Nalufarid artificial hapten, artificial antigen, and preparation method and application thereof

Abstract

The present application belongs to the technical field of bio-chemical industry, and particularly relates to a nafurexone artificial hapten, an artificial antigen, and a preparation method and application thereof. The nafurexone artificial hapten of the present application retains the characteristic structure of nafurexone to the greatest extent, and has an active group that can be coupled with a carrier protein, and can be used as an antigenic determinant. The nafurexone artificial antigen prepared further can be used to immunize to obtain an anti-nafurexone polyclonal antibody with high affinity, high sensitivity and high specificity. The titer of the immune serum obtained by immunizing a New Zealand white rabbit is as high as 1:128000, and can be used for rapid and accurate immunodetection and immunological analysis of nafurexone.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological chemical industry, and particularly relates to a nalfurafine artificial hapten, an artificial antigen, and a preparation method and application thereof. BACKGROUND

[0002] Nalfurafine is an antipruritic drug marketed in Japan, which is used for treating uremic pruritus in patients with chronic kidney disease undergoing hemodialysis. It is an effective, selective, and centrally penetrating kappa-opioid receptor (KOR) agonist, and is the first and currently the only selective KOR agonist approved for clinical use, which is informally referred to as the "first non-narcotic opioid drug" in history.

[0003] It has been found that nalfurafine is effective in various animal models related to drug abuse, addiction, and dependence, and can become a new potential treatment for these diseases. In rodents, the drug attenuates the discriminative and rewarding effects of cocaine and the rewarding and locomotor effects of morphine, and reduces the aversive effects of mecamylamine on nicotine withdrawal, but the drug needs to be strictly taken according to the doctor's advice. Therefore, for regulatory purposes, it is necessary to develop a method for rapid detection of nalfurafine.

[0004] At present, the detection of nalfurafine mainly relies on high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography (TLC), mass spectrometry (MS), etc., but there are disadvantages such as expensive instruments, time-consuming detection, and the need for professional technical personnel to operate, which cannot meet the requirements of modern detection for rapidness and accuracy.

[0005] Immunoassay can make up for all the above shortcomings. Immunoassay is an analysis method for detecting various substances (such as drugs, hormones, proteins, microorganisms, etc.) by using the specific binding reaction of antigen and antibody. The key to establishing an immunoassay method for small molecule compounds is to be able to manufacture nalfurafine antigens and antibodies with high affinity and high specificity to nalfurafine. However, most small molecule compounds (molecular weight less than 1000), including nalfurafine, are not immunogenic, i.e., lack T cell epitopes and cannot directly induce the animal body to produce specific antibodies, so small molecule substances are called haptens. Through appropriate chemical modification, a connecting arm with an active group at the end is brought to a certain position on the hapten molecular structure, and then combined with a large molecule carrier to generate a hapten-carrier conjugate (i.e., artificial antigen). Artificial antigens can indirectly induce the proliferation and differentiation of B cells with the help of T cell epitopes, and then produce specific antibodies. Therefore, the synthesis of efficient artificial antigens is the prerequisite and key to guarantee immunoassay.

[0006] However, there is no related report on nalfurafine artificial haptens and artificial antigens in the prior art. SUMMARY

[0007] In view of the deficiencies in the prior art, the present application provides a nalfurafine artificial hapten, an artificial antigen and a preparation method thereof, and applies the nalfurafine artificial antigen to the preparation of an anti-nalfurafine antibody with high detection sensitivity and strong specificity.

[0008] To achieve the above-mentioned application purposes, the present application is implemented by the following technical solutions.

[0009] A nalfurafine artificial hapten, whose molecular structure is shown in formula (I):

[0010]

[0011] The nalfurafine artificial hapten of the present application retains the characteristic structure of nalfurafine to the greatest extent, and has an active group that can be coupled with a carrier protein, and can be used as an antigenic determinant.

[0012] The preparation method of the nalfurafine artificial hapten as described above comprises the following steps:

[0013] (S.1) dilute concentrated ammonia water with deionized water to obtain a DAS solution, dissolve nalfurafine hydrochloride in deionized water, add the DAS solution to adjust the pH to alkaline, then extract with dichloromethane, collect the dichloromethane phase, dry, filter and dry to obtain colorless oil A;

[0014] (S.2) dissolve the colorless oil A obtained in step (S.1) in anhydrous acetone, add potassium carbonate and ethyl bromoacetate, and perform a room temperature stirring reaction under nitrogen protection, after the reaction is completed, filter and dry to obtain white foamy solid B;

[0015] (S.3) dissolve the white foamy solid B obtained in step (S.2) in tetrahydrofuran and anhydrous methanol, add sodium hydroxide solution, stir at room temperature until the reaction is complete, adjust the pH with hydrochloric acid solution, directly dry, extract with anhydrous ethanol, filter, dry and purify to obtain nalfurafine artificial hapten I.

[0016] The present application introduces a connecting arm at the phenolic hydroxyl group of nalfurafine, and the introduction of the connecting arm at this modification site can retain the characteristic structure of nalfurafine to the greatest extent, and this modification site is as far as possible from other characteristic functional groups of nalfurafine, so as to maximize the exposure of the characteristic site to avoid interference with the specific antigenic determinant, thereby maximizing the recognition by the immune system.

[0017] Compared with the connecting arm in ring shape, the connecting arm in chain shape is adopted, the length of the connecting arm is suitable, the nalfurafine hapten small molecule can be fully exposed on the surface of the artificial antigen, the recognition degree of T cell to the connecting arm during immunization is reduced as far as possible, and the specificity and affinity of the obtained antibody to nalfurafine are stronger.

[0018] As preferred, the DAS solution is added in step (S.1) to adjust the pH to 9.

[0019] As preferred, the ratio of the colorless oil A, anhydrous acetone, potassium carbonate and ethyl bromoacetate in step (S.2) is 95-105:32-40:495-505:32-38 mg / mL / μL / mg / μL.

[0020] As preferred, the ratio of the white foamy solid B, tetrahydrofuran, anhydrous methanol and sodium hydroxide solution in step (S.3) is 95-105:1.45-1.50:1.90-2.0:4-8 mg / mL / mL / mL.

[0021] As preferred, the pH is adjusted to 4-5 by using hydrochloric acid solution in step (S.3).

[0022] As preferred, thin layer chromatography is used for purification in step (S.3), and the chromatography solution is 95% ethanol:1,4-dioxane:dichloromethane:ammonia water=8:1:10:1 (v / v / v / v).

[0023] A nalfurafine artificial antigen, the molecular structural formula of which is shown in formula (II):

[0024]

[0025] BSA is bovine serum albumin.

[0026] A preparation method of the nalfurafine artificial antigen as described above, comprising the following steps:

[0027] The nalfurafine artificial hapten I as described above is combined with bovine serum albumin by using N-hydroxysuccinimide active ester method to obtain nalfurafine artificial antigen II.

[0028] As preferred, a preparation method of the nalfurafine artificial antigen comprises the following steps:

[0029] (a) the nalfurafine artificial hapten I is mixed with cyclohexyl carbodiimide and N-hydroxysuccinimide in N,N-dimethylformamide, and the reaction is stirred at room temperature, and after the reaction is completed, centrifugation is performed, and the supernatant is taken;

[0030] (b) adding the supernatant obtained in step (a) into a bovine serum albumin solution dropwise and mixing uniformly to obtain a mixture and standing, and after the reaction is completed, carrying out dialysis, centrifugation, taking the supernatant to obtain the nafurafe artificial antigen II.

[0031] As a preference, the molar ratio of the nafurafe artificial hapten I, the cyclohexyl carbonyl diimide and the N-hydroxysuccinimide added in step (a) is 1: (1.3-1.4) : (1.3-1.4).

[0032] As a preference, the concentration of the bovine serum albumin solution in step (b) is 9-12 mg / mL; the volume ratio of the supernatant added to the bovine serum albumin solution is 1:8-12.

[0033] As a further preference, the preparation method of the bovine serum albumin solution comprises the following steps:

[0034] The bovine serum albumin is dissolved in a 0.01M PBS buffer solution, and the pH of the PBS buffer solution is 7.2-7.4.

[0035] The bovine serum albumin (BSA) is selected as the macromolecular carrier in the present application, and compared with the bovine gamma globulin (BGG), the bovine serum albumin has the following advantages:

[0036] ①The bovine serum albumin has many lysine residues, stronger immunogenicity, many antigenic determinants and higher coupling efficiency, and can easily cause stronger immune response;

[0037] ②The bovine serum albumin has a highly conserved structure and high stability, and is not easily affected by external factors, while the bovine gamma globulin is easily affected by external factors and is prone to precipitation, thereby affecting the performance of the antigen;

[0038] ③The bovine serum albumin is coupled with the hapten, has high coupling efficiency, is not prone to produce polyclonal antibodies, and is easier to improve the specificity of the antibodies. The nafurafe artificial antigen formed by combining the bovine serum albumin with the nafurafe artificial hapten has better specificity of the anti-nafurafe polyclonal antibodies obtained by animal immunization.

[0039] The use of the nafurafe artificial antigen as described above in the preparation of an anti-nafurafe antibody.

[0040] An anti-nafurafe polyclonal antibody is a globulin obtained by animal immunization of the nafurafe artificial antigen as described above, and can specifically immunoreact with nafurafe.

[0041] The present application is found through repeated experiments that the potency of the immune serum obtained by immunizing New Zealand white rabbits with the nalfurafine artificial antigen is 1:128000. Therefore, the nalfurafine artificial antigen of the present application can be used to obtain the anti-nalfurafine polyclonal antibody with high affinity, high sensitivity and strong specificity, and the anti-nalfurafine polyclonal antibody can be used for the immunodetection and analysis of nalfurafine.

[0042] Therefore, the present application has the following beneficial effects:

[0043] The nalfurafine artificial hapten of the present application retains the characteristic structure of nalfurafine to the greatest extent, and has an active group that can be coupled with a carrier protein, which can be used as an antigenic determinant. The nalfurafine artificial antigen prepared therefrom can be used to obtain the anti-nalfurafine polyclonal antibody with high affinity, high sensitivity and strong specificity, and the potency of the immune serum obtained by immunizing New Zealand white rabbits is as high as 1:128000, which can be used for rapid and accurate immunodetection and immunological analysis of nalfurafine. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The preparation flow chart of the nalfurafine artificial antigen II in Example 1.

[0045] Figure 2 The high performance liquid chromatogram of the nalfurafine artificial hapten I in Example 1.

[0046] Figure 3 The high resolution mass spectrum of the nalfurafine artificial hapten I in Example 1.

[0047] Figure 4 The ultraviolet absorption spectrum of the bovine serum albumin, the nalfurafine artificial hapten I and the nalfurafine artificial antigen II in Example 1.

[0048] Figure 5 The preparation flow chart of the nalfurafine artificial antigen IV in Comparative Example 1.

[0049] Figure 6 The preparation flow chart of the nalfurafine artificial antigen V in Comparative Example 2.

[0050] Figure 7 The preparation flow chart of the nalfurafine artificial antigen VI in Comparative Example 3.

[0051] Figure 8 The preparation flow chart of the nalfurafine artificial antigen VII in Comparative Example 4.

[0052] Figure 9 The preparation flow chart of the nalfurafine artificial antigen VIII in Comparative Example 5.

[0053] Figure 10Flow chart for preparation of nafurexine artificial antigen IX in Comparative Example 6.

[0054] Figure 11 Flow chart for preparation of nafurexine artificial antigen X in Comparative Example 7. DETAILED DESCRIPTION

[0055] The present application will be further described by examples with reference to the accompanying drawings. Ordinary skill in the art can implement the present application based on these descriptions. In addition, the examples of the present application involved in the following descriptions are generally only examples of a part of the present application, rather than all examples. Therefore, all other examples obtained by ordinary skill in the art based on the examples in the present application without making creative efforts should belong to the protection scope of the present application.

[0056] Example 1

[0057] The present example provides a nafurexine artificial hapten I and a preparation method thereof, and a nafurexine artificial antigen II and a preparation method thereof.

[0058] A preparation method of a nafurexine artificial antigen, comprising the following steps:

[0059] (1) Preparation of nafurexine artificial hapten I:

[0060] (S.1) 5 mL of concentrated ammonia water was diluted to 13 mL with deionized water to obtain a DAS solution, 100 mg (0.195 mmol) of nafurexine hydrochloride was weighed and dissolved in 10 mL of deionized water, and then the pH was adjusted to 9 with the DAS solution. A white precipitate appeared in the solution, and the aqueous phase was extracted with 15 mL of dichloromethane each time, and the extraction was performed for 3 times. The dichloromethane phase was collected, dried with anhydrous magnesium sulfate, filtered, and dried to obtain 88 mg (0.185 mmol) of colorless oil A. TLC detection was performed on the colorless oil A, the developing solution was ethyl acetate:ammonia water=10 mL:200 μL, and the product Rf=0.5;

[0061] (S.2) 88 mg (0.185 mmol) of the colorless oil A obtained in step (S.1) was dissolved in a 50 mL round-bottom flask with 31 mL of anhydrous acetone, 440 mg of potassium carbonate and 31.7 μL of ethyl bromoacetate were added, and the reaction was stirred at room temperature under N2 protection for more than 17 h. After the reaction was completed, the reaction was stopped, filtered, and dried to obtain 93 mg (0.165 mmol) of white foamy solid B. TLC detection was performed on the white foamy solid B, the developing solution was ethyl acetate:ammonia water=10 mL:200 μL, and the product Rf=0.8;

[0062] (S.3) Dissolve 93 mg (0.165 mmol) of white bubbly solid B obtained in step (S.2) in 1.37 mL of tetrahydrofuran and 1.82 mL of anhydrous methanol. Place the solution in a 50 mL round-bottom flask and add 9.3 mL of 1 mol / L sodium hydroxide aqueous solution. The solution darkens and becomes turbid. Stir rapidly at room temperature for 4 h. The reaction is complete by TLC. The reaction is then stopped. Adjust the pH to ≈4 with 1 mol / L hydrochloric acid solution and dry the mixture to obtain a solid-oil mixture residue. Extract the residue three times with 10 mL of anhydrous ethanol each time. Filter and dry the residue to obtain a white bubbly solid. Purify the residue by thin-layer chromatography to obtain 50 mg (0.094 mmol) of nalfuraphene artificial hapten I. TLC analysis of the nalfuraphene artificial hapten I was performed using a chromatographic buffer of 95% ethanol:1,4-dioxane:dichloromethane:ammonia = 8:1:10:1. The product Rf = 0.6. The thin-layer chromatography method was used for purification. The chromatographic solution was 95% ethanol:1,4-dioxane:dichloromethane:ammonia water = 8:1:10:1. The product Rf = 0.6. The solvent and eluent were anhydrous ethanol.

[0063] The molecular structure of nalfuraphen artificial hapten I is shown in formula (Ⅰ):

[0064]

[0065] The high-performance liquid chromatogram of nalfuraphen artificial hapten I in this embodiment is as follows: Figure 2 As shown, the ultraviolet detector has a wavelength of 288 nm. The high-resolution mass spectrum of the nalfuraphen artificial hapten I in this embodiment is shown below. Figure 3 As shown.

[0066] from Figure 2 It can be seen that the purity of the purified nalfuraphen artificial hapten I reached over 98%. From... Figure 3 As can be seen, the characteristic peaks of the nalfuraphen artificial hapten I obtained in this embodiment are 535.25, 557.23, 558.24, and 579.26, corresponding to [M+H], respectively. + [M+Na] + [M+Na+H] + [M+HCOO] - Given that the theoretical molecular weight of nafpramine artificial hapten I is 534.24, it can be basically determined that the final compound obtained in step (S.3) is the nafpramine artificial hapten designed in this invention.

[0067] (2) Preparation of nalfuraphen artificial antigen II:

[0068] (a) 50 mg (0.094 mmol) of the nalfurafine artificial hapten I obtained in step (S.3) was placed in a 50 mL round-bottom flask, 2.5 mL of N,N-dimethylformamide (DMF) was added, followed by the addition of 14.6 mg (0.127 mmol) of N-hydroxysuccinimide (NHS) and 26.2 mg (0.127 mmol) of dicyclohexyl carbodiimide (DCC), and stirring was performed at 25°C for 20 h. After the reaction was completed, centrifugation was performed at 8000 rpm for 5 min, and the supernatant was taken and reserved for use;

[0069] (b) 14.5 g (0.0405 mol) of disodium hydrogen phosphate dodecahydrate, 43.875 g (0.75 mol) of sodium chloride, and 1.495 g (0.00958 mol) of sodium dihydrogen phosphate dihydrate were dissolved in deionized water and diluted to 5.0 L to obtain a 0.01 M PBS buffer having a pH of 7.4. 250 mg of bovine serum albumin was dissolved in 25 mL of the prepared PBS buffer to obtain a bovine serum albumin solution having a concentration of 10 mg / mL. The supernatant obtained in step (a) was slowly added dropwise to the prepared bovine serum albumin solution under rapid stirring, and the volume ratio of the supernatant to the bovine serum albumin solution was 1:10. The obtained mixture was stored overnight at 4°C to obtain an artificial antigen mixture. The artificial antigen mixture was moved into a dialysis bag and dialyzed against the prepared PBS buffer for 9 times. After the dialysis was completed, centrifugation was performed, and the supernatant was taken to obtain a nalfurafine artificial antigen II (i.e., a nalfurafine-bovine serum albumin conjugate). The preparation flowchart of the nalfurafine artificial antigen II in this example is shown in Figure 1 . The ultraviolet absorption spectrum of the bovine serum albumin, the nalfurafine artificial hapten I, and the nalfurafine artificial antigen II in this example is shown in Figure 4 . The molecular structural formula of the nalfurafine artificial antigen II is shown in formula (II):

[0070]

[0071] wherein BSA is bovine serum albumin.

[0072] Figure 4 . The ultraviolet absorption spectrum of the bovine serum albumin, the nalfurafine artificial hapten I, and the nalfurafine artificial antigen II in this example is shown in

[0073] Example 2

[0074] The difference between this embodiment and embodiment 1 is that:

[0075] This embodiment provides a nalfurafine artificial hapten I, a preparation method thereof, and a nalfurafine artificial antigen II and a preparation method thereof.

[0076] A preparation method of a nalfurafine artificial antigen, wherein,

[0077] (1) Preparation process of nalfurafine artificial hapten I:

[0078] In step (S.1), 100 mg (0.195 mmol) of nalfurafine hydrochloride was weighed; and 90 mg (0.189 mmol) of colorless oily substance A was obtained after being freed; in step (S.2), 30.3 mL of anhydrous acetone was used; 469 mg of potassium carbonate and 30.3 μL of ethyl bromoacetate were added; in step (S.3), 85 mg (0.151 mmol) of white foamy solid B obtained in step (S.2) was dissolved in 1.30 mL of tetrahydrofuran and 1.70 mL of anhydrous methanol; 3.58 mL of 1 mol / L sodium hydroxide aqueous solution was added; and the others were the same as in embodiment 1. Finally, nalfurafine artificial hapten I was obtained.

[0079] (2) Preparation process of nalfurafine artificial antigen II:

[0080] In step (a), 40 mg (0.0752 mmol) of nalfurafine artificial hapten I obtained in step (S.3) was placed in a 50 mL round-bottom flask; 2 mL of N,N-dimethylformamide (DMF) was added, and then 11.25 mg (0.0978 mmol) of N-hydroxysuccinimide (NHS) and 20.15 mg (0.0978 mmol) of dicyclohexylcarbodiimide (DCC) were added; in step (b), 144 mg of bovine serum albumin was dissolved in 16 mL of prepared PBS buffer to obtain a bovine serum albumin solution with a concentration of 9 mg / mL; the volume ratio of supernatant to bovine serum albumin solution was 1:8; and the others were the same as in embodiment 1. Finally, nalfurafine artificial antigen II was obtained.

[0081] Embodiment 3

[0082] The difference between this embodiment and embodiment 1 is that:

[0083] This embodiment provides a nalfurafine artificial hapten I, a preparation method thereof, and a nalfurafine artificial antigen II and a preparation method thereof.

[0084] A preparation method of a nalfurafine artificial antigen, wherein,

[0085] (1) Preparation process of nalfurafine artificial hapten I:

[0086] Step (S.1) weighed 100 mg (0.195 mmol) of nalfurafine hydrochloride; free base obtained 90 mg (0.189 mmol) of colorless oil A; in step (S.2) used 34.3 mL of anhydrous acetone; added 433 mg of potassium carbonate and 32.6 μL of ethyl bromoacetate; in step (S.3) 90 mg (0.160 mmol) of white foamy solid B obtained in step (S.2) was dissolved in 1.29 mL of tetrahydrofuran and 1.71 mL of anhydrous methanol; added 6.86 mL of 1 mol / L sodium hydroxide aqueous solution; the rest was the same as in Example 1. Finally, nalfurafine artificial hapten I was obtained.

[0087] (2) Preparation process of nalfurafine artificial antigen II:

[0088] In step (a), 46 mg (0.0864 mmol) of nalfurafine artificial hapten I obtained in step (S.3) was placed in a 50 mL round-bottom flask; 2.25 mL of N,N-dimethylformamide (DMF) was added, followed by 13.92 mg (0.121 mmol) of N-hydroxysuccinimide (NHS) and 24.93 mg (0.121 mmol) of cyclohexylcarbodiimide (DCC); in step (b), 324 mg of bovine serum albumin was dissolved in 27 mL of prepared PBS buffer to obtain a bovine serum albumin solution with a concentration of 12 mg / mL; the volume ratio of supernatant to bovine serum albumin solution was 1:12; the rest was the same as in Example 1. Finally, nalfurafine artificial antigen II was obtained.

[0089] Comparative Example 1

[0090] The difference between this comparative example and Example 1 is that:

[0091] This comparative example provides a nalfurafine artificial hapten III and a preparation method thereof, and a nalfurafine artificial antigen IV and a preparation method thereof.

[0092] A preparation method of a nalfurafine artificial antigen, wherein,

[0093] Preparation process of nalfurafine artificial hapten III:

[0094] Step (S.1) is the same as in Example 1, and step (S.2) is replaced by: 80 mg (0.168 mmol) of colorless oily substance A is dissolved in 8 mL of anhydrous ethanol, placed in a 50 mL round-bottom flask, 39.4 μL (0.336 mmol) of chlorobenzene is added, 20 mg (0.120 mmol) of potassium iodide, 40 mg (0.289 mmol) of potassium carbonate, and the reaction is refluxed at 80°C for 5 h. After the reaction is completed, it is directly transferred to dryness to obtain a colorless oily residue, which is dissolved in 10 mL of purified water, and the aqueous phase is extracted with 10 mL of ethyl acetate for 3 times. The organic phase is collected, washed with 10 mL of saturated sodium bicarbonate aqueous solution, 10 mL of purified water, and 10 mL of saturated sodium chloride aqueous solution in sequence, collected, dried, filtered, and transferred to dryness to obtain 120 mg of colorless oily substance. Purification is performed by thin layer chromatography to obtain 93 mg (0.164 mmol) of colorless oily substance C, with anhydrous ethanol as the solvent and eluent. TLC detection is performed on the colorless oily substance C, with ethyl acetate:ammonia water = 10 mL:200 μL as the developing agent, and the product Rf= 0.8. Step (S.3) is replaced by: 93 mg (0.164 mmol) of colorless oily substance C obtained in step (S.2) is dissolved in 9.3 mL of anhydrous pyridine, 32.8 mg (0.328 mmol) of succinic anhydride is added, and the reaction is refluxed at 100°C for 17 h. After the reaction is completed, it is directly transferred to dryness to obtain 168 mg of light yellow oily substance. Purification is performed by thin layer chromatography to obtain 128 mg (0.192 mmol) of light yellow oily substance D, with anhydrous ethanol as the solvent and eluent. TLC detection is performed on the light yellow oily substance D, with 95% ethanol:1,4-dioxane:dichloromethane:25% ammonia water = 8:1:10:1 (by volume) as the developing agent, and the product Rf= 0.5. A step (S.4) is added: 128 mg (0.192 mmol) of light yellow oily substance D obtained in step (S.3) is dissolved in 10 mL of anhydrous ethanol, placed in a 50 mL round-bottom flask, 50 mg of palladium-carbon (Pd content 10%) is added, hydrogen is introduced, and the reaction is performed at room temperature for 17 h. After the reaction is completed, it is filtered and transferred to dryness to obtain 105 mg of light yellow oily substance. Purification is performed by thin layer chromatography to obtain 62 mg (0.107 mmol) of nafarelin artificial hapten IV, with anhydrous ethanol as the solvent and eluent, 95% ethanol:1,4-dioxane:dichloromethane:25% ammonia water = 8:1:10:1 (by volume) as the developing agent, and the product Rf= 0.3.

[0095] (2) Preparation process of nafarelin artificial hapten IV:

[0096] Step (a) is replaced by: Put 62 mg (0.107 mmol) of nalfurafine artificial hapten III obtained in step (S.4) into a 50 mL round-bottom flask, add 3.1 mL of DMF, then add 16.6 mg (0.144 mmol) of NHS and 29.7 mg (0.144 mmol) of DCC, stir at room temperature overnight, centrifuge after the reaction is completed, and take the supernatant for standby use. In step (b), replace deionized water with double-distilled water to obtain a 0.01 M PBS buffer with a pH of 7.4; replace 250 mg of bovine serum albumin dissolved in 25 mL of the prepared PBS buffer with 0.31 g of bovine serum albumin dissolved in 31 mL of the prepared PBS buffer. The rest is the same as in Example 1. Finally, nalfurafine artificial antigen IV (i.e., a nalfurafine-bovine serum albumin conjugate) is obtained. The preparation process of nalfurafine artificial antigen IV in this comparative example is shown in Figure 5 .

[0097] Comparative Example 2

[0098] The difference between this comparative example and Comparative Example 1 is that:

[0099] This comparative example provides a nalfurafine artificial hapten III and a preparation method thereof, and a nalfurafine artificial antigen V and a preparation method thereof.

[0100] A preparation method of a nalfurafine artificial antigen, wherein,

[0101] (1) The preparation process of nalfurafine artificial hapten III is the same as in Comparative Example 1.

[0102] (2) The preparation process of nalfurafine artificial antigen V: in step (b), bovine serum albumin is replaced by bovine gamma globulin (as a carrier) to be coupled with nalfurafine artificial hapten III, and the coupling step is the same as in Comparative Example 1. The rest is the same as in Comparative Example 1. Finally, nalfurafine artificial antigen V is obtained. The preparation process of nalfurafine artificial antigen V in this comparative example is shown in Figure 6 .

[0103] Comparative Example 3

[0104] The difference between this comparative example and Comparative Example 1 is that:

[0105] This comparative example provides a nalfurafine artificial hapten III and a preparation method thereof, and a nalfurafine artificial antigen V and a preparation method thereof.

[0106] A preparation method of a nalfurafine artificial antigen, wherein,

[0107] (1) The preparation process of nalfurafine artificial hapten III is the same as in Comparative Example 1.

[0108] (2) Preparation of the nafurexine artificial antigen VI: step (a) is replaced by: 58 mg (0.100 mmol) of nafurexine artificial hapten III is weighed into a 50 mL round-bottom flask, 2.9 mL of DMF is added, followed by 13.8 μL (0.100 mmol) of triethylamine, and the mixture is stirred in an ice bath for 30 min, followed by the addition of 25.9 μL (0.2 mmol) of isobutyl chloroformate, and the reaction is continued to stir in an ice bath for 2 h. After the reaction is completed, the mixture is centrifuged, and the supernatant is taken for use. The other steps are the same as in Comparative Example 1. Finally, nafurexine artificial antigen VI is obtained. The preparation process of nafurexine artificial antigen VI in this comparative example is shown in Figure 7 .

[0109] Comparative Example 4

[0110] The difference between this comparative example and Comparative Example 3 is that:

[0111] This comparative example provides a nafurexine artificial hapten III, a preparation method thereof, a nafurexine artificial antigen VII, and a preparation method thereof.

[0112] A preparation method of a nafurexine artificial antigen, wherein,

[0113] (1) The preparation process of nafurexine artificial hapten III is the same as in Comparative Example 1.

[0114] (2) Preparation of the nafurexine artificial antigen VII: in step (b), bovine serum albumin is replaced by bovine gamma globulin (as a carrier) to couple with the nafurexine artificial hapten III, and the coupling step is the same as in Comparative Example 3. The other steps are the same as in Comparative Example 3. Finally, nafurexine artificial antigen VII is obtained. The preparation process of nafurexine artificial antigen VII in this comparative example is shown in Figure 8 .

[0115] Comparative Example 5

[0116] The difference between this comparative example and Example 1 is that:

[0117] This comparative example provides a nafurexine artificial hapten I, a preparation method thereof, a nafurexine artificial antigen VIII, and a preparation method thereof.

[0118] A preparation method of a nafurexine artificial antigen, wherein,

[0119] (1) The preparation process of nafurexine artificial hapten I is the same as in Example 1.

[0120] (2) Preparation process of nafurexine artificial antigen VIII: step (a) is replaced by: 42 mg (0.079 mmol) of nafurexine artificial hapten I obtained in step (S.3) is weighed into a 50 mL round-bottom flask, 2.1 mL of DMF is added, followed by 10.9 μL (0.079 mmol) of triethylamine, and the mixture is stirred in an ice bath for 30 min, followed by the addition of 20.4 μL (0.158 mmol) of isobutyl chloroformate, and the reaction is continued to stir in an ice bath for 2 h. After the reaction is completed, centrifugation is performed, and the supernatant is taken for use. In step (b), deionized water is replaced by double-distilled water to obtain a 0.01 M PBS buffer with a pH of 7.4; and 0.21 g of bovine serum albumin is dissolved in 21 mL of the prepared PBS buffer instead of 250 mg of bovine serum albumin dissolved in 25 mL of the prepared PBS buffer. The rest is the same as in Example 1. Finally, nafurexine artificial antigen VIII is obtained. The preparation process of nafurexine artificial antigen VIII in this comparative example is shown in Figure 9 .

[0121] Comparative Example 6

[0122] The difference between this comparative example and Comparative Example 5 is that:

[0123] This comparative example provides a nafurexine artificial hapten I, a preparation method thereof, and a nafurexine artificial antigen IX, and a preparation method thereof.

[0124] A preparation method of a nafurexine artificial antigen, wherein,

[0125] (1) The preparation process of nafurexine artificial hapten I is the same as in Example 1.

[0126] (2) Preparation process of nafurexine artificial antigen IX: in step (b), bovine serum albumin (as a carrier) is used instead of bovine serum albumin to couple with nafurexine artificial hapten I, and the coupling step is the same as in Comparative Example 5. The rest is the same as in Comparative Example 5. Finally, nafurexine artificial antigen IX is obtained. The preparation process of nafurexine artificial antigen IX in this comparative example is shown in Figure 10 .

[0127] Comparative Example 7

[0128] The difference between this comparative example and Example 1 is that:

[0129] This comparative example provides a nafurexine artificial hapten I, a preparation method thereof, and a nafurexine artificial antigen X, and a preparation method thereof.

[0130] A preparation method of a nafurexine artificial antigen, wherein,

[0131] (1) The preparation process of nafurexine artificial hapten I is the same as in Example 1.

[0132] (2) Preparation of nafurexine artificial antigen X: In step (b), bovine serum albumin was replaced by bovine gamma globulin (as a carrier) to couple with nafurexine artificial hapten I, and the coupling step was the same as in Example 1, and the others were the same as in Example 1. Finally, nafurexine artificial antigen X was obtained. The preparation process of nafurexine artificial antigen X in the present comparative example is shown in Figure 11 .

[0133]

Performance measurement and analysis of nafurexine artificial antigen

[0134] (1) Identification of nafurexine artificial antigen:

[0135] Molar absorption coefficient ε: nafurexine artificial hapten I and nafurexine artificial hapten III were prepared according to the methods in Example 1 and Comparative Example 1, respectively. Then, nafurexine artificial hapten solutions with concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, and 400 μg / mL were prepared using PBS buffer, respectively. The maximum absorption wavelength of nafurexine artificial hapten I was 288 nm, and the maximum absorption wavelength of nafurexine artificial hapten III was 292 nm, as shown by the ultraviolet scanning diagram. The absorbance values of the corresponding haptens were measured at 288 nm and 292 nm, respectively, and parallel samples were prepared for each concentration. The calculation formula of molar absorption coefficient (i.e. molar absorption coefficient) was: ε = absorbance / molar concentration. The calculation data results are shown in Table 1 below.

[0136] Table 1: Calculation table of molar absorption coefficient of nafurexine artificial hapten I and III

[0137]

[0138] Determination of the concentration of the conjugate protein: nafurexine artificial antigens were prepared according to the methods in Example 1 and Comparative Examples 1-7, respectively. One mL of bovine serum albumin solution with concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, and 120 μg / mL was prepared using PBS buffer, 3 mL of Coomassie brilliant blue staining solution was added, and it was immediately mixed, heated in a 30°C water bath for 5 min, parallel samples were prepared for each concentration, and the absorbance was measured at 655 nm. The relationship curve between protein concentration and absorbance was drawn. The nafurexine artificial antigen solution (prepared using PBS buffer) was diluted by a certain proportion, and the absorbance of the nafurexine artificial antigen was measured at 655 nm. The corresponding protein concentration value of the artificial antigen solution was read from the curve, and the calculation data results are shown in Table 2 below.

[0139] Coupling ratio determination: 100 μg / mL of BSA in PBS was prepared, and the conjugate (i.e. nalfurafine artificial antigen II) was diluted to 100 μg / mL with PBS, and the absorbance A1 was measured at 288 nm. The absorbance A2 was measured with BSA in PBS as blank. The calculation formula of the coupling ratio γ was: γ = [(A1-A2) / ε] / (100x10 -3 / 66400), wherein ε is the molar absorption coefficient (L / mol), 66400 is the molecular weight of BSA, and 100x10 -3 is the concentration of BSA (g / L). The calculation data results are shown in Table 2.

[0140] When bovine gamma globulin was used as the carrier, the calculation formula of the coupling ratio γ was: γ = [(A1-A2) / ε] / (100x10 -3 / 43000), wherein 43000 is the molecular weight of bovine gamma globulin, and 100x10 -3 is the concentration of bovine gamma globulin (g / L). The calculation data results are shown in Table 2.

[0141] Table 2: Coupling ratio and molar absorption coefficient of each nalfurafine artificial antigen

[0142]

[0143]

[0144] From Tables 1-2, it can be seen that the structure of the artificial hapten, the activation method of the artificial hapten, and the structure of the carrier protein all have an effect on the coupling ratio when the artificial hapten is crosslinked with the carrier protein.

[0145] Animal immunization

[0146] Nalfurafine artificial antigens were prepared according to the methods of Example 1 and Comparative Examples 1-7, respectively. Each of the prepared nalfurafine artificial antigens was used to immunize New Zealand white rabbits, and the obtained immune serum was detected for titer by ELISA. The detection results are shown in Table 3.

[0147] Table 3: Titer detection results of each immune serum

[0148] Number Naloxone artificial antigen Immune serum titer Example 1 Ⅱ 1:128000 Comparative Example 1 Ⅳ 1:64000 Comparative Example 2 Ⅴ 1:32000 Comparative Example 3 Ⅵ 1:16000 Comparative Example 4 Ⅶ / Comparative Example 5 Ⅷ 1:64000 Comparative Example 6 Ⅸ / Comparative Example 7 Ⅹ 1:32000

[0149] From Table 3, compared with Example 1, the titers of the immune serum obtained by immunizing animals with the nafurafe artificial antigen prepared by the method in each of the comparative examples are all low, and cannot be used in the immune analysis. Among them, the nafurafe artificial antigen VII prepared by the method in Comparative Example 4 and the nafurafe artificial antigen IX prepared by the method in Comparative Example 6 immediately appear a large amount of precipitate during dialysis, the protein concentration of the conjugate is very low, and the coupling effect is poor, so no titer determination is made. The nafurafe artificial antigen V obtained in Comparative Example 2 and the nafurafe artificial antigen X obtained in Comparative Example 7 appear a large amount of precipitate after freezing preservation, and the stability is very poor, so they cannot be used as the immunization antigen. The titer of the immune serum obtained by immunizing animals with the nafurafe artificial antigen II (Example 1) is 1:128000, and the property is stable, and it can be completely used in the immune analysis, and can provide a more convenient, rapid and accurate way for the detection of nafurafe.

[0150] The above only describes the preferred embodiments and principles of the present application in detail, and for those skilled in the art, the specific implementation manner can be changed according to the idea provided by the present application, and these changes should be considered as the protection scope of the present application.

Claims

1. A nalfurafine artificial hapten, characterized in that, The molecular structural formula is shown as formula (I):

2. A method of preparing a nafarelin artificial hapten according to claim 1, wherein, The method comprises the following steps: (S.1) After concentrated ammonia is diluted with deionized water to obtain a DAS solution, nafurexine hydrochloride is dissolved in deionized water, the DAS solution is added to adjust the pH to be alkaline, dichloromethane is added for extraction, the dichloromethane phase is collected, dried, filtered and dried to obtain colorless oil A; (S.2) The colorless oil A obtained in step (S.1) is dissolved in anhydrous acetone, potassium carbonate and ethyl bromoacetate are added, and stirring reaction is carried out at room temperature under nitrogen protection; after the reaction is completed, filtration and drying are carried out to obtain white foamy solid B; (S.3) The white foamy solid B obtained in step (S.2) is dissolved in tetrahydrofuran and anhydrous methanol, sodium hydroxide solution is added, and stirring reaction is carried out at room temperature until completion; hydrochloric acid solution is added to adjust the pH, and then direct drying is carried out; anhydrous ethanol is added for extraction, filtration, drying and purification to obtain nafurexine artificial hapten I.

3. A method of preparing a nafarelin artificial hapten according to claim 2, characterized by, In step (S.2), the liquid ratio of the colorless oil A, anhydrous acetone, potassium carbonate and ethyl bromoacetate is 95-105:32-40:495-505:32-38 mg / mL / mg / μL.

4. The method for preparing a nalfuraphen artificial hapten according to claim 2, characterized in that, In step (S.3), the liquid ratio of the white foamy solid B, tetrahydrofuran, anhydrous methanol and sodium hydroxide solution is 95-105:1.45-1.50:1.90-2.0:4-8 mg / mL / mL / mL.

5. A nalfurafine artificial antigen, characterized by, The molecular structural formula is shown as formula (II): Formula (II); BSA is bovine serum albumin.

6. The method of preparing a nafurafe artificial antigen according to claim 5, wherein, The method comprises the following steps: The nafurexine artificial hapten I is combined with bovine serum albumin by an N-hydroxysuccinimide active ester method to obtain nafurexine artificial antigen II.

7. The method for preparing a nalfurafine artificial antigen according to claim 6, wherein, The method comprises the following steps: (a) The nafurexine artificial hapten I, cyclohexyl carbodiimide and N-hydroxysuccinimide are mixed in N,N-dimethylformamide to carry out stirring reaction at room temperature; after the reaction is completed, centrifugation is carried out, and the supernatant is taken; (b) The supernatant obtained in step (a) is added dropwise into a bovine serum albumin solution to mix uniformly to obtain a mixed solution; after the reaction is completed, dialysis and centrifugation are carried out, and the supernatant is taken to obtain the nafurexine artificial antigen II.

8. The method for preparing a nalfurafine artificial antigen according to claim 7, wherein, In step (a), the molar ratio of the nafurexine artificial hapten I, cyclohexyl carbodiimide and N-hydroxysuccinimide is 1:(1.3-1.4):(1.3-1.4).

9. The method for preparing a nalfurafine artificial antigen according to claim 7, wherein, In step (b), the concentration of the bovine serum albumin solution is 9-12 mg / mL; the volume ratio of the supernatant to the bovine serum albumin solution is 1:8-12.

10. Use of the nafurexine artificial antigen according to claim 5 in the preparation of an anti-nafurexine antibody.

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