阅读说明：本技术 一种喹硫平人工半抗原、人工抗原及其制备方法和应用 (Quetiapine 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：本发明公开了一种喹硫平人工半抗原、人工抗原及其制备方法和应用。喹硫平人工半抗原的分子结构式如式(Ⅰ),喹硫平人工抗原的分子结构式如式(Ⅱ)。本发明的喹硫平人工半抗原最大程度地保留了喹硫平的特征结构,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；进一步制备获得的喹硫平人工抗原可免疫获得亲和力高、灵敏度高、特异性强的抗喹硫平抗体,经免疫Balb/c小鼠获得的免疫腹水的效价高达1∶78000,可用于对喹硫平进行快速、准确的免疫检测和免疫分析。(The invention discloses a quetiapine artificial hapten, an artificial antigen, a preparation method and application thereof. The molecular structural formula of the quetiapine artificial hapten is shown as a formula (I), and the molecular structural formula of the quetiapine artificial antigen is shown as a formula (II). The quetiapine artificial hapten disclosed by the invention furthest reserves the characteristic structure of quetiapine, has an active group which can be coupled with carrier protein and can be used as an antigenic determinant; the quetiapine artificial antigen further prepared can be used for immunizing to obtain an anti-quetiapine antibody with high affinity, high sensitivity and strong specificity, the titer of immune ascites obtained by immunizing Balb/c mice is as high as 1: 78000, and the quetiapine artificial antigen can be used for carrying out quick and accurate immunoassay and immunoassay on quetiapine.)

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

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

(1) weighing quetiapine fumarate, dissolving in purified water, adjusting pH to 9 with concentrated ammonia water, extracting with dichloromethane for three times, collecting organic phase, drying, filtering, and evaporating under reduced pressure to obtain yellowish oily substance A;

(2) mixing the light yellow oily substance A and succinic anhydride according to the molar ratio of 1: 1-1.5, adding pyridine for dissolving, and stirring and refluxing in an oil bath at 100 ℃ for reacting for 18 hours; and (3) after the reaction is finished, cooling to room temperature, evaporating the solvent under reduced pressure, adding purified water, extracting with dichloromethane for three times, collecting an organic phase, drying, filtering, and evaporating under reduced pressure to obtain a brownish black oily residue, and separating the residue by TLC (thin layer chromatography) (the solvent and an eluent are absolute ethyl alcohol, a developing agent is dichloromethane, 95% ethyl alcohol, 1, 4-dioxane and concentrated ammonia water are 10:8:1:1) to obtain a yellow oily substance I, namely the quetiapine artificial hapten.

3. An artificial antigen of quetiapine, which is characterized in that the molecular structural formula is shown as (II):

in the formula (II), BGG is bovine gamma globulin.

4. The quetiapine artificial antigen according to claim 3, wherein the quetiapine artificial antigen is obtained by combining the quetiapine artificial hapten according to claim 1 with bovine gamma globulin by an active ester method.

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

(a) dissolving quetiapine 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 the bovine gamma globulin solution, standing the mixed solution at 4 ℃ overnight, dialyzing, centrifuging and taking the supernatant to obtain the quetiapine artificial antigen.

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

7. Use of the quetiapine artificial antigen as claimed in claim 3 in the preparation of an anti-quetiapine antibody.

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

9. Use of an anti-quetiapine antibody according to claim 8 for detecting quetiapine.

Technical Field

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

Background

Quetiapine is an atypical antipsychotic drug useful in the maintenance therapy of schizophrenia, acute mania, bipolar depression and bipolar disorder. Along with the wide clinical application of the traditional Chinese medicine, the understanding of adverse reactions of the traditional Chinese medicine is deeper and deeper.

According to survey reports, certain adverse phenomena are also accompanied when quetiapine is taken, and common adverse reactions are dizziness, somnolence, postural hypotension, palpitation, dry mouth, inappetence and constipation. It also can cause prolactin level increase, body weight gain, abdominal pain, asymptomatic transaminase increase, total cholesterol and triglyceride increase, excitation and insomnia.

At present, the detection of quetiapine 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 method is expensive in instrument, time-consuming in detection, and needs to be operated by professional technicians, and cannot meet the requirements of modern detection on rapidness and accuracy. 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 quetiapine, are not immunogenic, i.e., lack of T cell epitopes and do not directly induce the production of specific antibodies in the animal body, small molecule substances are called haptens. Through appropriate chemical modification, a connecting arm with an active group at the upper end position is carried at a certain position of a hapten molecular structure, and then the connecting arm is combined with a macromolecular carrier to generate a hapten-carrier conjugate (namely an artificial antigen), and the artificial antigen can indirectly induce the proliferation and differentiation of B cells by means of T cell epitopes to further generate a specific antibody.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings in the prior art and provide a preparation method of the quetiapine-bovine gamma globulin artificial antigen.

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

A quetiapine artificial hapten, which has a molecular structural formula shown as (I):

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

(1) weighing quetiapine fumarate, dissolving in purified water, adjusting pH to 9 with 1N sodium hydroxide solution, extracting with dichloromethane for three times, collecting organic phase, drying, filtering, and evaporating under reduced pressure to obtain yellowish oily substance A;

(2) mixing the light yellow oily substance A and succinic anhydride according to the molar ratio of 1: 1-1.5, adding pyridine for dissolving, and stirring and refluxing in an oil bath at 100 ℃ for reacting for 18 hours; and (3) after the reaction is finished, cooling to room temperature, evaporating the solvent under reduced pressure, adding purified water, extracting with dichloromethane for three times, collecting an organic phase, drying, filtering, and evaporating under reduced pressure to obtain a brownish black oily residue, wherein the residue is separated by TLC (thin layer chromatography) (the solvent and a washing and dehydrating machine are absolute ethyl alcohol, a developing agent is dichloromethane, 95% ethyl alcohol and 1, 4-dioxane and concentrated ammonia water are 10:8:1:1) to obtain a yellow oily substance I, namely the quetiapine artificial hapten.

By the method, the connecting arm is introduced to the hydroxyl position of the quetiapine, and the connecting arm introduced to the modification site can retain the characteristic structure of the quetiapine to a greater extent and also provide an active site capable of being coupled with carrier protein.

Compared with the adoption of the annular connecting arm, the connecting arm adopted by the invention is in a chain shape, so that the recognition degree of T cells to the connecting arm in the immunization can be reduced as much as possible, and thus, the antibody obtained by immunization has stronger specificity and affinity to quetiapine.

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

in the formula (II), BGG is bovine gamma globulin.

The invention also provides a preparation method of the quetiapine artificial antigen, and the quetiapine artificial antigen is obtained by combining the quetiapine artificial hapten with bovine gamma globulin through an active ester method.

Specifically, when the quetiapine artificial antigen is prepared by an active ester method, the method comprises the following steps:

(a) dissolving quetiapine artificial hapten I, N-hydroxysuccinimide and dicyclohexylcarbodiimide in N, N-Dimethylformamide (DMF) at a 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 the bovine gamma globulin solution, standing the mixed solution at 4 ℃ overnight, dialyzing, centrifuging and taking the supernatant to obtain the quetiapine artificial antigen.

The bovine gamma globulin solution is prepared by dissolving bovine gamma globulin in PBS (pH 7.2-7.4) buffer solution with the phosphate ion concentration of 0.01 mol/L.

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

The invention selects Bovine Gamma Globulin (BGG) as a macromolecular carrier, and has the following advantages compared with Bovine Serum Albumin (BSA): the bovine gamma globulin can be combined with more quetiapine artificial haptens, and the immunogenicity is good; the combination of the bovine gamma globulin and the quetiapine artificial hapten is more stable in experiments, and after the bovine serum albumin is combined with the quetiapine artificial hapten, the quetiapine artificial hapten is easy to separate in subsequent treatment, so that the final artificial antigen is easy to precipitate in long-term storage and has poor stability; and the anti-quetiapine antibody obtained by animal immunization has better specificity by using the quetiapine artificial antigen formed by combining the bovine gamma globulin and the quetiapine artificial hapten.

The invention also provides application of the quetiapine artificial antigen in preparation of an anti-quetiapine antibody.

The invention also provides an anti-quetiapine antibody which is globulin obtained by immunizing animals by the quetiapine artificial antigen and capable of generating specific immune reaction with quetiapine.

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

Experiments show that the titer of the monoclonal antibody obtained by immunizing Balb/c mice with the quetiapine artificial antigen is 1: 78000. The artificial antigen of quetiapine can be used for immunizing to obtain an anti-quetiapine antibody with high affinity, high sensitivity and strong specificity, and the anti-quetiapine antibody can be used for immunodetection and analysis of quetiapine.

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

the quetiapine artificial hapten disclosed by the invention furthest reserves the characteristic structure of quetiapine, has an active group which can be coupled with carrier protein and can be used as an antigenic determinant; the quetiapine artificial antigen further prepared can be used for immunizing to obtain an anti-quetiapine antibody with high affinity, high sensitivity and strong specificity, the titer of immune ascites obtained by immunizing a new Balb/c mouse is as high as 1: 78000, and the quetiapine artificial antigen can be used for carrying out quick and accurate immunoassay and immunoassay on quetiapine.

Drawings

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

wherein THF represents tetrahydrofuran, MeOH represents methanol, DCC represents dicyclohexylcarbodiimide, DMF represents N, N-dimethylformamide, Et₃N 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 quetiapine artificial hapten of the present invention;

wherein mAU represents milliabsorbance units, min represents minutes;

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

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

FIG. 4 is a UV scan of the quetiapine artificial hapten, bovine gamma globulin, quetiapine artificial antigen;

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

FIG. 5 is a flow chart illustrating the preparation of an artificial antigen of quetiapine in comparative example 1;

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

FIG. 7 is a flow chart illustrating the preparation of an artificial antigen of quetiapine in comparative example 3;

FIG. 8 is a flow chart illustrating the preparation of an artificial antigen of quetiapine in comparative example 4;

FIG. 9 is a flow chart illustrating the preparation of an artificial antigen of quetiapine in comparative example 5;

FIG. 10 is a flow chart illustrating the preparation of an artificial antigen of quetiapine in comparative example 6;

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

Detailed Description

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