p-nitroaniline hydrophobic haptens, methods of synthesis and use thereof

Abstract

The present application relates to the technical field of biological chemical industry, especially to a kind of p-nitrophenylamine hydrophobic hapten and its preparation method and application.The present application is based on p-nitrophenylamine, by introducing polar atom (F, N or O) from spacer arm to adjust the hydrophobicity of target material.Then, hapten is coupled with carrier protein, and the immunogenicity of artificial antigen is evaluated.The present application explores the influence of the change of hapten hydrophobicity on immunogenicity, which provides theoretical support for rational design of hapten on one hand, and provides material basis for preparation of high-affinity antibody of p-nitrophenylamine on the other hand.The present application provides a new idea for rational design of small molecule compound hapten and preparation of high-affinity antibody.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, and in particular to a class of p-nitroaniline hydrophobic haptens, their synthesis methods, and applications. Background Technology

[0002] p-Nitroaniline, a member of the aniline family of compounds, is widely used in the production of pesticides, veterinary drugs, rubber, dyes, and pharmaceuticals. The excessive use and emission of this compound have caused significant environmental pollution, particularly to water bodies. p-Nitroaniline is highly chemically toxic and can cause harm through inhalation, ingestion, and skin contact, inducing diseases such as hypoxia, jaundice, and anemia. As a chemical with a high incidence of sudden environmental incidents and a characteristic pollutant, p-nitroaniline is a key focus of environmental risk prevention and control.

[0003] Antibodies are the core reagents in immunoassay methods. One of the key steps in preparing small molecule antibodies is the rational design and synthesis of haptens. The properties of haptens have a significant impact on their immunogenicity, directly determining the specificity and potency of the resulting antibodies. In traditional hapten design, hydrophobicity is usually an important consideration because it can significantly affect the stability and affinity of antigen-antibody interactions. In the fields of protein and nanovaccines, studies have shown that enhancing the hydrophobicity of immunogens can induce stronger immune responses; however, in the field of small molecule antibody preparation, the impact of hapten hydrophobicity on immune responses remains unknown. Because p-nitroaniline is a small molecule compound with a relatively low molecular weight, its monoclonal antibody preparation faces problems such as poor specificity and sensitivity, and difficulty in screening.

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

[0005] To address the aforementioned technical problems, this invention provides a class of p-nitroaniline hydrophobic haptens, their synthesis method, and applications.

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

[0007] In a first aspect, the present invention provides a class of p-nitroaniline haptens, said p-nitroaniline haptens containing a hydrophobic spacer arm, and having a structural formula of any one of the following:

[0008] I;

[0009] II;

[0010] III;

[0011] IV.

[0012] Based on the target structure of p-nitroaniline, this invention modulates the hydrophobicity of the target by introducing polar atoms (F, N or O) from the spacer arm, and finally screens out the above four hapten structures that have better performance in hydrophobicity and enhanced immune response.

[0013] In a second aspect, the present invention provides a p-nitroaniline artificial antigen, comprising the p-nitroaniline hapten and a carrier protein coupled to the p-nitroaniline hapten.

[0014] Preferably, the p-nitroaniline hapten is coupled to the carrier protein via an amide bond.

[0015] Preferably, the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole hemocyanin, thyroprotein, and human serum albumin.

[0016] Preferably, the carrier protein is coupled to the carboxyl group of the p-nitroaniline hapten using the active ester method.

[0017] Preferably, the molar ratio of the p-nitroaniline hapten to the carrier protein is 4.75 to 13.38:1.

[0018] This invention couples haptens to carrier proteins to evaluate the immunogenicity of artificial antigens. On one hand, it explores the effect of increased hydrophobicity of haptens on immunogenicity, providing theoretical support for the rational design of haptens. On the other hand, it provides a material basis for the preparation of high-affinity antibodies against p-nitroaniline.

[0019] Thirdly, the present invention provides any of the following applications of the p-nitroaniline hapten or the p-nitroaniline artificial antigen:

[0020] Used to evaluate the effect of hapten hydrophobicity on immunogenicity;

[0021] Applications in the preparation of p-nitroaniline-specific antibodies;

[0022] Application in the detection of specific antibodies against p-nitroaniline;

[0023] The application is for non-disease diagnosis purposes.

[0024] The four haptens provided by this invention are specially prepared to address the impact of hapten hydrophobicity on the preparation of monoclonal antibodies in immunoassay techniques, providing a new approach and method for the rational design of small molecule compound haptens and the preparation of high-affinity antibodies.

[0025] Fourthly, the present invention provides specific antibodies prepared from the artificial antigen, wherein the specific antibodies are polyclonal antibodies and / or monoclonal antibodies.

[0026] The specific antibody provided by this invention has a relatively high affinity.

[0027] Fifthly, the present invention provides a reagent or kit for detecting p-nitroaniline drugs prepared from the specific antibody.

[0028] Sixthly, the present invention provides any of the following applications of the specific antibody:

[0029] Application in a fluorescence polarization detection kit for detecting p-nitroaniline drugs;

[0030] Application in the preparation of enzyme-linked immunosorbent assay kits for p-nitroaniline drugs;

[0031] Application in the preparation of lateral flow chromatography test strips for p-nitroaniline drugs;

[0032] The application is for non-disease diagnosis purposes.

[0033] Beneficial effects:

[0034] This invention provides a class of p-nitroaniline hydrophobic haptens, their synthesis methods, and applications. Specifically, based on p-nitroaniline, the hydrophobicity of the target is adjusted by introducing polar atoms (F, N, or O) from the spacer arm. Subsequently, the hapten is coupled to a carrier protein, and the immunogenicity of the artificial antigen is evaluated. This invention explores the impact of changes in hapten hydrophobicity on immunogenicity, providing theoretical support for the rational design of haptens and a material basis for the preparation of high-affinity antibodies against p-nitroaniline. This invention offers a new approach for the rational design of small molecule compound haptens and the preparation of high-affinity antibodies. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0036] Figure 1 The above is the 1H NMR spectrum of the p-nitroaniline hapten shown in formula (Ⅰ) in an embodiment of the present invention.

[0037] Figure 2 The above is the 1H NMR spectrum of the p-nitroaniline hapten shown in formula (II) in this embodiment of the invention.

[0038] Figure 3 The above is the 1H NMR spectrum of the p-nitroaniline hapten shown in formula (III) in this embodiment of the invention.

[0039] Figure 4 The above is the 1H NMR spectrum of the p-nitroaniline hapten shown in formula (Ⅳ) in this embodiment of the invention.

[0040] Figure 5 The diagram shows the surface electrostatic potential distribution of the p-nitroaniline hapten as shown in formulas (I) to (IV) in the embodiments of the present invention.

[0041] Figure 6 The images shown are MALDI-TOF-MS images of the artificial antigens prepared by formulas (I) to (IV) in the embodiments of the present invention.

[0042] Figure 7 The antibody titer is the antibody titer after three immunizations with the artificial antigen of the p-nitroaniline hapten shown in formulas (I) to (IV) in the embodiments of the present invention.

[0043] Figure 8 The inhibition rates of the antibodies against (A) p-nitroaniline (10 μg / mL) and (B) the corresponding hapten (50 ng / mL) after triple immunization with the artificial antigens of the p-nitroaniline hapten shown in formulas (I) to (IV) in the embodiments of the present invention are shown. Detailed Implementation

[0044] Hydrophobicity is one of the most important physicochemical properties of molecules. In the fields of proteins and nanovaccines, studies have shown that enhancing the hydrophobicity of immunogens can induce stronger immune responses. However, in the field of small molecule antibody preparation, the effect of hapten hydrophobicity on immune responses remains unknown. The purpose of this invention is to address the shortcomings of existing detection technologies by providing a method for preparing p-nitroaniline haptens with different hydrophobicities and their applications. This hapten can be conjugated with bovine serum albumin or oval serum albumin to obtain artificial antigens. High-affinity, highly specific monoclonal antibodies can be obtained by immunizing animals with these artificial antigens, thereby establishing an immunoassay-based method for detecting p-nitroaniline residues.

[0045] This invention primarily modulates the hydrophobicity of the spacer arms by introducing polar atoms (F, N, and O) into them. The introduction of F atoms increases the molecule's hydrophobicity, while the introduction of N and O atoms may increase the hydrophobicity of the hapten. This invention utilizes the changes in molecular properties induced by polar atoms to regulate the stability and distribution of the hapten in vivo, thereby influencing the immune system's recognition of it. Through this strategy, this invention not only reveals the differences in immunogenicity of haptens with different levels of hydrophobicity but also provides new insights for the rational design of haptens.

[0046] This invention systematically evaluated the effects of haptens with different hydrophobicities on immunogenicity, and finally prepared a highly specific and high-affinity p-nitroaniline monoclonal antibody, and established an immunoassay method for detecting chemical risk factors associated with p-nitroaniline.

[0047] In a more specific and preferred embodiment, the present invention provides the following solution:

[0048] First, this invention provides a class of p-nitroaniline hydrophobic haptens, the structural formula of which is as follows:

[0049] I;

[0050] II;

[0051] III;

[0052] IV.

[0053] Furthermore, the present invention also provides methods for preparing the above-mentioned four different hydrophobic p-nitroaniline haptens.

[0054] The method for preparing the hapten represented by formula (Ⅰ) provided by the present invention includes the following steps:

[0055] p-Nitroaniline (13.8 g, 0.1 mol) and triethylamine (Et3N, 12.1 g, 0.1 mol) were dissolved in THF under ice bath conditions to form solution A. Then, hexanoyl chloride (16.2 g, 0.1 mol) was dissolved in THF and slowly added dropwise to solution A, with stirring at room temperature for 2 h. The resulting solution was then poured into water for post-treatment. After filtration and drying, 2.5 g of a white solid was obtained.

[0056] The method for preparing the hapten represented by formula (II) provided by the present invention includes the following steps:

[0057] 2.0 g of 3,6-dioxobenzoic acid (11.23 mmol) was dissolved in 40 mL of dry DCM, and 2.85 g of oxaloyl chloride (22.46 mmol) was added. After the addition was complete, 5 drops of anhydrous DMF were added dropwise to the system, and the resulting solution was stirred at room temperature for 1 h. After 1 h, 60 mL of anhydrous DCM solution containing 1.40 g of p-nitroaniline (10.11 mmol) was slowly added dropwise to the system, and the mixture was stirred at room temperature for another 0.5 h. The reaction was then stopped by TLC. The pH of the system was adjusted to 3-4 with 2 N HCl, and then 50 mL of saturated saline solution was added and stirred for 5 min. The mixture was allowed to stand and separate into layers. The aqueous phase was extracted with 50 mL × 2 ethyl acetate. The organic phases were combined, dissolved under reduced pressure, and the residue was purified by silica gel column chromatography (200-300 mesh, DCM / MeOH = 15 / 1-10 / 1) to obtain 0.82 g of a pale yellow solid, which was the target product.

[0058] The present invention provides a method for preparing the hapten represented by formula (III) comprising the following steps:

[0059] Step 1: Dissolve 5.0 g (66.57 mmol) methylethanolamine in 50 mL DCM. Add 8.0 g (79.21 mmol) Et3N and 15.9 g (72.85 mmol) (Boc)2O under ice bath conditions. After stirring for 15 min, move the mixture to room temperature and stir overnight. Monitor the reaction of the starting material by TLC until complete. Wash with 1 M hydrochloric acid solution and saturated NaHCO3 sequentially. Collect the organic phase, dry it with anhydrous Na2SO4, and concentrate it under reduced pressure to remove the solvent, yielding 11.3 g of a pale yellow oil. Yield: 96.9%. 6 g (34.24 mmol) of compound 1 was dissolved in 60 mL of MTBE, followed by the addition of 60 mL of 50% NaOH. After stirring for 30 min, 12.6 g (64.60 mmol) of tert-butyl bromoacetate was added. The mixture was stirred at room temperature, and after 3 h, the reaction was monitored by TLC until the starting material was completely reacted. The mixture was diluted with an appropriate amount of MTBE, and the system was poured into a separatory funnel. The MTBE phase was collected, and the aqueous phase was extracted once with MTBE. The organic phases were combined and washed with 1 M dilute hydrochloric acid. The organic phase was collected, dried over anhydrous Na2SO4, and concentrated under reduced pressure to remove the solvent, yielding 10.3 g of a pale yellow oil, which was compound 2.

[0060] Step 2: Dissolve 10.3 g (35.59 mmol) of compound 2 in 100 mL of DCM, add 53 mL (713.56 mmol) of TFA, stir at room temperature, and after 1 h, monitor the reaction of the starting material by TLC until it is complete. Concentrate under reduced pressure to remove the solvent and residual TFA. Dissolve the crude product in 50 mL of DCM, add 21.4 g (211.88 mmol) of Et3N and 8.5 g (38.95 mmol) of (Boc)2O, stir at room temperature, and after 3 h, monitor the reaction of the starting material by TLC until it is complete. Wash with 1 M HCl solution, collect the organic phase, dry it with anhydrous Na2SO4, concentrate under reduced pressure to remove the solvent, and obtain 5.7 g of a pale yellow oil, which is compound 4.

[0061] Step 3: Dissolve 5.7 g (24.44 mmol) of compound 4 in 50 mL of DCM, add 3.71 g (26.86 mmol) of 4-nitroaniline, 9.36 g (48.83 mmol) of EDCI·HCl and 6.0 g (49.11 mmol) of DMAP, stir overnight at room temperature, monitor the reaction of the starting material by TLC until the reaction is complete, wash twice with 1 M HCl solution, collect the organic phase, dry it with anhydrous Na2SO4 and concentrate it under reduced pressure to remove the solvent, purify it by silica gel column chromatography, and obtain 5.3 g of pale yellow oil, which is compound 5.

[0062] Step 4: Dissolve 5.0 g (14.15 mmol) of compound 5 in 60 mL of DCM, add 16.1 g (141.20 mmol) of TFA, stir at room temperature, and after 2 h, monitor the reaction of the starting material by TLC until it is complete. Concentrate under reduced pressure to remove the solvent and residual TFA. Dissolve the crude product (about 3.6 g, 14.22 mmol) in 40 mL of DMF, add 5.86 g (42.46 mmol) of K2CO3, stir for 10 min, then add 3.33 g (17.07 mmol) of tert-butyl bromoacetate, stir at room temperature overnight, monitor the reaction of the starting material by TLC until it is complete, remove the solvent by rotary evaporation, dilute with DCM, wash with water, collect the organic phase, dry with anhydrous Na2SO4, concentrate under reduced pressure to remove the solvent, and purify by silica gel column chromatography to obtain 3.2 g of a pale yellow oil, which is compound 7.

[0063] Step 5: Dissolve 2.7 g (7.35 mmol) of compound 7 in 15 mL of acetone, add 1.57 g (11.06 mmol) of iodomethane, stir at room temperature, and a white solid immediately precipitates. After stirring for 2 h, the reaction of the starting material is monitored by TLC until it is complete. Filter under reduced pressure, wash the white solid with acetone, collect the white solid, and dry it to obtain 3.2 g of compound 8.

[0064] Step 6: Dissolve 3.0 g (5.89 mmol) of compound 8 in 15 mL of DCM, add 15 mL of TFA, stir the reaction at RT, and after 2 h, monitor the reaction of the starting material by TLC until it is complete. Concentrate under reduced pressure to remove the solvent and residual TFA, then add 30 mL of DCM to dissolve it, add a small amount of solid NaHCO3 until no bubbles are generated, remove the solvent by rotary evaporation, and purify by silica gel column chromatography (DCM:MeOH=2:1) ​​to obtain 1.9 g of white solid, which is the target product.

[0065] The method for preparing the hapten represented by formula (Ⅳ) provided by the present invention includes the following steps:

[0066] 5.30 g (13.58 mmol) of perfluorooctanoic acid was dissolved in 50 mL of ultra-dry dichloromethane under nitrogen protection. 3.79 g (29.90 mmol) of oxaloyl chloride and 5 drops of ultra-dry DMF (producing numerous bubbles) were added to the solution, and the mixture was stirred at room temperature for 1 h. After 1 h, the system was cooled in an ice-water bath, and 13.5 mL (19.56 mmol) of N,N-diisopropylethylamine was added, followed by the fractional addition of 1.88 g (13.59 mmol) of p-nitroaniline. Stirring continued for 1 h after the addition was complete. TLC showed the reaction was complete. The pH of the system was adjusted to 2–3 with 2 NHCl, and the mixture was allowed to separate into layers. The aqueous layer was extracted twice with 50 mL × 2 dichloromethane. The organic layers were combined, dissolved, and the resulting oil was purified by silica gel column chromatography (200–300 M, DCM / MeOH = 10 / 1) to give 0.71 g of a brownish-yellow solid, which was the target product.

[0067] Furthermore, the present invention provides four artificial p-nitroaniline antigens obtained based on the above-mentioned haptens, which are specifically obtained by coupling p-nitroaniline hydrophobic haptens of formulas (I) to (IV) with a carrier protein.

[0068] The carrier protein is selected from bovine serum albumin, ovalbumin, keyhole hemocyanin, thyroprotein, and human serum albumin, etc.; bovine serum albumin and ovalbumin are preferred.

[0069] Furthermore, the present invention provides a method for preparing the artificial antigen, which can use an activated ester method to couple a carrier protein to the carboxyl carbon of the hapten of formula (I) to (IV).

[0070] Preferably, the molar ratio of the compounds shown in formulas (I) to (IV) to bovine serum albumin is 4.75 to 13.38:1.

[0071] Furthermore, this aspect provides for any of the following applications of the p-nitroaniline hapten or the p-nitroaniline artificial antigen:

[0072] Used to evaluate the effect of hapten hydrophobicity on immunogenicity.

[0073] Used to prepare specific antibodies against p-nitroaniline.

[0074] Used to detect specific antibodies against p-nitroaniline.

[0075] Furthermore, the present invention provides specific antibodies prepared from the aforementioned p-nitroaniline artificial antigen, including polyclonal antibodies and monoclonal antibodies, preferably monoclonal antibodies. The polyclonal antibodies are obtained by immunizing experimental animals (such as New Zealand white rabbits) with the p-nitroaniline artificial antigen and then purifying the collected serum. The monoclonal antibodies can be prepared using hybridoma technology.

[0076] Furthermore, the present invention provides a p-nitroaniline detection reagent or kit prepared from the specific antibody.

[0077] Furthermore, the present invention provides any of the following applications of the specific antibody:

[0078] Application in a fluorescence polarization detection kit for detecting p-nitroaniline.

[0079] Application in the preparation of flow-testing immunochromatographic strips for p-nitroaniline.

[0080] Application in the preparation of an enzyme-linked immunosorbent assay kit for p-nitroaniline.

[0081] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0082] This invention discloses for the first time four different hydrophobic p-nitroaniline haptens and artificial antigens, along with their preparation methods. By evaluating the impact of each hapten's hydrophobicity on immunogenicity, the mechanism by which hapten hydrophobicity affects the immune response is revealed, providing a theoretical basis for the rational design of haptens. By appropriately adjusting the hydrophobicity of the haptens, the immunogenicity of the artificial antigen is enhanced, thereby obtaining specific antibodies with high titers and high sensitivity. The p-nitroaniline haptens and the antibodies prepared in this study provide a new means for establishing a rapid, simple, inexpensive, sensitive, and specific method for the detection of p-nitroaniline.

[0083] In this study, different hydrophobic p-nitroaniline haptens were synthesized, and their calculated molecular polarity (MPI) ranged from 11.85 to 22.24. Formula (I) of the p-nitroaniline hapten, which exhibited strong hydrophobicity (MPI of 16.51), successfully induced the highest hapten-specific antibody titer after triple immunization, which was 2.3 to 320 times higher than other groups. It effectively induced antiserum and monoclonal antibodies with high affinity for p-nitroaniline, among which the monoclonal antibody showed an affinity 3.35 to 505.88 times higher than other haptens.

[0084] The artificial antigens provided by this invention are used to prepare p-nitroaniline antibodies (polyclonal and monoclonal antibodies). The preparation process is simple and economical, and the detection sensitivity of the antibodies against p-nitroaniline can reach 30.74 ng / mL. By improving the hydrophobicity of the p-nitroaniline hapten, the affinity of the nitroaniline antibody is enhanced, providing a new idea and method for the rational design of small molecule compound haptens and the preparation of high-affinity antibodies.

[0085] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0086] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0087] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0088] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0089] Example 1

[0090] (I) This embodiment provides different hydrophobic p-nitroaniline haptens and their preparation methods.

[0091] (1) Preparation of p-nitroaniline hapten as shown in formula (I)

[0092] p-Nitroaniline (13.8 g, 0.1 mol) and triethylamine (Et3N, 12.1 g, 0.1 mol) were dissolved in THF under ice bath conditions to form solution A. Then, hexanoyl chloride (16.2 g, 0.1 mol) was dissolved in THF and slowly added dropwise to solution A, with stirring at room temperature for 2 h. The resulting solution was then poured into water for post-treatment. After filtration and drying, 2.5 g of a white solid was obtained.

[0093] The synthesis process steps are as follows:

[0094] .

[0095] (2) Preparation of p-nitroaniline hapten as shown in formula (II)

[0096] 2.0 g of 3,6-dioxobenzoic acid (11.23 mmol) was dissolved in 40 mL of dry DCM, and 2.85 g of oxaloyl chloride (22.46 mmol) was added. After the addition was complete, 5 drops of anhydrous DMF were added dropwise to the system, and the resulting solution was stirred at room temperature for 1 h. After 1 h, 60 mL of anhydrous DCM solution containing 1.40 g of p-nitroaniline (10.11 mmol) was slowly added dropwise to the system, and the mixture was stirred at room temperature for another 0.5 h. The reaction was then stopped by TLC. The pH of the system was adjusted to 3-4 with 2 N HCl, and then 50 mL of saturated saline solution was added and stirred for 5 min. The mixture was allowed to stand and separate into layers. The aqueous phase was extracted with 50 mL × 2 ethyl acetate. The organic phases were combined, dissolved under reduced pressure, and the residue was purified by silica gel column chromatography (200-300 mesh, DCM / MeOH = 15 / 1-10 / 1) to obtain 0.82 g of a pale yellow solid, which was the target product.

[0097] The synthesis process steps are as follows:

[0098] .

[0099] (3) Preparation of p-nitroaniline hapten as shown in formula (III)

[0100] Step 1: Dissolve 5.0 g (66.57 mmol) methylethanolamine in 50 mL DCM. Add 8.0 g (79.21 mmol) Et3N and 15.9 g (72.85 mmol) (Boc)2O under ice bath conditions. After stirring for 15 min, move the mixture to room temperature and stir overnight. Monitor the reaction of the starting material by TLC until complete. Wash with 1 M hydrochloric acid solution and saturated NaHCO3 sequentially. Collect the organic phase, dry it with anhydrous Na2SO4, and concentrate it under reduced pressure to remove the solvent, yielding 11.3 g of a pale yellow oil. Yield: 96.9%. 6 g (34.24 mmol) of compound 1 was dissolved in 60 mL of MTBE, followed by the addition of 60 mL of 50% NaOH. After stirring for 30 min, 12.6 g (64.60 mmol) of tert-butyl bromoacetate was added. The mixture was stirred at room temperature, and after 3 h, the reaction was monitored by TLC until the starting material was completely reacted. An appropriate amount of MTBE was added to dilute the mixture, and the system was poured into a separatory funnel. The MTBE phase was collected, and the aqueous phase was extracted once with MTBE. The organic phases were combined and washed with 1 M dilute hydrochloric acid. The organic phase was collected, dried over anhydrous Na2SO4, and concentrated under reduced pressure to remove the solvent, yielding 10.3 g of a pale yellow oil, which was the target product.

[0101] Step 2: 10.3 g (35.59 mmol) of compound 2 was dissolved in 100 mL of DCM, and 53 mL (713.56 mmol) of TFA was added. The mixture was stirred at room temperature. After 1 h, the reaction was monitored by TLC until the starting material was completely reacted. The mixture was then concentrated under reduced pressure to remove the solvent and residual TFA. The crude product was dissolved in 50 mL of DCM, and 21.4 g (211.88 mmol) of Et3N and 8.5 g (38.95 mmol) of (Boc)2O were added. The mixture was stirred at room temperature. After 3 h, the reaction was monitored by TLC until the starting material was completely reacted. The mixture was washed with 1 M HCl solution, and the organic phase was collected. After drying with anhydrous Na2SO4, the mixture was concentrated under reduced pressure to remove the solvent, yielding 5.7 g of a pale yellow oily substance. Yield: 68.7%.

[0102] Step 3: 5.7 g (24.44 mmol) of compound 4 was dissolved in 50 mL of DCM, and 3.71 g (26.86 mmol) of 4-nitroaniline, 9.36 g (48.83 mmol) of EDCI·HCl and 6.0 g (49.11 mmol) of DMAP were added. The mixture was stirred overnight at room temperature. After the reaction was complete, the starting material was monitored by TLC. The mixture was washed twice with 1 M HCl solution, and the organic phase was collected. After drying with anhydrous Na2SO4, the mixture was concentrated under reduced pressure to remove the solvent. After purification by silica gel column chromatography, 5.3 g of a pale yellow oil was obtained, with a yield of 61.3%.

[0103] Step 4: Dissolve 5.0 g (14.15 mmol) of compound 5 in 60 mL of DCM, add 16.1 g (141.20 mmol) of TFA, stir at room temperature, and after 2 h, monitor the reaction of the starting material by TLC until it is complete. Concentrate under reduced pressure to remove the solvent and residual TFA. Dissolve the crude product (about 3.6 g, 14.22 mmol) in 40 mL of DMF, add 5.86 g (42.46 mmol) of K2CO3, stir for 10 min, then add 3.33 g (17.07 mmol) of tert-butyl bromoacetate, stir at room temperature overnight, monitor the reaction of the starting material by TLC until it is complete, remove the solvent by rotary evaporation, dilute with DCM, wash with water, collect the organic phase, dry with anhydrous Na2SO4, concentrate under reduced pressure to remove the solvent, and purify by silica gel column chromatography to obtain 3.2 g of a pale yellow oil, with a yield of 61.5%.

[0104] Step 5: Dissolve 2.7 g (7.35 mmol) of compound 7 in 15 mL of acetone, add 1.57 g (11.06 mmol) of iodomethane, stir at room temperature, and a white solid immediately precipitates. After stirring for 2 h, the reaction of the starting material is monitored by TLC until it is complete. Filter under reduced pressure, wash the white solid with acetone, collect the white solid, dry it to obtain 3.2 g, yield 85.4%.

[0105] Step 6: Dissolve 3.0 g (5.89 mmol) of compound 8 in 15 mL of DCM, add 15 mL of TFA, stir the reaction at RT, and after 2 h, monitor the reaction of the starting material by TLC until it is complete. Concentrate under reduced pressure to remove the solvent and residual TFA, then add 30 mL of DCM to dissolve it, add a small amount of solid NaHCO3 until no bubbles are generated, remove the solvent by rotary evaporation, and purify by silica gel column chromatography (DCM:MeOH=2:1) ​​to obtain 1.9 g of white solid, which is the target product.

[0106] The synthesis process steps are as follows:

[0107] .

[0108] (4) Preparation of the p-nitroaniline hapten shown in formula (Ⅳ):

[0109] 5.30 g (13.58 mmol) of perfluorooctanoic acid was dissolved in 50 mL of ultra-dry dichloromethane under nitrogen protection. 3.79 g (29.90 mmol) of oxaloyl chloride and 5 drops of ultra-dry DMF (producing numerous bubbles) were added to the solution, and the mixture was stirred at room temperature for 1 h. After 1 h, the system was cooled in an ice-water bath, and 13.5 mL (19.56 mmol) of N,N-diisopropylethylamine was added, followed by the fractional addition of 1.88 g (13.59 mmol) of p-nitroaniline. Stirring was continued for 1 h after the addition was complete. TLC showed the reaction was complete. The pH of the system was adjusted to 2-3 with 2N HCl, and then allowed to stand to separate into layers. The aqueous layer was extracted twice with 50 mL × 2 dichloromethane. The organic layers were combined, dissolved, and the resulting oily substance was purified by silica gel column chromatography (200-300M, DCM / MeOH=10 / 1) to obtain 0.71 g of brownish-yellow solid, which was the target product.

[0110] The synthesis process steps are as follows:

[0111] .

[0112] The four hydrophobic p-nitroaniline hapten synthesis methods provided by this invention are simple, have high purity and yield, and can be directly coupled with proteins to immunize animals to produce specific antibodies against p-nitroaniline hapten, which can be used for rapid detection of p-nitroaniline residues.

[0113] (ii) This embodiment characterizes the four different hydrophobic p-nitroaniline haptens synthesized above.

[0114] The structure of the synthesized p-nitroaniline hydrophobic hapten of formula (I) was determined by nuclear magnetic resonance, see [link to relevant documentation]. Figure 1 ¹H NMR (400 MHz, DMSO-d⁶) δ 10.47 (s, NH, ¹H), 8.17 (d, J = 9.1 Hz, ArH, 2H), 7.80 (d, J = 9.3 Hz, ArH, 2H), 2.35 (t, J = 7.2 Hz, CH₂, 2H), 2.21 (t, J = 7.2 Hz, CH₂, 2H), 1.62–1.44 (m, CH₂, 4H). This NMR data demonstrates the successful synthesis of the hydrophilic hapten of p-nitroaniline shown in formula (Ⅰ).

[0115] The structure of the synthesized p-nitroaniline hydrophobic hapten of formula (II) was determined by nuclear magnetic resonance, see [link to relevant documentation]. Figure 2¹H NMR (400 MHz, DMSO-d6) δ: 12.63 (s, 1H), 10.28 (s, 1H), 8.23 ​​(d, J = 9.1 Hz, 2H), 7.92 (d, J = 9.2 Hz, 2H), 4.18 (s, 2H), 4.08 (s, 2H), 3.69 (d, J = 3.5 Hz, 4H). The NMR results demonstrate the successful synthesis of the hydrophilic hapten of p-nitroaniline shown in formula (II).

[0116] (3) The structure of the synthesized p-nitroaniline hydrophobic hapten of formula (III) was determined by nuclear magnetic resonance, see below. Figure 3 ¹H NMR (600 MHz, Methanol-d⁴) δ: 8.23 ​​(d, J = 9.2 Hz, 1H), 7.95 (d, J = 9.2 Hz, 1H), 4.26 (s, 1H), 4.07 (d, J = 1.6 Hz, 1H), 4.05 (d, J = 4.1 Hz, 1H), 4.01 (dt, J = 6.4, 2.0 Hz, 1H), 3.39 (s, 4H). From the perspective of NMR, this demonstrates the successful synthesis of the hydrophobic hapten of p-nitroaniline shown in formula (III).

[0117] (4) The structure of the synthesized p-nitroaniline hydrophobic hapten of formula (IV) was determined by nuclear magnetic resonance, see below. Figure 4 1H NMR (400 MHz, DMSO-d6) δ 11.79 (s, 1H, COOH), 8.60 (s, 1H, CONH), 8.31 (m, 2H, Ph), 8.09 – 7.79 (m, 2H, Ph). [3.63–3.60 (m, 2H), 3.15–3.12 m, 2H), 1.50–1.10 (m, 18H). DIPEA]. The successful synthesis of the hydrophobic hapten of p-nitroaniline shown in formula (Ⅳ) is demonstrated from the perspective of nuclear magnetic resonance.

[0118] (III) In this embodiment, the geometry of the p-nitroaniline hydrophobic hapten is further optimized based on the M06-2X density functional theory and the TZVP basis set. The physicochemical parameters of the p-nitroaniline hapten shown in equations (I) to (IV) are extracted from the calculation results, and the molecular surface electrostatic potential distribution diagram is plotted. Figure 5(Table 1) Physicochemical parameters include molecular weight (MW), dipole moment (μ), molecular polarity index (MPI), lipid-water partition coefficient (cLogP), van der Waals surface area (SA), polar surface area (PSA), and solvent-accessible surface area (SASA). The results show that the polar molecular parameters and molecular surface electrostatic potential distributions of the four haptens differ to some extent, suggesting that the haptens designed and synthesized in this study have different hydrophobicities.

[0119] Table 1 shows the physicochemical parameters of the p-nitroaniline hapten (I) to (IV).

[0120] Hapten I Ⅱ Ⅲ Ⅳ MW 266.25 298.25 326.33 510.19 μ 2.8895 3.7960 9.3661 4.5973 MPI 16.51 16.89 22.24 11.85 cLogP 1.63 0.73 -1.56 4.89 SA 502.32 456.95 496.85 687.27 PSA 216.7 205.97 223.99 160.47

[0121] Example 2

[0122] This embodiment provides methods for preparing and characterizing artificial antigens of different hydrophobicities of p-nitroaniline. The difference between the immunogen and coating antigen preparation methods lies in the type of carrier protein used: BSA is mainly used as the carrier protein for the immunogen, while OVA is mainly used as the carrier protein for the coating antigen, and the coupling method is the active ester method.

[0123] (1) Preparation of immunogens with different hydrophobicity of p-nitroaniline

[0124] 9.03 μmol of the compounds of formulas (I) to (IV) prepared in Example 1 were dissolved in 1 mL of DMF, and 10.38 μmol of NHS and 10.38 μmol of EDC were added. The mixture was stirred at room temperature for 24 h to obtain solution I.

[0125] Add 20 mg BSA to 10 mL PBS buffer and dissolve thoroughly to obtain solution II.

[0126] Solution I was slowly added dropwise to solution II, and the mixture was stirred slowly at 4 °C for 24 h. The mixture was then placed in a dialysis bag and dialyzed in PBS at 4 °C for 72 h (with 6 water changes in between) to obtain the p-nitroaniline immunogen solution. This solution was aliquoted and stored at -20 °C. The p-nitroaniline immunogen synthesized from the compounds shown in formulas (I) to (IV) is abbreviated as PNA-BSA.

[0127] (2) Identification of immunogens of different hydrophobicity of p-nitroaniline

[0128] The binding ratio of BSA to hapten in PNA-BSA solution was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The results are shown in the figure. Figure 6 .

[0129] The binding ratios of the artificial antigens of the compounds shown in formulas (I) to (IV) are calculated using the following formulas (Table 2):

[0130] Binding ratio = {M (conjugate) - M (protein)} / M (hapten)

[0131] (3) Synthesis of different hydrophobic p-nitroaniline coating agents

[0132] Using OVA instead of BSA, the preparation method and steps of the coating agent are the same as those for the immunogen. The compounds shown in formulas (I) to (IV) are used to synthesize different hydrophobic p-nitroaniline coating agents, abbreviated as PNA-OVA.

[0133] Table 2 shows the binding ratios of different hydrophobic p-nitroaniline artificial antigens as indicated by formulas (I) to (IV).

[0134] Artificial antigen molecular weight Combination ratio BSA 66094 - I-BSA 71198 12.97 II-BSA 70399.78 13.38 Ⅲ-BSA 69147.4 10.40 -BSA 68516.89 4.75

[0135] Example 3

[0136] This embodiment provides polyclonal antibodies against different hydrophobic p-nitroaniline haptens and their preparation methods.

[0137] Thirty-two 6-8 week old female BALB / c mice (SPF grade) were immunized with PNA-BSA prepared in Example 2 and randomly divided into four groups of eight mice each. The immunization program consisted of one basic immunization and several booster immunizations.

[0138] For the first immunization, 100 μg of immunogen was mixed with an equal volume of Freund's complete adjuvant, emulsified, and injected subcutaneously into the neck and back of mice at multiple sites, with 200 μL injected into each mouse for basic immunization.

[0139] 100 μg of immunogen was mixed with an equal volume of Freund's incomplete adjuvant and emulsified. Booster immunizations were performed every 3 weeks after the initial immunization, with a volume of 200 μL per animal.

[0140] Blood was collected from the orbital rim on day 7 after each immunization. Serum was separated, and the titer of serum antibodies and the inhibition rate of p-nitroaniline 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.

[0141] Example 4

[0142] This embodiment measures the titers and affinities of polyclonal antibodies against different hydrophobic p-nitroaniline haptens.

[0143] (a) The antibody titer was detected using an indirect ELISA method. The specific operating steps are as follows:

[0144] (1) Coating: The coating material was serially diluted with 0.05 M, pH 9.6 carbonate buffer, 100 μL / well, and reacted at 4 ℃ for 16 h.

[0145] (2) Washing: Pour out the solution in the plate, shake dry, and wash once with washing solution for 3 minutes each time.

[0146] (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.

[0147] (4) Sample addition: Add 100 μL of serially diluted antibody to each well and react at 37 °C for 30 min. After thorough washing, add 100 μL of HRP-goat anti-mouse IgG diluted 1:5000 to each well and react at 37 °C for 30 min.

[0148] (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.

[0149] (6) Termination and measurement: Add 100 μL of stop solution to each well to terminate the reaction, and then measure the OD value of each well at 450 nm using an ELISA reader.

[0150] (7) Data processing: Antibody titer was defined as the reciprocal of the highest dilution with an OD value higher than the average value of the blank control wells ± 3 SD (Table 3, Figure 7 ).

[0151] (II) The antibody affinity was detected using an indirect competitive ELISA method. The specific operating steps are as follows:

[0152] (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.

[0153] (2) Washing: Pour out the solution in the plate, shake dry, and wash once with washing solution for 3 minutes each time.

[0154] (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.

[0155] (4) Prepare standard solutions: dilute p-nitroaniline standard to 10 μg / mL and p-nitroaniline hapten to 50 ng / mL for detection of antibodies prepared from different hydrophobic p-nitroaniline artificial antigens as shown in formulas (I) to (IV).

[0156] (5) Sample addition: Add 50 μL of diluted standard of each concentration to each well, and then add 50 μL of antibody at the optimal dilution factor per well. Incubate at 37 °C for 30 min. After thorough washing, add 100 μL of HRP-goat anti-mouse IgG diluted 1:5000 per well and incubate at 37 °C for 30 min.

[0157] (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.

[0158] (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.

[0159] (8) Data processing: Antibody affinity was assessed by inhibition rate. The inhibition rates of antibodies prepared from different hydrophobic p-nitroaniline artificial antigens were shown in Table 3 (Equations (I) to (IV)). Figure 8 ), calculated according to the following formula:

[0160] Inhibition rate (%) = B / B0 × 100%, where B0 and B are the OD values ​​at 450 nm when no standard is present and when a standard is present, respectively.

[0161] The results showed that the more hydrophobic p-nitroaniline hapten (Formula I) could improve the overall antibody titer induced by the body and the affinity for p-nitroaniline. The difference was more significant after the third immunization, with the highest hapten-specific antibody titer induced, which was 2.3 to 320 times higher than other groups; at the same time, it effectively induced antiserum with high affinity for p-nitroaniline, which was 1.81 to 2.09 times higher than that of the haptens (Formulas II and IV), with an average inhibition rate of 38.9%.

[0162] Table 3. Antibody titers and inhibition rates of serum antibodies prepared from artificial antigens with different hydrophobicities of p-nitroaniline (Equations (I) to (IV))

[0163] Hapten <![CDATA[PNA inhibition rate (%) a > <![CDATA[Corresponding hapten inhibition rate (%) a > <![CDATA[Antibody titer b > Ⅰ 38.90 59.00 1228800 Ⅱ 70.50 69.20 532480 Ⅲ 36.63 39.25 153600 Ⅳ 81.20 96.80 3840

[0164] Note: a The average inhibition rate of PNA (10 μg / mL) and the corresponding hapten (50 ng / mL) at different antibody dilutions after triple immunization represents the average inhibition rate of PNA (10 μg / mL) and the corresponding hapten (50 ng / mL). b This represents the average antibody titer against the hydrophobic hapten of nitroaniline after three immunizations.

[0165] Example 5

[0166] This embodiment provides monoclonal antibodies against different hydrophobic p-nitroaniline haptens and their preparation methods, and determines the properties of the monoclonal antibodies.

[0167] (I) Cell fusion and cloning

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

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

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

[0171] (II) Cell cryopreservation and thawing

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

[0173] (III) Preparation of Monoclonal Antibodies

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

[0175] (iv) Identification of monoclonal antibodies

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

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

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

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

[0180] (4) Prepare standard solutions: The p-nitroaniline standard solution is serially diluted 3 times to obtain a total of 8 concentrations. Each concentration is set up with 3 replicates for detection of antibodies prepared from the p-nitroaniline artificial antigens shown in formulas (I) to (IV).

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

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

[0183] (7) Termination and measurement: Add 100 μL of stop solution to each well to terminate the reaction, and then measure the OD450 value of each well using an ELISA reader.

[0184] (8) Data processing: The standard concentration was used as the log value on the x-axis and the OD value on the y-axis. The four-parameter equation of Origin 8.5 was used for fitting to establish a standard curve and obtain the IC. 50 The values ​​are shown in Table 4.

[0185] The results showed that the more hydrophobic formula (I) p-nitroaniline hapten significantly improved the affinity of the prepared monoclonal antibody for p-nitroaniline, and the IC50 of the monoclonal antibody was higher. 50 The concentration range was 30.74–58.20 ng / mL. The IC50 of the monoclonal antibody against the p-nitroaniline hapten was compared with that of other haptens. 50 The levels were reduced by 3.35 to 505.88 times, indicating that the p-nitroaniline hapten designed in this patent can be successfully used to prepare high-affinity monoclonal antibodies.

[0186] Table 4 IC50 of monoclonal antibodies prepared from artificial antigens with different hydrophobicities of p-nitroaniline (I) to (IV)

[0187]

[0188] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A p-nitroaniline hapten, characterized in that, The p-nitroaniline hapten contains a hydrophobic spacer arm, and its structural formula is any one of the following: Ⅱ； Ⅲ； Ⅳ。 2. A p-nitroaniline artificial antigen, characterized in that, It includes the p-nitroaniline hapten of claim 1 and the carrier protein coupled to the p-nitroaniline hapten.

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

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

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

6. The preparation method according to claim 5, characterized in that, The molar ratio of the p-nitroaniline hapten to the carrier protein was 4.75–13.38:

1.

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

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