Amantadine hydrophobic hapten and synthetic method and application thereof

Abstract

The invention relates to the technical field of biochemistry, in particular to amantadine hydrophobic haptens as well as a synthesis method and application thereof. Based on an amantadine target structure, different hydrophobic spacer arms are introduced, so that the hydrophobicity of the hapten is improved. And then coupling the hapten with a carrier protein, and evaluating the immunogenicity of the artificial antigen. The invention explores the influence of hapten hydrophobicity increase on immunogenicity, on one hand, theoretical support is provided for reasonable design of the hapten; on the other hand, a material basis is provided for preparing a high-affinity antibody of amantadine. The invention provides a new thought for reasonable design of small molecule compound hapten and preparation of high-affinity antibody.

Description

Technical Field

[0001] The invention relates to the technical field of biochemistry, and in particular to a class of adamantane amine hydrophobic hapten and a synthesis method and application thereof. Background Art

[0002] Amantadine (AMD) is a chemical risk factor, also known as tricyclodecylamine. It is the first antiviral drug approved by the U.S. Food and Drug Administration (FDA). This type of drug has been used clinically for many years. Due to the low price and good antiviral effect of AMD, domestic breeding companies have illegally used AMD to prevent and treat diseases caused by viral infections in animals. With the widespread use of amantadine, the side effects caused by it have gradually emerged, including drug resistance problems, promotion of strain mutation, neurotoxicity, etc., posing a serious potential threat to people's health. Therefore, establishing a highly sensitive, specific, simple and easy-to-use method for detecting banned drugs is an important technical support for ensuring food safety and is of great significance to protecting people's health. Rapid detection technology based on immunoassays with specific recognition of antigens and antibodies can effectively make up for the shortcomings of instrumental analysis and detection due to its low cost and batch screening advantages.

[0003] Antibodies are the core reagents of immunoassays. Since amantadine is a small molecule compound with a relatively small molecular weight, its monoclonal antibody preparation has problems such as poor specificity and sensitivity and difficulty in screening. In view of this, the present invention is proposed. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a class of adamantane amine hydrophobic haptens and a synthesis method and application thereof.

[0005] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides an amantadine hapten, wherein the amantadine hapten comprises a hydrophobic spacer arm, and the structural formula thereof is any one of the following structural formulas: I; II; III; IV; V; VI; VII; VIII.

[0006] The present invention is based on the target structure of adamantane amine, and introduces a spacer arm containing a saturated fatty chain, an unsaturated fatty chain, a benzene ring, and ethylene glycol to increase the hydrophobicity of the hapten. The present invention finally screened the above 8 hapten structures that have relatively outstanding performance in hydrophobicity and enhancing immune response.

[0007] In a second aspect, the present invention provides an amantadine artificial antigen, which comprises the amantadine hapten and a carrier protein coupled to the amantadine hapten.

[0008] Preferably, the amantadine hapten is coupled to the carrier protein via an amide bond.

[0009] Preferably, the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein and human serum albumin.

[0010] Preferably, the carrier protein is coupled to the carboxyl group of the adamantane hapten using an active ester method.

[0011] Preferably, the coupling molar ratio of the amantadine hapten to the carrier protein is 10-15:1.

[0012] The present invention couples the hapten with a carrier protein to evaluate the immunogenicity of the artificial antigen. On the one hand, the effect of increased hydrophobicity of the hapten on the immunogenicity is explored, providing theoretical support for the rational design of the hapten. On the other hand, it provides a material basis for the preparation of high-affinity antibodies against adamantane.

[0013] In a third aspect, the present invention provides any of the following uses of the adamantane hapten or the adamantane artificial antigen: Used to evaluate the effect of hapten hydrophobicity on immunogenicity; Use in the preparation of adamantane-specific antibodies; Used in detecting adamantane-specific antibodies; The application is for non-disease diagnosis purposes.

[0014] The eight haptens provided by the present invention are specially prepared according to the influence of the hydrophobicity of haptens on the preparation of monoclonal antibodies in immunoassay technology, providing a new idea and method for the rational design of small molecule compound haptens and the preparation of high-affinity antibodies.

[0015] In a fourth aspect, the present invention provides specific antibodies prepared from the artificial antigen, wherein the specific antibodies are polyclonal antibodies and / or monoclonal antibodies.

[0016] The specific antibodies provided by the present invention have a higher affinity.

[0017] In a fifth aspect, the present invention provides an amantadine drug detection reagent or kit prepared using the specific antibody.

[0018] In a sixth aspect, the present invention provides any of the following uses of the specific antibody: Application in fluorescence polarization detection kit for detecting amantadine drugs; Application in the preparation of enzyme-linked immunosorbent assay kits for amantadine drugs; Application in the preparation of lateral flow chromatography test strips for amantadine drugs; The application is for non-disease diagnosis purposes.

[0019] Beneficial effects: The present invention provides a class of hydrophobic haptens of adamantane and a synthesis method and application thereof. Specifically, based on adamantane, the hydrophobicity of the hapten is increased to varying degrees by introducing a spacer arm containing structures such as a saturated fatty chain, an unsaturated fatty chain, a benzene ring and ethylene glycol. Subsequently, the hapten is coupled to a carrier protein to evaluate the immunogenicity of the artificial antigen. The present invention explores the effect of increased hydrophobicity of the hapten on the immunogenicity, which provides theoretical support for the rational design of haptens on the one hand, and provides a material basis for the preparation of high-affinity antibodies to adamantane on the other hand. The present invention provides a new idea for the rational design of small molecule compound haptens and the preparation of high-affinity antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0021] Figure 1 The figure is the hydrogen nuclear magnetic resonance spectrum of the adamantane hapten represented by formula (I) in the embodiment of the present invention.

[0022] Figure 2 The figure is the hydrogen nuclear magnetic resonance spectrum of the adamantane hapten represented by formula (II) in the embodiment of the present invention.

[0023] Figure 3 The figure is the hydrogen nuclear magnetic resonance spectrum of the adamantane hapten represented by formula (III) in the embodiment of the present invention.

[0024] Figure 4 The figure is the hydrogen nuclear magnetic resonance spectrum of the adamantane hapten represented by formula (IV) in the embodiment of the present invention.

[0025] Figure 5 The figure is the hydrogen nuclear magnetic resonance spectrum of the adamantane hapten represented by formula (V) in the embodiment of the present invention.

[0026] Figure 6 This is a full-mode scanning mass spectrum of the adamantane amine hapten represented by formula (VI) in an embodiment of the present invention.

[0027] Figure 7 The figure is the hydrogen nuclear magnetic resonance spectrum of the hydrophobic hapten of adamantane amine represented by formula (VII) in the embodiment of the present invention.

[0028] Figure 8 This is a full-mode scanning mass spectrum of the hydrophobic hapten of adamantane amine represented by formula (VIII) in the embodiment of the present invention.

[0029] Fig. 9 The figure is a molecular surface electrostatic potential energy distribution diagram of the adamantane hapten represented by formula (I) to (VIII) in the embodiments of the present invention.

[0030] Fig.10 This is a MALDI-TOF-MS graph of the artificial antigen prepared from the adamantane amine hapten represented by formula (I) to (VIII) in the embodiments of the present invention.

[0031] Fig.11 The antibody titer after three immunizations with the artificial antigen of adamantane hapten represented by formula (I) to (VIII) in the embodiments of the present invention.

[0032] Fig.12 It is 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 antigen of adamantane hapten represented by formula (I) to (VIII) in the embodiments of the present invention. DETAILED DESCRIPTION

[0033] Hydrophobicity is one of the most important physical and chemical properties of molecules. In the field 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 compound antibody preparation, the effect of hapten hydrophobicity on immune response is still unknown. The hydrophobicity of haptens can be increased to varying degrees by introducing spacer arms with different hydrophobicities (containing saturated fatty chains, unsaturated fatty chains, benzene rings and ethylene glycol). Exploring the effect of hapten hydrophobicity on immunogenicity can, on the one hand, provide theoretical support for the rational design of haptens; on the other hand, it can provide a material basis for the preparation of high-affinity antibodies for small molecule compounds. This type of hapten has an active group while improving hydrophobicity, and can be conjugated with a carrier protein to prepare an artificial antigen with a suitable coupling ratio and high hydrophobicity; further, it promotes the immunogenicity of artificial antigens, prepares highly specific and high-affinity monoclonal antibodies, and establishes relevant immunoassay methods to detect chemical risk factors such as adamantane.

[0034] The purpose of the present invention is to improve the deficiencies of existing hapten design theory and immunoassay technology, and to provide a method for synthesizing haptens and artificial antigens with different hydrophobicity and their application. The hydrophobic hapten can be conjugated with a carrier protein such as bovine serum albumin or egg serum albumin to obtain an artificial antigen. By increasing the hydrophobicity of the hapten, the problem of weak immunogenicity of the artificial antigen is improved. Immunized animals are used to obtain monoclonal antibodies with high affinity and strong specificity, thereby providing a theoretical basis and technical support for establishing an immunoassay technology for detecting adamantane residues.

[0035] In order to achieve the object of the present invention, the present invention provides the following scheme: First, the present invention provides a class of adamantane amine hydrophobic hapten, the structural formula of which is as follows: I; II; III; IV; V; VI; VII; VIII.

[0036] Furthermore, the present invention also provides a method for preparing the above 8 kinds of adamantane amine hydrophobic haptens.

[0037] The method for preparing the hapten represented by formula (I) provided by the present invention comprises the following steps: Dissolve 3,6-dioxanedicarboxylic acid in DCM, add oxalyl chloride, and after the addition, add anhydrous DMF to the system, and stir the resulting solution at room temperature. Then slowly add anhydrous DCM solution containing adamantane amine to the system, and the reaction is complete by TLC detection. Adjust the system to acidity with 2 N HCl, then add saturated brine, stand and separate, extract the aqueous phase with ethyl acetate, combine the organic phases, desolventize under reduced pressure, and purify the residue by silica gel column chromatography to obtain the target product.

[0038] The present invention provides a method for preparing a hapten represented by formula (II), comprising the following steps: Dissolve suberic acid and DMA in a mixed solvent of dichloromethane and DMF, add adamantane hydrochloride and stir at room temperature for 1. Add EDC in batches and stir at room temperature overnight. Add dilute hydrochloric acid solution, extract three times with dichloromethane, combine the organic phases and wash twice with dilute hydrochloric acid solution. Extract the organic phase three times with NaOH solution, combine the aqueous phases, wash three times with dichloromethane, and then adjust the acidity with 1 N dilute hydrochloric acid solution. A white solid precipitates and the target product is collected by filtration.

[0039] The present invention provides a method for preparing a hapten represented by formula (III), comprising the following steps: Dissolve octenedioic acid in dry dichloromethane, add oxalyl chloride and DMF, and react the resulting solution at room temperature. Then slowly drop anhydrous dichloromethane solution containing adamantane amine into the system, and the reaction is complete when detected by TLC. Adjust the acidity with 2 M dilute hydrochloric acid aqueous solution, then add saturated brine and stir, stand and separate, extract the aqueous phase with ethyl acetate, combine the organic phases, desolventize under reduced pressure, and purify the residue with silica gel column chromatography to obtain the target product.

[0040] The present invention provides a method for preparing a hapten represented by formula (IV), comprising the following steps: Adipic acid and DMAP were dissolved in a mixed solvent of dichloromethane and DMF, and adamantane hydrochloride was added and stirred at room temperature for 10 minutes. EDCI was added in batches and stirred at room temperature overnight. After adding 1 N dilute hydrochloric acid aqueous solution, dichloromethane was extracted three times, and the organic phases were combined and washed twice with 1 N dilute hydrochloric acid aqueous solution. The organic phase was extracted three times with 1 N NaOH aqueous solution, and the aqueous phases were combined and washed three times with dichloromethane, and then the acidity was adjusted with 1 N dilute hydrochloric acid aqueous solution to obtain the precipitated target product.

[0041] The present invention provides a method for preparing a hapten represented by formula (V), comprising the following steps: Dissolve 1,3-butadiene-1,4-dicarboxylic acid, TBTU and triethylamine in DMF, then slowly drop a DMF solution containing adamantane into the solution, and continue stirring at room temperature overnight. After the reaction is completed, add water to the system to adjust the acidity of the system, then extract with ethyl acetate, combine the organic phases, desolventize, and purify the residue by silica gel column chromatography to obtain the target product.

[0042] The present invention provides a method for preparing a hapten represented by formula (VI), comprising the following steps: Amantadine and p-formylbenzoic acid were dissolved in ethanol, acetic acid was added dropwise to the system, the resulting solution was stirred at room temperature, and the reaction was completed by TLC detection. After desolventizing under reduced pressure, water was added, filtered, and the precipitated product was purified by silica gel column chromatography to obtain the target product.

[0043] The present invention provides a method for preparing a hapten represented by formula (VII), comprising the following steps: Dissolve adamantane in DMF, add ethyl bromooctanoate and K2CO3, stir and react at 60°C for 16 h, then dilute with water and extract with ethyl acetate; dry the sodium salt and filter out the solid, concentrate the filtrate in vacuum and purify to obtain 8-(adamantyl)aminocaprylic acid ethyl ester; dissolve 8-(adamantyl)aminocaprylic acid ethyl ester in ethanol aqueous solution, add NaOH, stir and react for 1 h; remove ethanol and adjust the solution pH to 5; finally obtain the target product after concentration and purification.

[0044] The present invention provides a method for preparing a hapten represented by formula (VIII), comprising the following steps: Dissolve adamantane in DMF, add ethyl bromooctanoate and K2CO3, stir at room temperature for 6 h, add 50 mL of water and stir for 5 min, filter, and purify the precipitated product by silica gel column chromatography to obtain the target product.

[0045] Furthermore, the present invention provides eight kinds of artificial adamantane antigens obtained based on the above haptens, which are specifically obtained by coupling the adamantane hydrophobic haptens of formula (I) to (VIII) with carrier proteins.

[0046] Wherein, the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein and human serum albumin, etc.; preferably bovine serum albumin and ovalbumin.

[0047] Furthermore, the present invention provides a method for preparing the artificial antigen, wherein the carrier protein can be coupled to the carboxyl carbon of the hapten of formula (I) to (VIII) by using the activated ester method.

[0048] Preferably, the coupling molar ratio of the compound represented by formula (I) to (VIII) to bovine serum albumin is 10 to 15:1.

[0049] Furthermore, the present invention provides any of the following uses of the amantadine hapten or the amantadine artificial antigen: Used to evaluate the effect of hapten hydrophobicity on immunogenicity.

[0050] Used for the preparation of adamantane-specific antibodies.

[0051] Used to detect antibodies specific for amantadine.

[0052] Furthermore, the present invention provides specific antibodies prepared from the artificial adamantane antigen, including polyclonal antibodies and monoclonal antibodies, preferably monoclonal antibodies. The polyclonal antibodies can be obtained by immunizing experimental animals (such as New Zealand white rabbits) with the artificial adamantane antigen and collecting and purifying the serum. The monoclonal antibodies can be prepared by hybridoma technology.

[0053] Furthermore, the present invention provides an amantadine detection reagent or kit prepared from the specific antibody.

[0054] Furthermore, the present invention provides any of the following applications of the specific antibody: Application in fluorescence polarization detection kit for detecting adamantane.

[0055] Application in the preparation of lateral flow immunochromatographic test strips for amantadine.

[0056] Application in the preparation of enzyme-linked immunosorbent assay kit for amantadine.

[0057] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention discloses for the first time 8 kinds of adamantane haptens and artificial antigens with different hydrophobicities and methods for preparing the same. By evaluating the effect of the hydrophobicity of each hapten on the immunogenicity, the mechanism of the effect of the hydrophobicity of the hapten on the immune response is revealed, providing a theoretical basis for the rational design of haptens. By appropriately adjusting the hydrophobicity of the hapten, the immunogenicity of the artificial antigen is enhanced, thereby obtaining specific antibodies with high titer and high sensitivity. The adamantane haptens and antibodies prepared therefrom provided in this study provide a new means for establishing a rapid, simple, inexpensive, sensitive and specific adamantane detection method.

[0058] In this study, different hydrophobic adamantane haptens were synthesized, and the polar molecular parameters MPI of the adamantane haptens were calculated to be in the range of 7.25 to 11.20. The adamantane haptens of formula (VII) and formula (VIII) were the most hydrophobic haptens (MPI of 7.25 and 7.82), and successfully induced the highest hapten-specific antibody titers after three immunizations, which were 1.3 to 3.9 times higher than those of other groups; at the same time, they effectively induced antiserum and monoclonal antibodies with high affinity for adamantane, which were 2.3 to 5.0 times and 303.4 to 143413.7 times higher than those of other haptens, respectively.

[0059] The artificial antigen provided by the present invention is used to prepare amantadine antibodies (polyclonal antibodies and monoclonal antibodies). The preparation process is simple and economical. The detection sensitivity of the antibody to amantadine can reach 1.5 ng / mL. The affinity of the amantadine antibody is enhanced by improving the hydrophobicity of the amantadine hapten, which provides a new idea and method for the rational design of small molecule compound haptens and the preparation of high-affinity antibodies.

[0060] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0061] 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0062] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0063] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0064] Example 1

[0065] (I) This example provides different hydrophobic adamantane amine haptens and their synthesis methods, as follows.

[0066] (1) Preparation of the hydrophobic hapten of adamantane amine represented by formula (I)

[0067] Dissolve 2.00 g of 3,6-dioxanoic acid (11.22 mmol) in 100 mL of dry DCM, add 2.85 g of oxalyl chloride (22.45 mmol), add 5 drops of anhydrous DMF to the system, and stir the resulting solution at room temperature for 1 h. After 1 h, slowly add 150 mL of anhydrous DCM solution containing 1.53 g of adamantane amine (10.10 mmol) to the system, continue stirring at room temperature for 0.5 h, and the reaction is complete by TLC detection. Adjust the pH of the system to 3-4 with 2 N HCl, then add 150 mL of saturated brine and stir for 5 min, let stand and separate, extract the aqueous phase with 150 mL×2 ethyl acetate, combine the organic phases, desolventize under reduced pressure, and purify the residue by silica gel column chromatography (200-300 mesh, DCM / MeOH=15 / 1-10 / 1) to obtain 0.33 g of light yellow solid, which is the target product.

[0068] The synthesis process steps are as follows: .

[0069] (2) Preparation of the hydrophobic hapten of adamantane amine represented by formula (II)

[0070] Suberic 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. After adding EDCI (3.83 g, 20 mmol) in batches, 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 organic phases were combined and washed twice with 2×200 mL of 1 N dilute hydrochloric acid aqueous solution. The organic phase was extracted three times with 3×100 mL of 1N NaOH aqueous solution, and the aqueous phases were combined and washed three times with 3×200 mL of dichloromethane. Then, the pH was adjusted to 2~3 acidic 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.

[0071] The synthesis process steps are as follows: .

[0072] (3) Preparation of the hydrophobic hapten of adamantane amine represented by formula (III)

[0073] Dissolve 2.40 g of octenedioic acid (13.94 mmol) in 150 mL of dry dichloromethane, add 3.54 g of oxalyl chloride (22.46 mmol), add 5 drops of anhydrous DMF to the system, and stir the resulting solution at room temperature for 1 h. After 1 h, slowly add 200 mL of anhydrous dichloromethane solution containing 1.90 g of adamantane amine (12.55 mmol) to the system, continue stirring at room temperature for 0.5 h, and the reaction is complete by TLC detection. Adjust the pH of the system to 3-4 with 2 M HCl, then add 150 mL of saturated brine and stir for 5 min, let stand and separate, extract the aqueous phase with 150 mL×2 ethyl acetate, combine the organic phases, desolventize under reduced pressure, and purify the residue by silica gel column chromatography (200-300 mesh, DCM / MeOH=15 / 1-10 / 1) to obtain 1.64 g of light yellow solid, which is the target product.

[0074] The synthesis process steps are as follows: .

[0075] (4) Preparation of the hydrophobic hapten of adamantane amine represented by formula (IV)

[0076] 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. After adding EDCI (3.83 g, 20 mmol) in batches, 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 organic phases were combined and washed twice 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, and the aqueous phases were combined and washed three times with 3×200 mL of dichloromethane. Then, the pH was adjusted to 2~3 acidic with 1 N dilute hydrochloric acid aqueous solution. A white solid precipitated and 1.52 g of the white solid was collected by filtration.

[0077] The synthesis process steps are as follows: .

[0078] (5) Preparation of the hydrophobic hapten of adamantane amine represented by formula (V)

[0079] 2.50 g 1,3-butadiene-1,4-dicarboxylic acid (1.76 mmol), 7.35 g TBTU (22.88 mmol) and 5.69 g triethylamine (44.00 mmol) were dissolved in 40 mL DMF in sequence and stirred for 30 min. Then 60 mL DMF solution containing 2.39 g adamantane amine (15.8 mmol) was slowly added to the solution and stirred overnight at room temperature. After the reaction was completed, 150 mL water was added to the system and stirred for 5 min. The pH of the system was adjusted to 3-4. Then, the system was extracted with 100 mL × 3 ethyl acetate. The organic phases were combined and desolventized. The residue was purified by silica gel column chromatography (200-300 mesh, DCM / MeOH=15 / 1-10 / 1) to obtain 2.23 g of white solid, which was the target product.

[0080] The synthesis process steps are as follows: .

[0081] (6) Preparation of the hydrophobic hapten of adamantane amine represented by formula (VI)

[0082] Weigh 500 mg of adamantane amine (3.31 mmol) and 546 mg of p-formylbenzoic acid (3.64 mmol) and dissolve them in 2 mL of ethanol. After the addition, add 5 drops of 100 uL of acetic acid to the system. Stir the resulting solution at room temperature for 6 h. TLC detection shows that the reaction is complete. After desolventizing under reduced pressure, add 50 mL of water and stir for 5 minutes. Filter with suction, and precipitate the product and purify it by silica gel column chromatography to obtain 161 mg of a white solid, which is the target product.

[0083] The synthesis process steps are as follows: .

[0084] (7) Preparation of the hydrophobic hapten of adamantane amine represented by formula (VI)

[0085] 5.04 g of adamantane amine (33.38 mmol) was dissolved in 15 mL of DMF, and 9.1 g of ethyl bromooctanoate (36.23 mmol) and 9.1 g of K2CO3 (65.84 mmol) were added. The mixture was stirred at 60 °C for 16 h. Then, 100 mL of water was added for dilution, and the mixture was extracted with ethyl acetate (3×50 mL). After the organic phase was combined, the mixture was dried with sodium, and then the solid was filtered out. The filtrate was vacuum concentrated and purified to obtain 8-(adamantyl)aminocaprylic acid ethyl ester. Preparation of 8-(adamantyl)aminocaprylic acid: 4 g of 8-(adamantyl)aminocaprylic acid ethyl ester was dissolved in ethanol aqueous solution (ethanol: water = 1:1), 0.9 g of NaOH was added, and the mixture was stirred for 1 h. After removing ethanol, the pH of the solution was adjusted to 5 with 3M HCl. The mixed solution was vacuum concentrated and purified to finally obtain 1.3 g of a white solid, which was the target product.

[0086] The synthesis process steps are as follows: .

[0087] (8) Preparation of the hydrophobic hapten of adamantane amine represented by formula (VIII)

[0088] Weigh 500 mg of adamantane amine (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 resulting solution at room temperature for 6 h. TLC indicates that the reaction is complete. Then add 50 mL of water and stir for 5 min. Filter with suction. The precipitated product is purified by silica gel column chromatography to obtain 120 mg of a white solid, which is the target product.

[0089] The synthesis process steps are as follows: .

[0090] The eight hydrophobic amantadine haptens provided by the invention have simple synthesis methods, high purity and high yield, can be directly coupled with proteins to immunize animals to produce specific antibodies against the amantadine haptens, and can be used for rapid detection of amantadine residues.

[0091] (II) In this example, the 8 hydrophobic haptens of adamantane amine synthesized above are characterized as follows.

[0092] (1) The structure of the hydrophobic hapten of adamantane amine represented by formula (I) was determined by nuclear magnetic resonance. Figure 1 :1H NMR (300 MHz, DMSO-d6) δ: 11.96 (s, COOH, 1H), 10.46 (s, NH, 1H), 8.18 (d, J = 9.1Hz, 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, it can be seen that the hydrophobic hapten of adamantane amine shown in formula (I) was successfully synthesized.

[0093] (2) The structure of the hydrophobic hapten of amantadine shown in formula (II) was determined by nuclear magnetic resonance. Figure 2 :1HNMR (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 nuclear magnetic resonance, it can be shown that the hydrophobic hapten of adamantane amine shown in formula (II) was successfully synthesized.

[0094] (3) The structure of the hydrophobic hapten of adamantane amine represented by formula (III) was determined by nuclear magnetic resonance. Figure 3:1HNMR(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, it can be seen that the hydrophobic hapten of adamantane amine shown in formula (III) was successfully synthesized.

[0095] (4) The structure of the hydrophobic hapten of adamantane amine represented by formula (IV) was determined by nuclear magnetic resonance. Figure 4 :1HNMR (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). From the perspective of nuclear magnetic resonance, it can be seen that the hydrophobic hapten of adamantane amine shown in formula (IV) was successfully synthesized.

[0096] (5) The structure of the hydrophobic hapten of adamantane amine represented by formula (V) was determined by nuclear magnetic resonance. Figure 5 :1HNMR(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). From the perspective of nuclear magnetic resonance, it can be shown that the hydrophobic hapten of adamantane amine shown in formula (V) was successfully synthesized.

[0097] (6) The molecular weight of the synthesized hydrophobic hapten of formula (VI) was determined by mass spectrometry. Figure 6 : In full scan mode, the molecular ion peak is 284, which matches the theoretical molecular weight of the hapten of formula (VI). From the perspective of mass spectrometry, it can be seen that the hydrophobic hapten of adamantaneamine shown in formula (VI) has been successfully synthesized.

[0098] (7) The structure of the hydrophobic hapten of adamantane amine represented by formula (VII) was determined by nuclear magnetic resonance. 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. From the perspective of nuclear magnetic resonance, it was shown that the hydrophobic hapten of adamantane amine represented by formula (VII) was successfully synthesized.

[0099] (8) The molecular weight of the synthesized hydrophobic hapten of formula (VIII) was determined by mass spectrometry. Figure 8 : In full scan mode, the molecular ion peak is 266, which matches the theoretical molecular weight of the hapten of formula (VIII). From the perspective of mass spectrometry, it can be seen that the hydrophobic hapten of adamantaneamine represented by formula (VIII) has been successfully synthesized.

[0100] (III) This example further performs geometry optimization on the hydrophobic hapten of adamantane amine based on the M06-2X density functional theory and the TZVP basis set, extracts the physicochemical parameters of the hapten of adamantane amine shown in formula (I) to (VIII) from the calculation results, and plots the distribution diagram of the molecular surface electrostatic potential ( Fig. 9 , Table 1), the 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 showed that the polar molecular parameters and molecular surface electrostatic potential distribution of the eight haptens were different, suggesting that the haptens designed and synthesized in this study have different hydrophobicity.

[0101] Table 1 Physicochemical parameters of adamantane haptens shown in (I) to (VIII)

[0102] Example 2

[0103] This example provides artificial antigens of different hydrophobic amantadine and their preparation methods and characterization results. The difference between the immunogen and the coating in the preparation method lies in the type of carrier protein used. The immunogen carrier protein mainly uses BSA, and the coating carrier protein mainly uses OVA. The coupling method is the active ester method.

[0104] (1) Preparation of different hydrophobic amantadine immunogens

[0105] 9.03 μmol of the compounds of formula (I) to (VIII) prepared in Example 1 were dissolved in 1 mL of DMF, 10.38 μmol of NHS and 10.38 μmol of EDC were added, and the mixture was stirred at room temperature for 24 h to obtain solution I.

[0106] Add 20 mg of BSA into 10 mL of PBS buffer and fully dissolve to obtain Solution II.

[0107] Solution I was slowly added dropwise to solution II, and the mixture was slowly stirred at 4°C for 24 h, then placed in a dialysis bag and dialyzed in PBS at 4°C for 72 h (with water changed 6 times in between) to obtain an amantadine immunogen solution, which was then aliquoted and stored at -20°C. The amantadine immunogen synthesized from the compounds represented by formula (I) to (VIII) is referred to as AMD-BSA.

[0108] (2) Identification of different hydrophobic amantadine immunogens

[0109] The binding ratios of BSA and hapten in AMD-BSA solution were determined by matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF-MS). The results are shown in Fig.10 .

[0110] The binding ratio of the artificial antigen of the compound represented by formula (I) to (VIII) was calculated by the following formula (Table 2): Binding ratio = {M (conjugate) - M (protein)} / M (hapten) .

[0111] (3) Synthesis of different hydrophobic adamantane coatings

[0112] OVA is used to replace BSA, and the preparation method and steps of the coating are the same as those of the immunogen. The compounds shown in formula (I) to (VIII) are used to synthesize different hydrophobic amantadine coatings, which are referred to as AMD-OVA.

[0113] Table 2 Binding ratios of artificial antigens of different hydrophobicity of adamantane amines represented by formula (I) to (VIII)

[0114] Example 3

[0115] This example provides polyclonal antibodies against different hydrophobic amantadine haptens and methods for preparing the same.

[0116] 64 6-8 week old female BALB / c mice (SPF level) were immunized with the AMD-BSA prepared in Example 2 and randomly divided into 8 groups, 8 mice in each group. The immunization procedure was one basic immunization and several booster immunizations.

[0117] For the first immunization, 100 μg of the immunogen was mixed with an equal volume of Freund's complete adjuvant, emulsified, and injected subcutaneously at the back of the neck of the mouse at multiple points, with 200 μL per mouse for basic immunization.

[0118] 100 μg of immunogen was mixed with an equal volume of Freund's incomplete adjuvant for emulsification. After the first immunization, booster immunization was performed every 3 weeks, with a volume of 200 μL per mouse.

[0119] On the 7th day after each immunization, blood was collected from the eye sockets, and serum was separated. The titer of serum antibodies and the inhibition rate of recognition of amantadine were detected by indirect competitive ELISA. After blood collection, the blood was kept at 37 °C for 30 min, then centrifuged at 3000 rpm for 20 min, and the supernatant was collected, which was the polyclonal antibody and stored in aliquots at -20 °C.

[0120] Example 4

[0121] In this example, the titer and affinity of polyclonal antibodies to different hydrophobic amantadine haptens were determined.

[0122] (I) The antibody titer was detected by indirect ELISA method. The specific steps are as follows: (1) Coating: Dilute the coating agent in series with 0.05 M, pH 9.6 carbonate buffer, 100 μL / well, and react at 4 °C for 16 h.

[0123] (2) Washing: Pour off the solution in the plate, spin dry, and wash once with detergent for 3 min each time.

[0124] (3) Blocking: After patting dry, add 150 μL / well blocking solution and react at 37°C for 1 h. After washing, pat dry and set aside.

[0125] (4) Sample addition: Add 100 μL / well of serially diluted antibody to each well and react at 37°C for 30 min. After thorough washing, add 1:5000 diluted HRP-goat anti-mouse IgG, 100 μL / well, and react at 37°C for 30 min.

[0126] (5) Color development reaction: Take out the ELISA plate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37 °C in the dark for 15 min.

[0127] (6) Termination and measurement: Add 100 μL of stop solution to each well to terminate the reaction, and then use an ELISA reader to measure the OD value of each well at 450 nm.

[0128] (7) Data processing: The antibody titer was defined as the reciprocal of the highest dilution whose OD value was higher than the mean ± 3SD of the blank control wells (Table 3, Fig.11 ).

[0129] (II) The antibody affinity was detected by indirect competitive ELISA method. The specific steps are as follows: (1) Coating: Dilute the coating agent in series with 0.05 M, pH 9.6 carbonate buffer, 100 μL / well, and react at 37 °C for 2 h.

[0130] (2) Washing: Pour off the solution in the plate, spin dry, and wash once with detergent for 3 min each time.

[0131] (3) Blocking: After patting dry, add 150 μL / well blocking solution and react at 37°C for 1 h. After washing, pat dry and set aside.

[0132] (4) Prepare standard solution: dilute the adamantane standard to 10 μg / mL and dilute the adamantane hapten to 50 ng / mL for the detection of antibodies prepared from the adamantane artificial antigens of different hydrophobicities as shown in formulas (I) to (VIII).

[0133] (5) Sample addition: Add 50 μL of diluted standard of each concentration to each well, and then add 50 μL / well of the optimal dilution of antibody, and react at 37°C for 30 min. After thorough washing, add 1:5000 diluted HRP-goat anti-mouse IgG, 100 μL / well, and react at 37°C for 30 min.

[0134] (6) Color development reaction: Take out the ELISA plate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37 °C in the dark for 15 min.

[0135] (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.

[0136] (8) Data processing: Antibody affinity was evaluated by inhibition rate. The inhibition rates of antibodies prepared with different hydrophobic adamantane artificial antigens in formulas (I) to (VIII) were as follows (Table 3, Fig.12 ) is calculated according to the following formula: Inhibition rate (%) = B / B0 × 100%, where B0 and B are the OD values ​​at 450 nm in the absence and presence of the standard, respectively.

[0137] The results showed that the most hydrophobic amantadine haptens of formula (VII) and formula (VIII) could improve the antibody titer and affinity for amantadine induced by the body as a whole. The difference was more significant after the third immunization, and the highest hapten-specific antibody titer was induced, which was 1.3 to 3.9 times higher than that of other groups; at the same time, antiserum with high affinity for amantadine was effectively induced, and the affinity was 2.3 to 5.0 times higher than that of other haptens.

[0138] Table 3 Serum antibody titers and inhibition rates of artificial antigens prepared from hydrophobic amantadine of formula (I) to (VIII)

[0139] Note: a Represents the average inhibition rate of AMD (10 μg / mL) and corresponding hapten (50 ng / mL) at different antibody dilutions after three immunizations. b Represents the mean antibody titer to the hydrophobic hapten of amantadine after three immunizations.

[0140] Example 5

[0141] This example provides monoclonal antibodies to amantadine haptens of different hydrophobicity and preparation methods thereof, and the properties of the monoclonal antibodies are measured.

[0142] 1. Cell fusion and cloning

[0143] (1) BALB / c mice with the highest serum suppression rate in each group were selected for shock immunization and cell fusion experiments. The shock immunization was performed by intraperitoneal injection of 3 times the immune dose of the immunogen.

[0144] (2) Three days after the shock immunization, spleen cells were collected and fused with SP2 / 0 myeloma cells at a ratio of 5:1 (quantity ratio). The cell supernatant was measured by indirect competitive ELISA to screen the positive wells.

[0145] (3) The positive wells were cloned using the limiting dilution method to obtain hybridoma cell lines that can secrete monoclonal antibodies against the drug adamantane.

[0146] 2. Cell cryopreservation and thawing

[0147] Hybridoma cells were prepared into 1×10 6 Cell suspensions of 100 cells / mL were stored in liquid nitrogen for a long time. When thawing, the cryopreserved tubes were taken out 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 bottles for culture.

[0148] 3. Preparation of monoclonal antibodies

[0149] BALB / c mice were intraperitoneally injected with sterile paraffin oil (0.5 mL / mouse). Seven days later, the hybridoma cells prepared above (5×10 5 After 7 days, the ascites was collected to prepare the monoclonal antibody, which was stored at -20 ℃.

[0150] 4. Identification of monoclonal antibodies

[0151] The monoclonal antibody solution obtained in step 3 was used to detect antibody affinity using the indirect competitive ELISA method. The specific operation steps are as follows: (1) Coating: Dilute the coating agent in series with 0.05 M, pH 9.6 carbonate buffer, 100 μL / well, and react at 37 °C for 2 h.

[0152] (2) Washing: Pour off the solution in the plate, spin dry, and wash once with detergent for 3 min each time.

[0153] (3) Blocking: After patting dry, add 150 μL / well blocking solution and react at 37°C for 1 h. After washing, pat dry and set aside.

[0154] (4) Preparation of standard solution: The standard solution of adamantane was diluted 3-fold to a total of 8 concentrations, and 3 replicate wells were set for each concentration for the detection of antibodies prepared against the adamantane artificial antigens shown in formulas (I) to (VIII).

[0155] (5) Sample addition: Add 50 μL of diluted standard of each concentration to each well, and then add 50 μL / well of the optimal dilution of antibody, and react at 37°C for 30 min. After thorough washing, add 1:5000 diluted HRP-goat anti-mouse IgG, 100 μL / well, and react at 37°C for 30 min.

[0156] (6) Color development reaction: Take out the ELISA plate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37 °C in the dark for 15 min.

[0157] (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.

[0158] (8) Data processing: The standard concentration was taken as the Log value as the horizontal axis and the OD value as the vertical 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.

[0159] The results showed that the hydrophobicity of the adamantane haptens of formula (VII) and formula (VIII) significantly increased the affinity of the prepared monoclonal antibody for adamantane. The IC 50Compared with other haptens, the IC values ​​of the monoclonal antibodies of the adamantane hapten of formula (VII) and formula (VIII) are 1.5~13.2 ng / mL. 50 The reduction was 303.4 to 143413.7 times, indicating that the adamantane hapten designed in this patent can be used to prepare high-affinity monoclonal antibodies.

[0160] Table 4 IC of monoclonal antibodies prepared from artificial antigens of different hydrophobicity of adamantane amine of formula (I) to (VIII) 50

[0161] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions 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 structural formulas: I； Ⅱ； Ⅲ； Ⅳ； Ⅴ； Ⅵ； Ⅶ； Ⅷ。 2. The artificial antigen of amantadine, characterized in that The invention comprises the adamantane hapten according to claim 1 and a carrier protein coupled to the adamantane hapten.

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

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

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

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

1.

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

8. A specific antibody prepared from the artificial antigen according to any one of claims 2 to 4, wherein the specific antibody is a polyclonal antibody and / or a monoclonal antibody.

9. An amantadine drug detection reagent or kit prepared from the specific antibody according to claim 8.

10. Any of the following uses of the specific antibody according to claim 8: Application in fluorescence polarization detection kit for detecting amantadine drugs; Application in the preparation of enzyme-linked immunosorbent assay kits for amantadine drugs; Application in the preparation of lateral flow chromatography test strips for amantadine drugs; The application is for non-disease diagnosis purposes.

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