阅读说明：本技术 一种巴氯芬人工半抗原、人工抗原及其制备方法和应用 (Baclofen artificial hapten, artificial antigen, preparation method and application thereof ) 是由 王镇 邵越水 于 2020-06-30 设计创作，主要内容包括：本发明公开了一种巴氯芬人工半抗原、人工抗原及其制备方法和应用。巴氯芬人工半抗原的分子结构式如式(Ⅰ),巴氯芬人工抗原的分子结构式如式(Ⅱ)。本发明的巴氯芬人工半抗原最大程度地保留了巴氯芬的特征结构,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；进一步制备获得的巴氯芬人工抗原可免疫获得亲和力高、灵敏度高、特异性强的抗巴氯芬抗体,经免疫新西兰白兔获得的免疫血清的效价高达1∶80000,可用于对巴氯芬进行快速、准确的免疫检测和免疫分析。(The invention discloses a baclofen artificial hapten, an artificial antigen, a preparation method and application thereof. The molecular structural formula of the baclofen artificial hapten is shown as a formula (I), and the molecular structural formula of the baclofen artificial antigen is shown as a formula (II). The baclofen artificial hapten of the invention furthest reserves the characteristic structure of baclofen, has an active group which can be coupled with carrier protein and can be used as an antigenic determinant; the baclofen artificial antigen further prepared can be immunized to obtain an anti-baclofen antibody with high affinity, high sensitivity and strong specificity, the titer of immune serum obtained by immunizing a New Zealand white rabbit is as high as 1: 80000, and the baclofen artificial antigen can be used for carrying out rapid and accurate immunoassay and immunoassay on baclofen.)

1. The baclofen artificial hapten is characterized in that the molecular structural formula is shown as (I):

2. the method of preparing the baclofen artificial hapten as claimed in claim 1, comprising the steps of:

(1) mixing the baclofen and the trifluoroacetic anhydride according to the mol ratio of 1: 1.1, adding benzene dried by a 3A molecular sieve, stirring and reacting for 1 hour at room temperature, then transferring the mixture into an oil bath at 80 ℃, continuously stirring and refluxing for 3 hours, cooling to room temperature after the reaction is finished, and evaporating the solvent to dryness under reduced pressure to obtain a light yellow oily substance A;

(2) mixing the light yellow oily matter A, N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride according to the mol ratio of 1: 1.35-1.5: 2.45-3.0, adding dichloromethane for dissolving, and stirring and reacting for 18 hours at room temperature; after the reaction is finished, transferring the reaction solution into a separating funnel, washing an organic phase by respectively using 0.1N hydrochloric acid, purified water, a saturated sodium bicarbonate solution and a saturated saline solution, drying, filtering and evaporating the organic phase under reduced pressure to obtain an off-white solid B;

(3) dissolving p-aminomethyl benzoic acid in a mixed solvent of tetrahydrofuran and purified water in a volume ratio of 2: 1, uniformly stirring at room temperature, and then adding an off-white solid B tetrahydrofuran solution, wherein the reaction solution is turbid; then adding 1N sodium hydroxide aqueous solution, and stirring and reacting for 3 hours at room temperature; and (3) ending the reaction, evaporating the solvent to dryness under reduced pressure, adding double distilled water, adjusting the pH value to 6 by using 6N hydrochloric acid, extracting for three times by using ethyl acetate, collecting an organic phase, drying, filtering, evaporating to dryness under reduced pressure, and separating by TLC (thin layer chromatography) to obtain a yellow oily substance I, namely baclofen artificial hapten.

3. A baclofen artificial antigen is characterized in that the molecular structural formula is shown as (II):

in the formula (II), BSA is bovine serum albumin.

4. The baclofen artificial antigen of claim 3 wherein the baclofen artificial antigen is obtained by combining a baclofen artificial hapten with bovine serum albumin by an active ester method.

5. The baclofen artificial antigen of claim 4, comprising the steps of:

(a) dissolving baclofen artificial hapten I, N-hydroxysuccinimide and dicyclohexylcarbodiimide in N, N-Dimethylformamide (DMF) according to the molar ratio of 1: 1.35-1.5, stirring at room temperature for reacting for 18 hours, and centrifuging to obtain a supernatant after the reaction is finished;

(b) and (3) dropwise adding the supernatant into a bovine serum albumin solution, quickly and uniformly mixing, standing overnight at 4 ℃, dialyzing by a 0.5% sodium carbonate aqueous solution with the pH value of 12.00 and a Phosphate Buffer Solution (PBS), and centrifuging to obtain the supernatant, thereby obtaining the baclofen artificial antigen.

6. The baclofen artificial antigen of claim 5 wherein in step (b) the concentration of the bovine serum albumin solution is 5mg/ml and the volume ratio of the supernatant to the bovine serum albumin solution is 1: 10.

7. Use of the baclofen artificial antigen of claim 3 in the preparation of an anti-baclofen antibody.

8. An anti-baclofen antibody, which is a globulin obtained by immunizing an animal with the baclofen artificial antigen of claim 3 and specifically immunoreactive with baclofen.

9. Use of the anti-baclofen antibody of claim 8 for detecting baclofen.

Technical Field

The invention belongs to the technical field of biochemical engineering, and particularly relates to a baclofen artificial hapten, an artificial antigen, and preparation methods and applications thereof.

Background

Baclofen is a skeletal muscle relaxant and is used for improving spasm symptoms of muscle tension increase caused by pyramidal tract injury, spastic hemiplegia and paraplegia caused by different reasons, such as multiple sclerosis, cerebrovascular disease, spinal cord injury and myelitis sequelae, cerebral palsy of children, tetanus, intractable hiccup and the like.

The medicine is absorbed quickly and completely by the gastrointestinal tract. After the oral administration for 1-3 h, the plasma concentration reaches the peak, but the individual difference is large. 70-80% of the drug is excreted as it is from the stool and about 15% is metabolized by the liver.

Sudden withdrawal from prolonged use can cause: withdrawal syndrome, dizziness, headache, debilitation, hallucination, epilepsy, diarrhea, constipation, skin rash, anorexia, hypotension, nausea, emesis, euphoria, melancholy, insomnia, tinnitus, numbness, conversation ambiguity, liver function damage, tremor, etc.

At present, the detection of baclofen mainly depends on High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Thin Layer Chromatography (TLC), Mass Spectrometry (MS) and the like, but the existing detection methods are expensive in instruments, time-consuming in detection, and operation is required by professional technicians, and the requirements of modern detection on rapidness and accuracy cannot be met. Therefore, it is necessary to establish a rapid, sensitive and accurate detection technique.

The immunoassay method, which is an assay method for detecting various substances (drugs, hormones, proteins, microorganisms, etc.) using an antigen-antibody specific binding reaction, can make up all of the above disadvantages, and the key to establishing an immunoassay method for small-molecule compounds is the ability to produce antibodies having high affinity and high specificity for small-molecule compounds. However, since most small molecule compounds (molecular weight less than 1000), including baclofen, are not immunogenic, i.e., lack T cell epitopes and cannot directly induce the production of specific antibodies in animal bodies, small molecule substances are called haptens. Through appropriate chemical modification, a connecting arm is connected to a certain position of a hapten molecular structure, and then the connecting arm is combined with a macromolecular carrier to synthesize a hapten-carrier conjugate (i.e. an artificial antigen), wherein the artificial antigen can indirectly induce the proliferation and differentiation of B cells by means of T cell epitopes so as to generate specific antibodies.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings in the prior art and provide a preparation method of a baclofen-bovine serum albumin artificial antigen.

The invention firstly provides a baclofen artificial hapten, which furthest reserves the characteristic structure of baclofen, has an active group which can be coupled with carrier protein and can be used as an antigenic determinant.

A baclofen artificial hapten, the molecular structural formula of which is shown as (I):

the invention discloses a preparation method of baclofen artificial hapten, which comprises the following steps:

(1) mixing the baclofen and the trifluoroacetic anhydride according to the mol ratio of 1: 1.1, adding benzene dried by a 3A molecular sieve, stirring and reacting for 1 hour at room temperature, then transferring the mixture into an oil bath at 80 ℃, continuously stirring and refluxing for 3 hours, cooling to room temperature after the reaction is finished, and evaporating the solvent to dryness under reduced pressure to obtain a light yellow oily substance A;

(2) Mixing the light yellow oily matter A, N-hydroxysuccinimide and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride according to the mol ratio of 1: 1.35-1.5: 2.45-3.0, adding dichloromethane for dissolving, and stirring and reacting for 18 hours at room temperature; after the reaction is finished, transferring the reaction solution into a separating funnel, washing an organic phase by respectively using 0.1N hydrochloric acid, purified water, a saturated sodium bicarbonate solution and a saturated saline solution, drying, filtering and evaporating the organic phase under reduced pressure to obtain an off-white solid B;

(3) dissolving p-aminomethyl benzoic acid in a mixed solvent of tetrahydrofuran and purified water in a volume ratio of 2: 1, uniformly stirring at room temperature, and then adding an off-white solid B tetrahydrofuran solution, wherein the reaction solution is turbid; then adding 1N sodium hydroxide aqueous solution until the reaction solution is just clear, and stirring and reacting for 3 hours at room temperature; and (3) ending the reaction, evaporating the solvent to dryness under reduced pressure, adding double distilled water, adjusting the pH value to 6 by using 6N hydrochloric acid, extracting the mixture for three times by using ethyl acetate, collecting an organic phase, drying, filtering, evaporating to dryness under reduced pressure, and separating by TLC (thin layer chromatography) to obtain a yellow oily substance I, namely the baclofen artificial hapten as claimed in claim 1.

By the method, the connecting arm is introduced to the carboxyl of baclofen, and the characteristic structure of baclofen can be retained to a greater extent by introducing the connecting arm to the modification site, and the active site capable of being coupled with carrier protein is provided.

Compared with the method adopting general saturated chain hydrocarbon as the connecting arm, the connecting arm adopted by the invention contains a benzene ring structure, so that the specificity of the artificial hapten is enhanced, the conformation is kept stable in a solution, and the immunological characteristic of the formed artificial antigen (II) is improved.

The invention also provides a baclofen artificial antigen, the molecular structure of which is shown as (II):

in the formula (II), BSA is bovine serum albumin.

The invention also provides a preparation method of the baclofen artificial antigen, wherein the baclofen artificial antigen is obtained by combining the baclofen artificial hapten with bovine serum albumin through an active ester method.

Specifically, the method for preparing the baclofen artificial antigen by adopting the active ester method comprises the following steps:

(a) dissolving baclofen artificial hapten I, N-hydroxysuccinimide and dicyclohexylcarbodiimide in N, N-dimethylformamide according to the molar ratio of 1: 1.35-1.5, stirring at room temperature for reacting for 18 hours, and centrifuging to obtain a supernatant after the reaction is finished;

(b) and (3) dropwise adding the supernatant into a bovine serum albumin solution, standing the mixed solution at 4 ℃ overnight, dialyzing by a 0.5% sodium carbonate aqueous solution with the pH value of 12.00 and a Phosphate Buffer Solution (PBS), and centrifuging to obtain the supernatant, thereby obtaining the baclofen artificial antigen.

The bovine serum albumin solution of the present invention is prepared by dissolving bovine serum albumin in a PBS (pH 7.2-7.4) buffer solution having a phosphate ion concentration of 0.01 mol/L.

In the step (b), the concentration of the bovine serum albumin solution is 5mg/ml, and the volume ratio of the supernatant to the bovine serum albumin solution is 1: 10.

The Bovine Serum Albumin (BSA) selected by the invention is taken as a macromolecular carrier, and compared with other carrier proteins, the bovine serum albumin has the following advantages: the BSA has 583 amino acid residues, is easy to couple with baclofen hapten, can prepare baclofen artificial antigens with different coupling ratios, and has higher immunogenicity; secondly, the BSA is economical and practical and has low cost; and the BSA has stable chemical properties, good solubility and stability in acidic and weakly alkaline environments, and is suitable for long-term storage.

The invention also provides application of the baclofen artificial antigen in preparing an anti-baclofen antibody.

The invention also provides an anti-baclofen antibody, which is globulin obtained by animal immunization of baclofen artificial antigen and capable of generating specific immunoreaction with baclofen.

The invention also provides application of the anti-baclofen antibody in baclofen detection.

Experiments show that the titer of immune serum obtained by immunizing new zealand white rabbits with the baclofen artificial antigen is 1: 80000. The baclofen artificial antigen can be used for immunizing to obtain the baclofen-resistant antibody with high affinity, high sensitivity and strong specificity, and the baclofen-resistant antibody can be used for immune detection and analysis of baclofen.

Compared with the prior art, the invention has the beneficial effects that:

the baclofen artificial hapten of the invention furthest reserves the characteristic structure of baclofen, has an active group which can be coupled with carrier protein and can be used as an antigenic determinant; the baclofen artificial antigen further prepared can be immunized to obtain an anti-baclofen antibody with high affinity, high sensitivity and strong specificity, the titer of immune serum obtained by immunizing a New Zealand white rabbit is as high as 1: 80000, and the baclofen artificial antigen can be used for carrying out rapid and accurate immunoassay and immunoassay on baclofen.

Drawings

FIG. 1 is a flow chart of the preparation of baclofen artificial antigen of the present invention;

wherein THF represents tetrahydrofuran, EDCI represents 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, MeOH represents methanol, DCM represents dichloromethane, DCC represents dicyclohexylcarbodiimide, DMF represents N, N-dimethylformamide, Et ₃N represents triethylamine, BSA represents bovine serum albumin, BGG represents bovine gamma globulin, the same as the following;

FIG. 2 is a liquid chromatogram of the baclofen artificial hapten of the invention;

wherein mAU represents milliabsorbance units, min represents minutes;

FIG. 3 is a mass spectrum of baclofen hapten of the present invention;

wherein Relative Absundance represents Relative Abundance; m/z represents a charge-to-mass ratio;

FIG. 4 is an ultraviolet scan of bovine serum albumin, baclofen artificial hapten and baclofen artificial antigen;

wherein A represents ultraviolet-visible absorbance, and λ (nm) represents wavelength (nm);

FIG. 5 is a flow chart of the preparation of a baclofen artificial antigen of comparative example 1;

FIG. 6 is a flow chart of the preparation of a comparative example 2 baclofen artificial antigen;

FIG. 7 is a flow chart of the preparation of a comparative example 3 baclofen artificial antigen;

FIG. 8 is a flow chart of the preparation of a comparative example 4 baclofen artificial antigen;

FIG. 9 is a flow chart of the preparation of a comparative example 5 baclofen artificial antigen;

FIG. 10 is a flow chart of the preparation of a comparative example 6 baclofen artificial antigen;

FIG. 11 is a flow chart of the preparation of the artificial antigen of baclofen in comparative example 7.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.