapten and its synthesis method and application.The present application is based on adamantylamine target structure, introduces different hydrophobic spacer, improves the hydrophobicity of hapten.Then, hapten is coupled with carrier protein, and the immunogenicity of artificial antigen is evaluated.The present application explores the influence of the increase of hapten hydrophobicity on immunogenicity, which provides theoretical support for the rational design of hapten on one hand, and provides material basis for the preparation of high-affinity antibody of adamantylamine on the other hand.The present application provides a new idea for the rational design of small molecule compound hapten and the preparation of high-affinity antibody.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and in particular to a class of adamantane hydrophobic haptens, their synthesis methods, and applications. Background Technology

[0002] Amantadine (AMD), also known as tricyclic decylamine, is a chemical risk factor and the first antiviral drug approved by the U.S. Food and Drug Administration (FDA). This class of drugs has been used clinically for many years. Due to its low price and good antiviral efficacy, AMD has been illegally used by domestic livestock farms for the prevention and treatment of viral diseases in animals. With the widespread use of amantadine, side effects have gradually emerged, including drug resistance, promotion of strain mutation, and neurotoxicity, posing a serious potential threat to human health. Therefore, establishing a highly sensitive, specific, and easy-to-use method for detecting prohibited drugs is crucial for ensuring food safety and is of great significance for protecting public health. Rapid detection technologies based on antigen and antibody specific recognition through immunoassay, due to their low cost and ability to perform batch screening, can effectively compensate for the shortcomings of instrumental analysis.

[0003] Antibodies are core reagents in immunoassay methods. Because adamantane is a small molecule compound with a low molecular weight, the preparation of monoclonal antibodies against it suffers from poor specificity and sensitivity, and is difficult to screen. Therefore, this invention is proposed. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a class of adamantane hydrophobic haptens, their synthesis method, and applications.

[0005] Specifically, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides an adamantane hapten, said adamantane hapten containing a hydrophobic spacer arm, having a structural formula as any one of the following:

[0007] I;

[0008] II;

[0009] III;

[0010] IV;

[0011] V;

[0012] VI;

[0013] VII;

[0014] VIII.

[0015] This invention, based on the target structure of adamantane, aims to increase the hydrophobicity of the hapten by introducing spacer arms containing saturated fatty chains, unsaturated fatty chains, benzene rings, and ethylene glycol structures. Ultimately, this invention screened eight hapten structures that demonstrated superior performance in hydrophobicity and enhanced immune response.

[0016] In a second aspect, the present invention provides an adamantane artificial antigen comprising the adamantane hapten and a carrier protein coupled to the adamantane hapten.

[0017] Preferably, the adamantane hapten is coupled to the carrier protein via an amide bond.

[0018] Preferably, the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole hemocyanin, thyroprotein, and human serum albumin.

[0019] Preferably, the carrier protein is coupled to the carboxyl group of the adamantane hapten using the active ester method.

[0020] Preferably, the molar ratio of the adamantane hapten to the carrier protein is 10-15:1.

[0021] This invention couples haptens to carrier proteins to evaluate the immunogenicity of artificial antigens. On one hand, it explores the effect of increased hydrophobicity of haptens on immunogenicity, providing theoretical support for the rational design of haptens. On the other hand, it provides a material basis for the preparation of high-affinity antibodies against amantadine.

[0022] Thirdly, the present invention provides any of the following applications of the amantadine hapten or the amantadine artificial antigen:

[0023] Used to evaluate the effect of hapten hydrophobicity on immunogenicity;

[0024] Applications in the preparation of adamantane-specific antibodies;

[0025] Application in the detection of adamantane-specific antibodies;

[0026] The application is for non-disease diagnosis purposes.

[0027] The eight haptens provided in this invention are specially prepared to address the impact of hapten hydrophobicity on monoclonal antibody preparation in immunoassay techniques, offering a new approach and method for the rational design of small molecule compound haptens and the preparation of high-affinity antibodies.

[0028] Fourthly, the present invention provides specific antibodies prepared from the artificial antigen, wherein the specific antibodies are polyclonal antibodies and / or monoclonal antibodies.

[0029] The specific antibody provided by this invention has a relatively high affinity.

[0030] Fifthly, the present invention provides an amantadine drug detection reagent or kit prepared from the specific antibody.

[0031] Sixthly, the present invention provides any of the following applications of the specific antibody:

[0032] Application in a fluorescence polarization detection kit for detecting amantadine;

[0033] Application in the preparation of enzyme-linked immunosorbent assay kits for amantadine;

[0034] Application in the preparation of lateral flow chromatography test strips for the preparation of amantadine;

[0035] The application is for non-disease diagnosis purposes.

[0036] Beneficial effects:

[0037] This invention provides a class of adamantane-based hydrophobic haptens, their synthesis methods, and applications. Specifically, based on adamantane, the hydrophobicity of the hapten is increased to varying degrees by introducing spacer arms containing saturated fatty chains, unsaturated fatty chains, benzene rings, and ethylene glycol structures. Subsequently, the hapten is coupled to a carrier protein, and the immunogenicity of the artificial antigen is evaluated. This invention explores the effect of increased hapten hydrophobicity on immunogenicity, providing theoretical support for the rational design of haptens and a material basis for the preparation of high-affinity antibodies against adamantane. This invention offers a new approach for the rational design of small molecule compound haptens and the preparation of high-affinity antibodies. Attached Figure Description

[0038] 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.

[0039] Figure 1 The above is the 1H NMR spectrum of the adamantane hapten shown in formula (Ⅰ) in this embodiment of the invention.

[0040] Figure 2 The above is the 1H NMR spectrum of the adamantane hapten shown in formula (II) in this embodiment of the invention.

[0041] Figure 3 The above is the 1H NMR spectrum of the adamantane hapten shown in formula (III) in this embodiment of the invention.

[0042] Figure 4 The above is the 1H NMR spectrum of the adamantane hapten shown in formula (Ⅳ) in this embodiment of the invention.

[0043] Figure 5 The image shows the 1H NMR spectrum of the adamantane hapten shown in formula (V) in this embodiment of the invention.

[0044] Figure 6 This is a full-mode mass spectrum of the adamantane hapten shown in formula (VI) in an embodiment of the present invention.

[0045] Figure 7 The image shows the 1H NMR spectrum of the adamantane hydrophobic hapten shown in formula (VII) in this embodiment of the invention.

[0046] Figure 8 This is a full-mode mass spectrum of the adamantane hydrophobic hapten shown in formula (VIII) in an embodiment of the present invention.

[0047] Figure 9 The diagram shows the surface electrostatic potential energy distribution of the adamantane hapten shown in formulas (Ⅰ) to (Ⅷ) in the embodiments of the present invention.

[0048] Figure 10 The images show MALDI-TOF-MS plots of the artificial antigens prepared from the adamantane haptens shown in formulas (Ⅰ) to (Ⅷ) in the embodiments of the present invention.

[0049] Figure 11 The antibody titer is the antibody titer after three immunizations with the artificial antigen of the adamantane hapten shown in formulas (Ⅰ) to (Ⅷ) in the embodiments of the present invention.

[0050] Figure 12 The inhibition rate of the antibody against (A) adamantane (10 μg / mL) and (B) the corresponding hapten (50 ng / mL) after three immunizations with the artificial antigens shown in formulas (Ⅰ) to (Ⅷ) in the embodiments of the present invention is shown. Detailed Implementation

[0051] Hydrophobicity is one of the most important physicochemical properties of molecules. In the fields of proteins and nanovaccines, studies have shown that enhancing the hydrophobicity of immunogens can induce stronger immune responses. However, in the field of small molecule antibody preparation, the effect of hapten hydrophobicity on immune responses remains unknown. Introducing spacer arms with different hydrophobicities (containing saturated fatty chains, unsaturated fatty chains, benzene rings, and ethylene glycol) can increase the hydrophobicity of haptens to varying degrees. Exploring the influence of hapten hydrophobicity on immunogenicity can provide theoretical support for the rational design of haptens and provide a material basis for the preparation of high-affinity antibodies from small molecule compounds. Such haptens, while improving hydrophobicity, possess active groups that can conjugate with carrier proteins, resulting in artificial antigens with suitable conjugation ratios and high hydrophobicity. This, in turn, promotes the immunogenicity of artificial antigens, enabling the preparation of highly specific, high-affinity monoclonal antibodies and the establishment of related immunoassay methods for detecting chemical risk factors such as amantadine.

[0052] The purpose of this invention is to address the shortcomings of existing hapten design theories and immunoassay techniques by providing a method for synthesizing different hydrophobic haptens and artificial antigens, and their applications. Hydrophobic haptens can be conjugated with carrier proteins such as bovine serum albumin or oval serum albumin to obtain artificial antigens. By increasing the hydrophobicity of the hapten, the problem of weak immunogenicity of artificial antigens is improved. Immunizing animals yields monoclonal antibodies with high affinity and strong specificity, thus providing a theoretical basis and technical support for establishing an immunoassay technique for detecting amantadine residues.

[0053] To achieve the objectives of this invention, the following solution is provided:

[0054] First, this invention provides a class of adamantane hydrophobic haptens with the following structural formula:

[0055] I;

[0056] II;

[0057] III;

[0058] IV;

[0059] V;

[0060] VI;

[0061] VII;

[0062] VIII.

[0063] Furthermore, the present invention also provides methods for preparing the above-mentioned eight adamantane hydrophobic haptens.

[0064] The method for preparing the hapten represented by formula (Ⅰ) provided by the present invention includes the following steps:

[0065] 3,6-Dioxanoic acid was dissolved in DCM, oxalyl chloride was added, and anhydrous DMF was added dropwise to the system. The resulting solution was stirred at room temperature. Then, anhydrous DCM solution containing adamantane was slowly added dropwise to the system, and the reaction was stopped by TLC. The system was adjusted to acidity with 2 NHCl, and then saturated brine was added. The mixture was allowed to stand for separation, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to obtain the target product.

[0066] The present invention provides a method for preparing the hapten shown in formula (II), comprising the following steps:

[0067] Succinic acid and DMA were dissolved in a mixed solvent of dichloromethane and DMF. After adding adamantane hydrochloric acid, the mixture was stirred at room temperature for 1 minute. EDC was added in portions, and the mixture was stirred overnight at room temperature. A dilute hydrochloric acid aqueous solution was added, and the mixture was extracted three times with dichloromethane. The combined organic phases were washed twice with dilute hydrochloric acid aqueous solution. The organic phase was extracted three times with NaOH aqueous solution. The combined aqueous phases were washed three times with dichloromethane, and the acidity was adjusted with 1 N dilute hydrochloric acid aqueous solution. A white solid precipitated, and the target product was collected by filtration.

[0068] The present invention provides a method for preparing the hapten shown in formula (III), comprising the following steps:

[0069] Octenic acid was dissolved in dry dichloromethane, and oxaloyl chloride and DMF were added. The resulting solution was reacted at room temperature. Then, an anhydrous dichloromethane solution containing adamantane was slowly added dropwise to the system, and the reaction was stopped by TLC. The acidity was adjusted with 2 M dilute hydrochloric acid aqueous solution, followed by the addition of saturated brine and stirring. The mixture was allowed to stand for separation, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, dissolved under reduced pressure, and the residue was purified by silica gel column chromatography to obtain the target product.

[0070] The present invention provides a method for preparing the hapten shown in formula (Ⅳ), comprising the following steps:

[0071] Adipic acid and DMAP were dissolved in a mixed solvent of dichloromethane and DMF. After adding adamantane hydrochloric acid, the mixture was stirred at room temperature for 10 minutes. EDCI was added in portions, and the mixture was stirred overnight at room temperature. After adding 1 N dilute hydrochloric acid aqueous solution, the mixture was extracted three times with dichloromethane. The combined organic phases were washed twice again with 1 N dilute hydrochloric acid aqueous solution. The organic phase was then extracted three times with 1 N NaOH aqueous solution. The combined aqueous phases were washed three times with dichloromethane, and the acidity was adjusted with 1 N dilute hydrochloric acid aqueous solution to obtain the precipitated target product.

[0072] This invention provides a method for preparing the hapten shown in formula (V), comprising the following steps:

[0073] 1,3-Butadiene-1,4-dicarboxylic acid, TBTU, and triethylamine were dissolved in DMF. Then, a DMF solution containing adamantane was slowly added dropwise to the solution. After the addition was complete, the mixture was stirred overnight at room temperature. After the reaction was complete, water was added to adjust the acidity of the system. The mixture was then extracted with ethyl acetate. The organic phases were combined, the solvent was removed, and the residue was purified by silica gel column chromatography to obtain the target product.

[0074] This invention provides a method for preparing the hapten shown in formula (VI), comprising the following steps:

[0075] Amantadine and p-aldehyde benzoic acid were dissolved in ethanol, and acetic acid was added dropwise to the system. The resulting solution was stirred at room temperature, and the reaction was monitored by TLC until completion. After desolvation under reduced pressure, water was added, and the mixture was filtered. The precipitated product was purified by silica gel column chromatography to obtain the target product.

[0076] This invention provides a method for preparing the hapten shown in formula (VII), comprising the following steps:

[0077] Adamantane was dissolved in DMF, and ethyl bromide and K2CO3 were added. The mixture was stirred at 60 °C for 16 h, diluted with water, and extracted with ethyl acetate. The sodium salt was dried and the solid was filtered off. The filtrate was concentrated and purified under vacuum to obtain ethyl 8-(adamantyl)aminooctanoate. ethyl 8-(adamantyl)aminooctanoate was dissolved in an aqueous ethanol solution, and NaOH was added. The mixture was stirred for 1 h. After removing the ethanol, the pH of the solution was adjusted to 5. The final product was obtained after concentration and purification.

[0078] This invention provides a method for preparing the hapten shown in formula (VIII), comprising the following steps:

[0079] Amantadine was dissolved in DMF, ethyl bromide and K2CO3 were added, and the mixture was stirred at room temperature for 6 h. 50 mL of water was added and stirred for 5 min. The mixture was filtered, and the precipitated product was purified by silica gel column chromatography to obtain the target product.

[0080] Furthermore, the present invention provides eight artificial adamantane antigens obtained based on the above-mentioned haptens, which are specifically obtained by coupling the adamantane hydrophobic haptens of formulas (I) to (VIII) with a carrier protein.

[0081] The carrier protein is selected from bovine serum albumin, ovalbumin, keyhole hemocyanin, thyroprotein, and human serum albumin, etc.; preferably bovine serum albumin and ovalbumin.

[0082] Furthermore, the present invention provides a method for preparing the artificial antigen, which can use the activated ester method to couple the carrier protein to the carboxyl carbon of the haptens of formula (I) to (VIII).

[0083] Preferably, the molar ratio of the compounds shown in formulas (I) to (VIII) to bovine serum albumin is 10 to 15:1.

[0084] Furthermore, this aspect provides for any of the following applications of the amantadine hapten or the amantadine artificial antigen:

[0085] Used to evaluate the effect of hapten hydrophobicity on immunogenicity.

[0086] Used to prepare adamantane-specific antibodies.

[0087] Used to detect adamantane-specific antibodies.

[0088] Furthermore, the present invention provides specific antibodies prepared from the amantadine artificial antigen, including polyclonal antibodies and monoclonal antibodies, preferably monoclonal antibodies. The polyclonal antibodies are obtained by immunizing experimental animals (such as New Zealand white rabbits) with the amantadine artificial antigen and then purifying the serum. The monoclonal antibodies are prepared using hybridoma technology.

[0089] Furthermore, the present invention provides an amantadine detection reagent or kit prepared from the aforementioned specific antibody.

[0090] Furthermore, the present invention provides any of the following applications of the specific antibody:

[0091] Application in a fluorescence polarization detection kit for the detection of adamantane.

[0092] Application in the preparation of flow-testing immunochromatographic strips for adamantane.

[0093] Application in the preparation of an enzyme-linked immunosorbent assay kit for amantadine.

[0094] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0095] This invention discloses for the first time eight amantadine haptens with different hydrophobicities and artificial antigens, along with their preparation methods. By evaluating the impact of each hapten's hydrophobicity on immunogenicity, the mechanism by which hapten hydrophobicity affects the immune response is revealed, providing a theoretical basis for the rational design of haptens. By appropriately adjusting the hydrophobicity of the haptens, the immunogenicity of the artificial antigens is enhanced, thereby obtaining specific antibodies with high titers and high sensitivity. The amantadine haptens and the antibodies prepared in this study provide a new means for establishing a rapid, simple, inexpensive, sensitive, and specific method for the detection of amantadine.

[0096] In this study, different hydrophobic adamantane haptens were synthesized, and their calculated molecular polarity (MPI) ranged from 7.25 to 11.20. The adamantane haptens of formulas (VII) and (VIII) were the most hydrophobic (MPI 7.25 and 7.82, respectively), successfully inducing the highest hapten-specific antibody titers after triple immunization, which were 1.3–3.9 times higher than other groups. Simultaneously, they effectively induced high-affinity antiserum and monoclonal antibodies against adamantane, with affinities 2.3–5.0 times and 303.4–143413.7 times higher than other haptens, respectively.

[0097] The artificial antigens provided by this invention are used to prepare amantadine antibodies (polyclonal and monoclonal antibodies). The preparation process is simple and economical, and the detection sensitivity of the antibodies for amantadine can reach 1.5 ng / mL. By improving the hydrophobicity of the amantadine hapten, the affinity of the amantadine antibody is enhanced, providing a new idea 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

[0103] (I) This embodiment provides different hydrophobic adamantane haptens and their synthesis methods, as detailed below.

[0104] (1) Preparation of the adamantane hydrophobic hapten shown in formula (Ⅰ)

[0105] 2.00 g of 3,6-dioxanoic acid (11.22 mmol) was dissolved in 100 mL of dry DCM. 2.85 g of oxaloyl chloride (22.45 mmol) was added, followed by 5 drops of anhydrous DMF. The resulting solution was stirred at room temperature for 1 h. After 1 h, 150 mL of anhydrous DCM solution containing 1.53 g of adamantane (10.10 mmol) was slowly added dropwise. The mixture was stirred at room temperature for another 0.5 h, and the reaction was stopped by TLC. The pH of the system was adjusted to 3–4 with 2 N HCl, and then 150 mL of saturated saline solution was added and stirred for 5 min. The mixture was allowed to stand and separate into layers. The aqueous phase was extracted with 150 mL × 2 ethyl acetate. The organic phases were combined, dissolved under reduced pressure, and the residue was purified by silica gel column chromatography (200–300 mesh, DCM / MeOH = 15 / 1–10 / 1) to obtain 0.33 g of a pale yellow solid, which was the target product.

[0106] The synthesis process steps are as follows:

[0107] .

[0108] (2) Preparation of the adamantane hydrophobic hapten shown in formula (II)

[0109] Oxalic acid (3.48 g, 20 mmol) and DMAP (4.88 g, 40 mmol) were dissolved in a mixed solvent of dichloromethane (200 mL) and DMF (20 mL). After adding adamantane hydrochloride (3.75 g, 20 mmol), the mixture was stirred at room temperature for 10 minutes. EDCI (3.83 g, 20 mmol) was added in portions, and the mixture was stirred overnight at room temperature. 200 mL of 1 N dilute hydrochloric acid aqueous solution was added to the reaction system, and the mixture was extracted three times with 3 × 100 mL of dichloromethane. The combined organic phases were washed twice again with 2 × 200 mL of 1 N dilute hydrochloric acid aqueous solution. The organic phase was extracted three times with 3 × 100 mL of 1 N NaOH aqueous solution. The combined aqueous phases were washed three times with 3 × 200 mL of dichloromethane, and the pH was adjusted to 2–3 with 1 N dilute hydrochloric acid aqueous solution. A white solid precipitated, and 1.81 g of the white solid was collected by filtration, which was the target product.

[0110] The synthesis process steps are as follows:

[0111] .

[0112] (3) Preparation of the adamantane hydrophobic hapten shown in formula (III)

[0113] 2.40 g of octenedioic acid (13.94 mmol) was dissolved in 150 mL of dry dichloromethane. 3.54 g of oxaloyl chloride (22.46 mmol) was added, followed by 5 drops of anhydrous DMF. The resulting solution was stirred at room temperature for 1 h. After 1 h, 200 mL of anhydrous dichloromethane solution containing 1.90 g of adamantane (12.55 mmol) was slowly added dropwise. The mixture was stirred at room temperature for another 0.5 h, and the reaction was stopped by TLC. The pH of the system was adjusted to 3–4 with 2 M HCl, and then 150 mL of saturated saline solution was added and stirred for 5 min. The mixture was allowed to stand and separate into layers. The aqueous phase was extracted with 150 mL × 2 ethyl acetate. The organic phases were combined, dissolved under reduced pressure, and the residue was purified by silica gel column chromatography (200–300 mesh, DCM / MeOH = 15 / 1–10 / 1) to obtain 1.64 g of a pale yellow solid, which was the target product.

[0114] The synthesis process steps are as follows:

[0115] .

[0116] (4) Preparation of the adamantane hydrophobic hapten shown in formula (Ⅳ)

[0117] Adipic acid (2.92 g, 20 mmol) and DMAP (4.88 g, 40 mmol) were dissolved in a mixed solvent of dichloromethane (200 mL) and DMF (20 mL). After adding adamantane hydrochloride 1 (3.75 g, 20 mmol), the mixture was stirred at room temperature for 10 minutes. EDCI (3.83 g, 20 mmol) was added in portions, and the mixture was stirred at room temperature overnight. 200 mL of 1 N dilute hydrochloric acid aqueous solution was added to the reaction system, and the mixture was extracted three times with 3 × 100 mL of dichloromethane. The combined organic phases were washed twice again with 2 × 200 mL of 1 N dilute hydrochloric acid aqueous solution. The organic phase was extracted three times with 3 × 100 mL of 1 N NaOH aqueous solution. The combined aqueous phases were washed three times with 3 × 200 mL of dichloromethane, and the pH was adjusted to 2–3 with 1 N dilute hydrochloric acid aqueous solution. A white solid precipitated, and 1.52 g of the white solid was collected by filtration.

[0118] The synthesis process steps are as follows:

[0119] .

[0120] (5) Preparation of the adamantane hydrophobic hapten shown in formula (V)

[0121] 2.50 g of 1,3-butadiene-1,4-dicarboxylic acid (1.76 mmol), 7.35 g of TBTU (22.88 mmol), and 5.69 g of triethylamine (44.00 mmol) were dissolved sequentially in 40 mL of DMF and stirred for 30 min. Then, 60 mL of DMF solution containing 2.39 g of adamantane (15.8 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred overnight at room temperature. After the reaction was complete, 150 mL of water was added to the system, and the mixture was stirred for 5 min to adjust the pH to 3-4. The mixture was then extracted with 100 mL × 3 ethyl acetate. The organic phases were combined, the solvent was removed, and the residue was purified by silica gel column chromatography (200-300 mesh, DCM / MeOH = 15 / 1-10 / 1) to give 2.23 g of white solid, which was the target product.

[0122] The synthesis process steps are as follows:

[0123] .

[0124] (6) Preparation of the adamantane hydrophobic hapten shown in formula (VI)

[0125] Weigh 500 mg of adamantane (3.31 mmol) and dissolve 546 mg (3.64 mmol) of p-aldehyde benzoic acid in 2 mL of ethanol. After adding the ethanol, add 5 drops of 100 μL of acetic acid to the system. Stir the resulting solution at room temperature for 6 h, and the reaction is detected by TLC. After desolvation under reduced pressure, add 50 mL of water and stir for 5 minutes. Filter the solution, and purify the precipitated product by silica gel column chromatography to obtain 161 mg of white solid, which is the target product.

[0126] The synthesis process steps are as follows:

[0127] .

[0128] (7) Preparation of the adamantane hydrophobic hapten shown in formula (VII)

[0129] 5.04 g of adamantane (33.38 mmol) was dissolved in 15 mL of DMF, and 9.1 g of ethyl bromide (36.23 mmol) and 9.1 g of K2CO3 (65.84 mmol) were added. The mixture was stirred at 60 °C for 16 h. After dilution with 100 mL of water, the mixture was extracted with ethyl acetate (3 × 50 mL). After the organic compounds were combined, the mixture was dried with sodium hydroxide, and the solid was filtered off. The filtrate was concentrated and purified under vacuum to obtain ethyl 8-(adamantyl)aminooctanoate. Preparation of 8-(adamantyl)aminooctanoic acid: 4 g of ethyl 8-(adamantyl)aminooctanoate was dissolved in an aqueous ethanol solution (ethanol:water = 1:1), and 0.9 g of NaOH was added. The mixture was stirred for 1 h. After removing the ethanol, the pH of the solution was adjusted to 5 with 3M HCl. The mixture was concentrated and purified under vacuum to obtain 1.3 g of white solid, which was the target product.

[0130] The synthesis process steps are as follows:

[0131] .

[0132] (8) Preparation of the adamantane hydrophobic hapten shown in formula (VIII)

[0133] Weigh 500 mg of amantadine (3.31 mmol), 760 mg of methyl 6-bromohexanoate (3.36 mmol), and 914 mg of K₂CO₃ (6.81 mmol) and dissolve them in 5 mL of anhydrous DMF. Stir the solution at room temperature for 6 h, and the reaction is confirmed by TLC. Then, add 50 mL of water and stir for 5 min. Filter the solution, and purify the precipitated product by silica gel column chromatography to obtain 120 mg of a white solid, which is the target product.

[0134] The synthesis process steps are as follows:

[0135] .

[0136] The eight methods for synthesizing hydrophobic amantadine haptens provided by this invention are simple, have high purity and yield, and can be directly coupled with proteins to immunize animals to produce specific antibodies against amantadine haptens. These methods can be used for the rapid detection of amantadine residues.

[0137] (II) This embodiment characterizes the eight adamantane hydrophobic haptens synthesized above, as follows.

[0138] (1) The structure of the synthesized adamantane hydrophobic hapten of formula (Ⅰ) was determined by nuclear magnetic resonance, see below. Figure 1 ¹H NMR (300 MHz, DMSO-d6) δ: 11.96 (s, COOH, 1H), 10.46 (s, NH, 1H), 8.18 (d, J = 9.1 Hz, ArH, 2H), 7.81 (d, J = 9.2 Hz, ArH, 2H), 2.34 (t, J = 7.4 Hz, CH₂, 2H), 2.17 (t, J = 7.3 Hz, CH₂, 2H), 1.61–1.51 (m, CH₂, 2H), 1.51–1.42 (m, CH₂, 2H), 1.27 (m, CH₂, 4H). From the perspective of NMR, this demonstrates the successful synthesis of the adamantane hydrophobic hapten shown in formula (Ⅰ).

[0139] (2) The structure of the synthesized adamantane hydrophobic hapten, as shown in formula (II), was determined by nuclear magnetic resonance, see [reference needed]. Figure 2 ¹H NMR (400 MHz, DMSO-D6) δ: 11.95 (s, 1H), 7.18 (s, 1H), 2.18 (t, J = 7.4 Hz, 2H), 2.04–1.96 (m, 5H), 1.93–1.86 (m, 6H), 1.63–1.57 (m, 6H), 1.54–1.36 (m, 4H), 1.31–1.13 (m, 4H). From the perspective of NMR, this demonstrates the successful synthesis of the adamantane hydrophobic hapten shown in formula (II).

[0140] (3) The structure of the synthesized adamantane hydrophobic hapten of formula (III) was determined by nuclear magnetic resonance, see [reference needed]. Figure 3¹H NMR (400 MHz, DMSO-d6) δ: 7.21 (s, 1H), 5.41 (q, J = 3.5, 2.6 Hz, 2H), 2.24 (dd, J = 8.0, 5.5 Hz, 2H), 2.17 (td, J = 6.6, 3.7 Hz, 2H), 2.12 (ddd, J = 8.4, 5.6, 3.5 Hz, 2H), 2.07 – 2.01 (m, 2H), 2.01 – 1.97 (m, 4H), 1.90 (s, 6H), 1.61 (s, 6H). From the perspective of NMR, this demonstrates the successful synthesis of the adamantane hydrophobic hapten shown in formula (III).

[0141] (4) The structure of the synthesized adamantane hydrophobic hapten of formula (IV) was determined by nuclear magnetic resonance, see below. Figure 4 ¹H NMR (400 MHz, DMSO-D6) δ: 11.95 (s, 1H), 7.21 (s, 1H), 2.25 - 2.12 (m, 2H), 2.04-1.94 (m, 5H), 1.92-1.88 (m, 6H), 1.63-1.57 (m, 6H), 1.48-1.41 (m, 4H). The NMR results demonstrate the successful synthesis of the adamantane hydrophobic hapten shown in formula (Ⅳ).

[0142] (5) The structure of the synthesized adamantane hydrophobic hapten of formula (V) was determined by nuclear magnetic resonance, see below. Figure 5 ¹H NMR (400 MHz, Chloroform-d) δ: 7.38–7.29 (m, ¹H), 7.20 (dd, J = 14.7, 11.5 Hz, ¹H), 6.15 (dd, J = 14.9, 10.6 Hz, ³H), 2.09 (s, ³H), 2.04 (s, ⁶H), 1.69 (s, ⁶H). NMR results demonstrate the successful synthesis of the adamantane hydrophobic hapten shown in formula (Ⅴ).

[0143] (6) The molecular weight of the synthesized adamantane hydrophobic hapten of formula (VI) was determined by mass spectrometry, see [link to relevant documentation]. Figure 6 The molecular ion peak at 284 in full scan mode matches the theoretical molecular weight of the hapten of formula (VI). This demonstrates from a mass spectrometry perspective that the adamantane hydrophobic hapten shown in formula (VI) was successfully synthesized.

[0144] (7) The structure of the synthesized adamantane hydrophobic hapten of formula (VII) was determined by nuclear magnetic resonance, see [link to relevant documentation]. Figure 7: 1HNMR (400 MHz, DMSO-d6): δ 1.29 (s, 6H), 1.48-1.53 ​​(m, 2H), 1.58-1.67 (s, 8H), 1.87 (s, 6H), 2.11 (s, 3H), 2.21 (t, J = 8.0 Hz,2H), 2.81 (t, J = 8.0 Hz,2H), 8.71 (s,1H), 11.91 (s,1H); 13CNMR (400 MHz, CD3OD): δ 24.85, 26.53, 26.58, 28.73,28.87, 34.11, 5.70, 38.00, 56.39, 174.93. The successful synthesis of the adamantane hydrophobic hapten shown in formula (Ⅶ) is demonstrated from the perspective of nuclear magnetic resonance.

[0145] (8) The molecular weight of the synthesized adamantane hydrophobic hapten of formula (VIII) was determined by mass spectrometry, see [reference needed]. Figure 8 The molecular ion peak at 266 in full scan mode matches the theoretical molecular weight of the hapten of formula (VIII). This demonstrates from a mass spectrometry perspective that the adamantane hydrophobic hapten shown in formula (VIII) was successfully synthesized.

[0146] (III) In this embodiment, the geometry of the adamantane hydrophobic hapten is further optimized based on the M06-2X density functional theory and the TZVP basis set. The physicochemical parameters of the adamantane hapten shown in equations (I) to (VIII) are extracted from the calculation results, and the molecular surface electrostatic potential distribution diagram is plotted. Figure 9 (Table 1) Physicochemical parameters include molecular weight (MW), dipole moment (μ), molecular polarity index (MPI), lipid-water partition coefficient (cLogP), van der Waals surface area (SA), polar surface area (PSA), and solvent-accessible surface area (SASA). The results show that the polar molecular parameters and molecular surface electrostatic potential distributions of the eight haptens differ to some extent, suggesting that the haptens designed and synthesized in this study have different hydrophobicities.

[0147] The physicochemical parameters of the adamantane hapten shown in Table 1 (I) to (VIII)

[0148]

[0149] Example 2

[0150] This embodiment provides different hydrophobic amantadine artificial antigens, their preparation methods, and characterization results. The difference between the immunogen and the coating antigen lies in the type of carrier protein used: the immunogen carrier protein is mainly BSA, while the coating antigen carrier protein is mainly OVA, and the coupling method is the active ester method.

[0151] (1) Preparation of different hydrophobic amantadine immunogens

[0152] 9.03 μmol of the compounds of formulas (I) to (VIII) prepared in Example 1 were dissolved in 1 mL of DMF, and 10.38 μmol of NHS and 10.38 μmol of EDC were added. The mixture was stirred at room temperature for 24 h to obtain solution I.

[0153] Add 20 mg BSA to 10 mL PBS buffer and dissolve thoroughly to obtain solution II.

[0154] Solution I was slowly added dropwise to solution II, and the mixture was stirred slowly at 4 °C for 24 h. The mixture was then transferred to a dialysis bag and dialyzed in PBS at 4 °C for 72 h (with 6 water changes in between) to obtain the amantadine immunogen solution. This solution was aliquoted and stored at -20 °C. The amantadine immunogen synthesized from the compounds shown in formulas (I) to (VIII) is abbreviated as AMD-BSA.

[0155] (2) Identification of different hydrophobic amantadine immunogens

[0156] The binding ratio of BSA to hapten in AMD-BSA solution was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results are shown in the figure. Figure 10 .

[0157] The binding ratios of the artificial antigens represented by compounds in formulas (I) to (VIII) are calculated using the following formulas (Table 2):

[0158] Binding ratio = {M (conjugate) - M (protein)} / M (hapten).

[0159] (3) Synthesis of different hydrophobic adamantane coating agents

[0160] 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) to (VIII) are used to synthesize different hydrophobic adamantane-coated agents, abbreviated as AMD-OVA.

[0161] Table 2 shows the binding ratios of different hydrophobic adamantane artificial antigens as indicated by formulas (I) to (VIII).

[0162]

[0163] Example 3

[0164] This embodiment provides polyclonal antibodies against different hydrophobic adamantane haptens and their preparation methods.

[0165] Sixty-four 6-8 week old female BALB / c mice (SPF grade) were immunized with the AMD-BSA prepared in Example 2 and randomly divided into 8 groups of 8 mice each. The immunization program consisted of one basic immunization and several booster immunizations.

[0166] 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.

[0167] 100 μg of immunogen was mixed with an equal volume of Freund's incomplete adjuvant and emulsified. Booster immunizations were performed every 3 weeks after the initial immunization, with a volume of 200 μL per animal.

[0168] Blood was collected from the orbital rim on day 7 after each immunization. Serum was separated, and the titer of serum antibodies and the inhibition rate of amantadine recognition were detected by indirect competitive ELISA. After blood collection, the blood was allowed to stand at 37 °C for 30 min, then centrifuged at 3000 rpm for 20 min. The supernatant was collected as polyclonal antibody and aliquoted and stored at -20 °C.

[0169] Example 4

[0170] This embodiment measures the titers and affinities of polyclonal antibodies against different hydrophobic amantadine haptens.

[0171] (a) The antibody titer was detected using an indirect ELISA method. The specific operating steps are as follows:

[0172] (1) Coating: The coating material was serially diluted with 0.05 M, pH 9.6 carbonate buffer, 100 μL / well, and reacted at 4 ℃ for 16 h.

[0173] (2) Washing: Pour out the solution in the plate, shake dry, and wash once with washing solution for 3 minutes each time.

[0174] (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.

[0175] (4) Sample addition: Add 100 μL of serially diluted antibody to each well and react at 37 °C for 30 min. After thorough washing, add 100 μL of HRP-goat anti-mouse IgG diluted 1:5000 to each well and react at 37 °C for 30 min.

[0176] (5) 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.

[0177] (6) Termination and measurement: Add 100 μL of stop solution to each well to terminate the reaction, and then measure the OD value of each well at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0178] (7) Data processing: Antibody titer was defined as the reciprocal of the highest dilution with an OD value higher than the average value of the blank control wells ± 3 SD (Table 3, Figure 11 ).

[0179] (II) The antibody affinity was detected using an indirect competitive ELISA method. The specific operating steps are as follows:

[0180] (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.

[0181] (2) Washing: Pour out the solution in the plate, shake dry, and wash once with washing solution for 3 minutes each time.

[0182] (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.

[0183] (4) Prepare standard solutions: Dilute the adamantane standard to 10 μg / mL and the adamantane hapten to 50 ng / mL for the detection of antibodies prepared from different hydrophobic adamantane artificial antigens as shown in formulas (I) to (VIII).

[0184] (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.

[0185] (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.

[0186] (7) Termination and measurement: Add 100 μL of stop solution to each well to terminate the reaction, and then measure the OD450 value of each well using an ELISA reader.

[0187] (8) Data processing: Antibody affinity was assessed by inhibition rate. The inhibition rates of antibodies prepared from different hydrophobic amantadine artificial antigens were shown in Table 3 (I) to (VIII). Figure 12 Calculate according to the following formula:

[0188] Inhibition rate (%) = B / B0 × 100%, where B0 and B are the OD values ​​at 450 nm when no standard is present and when a standard is present, respectively.

[0189] The results showed that the amantadine haptens of formulas (VII) and (VIII), which have the highest hydrophobicity, could increase the overall antibody titer induced by the body and the affinity for amantadine. The difference was more significant after the third immunization, with the highest hapten-specific antibody titer induced, which was 1.3 to 3.9 times higher than other groups; at the same time, it effectively induced antiserum with high affinity for amantadine, which was 2.3 to 5.0 times higher than the affinity of other haptens.

[0190] Table 3. Serum antibody titers and inhibition rates prepared from different hydrophobic adamantane artificial antigens according to formulas (I) to (VIII).

[0191]

[0192] Note: a The average inhibition rate of AMD (10 μg / mL) and the corresponding hapten (50 ng / mL) at different antibody dilutions after triple immunization represents the average inhibition rate of AMD (10 μg / mL) and the corresponding hapten (50 ng / mL). b This represents the average antibody titer of the amantadine hydrophobic hapten after triple immunization.

[0193] Example 5

[0194] This embodiment provides monoclonal antibodies against different hydrophobic adamantane haptens and their preparation methods, and determines the properties of the monoclonal antibodies.

[0195] (I) Cell fusion and cloning

[0196] (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.

[0197] (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.

[0198] (3) The positive wells were cloned using the limiting dilution method to obtain hybridoma cell lines that can secrete monoclonal antibodies against amantadine.

[0199] (II) Cell cryopreservation and thawing

[0200] 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.

[0201] (III) Preparation of Monoclonal Antibodies

[0202] 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 ℃.

[0203] (iv) Identification of monoclonal antibodies

[0204] 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:

[0205] (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.

[0206] (2) Washing: Pour out the solution in the plate, shake dry, and wash once with washing solution for 3 minutes each time.

[0207] (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.

[0208] (4) Prepare standard solutions: Dilute the adamantane standard solution three times to obtain a total of eight concentrations. Set up three replicates for each concentration for the detection of antibodies prepared from the adamantane artificial antigens shown in formulas (I) to (VIII).

[0209] (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.

[0210] (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.

[0211] (7) Termination and measurement: Add 100 μL of stop solution to each well to terminate the reaction, and then measure the OD450 value of each well using an ELISA reader.

[0212] (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 Table 4.

[0213] The results showed that the amantadine haptens of formulas (VII) and (VIII), which have the highest hydrophobicity, significantly improved the affinity of the prepared monoclonal antibodies for amantadine, and the IC50 of the monoclonal antibodies was significantly increased.50 The concentration ranges from 1.5 to 13.2 ng / mL. Compared to other haptens, the IC50 values ​​of the amantadine hapten monoclonal antibodies of formulas (VII) and (VIII) are [not specified]. 50 The concentration was reduced by 303.4 to 143413.7 times, indicating that the amantadine hapten designed in this patent can be used to prepare monoclonal antibodies with high affinity.

[0214] Table 4. IC50 of monoclonal antibodies prepared from artificial antigens with different hydrophobicities of amantadine (I) to (VIII) 50

[0215]

[0216] 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. An amantadine hapten, characterized in that, The adamantane hapten contains a hydrophobic spacer arm, and its structural formula is any one of the following: I； Ⅵ。 2. Amantadine artificial antigen, characterized in that, It includes the adamantane hapten of claim 1 and the carrier protein coupled to the adamantane hapten.

3. The amantadine artificial antigen according to claim 2, characterized in that, The adamantane hapten is coupled to the carrier protein via an amide bond.

4. The adamantane artificial antigen according to claim 2 or 3, characterized in that, The carrier protein is selected from bovine serum albumin, ovalbumin, keyhole hemocyanin, thyroprotein, and human serum albumin.

5. The method for preparing the adamantane artificial antigen according to any one of claims 2-4, characterized in that, The carrier protein was coupled to the carboxyl group of the adamantane hapten using the active ester method.

6. The preparation method according to claim 5, characterized in that, The molar ratio of the amantadine hapten to the carrier protein is 10-15:

1.

7. Any of the following applications of the adamantane hapten of claim 1 or the adamantane artificial antigen of any one of claims 2-4: Used to evaluate the effect of hapten hydrophobicity on immunogenicity; Applications in the preparation of adamantane-specific antibodies; Application in the detection of adamantane-specific antibodies; The application is for non-disease diagnosis purposes.

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