P-nitroaniline hydrophobic hapten as well as synthesis method and application thereof

Abstract

The invention relates to the technical field of biochemical engineering, in particular to paranitroaniline hydrophobic haptens as well as a preparation method and application thereof. Specifically, on the basis of paranitroaniline, the hydrophobicity of a target object is adjusted by introducing a polar atom (F, N or O) from a spacer arm. And then coupling the hapten with a carrier protein, and evaluating the immunogenicity of the artificial antigen. The invention explores the influence of hapten hydrophobic change on immunogenicity, on one hand, theoretical support is provided for reasonable design of hapten; on the other hand, a material basis is provided for preparing a high-affinity antibody of paranitroaniline. The invention provides a new thought for reasonable design of small molecule compound hapten and preparation of high-affinity antibody.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical engineering, and in particular to a p-nitroaniline hydrophobic hapten and a synthesis method and application thereof. Background Art

[0002] p-Nitroaniline belongs to the aniline class of compounds and is widely used in the production of pesticides, veterinary drugs, rubber, dyes, pharmaceuticals and other chemicals. Excessive use and discharge of such compounds have caused a great deal of pollution to the environment (especially water bodies). p-Nitroaniline is highly chemically toxic and can produce toxic effects through inhalation, oral ingestion, skin contact and other means, inducing diseases such as hypoxia, jaundice and anemia. As a chemical with a high incidence of sudden environmental events and a characteristic pollutant, p-Nitroaniline is the focus of environmental risk prevention and control.

[0003] Antibodies are the core reagents of immunoassay methods. One of the key steps in the preparation of small molecule antibodies is the rational design and synthesis of haptens. The properties of haptens have an important influence on their immunogenicity, which directly determines the specificity and efficacy of the produced 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 proteins and nanovaccines, studies have shown that enhancing the hydrophobicity of immunogens can induce stronger immune responses; however, in the field of small molecule compound antibody preparation, the effect of hapten hydrophobicity on immune response is still unknown. Since p-nitroaniline is a small molecule compound with a small molecular weight, its monoclonal antibody preparation has problems such as poor specificity and sensitivity and difficult screening.

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

[0005] In order to solve the above technical problems, the present invention provides a type of p-nitroaniline hydrophobic hapten and a synthesis method and application thereof.

[0006] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a type of p-nitroaniline hapten, wherein the p-nitroaniline hapten contains a hydrophobic spacer arm, and its structural formula is any one of the following structural formulas: I; II; III; IV.

[0007] The present invention is based on the target structure of p-nitroaniline, and adjusts the hydrophobicity of the target by introducing polar atoms (F, N or O) from the spacer arm, and finally screens the above four hapten structures that have relatively outstanding performance in hydrophobicity and enhanced immune response.

[0008] In a second aspect, the present invention provides an artificial p-nitroaniline antigen, which comprises the p-nitroaniline hapten and a carrier protein coupled to the p-nitroaniline hapten.

[0009] Preferably, the p-nitroaniline hapten is coupled to the carrier protein via an amide bond.

[0010] Preferably, the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein and human serum albumin.

[0011] Preferably, the carrier protein is coupled to the carboxyl group of the p-nitroaniline hapten using an active ester method.

[0012] Preferably, the coupling molar ratio of p-nitroaniline hapten to carrier protein is 4.75-13.38:1.

[0013] The present invention couples the hapten with a carrier protein to evaluate the immunogenicity of the artificial antigen. On the one hand, the effect of increased hydrophobicity of the hapten on the immunogenicity is explored, providing theoretical support for the rational design of the hapten. On the other hand, it provides a material basis for the preparation of high-affinity antibodies against p-nitroaniline.

[0014] In a third aspect, the present invention provides any of the following uses of the p-nitroaniline hapten or the p-nitroaniline artificial antigen: Used to evaluate the effect of hapten hydrophobicity on immunogenicity; Used in the preparation of p-nitroaniline-specific antibodies; Used in the detection of p-nitroaniline-specific antibodies; The application is for non-disease diagnosis purposes.

[0015] The four haptens provided by the present invention are specially prepared in view of the influence of the hydrophobicity of haptens on the preparation of monoclonal antibodies in immunoassay technology, providing a new idea and method for the rational design of small molecule compound haptens and the preparation of high-affinity antibodies.

[0016] In a fourth aspect, the present invention provides specific antibodies prepared from the artificial antigen, wherein the specific antibodies are polyclonal antibodies and / or monoclonal antibodies.

[0017] The specific antibodies provided by the present invention have a higher affinity.

[0018] In a fifth aspect, the present invention provides a p-nitroaniline drug detection reagent or kit prepared using the specific antibody.

[0019] In a sixth aspect, the present invention provides any of the following uses of the specific antibody: Application in fluorescence polarization detection kit for detecting p-nitroaniline drugs; Application in the preparation of enzyme-linked immunosorbent assay kits for p-nitroaniline drugs; Application in the preparation of lateral flow chromatography test strips for p-nitroaniline drugs; The application is for non-disease diagnosis purposes.

[0020] Beneficial effects: The present invention provides a type of p-nitroaniline hydrophobic hapten and its synthesis method and application. 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 with a carrier protein to evaluate the immunogenicity of the artificial antigen. The present invention explores the effect of changes in the hydrophobicity of the hapten on the immunogenicity, which provides theoretical support for the rational design of haptens on the one hand, and provides a material basis for the preparation of high-affinity antibodies against p-nitroaniline on the other hand. The present invention provides a new idea for the rational design of small molecule compound haptens and the preparation of high-affinity antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0022] Figure 1 The figure is the hydrogen nuclear magnetic resonance spectrum of the p-nitroaniline hapten represented by formula (I) in the embodiment of the present invention.

[0023] Figure 2 The figure is the hydrogen nuclear magnetic resonance spectrum of the p-nitroaniline hapten represented by formula (II) in the embodiment of the present invention.

[0024] Figure 3 The figure is the hydrogen nuclear magnetic resonance spectrum of the p-nitroaniline hapten represented by formula (III) in the embodiment of the present invention.

[0025] Figure 4 The figure is the hydrogen nuclear magnetic resonance spectrum of the p-nitroaniline hapten represented by formula (IV) in the embodiment of the present invention.

[0026] Figure 5 The figure is a graph showing the distribution of the molecular surface electrostatic potential energy of the p-nitroaniline haptens represented by formulas (I) to (IV) in the embodiments of the present invention.

[0027] Figure 6This is a MALDI-TOF-MS graph of the artificial antigen for the preparation of the p-nitroaniline hapten represented by formula (I) to (IV) in the embodiments of the present invention.

[0028] Figure 7 The antibody titer after three immunizations with the artificial antigen of p-nitroaniline hapten represented by formula (I) to (IV) in the embodiment of the present invention.

[0029] Figure 8 It is the inhibition rate of the antibody against (A) p-nitroaniline (10 μg / mL) and (B) the corresponding hapten (50 ng / mL) after three immunizations with the p-nitroaniline hapten artificial antigen shown in formula (I) to (IV) in the embodiments of the present invention. DETAILED DESCRIPTION

[0030] Hydrophobicity is one of the most important physical and chemical properties of molecules. In the field of proteins and nano-vaccines, studies have shown that enhancing the hydrophobicity of immunogens can induce stronger immune responses. However, in the field of small molecule compound antibody preparation, the effect of hapten hydrophobicity on immune response is still unknown. The purpose of the present invention is to provide a preparation method and application of p-nitroaniline haptens with different hydrophobicity in view of the shortcomings of existing detection technology. The hapten can be coupled with bovine serum albumin or egg serum albumin to obtain an artificial antigen, and a high-affinity and highly specific monoclonal antibody can be obtained from an animal immunized with an artificial antigen, thereby establishing a detection method for p-nitroaniline residues based on immunological technology.

[0031] The present invention mainly adjusts the hydrophobicity of the spacer arm by introducing polar atoms (F, N and O) on the spacer arm of the hapten. The introduction of F atoms can increase the hydrophobicity of the molecule, while the introduction of N and O may increase the hydrophobicity of the hapten. The present invention uses polar atoms to induce changes in molecular properties, adjust the stability and distribution of haptens in the body, and then affect the recognition of the immune system. Through the above strategy, the present invention not only reveals the difference in immunogenicity of haptens with different hydrophobicity levels, but also provides new ideas for the rational design of haptens.

[0032] The present invention systematically evaluates the effects of haptens of different hydrophobicity on immunogenicity, ultimately prepares a p-nitroaniline monoclonal antibody with high specificity and high affinity, and establishes an immunoassay method for detecting p-nitroaniline chemical risk factors.

[0033] In a more specific and preferred embodiment, the solution provided by the present invention is as follows: First, the present invention provides a type of p-nitroaniline hydrophobic hapten, the structural formula of which is as follows: I; II; III; IV.

[0034] Furthermore, the present invention also provides a method for preparing the above four p-nitroaniline haptens with different hydrophobicity.

[0035] The method for preparing the hapten represented by formula (I) provided by the present invention comprises the following steps: Dissolve p-nitroaniline (13.8 g, 0.1 mol) and triethylamine (Et3N, 12.1 g, 0.1 mol) in THF under ice bath conditions to obtain liquid A. Then, dissolve hexanoyl chloride (16.2 g, 0.1 mol) in THF, slowly dropwise add to liquid A, and stir at room temperature for 2 h. Pour the reacted solution into water for post-treatment. After filtration, drying, and other steps, 2.5 g of white solid was obtained.

[0036] The method for preparing the hapten represented by formula (II) provided by the present invention comprises the following steps: Dissolve 2.0 g of 3, 6-dioxybenzene acid (11.23 mmol) in 40 mL of dry DCM, add 2.85 g of oxalyl chloride (22.46 mmol), add 5 drops of anhydrous DMF to the system, and stir the resulting solution at room temperature for 1 h. After 1 h, slowly add 60 mL of anhydrous DCM solution containing 1.40 g of p-nitroaniline (10.11 mmol) to the system, continue stirring at room temperature for 0.5 h, and the reaction is complete by TLC detection. Adjust the pH of the system to 3-4 with 2 N HCl, then add 50 mL of saturated brine and stir for 5 min, let stand and separate, extract the aqueous phase with 50 mL×2 ethyl acetate, combine the organic phases, desolventize under reduced pressure, and purify the residue by silica gel column chromatography (200~300 mesh, DCM / MeOH=15 / 1~10 / 1) to obtain 0.82 g of light yellow solid, which is the target product.

[0037] The present invention provides a method for preparing a hapten represented by formula (III) comprising the following steps: Step 1: Dissolve 5.0 g (66.57 mmol) of methylethanolamine in 50 mL of DCM, add 8.0 g (79.21 mmol) of Et3N and 15.9 g (72.85 mmol) of (Boc)2O under ice bath, stir for 15 min and then move to room temperature to react, stir overnight, monitor the reaction of the raw material by TLC, wash with 1 M hydrochloric acid solution and saturated NaHCO3 in turn, collect the organic phase, dry over anhydrous Na2SO4, and concentrate under reduced pressure to remove the solvent to obtain 11.3 g of light yellow oil, yield: 96.9%. 6 g (34.24 mmol) of compound 1 was dissolved in 60 mL of MTBE, and then 60 mL of 50% NaOH was added. After stirring for 30 min, 12.6 g (64.60 mmol) of tert-butyl bromoacetate was added and stirred at room temperature. After 3 h, TLC monitored the completion of the reaction of the raw materials. An appropriate amount of MTBE was added for dilution, 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 1M dilute hydrochloric acid. The organic phases were collected, dried over anhydrous Na2SO4, and concentrated under reduced pressure to remove the solvent to obtain 10.3 g of a light yellow oily substance, thereby obtaining compound 2.

[0038] 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 to react, monitor the reaction of the raw material by TLC after 1 h, and remove the solvent and the remaining TFA by vacuum concentration. Dissolve the obtained 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 to react, monitor the reaction of the raw material by TLC after 3 h, wash with 1 M HCl solution, collect the organic phase, dry it over anhydrous Na2SO4, and concentrate under reduced pressure to remove the solvent to obtain 5.7 g of light yellow oil, namely compound 4.

[0039] 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 at room temperature and react overnight. After the reaction of the raw material is completed, monitor by TLC, wash twice with 1 M HCl solution, collect the organic phase, dry over anhydrous Na2SO4, and concentrate under reduced pressure. After removing the solvent, purify by silica gel column to obtain 5.3 g of light yellow oil, i.e., compound 5.

[0040] Step 4: 5.0 g (14.15 mmol) of compound 5 was dissolved in 60 mL of DCM, 16.1 g (141.20 mmol) of TFA was added, and the reaction was stirred at room temperature. After 2 h, the reaction of the raw material was completed by TLC monitoring. The solvent and the remaining TFA were removed by vacuum concentration. The crude product (about 3.6 g, 14.22 mmol) was dissolved in 40 mL of DMF, 5.86 g (42.46 mmol) of K2CO3 was added, and after stirring for 10 min, 3.33 g (17.07 mmol) of tert-butyl bromoacetate was added. The mixture was stirred at room temperature overnight. After the reaction of the raw material was completed by TLC monitoring, the solvent was removed by vortexing, and DCM was added for dilution. The mixture was washed with water, and the organic phase was collected. After drying over anhydrous Na2SO4, the mixture was concentrated under reduced pressure to remove the solvent. After purification by silica gel column, 3.2 g of light yellow oil, i.e., compound 7, was obtained.

[0041] 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, and stir at room temperature. A white solid will precipitate out of the system immediately. After stirring for 2 h, TLC monitoring shows that the reaction of the raw material is complete. Filter under reduced pressure, wash the white solid with acetone, collect the white solid, and dry to obtain 3.2 g of compound 8.

[0042] Step 6: Dissolve 3.0 g (5.89 mmol) of compound 8 in 15 mL of DCM, add 15 mL of TFA, stir at RT to react, monitor the reaction of the raw material by TLC after 2 h, concentrate under reduced pressure to remove the solvent and the remaining TFA, then add 30 mL of DCM to dissolve, add a small amount of solid NaHCO 3 The solvent was removed by vortexing until no bubbles were generated, and the residue was purified by silica gel column (DCM:MeOH=2:1) ​​to obtain 1.9 g of white solid, which was the target product.

[0043] The method for preparing the hapten represented by formula (IV) provided by the present invention comprises the following steps: Dissolve 5.30 g (13.58 mmol) of perfluorooctanedioic acid in 50 mL of ultra-dry dichloromethane under nitrogen protection. Add 3.79 g (29.90 mmol) of oxalyl chloride and 5 drops of ultra-dry DMF (a large number of bubbles are generated) to the solution, and stir the resulting solution at room temperature for 1 h. After 1 h, cool the system in an ice-water bath, add 13.5 mL (19.56 mmol) of N,N-diisopropylethylamine, and then add 1.88 g (13.59 mmol) of p-nitroaniline in batches, and continue stirring for 1 h after the addition. TLC shows that the reaction is complete. Adjust the pH of the system to 2~3 with 2 NHCl, then stand and separate, extract the aqueous layer twice with 50 mL×2 dichloromethane, combine the organic layers, desolventize, and purify the resulting oil by silica gel column chromatography (200~300M, DCM / MeOH=10 / 1) to obtain 0.71 g of brown-yellow solid, which is the target product.

[0044] Furthermore, the present invention provides four p-nitroaniline artificial antigens obtained based on the above haptens, which are specifically obtained by coupling the p-nitroaniline hydrophobic haptens of formula (I) to (IV) with carrier proteins.

[0045] Wherein, the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, thyroid protein and human serum albumin, etc.; preferably bovine serum albumin and ovalbumin.

[0046] Furthermore, the present invention provides a method for preparing the artificial antigen, wherein the carrier protein can be coupled to the carboxyl carbon of the hapten of formula (I) to (IV) by using the activated ester method.

[0047] Preferably, the coupling molar ratio of the compound represented by formula (I) to (IV) to bovine serum albumin is 4.75 to 13.38:1.

[0048] Furthermore, the present invention provides any of the following uses of the p-nitroaniline hapten or the p-nitroaniline artificial antigen: Used to evaluate the effect of hapten hydrophobicity on immunogenicity.

[0049] Used to prepare p-nitroaniline-specific antibodies.

[0050] Used to detect para-nitroaniline-specific antibodies.

[0051] Furthermore, the present invention provides specific antibodies prepared from the p-nitroaniline artificial antigen, including polyclonal antibodies and monoclonal antibodies, preferably monoclonal antibodies. The polyclonal antibodies can be obtained by immunizing experimental animals (such as New Zealand white rabbits) with p-nitroaniline artificial antigens and collecting and purifying serum. The monoclonal antibodies can be prepared by hybridoma technology.

[0052] Furthermore, the present invention provides a p-nitroaniline detection reagent or kit prepared from the specific antibody.

[0053] Furthermore, the present invention provides any of the following applications of the specific antibody: Application in fluorescence polarization detection kit for detecting p-nitroaniline.

[0054] Application in the preparation of flow-through immunochromatographic test strips for p-nitroaniline.

[0055] Application in the preparation of enzyme-linked immunosorbent assay kit for p-nitroaniline.

[0056] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention discloses for the first time four kinds of p-nitroaniline haptens and artificial antigens with different hydrophobicities and methods for preparing the same. By evaluating the effect of the hydrophobicity of each hapten on the immunogenicity, the mechanism of the effect of the hydrophobicity of the hapten on the immune response is revealed, providing a theoretical basis for the rational design of haptens. By appropriately adjusting the hydrophobicity of the hapten, the immunogenicity of the artificial antigen is enhanced, thereby obtaining a specific antibody with high titer and high sensitivity. The p-nitroaniline hapten and the antibody prepared therefrom provided in this study provide a new means for establishing a rapid, simple, inexpensive, sensitive and specific method for detecting p-nitroaniline.

[0057] In this study, different hydrophobic p-nitroaniline haptens were synthesized, and the polar molecular parameter MPI ranged from 11.85 to 22.24 after computational chemistry. The p-nitroaniline hapten of formula (I) with a strong hydrophobicity (MPI of 16.51) successfully induced the highest hapten-specific antibody titer after three immunizations, which was 2.3 to 320 times higher than that of other groups, and effectively induced antiserum and monoclonal antibodies with high affinity for p-nitroaniline, among which the monoclonal antibody had an affinity 3.35 to 505.88 times higher than that of other haptens.

[0058] The artificial antigen provided by the present invention is used to prepare p-nitroaniline antibodies (polyclonal antibodies and monoclonal antibodies). The preparation process is simple and economical. The detection sensitivity of the antibody to p-nitroaniline can reach 30.74 ng / mL. The affinity of the nitroaniline antibody is enhanced by increasing the hydrophobicity of the p-nitroaniline hapten, which provides a new idea and method for the rational design of small molecule compound haptens and the preparation of high-affinity antibodies.

[0059] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0060] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0061] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0062] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0063] Example 1

[0064] (I) This example provides p-nitroaniline haptens of different hydrophobicity and methods for preparing the same.

[0065] (1) Preparation of p-nitroaniline hapten represented by formula (I)

[0066] Dissolve p-nitroaniline (13.8 g, 0.1 mol) and triethylamine (Et3N, 12.1 g, 0.1 mol) in THF under ice bath conditions to obtain liquid A. Then, dissolve hexanoyl chloride (16.2 g, 0.1 mol) in THF, slowly dropwise add to liquid A, and stir at room temperature for 2 h. Pour the reacted solution into water for post-treatment. After filtration, drying, and other steps, 2.5 g of white solid was obtained.

[0067] The synthesis process steps are as follows: .

[0068] (2) Preparation of p-nitroaniline hapten represented by formula (II)

[0069] Dissolve 2.0 g of 3, 6-dioxobenzoic acid (11.23 mmol) in 40 mL of dry DCM, add 2.85 g of oxalyl chloride (22.46 mmol), add 5 drops of anhydrous DMF to the system, and stir the resulting solution at room temperature for 1 h. After 1 h, slowly add 60 mL of anhydrous DCM solution containing 1.40 g of p-nitroaniline (10.11 mmol) to the system, continue stirring at room temperature for 0.5 h, and the reaction is complete by TLC detection. Adjust the pH of the system to 3~4 with 2 N HCl, then add 50 mL of saturated brine and stir for 5 min, let stand and separate, extract the aqueous phase with 50 mL×2 ethyl acetate, combine the organic phases, desolventize under reduced pressure, and purify the residue by silica gel column chromatography (200~300 mesh, DCM / MeOH=15 / 1~10 / 1) to obtain 0.82 g of light yellow solid, which is the target product.

[0070] The synthesis process steps are as follows: .

[0071] (3) Preparation of p-nitroaniline hapten represented by formula (III)

[0072] Step 1: Dissolve 5.0 g (66.57 mmol) of methylethanolamine in 50 mL of DCM, add 8.0 g (79.21 mmol) of Et3N and 15.9 g (72.85 mmol) of (Boc)2O under ice bath, stir for 15 min and then move to room temperature to react, stir overnight, monitor the reaction of the raw material by TLC, wash with 1 M hydrochloric acid solution and saturated NaHCO3 in turn, collect the organic phase, dry over anhydrous Na2SO4, and concentrate under reduced pressure to remove the solvent to obtain 11.3 g of light yellow oil, yield: 96.9%. 6 g (34.24 mmol) of compound 1 was dissolved in 60 mL of MTBE, and then 60 mL of 50% NaOH was added. After stirring for 30 min, 12.6 g (64.60 mmol) of tert-butyl bromoacetate was added and stirred at room temperature. After 3 h, TLC monitored the reaction of the raw materials. An appropriate amount of MTBE was added for dilution. The system was poured into a separatory funnel, and the MTBE phase was collected. The aqueous phase was extracted once with MTBE, the organic phases were combined and washed with 1M dilute hydrochloric acid, the organic phases were collected, dried over anhydrous Na2SO4, and concentrated under reduced pressure to remove the solvent to obtain 10.3 g of a light yellow oil, which was the target product.

[0073] 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 to react, monitor the reaction of the raw material by TLC after 1 h, and remove the solvent and the remaining TFA by vacuum concentration. Dissolve the obtained 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 to react, monitor the reaction of the raw material by TLC after 3 h, wash with 1 M HCl solution, collect the organic phase, dry it over anhydrous Na2SO4, and concentrate under reduced pressure to remove the solvent to obtain 5.7 g of light yellow oil, with a yield of 68.7%.

[0074] 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 at room temperature and react overnight. After the reaction of the raw material is completed, monitor by TLC, wash twice with 1 M HCl solution, collect the organic phase, dry over anhydrous Na2SO4, and concentrate under reduced pressure. After removing the solvent, purify by silica gel column to obtain 5.3 g of light yellow oil, yield: 61.3%.

[0075] Step 4: 5.0 g (14.15 mmol) of compound 5 was dissolved in 60 mL of DCM, 16.1 g (141.20 mmol) of TFA was added, and the reaction was stirred at room temperature. After 2 h, the reaction of the raw material was completed as monitored by TLC. The solvent and the remaining TFA were removed by concentration under reduced pressure. The crude product (about 3.6 g, 14.22 mmol) was dissolved in 40 mL of DMF, 5.86 g (42.46 mmol) of K2CO3 was added, and after stirring for 10 min, 3.33 g (17.07 mmol) of tert-butyl bromoacetate was added. The mixture was stirred at room temperature overnight. The reaction of the raw material was completed as monitored by TLC. The solvent was removed by rotation, and DCM was added for dilution. The mixture was washed with water, and the organic phase was collected. After drying over anhydrous Na2SO4, the mixture was concentrated under reduced pressure to remove the solvent. After purification by silica gel column, 3.2 g of a light yellow oil was obtained with a yield of 61.5%.

[0076] 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 will precipitate out of the system immediately. After stirring for 2 h, TLC monitoring shows that the reaction of the raw material is complete. Filter under reduced pressure, wash the white solid with acetone, collect the white solid, and dry to obtain 3.2 g, with a yield of 85.4%.

[0077] Step 6: Dissolve 3.0 g (5.89 mmol) of compound 8 in 15 mL of DCM, add 15 mL of TFA, and react by stirring at RT. After 2 h, the reaction of the raw material was completed by monitoring by TLC. The solvent and the remaining TFA were removed by concentration under reduced pressure, and then 30 mL of DCM was added to dissolve. A small amount of solid NaHCO3 was added until no bubbles were generated. The solvent was removed by rotation, and the mixture was purified by silica gel column (DCM:MeOH=2:1) ​​to obtain 1.9 g of white solid, which was the target product.

[0078] The synthesis process steps are as follows: .

[0079] (4) Preparation of p-nitroaniline hapten represented by formula (IV): Dissolve 5.30 g (13.58 mmol) of perfluorooctanedioic acid in 50 mL of ultra-dry dichloromethane under nitrogen protection. Add 3.79 g (29.90 mmol) of oxalyl chloride and 5 drops of ultra-dry DMF (with a lot of bubbles), and stir the resulting solution at room temperature for 1 h. After 1 h, cool the system in an ice-water bath, add 13.5 mL (19.56 mmol) of N,N-diisopropylethylamine, and then add 1.88 g (13.59 mmol) of p-nitroaniline in batches. Continue stirring for 1 h after the addition. TLC shows that the reaction is complete. The pH of the system was adjusted to 2-3 with 2N HCl, and then the mixture was allowed to stand for stratification. The aqueous layer was extracted twice with 50 mL×2 dichloromethane, the organic layers were combined, and the solvent was removed. The resulting oil was purified by silica gel column chromatography (200-300 M, DCM / MeOH=10 / 1) to obtain 0.71 g of a brown-yellow solid, which was the target product.

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

[0081] The four p-nitroaniline haptens with different hydrophobicity provided by the invention are simple to synthesize, have high purity and yield, can be directly coupled with proteins to immunize animals to produce specific antibodies against p-nitroaniline haptens, and can be used for rapid detection of p-nitroaniline residues.

[0082] (ii) This example characterizes the four p-nitroaniline haptens with different hydrophobicities synthesized above.

[0083] The structure of the synthesized p-nitroaniline hydrophobic hapten represented by formula (I) was determined by nuclear magnetic resonance. Figure 1 :1HNMR (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). From the perspective of nuclear magnetic resonance, it can be shown that the hydrophilic hapten of p-nitroaniline shown in formula (Ⅰ) was successfully synthesized.

[0084] The structure of the synthesized p-nitroaniline hydrophobic hapten represented by formula (II) was determined by nuclear magnetic resonance. Figure 2:1HNMR (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 = 3.5Hz, 4H). From the perspective of nuclear magnetic resonance, it can be shown that the hydrophilic hapten of p-nitroaniline shown in formula (II) was successfully synthesized.

[0085] (3) The structure of the synthesized p-nitroaniline hydrophobic hapten represented by formula (III) was determined by nuclear magnetic resonance. Figure 3 :1HNMR (600 MHz, Methanol-d4) δ: 8.23 ​​(d, J = 9.2 Hz, 1H), 7.95 (d, J = 9.2Hz, 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 nuclear magnetic resonance, it can be seen that the hydrophobic hapten of p-nitroaniline represented by formula (III) was successfully synthesized.

[0086] (4) The structure of the synthesized p-nitroaniline hydrophobic hapten represented by formula (IV) was determined by nuclear magnetic resonance. 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]. From the perspective of nuclear magnetic resonance, it can be shown that the hydrophobic hapten of p-nitroaniline represented by formula (IV) was successfully synthesized.

[0087] (III) This example further performs geometry optimization on the p-nitroaniline hydrophobic hapten based on the M06-2X density functional theory and the TZVP basis set, extracts the physicochemical parameters of the p-nitroaniline hapten shown in formulas (I) to (IV) from the calculation results, and plots the molecular surface electrostatic potential distribution diagram ( Figure 5, Table 1), the physicochemical parameters include molecular weight (MW), dipole moment (μ), molecular polarity index (MPI), lipid-water partition coefficient (cLogP), van der Waals surface area (SA), polar surface area (PSA) and solvent accessible surface area (SASA). The results showed that the polar molecular parameters and molecular surface electrostatic potential distribution of the four haptens were different, suggesting that the haptens designed and synthesized in this study have different hydrophobicity.

[0088] Table 1 Physicochemical parameters of p-nitroaniline haptens shown in (I) to (IV)

[0089] Example 2

[0090] This example provides the preparation method and characterization results of artificial p-nitroaniline antigens with different hydrophobicity. Among them, in the preparation method of the immunogen and the coating source, the difference between the two lies in the type of carrier protein used. The immunogen carrier protein mainly uses BSA, and the coating source carrier protein mainly uses OVA. The coupling method is the active ester method.

[0091] (1) Preparation of p-nitroaniline immunogens with different hydrophobicity

[0092] 9.03 μmol of the compounds of formula (I) to (IV) prepared in Example 1 were dissolved in 1 mL of DMF, 10.38 μmol of NHS and 10.38 μmol of EDC were added, and the mixture was stirred at room temperature for 24 h to obtain solution I.

[0093] Add 20 mg of BSA into 10 mL of PBS buffer and fully dissolve to obtain Solution II.

[0094] Solution I was slowly added dropwise to solution II, and the mixture was slowly stirred at 4 °C for 24 h, then placed in a dialysis bag and dialyzed in PBS at 4 °C for 72 h (with water changed 6 times in between) to obtain a p-nitroaniline immunogen solution, which was then aliquoted and stored at -20 °C. The p-nitroaniline immunogen synthesized from the compounds shown in formula (I) to (IV) is referred to as PNA-BSA.

[0095] (2) Identification of p-nitroaniline immunogens with different hydrophobicity

[0096] The binding ratios of BSA and hapten in PNA-BSA solution were determined by matrix-assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF-MS). The results are shown in Figure 6 .

[0097] The binding ratio of the artificial antigen of the compound represented by formula (I) to (IV) was calculated by the following formula (Table 2): Binding ratio = {M (conjugate) - M (protein)} / M (hapten)

[0098] (3) Synthesis of p-nitroaniline coatings with different hydrophobicity

[0099] OVA is used to replace BSA, and the preparation method and steps of the coating agent are the same as those of the immunogen. The compounds shown in formula (I) to (IV) are used to synthesize the p-nitroaniline coating agents with different hydrophobicity, which are referred to as PNA-OVA.

[0100] Table 2 Binding ratios of p-nitroaniline artificial antigens with different hydrophobicity shown in formula (I) to (IV)

[0101] Example 3

[0102] This example provides polyclonal antibodies against p-nitroaniline haptens of different hydrophobicity and methods for preparing the same.

[0103] The PNA-BSA prepared in Example 2 was used to immunize 32 female BALB / c mice (SPF grade) aged 6-8 weeks and randomly divided into 4 groups, 8 mice in each group. The immunization procedure was one basic immunization and several booster immunizations.

[0104] For the first immunization, 100 μg of the immunogen was mixed with an equal volume of Freund's complete adjuvant, emulsified, and injected subcutaneously at the back of the neck of the mouse at multiple points, with 200 μL per mouse for basic immunization.

[0105] 100 μg of immunogen was mixed with an equal volume of Freund's incomplete adjuvant for emulsification. After the first immunization, booster immunization was performed every 3 weeks, with a volume of 200 μL per mouse.

[0106] On the 7th day after each immunization, blood was collected from the eye sockets, and serum was separated. The titer of serum antibodies and the inhibition rate of recognition of p-nitroaniline were detected by indirect competitive ELISA. After blood collection, the blood was kept at 37 °C for 30 min, then centrifuged at 3000 rpm for 20 min, and the supernatant was collected, which was the polyclonal antibody and stored in aliquots at -20 °C.

[0107] Example 4

[0108] In this example, the titer and affinity of polyclonal antibodies to p-nitroaniline haptens of different hydrophobicity were determined.

[0109] (I) The antibody titer was detected by indirect ELISA method. The specific steps are as follows: (1) Coating: Dilute the coating agent in series with 0.05 M, pH 9.6 carbonate buffer, 100 μL / well, and react at 4 °C for 16 h.

[0110] (2) Washing: Pour off the solution in the plate, spin dry, and wash once with detergent for 3 min each time.

[0111] (3) Blocking: After patting dry, add 150 μL / well blocking solution and react at 37°C for 1 h. After washing, pat dry and set aside.

[0112] (4) Sample addition: Add 100 μL / well of serially diluted antibody to each well and react at 37°C for 30 min. After thorough washing, add 1:5000 diluted HRP-goat anti-mouse IgG, 100 μL / well, and react at 37°C for 30 min.

[0113] (5) Color development reaction: Take out the ELISA plate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37 °C in the dark for 15 min.

[0114] (6) Termination and measurement: Add 100 μL of stop solution to each well to terminate the reaction, and then use an ELISA reader to measure the OD value of each well at 450 nm.

[0115] (7) Data processing: The antibody titer was defined as the reciprocal of the highest dilution whose OD value was higher than the mean ± 3SD of the blank control wells (Table 3, Figure 7 ).

[0116] (II) The antibody affinity was detected by indirect competitive ELISA method. The specific steps are as follows: (1) Coating: Dilute the coating agent in series with 0.05 M, pH 9.6 carbonate buffer, 100 μL / well, and react at 37 °C for 2 h.

[0117] (2) Washing: Pour off the solution in the plate, spin dry, and wash once with detergent for 3 min each time.

[0118] (3) Blocking: After patting dry, add 150 μL / well blocking solution and react at 37°C for 1 h. After washing, pat dry and set aside.

[0119] (4) Prepare standard solution: dilute the p-nitroaniline standard to 10 μg / mL and the p-nitroaniline hapten to 50 ng / mL for the detection of antibodies prepared from p-nitroaniline artificial antigens of different hydrophobicities as shown in formulas (I) to (IV).

[0120] (5) Sample addition: Add 50 μL of diluted standard of each concentration to each well, and then add 50 μL / well of the optimal dilution of antibody, and react at 37°C for 30 min. After thorough washing, add 1:5000 diluted HRP-goat anti-mouse IgG, 100 μL / well, and react at 37°C for 30 min.

[0121] (6) Color development reaction: Take out the ELISA plate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37 °C in the dark for 15 min.

[0122] (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.

[0123] (8) Data processing: Antibody affinity was evaluated by inhibition rate. The inhibition rates of antibodies prepared with different hydrophobic p-nitroaniline artificial antigens (Table 3) were given in formulas (I) to (IV). Figure 8 ), calculated according to the following formula: Inhibition rate (%) = B / B 0 × 100%, of which B 0 A and B are the OD values ​​at 450 nm in the absence and presence of the standard, respectively.

[0124] The results showed that the p-nitroaniline hapten of formula (I) with higher hydrophobicity can improve the antibody titer and affinity for p-nitroaniline induced by the body as a whole. The difference was more significant after the third immunization, inducing the highest hapten-specific antibody titer, which was 2.3 to 320 times higher than that of 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 haptens of formula (II) and formula (IV), and the average inhibition rate was 38.9%.

[0125] Table 3 Antibody titers and inhibition rates of serum antibodies prepared from artificial p-nitroaniline antigens with different hydrophobicity of formula (I) to (IV)

[0126] Note: a Represents the average inhibition rate of PNA (10 μg / mL) and corresponding hapten (50 ng / mL) at different antibody dilutions after three immunizations. b Represents the average antibody titer against the p-nitroaniline hydrophobic hapten after three immunizations.

[0127] Example 5

[0128] This example provides monoclonal antibodies to p-nitroaniline haptens of different hydrophobicity and preparation methods thereof, and the properties of the monoclonal antibodies are measured.

[0129] 1. Cell fusion and cloning

[0130] (1) BALB / c mice with the highest serum suppression rate in each group were selected for shock immunization and cell fusion experiments. The shock immunization was performed by intraperitoneal injection of 3 times the immune dose of the immunogen.

[0131] (2) Three days after the shock immunization, spleen cells were collected and fused with SP2 / 0 myeloma cells at a ratio of 5:1 (quantity ratio). The cell supernatant was measured by indirect competitive ELISA to screen the positive wells.

[0132] (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.

[0133] 2. Cell cryopreservation and thawing

[0134] Hybridoma cells were prepared into 1×10 6 Cell suspensions of 100 cells / mL were stored in liquid nitrogen for a long time. When thawing, the cryopreserved tubes were taken out and immediately placed in a 37°C water bath for rapid thawing. After centrifugation to remove the cryopreservation solution, the cells were transferred to culture bottles for culture.

[0135] 3. Preparation of monoclonal antibodies

[0136] BALB / c mice were intraperitoneally injected with sterile paraffin oil (0.5 mL / mouse). Seven days later, the hybridoma cells prepared above (5×10 5 After 7 days, the ascites was collected to prepare the monoclonal antibody, which was stored at -20 ℃.

[0137] 4. Identification of monoclonal antibodies

[0138] The monoclonal antibody solution obtained in step 3 was used to detect antibody affinity using the indirect competitive ELISA method. The specific operation steps are as follows: (1) Coating: Dilute the coating agent in series with 0.05 M, pH 9.6 carbonate buffer, 100 μL / well, and react at 37 °C for 2 h.

[0139] (2) Washing: Pour off the solution in the plate, spin dry, and wash once with detergent for 3 min each time.

[0140] (3) Blocking: After patting dry, add 150 μL / well blocking solution and react at 37°C for 1 h. After washing, pat dry and set aside.

[0141] (4) Preparation of standard solution: The p-nitroaniline standard solution was diluted 3-fold to a total of 8 concentrations, and 3 replicate wells were set for each concentration for the detection of antibodies prepared against the p-nitroaniline artificial antigens shown in formulas (I) to (IV).

[0142] (5) Sample addition: Add 50 μL of diluted standard of each concentration to each well, and then add 50 μL / well of the optimal dilution of antibody, and react at 37°C for 30 min. After thorough washing, add 1:5000 diluted HRP-goat anti-mouse IgG, 100 μL / well, and react at 37°C for 30 min.

[0143] (6) Color development reaction: Take out the ELISA plate, wash it thoroughly, add 100 μL of TMB color development solution to each well, and react at 37 °C in the dark for 15 min.

[0144] (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.

[0145] (8) Data processing: The standard concentration was taken as the Log value as the horizontal axis and the OD value as the vertical axis. The four-parameter equation of Origin 8.5 was used for fitting to establish a standard curve and obtain the IC 50 The values ​​are shown in Table 4.

[0146] The results showed that the hydrophobic p-nitroaniline hapten of formula (I) significantly increased the affinity of the prepared monoclonal antibody to p-nitroaniline, and the IC 50 The IC values ​​of monoclonal antibody against nitroaniline hapten were 30.74~58.20 ng / mL. 50 It was reduced by 3.35~505.88 times, indicating that the p-nitroaniline hapten designed in this patent can successfully prepare high-affinity monoclonal antibodies.

[0147] Table 4 IC50 of monoclonal antibodies prepared from p-nitroaniline artificial antigens with different hydrophobicity of formula (I) to (IV)

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. 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 structural formulas: I； Ⅱ； Ⅲ； Ⅳ。 2. The p-nitroaniline artificial antigen is characterized by: The invention comprises the p-nitroaniline hapten according to claim 1 and a 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 limpet hemocyanin, thyroid protein and human serum albumin.

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

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

1.

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

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

9. A p-nitroaniline drug detection reagent or kit prepared from the specific antibody according to claim 8.

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

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