CN110922400A - Ziprasidone artificial antigen and preparation method thereof - Google Patents
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Abstract
The invention discloses a ziprasidone artificial antigen and a preparation method thereof.A glutaric anhydride connecting arm is directly introduced to a secondary amino group of ziprasidone to prepare a ziprasidone hapten, and the introduced connecting arm has no other characteristic structure, so that nonspecific combination caused by the connecting arm can be effectively reduced, and the prepared artificial antigen has better specificity; the hapten reacts with N-hydroxysuccinimide and N, N-cyclohexyl carbodiimide to obtain active ester, and the active ester and carrier protein undergo coupling reaction to obtain ziprasidone artificial antigen; the preparation process of the ziprasidone artificial antigen is simple, the production cost is low, and the prepared ziprasidone artificial antigen can be used for animal immunization to obtain a corresponding antibody; the prepared ziprasidone artificial antigen and antibody can be used for detecting whether a sample contains ziprasidone and derived drugs thereof by using an immunoassay method.
Description
Technical Field
The invention particularly relates to a ziprasidone artificial antigen and a preparation method thereof.
Background
Ziprasidone is an atypical antipsychotic with the structural formula:
in vitro studies showed that ziprasidone has a high affinity for dopamine D2, D3, 5-hydroxytryptamine 5HT2A,5HT2C, 5HT1A, 5HT1D, a-adrenergic receptors, a moderate affinity for histamine H1 receptors, and no affinity for other tested receptors/binding sites including the M cholinergic receptor. Ziprasidone has antagonistic effect on dopamine D2, 5-HT2A, 5-HT1D and has agonistic effect on 5-HT1A receptor. Ziprasidone inhibits the reuptake of 5-HT and norepinephrine by synapses.
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 are considerable individual differences in the ability of patients to absorb, distribute, metabolize, and excrete drugs. Such differences may be caused by complications, age, concomitant medication or genetic characteristics. Different drug formulations can also affect the metabolism of the drug. TDM allows for dose optimization for individual patients, improving therapeutic and functional outcomes.
Methods for determining serum or plasma concentration levels of a drug, such as ziprasidone, involve detection using High Performance Liquid Chromatography (HPLC) with UV or mass spectrometric detection, gas chromatography-mass spectrometry (GC-MS), immunoassays and the like, where High Performance Liquid Chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and the like require large analytical instruments, and these analytical methods suffer from the disadvantages of expensive instruments, need for professional technicians to operate, need to be performed in specialized laboratories, and the like.
Because ziprasidone is a small molecule with immunoreactivity and no immunogenicity, the ziprasidone must be linked to a protein macromolecule and the T cell epitope of the macromolecule is used for stimulating the body to generate antibody specific immune response. Therefore, the development and development of a new and effective synthetic method of ziprasidone artificial antigen are very necessary.
Disclosure of Invention
Aiming at the situation and overcoming the defects of the prior art, the invention provides a ziprasidone artificial antigen and a preparation method thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
a ziprasidone hapten having the structure shown in formula (I):
a ziprasidone artificial antigen having a structure as shown in formula (II): wherein p is a carrier protein, and n represents the number of ziprasidone haptens coupled to one carrier protein; the value range of n is 5-60; wherein-CONH-represents an amide group, i.e.
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 process for preparing a ziprasidone hapten, which is useful for preparing a hapten as described above, comprising the steps of:
adding ziprasidone into a reaction container, then adding pyridine and glutaric anhydride, and stirring and reacting for 2-24 hours at room temperature; after the reaction is finished, evaporating the solvent to dryness, and purifying by using a thin-layer chromatography to obtain ziprasidone hapten; wherein, ziprasidone: glutaric anhydride is 1:1 to 1:4, the ratio being the ratio of the amounts of the substances.
A method for preparing a ziprasidone artificial antigen for use in preparing said ziprasidone artificial antigen comprising the steps of:
(1) preparation of ziprasidone hapten:
adding ziprasidone into a reaction container, then adding pyridine and glutaric anhydride, and stirring and reacting for 2-24 hours at room temperature; after the reaction is finished, evaporating the solvent to dryness, and purifying by using a thin-layer chromatography to obtain ziprasidone hapten; wherein, ziprasidone: glutaric anhydride is 1:1-1:4, the ratio is the ratio of the amounts of the substances;
(2) preparation of ziprasidone artificial antigen:
a. weighing ziprasidone hapten in another reaction container, adding N, N-dimethylformamide, adding N-hydroxysuccinimide and N, N-dicyclohexylcarbodiimide, stirring at room temperature for reacting overnight, centrifuging after the reaction is finished, and taking supernatant as solution A; wherein, ziprasidone hapten: n-hydroxysuccinimide: n, N-dicyclohexylcarbodiimide in a ratio of 1:1:1 to 1:7:7, 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 carrier protein, dissolving the carrier protein 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. dropwise adding the A liquid to the B liquid under stirring, and standing the obtained mixed liquid at 4 ℃ overnight to obtain ziprasidone artificial antigen mixed liquid; 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 ziprasidone artificial antigen mixed solution into a dialysis bag, dialyzing by using the PBS buffer solution in the step (b), and centrifuging to obtain a supernatant after dialysis is finished to obtain the ziprasidone artificial antigen.
An antibody, which is obtained by immunizing an animal with the ziprasidone artificial antigen.
Further, the antibody is a monoclonal antibody or a polyclonal antibody.
The invention has the beneficial effects that:
(1) the invention takes ziprasidone as a raw material to produce an artificial antigen, glutaric anhydride connecting arms are directly introduced on secondary amino groups to prepare ziprasidone hapten, the hapten reacts with N-hydroxysuccinimide and N, N-cyclohexyl carbodiimide to obtain active ester, and the active ester and protein undergo coupling reaction to obtain the ziprasidone artificial antigen. The hapten can be obtained only by one step, so that the time cost and the labor cost are greatly saved, the process is simple, the production cost is low, the raw material source is not limited, in addition, the connecting arm introduced by the invention has no other characteristic structures, the nonspecific combination caused by the connecting arm can be effectively reduced, the prepared artificial antigen has higher specificity, and the animal immunization can be carried out to obtain the corresponding antibody.
(2) The ziprasidone artificial antigen and antibody prepared by the invention can be used for detecting whether a sample contains ziprasidone 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 ziprasidone artificial antigen and the ziprasidone 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 ziprasidone hapten.
FIG. 2 is a synthetic roadmap for ziprasidone artificial antigen.
FIG. 3 is a UV scanning spectrum of ziprasidone hapten, ziprasidone artificial antigen, 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 ziprasidone hapten
Weighing 100mg (0.24mmol) of ziprasidone, adding the ziprasidone into a 50ml single-neck round-bottom flask, then adding 5ml of pyridine, 50mg (0.44mmol) of glutaric anhydride, adding a stirrer, and stirring at room temperature for reaction for 20 hours; after the reaction was completed, the solvent was evaporated under reduced pressure and purified by thin layer chromatography, TLC: the chromatographic solution is ethyl acetate, and the specific transfer value Rf of the product is 0.2-0.3; 75mg of hapten (2) is obtained, and the specific synthetic route is shown in figure 1.
(2) Preparation of ziprasidone artificial antigen
a. Weighing 50mg (0.09mmol) of ziprasidone hapten into a 50ml round-bottom flask, adding 3ml of N, N-Dimethylformamide (DMF), adding 64mg (0.57mmol) of N-hydroxysuccinimide (NHS) and 114mg (0.55mmol) of N, N-Dicyclohexylcarbodiimide (DCC), stirring at room temperature for reacting overnight, centrifuging after the reaction is finished, and taking a 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 0.25g of bovine serum albumin, and dissolving the bovine serum albumin in 50ml of the PBS buffer solution with the pH value of 7.2-7.4 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, standing and storing the obtained mixed solution at 4 ℃ overnight to obtain ziprasidone artificial antigen mixed solution.
e. And (b) transferring the ziprasidone 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 ziprasidone artificial antigen (4), wherein the specific synthetic route is shown in figure 2.
By performing the above-described steps using 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), different ziprasidone artificial antigens can be prepared.
(3) Identification of ziprasidone artificial antigen:
(a) ultraviolet scanning atlas
An ultraviolet scan of ziprasidone hapten, ziprasidone artificial antigen and bovine serum albumin is shown in figure 3. As can be seen from FIG. 3, the ziprasidone artificial antigen was successfully prepared by the present invention.
(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: PBS buffer solution with pH of 7.2-7.4 is used for preparing ziprasidone hapten solution to obtain PBS solution with ziprasidone hapten concentrations of 0,5,10,20,30 and 40 mu g/ml respectively, the maximum absorption wavelength of the ziprasidone hapten is 295nm through an ultraviolet scanning figure 3, the absorbance value is measured at 295nm, and each concentration is used as a parallel sample. 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 1514.3L/(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. Diluting the ziprasidone artificial antigen solution according to a certain proportion, measuring the absorbance value of the ziprasidone artificial antigen at 655nm, and obtaining the corresponding protein concentration value in the ziprasidone artificial antigen solution from the curve. The protein concentration in the ziprasidone artificial antigen solution calculated by the invention is 3.118 mg/ml.
And (3) coupling ratio determination: diluting the conjugate, namely ziprasidone artificial antigen solution, with PBS 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 283nm1(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 ziprasidone hapten, 65000 is the molecular weight of bovine serum albumin, c 100X 10-3The concentration (mu g/ml) of bovine serum albumin in the ziprasidone artificial antigen solution is calculated to be gamma about 15.
Example 2
(1) Preparation of ziprasidone hapten
Weighing 100mg (0.24mmol) of ziprasidone, adding the ziprasidone into a 50ml single-neck round-bottom flask, then adding 5ml of pyridine and 0.24mmol of glutaric anhydride, adding a stirrer, and stirring at room temperature for reaction for 22 hours; after the reaction was completed, the solvent was evaporated under reduced pressure and purified by thin layer chromatography, TLC: the chromatographic solution is ethyl acetate, and the specific transfer value Rf of the product is 0.2-0.3; 85mg of hapten (2) is obtained, and the specific synthetic route is shown in figure 1.
(2) Preparation of ziprasidone artificial antigen
a. Weighing 0.15mmol of hapten into a 50ml round-bottom flask, adding 4ml of N, N-Dimethylformamide (DMF), adding 0.15mmol of N-hydroxysuccinimide (NHS) and 0.15mmol 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 0.20g of bovine serum albumin, and dissolving the bovine serum albumin in 50ml of the PBS buffer solution with the pH value of 7.2-7.4 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, standing and storing the obtained mixed solution at 4 ℃ overnight to obtain ziprasidone artificial antigen mixed solution.
e. And (b) transferring the ziprasidone 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 ziprasidone artificial antigen (4), wherein the specific synthetic route is shown in figure 2. As can be seen from the ultraviolet scanning spectrum, the ziprasidone artificial antigen is successfully prepared in the embodiment.
By performing the above-described steps using 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), different ziprasidone artificial antigens can be prepared.
Example 3
(1) Preparation of ziprasidone hapten
Weighing 0.20mmol of ziprasidone, adding the ziprasidone into a 50ml single-neck round-bottom flask, then adding 5ml of pyridine and 0.80mmol of glutaric anhydride, adding a stirrer, and stirring and reacting for 10 hours at room temperature; after the reaction was completed, the solvent was evaporated under reduced pressure and purified by thin layer chromatography, TLC: the chromatographic solution is ethyl acetate, and the specific transfer value Rf of the product is 0.2-0.3; hapten (2) is obtained, and the specific synthetic route is shown in figure 1.
(2) Preparation of ziprasidone artificial antigen
a. Weighing 0.10mmol of hapten into a 50ml round-bottom flask, adding 3ml of N, N-Dimethylformamide (DMF), adding 0.28mmol of N-hydroxysuccinimide (NHS) and 0.48mmol 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 0.50g of bovine serum albumin, and dissolving the bovine serum albumin in 50ml of the PBS buffer solution with the pH value of 7.2-7.4 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, standing and storing the obtained mixed solution at 4 ℃ overnight to obtain ziprasidone artificial antigen mixed solution.
e. And (b) transferring the ziprasidone 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 ziprasidone artificial antigen (4), wherein the specific synthetic route is shown in figure 2. As can be seen from the ultraviolet scanning spectrum, the ziprasidone artificial antigen is successfully prepared in the embodiment.
By performing the above-described steps using 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), different ziprasidone artificial antigens can be prepared.
Preparation of antibodies
Animals, such as mice, rabbits, or other mammals, may be immunized with the ziprasidone 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 for obtaining antibodies or antibody fragments of the invention (preparation AND use of antibodies-utility manual (crcpres, 2007). The antibody prepared from the ziprasidone artificial antigen can specifically recognize the artificial antigen or hapten or micromolecular substance, but cannot recognize other unrelated antigens or micromolecular 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 ziprasidone artificial antigen and antibody
The ziprasidone hapten or ziprasidone artificial antigen synthesized by the invention and an antibody (the antibody refers to a monoclonal antibody or a polyclonal antibody) generated by the antigen and the antibody can be used for detecting whether the sample contains ziprasidone and derivatives thereof 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 ziprasidone artificial antigen can be used for immunotherapy of various diseases caused by ziprasidone, for example, an immunological reagent for treating ziprasidone addiction. The antibody is thus used as a pharmaceutical agent.
The ziprasidone artificial antigen and antibody prepared in the embodiment 1 are used as main raw materials to prepare a colloidal gold lateral chromatography detection test strip, and 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 ziprasidone 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 ziprasidone concentration of 0ng/mL indicates negative, all the 30 detection results are negative, that is, the negative detection times are 30, and the accuracy is 100%.
TABLE 1 test results
The detection results in table 1 show that the ziprasidone artificial antigen and antibody prepared by the invention have higher specificity, and the test strip has very high sensitivity in the detection of ziprasidone, and in the embodiment, the sensitivity is 100ng/ml, so that the requirements of market detection are completely met.
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 ziprasidone artificial antigen and 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 substances in table 2 is detected, wherein the use amount of a sample is 50 mu l.
TABLE 2 Cross-reaction results
Note: "+" indicates cross-reaction and "-" indicates no reaction.
The result shows that the ziprasidone artificial antigen prepared by the invention has high specificity and no cross reaction with other medicines.
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 ziprasidone hapten, having a structure according to formula (i):
2. a ziprasidone artificial antigen is characterized by having a structure shown in formula (II): wherein p is a carrier protein, and n represents the number of ziprasidone haptens coupled to one carrier protein;
3. the ziprasidone artificial antigen of 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 process for preparing a ziprasidone hapten, wherein the process is used to prepare the hapten of claim 1, comprising the steps of:
adding ziprasidone into a reaction container, then adding pyridine and glutaric anhydride, and stirring and reacting for 2-24 hours at room temperature; after the reaction is finished, evaporating the solvent to dryness, and purifying by using a thin-layer chromatography to obtain ziprasidone hapten; wherein, ziprasidone: glutaric anhydride is 1:1 to 1:4, the ratio being the ratio of the amounts of the substances.
5. A method for preparing a ziprasidone artificial antigen that is used to prepare a ziprasidone artificial antigen of any one of claims 2-3, comprising the steps of:
(1) preparation of ziprasidone hapten:
adding ziprasidone into a reaction container, then adding pyridine and glutaric anhydride, and stirring and reacting for 2-24 hours at room temperature; after the reaction is finished, evaporating the solvent to dryness, and purifying by using a thin-layer chromatography to obtain ziprasidone hapten; wherein, ziprasidone: glutaric anhydride is 1:1-1:4, the ratio is the ratio of the amounts of the substances;
(2) preparation of ziprasidone artificial antigen:
a. weighing ziprasidone hapten in another reaction container, adding N, N-dimethylformamide, adding N-hydroxysuccinimide and N, N-dicyclohexylcarbodiimide, stirring at room temperature for reacting overnight, centrifuging after the reaction is finished, and taking supernatant as solution A; wherein, ziprasidone hapten: n-hydroxysuccinimide: n, N-dicyclohexylcarbodiimide in a ratio of 1:1:1 to 1:7:7, 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 carrier protein, dissolving the carrier protein 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. dropwise adding the A liquid to the B liquid under stirring, and standing the obtained mixed liquid at 4 ℃ overnight to obtain ziprasidone artificial antigen mixed liquid; 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 ziprasidone artificial antigen mixed solution into a dialysis bag, dialyzing by using the PBS buffer solution in the step (b), and centrifuging to obtain a supernatant after dialysis is finished to obtain the ziprasidone artificial antigen.
6. An antibody obtained by immunizing an animal with the ziprasidone artificial antigen of any one of claims 2 to 3.
7. An antibody according to claim 6, wherein said antibody is a monoclonal antibody or a polyclonal antibody.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1898236A (en) * | 2003-10-24 | 2007-01-17 | 特瓦制药工业有限公司 | Processes for preparation of ziprasidone |
| CN101538307A (en) * | 2009-04-27 | 2009-09-23 | 华南农业大学 | Dehydroepiandrosterone hapten, artificial antigen and antibody as well as preparation method and application thereof |
| CN102917592A (en) * | 2010-03-16 | 2013-02-06 | 萨拉戴克斯生物医学公司 | Risperidone immunoassay |
| CN103951577A (en) * | 2014-04-25 | 2014-07-30 | 中国农业科学院油料作物研究所 | Artificial hapten and artificial antigen of capsaicine, as well as preparation methods thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1898236A (en) * | 2003-10-24 | 2007-01-17 | 特瓦制药工业有限公司 | Processes for preparation of ziprasidone |
| CN101538307A (en) * | 2009-04-27 | 2009-09-23 | 华南农业大学 | Dehydroepiandrosterone hapten, artificial antigen and antibody as well as preparation method and application thereof |
| CN102917592A (en) * | 2010-03-16 | 2013-02-06 | 萨拉戴克斯生物医学公司 | Risperidone immunoassay |
| CN103951577A (en) * | 2014-04-25 | 2014-07-30 | 中国农业科学院油料作物研究所 | Artificial hapten and artificial antigen of capsaicine, as well as preparation methods thereof |
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Application publication date: 20200327 |