WO2022188302A1 - 一种普瑞巴林人工半抗原、人工抗原及其制备方法和应用 - Google Patents

一种普瑞巴林人工半抗原、人工抗原及其制备方法和应用 Download PDF

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WO2022188302A1
WO2022188302A1 PCT/CN2021/102822 CN2021102822W WO2022188302A1 WO 2022188302 A1 WO2022188302 A1 WO 2022188302A1 CN 2021102822 W CN2021102822 W CN 2021102822W WO 2022188302 A1 WO2022188302 A1 WO 2022188302A1
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pregabalin
artificial
hapten
artificial antigen
reaction
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French (fr)
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邵越水
王镇
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杭州同舟生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/70—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/72—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms
    • C07C235/76—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/30—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by doubly-bound oxygen atoms
    • C07C233/31—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by doubly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/14—Preparation of carboxylic acid amides by formation of carboxamide groups together with reactions not involving the carboxamide groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9473—Anticonvulsants, e.g. phenobarbitol, phenytoin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/948—Sedatives, e.g. cannabinoids, barbiturates

Definitions

  • the invention belongs to the technical field of biochemical industry, and in particular relates to a pregabalin artificial hapten, an artificial antigen and a preparation method and application thereof.
  • pregabalin is mainly used for the treatment of partial-onset epilepsy, diabetic neuralgia, postherpetic neuralgia, and anxiety disorders in adults. It is a GABA analog that reduces the release of neurotransmitters such as glutamate, norepinephrine, substance P, and calcitonin gene-related peptide. Recent studies have found it may also play a role in the treatment of addiction to tobacco, alcohol and benzodiazepines.
  • pregabalin abuse Although reports of pregabalin abuse are currently very rare, caution is warranted, especially in those prone to substance dependence. Substance abuse symptoms are more likely to occur if pregabalin is taken in excess of the recommended dose or if the drug is administered the wrong way (eg, rectal or intranasal). Skopp et al. have reported a patient with paradoxical agitation, anxiety attacks, and abnormal thinking following pregabalin abuse. There is currently no specific antidote for the abuse of pregabalin, and insomnia, nausea, headache, diarrhea and other discomforts can occur after rapid withdrawal. Therefore, there is an urgent need to establish a fast, sensitive and accurate detection technology.
  • pregabalin mainly relies on high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography (TLC), mass spectrometry (MS), etc., but there are expensive instruments, time-consuming detection, and It requires professional and technical personnel to operate, and cannot meet the fast and accurate requirements of modern testing.
  • HPLC high performance liquid chromatography
  • GC gas chromatography
  • TLC thin layer chromatography
  • MS mass spectrometry
  • Immunoassay can make up for all the above shortcomings.
  • Immunoassay is an analytical method that uses the specific binding reaction of antigen and antibody to detect various substances (drugs, hormones, proteins, microorganisms, etc.), and the key to establishing immunoassay methods for small molecule compounds It is capable of producing antibodies with high affinity and high specificity for small molecule compounds.
  • most small-molecule compounds molecular weight less than 1000
  • pregabalin are not immunogenic, that is, they lack T cell epitopes and cannot directly induce the production of specific antibodies in animals, so small-molecule substances are called for the hapten.
  • a linking arm with an active group at the upper end is attached to a certain position of the hapten molecular structure, and then combined with a macromolecular carrier to generate a hapten-carrier conjugate (ie, an artificial antigen), an artificial antigen.
  • a hapten-carrier conjugate ie, an artificial antigen
  • the proliferation and differentiation of B cells can be induced indirectly by means of T cell epitopes, which in turn produce specific antibodies.
  • the invention provides a pregabalin artificial hapten, the pregabalin artificial hapten retains the characteristic structure of pregabalin to the greatest extent, and has an active group that can be coupled with a carrier protein, and can be used as an antigenic determination cluster.
  • a pregabalin artificial hapten the molecular structure of which is shown in (I):
  • the present invention also provides a preparation method of the pregabalin artificial hapten, comprising the following steps:
  • TLC thin layer chromatography
  • introducing a linker arm at the N-position of pregabalin, and introducing a linker arm at this modification site can preserve the characteristic structure of pregabalin to the greatest extent.
  • the linking arm used in the present invention contains a conjugated double bond structure, so that the hapten has ultraviolet absorption characteristics, not only can make the ordinary colorless under 254nm light wave.
  • Regabalin becomes a pregabalin artificial hapten that develops color under the light wave, which greatly reduces the difficulty of purification of the artificial hapten; and can enhance the specificity of the artificial hapten, maintain a stable conformation in the solution, and improve the performance of the artificial hapten.
  • Immunological properties of the resulting artificial antigen II is a conjugated double bond structure, so that the hapten has ultraviolet absorption characteristics, not only can make the ordinary colorless under 254nm light wave.
  • Regabalin becomes a pregabalin artificial hapten that develops color under the light wave, which greatly reduces the difficulty of purification of the artificial hapten; and can enhance the specificity of the artificial hapten, maintain a stable conformation in the solution, and improve the performance of the artificial hapten.
  • the present invention also provides a pregabalin artificial antigen whose molecular structural formula is shown in (II):
  • BSA bovine serum albumin
  • the present invention also provides a preparation method of the pregabalin artificial antigen, comprising: combining the pregabalin artificial hapten II with bovine serum albumin by an active ester method to obtain the pregabalin artificial antigen II.
  • step (b) the concentration of the bovine serum albumin solution is 20 mg/ml, and the volume ratio of the supernatant to the bovine serum albumin solution is 1:5.
  • the bovine serum albumin (BSA) selected in the present invention has the following advantages: 1. BSA has 583 amino acid residues, which can be easily coupled with the pregabalin hapten, and can produce different Pregabalin artificial antigen with coupling ratio, and has high immunogenicity; 2BSA is economical and low cost; 3BSA has relatively stable chemical properties, and also has good solubility and stability in acidic and weakly alkaline environments , suitable for long-term storage.
  • the present invention also provides the application of the pregabalin artificial antigen II in preparing anti-pregabalin antibody.
  • the present invention also provides an anti-pregabalin antibody, which is a globulin which is obtained by immunizing animals with the pregabalin artificial antigen and can specifically immunoreact with pregabalin.
  • the present invention also provides the application of the anti-pregabalin antibody in the detection of pregabalin.
  • the pregabalin artificial hapten of the present invention retains the characteristic structure of pregabalin 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 pregabalin artificial antigen obtained by further preparation
  • Anti-pregabalin antibodies with high affinity, high sensitivity and specificity can be obtained by immunization.
  • the titer of the immune serum obtained by immunizing New Zealand white rabbits is as high as 1:90,000, which can be used for rapid and accurate immunodetection and detection of pregabalin. Immunoassay.
  • Fig. 1 is the preparation flow chart of pregabalin artificial antigen of the present invention
  • DMAP 4-dimethylaminopyridine
  • NHS stands for N-hydroxysuccinimide
  • DCC stands for dicyclohexylcarbodiimide
  • BSA stands for bovine serum albumin
  • DMF stands for N,N-dimethylformamide, and the following same;
  • Fig. 2 is the NMR spectrum of pregabalin artificial hapten
  • Fig. 3 is the carbon nuclear magnetic resonance spectrum of pregabalin artificial hapten
  • Fig. 4 is the liquid chromatogram of pregabalin artificial hapten
  • mAU milliabsorbance units
  • min minutes
  • Fig. 5 is the mass spectrogram of pregabalin hapten
  • Fig. 6 is the ultraviolet scanning diagram of bovine serum albumin, pregabalin artificial hapten, and pregabalin artificial antigen;
  • Abs represents the ultraviolet-visible absorption spectrum
  • WL (nm) represents the wavelength (nm);
  • Fig. 7 is the preparation flow chart of comparative example 1 pregabalin artificial antigen
  • Fig. 8 is the preparation flow chart of comparative example 2 pregabalin artificial antigen
  • Fig. 9 is the preparation flow chart of comparative example 3 pregabalin artificial antigen
  • Fig. 10 is the preparation flow chart of comparative example 4 pregabalin artificial antigen
  • EDCI represents 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the same below;
  • Fig. 11 is the preparation flow chart of comparative example 5 pregabalin artificial antigen
  • BGG represents bovine gamma globulin
  • Fig. 12 is the preparation flow chart of comparative example 6 pregabalin artificial antigen
  • Figure 13 is a flow chart of the preparation of the pregabalin artificial antigen in Comparative Example 7.
  • Figure 14 is a flow chart of the preparation of the pregabalin artificial antigen in Comparative Example 8.
  • Figure 15 is a flow chart of the preparation of the pregabalin artificial antigen in Comparative Example 9.
  • Figure 16 is a flow chart of the preparation of the pregabalin artificial antigen in Comparative Example 10.
  • TLC thin layer chromatography
  • the NMR data for the pregabalin artificial hapten are:
  • the compounds deduced from the H NMR and C NMR data are basically consistent with the pregabalin artificial hapten.
  • the liquid chromatogram of the pregabalin artificial hapten is shown in FIG. 4 (ultraviolet detector, wavelength 215 nm), and the mass spectrum of the pregabalin hapten is shown in FIG. 5 .
  • the purity of the pregabalin artificial hapten obtained through purification reaches more than 99.9%, and as can be seen from Figure 5, the main mass-to-charge ratio ( m/z) are 272.1498 (M+H), 294.1321 (M+Na) and 310.0986 (M+K), which are consistent with their theoretical molecular weights of 271.14. Combined with the NMR data, it can be determined that the final compound obtained in step 2 is the artificial semi-pregabalin. Antigen I.
  • step 7 Under rapid stirring, slowly add the supernatant of step 3 to the bovine serum albumin solution in the previous step, the volume ratio of the supernatant to the bovine serum albumin solution is 1:5, and the obtained mixed solution is at 4°C. The mixture was kept at rest overnight to obtain an artificial antigen mixture.
  • curve a is the UV scanning image of the pregabalin artificial hapten
  • curve b is the UV scanning image of the pregabalin artificial antigen
  • curve c is the UV scanning image of the bovine serum albumin.
  • the maximum absorption wavelength of pregabalin artificial hapten is 270nm
  • the maximum absorption wavelength of pregabalin artificial antigen is 273nm.
  • the maximum absorption wavelength of pregabalin artificial antigen changes significantly , indicating the successful coupling of pregabalin artificial hapten and bovine serum albumin.
  • the preparation method of the pregabalin artificial antigen of this comparative example comprises the following steps:
  • step 8 Under rapid stirring, slowly drop the supernatant of step 4 into the bovine serum albumin solution in the previous step, the volume ratio of the supernatant to the bovine serum albumin solution is 1:5, and the obtained mixed solution is at 4 °C. The mixture was kept at rest overnight to obtain an artificial antigen mixture.
  • the preparation method of the pregabalin artificial antigen of this comparative example comprises the following steps:
  • the reddish-brown oil was separated by TLC thin layer chromatography.
  • step 8 Under rapid stirring, slowly drop the supernatant of step 4 into the bovine serum albumin solution in the previous step, the volume ratio of the supernatant to the bovine serum albumin solution is 1:5, and the obtained mixed solution is at 4 °C. The mixture was kept at rest overnight to obtain an artificial antigen mixture.
  • the preparation method of the pregabalin artificial antigen of this comparative example comprises the following steps:
  • step 6 Under rapid stirring, slowly add the supernatant of step 2 to the bovine serum albumin solution in the previous step, the volume ratio of the supernatant to the bovine serum albumin solution is 1:5, and the obtained mixed solution is at 4 °C. The mixture was kept at rest overnight to obtain an artificial antigen mixture.
  • the preparation method of the pregabalin artificial antigen of this comparative example comprises the following steps:
  • the reaction solution was cooled to room temperature, the stirring bar was washed with 2 ml of benzene, and the solvent was evaporated to dryness under reduced pressure to obtain 159 mg (0.624 mmol) of a light yellow oily substance, namely the pregabalin artificial hapten VII.
  • TLC detection under the ultraviolet light of 254nm, neither the raw material spot nor the product spot developed color.
  • reaction solution was transferred into a 60ml separatory funnel, washed successively with 15ml of 0.1N hydrochloric acid, 15ml of double distilled water, 15ml of saturated NaHCO solution and 15ml of saturated brine, and the organic phase was washed with anhydrous sulfuric acid. It was dried over magnesium, filtered, and dried to obtain 198 mg (0.563 mmol) of yellow oily product D;
  • pregabalin artificial hapten IX was obtained by TLC purification.
  • step 8 Under rapid stirring, slowly drop the supernatant of step 4 into the bovine serum albumin solution in the previous step, the volume ratio of the supernatant to the bovine serum albumin solution is 1:5, and the obtained mixed solution is at 4 °C. The mixture was left standing and stored overnight to obtain an artificial antigen mixture.
  • PBS was used to prepare cattle with concentrations of 0 ⁇ g/ml, 10 ⁇ g/ml, 20 ⁇ g/ml, 30 ⁇ g/ml, 40 ⁇ g/ml, 60 ⁇ g/ml, 80 ⁇ g/ml, 100 ⁇ g/ml, 120 ⁇ g/ml
  • Add 3 ml of Coomassie brilliant blue staining solution to each serum protein solution mix immediately, warm in a 30°C water bath for 5 minutes, make parallel samples for each concentration, measure the absorbance at 655nm, and draw the relationship between the protein concentration and absorbance.
  • Dilute the artificial antigen solution (prepared with PBS) according to a certain ratio, measure the absorbance value of the artificial antigen at 655 nm, and read the corresponding protein concentration value of the artificial antigen solution from the curve.
  • ⁇ is the molar absorption coefficient (L/mol)
  • 65000 is the molecular weight of bovine serum albumin
  • 100 ⁇ 10 -3 is the concentration of bovine serum albumin ( ⁇ g/ml).
  • New Zealand white rabbits were immunized with pregabalin artificial antigens II, IV, VI, VIII, and X, and the titers of the obtained immune sera were tested by ELISA. The test results are shown in Table 2.
  • Example 2 compared with Example 1 and Comparative Example 3, the titers of the immune sera obtained by immunizing animals with pregabalin artificial antigens of each comparative example were all low and could not be used in immunoassays.
  • the titers of the immune serum obtained from animal immunization with pregabalin artificial antigens II and VIII are 1:90,000 and 76,000 respectively, which can be used in immunoassays and provide a more convenient, fast and accurate way for the detection of pregabalin.
  • the preparation method of the pregabalin artificial antigen of this comparative example comprises the following steps:
  • Example 1-22 are the same as in Example 1.
  • Example 3-8 Similar to Example 1, the bovine serum albumin was replaced with bovine gamma globulin as the carrier, and the artificial hapten I of pregabalin was coupled to obtain the artificial antigen XI of pregabalin.
  • the preparation method of the pregabalin artificial antigen of this comparative example comprises the following steps:
  • Example 1 is the same as in Example 1.
  • Example 4-8 are the same as those in Example 1, to obtain pregabalin artificial antigen XII.
  • the preparation method of the pregabalin artificial antigen of this comparative example comprises the following steps:
  • Example 1 is the same as in Example 1.
  • 4-8 is similar to Comparative Example 6, except that bovine serum albumin is replaced with bovine gamma globulin,
  • This implements a preparation method of a pregabalin artificial antigen (reaction process is shown in Figure 14), comprising the following steps:
  • the preparation method of the pregabalin artificial antigen of this comparative example comprises the following steps:
  • the preparation method of the pregabalin artificial antigen of this comparative example comprises the following steps:
  • 3-7 is similar to Comparative Example 3, except that bovine serum albumin is replaced with bovine gamma globulin,
  • Test Example 2 Performance determination of pregabalin artificial antigens XI, XII, XIII, XIV, XV, XVI (1) According to the method of Test Example 1, measure the pregabalin artificial antigens XI, XII, XIII, XIV, XV, XVI The coupling ratio and concentration. See Table 3.
  • Example 1 II twenty one 11.32mg/ml 5134.25 Comparative Example 5 XI 19 14.18mg/ml 5134.25 Comparative Example 6 XII 27 12.67mg/ml 5134.25 Comparative Example 7 XIII 12 10.17mg/ml 5134.25 Comparative Example 3 VIII 19 2.95mg/ml 4721.16 Comparative Example 8 XIV 29 2.16mg/ml 4721.16 Comparative Example 9 XV 27 3.38mg/ml 4721.16 Comparative Example 10 XVI twenty one 3.07mg/ml 4721.16
  • New Zealand white rabbits were immunized with the prepared pregabalin artificial antigens XI, XII, XIII, XIV, XV, and XVI, and the titers of the obtained immune serum were detected by ELISA.
  • the detection results are shown in Table 4.
  • the titer of the immune serum obtained by the artificial antigen II obtained in Example 1 from animal immunization is the highest, and is better than the titer of the immune serum corresponding to the artificial antigen VIII, and the obtained polyclonal antibody is matched with the artificial antigen II. It can obtain better effect, can be better used in immunoassay, and can provide a more convenient and rapid way for the detection of pregabalin.

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Abstract

公开了一种普瑞巴林人工半抗原、人工抗原及其制备方法和应用。普瑞巴林人工半抗原的分子结构式如式(I)，普瑞巴林人工抗原的分子结构式如式(II)。所述应用是所述普瑞巴林人工抗原在制备抗普瑞巴林抗体中的应用。所述普瑞巴林人工半抗原最大程度地保留了普瑞巴林的特征结构，且具有可以与载体蛋白发生偶联的活性基团，可作为抗原决定簇；进一步制备获得的普瑞巴林人工抗原可免疫获得亲和力高、灵敏度高、特异性强的抗普瑞巴林抗体，经免疫新西兰白兔获得的免疫血清的效价高达1:90000，可用于对普瑞巴林进行快速、准确的免疫检测和免疫分析。

Description

一种普瑞巴林人工半抗原、人工抗原及其制备方法和应用 技术领域
本发明属于生物化工技术领域，具体涉及一种普瑞巴林人工半抗原、人工抗原及其制备方法和应用。
背景技术
普瑞巴林作为一种抗惊厥药，主要用于成人癫痫部分性发作、糖尿病性神经痛、疱疹后神经痛和焦虑障碍治疗。它是一种GABA类似物，能降低谷氨酸、去甲肾上腺素、P物质和降钙素基因相关肽等神经递质的释放。最近的研究发现其在治疗烟草、酒精和苯二氮卓类药物成瘾方面也能起到一定的作用。
虽然目前对普瑞巴林滥用的报道非常罕见，但是仍需引起注意尤其是那些有物质依赖倾向的患者。若服用超出推荐剂量的普瑞巴林或采用错误的用药方式(如直肠给药或鼻内给药)，较易出现药物滥用症状。Skopp等人曾报道过一例患者在滥用普瑞巴林后出现反常激越、焦虑发作和异常思维。目前对于普瑞巴林滥用并没有特定的解毒剂，而快速撤药后会出现失眠、恶心、头痛、腹泻等不适。因此，对建立快速、灵敏、准确的检测技术提出了迫切需求。
目前，对普瑞巴林的检测主要依靠高效液相色谱法(HPLC)、气相色谱法(GC)、薄层色谱法(TLC)、质谱法(MS)等，但是存在仪器昂贵，检测费时，并且需要专业技术人员进行操作，不能达到现代检测对快速、准确的要求。
免疫分析法可以弥补以上所有缺点，免疫分析法是一种利用抗原抗体特异性结合反应检测各种物质(药物、激素、蛋白质、微生物等)的分析方法，建立小分子化合物的免疫分析方法的关键是能够制造出对小分子化合物具有高亲和力和高特异性的抗体。但是，由于包括普瑞巴林在内的大多数小分子化合物(分子量小于1000)，不具有免疫原性，即缺乏T细胞表位而无法直接诱导动物机体产生特异性抗体，故小分子物质被称为半抗原。通过适当的化学修饰，在半抗原分子结构的某个位置上带上端部为活性基团的连接臂，再与大分子载体结合，生成半抗原-载体偶联物(即人工抗原)，人工抗原可以借助T细胞表位来间接诱导B细胞的增殖和分化，继而产生特异性抗体。
发明内容
本发明提供了一种普瑞巴林人工半抗原，该普瑞巴林人工半抗原最大程度地保留了普瑞巴林的特征结构，且具有可以与载体蛋白发生偶联的活性基团，可作为抗原决定簇。
一种普瑞巴林人工半抗原，其分子结构式如(Ⅰ)所示：
Figure PCTCN2021102822-appb-000001
本发明还提供了该普瑞巴林人工半抗原的制备方法，包括以下步骤：
(1)将普瑞巴林溶于甲醇中，再加入浓硫酸，于68℃下回流搅拌反应24小时，反应产物经碳酸氢钠固体中和，转干，再用四氢呋喃提取，得略黄色油状物A。
(2)将略黄色油状物A与顺丁烯二酸酐按摩尔比1:2～3混合于二氯甲烷中，再加入4-二甲氨基吡啶，在室温下搅拌反应20小时，反应产物经纯化水洗涤，取有机相，干燥、过滤得棕黄色油状物；
将上述黄色油状物通过薄层色谱(TLC)分离得淡黄色油状物，即普瑞巴林半抗原Ⅰ；TLC：层析液为95％乙醇：二氯甲烷：1,4-二氧六环：氨水＝8:10:1:1，产物R f＝0.4。
通过上述方法，在普瑞巴林的N位上引入连接臂，在该修饰位点上引入连接臂能最大程度地保留普瑞巴林的特征结构。
与采用一般的饱和链烃作为连接臂相比，本发明所采用的连接臂中含有共轭双键结构，使得该半抗原具备紫外吸收特性，不仅能够使原本在254nm光波下不显色的普瑞巴林变成在该光波下显色的普瑞巴林人工半抗原，大大降低了该人工半抗原提纯的难度；而且可使该人工半抗原的特异性增强，在溶液中保持构象稳定，提高所得到的人工抗原II的免疫特性。
本发明还提供了一种普瑞巴林人工抗原，其分子结构式如(Ⅱ)所示：
Figure PCTCN2021102822-appb-000002
其中，BSA为牛血清蛋白。
本发明还提供了一种所述普瑞巴林人工抗原的制备方法，包括：通过活泼酯法使所述普瑞巴林人工半抗原Ⅱ与牛血清蛋白结合，获得所述普瑞巴林人工抗原Ⅱ。
具体地，包括以下步骤：
(a)将所述普瑞巴林人工半抗原Ⅰ、与N-羟基琥珀酰亚胺、二环己基碳二亚胺以摩尔比为1∶1.35～1.5∶1.35～1.5溶解于N，N-二甲基甲酰胺中，室温搅拌反应18小时，反应结束后离心取上清液；
(b)将上清液滴加到牛血清蛋白溶液中，混合液于4℃下静置过夜，经pH＝12.00的0.5％碳酸钠水溶液和磷酸盐缓冲溶液(PBS)透析、离心取上清液，获得普瑞巴林人工抗原Ⅱ。
本发明中牛血清蛋白溶液是将牛血清蛋白溶于磷酸根离子浓度为0.01mol/L，pH＝7.2～7.4的磷酸盐缓冲液中制成的。
步骤(b)中，所述牛血清蛋白溶液的浓度为20mg/ml，上清液与牛血清蛋白溶液的体积比为1∶5。
本发明选用的牛血清蛋白(BSA)作为大分子载体，与其他载体蛋白相比，具有以下优点：①BSA具有583个氨基酸残基，较容易与普瑞巴林半抗原发生偶联，可制得不同偶联比的普瑞巴林人工抗原，且具有较高的免疫原性；②BSA经济实惠，成本低；③BSA的化学性质比较稳定，在酸性和弱碱性的环境下也有很好的溶解度和稳定性，适合长期储存。
本发明还提供了所述普瑞巴林人工抗原Ⅱ在制备抗普瑞巴林抗体中的应用。
本发明还提供了一种抗普瑞巴林抗体，是由所述普瑞巴林人工抗原经动物免疫得到的、可与普瑞巴林发生特异性免疫反应的球蛋白。
本发明还提供了所述抗普瑞巴林抗体在检测普瑞巴林中的应用。
试验发现，将所述普瑞巴林人工抗原免疫新西兰白兔，获得的免疫血清的效价为1:90000。表明本发明的普瑞巴林人工抗原可免疫获得亲和力高、灵敏度高、特异性强的抗普瑞巴林抗体，该抗普瑞巴林抗体可用于普瑞巴林的免疫检测和分析。
与现有技术相比，本发明的有益效果为：
本发明的普瑞巴林人工半抗原最大程度地保留了普瑞巴林的特征结构，且具有可以与载体蛋白发生偶联的活性基团，可作为抗原决定簇；进一步制备获得的普瑞巴林人工抗原可免疫获得亲和力高、灵敏度高、特异性强的抗普瑞巴林抗体，经免疫新西兰白兔获得的免疫血清的效价高达1:90000，可用于对普瑞巴林进行快速、准确的免疫检测和免疫分析。
附图说明
图1为本发明普瑞巴林人工抗原的制备流程图；
其中，DMAP表示4-二甲氨基吡啶，NHS表示N-羟基琥珀酰亚胺，DCC表示二环己基碳二亚胺，BSA表示牛血清蛋白，DMF表示N,N-二甲基甲酰胺，下同；
图2为普瑞巴林人工半抗原的核磁共振氢谱图；
图3为普瑞巴林人工半抗原的核磁共振碳谱图；
图4为普瑞巴林人工半抗原的液相色谱图；
其中，mAU表示毫吸光度单位，min表示分钟；
图5为普瑞巴林半抗原的质谱图；
图6为牛血清蛋白、普瑞巴林人工半抗原、普瑞巴林人工抗原的紫外扫描图；
其中，Abs表示紫外-可见吸收光谱，WL(nm)表示波长(nm)；
图7为对比例1普瑞巴林人工抗原的制备流程图；
图8为对比例2普瑞巴林人工抗原的制备流程图；
图9为对比例3普瑞巴林人工抗原的制备流程图；
图10为对比例4普瑞巴林人工抗原的制备流程图；
其中，EDCI表示表示1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，下同；
图11为对比例5普瑞巴林人工抗原的制备流程图；
其中，BGG表示牛丙种球蛋白；
图12为对比例6普瑞巴林人工抗原的制备流程图；
图13为对比例7普瑞巴林人工抗原的制备流程图。
图14为对比例8普瑞巴林人工抗原的制备流程图。
图15为对比例9普瑞巴林人工抗原的制备流程图。
图16为对比例10普瑞巴林人工抗原的制备流程图。
实施例1
本发明实施例普瑞巴林人工抗原的制备方法(反应历程如图1)，包括以下步骤：
(1)人工半抗原的制备：
①称取200mg(1.258mmol)普瑞巴林置于50ml单口圆底烧瓶中，加入10ml甲醇和0.5ml浓硫酸，加入搅拌子，置于68℃油浴中搅拌回流反应24小时；反应结束后，冷却至室温，向反应体系中慢慢加入NaHCO 3固体调节pH＝7-8，将残液减压蒸干，用15ml四氢呋喃提取、过滤、转干后得到210mg(1.214mmol)略黄色油状产物A；
②在50ml单口圆底烧瓶中，将上步210mg(1.214mmol)略黄色油状产物A溶于10ml的二氯甲烷中，加入238mg(2.428mmol)的顺丁烯二酸酐和20mg4-二甲氨基吡啶(DMAP)，在室温下搅拌反应20小时；反应结束后，将反应液转入60ml的分液漏斗中，用15ml纯化水洗涤两次，有机相经无水硫酸镁干燥，过滤，转干后得粗产物160mg为棕黄色油状物。
该粗产物再经过薄层色谱(TLC)分离，溶剂和洗脱剂是无水乙醇，层析液是95％乙醇：二氯甲烷：1,4-二氧六环：氨水＝8:10:1:1，收集Rf＝0.4的点，用150ml无水乙醇洗脱，过滤，浓缩得淡黄色油状产物72mg(0.266mmol)，即普瑞巴林人工半抗原Ⅰ。
普瑞巴林人工半抗原的核磁共振氢谱见图2，核磁共振碳谱见图3。
普瑞巴林人工半抗原的核磁共振数据为：
1H NMR(400MHz,Chloroform-d)δ9.13(s,1H)，6.27–6.19(m,2H)，3.62(s,3H)，3.35–3.14(m,2H)，2.37–2.11(m,3H)，1.60(dq,J＝13.2,6.4Hz,1H)，1.23–1.06(m,2H)，0.85(dd,J＝10,6.4Hz,6H)。
13C NMR(101MHz,Chloroform-d)δ173.93，166.87，136.45，65.82，58.04，51.73，43.34，41.56，37.27，32.98，25.14，22.61，22.56，18.34，15.24。
根据核磁共振氢谱和碳谱数据推测出的化合物基本与普瑞巴林人工半抗原吻合。
普瑞巴林人工半抗原的液相色谱图见图4(紫外检测器，波长215nm)，普瑞巴林半抗原的质谱图见图5。
从图4可以看出经过纯化得到的普瑞巴林人工半抗原的纯度达到99.9％以上，从图5可以看出本实施例得到的普瑞巴林人工半抗原的分子离子峰的主要质荷比(m/z)为272.1498(M+H)、294.1321(M+Na)和310.0986(M+K)，与其理论分子量271.14吻合，结合核磁共振数据可以确定步骤②得到的最终化合物就是普瑞巴林人工半抗原Ⅰ。
(2)普瑞巴林人工抗原的制备：
③将72mg(0.266mmol)普瑞巴林人工半抗原置于50ml圆底烧瓶中，加搅拌子，加入3.6ml N，N-二甲基甲酰胺(DMF)溶解，再加入46mg(0.400mmol)N-羟基琥珀酰亚胺(NHS)和82mg(0.398mmol)二环己基碳二亚胺(DCC)，25℃搅拌反应18小时，反应结束后离心，取上清液，备用。
④称取14.500g(0.0405mol)十二水磷酸氢二钠，43.875g(0.75mol)氯化钠，1.495g(0.00958mol)二水磷酸二氢钠用双蒸水溶解并定容至5.0L，得到磷酸根离子浓度为0.01mol/L，钠离子浓度为0.17mol/L的磷酸盐缓冲液(PBS)。
⑤称取25gNa 2CO 3固体粉末溶于纯化水中并定容至5L，用2N的NaOH水溶液调pH＝12.00±0.01，制得碱性透析液。
⑥称取0.36g牛血清蛋白溶于18ml步骤④的PBS中，得到浓度为20mg/ml的牛血清蛋白溶液。
⑦在快速搅拌下，将步骤③的上清液缓慢滴加到上步的牛血清蛋白溶液中，上清液与牛血清蛋白溶液的体积比为1:5，得到的混合液在4℃条件下静置保存过夜，得到人工抗原混合液。
⑧将上步人工抗原混合液转入透析袋中，用步骤⑤的碱性透析液透析两天，每天各一次，再将透析袋转入④中得PBS缓冲液中透析7次，每两次时间间隔不少于两小时，透析结束后，离心取上清液即得到人工抗原：普瑞巴林-牛血清蛋白偶联物(如式Ⅱ)。普瑞巴林人工抗原制备前后的紫外扫描图见图6。
图6中，曲线a为普瑞巴林人工半抗原的紫外扫描图，曲线b为普瑞巴林人工抗原的紫外扫描图，曲线c为牛血清蛋白的紫外扫描图。普瑞巴林人工半抗原的最大吸收波长为270nm，普瑞巴林人工抗原的最大吸收波长为273nm，与普瑞巴林半抗原、牛血清蛋白相比，普瑞巴林人工抗原的最大吸收波长出现明显变化，说明普瑞巴林人工半抗原和牛血清蛋白偶联成功。
对比例1
本对比例的普瑞巴林人工抗原的制备方法(反应历程如图7)，包括以下步骤：
(1)普瑞巴林人工半抗原的制备：
①称取200mg(1.258mmol)普瑞巴林置于50ml单口圆底烧瓶中，加入10ml甲醇和0.5ml浓硫酸，加入搅拌子，置于68℃油浴中搅拌回流反应24小时；反应结束后，冷却至室温，向反应体系中慢慢加入NaHCO 3固体调节pH＝7-8，将残液减压蒸干，用15ml四氢呋喃提取、过滤、转干后得到195mg(1.127mmol)略黄色油状产物A；
②将上步195mg(1.127mmol)略黄色油状产物A溶于5mlN,N-二甲基甲酰胺(DMF)中，置于50ml三口圆底烧瓶中，中间口接冷凝管和装有无水CaCl 2的干燥管，左口通入氮气作为保护气，右口用橡皮塞塞紧，从右口加入54mg(1.350mmol)NaH(质量分数为60％)固体，将反应瓶置于60℃油浴中搅拌反应30分钟，然后用注射器逐滴加入246μL(1.688mmol)溴乙酸叔丁酯和2mlN,N-二甲基甲酰胺的混合溶液，继续保持60℃反应17小时；
结束反应，冷却至室温，用DMF将反应液转入50ml单口圆底烧瓶中，减压蒸干溶剂得黄色粘稠物，向瓶中加入15ml水，用15ml*2乙酸乙酯萃取，合并有机相，分别用20ml纯化水和20ml饱和的食盐水洗涤有机相，取有机相，用无水Na 2SO 4干燥、过滤、减压蒸干得棕黄色油状物B285mg(0.993mmol)。
③将上步棕黄色油状物B285mg(0.993mmol)溶于15ml二氯甲烷和5ml 三氟乙酸中，25℃下快速搅拌反应5小时；反应结束，减压蒸干溶剂，加入10ml10％的碳酸钾溶液，用15ml*3的乙酸乙酯洗涤水相，收集水相，用6N的盐酸调pH＝3，再用20ml*3的乙酸乙酯萃取，收集有机相，用无水硫酸镁干燥，过滤，减压蒸干得红棕色油状物；
该红棕色油状物再经过TLC分离，溶剂和洗脱剂是无水乙醇，层析液是95％乙醇：二氯甲烷：1,4-二氧六环：氨水＝8:10:1:1，收集Rf＝0.1～0.5的点，用150ml无水乙醇洗脱，过滤，浓缩得棕黄色油状物63mg(0.273mmol)，即普瑞巴林人工半抗原Ⅲ。
(2)普瑞巴林人工抗原的制备：
④将上步普瑞巴林人工半抗原Ⅲ63mg(0.273mmol)置于50ml圆底烧瓶中，加入3.15ml N,N-二甲基甲酰胺(DMF)，再加入47mg(0.409mmol)N-羟基琥珀酰亚胺(NHS)和84mg(0.409mmol)环己基碳酰二亚胺(DCC)，室温搅拌反应过夜，反应结束离心，取上清液，备用。
⑤称取14.5g十二水磷酸氢二钠，43.875g氯化钠，1.495g二水磷酸二氢钠用双蒸水溶解定容至5.0L，得到磷酸盐缓冲液(PBS)，pH为7.2～7.4。
⑥称取25gNa 2CO 3粉末溶于纯化水中并定容至5L，用2N的NaOH水溶液调pH＝12.00±0.01，制得碱性透析液。
⑦称取0.320g牛血清蛋白溶于16ml步骤⑤的PBS中，得到浓度为20mg/ml的牛血清蛋白溶液。
⑧在快速搅拌下，将步骤④的上清液缓慢滴加到上步的牛血清蛋白溶液中，上清液与牛血清蛋白溶液的体积比为1:5，得到的混合液在4℃条件下静置保存过夜，得到人工抗原混合液。
⑨将上步人工抗原混合液转入透析袋中，用步骤⑥的碱性透析液透析两天，每天各一次，再将透析袋转入⑤中得PBS中透析7次，每两次时间间隔不少于两小时，透析结束后，离心取上清液即得到人工抗原Ⅳ。
对比例2
本对比例的普瑞巴林人工抗原的制备方法(反应历程如图8)，包括以下步骤：
(1)普瑞巴林人工半抗原的制备：
①称取200mg(1.258mmol)普瑞巴林置于50ml单口圆底烧瓶中，加入10ml甲醇和0.5ml浓硫酸，加入搅拌子，将油浴升温至68℃搅拌回流反应24小时；反应结束后，冷却至室温，向反应体系中慢慢加入NaHCO 3固体调节pH＝7-8，将残液减压蒸干，用15ml四氢呋喃提取、过滤、转干后得到202mg(1.168mmol)略黄色油状产物A；
②将上步202mg(1.168mmol)略黄色油状产物A溶于5mlN,N-二甲基甲酰胺(DMF)中，置于50ml三口圆底烧瓶中，中间口接冷凝管和装有无水CaCl 2的干燥管，左口通入氮气，右口塞橡皮塞，从右口加入56mg(1.402mmol)NaH(质量分数为60％)固体，将反应瓶置于60℃油浴中回流搅拌反应30分钟，然后用注射器逐滴加入368μL(1.752mmol)溴己酸叔丁酯和2mlN,N-二甲基甲酰胺的混合溶液，继续保持60℃反应20小时；
结束反应，冷却至室温，用DMF将反应液转入50ml单口圆底烧瓶中，减压蒸干溶剂得黄色粘稠物，向瓶中加入15ml水，用15ml*2乙酸乙酯萃取，合并有机相，分别用20ml纯化水和20ml饱和的食盐水洗涤有机相，取有机相，用无水Na 2SO 4干燥、过滤、减压蒸干得棕黄色油状物B293mg(0.854mmol)。
③将上步棕黄色油状物B293mg(0.854mmol)溶于15ml二氯甲烷和5ml三氟乙酸中，25℃下快速搅拌反应5小时；反应结束，减压蒸干溶剂，加入10ml10％的碳酸钾溶液，用15ml*3的乙酸乙酯洗涤水相，收集水相，用6N的盐酸调pH＝3，再用20ml*3的乙酸乙酯萃取，收集有机相，用无水硫酸镁干燥，过滤，减压蒸干得红棕色油状物；
该红棕色油状物再经过TLC薄层色谱分离，溶剂和洗脱剂是无水乙醇，层析液是95％乙醇：二氯甲烷：1,4-二氧六环：氨水＝8:10:1:1，收集Rf＝0.1～0.5的点，用150ml无水乙醇洗脱，过滤，浓缩得棕黄色油状产物82mg(0.286mmol)，即普瑞巴林人工半抗原Ⅴ。
(2)普瑞巴林人工抗原的制备：
④将上步普瑞巴林人工半抗原Ⅴ82mg(0.286mmol)置于50ml圆底烧瓶中，加入4.10ml N,N-二甲基甲酰胺(DMF)，再加入49mg(0.429mmol)N-羟基琥珀酰亚胺(NHS)和88mg(0.429mmol)环己基碳酰二亚胺(DCC)，室温搅拌反应过夜，反应结束离心，取上清液，备用。
⑤称取14.5g十二水磷酸氢二钠，43.875g氯化钠，1.495g二水磷酸二氢钠用双蒸水溶解定容至5.0L，得到PBS，pH为7.2～7.4。
⑥称取25gNa 2CO 3固体粉末溶于纯化水中并定容至5L，用2N的NaOH水溶液调节pH＝12.00±0.01，制得碱性透析液。
⑦称取0.420g牛血清蛋白溶于21ml步骤⑤的PBS中，得到浓度为20mg/ml的牛血清蛋白溶液。
⑧在快速搅拌下，将步骤④的上清液缓慢滴加到上步的牛血清蛋白溶液中，上清液与牛血清蛋白溶液的体积比为1:5，得到的混合液在4℃条件下静置保存过夜，得到人工抗原混合液。
⑨将上步人工抗原混合液转入透析袋中，用步骤⑥的碱性透析液透析两天，每天各一次，再将透析袋转入⑤中得PBS缓冲液中透析7次，每两次时间间隔不少于两小时，透析结束后，离心取上清液即得到人工抗原Ⅵ。
对比例3
本对比例的普瑞巴林人工抗原的制备方法(反应历程如图9)，包括以下步骤：
(1)普瑞巴林人工半抗原的制备：
①称取普瑞巴林100mg(0.628mmol)置于50ml单口圆底烧瓶中，加入2ml3A分子筛干燥过的苯，加入搅拌子，在0℃的冰浴中，搅拌溶解，将三氟乙酸酐0.133ml(0.942mmol)缓慢滴加到上述反应液中，然后撤去冰浴，室温搅拌反应1hrs，之后转入78℃油浴中搅拌回流反应3hrs；
反应结束，将反应液冷却至室温，搅拌子用2ml苯洗涤，减压蒸干溶剂，得到152mg(0.596mmol)淡黄色油状物即普瑞巴林人工半抗原Ⅶ。TLC检测，在254nm的紫外光下原料点和产物点都不显色。
(2)普瑞巴林人工抗原的制备：
②将普瑞巴林人工半抗原152mg(0.596mmol)溶于7.6mlN,N-二甲基甲酰胺(DMF)，再加入93mg(0.805mmol)N-羟基琥珀酰亚胺(NHS)和166mg(0.805mmol)二环己基碳酰二亚胺(DCC)，25℃搅拌反应过夜，反应结束，离心，取上清液备用。
③称取14.5g十二水磷酸氢二钠，43.875g氯化钠，1.495g二水磷酸二氢钠用双蒸水溶解定容至5.0L，得到PBS，pH为7.2～7.4。
④称取25gNa 2CO 3固体粉末溶于纯化水中并定容至5L，用2N的NaOH水溶液调节pH＝12.00±0.01，制得碱性透析液。
⑤称取0.19g牛血清蛋白溶于38ml步骤③的PBS中，得到浓度为5mg/ml牛血清蛋白溶液。
⑥在快速搅拌下，将步骤②的上清液缓慢滴加到上步的牛血清蛋白溶液中，上清液与牛血清蛋白溶液的体积比为1:5，得到的混合液在4℃条件下静置保存过夜，得到人工抗原混合液。
⑦将上步人工抗原混合液转入透析袋中，用步骤④的碱性透析液透析两天，每天各一次，再将透析袋转入③中得PBS缓冲液中透析7次，每两次时间间隔不少于两小时，透析结束后，离心取上清液即得到人工抗原Ⅷ。
对比例4
本对比例的普瑞巴林人工抗原的制备方法(反应历程如图10)，包括以下步骤：
(1)普瑞巴林人工半抗原的制备：
①称取普瑞巴林100mg(0.628mmol)置于50ml单口圆底烧瓶中，加入2ml3A分子筛干燥过的苯，加入搅拌子，在0℃的冰浴中，搅拌溶解，将三氟乙酸酐0.133ml(0.942mmol)缓慢滴加到上述反应液中，然后撤去冰浴，室温搅拌反应1hrs，之后转入78℃油浴中搅拌回流反应3hrs；
反应结束，将反应液冷却至室温，搅拌子用2ml苯洗涤，减压蒸干溶剂，得到159mg(0.624mmol)淡黄色油状物即普瑞巴林人工半抗原Ⅶ。TLC检测，在254nm的紫外光下原料点和产物点都不显色。
②在50ml单口圆底烧瓶中，将159mg(0.624mmol)淡黄色油状物Ⅶ溶于15ml的二氯甲烷中，加入108mg的N-羟基琥珀酰亚胺(NHS，0.936mmol)和179mg的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI，0.936mmol)，26℃搅拌反应20小时；
反应结束后，将反应液转入60ml的分液漏斗中，依次用15ml0.1N的盐酸、 15ml的双蒸水、15ml的饱和NaHCO 3溶液和15ml的饱和食盐水洗涤，有机相经无水硫酸镁干燥，过滤，转干后得198mg(0.563mmol)黄色油状产物D；
③将111mg(0.732mmol)对氨甲基苯甲酸溶于3ml双蒸水和12ml四氢呋喃的混合液中，获得对氨甲基苯甲酸溶液，待用；将198mg(0.563mmol)黄色油状产物D溶于6ml的四氢呋喃中，获得产物D溶液；将对甲氨基苯甲酸溶液逐滴加入到搅拌着的产物D溶液中，再加入1.1ml1N的NaOH水溶液，此时溶液pH＝10，26℃搅拌反应4小时；
反应结束后滴加6N的盐酸调pH＝6，用3×15ml乙酸乙酯萃取，合并有机相，经干燥、过滤、转干后得到淡黄色泡状固体，将该淡黄色泡状固体溶于2ml无水乙醇中，用TLC纯化得到普瑞巴林人工半抗原Ⅸ128mg(0.330mmol)。TLC采用的层析液为95％乙醇：二氯甲烷：1,4-二氧六环：氨水＝8:10:1:1，收集Rf＝0.5的点，用150ml无水乙醇洗脱、过滤、浓缩得产物，即普瑞巴林人工半抗原Ⅸ。
(2)普瑞巴林人工抗原的制备：
④将普瑞巴林人工半抗原Ⅸ128mg(0.330mmol)溶于6.4mlN,N-二甲基甲酰胺(DMF)，再加入51mg(0.446mmol)N-羟基琥珀酰亚胺(NHS)和92mg(0.446mmol)二环己基碳酰二亚胺(DCC)，25℃搅拌反应过夜，反应结束，离心，取上清液备用。
⑤称取14.5g十二水磷酸氢二钠，43.875g氯化钠，1.495g二水磷酸二氢钠用双蒸水溶解定容至5.0L，得到PBS，pH为7.2～7.4。
⑥称取25gNa 2CO 3固体粉末溶于纯化水中并定容至5L，用2N的NaOH水溶液调节pH＝12.00±0.01，制得碱性透析液。
⑦称取0.16g牛血清蛋白溶于32ml步骤⑤的PBS缓冲液中，得到浓度为5mg/ml牛血清蛋白溶液。
⑧在快速搅拌下，将步骤④的上清液缓慢滴加到上步的牛血清蛋白溶液中，上清液与牛血清蛋白溶液的体积比为1:5，得到的混合液在4℃条件下静置保存过夜，得到人工抗原混合液。
⑨将上步人工抗原混合液转入透析袋中，用步骤⑥的碱性透析液透析两天，每天各一次，再将透析袋转入⑤中得PBS缓冲液中透析7次，每两次时间间隔不少于两小时，透析结束后，离心取上清液即得到人工抗原Ⅹ。
检测例1普瑞巴林人工抗原Ⅱ、Ⅳ、Ⅵ、Ⅷ、Ⅹ的性能测定
(1)普瑞巴林人工抗原的鉴定：
摩尔吸收系数ε：用步骤⑤的PBS配制配制浓度为0μg/ml、5μg/ml、10μg/ml、20μg/ml、30μg/ml、40μg/ml的普瑞巴林人工半抗原溶液，通过紫外扫描图可知普瑞巴林半抗原的最大吸收波长为270nm，在270nm处测吸光值，每个浓度做平行样。摩尔吸光系数(即摩尔吸收系数)的计算公式为：ε＝吸光值/摩尔浓度。
偶联物蛋白浓度的测定：用PBS配制浓度为0μg/ml、10μg/ml、20μg/ml、30μg/ml、40μg/ml、60μg/ml、80μg/ml、100μg/ml、120μg/ml的牛血清蛋白溶液各1ml，加入3ml考马斯亮蓝染色液，立即混匀，30℃水浴温热5分钟，每个浓度做平行样，在655nm处测吸光值，绘制蛋白浓度与吸光值的关系曲线。将人工抗原溶液(用PBS配制)按一定比例稀释，在655nm处测得人工抗原的吸光值，从曲线上读取人工抗原溶液的相应蛋白浓度值。
偶联比测定：配制100μg/ml的牛血清蛋白的PBS溶液，将偶联物(即普瑞巴林人工抗原)用PBS稀释到100μg/ml，在292nm处测得吸光值A 1，以PBS为空白测得吸光值A 2，则偶联比率γ为：γ＝[(A 1－A 2)/ε]/(100×10 -3/65000)。
其中ε为摩尔吸光系数(L/mol)，65000为牛血清蛋白的分子量，100×10 -3为牛血清蛋白浓度(μg/ml)。
采用牛丙种球蛋白作载体时，偶联比的计算式为：γ＝[(A 1－A 2)/ε]/(100×10 -3/150000)；其中，150000为牛丙种球蛋白的分子量。
表1各普瑞巴林人工抗原的偶联比和摩尔吸收系数
编号 人工抗原 偶联比 偶联物蛋白浓度 摩尔吸收系数
实施例1 Ⅱ 21 11.32mg/ml 5134.25
对比例1 Ⅳ 17 12.60mg/ml 5328.46
对比例2 Ⅵ 20 15.35mg/ml 4678.23
对比例3 Ⅷ 19 2.95mg/ml 4721.16
对比例4 Ⅹ 26 3.17mg/ml 5812.44
由表1可见，人工半抗原的结构，对人工半抗原与载体蛋白偶联时的结合比 影响很大。
(2)动物免疫
将普瑞巴林人工抗原Ⅱ、Ⅳ、Ⅵ、Ⅷ、Ⅹ免疫新西兰白兔，得到的免疫血清经ELISA方法检测其效价，检测结果见表2。
表2各免疫血清的效价检测结果
编号 普瑞巴林人工抗原 免疫血清效价
实施例1 Ⅱ 90000
对比例1 Ⅳ 20000
对比例2 Ⅵ 30000
对比例3 Ⅷ 76000
对比例4 Ⅹ 50000
由表2可见，与实施例1和对比例3相比，利用各对比例普瑞巴林人工抗原进行动物免疫获得的免疫血清，其效价均偏低，不能用于免疫分析中。而利用普瑞巴林人工抗原Ⅱ和VIII进行动物免疫获得的免疫血清，其效价分别达1:90000和76000，可用于免疫分析中，能为普瑞巴林的检测提供更加方便快速准确的途径。
针对实施例1和对比例3，仍需进一步探索不同的载体蛋白和不同的偶联方法是否能提高抗原的性能。故实施了对比例5到对比例10。
对比例5
本对比例的普瑞巴林人工抗原的制备方法(反应历程如图11)，包括以下步骤：
(1)普瑞巴林人工半抗原的制备：
①-②②与实施例1相同。
(2)普瑞巴林人工抗原的制备：
③-⑧与实施例1相似，将牛血清蛋白换成牛丙种球蛋白作为载体，与普瑞巴林人工半抗原Ⅰ进行偶联，得到普瑞巴林人工抗原Ⅺ。
对比例6
本对比例的普瑞巴林人工抗原的制备方法(反应历程如图12)，包括以下步骤：
(1)普瑞巴林人工半抗原的制备：
①-②与实施例1相同。
(2)普瑞巴林人工抗原的制备：
③称取72mg(0.266mmol)普瑞巴林人工半抗原Ⅰ置于50ml圆底烧瓶中，加入4ml N,N-二甲基甲酰胺(DMF)，再加入37μL(0.266mmol)三乙胺和34μL(0.266mmol)氯甲酸异丁酯，室温搅拌反应18小时，反应结束后离心，取上清液，备用。
④-⑧与实施例1相同，得到普瑞巴林人工抗原Ⅻ。
对比例7
本对比例的普瑞巴林人工抗原的制备方法(反应历程如图13)，包括以下步骤：
(1)普瑞巴林人工半抗原的制备：
①-②与实施例1相同。
(2)普瑞巴林人工抗原的制备：
③与对比例6相同
④-⑧与对比例6相似，只是将牛血清蛋白换成牛丙种球蛋白，
与普瑞巴林人工半抗原Ⅰ进行偶联，得普瑞巴林人工抗原XIII。
对比例8
本实施一种普瑞巴林人工抗原的制备方法(反应历程如图14)，包括以下步骤：
(1)普瑞巴林人工半抗原的制备：
①与对比例3相同。
(2)普瑞巴林人工抗原的制备：
②-⑦与对比例3相似，只是将牛血清蛋白换成牛丙种球蛋白，
与普瑞巴林人工半抗原Ⅶ进行偶联，得普瑞巴林人工抗原XIV。
对比例9
本对比例的普瑞巴林人工抗原的制备方法(反应历程如图15)，包括以下步骤：
(1)普瑞巴林人工半抗原的制备：
①与对比例3相同。
(2)普瑞巴林人工抗原的制备：
②称取152mg(0.596mmol)普瑞巴林人工半抗原Ⅶ置于50ml圆底烧瓶中，加入7.6ml N,N-二甲基甲酰胺(DMF)，再加入83μL(0.596mmol)三乙胺和76μL(0.596mmol)氯甲酸异丁酯，室温搅拌反应18小时，反应结束后离心，取上清液，备用。
③-⑦与对比例3相同，得普瑞巴林人工抗原XV。
对比例10
本对比例的普瑞巴林人工抗原的制备方法(反应历程如图16)，包括以下步骤：
(1)普瑞巴林人工半抗原的制备：
①与对比例3相同。
(2)普瑞巴林人工抗原的制备：
②称取152mg(0.596mmol)普瑞巴林人工半抗原Ⅶ置于50ml圆底烧瓶中，加入7.6ml N,N-二甲基甲酰胺(DMF)，再加入83μL(0.596mmol)三乙胺和76μL(0.596mmol)氯甲酸异丁酯，室温搅拌反应18小时，反应结束后离心，取上清液，备用。
③-⑦与对比例3相似，只是将牛血清蛋白换成牛丙种球蛋白，
与普瑞巴林人工半抗原Ⅶ进行偶联，得普瑞巴林人工抗原XVI。
检测例2普瑞巴林人工抗原XI、XII、XIII、XIV、XV、XVI的性能测定(1)根据检测例1的方法，测得普瑞巴林人工抗原XI、XII、XIII、XIV、XV、XVI的偶联比和浓度。见表3。
表3各普瑞巴林人工抗原的偶联比和浓度
编号 人工抗原 偶联比 偶联物蛋白浓度 摩尔吸收系数
实施例1 Ⅱ 21 11.32mg/ml 5134.25
对比例5 Ⅺ 19 14.18mg/ml 5134.25
对比例6 Ⅻ 27 12.67mg/ml 5134.25
对比例7 XIII 12 10.17mg/ml 5134.25
对比例3 Ⅷ 19 2.95mg/ml 4721.16
对比例8 XIV 29 2.16mg/ml 4721.16
对比例9 XV 27 3.38mg/ml 4721.16
对比例10 XVI 21 3.07mg/ml 4721.16
(2)动物免疫
将制备的普瑞巴林人工抗原XI、XII、XIII、XIV、XV、XVI免疫新西兰白兔，得到的免疫血清经ELISA方法检测其效价，检测结果见表4。
表4各免疫血清的效价检测结果
编号 人工抗原 免疫血清效价
实施例1 Ⅱ 90000
对比例5 Ⅺ 70000
对比例6 Ⅻ 45000
对比例7 XIII 55000
对比例3 Ⅷ 76000
对比例8 XIV 65000
对比例9 XV 60000
对比例10 XVI 30000
由表4可见，实施例1得到的人工抗原Ⅱ进行动物免疫获得的免疫血清效价最高，且更优于人工抗原Ⅷ对应的免疫血清的效价，其得到的多克隆抗体与人工抗原Ⅱ搭配能获得更好的效果，可以更好的用于免疫分析中，能为普瑞巴林的检测提供更加方便快速的途径。

Claims (9)

  1. 一种普瑞巴林人工半抗原，其特征在于，其分子结构式如(Ⅰ)所示：
    Figure PCTCN2021102822-appb-100001
  2. 权利要求1所述普瑞巴林人工半抗原的制备方法，其特征在于，包括以下步骤：
    (1)将普瑞巴林溶于甲醇，加入浓硫酸，于68℃下搅拌反应24h，反应产物经调碱、蒸干、提取得略黄色油状产物A；
    (2)将上一步所获略黄色油状产物A与顺丁烯二酸酐按摩尔比1:2～3混合于二氯甲烷中，于室温下搅拌反应20h，反应产物经洗涤、干燥、过滤、转干后，再经薄层色谱分离得淡黄色油状产物，即普瑞巴林人工半抗原Ⅰ。
  3. 一种普瑞巴林人工抗原，其特征在于，其分子结构式如(Ⅱ)所示：
    Figure PCTCN2021102822-appb-100002
    其中，BSA为牛血清蛋白。
  4. 权利要求3所述普瑞巴林人工抗原的制备方法，其特征在于，包括：通过活泼酯法使权利要求1所述的普瑞巴林人工半抗原与牛血清蛋白结合，获得如权利要求3所述的普瑞巴林人工抗原Ⅱ。
  5. 如权利要求4所述普瑞巴林人工抗原的制备方法，其特征在于，包括以下步骤：
    (a)将所述普瑞巴林人工半抗原、N-羟基琥珀酰亚胺、二环己基碳二亚胺按摩尔比1:1.35～1.5:1.35～1.5混合于N，N-二甲基甲酰胺中，25℃下搅拌反应18h，反应结束后离心取上清液；
    (b)将上清液滴加到牛血清蛋白溶液中，将所得混合液于4℃下静置过夜，经透析、离心取上清液，获得如权利要求3所述的普瑞巴林人工抗原。
  6. 如权利要求5所述普瑞巴林人工抗原的制备方法，其特征在于，步骤(b)中，所述牛血清蛋白溶液的浓度为20mg/mL，上清液与牛血清蛋白溶液的体积比为1:5。
  7. 权利要求3所述普瑞巴林人工抗原在制备抗普瑞巴林抗体中的应用。
  8. 一种抗普瑞巴林抗体，其特征在于，是由权利要求3所述普瑞巴林人工抗原经动物免疫得到的、可与普瑞巴林发生特异性免疫反应的球蛋白。
  9. 权利要求8所述抗普瑞巴林抗体在检测普瑞巴林中的应用。
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