Halogen modified hapten for enhancing titer and affinity of antibody as well as preparation method and application of halogen modified hapten

Abstract

The invention discloses a halogen modified hapten for enhancing antibody titer and affinity as well as a preparation method and application of the halogen modified hapten, and belongs to the technical field of biochemical engineering. According to the method, an amantadine structure is innovatively and chemically modified, various halogens such as fluorine, chlorine and bromine are introduced, the AMA halogen modified hapten shown in the formula (I) is formed, and the traditional'most similar 'design principle is broken through. Compared with an unmodified hapten, the hapten modified by halogen in the invention has the advantages that the titer of the polyclonal antibody on amantadine can be obviously improved by 3.4-14 times; wherein the haptens A5 and A6 can obviously improve the titer of the monoclonal antibody to amantadine by 3-18 times, and the affinity is improved by 10.8-16.1 times. The invention provides a new thought and a new method for the rational design of the hapten of the small molecule compound and the preparation of the high-affinity antibody, and the related halogen modification type and quantity strategy can also provide valuable reference for the design of the hapten of other small molecule compounds.

Description

Technical Field

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

[0002] Amantadine (AMA) is a small-molecule antiviral compound, and its residues in animal-derived foods may pose a potential risk to consumer health. Therefore, developing rapid and sensitive methods for detecting amantadine residues is of great significance for ensuring food safety.

[0003] Immunoassays based on antigen-antibody specific recognition have become the mainstream technology for rapid detection of veterinary drug residues due to their advantages such as low cost, simple operation, and large sample throughput. Immunoassays for detecting small molecule compounds fall under the category of "reagent-limited" analysis, where antibody affinity plays a crucial role in detection sensitivity. The primary principle of traditional hapten design theory is "maximum similarity between the hapten and the target." Existing research typically does not alter the original characteristic structure of the small molecule target but introduces a benzene ring or other larger groups as spacers in the hapten design to increase its molecular weight and structural complexity, thereby enhancing antibody response. However, the benzene ring, as a planar rigid structure, is relatively large. Introducing it into the hapten is highly likely to cause steric hindrance, affecting the BCR's recognition of the target antigenic determinant's characteristic structure and hindering the production of high-affinity specific antibodies. Therefore, this irrational design strategy based on the "similarity principle" and "empirical" has low accuracy, controllability, and success rate, making it difficult to prepare high-affinity antibodies that meet detection requirements and thus restricting the development of immunoassay methods.

[0004] In summary, existing immunoassay methods based on traditional hapten design strategies still face technical bottlenecks when targeting small molecule compounds such as amantadine, including insufficient antibody affinity, limited detection sensitivity, and low method reliability. Therefore, there is an urgent need in this field for a more rational and controllable new hapten design strategy, as well as high-affinity and specific antibody preparation technology based on this strategy and corresponding highly sensitive immunoassay methods, to overcome existing technical obstacles, improve the overall efficiency of veterinary drug residue detection, and better meet the pressing needs of food safety supervision. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a halogen-modified adamantane hapten with enhanced antibody titer and affinity, its preparation method, and its applications. Obtained using halogen-modified adamantane, the prepared antibody exhibits relatively high titer and affinity, providing a new and effective approach for preparing high-titer and high-affinity adamantane antibodies.

[0006] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a halogen-modified adamantane hapten, the general structural formula of which is shown in formula (I): Formula (I); Wherein, R is an adamantyl alkyl group containing one or more halogen atoms, wherein the halogen atoms are selected from any one or more of fluorine, chlorine and bromine.

[0007] Halogen modification can lead to subtle differences in conformation, charge distribution, and physicochemical properties between haptens and target molecules. This invention utilizes halogen-modified adamantane to obtain haptens, resulting in antibodies with relatively high titers and affinities. This may be related to the high electronegativity and hydrophobicity of halogens, which effectively increase electrostatic and hydrophobic interactions between the hapten and antibody, thereby enhancing antibody titers and affinities. Therefore, this invention provides an effective route for preparing high-titer and high-affinity antibodies using adamantane.

[0008] Preferably, the R group is selected from any one of the following groups: .

[0009] The second objective of this invention is to provide a method for preparing the halogen-modified adamantane hapten as described above, comprising the following steps: (1) Halogen-modified adamantane carboxylic acid with the structural formula R-COOH was dissolved in an aprotic polar solvent with an organic base, activated by adding a coupling reagent, and then reacted with methyl 6-aminohexanoate hydrochloride to obtain intermediate compound 3. (2) Compound 3 was hydrolyzed in an alcohol solvent with an aqueous solution of an alkali metal hydroxide. After the reaction was completed, the mixture was acidified and purified to obtain the halogen-modified adamantane hapten. The general structural formula of compound 3 is: .

[0010] Preferably, the aprotic polar solvent in step (1) is N,N-dimethylformamide (DMF), the organic base is N,N-diisopropylethylamine (DIPEA), and the coupling agent is O-benzotriazole-tetramethylurea hexafluorophosphate (TBTU).

[0011] Preferably, the alcohol solvent in step (2) is ethanol, the alkali metal hydroxide is sodium hydroxide or potassium hydroxide, and the hydrolysis reaction temperature is 30~50℃.

[0012] The third objective of this invention is to provide a halogen-modified adamantane hapten, which is prepared by covalently coupling any of the above-described halogen-modified adamantane haptens with a carrier protein.

[0013] The halogen-modified adamantane artificial antigen is obtained by conjugating the halogen-modified adamantane hapten with a carrier protein. The halogen-modified adamantane artificial antigen can function as either an immunogen or a coating antigen.

[0014] Preferably, the carrier protein is selected from bovine serum albumin and / or ovalbumin.

[0015] Preferably, the molar ratio of the halogen-modified adamantane hapten to the carrier protein is 9.2:1 to 11.7:1.

[0016] The present invention also provides a method for preparing the halogen-modified adamantane hapten, wherein a carrier protein is coupled to the carboxyl carbon of the halogen-modified adamantane hapten using an activated ester method.

[0017] The fourth objective of this invention is to provide a specific antibody, which is obtained by immunizing animals with the halogen-modified amantadine artificial antigen described above, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

[0018] The fifth objective of this invention is to provide the application of any of the above-described halogen-modified adamantane haptens, any of the above-described halogen-modified adamantane artificial antigens, and / or the described specific antibodies in the preparation of immunoassay products for detecting adamantane.

[0019] The present invention also provides immunoassay products prepared from the specific antibodies, such as amantadine detection reagents or kits.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects: 1. This invention discloses for the first time a novel set of halogen-modified adamantane haptens, artificial antigens, and their preparation methods. The adamantane (a cyclic tetrahedral hydrocarbon) in adamantane is chemically modified structurally to introduce various halogen-modified haptens such as fluorine, chlorine, and bromine, resulting in formula (I)AMA halogen-modified haptens, breaking through the traditional "most similarity" design principle. The effects of halogen modification type and quantity on antibody titer and affinity were preliminarily explored in both polyclonal and monoclonal antibodies. Subsequent experimental results showed that, compared with the unmodified hapten, the formula (I)AMA halogen-modified hapten enhanced the titer of polyclonal antibodies against adamantane, increasing it by up to 3.4 to 14 times. When the hapten was modified with two or three fluorine atoms, the prepared monoclonal antibodies showed a 3 to 18-fold increase in titer against adamantane and a 10.8 to 16.1-fold increase in affinity, with the titer and affinity increasing with the number of modified fluorine atoms.

[0021] 2. The halogen-modified hapten provided by this invention significantly enhances antibody titer and affinity, offering a new approach and method for the rational design of small molecule compound haptens and the preparation of high-titer and high-affinity antibodies.

[0022] 3. Furthermore, the types and quantities of halogen-modified haptens provided by this invention can also serve as a reference for the design of other small molecule compound haptens. Attached Figure Description

[0023] Figure 1 This is a flowchart of the preparation of the AMA halogen-modified hapten shown in formula (I) of Example 1.

[0024] Figure 2 The NMR spectrum of the AMA halogen-modified hapten A1 (AMA-1) shown in formula (I) in Example 1 is shown. 1 HNMR).

[0025] Figure 3 The mass spectrometry identification diagram of the AMA halogen-modified hapten A1 (AMA-1) shown in formula (I) in Example 1 is shown.

[0026] Figure 4 The NMR spectrum of the AMA halogen-modified hapten A2 (AMA-1F) shown in formula (I) in Example 1 is shown. 1 HNMR).

[0027] Figure 5 The mass spectrometry identification diagram of the AMA halogen-modified hapten A2 (AMA-1F) shown in formula (I) in Example 1 is shown.

[0028] Figure 6 The NMR spectrum of the AMA halogen-modified hapten A3 (AMA-1Br) shown in formula (I) in Example 1 is shown below. 1 HNMR).

[0029] Figure 7 The mass spectrometry identification diagram of the AMA halogen-modified hapten A3 (AMA-1Br) shown in formula (I) in Example 1 is shown.

[0030] Figure 8 The NMR spectrum of the AMA halogen-modified hapten A4 (AMA-1Cl) shown in formula (I) in Example 1 is shown. 1 HNMR).

[0031] Figure 9 The mass spectrometry identification diagram of the AMA halogen-modified hapten A4 (AMA-1Cl) shown in formula (I) in Example 1 is shown.

[0032] Figure 10The NMR spectrum of the AMA halogen-modified hapten A5 (AMA-2F) shown in formula (I) in Example 1 is shown. 1 HNMR).

[0033] Figure 11 The mass spectrometry identification diagram of the AMA halogen-modified hapten A5 (AMA-2F) shown in formula (I) in Example 1 is shown.

[0034] Figure 12 The NMR spectrum of the AMA halogen-modified hapten A6 (AMA-3F) shown in formula (I) in Example 1 is shown. 1 HNMR).

[0035] Figure 13 The mass spectrometry identification chromatogram of AMA halogen-modified hapten A6 (AMA-3F) shown in formula (I) in Example 1 is shown.

[0036] Figure 14 The AMA and A1~A6 (A), Log P (B), charge distribution (C), and van der Waals surface ESP distribution (D) of AMA and A1~A6 are shown in Formula (I) of Example 1.

[0037] Figure 15 The image shown is a MALDI-TOF-MS image of the artificial antigen prepared from the AMA halogen-modified hapten shown in Formula (I) of Example 2.

[0038] Figure 16 The images shown are: (A) confocal image of RAW 264.7 after incubation with FITC-labeled halogen-modified hapten for 4 h, (B) histogram by flow cytometry, and (C) quantitative fluorescence intensity measured by flow cytometry in Example 3.

[0039] Figure 17 The effect of halogen-modified hapten on RAW 264.7 proliferation in Example 3.

[0040] Figure 18 This refers to the differentiation of CD19 cells from the spleen of mice immunized with the halogen-modified hapten in Example 3. + Flow cytometry (A) and histogram (B) of B cells.

[0041] Figure 19 The CD3+ differentiation of mouse spleen lymphocytes after immunization with the halogen-modified hapten in Example 3. + Flow cytometry (A) and histogram (B) of T cells.

[0042] Figure 20 This refers to the differentiation of CD4+ splenic lymphocytes from mice immunized with the halogen-modified hapten in Example 3. + and CD8 +Flow cytometry (A) and histograms (B and C) of T cells.

[0043] Figure 21 This study investigates the effect of halogen-modified hapten immunization on the proliferation of splenic lymphocytes in mice after immunization.

[0044] Figure 22 The diagram shows the clonal diversity (AC), CDR3 length distribution (DF), and CDR3 abundance (GI) of the BCR immune repertoire gene characteristics of the halogen-modified hapten in Example 3.

[0045] Figure 23 Pearson correlation coefficient heatmap of the CDR3 amino acid sequences of IGH(A), IGK(B), and IGL(C) in the BCR immune repertoire gene features of the halogen-modified hapten in Example 3.

[0046] Figure 24 This study analyzes the characteristic mutation frequencies of the BCR immune repertoire genes in response to the halogen-modified hapten in Example 3. The mutation frequencies are as follows: IGH (A and D), IGK (B and E), and IGL (C and F).

[0047] Figure 25 This is a graph showing the frequency of use of heavy chain germline genes IGHV (A), IGHD (B), and IGHJ (C) in the gene characteristics of the BCR immune repositories of halogenated haptens in Example 3.

[0048] Figure 26 This is a frequency diagram of the use of light chain germline genes IGKV (A), IGKJ (B), IGLV (C), and IGLJ (D) in the gene characteristics of the BCR immune repositories of halogenated haptens in Example 3.

[0049] Figure 27 This is a graph showing the frequency of VJ germline gene combinations used in the heavy chain of the BCR immune repertoire gene feature of the halogen-modified hapten in Example 3.

[0050] Figure 28 This is a frequency diagram of the VJ germline gene combination of light chain IGK (A) and IGL (B) in the gene characteristics of the BCR immune repertoire of the halogen-modified hapten in Example 3.

[0051] Figure 29 The titers of antibodies after primary (A), secondary (B), tertiary (C), and quaternary (D) immunizations with AMA halogen-modified artificial antigens as shown in Formula (I) in Example 5 are given.

[0052] Figure 30The typing of polyclonal antibodies after four immunizations with AMA halogen-modified artificial antigens A1-BSA (A), A2-BSA (B), A3-BSA (C), A4-BSA (D), A5-BSA (E) and A6-BSA (F) as shown in Formula (I) in Example 5.

[0053] Figure 31 The affinity of the antibody for 1 μg / mL AMA after primary immunization (A), secondary immunization (B), tertiary immunization (C), and quaternary immunization (D) of the AMA halogen-modified artificial antigen shown in Formula (I) in Example 5 is given.

[0054] Figure 32 The affinity of the antibody against 1 μg / mL of self-hapten after primary immunization (A), secondary immunization (B), tertiary immunization (C), and quaternary immunization (D) of the AMA halogen-modified artificial antigen shown in Formula (I) of Example 5 is given. Detailed Implementation

[0055] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the invention without departing from its spirit and essence are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0056] This invention discloses a halogen-modified adamantane hapten structure that enhances antibody titer and affinity, its preparation method, and its application. The general structural formula of the halogen-modified adamantane hapten is shown in formula (I): Formula (I); Wherein, R is an adamantyl alkyl group containing one or more halogen atoms, wherein the halogen atoms are selected from any one or more of fluorine, chlorine and bromine.

[0057] This invention chemically modifies the structure of amantadine (AMA), designing three typical halogen-modified amantadine haptens containing fluorine, chlorine, and bromine at the adamantane position, as shown in formula (I), and naming them AMA halogen-modified haptens. Here, R represents adamantane groups containing different types and quantities of halogen-modified groups. This invention utilizes quantum chemistry and molecular simulation techniques to characterize the above-mentioned halogen-modified haptens, clarifying the influence of halogen modification on the hapten's structure and properties. Through immunogen synthesis and animal immunization processes, antibodies prepared from halogen-modified haptens can effectively enhance titer and affinity, providing a new approach and method for the rational design of small molecule compound haptens and the preparation of high-titer and high-affinity antibodies.

[0058] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0059] Example 1: Preparation and characterization of halogen-modified adamantane hapten 1. Preparation of the AMA halogen-modified hapten shown in formula (I) Hapten A1: (1) Adamantane-1-carboxylic acid (compound 1, 500 mg, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: A1500576, CAS number: 828-51-3) was dissolved in 2.5 mL of thionyl chloride solution (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: T433841-100 ml, CAS number: 7719-09-7). The resulting solution was stirred at room temperature and reacted for 12 h. After desolvation under reduced pressure, the product was dissolved in tetrahydrofuran (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: T407518-100 ml, CAS number: 109-99-9). Methyl 6-aminohexanoate hydrochloride (compound 2, 555 mg, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: M138421, CAS number: 1926-80-3) was added to the above solution and stirred at room temperature for 6 h to obtain compound 3. (2) Compound 3 was desolvated under reduced pressure, resolvated with methanol, and then a sufficient amount of 2 mol / L NaOH solution was added. The mixture was then placed in an oil bath for 2 h. The reaction was detected by TLC. The pH of the system was adjusted to 7 using 2 mol / L HCl solution. The solvent was evaporated, and then 30 ml of pure water was added and stirred for 5 minutes. The mixture was then filtered to obtain compound (Ⅰ).

[0060] Hapten A2: (1) 3-fluoroadamantane-1-carboxylic acid (compound 1, 1.56 g, Aqicon Pharmaceutical Technology (Shanghai) Co., Ltd., catalog number: ABF14120-1 g, CAS number: 880-50-2), DIPEA (19.65 mmol, 2.54 g, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: D109321, CAS number: 7087-68-5) were dissolved in 20 ml of DMF (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: D742341, CAS number: 68-12-2). TBTU (11.80 mmol, 3.79 g, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: T109338, CAS number: 125700-67-6) was added to the solution. After stirring for 15 minutes, methyl 6-aminohexanoate hydrochloride (compound 2, 1.72 g) was added to the system. g, Shanghai Aladdin Biochemical Technology Co., Ltd., item number: M138421, CAS number: 1926-80-3), after addition, continue stirring for 3 hours, TLC (DCM / MeOH=60 / 1) shows that the reaction is over. Add 30 ml water and 50 ml ethyl acetate to the system, stir for 5 minutes, let stand for layering, extract the aqueous layer twice with 50 ml × 2 ethyl acetate, combine the organic phases, remove solvent, and purify the obtained oil by silica gel column chromatography (200~300 mesh, DCM / MeOH=100 / 1~60 / 1), and the reaction yields compound 3; (2) Dissolve 2.35 g of compound 3 in 20 ml ethanol, add 20 ml of 10% sodium hydroxide solution to the solution, after addition, heat the system to 40 ℃ and stir for 3 hours, TLC shows that the reaction is over. The reaction solution was cooled to room temperature and acidified to pH 3-4 with 2 mol / L NHCl. Then it was extracted with 50 ml × 3 ethyl acetate. The organic phases were combined, dissolved, and the concentrate was purified by silica gel column chromatography (200-300 mesh, DCM / MeOH = 30 / 1-10 / 1) to obtain compound (Ⅰ).

[0061] Hapten A3: (1) 3-bromoadamantane-1-carboxylic acid (compound 1, 1.00 g, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: W132082, CAS number: 21816-08-0), DIPEA (9.65 mmol, 1.25 g, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: D109321, CAS number: 7087-68-5) were dissolved in 15 ml DMF (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: D742341, CAS number: 68-12-2), and TBTU (5.79 mmol, 1.86 g, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: T109338, CAS number: 125700-67-6) was added to the system. After stirring at 60 °C for 60 minutes, methyl 6-aminohexanoate hydrochloride (compound 2, 0.84 g) was added to the system. g, Shanghai Aladdin Biochemical Technology Co., Ltd., item number: M138421, CAS number: 1926-80-3), after addition, continue stirring at room temperature for 3 hours. Add 50 ml of water to the system, stir for 5 minutes, extract with 50 ml × 3 ethyl acetate, combine the organic phases, remove solvent, and purify the residue by silica gel column chromatography (200~300 mesh, PE / EA=3 / 1-1 / 1), and the reaction yields compound 3; (2) Dissolve 0.71 g of compound 3 in 20 ml of ethanol, add 20 ml of 10% sodium hydroxide solution to the solution, after addition, heat the system to 40 ℃ and stir for 3 hours, and TLC shows that the reaction is over. Cool the reaction solution to room temperature, acidify with 2 mol / L NHCl to pH=3~4, then extract with 60 ml × 3 ethyl acetate, combine the organic phases, remove solvent, and purify the concentrate by silica gel column chromatography (200~300 mesh, DCM / MeOH=30 / 1~10 / 1) to obtain compound (Ⅰ).

[0062] Hapten A4: (1) 3-chloroadamantane-1-carboxylic acid (compound 1, 1.00 g, Shanghai Maclean Biochemical Technology Co., Ltd., catalog number: 786095, CAS number: 34859-74-0), DIPEA (11.65 mmol, 1.51 g, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: D109321, CAS number: 7087-68-5) were dissolved in 20 ml DMF (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: D742341, CAS number: 68-12-2), and TBTU (6.99 mmol, 2.24 g, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: T109338, CAS number: 125700-67-6) was added to the system. After stirring at 60 ℃ for 30 minutes, methyl 6-aminohexanoate hydrochloride (compound 2, 0.84 g) was added to the system. g, Shanghai Aladdin Biochemical Technology Co., Ltd., item number: M138421, CAS number: 1926-80-3), after addition, continue stirring at room temperature for 3 hours. Add 50 ml of water to the system, stir for 5 minutes, extract with 50 ml × 3 ethyl acetate, combine the organic phases, remove solvent, and purify the residue by silica gel column chromatography (200~300 mesh, PE / EA=3 / 1-1 / 1), and the reaction yields compound 3; (2) Dissolve 0.64 g of compound 3 in 20 ml of ethanol, add 20 ml of 10% sodium hydroxide solution to the solution, after addition, heat the system to 40 ℃ and stir for 3 hours, and TLC shows that the reaction is over. Cool the reaction solution to room temperature, acidify with 2 mol / L NHCl to pH=3~4, then extract with 60 ml × 3 ethyl acetate, combine the organic phases, remove solvent, and purify the concentrate by silica gel column chromatography (200~300 mesh, DCM / MeOH=30 / 1~10 / 1) to obtain compound (Ⅰ).

[0063] Hapten A5: (1) 4,4-Difluoro-1-adamantanecarboxylic acid (compound 1, 1.00 g, Aqicon Pharmaceutical Technology (Shanghai) Co., Ltd., catalog number: ABA70639-1 g, CAS number: 438017-43-7), DIPEA (11.55 mmol, 1.45 g, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: D109321, CAS number: 7087-68-5) were dissolved in 15 ml of DMF (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: D742341, CAS number: 68-12-2). TBTU (5.08 mmol, 1.63 g, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: T109338, CAS number: 125700-67-6) was added to the solution. After stirring for 15 minutes, methyl 6-aminohexanoate hydrochloride (compound 2, 1.01 g) was added to the system. g, Shanghai Aladdin Biochemical Technology Co., Ltd., item number: M138421, CAS number: 1926-80-3), after addition, continue stirring for 3 hours, TLC (DCM / MeOH=60 / 1) showed that the reaction was over. Add 30 ml water and 50 ml ethyl acetate to the system, stir for 5 minutes, let stand for layering, extract the aqueous layer twice with 50 ml × 2 ethyl acetate, combine the organic phases, remove solvent, and purify the obtained oil by silica gel column chromatography (200~300 mesh, DCM / MeOH=100 / 1~60 / 1), and the reaction yielded compound 3; (2) Dissolve 1.40 g of compound 3 in a mixed solvent of 15 ml ethanol and 15 ml water, add KOH solution (12.66 mmol, 0.71 g) to the solution, heat the system to 50 ℃ and stir for 3 hours, TLC showed that the reaction was over. The reaction solution was cooled to room temperature and acidified to pH 3-4 with 2 mol / L NHCl. Then it was extracted with 30 ml × 4 ethyl acetate. The organic phases were combined, dissolved, and the concentrate was purified by silica gel column chromatography (200-300 mesh, DCM / MeOH=100 / 1-30 / 1) to obtain compound (Ⅰ).

[0064] Hapten A6: (1) 3,5,7-trifluoroadamantane-1-carboxylic acid (compound 1, 0.50 g, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: T726435, CAS number: 214557-89-8), DIPEA (5.33 mmol, 0.69 g, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: D109321, CAS number: 7087-68-5) were dissolved in 20 ml DMF (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: D742341, CAS number: 68-12-2). TBTU (3.20 mmol, 1.03 g, Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number: T109338, CAS number: 125700-67-6) was added to the solution. After stirring for 15 minutes, methyl 6-aminohexanoate hydrochloride (compound 2, 0.46 g) was added to the system. g, Shanghai Aladdin Biochemical Technology Co., Ltd., item number: M138421, CAS number: 1926-80-3), after addition, continue stirring at room temperature for 3 hours. Add 50 ml of water to the system, stir for 5 minutes, extract with 50 ml × 3 ethyl acetate, combine the organic phases, remove solvent, and purify the residue by silica gel column chromatography (200~300 mesh, PE / EA=3 / 1-1 / 1), and the reaction yields compound 3; (2) Dissolve 0.30 g of compound 3 in 10 ml of ethanol, add 10 ml of 10% sodium hydroxide solution to the solution, after addition, heat the system to 40 ℃ and stir for 3 hours, and TLC shows that the reaction is over. Cool the reaction solution to room temperature, acidify with 2 mol / L NHCl to pH=3~4, then extract with 30 ml × 3 ethyl acetate, combine the organic phases, remove solvent, and purify the concentrate by silica gel column chromatography (200~300 mesh, DCM / MeOH=30 / 1~10 / 1) to obtain compound (Ⅰ).

[0065] For the specific synthesis route, please refer to... Figure 1 The synthesized AMA halogen-modified hapten was subjected to proton nuclear magnetic resonance spectroscopy (NMR spectroscopy). 1 H-NMR and mass spectrometry determination, structure as follows Figures 2-13 As shown.

[0066] 2. Characterization of the halogen-modified hapten shown in formula (I) Using AMA as the target, computational chemistry was employed to optimize the lowest energy conformation of the AMA halogen-modified hapten based on the traditional AMA hapten, obtaining various molecular parameters characterizing its structure and properties (the figure only shows the Log P values ​​of different algorithms). The rationality of the halogenated hapten design was evaluated, and molecular superposition was performed using Discovery Studio software. The charge distribution and van der Waals surface ESP distribution of the AMA halogen-modified hapten shown in equation (I) were calculated. Figure 14 ).

[0067] The results show that, based on the main characteristic structure of AMA, the halogen-modified hapten of formula (I) can completely overlap, proving that halogenation can have a minimal impact on the characteristic structure of the hapten; halogen modification can enhance the hydrophobicity of the hapten; the effect of AMA halogen-modified hapten on the electrical distribution is limited to the location where halogen is introduced.

[0068] Example 2: Preparation and characterization of halogen-modified adamantaneamine artificial antigen In this embodiment, the difference between the preparation methods of the immunogen and the coating agent lies in the type of carrier protein used. The immunogen carrier protein mainly uses BSA, while the coating agent carrier protein mainly uses OVA. The coupling method used is the active ester method.

[0069] I. Synthesis and Identification of Halogen-Modified Amantadine Immunogen 1. Preparation of halogen-modified adamantane immunogen (1) Dissolve 0.2 mmol of the compound (I) prepared in Example 1 in 1.0 mL of DMF, add 0.3 mmol NHS and 0.3 mmol EDC, and stir at room temperature in the dark for 8 h with a magnetic stirrer to obtain solution I.

[0070] (2) Add 23 mg BSA to 10 mL PBS buffer and dissolve it completely to obtain solution II.

[0071] (3) According to the feeding ratio of 1:100, solution I was slowly added dropwise to solution II, stirred in the dark for 8 h, and then put into a dialysis bag. Dialyzed in PBS at 4 ℃ for 48 h (with 6 water changes in between) to obtain halogen-modified adamantane immunogen solution. After aliquoting, it was stored at -20℃. The halogen-modified adamantane immunogen synthesized by the compound shown in formula (I) is abbreviated as AMA-BSA.

[0072] 2. Identification of halogen-modified amantadine immunogen The binding ratio of BSA to hapten in AMA-BSA solution was determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Results are shown below. Figure 15 .

[0073] The binding ratio of the artificial antigen of the compound shown in formula (I) is calculated using the following formula (Table 1): Binding ratio = {M(conjugate) - M(protein)} / M(hapten) II. Synthesis of Halogen-Modified Amantadine Coated Agents Using OVA instead of BSA, the preparation method and steps of the coating agent are the same as those for the immunogen. The compound shown in formula (I) is used to synthesize the halogen-modified adamantaneamine coating agent, abbreviated as AMA-OVA.

[0074] Table 1 shows the binding ratios of AMA halogen-modified adamantaneamine artificial antigens according to formula (I). Example 3: Study on the effect of halogen-modified amantadine immunogen on antibody response I. Study on the effects of halogen-modified amantadine immunogen on phagocytosis, proliferation and differentiation of immune cells 1. Study on the effects of halogen-modified amantadine immunogen on macrophage phagocytosis and proliferation (1) Macrophage (RAW 264.7) cells were stored at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per laser confocal microscopy dish. After cell adhesion, AMA-BSA (50 μg / mL) prepared in Example 2 (FITC-labeled) was added, and the cells were incubated for 4 h. After removing the cell supernatant and staining with Hoechst 33342, the slides were removed and the distribution of halogen-modified amantadine immunogen in macrophages was observed and evaluated under a laser scanning confocal microscope. To obtain quantitative statistical data on phagocytosis, flow cytometry was used to assess phagocytosis after 4 h of incubation with halogen-modified amantadine immunogen. Figure 16 ).

[0075] (2) RAW 264.7 cells were seeded at a density of 5000 cells per well in 96-well culture plates. After cell attachment, the supernatant was discarded. Then, 100 µL of culture medium containing different concentrations of AMA-BSA (12.5, 25, and 50 µg / mL) prepared in Example 2 was added to each well, with three replicates for each concentration. An LPS positive control, a BSA control, and a blank control (BC) were also included. After culturing at 37 ℃ and 5% CO2 for 24 h, 10 µL of CCK-8 reagent was added to each well. After color development, the OD value was measured at 450 nm using a microplate reader to evaluate the effect of halogen-modified amantadine immunogen on macrophage proliferation. Figure 17 ).

[0076] The results showed that, compared with the unmodified hapten, the halogen-modified amantadine immunogen significantly improved the phagocytic and proliferation efficiency of macrophages, and both phagocytosis and proliferation results showed that the phagocytic and proliferation efficiency of macrophages increased with the increase of the number of modified fluorine atoms.

[0077] 2. Identification test of lymphocyte proliferation and differentiation by halogen-modified amantadine immunogen. (1) Forty-eight 6-8 week old female BALB / c mice (SPF grade) were immunized with the AMA-BSA prepared in Example 2 and randomly divided into 6 groups of 10 mice each. The immunization program consisted of one basic immunization and several booster immunizations. For the first immunization, 100 μg of immunogen was mixed with an equal volume of Freund's complete adjuvant, emulsified, and injected subcutaneously at multiple points on the back of the neck of the mice, 200 μL per mouse for basic immunization. 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 first immunization, with a volume of 200 μL per mouse each time.

[0078] (2) Twenty-one days after the third immunization of mice, mice were euthanized by cervical dislocation. Three mice from each group were randomly selected, and their spleens were isolated under aseptic conditions to prepare single-cell suspensions of spleen lymphocytes. Cells were stained with FITC-labeled anti-mouse CD3, APC-labeled anti-mouse CD8a, PerCP / Cyanine 5.5-labeled anti-mouse CD4, and PE-labeled anti-mouse CD19 antibodies under light-protected conditions and incubated at room temperature for 30 min. After staining, the supernatant was discarded by centrifugation, and the cells were washed once with PBS, followed by flow cytometry analysis. Figures 18-20 ).

[0079] (3) Adjust the density of the prepared spleen lymphocyte single-cell suspension to 5.0 × 10⁻⁶. 6 Cells / mL. 100 μL of cell suspension was seeded into 96-well plates, and 10 μL of halogen-modified hapten-BSA (100 μg / mL), identical to the immunogen, was added. The hapten-BSA was diluted to six concentration gradients, with 50 μg / mL LPS as a positive control. Each concentration was used in quadruple wells to assess antigen-specific proliferation. After culturing the cells at 37 °C and 5% CO2 for 72 h, 10 μL of CCK-8 reagent was added to each well, and incubation continued until color development was complete. OD was then measured using a microplate reader. 450 nm value to assess lymphocyte proliferation level ( Figure 21 The lymphocyte proliferation index is calculated using the following formula: Proliferation index = (OD) s -OD b ) / (OD c -OD b ), where OD s The absorbance value of the experimental well is represented by OD. c The absorbance value of the control well is represented by OD. b This represents the absorbance value of the blank well.

[0080] The results showed that, except for the A4-BSA immunization group, the CD19 of other AMA halogen-modified immunogen groups... + The levels of B cells were slightly higher in the group with the unmodified immunogen than in the group with the same immunogen, but the difference was not statistically significant (P>0.05), suggesting that the AMA halogen-modified hapten may have enhanced B cell activation to some extent. Figure 18 A and B); CD3 in the AMA halogen-modified immunogen group + The levels of T cells were slightly higher in the group with the unmodified immunogen than in the group with the same immunogen, but the difference was not statistically significant (P>0.05), suggesting that the AMA halogen-modified hapten may have enhanced T cell activation to some extent. Figure 19 A and B); CD3 in the AMA halogen-modified immunogroup + CD8 + The proportion of T cells was significantly higher in the A6-BSA group than in the unmodified immune group (P<0.05), suggesting that polyhalogenated haptens may stimulate T cell activation, leading to a stronger cellular immune response. Figure 20 (A, B, and C).

[0081] Lymphocyte proliferation level results as follows Figure 21 As shown, compared with the unmodified hapten, the fluorinated immunogen significantly improved the proliferation efficiency of lymphocytes. The results showed that the phagocytic proliferation efficiency of lymphocytes increased with the increase of the number of modified fluorine atoms.

[0082] II. Study on the Influence of Halogen-Modified Amantadine Hapten on Gene Characteristics of BCR Immune Reservoir (1) Mice that underwent three immunizations were randomly selected. Twenty-one days after the third immunization, spleens were isolated under sterile conditions. Hapten-specific memory B cells were sorted by flow cytometry. Total RNA was extracted from lysed cell samples and cDNA was synthesized by reverse transcription using the 5'-RACE method. The variable region of the BCR was amplified by PCR. The PCR products were end-repaired using End Prep Enzyme, A-linker was added, and indexes were added. PCR amplification was performed again to construct the BCR library. The sequencing library was quantitatively analyzed using Qubit 3.0 and Agilent 2100.

[0083] (2) The library was sequenced at both ends using the Illumine HiSeq platform, the Bcl2fastq (v2.20.0.422) software was used for image base identification, the Cutadapt (version 1.9.1) software was used for raw data filtering, and the FLASH (v2.2.00) software was used for filtered data splicing.

[0084] (3) The spliced ​​and filtered BCR variable region sequences were compared with mouse germline gene sequences in the IMGT database using MiXCR to determine the usage frequency of the corresponding V, D, and J germline genes, and CDR and FR regions were annotated for the BCR. MiXCR (v3.0.13) software was used to perform a two-step alignment between the sequences and the database. After the first alignment, invalid sequences were removed based on the alignment results. After the second alignment, the BCR sequences were clustered based on sequence similarity to obtain information such as the abundance of each clone and the usage frequency of germline genes. Finally, statistical analysis, clonal diversity analysis, and inter-sample similarity analysis were performed. Figures 22-28 )wait.

[0085] The results showed that the number of BCR clonal types could characterize the diversity of the BCR immune repertoire. As shown in Figure (22A), compared with the unmodified hapten A1, except for the trifluoro hapten A6, the number of IGH clonal types in the specific BCR immune repertoire of other AMA halogen-modified haptens decreased, with the monochloro hapten A4 having the fewest IGH clonal types. As shown in Figure (22B), the number of clonal types of the IGK light chain of the halogen-modified hapten was lower than that of the unmodified hapten A1. As shown in Figure (22C), the number of clonal types of the IGL light chain of the fluoro hapten was higher than that of the unmodified hapten A1, while the number of clonal types of the brominated and chlorohydrated haptens A3 and A4 was lower than that of the unmodified hapten A1, with the trifluoro hapten A6 having the most IGL clonal types. The immune repertoire induced by the fluoro hapten showed greater diversity in both the number of heavy chain and light chain clonal types than that of the brominated and chlorohydrated haptens.

[0086] CDR3 is the most important region for binding antigens in the BCR variable region, and its diversity characteristics can characterize the diversity of the BCR immune repertoire. The curves for CDR3 length are basically consistent across groups, with the CDR3 length diversity of the light chain being significantly lower than that of the heavy chain. Figure 22 (D and 22E).

[0087] The rank-abundance curve of CDR3 can also characterize antibody library diversity. As shown in Figure (22G), CDRH3 high-frequency clones are more abundant in monochloroheptane A4, followed by trifluoroheptane A6 and difluoroheptane A5, with no significant differences among other samples. As shown in Figure (22H), CDRK3 high-frequency clones are more abundant in difluoroheptane A5, with no significant differences among other samples. As shown in Figure (22I), the rank-abundance curve of CDRL3 does not change significantly, indicating relatively consistent clonal evenness. The lack of significant differences in the rank-abundance curves of CDR3 among samples immunized with different haptens suggests that the clonal frequency evenness of CDR3 is relatively consistent.

[0088] Inter-sample similarity analysis compares the similarity of different samples in immune components. The method involves calculating the Pearson correlation coefficient based on the abundance of CDR3 amino acid sequences on different chains in different samples and plotting a heatmap. As shown in Figure (23A), the IGH chain correlation analysis results show that the Pearson correlation coefficient for each sample is below 0.3, indicating very low correlation between samples. As shown in Figure (23B), the IGK chain correlation analysis results show that the correlation between samples is much higher than that of the IGH chain. Unmodified hapten A1 has a high correlation with other samples, except for monochlorohapten A4 and difluorohapten A5. Fluorinated haptens show high similarity. As shown in Figure (23C), the IGL chain correlation analysis results show that the correlation between samples before and after immunization, as well as after immunization, is much higher than that of the IGH chain. Trifluorohapten A6 has a high correlation with other samples, and unmodified hapten A1 has high similarity with difluorohapten A5 and trifluorohapten A6. The heavy chains of the BCR immune repertoire of samples before and after immunization show large differences, while the light chains show small differences.

[0089] High mutation rates in BCR somatic cells are fundamental to enhanced BCR affinity. The BCR mutation level can reflect the level of immune response to some extent; the higher the mutation frequency, the higher the affinity of the immune repertoire's BCRs. As shown in Figures (24A and 24D), the base mutation level of IGHV for monochloro hapten A4 is significantly higher than other samples, while the base mutation level of monofluoro hapten A2 is lower, with no significant differences among other samples. The base mutation level of IGHJ for halogen-free hapten A1 is higher than that of halogenated hapten samples, while the base mutation level of IGHJ for monobromo hapten A3 is significantly lower than that of other samples. As shown in Figures (24B and 24E), the base mutation level of IGKV for monobromo hapten A3 is slightly higher than other samples, and the base mutation level of IGKJ for monochloro hapten A4 is slightly higher than other samples. With the increase in the number of fluorine atoms in the hapten structure, the base mutation levels of both IGKV and IGKJ increase. As shown in Figures (24C and 24F), the base mutation level of IGLJ in the trifluoro hapten A6 was slightly higher than that in other samples; the base mutation level of IGLJ in the monochloro hapten A4 was significantly higher than that in other samples; the base mutation levels of IGLJ and IGLV in the halogenated samples were higher than those in the unmodified hapten A1, and the base mutation levels of IGLJ and IGLV increased with the increase of the number of fluorine atoms in the hapten structure.

[0090] The frequency of heavy chain V(D)J germline genes is shown in Figure (25A). The top 20 IGHV germline genes in each sample account for approximately 70% of the total. The proportion of IGHV germline genes in the top 20 AMA halogenated hapten samples is higher than that in the unmodified hapten A1, indicating a decrease in IGHV germline gene diversity after immunization. This may suggest an increase in dominant clones after immunization. As shown in Figure (25B), the top 20 IGHD germline genes in each sample account for approximately 80% of the total. The frequency of IGHD germline genes is relatively consistent across samples, with the monochloro hapten A4 sample showing the highest proportion. There are no significant differences among other haptens. As shown in Figure (25C), compared to the unmodified hapten A1, the frequency of IGHJ1 in the AMA halogenated hapten samples is lower, while the frequency of IGHJ3 is higher.

[0091] The usage frequency of light chain V and J germline genes is shown in Figure (26A). The proportion of the top 20 IGKV germline genes in each sample was higher than 60%. Among them, the proportion of the top 20 IGKV germline genes of AMA halogenated haptens was higher than that of unmodified haptens A1, indicating that the diversity of IGKV germline genes decreased after immunization, which may indicate an increase in dominant clones after immunization. As shown in Figure (26B), the usage frequency of the four IGKJ germline genes was relatively consistent in each sample. As shown in Figures (26C and 26D), compared with unmodified hapten A1, the usage frequency of IGLV 1 and IGLJ 1 increased in AMA halogenated hapten samples, but there was no significant difference among other AMA halogenated hapten samples.

[0092] The overall distribution of the heavy chain VJ germline gene combination frequency heatmap, such as Figure 27 As shown, there are some differences in the frequency of VJ germline gene usage among the samples. However, there are also some similar characteristics among different samples. For example, the frequency of IGHV 4-1 and IGHJ 2 / 3 combinations is relatively high in all samples. In particular, the frequency of IGHV 4-2 and IGHJ 1 combinations in the difluoro hapten A5 sample is much higher than that of other VJ germline gene combinations in the unmodified hapten sample, and also higher than that of the same VJ germline gene combination in other samples.

[0093] The overall distribution of VJ germline gene combination frequency heatmaps, from the perspective of the IGK chain, is shown in Figure (28A). There is a relatively consistent VJ germline gene usage frequency characteristic across the samples. IGHV 6-15 and IGHV 8-30 show relatively high usage frequencies in the samples, while IGKJ 2 and IGKJ 4 germline genes also exhibit high usage frequencies. Furthermore, their frequency characteristics in combination with IGKV germline genes are relatively consistent across the samples and remain at a relatively high usage frequency level.

[0094] From the perspective of the IGL chain, as shown in Figure (28B), the frequency of VJ germline genes used in each sample is basically the same, with the IGLV1-IGLJ 1 combination having the highest frequency of use, followed by the IGLV3-IGLJ 2 combination.

[0095] In summary, the analysis of V(D)J germline gene usage frequency and VJ germline gene combination usage frequency showed that there were large differences in heavy chains and small differences in light chains among the BCR immune repertoires of the samples. After AMA halogen-modified hapten, the use preference of IGHV, IGLV and IGLJ germline genes increased, and the dominant germline gene clones were amplified.

[0096] Example 4 Preparation of halogen-modified amantadine antibody Mice subjected to three immunizations had blood collected from their orbital sinuses on day 7 after each immunization, starting with the first immunization. Serum was separated, and the titer of serum antibodies and the inhibition rate against amantadine 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.

[0097] Example 5: Preparation and determination of halogen-modified amantadine polyclonal antibody I. The antibody titer and subtype were detected using an indirect, non-competitive ELISA method. The specific operating steps are as follows: (1) Coating: The coating material was diluted with 0.05 M, pH 9.6 carbonate buffer to 10 μg / ml, 100 μL / well, and reacted at 37 ℃ for 2 h.

[0098] (2) Washing: Pour off the solution in the plate, spin dry, and wash once with washing solution, 280 μL / well each time.

[0099] (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.

[0100] (4) Sample addition: Dilute the serum to be tested with PBS and react at 37 ℃ for 30 min. After thorough washing, add 100 μL / well of HRP-goat anti-mouse IgG or HRP-labeled goat anti-mouse IgG of each antibody subtype diluted 1:5000 and react at 37 ℃ for 30 min.

[0101] (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.

[0102] (6) Termination and measurement: Add 100 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0103] The absorbance (OD) of each well was measured at the specified wavelength. 450 (nm). Serum titer is defined as the OD value when the concentration of the coating agent is fixed at 10 μg / ml. 450 The serum dilution factors corresponding to nm values ​​between 1.5 and 2.0. Under the same coating agent concentration, a higher serum dilution factor indicates a higher serum antibody titer. Results are shown in [Figure Number]. Figures 29-30 .

[0104] II. The indirect competitive ELISA method was used to detect antibody affinity. The specific operating steps are as follows: (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.

[0105] (2) Washing: Pour off the solution in the plate, spin dry, and wash once with washing solution, 280 μL / well each time.

[0106] (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.

[0107] (4) Prepare standard solution: Dilute the adamantane standard or self-hapten to 1 μg / mL for the detection of antibodies prepared from the AMA halogen-modified adamantane artificial antigen shown in formula (I); (5) Sample addition: Add 50 μL of diluted amantadine standard or auto-hapten to each well, then add 50 μL of antibody at the optimal dilution factor per well, and react at 37 ℃ for 30 min. After thorough washing, add 100 μL of HRP-goat anti-mouse IgG diluted 1:5000 per well, and react at 37 ℃ for 30 min.

[0108] (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.

[0109] (7) Termination and measurement: Add 100 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0110] (8) Data processing: Formula (I) AMA halogen-modified amantadine artificial antigen (Table 3, Figures 29-30 The affinity of the antibody is calculated according to the following formula: Inhibition rate (%) = B / B0 × 100%, where B and B0 are the OD values ​​with and without the standard, respectively (Table 2). Figure 31 and Figure 32 ).

[0111] The results showed that the titer of the polyclonal antibody was as follows: Figure 29 As shown, all six AMA halogen-modified amantadine immunogens exhibited high antibody titers (>1.5 W). After introducing halogen atoms into the hapten structure, the antibody titers were significantly increased (3.4-14 times) compared with the unmodified hapten. The monofluorohalogen A2-BSA induced the highest antibody titers (>23 W). The trifluorohalogen A6-BSA and monochlorohalogen A4-BSA could also induce relatively high antibody titers (>10 W).

[0112] Polyclonal antibody subtypes such as Figure 30 As shown, the heavy chains of the antibody subtypes induced by unmodified immunogen A1-BSA and various halogenated immunogens A2-BSA, A3-BSA, A4-BSA, A5-BSA and A6-BSA may be IgG1, IgG2a or IgG2b, and the light chains may be Lambda. The antibodies in the serum of immunized mice are polyclonal antibodies, and further subtype detection is needed in the antibodies.

[0113] Polyclonal antibody affinity: Compared with the unmodified hapten, the halogen-modified hapten of formula (I) AMA, with modifications of two and three fluorine atoms, can enhance the antibody affinity for amantadine. After three and four immunizations, the antibody affinity for amantadine is slightly enhanced compared with the unmodified group. Figure 31 Furthermore, compared with unmodified haptens, halogen-modified haptens of formula (I) AMA can increase the antibody affinity for the corresponding hapten by 0.81 to 6.34 times, and the inhibition rate of the self-hapten increases with the number of fluorine atoms in the hapten structure, while the affinity also increases. Figure 32 ).

[0114] Table 2. Affinity of polyclonal antibodies prepared from AMA halogen-modified adamantaneamine artificial antigen (Equation (I)). Note: a represents the inhibition rate of AMA (1 μg / mL) and the corresponding hapten (1 μg / mL) at a fixed antibody dilution. b represents the fold change in the average inhibition rate of the AMA halogen-modified group compared to the unmodified group.

[0115] Example 6: Preparation and determination of halogen-modified amantadine monoclonal antibody I. Cell Fusion and Cloning (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.

[0116] (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.

[0117] (3) The positive wells were cloned using the limiting dilution method to obtain hybridoma cell lines that can secrete amantadine monoclonal antibodies.

[0118] II. Cell cryopreservation and thawing 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.

[0119] III. Preparation of Monoclonal Antibodies 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 ℃.

[0120] IV. Identification of Monoclonal Antibodies The monoclonal antibody solution obtained in step three was used to detect antibody titer, subtype, and affinity using an indirect competitive ELISA method. The specific steps are as follows: (1) Coating: The coating material was serially diluted with 0.05 M, pH 9.6 carbonate buffer to 10 μg / ml, 100 μL / well, and reacted at 37 ℃ for 2 h.

[0121] (2) Washing: Pour off the solution in the plate, spin dry, and wash once with washing solution, 280 μL / well.

[0122] (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.

[0123] (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. Used for the determination of monoclonal antibodies prepared from the AMA halogen-modified adamantane artificial antigen as shown in formula (I).

[0124] (5) Sample addition: Add 50 μL of diluted standard of each concentration to each well, and then add 50 μL of antibody of 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 or HRP-labeled goat anti-mouse IgG of each antibody subtype diluted 1:5000 to each well. Incubate at 37 °C for 30 min.

[0125] (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.

[0126] (7) Termination and measurement: Add 100 μL of stop solution to each well to terminate the reaction, and then measure the OD of each well using a microplate reader. 450 value.

[0127] (8) Data processing: The product of the original dilution factor and the antibody dilution factor is taken as the antibody titer; the standard concentration is used as the log value on the x-axis and the OD value on the y-axis. The standard curve is obtained by fitting the standard curve using the four-parameter equation of Origin 8.5 to obtain the IC50. 50 The values ​​are shown in Table 3.

[0128] The results showed that, compared with the unmodified hapten, the halogen-modified hapten of formula (I) AMA, when modified with halogen, resulted in monoclonal antibodies with 1 to 18-fold increased titer and 1.2 to 16.1-fold increased affinity for adamantane. Specifically, the monoclonal antibodies modified with two and three fluorine atoms showed significantly increased affinity for adamantane compared with the unmodified hapten, ranging from 10.8 to 16.1-fold, and the titer and affinity for adamantane increased with increasing number of fluorine atoms. The heavy chain isotype of monoclonal antibodies A1-5G4, A3-7B4, A4-10G4, and A5-7B10 was lgG1; the heavy chain isotype of A2-10G2 was lgG3; and the heavy chain isotype of A6-10G2 was lgG2b. The light chain isotype of all monoclonal antibodies was Lambda.

[0129] Table 3. Titer, Isotype, and Affinity IC50 of Monoclonal Antibodies Prepared from AMA Halogen-Modified Amantadine Artificial Antigens (I) 50 (ng / mL) Note: a represents the fold difference in titer of the monoclonal antibody prepared in the AMA halogen-modified group compared to the unmodified group; b represents the IC50 value of the monoclonal antibody prepared in the AMA halogen-modified group compared to the unmodified group. 50 The difference multiple.

[0130] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A halogen-modified adamantane hapten, characterized in that, Its general structural formula is shown in formula (I): Equation (I); Wherein, R is an adamantyl alkyl group containing one or more halogen atoms, wherein the halogen atoms are selected from any one or more of fluorine, chlorine and bromine.

2. The halogen-modified adamantane hapten according to claim 1, characterized in that, The R group is selected from any one of the following groups: 。 3. A method for preparing a halogen-modified adamantane hapten as described in any one of claims 1-2, characterized in that, Includes the following steps: (1) Halogen-modified adamantane carboxylic acid with the structural formula R-COOH was dissolved in an aprotic polar solvent with an organic base, activated by adding a coupling reagent, and then reacted with methyl 6-aminohexanoate hydrochloride to obtain intermediate compound 3. (2) Compound 3 was hydrolyzed in an alcohol solvent with an aqueous solution of an alkali metal hydroxide. After the reaction was completed, the mixture was acidified and purified to obtain the halogen-modified adamantane hapten. The general structural formula of compound 3 is: 。 4. The preparation method according to claim 3, characterized in that, The aprotic polar solvent in step (1) is N,N-dimethylformamide (DMF), the organic base is N,N-diisopropylethylamine (DIPEA), and the coupling agent is O-benzotriazole-tetramethylurea hexafluorophosphate (TBTU).

5. The preparation method according to claim 3, characterized in that, The alcohol solvent in step (2) is ethanol, the alkali metal hydroxide is sodium hydroxide or potassium hydroxide, and the hydrolysis reaction temperature is 30~50℃.

6. A halogen-modified amantadine artificial antigen, characterized in that, It is prepared by covalent coupling of the halogen-modified adamantane hapten as described in any one of claims 1 to 2 with a carrier protein.

7. The halogen-modified adamantane artificial antigen according to claim 6, characterized in that, The carrier protein is selected from bovine serum albumin and / or ovalbumin.

8. The halogen-modified adamantane artificial antigen according to any one of claims 6-7, characterized in that, The molar ratio of the halogen-modified adamantane hapten to the carrier protein is from 9.2:1 to 11.7:

1.

9. A specific antibody, characterized in that, It is obtained by immunizing animals with the halogen-modified adamantane artificial antigen as described in any one of claims 6 to 8, wherein the antibody is a polyclonal antibody or a monoclonal antibody.

10. The use of the halogen-modified adamantane hapten of any one of claims 1 to 2, the halogen-modified adamantane artificial antigen of any one of claims 6 to 8, and / or the specific antibody of claim 9 in the preparation of an immunoassay product for detecting adamantane.

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