CN110950808A - Clozapine artificial antigen and preparation method thereof - Google Patents

Clozapine artificial antigen and preparation method thereof Download PDF

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CN110950808A
CN110950808A CN201911043573.1A CN201911043573A CN110950808A CN 110950808 A CN110950808 A CN 110950808A CN 201911043573 A CN201911043573 A CN 201911043573A CN 110950808 A CN110950808 A CN 110950808A
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王百龙
王晨
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Hangzhou Biotest Biotech Co Ltd
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Abstract

The invention discloses a clozapine artificial antigen and a preparation method thereof.A succinic anhydride connecting arm is introduced to clozapine to obtain a clozapine hapten, the introduced connecting arm has no other characteristic structure, so that nonspecific combination caused by the connecting arm can be effectively reduced, and the site of the introduced connecting arm is far away from the characteristic structure of the clozapine, so that the prepared artificial antigen has better specificity; reacting the hapten with N-hydroxysuccinimide and N, N-cyclohexyl carbodiimide to obtain active ester, and performing coupling reaction on the active ester and carrier protein to obtain clozapine artificial antigen; the preparation process of the clozapine artificial antigen is simple, the production cost is low, and the prepared clozapine artificial antigen can be used for animal immunization to obtain a corresponding antibody; the prepared clozapine artificial antigen and antibody can be used for detecting whether a sample contains clozapine and derivative drugs thereof by using an immunoassay method.

Description

Clozapine artificial antigen and preparation method thereof
Technical Field
The invention particularly relates to a clozapine artificial antigen and a preparation method thereof.
Background
Clozapine is a light yellow crystalline powder; no odor and no smell, and the structural formula is as follows:
Figure BDA0002253508830000011
the product is a benzodiazepine, has strong blocking effect on 5-hydroxytryptamine (5-HT2A) receptors and dopamine (DA1) receptors in brain, also has blocking effect on dopamine (DA4) receptors, has weak blocking effect on dopamine (DA2) receptors, and also has the effects of resisting choline (M1), histamine (H1) and alpha-adrenergic receptors, and has light extrapyramidal system reaction and tardive dyskinesia, and does not generally cause increase of prolactin in blood. Can directly inhibit the brain stem network structure ascending activation system, has strong sedative and hypnotic effects, and is used for treating various types of schizophrenia.
Therapeutic drug monitoring (TDM for short) refers to the process of observing the curative effect of a drug in clinical practice, collecting blood of a patient at regular time (sometimes collecting urine, saliva and other liquids), measuring the concentration of the drug therein, and discussing the in vivo process of the drug, so as to individualize a drug administration scheme according to the specific situation of the patient by using the basic theory of pharmacokinetics and pharmacodynamics as guidance, by means of advanced analysis technology and electronic computer means, and by using the principle and formula of pharmacokinetics. Thereby achieving satisfactory curative effect and avoiding toxic and side effects, providing valuable laboratory basis for diagnosis and treatment of drug excessive poisoning, and improving clinical medication from traditional experience mode to more scientific level. Such monitoring allows, for example, identifying patients who do not comply with their dosing regimen, patients who have not reached a therapeutic dose, patients who are non-responsive at a therapeutic dose, patients with suboptimal drug resistance, patients with pharmacokinetic drug-drug interactions, or patients with abnormal metabolism leading to poor plasma concentrations. There is considerable individual variation in the ability of patients to absorb, distribute, metabolize, and excrete neuroleptic drugs. Such differences may be caused by complications, age, concomitant medication or genetic characteristics. Different drug formulations can also affect the metabolism of the neuroleptic agent. TDM allows for dose optimization for individual patients, improving therapeutic and functional outcomes.
Methods for determining serum or plasma concentration levels of a drug (e.g., clozapine) involve the use of High Performance Liquid Chromatography (HPLC) with UV or mass spectrometric detection, gas chromatography-mass spectrometry (GC-MS) for detection, immunoassays and the like, wherein High Performance Liquid Chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and the like require large analytical instruments, and such analytical methods suffer from the disadvantages of expensive instruments, need for skilled technician handling, need to be performed in specialized laboratories, and the like.
Since clozapine is a small molecule that is immunoreactive only and not immunogenic, it must be linked to a protein macromolecule to stimulate the body with the help of the T cell epitope of the macromolecule in order to generate an antibody-specific immune response. Therefore, it is necessary to develop a new and effective synthetic method for clozapine artificial antigen.
Disclosure of Invention
Aiming at the situation, the invention provides a clozapine artificial antigen and a preparation method thereof to overcome the defects of the prior art.
In order to achieve the purpose, the invention provides the following technical scheme:
a clozapine hapten having the structure shown in formula (i):
Figure BDA0002253508830000021
a clozapine artificial antigen has a structure shown in a formula (II): wherein, p is carrier protein, n represents the number of hapten coupled on one carrier protein; the value range of n is 15-70; wherein-CONH-represents an amide group, i.e.
Figure BDA0002253508830000022
Figure BDA0002253508830000031
Further, the carrier protein is selected from any one of bovine serum albumin, bovine gamma globulin, bovine thyroglobulin, keyhole limpet hemocyanin and chicken ovalbumin.
A method for preparing a hapten for clozapine, the method for preparing the hapten comprising the steps of:
adding clozapine into a reaction vessel, then adding dichloromethane to completely dissolve the clozapine, controlling the temperature at 0 ℃, adding succinic anhydride, then adding aluminum trichloride every 1 hour, and adding for three times in total; returning to room temperature, and stirring for reaction for 1-20 hours; after the reaction is finished, adding water and concentrated hydrochloric acid, stirring and reacting for 0.5-2 hours, then extracting with dichloromethane, collecting an organic phase, and evaporating the solvent under reduced pressure to obtain a product; and then purifying by thin layer chromatography to obtain clozapine hapten, wherein in the whole reaction, the ratio of clozapine: succinic anhydride: adding aluminum trichloride in a ratio of 1:1:1 to 1:5:5 in total, wherein the aluminum trichloride is added in three times, and the mass of the aluminum trichloride added each time is the same. The temperature is controlled at 0 ℃ and the aluminum trichloride is added in batches, so that the generation of byproducts can be reduced.
A method for preparing a clozapine artificial antigen for use in preparing the clozapine artificial antigen described above, comprising the steps of:
(1) preparation of clozapine hapten:
adding clozapine into a reaction vessel, then adding dichloromethane to completely dissolve the clozapine, controlling the temperature at 0 ℃, adding succinic anhydride, then adding aluminum trichloride every 1 hour, and adding for three times in total; returning to room temperature, and stirring for reaction for 1-20 hours; after the reaction is finished, adding water and concentrated hydrochloric acid, stirring and reacting for 0.5-2 hours, then extracting with dichloromethane, collecting an organic phase, and evaporating the solvent under reduced pressure to obtain a product; and then purifying by thin layer chromatography to obtain clozapine hapten, wherein in the whole reaction, the ratio of clozapine: succinic anhydride: adding aluminum trichloride in a ratio of 1:1:1 to 1:5:5 in total, wherein the aluminum trichloride is added in three times, and the mass of the aluminum trichloride added each time is the same.
(2) Preparation of clozapine artificial antigen:
a. weighing clozapine hapten in another reaction container, adding N, N-dimethylformamide to completely dissolve the clozapine hapten, adding N-hydroxysuccinimide and N, N-dicyclohexylcarbodiimide, stirring at room temperature for reacting overnight, centrifuging after the reaction is finished, and taking the supernatant as solution A; wherein, clozapine hapten: n-hydroxysuccinimide: n, N-dicyclohexylcarbodiimide in a ratio of 1:1:1 to 1:5:5, the ratio being the amount of the substance;
b. preparing a PBS buffer solution with the pH value of 7.2-7.4;
c. weighing bovine serum albumin, dissolving the bovine serum albumin in the PBS buffer solution in the step B, and recording the obtained solution as solution B; in the solution B, the concentration of the carrier protein is 2-20 mg/ml;
d. slowly dripping the solution A into the solution B under the condition of rapid stirring, standing and storing the obtained mixed solution at 4 ℃ overnight to obtain an artificial antigen mixed solution, wherein the solution A: the ratio of the solution B to the solution B is (1-5000) 1000, and the volume ratio is;
e. and (c) transferring the artificial antigen mixed solution into a dialysis bag, dialyzing by using the PBS buffer solution in the step b, and centrifuging and taking supernate after dialysis is finished to obtain the clozapine artificial antigen.
An antibody obtained by immunizing an animal with the clozapine artificial antigen described above.
Further, the antibody is a monoclonal antibody or a polyclonal antibody.
The invention has the beneficial effects that:
(1) the invention takes clozapine as a raw material to synthesize an artificial antigen, and introduces a succinic anhydride connecting arm on the clozapine to obtain a clozapine hapten; the hapten can be obtained only by one step, the yield is high, the time cost and the labor cost are greatly saved, in addition, the connecting arm introduced by the invention has no other characteristic structure, the nonspecific combination caused by the connecting arm can be effectively reduced, the site of the introduced connecting arm is far away from the characteristic structure of the clozapine, and the prepared artificial antigen has higher specificity; reacting the hapten with N-hydroxysuccinimide and N, N-cyclohexyl carbodiimide to obtain active ester, and performing coupling reaction on the active ester and carrier protein to obtain clozapine artificial antigen; the preparation process of the clozapine artificial antigen is simple, raw materials are easy to obtain, the production cost is low, and the prepared clozapine artificial antigen can be used for animal immunization to obtain a corresponding antibody.
(2) The clozapine artificial antigen and the antibody prepared by the invention can be used for detecting whether a sample contains clozapine and derivatives thereof by an immunoassay method, wherein the sample refers to a body fluid sample of a mammal or a human, such as saliva, urine and sweat, or a tissue sample of the mammal or the human, such as tissues of liver, spleen and the like. The clozapine artificial antigen and the clozapine artificial antibody can also be widely applied to detection of drug dependence and monitoring (TDM) and optimization of therapeutic drugs; the immunoassay is convenient and accurate, has low cost and simple operation, can well replace methods such as liquid chromatography HPLC and gas chromatography-mass spectrometry combined analyzer GC/MS, and the like, and has good application value.
Drawings
FIG. 1 is a scheme showing the synthesis of clozapine hapten.
FIG. 2 is a scheme showing the synthesis of clozapine artificial antigen.
FIG. 3 is a UV scanning spectrum of clozapine hapten, clozapine artificial antigen and bovine serum albumin.
FIG. 4 is a schematic diagram of the structure of a lateral flow test strip.
Detailed Description
The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, and it should be noted that the detailed description is only for describing the present invention, and should not be construed as limiting the present invention.
The materials (including reaction materials, other reagents, and the like) and instruments used in the following examples are commercially available.
Example 1
(1) Preparation of clozapine hapten
200mg (0.61mmol) clozapine are weighed into a 50ml single neck round bottom flask, followed by 10ml more methylene chloride to dissolve clozapine completely, the temperature is controlled at 0 ℃ and 100mg (1mmol) succinic anhydride is added, followed by 75mg (0.56mmol) aluminium trichloride every 1 hour for three times. Returning to room temperature, and stirring for reaction for 18 hours; after the reaction, 5ml of water and 1ml of concentrated hydrochloric acid were added, and the mixture was stirred for half an hour and then extracted 3 times with 10ml of dichloromethane. Collecting organic phase, and evaporating the solvent under reduced pressure to obtain 178mg of white solid product; then purified by thin layer chromatography, TLC: the chromatographic solution is ethyl acetate, and the specific transfer value Rf of the product is 0.4-0.5; obtaining 102mg of clozapine hapten, wherein the specific synthetic route is shown in figure 1;
(2) preparation of clozapine artificial antigen
a. 50mg (0.12mmol) of hapten is weighed into a 50ml round-bottom flask, 3ml of N, N-Dimethylformamide (DMF) is added to dissolve the clozapine hapten completely, 34mg (0.3mmol) of N-hydroxysuccinimide (NHS) and 64mg (0.3mmol) of N, N-Dicyclohexylcarbodiimide (DCC) are added, the mixture is stirred at room temperature for reaction overnight, after the reaction is finished, the mixture is centrifuged, and the supernatant is taken as solution A.
b. 14.5g of disodium hydrogen phosphate dodecahydrate, 43.875g of sodium chloride and 1.495g of sodium dihydrogen phosphate dihydrate are dissolved by double distilled water to a constant volume of 5.0L to obtain a PBS buffer solution with the pH of 7.2-7.4.
c. 0.25g bovine serum albumin was weighed and dissolved in 50ml PBS buffer solution described in step B, and the obtained solution was designated as solution B.
d. Slowly dripping the solution A into the solution B under the condition of rapid stirring, and standing and storing the obtained mixed solution at the temperature of 4 ℃ overnight to obtain the artificial antigen mixed solution.
e. And (b) transferring the artificial antigen mixed solution into a dialysis bag, dialyzing for 9 times by using the PBS buffer solution in the step b, centrifuging after dialysis is finished, and taking supernate to obtain the clozapine artificial antigen, wherein the specific synthetic route is shown in figure 2.
By carrying out the above-described steps with any one of Bovine Gamma Globulin (BGG), Bovine Thyroglobulin (BTG), Keyhole Limpet Hemocyanin (KLH) and chicken Ovalbumin (OVA) in place of Bovine Serum Albumin (BSA), various clozapine artificial antigens can be prepared.
(3) Identification of clozapine artificial antigen:
(a) ultraviolet scanning atlas
Ultraviolet scan of clozapine hapten, clozapine artificial antigen and bovine serum albumin as shown in figure 3; as can be seen from FIG. 3, the invention successfully prepares clozapine artificial antigen.
(b) And (3) coupling ratio determination: methods for estimating the ratio of two molecules coupled (coupling ratio) in a conjugate are based on the principle of detecting the amount (or relative amount) of the two molecules coupled in the conjugate, although many methods are available. Spectrophotometry is a method for measuring the concentrations of two coupled molecules respectively by using the principle that the absorption of light by a substance is in a proportional relationship with the concentrations of the two coupled molecules. In the macromolecular and micromolecular conjugate, the two molecules have different ultraviolet scanning spectrums respectively and show the property of superposing the spectrums.
Molar absorptivity epsilon: and (3) preparing a clozapine hapten solution by using a PBS buffer solution with the pH value of 7.2-7.4 to obtain the PBS solution with the clozapine hapten concentrations of 0,5,10,20,30 and 40 mu g/ml respectively, and obtaining an ultraviolet scanning figure 3 that the maximum absorption wavelength of the clozapine hapten is 288nm, measuring the absorbance value at 288nm and taking a parallel sample at each concentration. The molar absorption coefficient epsilon is A/(bc), wherein A is the absorbance, b is the thickness of the absorption cell and is expressed in cm, and b is 1 cm; c is the molar concentration of the light absorbing substance, and the unit is mol/L; the present invention calculates ε as 3414.3L/(mol cm).
Determination of conjugate protein concentration: and (3) respectively preparing 1ml of bovine serum albumin PBS solution with the concentration of 0, 1, 2, 4, 8, 10 and 20mg/ml by using PBS buffer solution with the pH value of 7.2-7.4, adding 3ml of Coomassie brilliant blue staining solution, immediately mixing uniformly, heating in a water bath at 30 ℃ for 5 minutes, taking parallel samples at each concentration, measuring the absorbance value at 655nm, and drawing a relation curve of the concentration of the bovine serum albumin and the absorbance value. And (3) diluting the clozapine artificial antigen solution according to a certain proportion, measuring the absorbance value of the clozapine artificial antigen at 655nm, and obtaining the corresponding protein concentration value in the clozapine artificial antigen solution from the curve. The protein concentration of the clozapine artificial antigen solution calculated by the invention is 2.215 mg/ml.
And (3) coupling ratio determination: diluting a conjugate, namely clozapine artificial antigen solution, with PBS (phosphate buffer solution) with the pH of 7.2-7.4 to the concentration of 100 mu g/ml, and measuring the absorbance value A at 288nm1(ii) a Diluting bovine serum albumin with PBS buffer solution with pH of 7.2-7.4 to 100 μ g/ml, and measuring absorbance value A at 278nm2The coupling ratio γ is then: γ ═ a1－A2)/ε]/(c*100×10-365000) where ε is the molar absorptivity L/(mol cm) of the hapten of clozapine, 65000 is the molecular weight of bovine serum albumin, c 100X 10-3For the protein concentration in clozapine artificial antigen solution (. mu.g/ml), the invention calculated γ ≈ 28.
Example 2
(1) Preparation of clozapine hapten
1mmol clozapine is weighed into a 100ml single neck round bottom flask and then 17ml dichloromethane is added to dissolve clozapine completely, the temperature is controlled at 0 ℃ and 1mmol succinic anhydride is added followed by three additions of 0.35mmol aluminium trichloride every 1 hour. Returning to room temperature, and stirring for reaction for 15 hours; after the reaction is finished, adding a small amount of water and concentrated hydrochloric acid to stop the reaction, in the embodiment, adding 5ml of water and 1ml of concentrated hydrochloric acid; the reaction was stirred for half an hour and then extracted 3 times with 15ml dichloromethane. Collecting an organic phase, and evaporating the solvent to dryness under reduced pressure to obtain a solid product; then purified by thin layer chromatography, TLC: the chromatographic liquid is ethyl acetate, and the product has a specific displacement value Rf0.4-0.5; obtaining clozapine hapten (2), wherein the specific synthetic route is shown in figure 1;
(2) preparation of clozapine artificial antigen
a. Weighing 1mmol of hapten into a 150ml round-bottom flask, adding 30ml of N, N-Dimethylformamide (DMF) to dissolve the clozapine hapten completely, adding 1mmol of N-hydroxysuccinimide (NHS) and 1mmol of N, N-Dicyclohexylcarbodiimide (DCC), stirring at room temperature for reacting overnight, centrifuging after the reaction is finished, and taking the supernatant as solution A.
b. 14.5g of disodium hydrogen phosphate dodecahydrate, 43.875g of sodium chloride and 1.495g of sodium dihydrogen phosphate dihydrate are dissolved by double distilled water to a constant volume of 5.0L to obtain a PBS buffer solution with the pH of 7.2-7.4.
c. Weighing 2.5g bovine serum albumin dissolved in 250ml PBS buffer solution in step B, and marking the obtained solution as solution B.
d. Slowly dripping the solution A into the solution B under the condition of rapid stirring, and standing and storing the obtained mixed solution at the temperature of 4 ℃ overnight to obtain the artificial antigen mixed solution.
e. And (3) transferring the artificial antigen mixed solution into a dialysis bag, dialyzing for 6 times by using the PBS buffer solution in the step b, centrifuging after dialysis is finished, and taking supernate to obtain clozapine artificial antigen (4), wherein the specific synthetic route is shown in figure 2. Through ultraviolet scanning spectrum identification, the clozapine artificial antigen is successfully prepared in the embodiment.
Example 3
(1) Preparation of clozapine hapten
1mmol clozapine is weighed into a 150ml single neck round bottom flask, followed by 17ml dichloromethane to dissolve clozapine completely, the temperature is controlled at 0 ℃, 5mmol succinic anhydride is added, followed by 1.6mmol aluminium trichloride every 1 hour for three times. Returning to room temperature, and stirring for reaction for 20 hours; after the reaction, adding a small amount of water and concentrated hydrochloric acid to terminate the reaction, in this example, adding 10ml of water and 2ml of concentrated hydrochloric acid; the reaction was stirred for 1 hour and then extracted 3 times with 30ml of dichloromethane. Collecting an organic phase, and evaporating the solvent to dryness under reduced pressure to obtain a solid product; then purified by thin layer chromatography, TLC: the chromatographic liquid is ethyl acetate, and the product has a specific displacement value Rf0.4-0.5; obtaining clozapine hapten (2), wherein the specific synthetic route is shown in figure 1;
(2) preparation of clozapine artificial antigen
a. Weighing 1mmol of clozapine hapten into a 150ml round-bottom flask, adding 50ml of N, N-Dimethylformamide (DMF) to completely dissolve the clozapine hapten, adding 5mmol of N-hydroxysuccinimide (NHS) and 5mmol of N, N-Dicyclohexylcarbodiimide (DCC), stirring at room temperature for reacting overnight, centrifuging after the reaction is finished, and taking the supernatant as A liquid.
b. 14.5g of disodium hydrogen phosphate dodecahydrate, 43.875g of sodium chloride and 1.495g of sodium dihydrogen phosphate dihydrate are dissolved by double distilled water to a constant volume of 5.0L, and a PBS buffer solution with a pH value of 7.2-7.4 is obtained.
c. Weighing 5g of bovine serum albumin, and dissolving in 250ml of the PBS buffer solution obtained in the step B to obtain a solution which is marked as solution B.
d. Slowly dripping the solution A into the solution B under the condition of rapid stirring, and standing and storing the obtained mixed solution at the temperature of 4 ℃ overnight to obtain the artificial antigen mixed solution.
e. And (3) transferring the artificial antigen mixed solution into a dialysis bag, dialyzing for 9 times by using the PBS buffer solution in the step b, centrifuging after dialysis is finished, and taking supernate to obtain clozapine artificial antigen (4), wherein the specific synthetic route is shown in figure 2. Through ultraviolet scanning spectrum identification, the clozapine artificial antigen is successfully prepared in the embodiment.
Preparation of antibodies
Animals, such as mice, rabbits, or other mammals, may be immunized with the clozapine artificial antigen prepared above to produce polyclonal Antibodies, or hybridoma cells may be used to produce monoclonal Antibodies, which are well known in the art AND will not be described herein, AND reference may be made to some textbooks or immunization manuals to obtain Antibodies or antibody fragments of the invention (preparation AND use of Antibodies-utility handbook. crppress, 2007). The antibody prepared from the clozapine artificial antigen can specifically recognize the artificial antigen or hapten or small molecular substances, but can not recognize other unrelated antigens or small molecular substances. By "specific" is meant that the antibody recognizes or binds only a particular type of antigen and does not recognize or bind other types of antigens.
Application of clozapine artificial antigen and antibody
The synthesized clozapine hapten or clozapine artificial antigen and the antibody (the antibody refers to a monoclonal antibody or a polyclonal antibody) generated by the clozapine hapten or the clozapine artificial antigen can be used for detecting whether a sample contains clozapine and a derivative drug by an immunoassay method, wherein the sample generally refers to a body fluid sample of a mammal or a human, such as saliva, urine and sweat, or a tissue sample of the mammal or the human, such as tissues of liver, spleen and the like. The "immunoassay method" is generally an immunoassay or detection method using the principle of binding antibody and antigen, and the immunoassay method includes an enzyme-linked immunosorbent assay (ELISA) method, and also includes a lateral flow assay strip (including fluorescence method and colloidal gold method), and may also include a competition method.
The lateral flow test strip may be made of absorbent or non-absorbent materials, and a single test strip may be made of a variety of materials for fluid transfer. One material of the test strip may be superimposed on another test strip material, for example, filter paper superimposed on nitrocellulose. Alternatively, one region of the test strip containing at least one material is positioned behind another region containing at least one different material. In this case, the liquid flows between the zones, which may or may not be superimposed on each other. The material on the test strip may be immobilized on a support such as a plastic backing or a hard surface to enhance the test strip holding power. The test strip regions may be arranged as follows: a sample addition zone, at least one reagent zone, at least one detection result zone, at least one control zone, at least one adulteration detection zone and a liquid absorption zone. If the detection zone comprises a control zone, it is preferred that the control zone is located after the analyte detection zone in the detection result zone. All of these zones or combinations thereof may be on a single strip containing one material. In addition, the zones are made of different materials and are joined together in the direction of liquid transfer. For example, the different zones may be in direct or indirect fluid communication. In this embodiment, the different zones may be connected in the direction of liquid transfer, at the end of one zone to the beginning of another zone, or superimposed on each other in the direction of liquid transfer, or connected by other materials, such as a connecting medium material (preferably a water-absorbing material such as filter paper, glass fibers, non-woven fabric or nitrocellulose). The connecting material can be a material including regions in which the ends are in contact with the beginning, a material including regions in which the ends are in contact with the beginning but liquid does not flow, or a material including regions in which the regions overlap with each other (for example, but not limited to, overlapping from end to end) but liquid does not flow, and liquid flow is formed. The general structure of a lateral flow test strip is shown in FIG. 4.
In addition, the antibody prepared from the clozapine artificial antigen of the present invention can also be used for immunotherapy of various diseases caused by clozapine, for example, an immunological reagent for treating clozapine addiction. The antibody is thus used as a pharmaceutical agent.
Preparing a colloidal gold lateral chromatography detection test strip by using the clozapine artificial antigen and the antibody prepared in the example 1 as main raw materials, wherein the colloidal gold lateral chromatography detection test strip is prepared by adopting the existing known technology in the field; the test strip is used for detecting the clozapine standard substance, and the using amount of a detection sample is 50 mu l; specific detection data are shown in table 1, wherein m represents the number of repeated detections of a single sample; the numbers of the two columns of negative and positive results respectively represent: the number of times the detection result is negative and the number of times the detection result is positive, thereby further obtaining the accuracy of the detection result; for example, a concentration of clozapine of 0ng/mL indicates negative, and all of the 30 tests are negative, i.e., the number of negative tests is 30, and the accuracy is 100%.
TABLE 1 test results
Figure BDA0002253508830000111
The detection results in table 1 show that the clozapine artificial antigen and the clozapine artificial antibody prepared by the invention have higher specificity, and the test strip has very high sensitivity in the detection of clozapine, wherein in the embodiment, the sensitivity is 100ng/ml, and completely meets the market detection requirements.
The concentration of the medicine in the body fluid of a patient can be conveniently judged clinically according to the detection result, so that the dosage and the period of medicine application are adjusted, the medicine is applied more accurately, and adverse reactions such as headache, anxiety, insomnia, somnolence, urinary incontinence and the like can be prevented; drug dependence studies can also be performed.
Cross-reaction experiments: the clozapine artificial antigen and the antibody prepared in example 1 are used as main raw materials to prepare a colloidal gold lateral chromatography detection test strip, and a standard substance of the substance in table 2 is detected, wherein the use amount of the sample is 50 mu l.
TABLE 2 Cross-reaction results
Figure BDA0002253508830000121
Note: "+" indicates cross-reaction and "-" indicates no reaction.
The result shows that the clozapine artificial antigen prepared by the invention has high specificity and has no cross reaction with other drugs.
It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims (7)

1. A clozapine hapten having the structure shown in formula (i):
Figure FDA0002253508820000011
2. a clozapine artificial antigen is characterized by having a structure shown in a formula (II): wherein, p is carrier protein, n represents the number of hapten coupled on one carrier protein;
Figure FDA0002253508820000012
3. the artificial antigen of clozapine as claimed in claim 2, wherein the carrier protein is selected from any one of bovine serum albumin, bovine gamma globulin, bovine thyroglobulin, keyhole limpet hemocyanin and chicken ovalbumin.
4. A method for preparing a hapten for clozapine, the method comprising the steps of:
adding clozapine into a reaction vessel, then adding dichloromethane to completely dissolve the clozapine, controlling the temperature at 0 ℃, adding succinic anhydride, then adding aluminum trichloride every 1 hour, and adding for three times in total; returning to room temperature, and stirring for reaction for 1-20 hours; after the reaction is finished, adding water and concentrated hydrochloric acid, stirring and reacting for 0.5-2 hours, then extracting with dichloromethane, collecting an organic phase, and evaporating the solvent under reduced pressure to obtain a product; and then purifying by thin layer chromatography to obtain clozapine hapten, wherein in the whole reaction, the ratio of clozapine: succinic anhydride: adding aluminum trichloride in a ratio of 1:1:1 to 1:5:5 in total, wherein the aluminum trichloride is added in three times, and the mass of the aluminum trichloride added each time is the same.
5. A method for preparing a clozapine artificial antigen for use in the preparation of a clozapine artificial antigen according to any one of claims 2-3, comprising the steps of:
(1) preparation of clozapine hapten:
adding clozapine into a reaction vessel, then adding dichloromethane to completely dissolve the clozapine, controlling the temperature at 0 ℃, adding succinic anhydride, and then adding aluminum trichloride every 1 hour for three times; returning to room temperature, and stirring for reaction for 1-20 hours; after the reaction is finished, adding water and concentrated hydrochloric acid, stirring and reacting for 0.5-2 hours, then extracting with dichloromethane, collecting an organic phase, and evaporating the solvent under reduced pressure to obtain a product; and then purifying by thin layer chromatography to obtain clozapine hapten, wherein in the whole reaction, the ratio of clozapine: succinic anhydride: adding aluminum trichloride in a ratio of 1:1:1 to 1:5:5 in total, wherein the aluminum trichloride is added in three times, and the mass of the added aluminum trichloride is the same each time;
(2) preparation of clozapine artificial antigen:
a. weighing clozapine hapten in another reaction container, adding N, N-dimethylformamide to completely dissolve the clozapine hapten, adding N-hydroxysuccinimide and N, N-dicyclohexylcarbodiimide, stirring at room temperature for reacting overnight, centrifuging after the reaction is finished, and taking the supernatant as solution A; wherein, clozapine hapten: n-hydroxysuccinimide: n, N-dicyclohexylcarbodiimide in a ratio of 1:1:1 to 1:5:5, the ratio being the amount of the substance;
b. preparing a PBS buffer solution with the pH value of 7.2-7.4;
c. weighing bovine serum albumin, dissolving the bovine serum albumin in the PBS buffer solution in the step B, and recording the obtained solution as solution B; in the solution B, the concentration of the carrier protein is 2-20 mg/ml;
d. slowly dripping the solution A into the solution B under the condition of rapid stirring, standing and storing the obtained mixed solution at 4 ℃ overnight to obtain an artificial antigen mixed solution, wherein the solution A: the ratio of the solution B to the solution B is (1-5000) 1000, and the volume ratio is;
e. and (c) transferring the artificial antigen mixed solution into a dialysis bag, dialyzing by using the PBS buffer solution in the step b, and centrifuging and taking supernate after dialysis is finished to obtain the clozapine artificial antigen.
6. An antibody obtained by immunizing an animal with the clozapine artificial antigen of any one of claims 2-3.
7. An antibody according to claim 6, wherein said antibody is a monoclonal antibody or a polyclonal antibody.
CN201911043573.1A 2019-10-30 2019-10-30 Clozapine artificial antigen and preparation method thereof Pending CN110950808A (en)

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Publication number Priority date Publication date Assignee Title
CN116444647A (en) * 2022-12-19 2023-07-18 浙江准策生物技术有限公司 Clozapine complete antigen and antibody, and preparation method and application thereof

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CN101987869A (en) * 2010-08-30 2011-03-23 南京工业大学 Anthracene artificial immunogen AN-L-Protein
CN103781915A (en) * 2011-05-24 2014-05-07 萨拉戴克斯生物医学公司 Clozapine immunoassay
CN104854110A (en) * 2012-08-21 2015-08-19 詹森药业有限公司 Haptens of olanzipine

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
CN101987869A (en) * 2010-08-30 2011-03-23 南京工业大学 Anthracene artificial immunogen AN-L-Protein
CN103781915A (en) * 2011-05-24 2014-05-07 萨拉戴克斯生物医学公司 Clozapine immunoassay
CN104854110A (en) * 2012-08-21 2015-08-19 詹森药业有限公司 Haptens of olanzipine

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116444647A (en) * 2022-12-19 2023-07-18 浙江准策生物技术有限公司 Clozapine complete antigen and antibody, and preparation method and application thereof
CN116444647B (en) * 2022-12-19 2024-04-30 浙江准策生物技术有限公司 Clozapine complete antigen and antibody, and preparation method and application thereof

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