CN110922357A - Aripiprazole artificial antigen and preparation method thereof - Google Patents
Aripiprazole artificial antigen and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an aripiprazole artificial antigen and a preparation method thereof, the invention takes aripiprazole as a raw material to synthesize the artificial antigen, a carboxymethyl hydroxylamine connecting arm is introduced on a carbonyl group to obtain the aripiprazole hapten, the yield is higher, the carbonyl group is far away from the characteristic structure of the aripiprazole, 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 aripiprazole artificial antigen; the aripiprazole artificial antigen has simple preparation process and low production cost, and the prepared aripiprazole artificial antigen can be used for animal immunization to obtain a corresponding antibody; the prepared aripiprazole artificial antigen and the prepared aripiprazole antibody can be used for detecting whether a sample contains aripiprazole and metabolic drugs thereof by an immunoassay method.
Description
Technical Field
The invention particularly relates to an aripiprazole artificial antigen and a preparation method thereof.
Background
Aripiprazole is a novel atypical anti-schizophrenia drug having the structural formula:
it has bidirectional regulating effect on DA energy nervous system, and is a stabilizer of DA transmitter. Has high affinity with D2, D3, 5-HT1A and 5-HT2A receptors. The anti-schizophrenia effect is produced by partial agonism of D2 and 5-HT1A receptors and antagonism of 5-HT2A receptors. After the aripiprazole is orally taken, the peak time of the blood concentration is 3-5 hours, and the half-life period is 48-68 hours. Dehydroaripiprazole is the main active metabolite. Aripiprazole can be used to treat various types of schizophrenia. The foreign clinical test shows that the aripiprazole has obvious curative effect on positive and negative symptoms of schizophrenia, can also improve accompanying emotional symptoms and reduce the recurrence rate of schizophrenia.
Therapeutic drug monitoring (TDM for short) refers to the process of observing the therapeutic effect of a drug, 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, guided by the basic theory of pharmacokinetics and pharmacodynamics, by means of advanced analysis techniques 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 neuroleptic drugs, such as aripiprazole, involve the use of High Performance Liquid Chromatography (HPLC) with UV or mass spectrometric detection, gas chromatography-mass spectrometry (GC-MS) combined analysis, immunoassays and the like, wherein High Performance Liquid Chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) combined analysis and the like require large-scale analytical equipment, and these analytical methods have the disadvantages of expensive equipment, need for professional technicians to operate, need to be performed in professional laboratories and the like.
Because the aripiprazole is a small molecule, only has immunoreactivity and no immunogenicity, the aripiprazole must be connected to protein macromolecules, and the T cell epitope of the macromolecules stimulates the organism to generate antibody specific immune response. Therefore, it is necessary to develop and develop a new and effective synthetic method of aripiprazole artificial antigen.
Disclosure of Invention
Aiming at the situation, in order to overcome the defects of the prior art, the invention provides an aripiprazole artificial antigen and a preparation method thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
an aripiprazole hapten having the structure shown in formula (I):
an aripiprazole artificial antigen having a structure represented by formula (II): wherein, p is carrier protein, n represents the number of hapten coupled on one carrier protein; the value range of n is 5-50, wherein-CONH-represents an amide group, namely
Further, the carrier protein p is selected from any one of Bovine Serum Albumin (BSA), Bovine Gamma Globulin (BGG), Bovine Thyroglobulin (BTG), Keyhole Limpet Hemocyanin (KLH) and chicken Ovalbumin (OVA).
A process for preparing an aripiprazole hapten, which is used for preparing the hapten as described above, comprising the steps of:
weighing aripiprazole, adding the aripiprazole into a reaction container, then adding pyridine and carboxymethyl hydroxylamine hemihydrochloride, and stirring and reacting for 2-20 hours at room temperature; after the reaction is finished, evaporating the solvent to dryness, filtering, extracting the filtrate with ethanol, filtering again, collecting the filtrate, spin-drying to obtain a product, and purifying by thin-layer chromatography to obtain the hapten; wherein, the aripiprazole: the ratio of the amount of the substance to the amount of the carboxymethylhydroxylamine hemihydrochloride is 1:1 to 1: 4.
A method for preparing an aripiprazole artificial antigen for preparing the above artificial antigen, comprising the steps of:
(1) preparation of aripiprazole artificial hapten:
weighing aripiprazole, adding the aripiprazole into a reaction container, then adding pyridine and carboxymethyl hydroxylamine hemihydrochloride, and stirring and reacting for 2-20 hours at room temperature; after the reaction is finished, evaporating the solvent to dryness, filtering, extracting the filtrate with ethanol, filtering again, collecting the filtrate, spin-drying to obtain a product, and purifying by thin-layer chromatography to obtain the hapten;
aripiprazole: the ratio of the amount of the substance to the amount of the carboxymethylhydroxylamine hemihydrochloride is 1:1 to 1: 4.
(2) Preparation of aripiprazole artificial antigen:
a. weighing the prepared aripiprazole hapten into a 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 the supernatant as solution A; aripiprazole hapten: n-hydroxysuccinimide: n, N-dicyclohexylcarbodiimide in a ratio of 1:1 to 3, the ratio being a ratio of the amounts of the substances;
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, wherein the protein concentration is 2-20mg/ml, and recording the obtained solution as solution B; the carrier protein is selected from any one of bovine serum albumin, bovine gamma globulin, bovine thyroglobulin, keyhole limpet hemocyanin and chicken egg white albumin;
d. dripping the A liquid into the B liquid under stirring, and standing the obtained mixed liquid at 4 ℃ overnight to obtain artificial antigen mixed liquid; wherein, the liquid 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 aripiprazole artificial antigen (II).
An antibody obtained by immunizing an animal with the aripiprazole artificial antigen described above.
Further, the antibody is a monoclonal antibody or a polyclonal antibody.
The invention has the beneficial effects that:
(1) the aripiprazole is used as a raw material to synthesize an artificial antigen, and a carboxymethyl hydroxylamine connecting arm is introduced to a carbonyl group to obtain an aripiprazole hapten, so that the aripiprazole hapten has high yield, the carbonyl group is far away from the characteristic structure of the aripiprazole, 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 aripiprazole artificial antigen; the aripiprazole artificial antigen provided by the invention has the advantages of simple preparation process, low production cost and unlimited raw material source, and the prepared aripiprazole artificial antigen can be used for animal immunization to obtain a corresponding antibody.
(2) The aripiprazole artificial antigen and the aripiprazole artificial antibody prepared by the invention can be used for detecting whether a sample contains aripiprazole and metabolic drugs thereof by an immunoassay method, and can 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 scheme for synthesis of aripiprazole hapten.
Figure 2 is a scheme of the synthesis of aripiprazole artificial antigen.
FIG. 3 is the UV scanning spectrum of aripiprazole hapten, aripiprazole 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) The preparation of the aripiprazole hapten comprises the following steps:
weighing 500mg (1.12mmol) of aripiprazole, adding into a 150ml single-neck round-bottom flask, then adding 25ml of pyridine and 437mg (2mmol) of carboxymethyl hydroxylamine hemihydrochloride, adding a stirrer, and stirring at room temperature for reaction for 20 hours; after the reaction is finished, the solvent is evaporated to dryness under reduced pressure, the filtrate is filtered by quick filter paper, the filtrate is extracted by 3X 10ml of ethanol and filtered again, the filtrate is collected and dried in a spinning mode, and 678mg of white solid product is obtained; purification by thin layer chromatography, TLC: the chromatographic liquid is ethyl acetate, and the product has a specific displacement value Rf0.4-0.5; obtaining 425mg aripiprazole hapten (2); the specific synthetic route is shown in figure 1;
(2) the preparation method of the aripiprazole artificial antigen comprises the following steps:
a. 200mg (0.4mmol) of the hapten (2) prepared above is weighed into a 50ml round-bottom flask, 10ml of N, N-Dimethylformamide (DMF) is added, 64mg (0.56mmol) of N-hydroxysuccinimide (NHS) and 114mg (0.55mmol) 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 aripiprazole artificial antigen (4), wherein the specific synthetic route is shown in figure 2.
Different aripiprazole artificial antigens can be prepared by carrying out the above-described procedure in place of Bovine Serum Albumin (BSA) by any one of Bovine Gamma Globulin (BGG), Bovine Thyroglobulin (BTG), Keyhole Limpet Hemocyanin (KLH) and chicken Ovalbumin (OVA).
(3) Identification of aripiprazole artificial antigen:
(a) ultraviolet scanning atlas
An ultraviolet scan of the aripiprazole hapten, aripiprazole artificial antigen and bovine serum albumin, as shown in figure 3; as can be seen from FIG. 3, the present invention successfully prepared an aripiprazole 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 absorption coefficient ε: the method comprises the steps of preparing an aripiprazole hapten solution by using a PBS buffer solution with the pH value of 7.2-7.4, wherein the aripiprazole hapten concentrations are 0,5,10,20,30 and 40 mu g/ml respectively, the maximum absorption wavelength of the aripiprazole hapten is 290nm through an ultraviolet scanning graph, the absorbance value is measured at 290nm, and each concentration is used as a parallel sample. The molar absorption coefficient ∈ 2514.3L/(mol cm) was calculated.
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 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 for each concentration, measuring the light absorption value at 655nm, and drawing a relation curve of the concentration of the bovine serum albumin and the light absorption value. Diluting the aripiprazole artificial antigen solution according to a certain proportion, measuring the absorbance value of the aripiprazole artificial antigen at 655nm, and obtaining the concentration value of bovine serum albumin in the aripiprazole artificial antigen solution from the curve. The protein concentration in the aripiprazole artificial antigen solution calculated by the invention is 5.225 mg/ml.
And (3) coupling ratio determination: diluting the conjugate, namely aripiprazole artificial antigen solution, with PBS buffer solution with pH of 7.2-7.4 to 100 mu g/ml, and measuring the absorbance A at 276nm1(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 A at 278nm2The coupling ratio γ is then: γ ═ a1－A2)/ε]/(c*100×10-3/65000), the invention calculates γ ≈ 19.
Wherein ε is the molar absorptivity of hapten L/(mol cm), 65000 is the molecular weight of bovine serum albumin, c 100 × 10-3The concentration of the protein in the aripiprazole artificial antigen solution (μ g/ml).
Example 2
(1) The preparation of the aripiprazole hapten comprises the following steps:
weighing 1mmol aripiprazole, adding into 150ml single-neck round-bottom flask, and heatingThen adding 25ml of pyridine and 1mmol of carboxymethyl hydroxylamine hemihydrochloride, adding a stirrer, and stirring and reacting for 15 hours at room temperature; after the reaction is finished, evaporating the solvent to dryness under reduced pressure, filtering by using quick filter paper, extracting the filtrate by using 3X 10ml of ethanol, filtering again, collecting the filtrate, and spin-drying to obtain a crude product; purification by thin layer chromatography, TLC: the chromatographic liquid is ethyl acetate, and the product has a specific displacement value Rf0.4-0.5; obtaining the aripiprazole hapten (2); the specific synthetic route is shown in figure 1;
(2) the preparation method of the aripiprazole artificial antigen comprises the following steps:
a. 0.4mmol of the hapten (2) prepared above is weighed into a 50ml round-bottom flask, 10ml of N, N-Dimethylformamide (DMF) is added, 0.4mmol of N-hydroxysuccinimide (NHS) and 0.4mmol of N, N-Dicyclohexylcarbodiimide (DCC) are added, stirring reaction is carried out at room temperature overnight, centrifugation is carried out after the reaction is finished, and the supernatant is taken as liquid 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. 0.20g 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 aripiprazole artificial antigen (4), wherein the specific synthetic route is shown in figure 2. As can be seen from the ultraviolet scanning spectrum, the aripiprazole artificial antigen was successfully prepared in this example.
Different aripiprazole artificial antigens can be prepared by carrying out the above-described procedure in place of Bovine Serum Albumin (BSA) by any one of Bovine Gamma Globulin (BGG), Bovine Thyroglobulin (BTG), Keyhole Limpet Hemocyanin (KLH) and chicken Ovalbumin (OVA).
Example 3
(1) The preparation of the aripiprazole hapten comprises the following steps:
weighing 1mmol of aripiprazole, adding the aripiprazole into a 150ml single-neck round-bottom flask, then adding 25ml of pyridine and 4mmol of carboxymethyl hydroxylamine hemihydrochloride, adding a stirrer, and stirring at room temperature for reaction for 8 hours; after the reaction is finished, evaporating the solvent to dryness under reduced pressure, filtering by using quick filter paper, extracting the filtrate by using 3X 10ml of ethanol, filtering again, collecting the filtrate, and spin-drying to obtain a crude product; purification by thin layer chromatography, TLC: the chromatographic liquid is ethyl acetate, and the product has a specific displacement value Rf0.4-0.5; obtaining the aripiprazole hapten (2); the specific synthetic route is shown in figure 1;
(2) the preparation method of the aripiprazole artificial antigen comprises the following steps:
a. 0.4mmol of the hapten (2) prepared above is weighed into a 50ml round-bottom flask, 10ml of N, N-Dimethylformamide (DMF) is added, 1.2mmol of N-hydroxysuccinimide (NHS) and 1.2mmol of N, N-Dicyclohexylcarbodiimide (DCC) are added, stirring reaction is carried out at room temperature overnight, centrifugation is carried out after the reaction is finished, and the supernatant is taken as liquid 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. 0.20g 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 aripiprazole artificial antigen (4), wherein the specific synthetic route is shown in figure 2. The aripiprazole artificial antigen was successfully prepared by uv-scan pattern identification in this example.
Different aripiprazole artificial antigens can be prepared by carrying out the above-described procedure in place of Bovine Serum Albumin (BSA) by any one of Bovine Gamma Globulin (BGG), Bovine Thyroglobulin (BTG), Keyhole Limpet Hemocyanin (KLH) and chicken Ovalbumin (OVA).
Preparation of antibodies
Animals, such as mice, rabbits, or other mammals, may be immunized with the aripiprazole 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 the Antibodies or antibody fragments of the invention (preparation AND use of Antibodies-utility handbook. CRC Press, 2007). The antibody prepared from the aripiprazole artificial antigen can specifically recognize the artificial antigen or hapten or small molecular substance, but can not recognize other irrelevant 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 aripiprazole artificial antigen and antibody
The synthesized aripiprazole hapten or aripiprazole artificial antigen and the antibody generated by the same (the antibody refers to a monoclonal antibody or a polyclonal antibody) can be used for detecting whether the sample contains the aripiprazole and the derivative drug 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" 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, a lateral flow assay (fluorescence method and colloidal gold method), and 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. The different zones may be joined along the direction of liquid transfer, with the end of one zone being joined to the beginning of the other zone, or superimposed on each other along the direction of liquid transfer, or joined by other materials, such as a joining medium material (preferably a water-absorbent material such as filter paper, glass fibers, non-woven fabric or nitrocellulose). The connecting medium material can be in fluid communication with a material that includes the regions with their ends in contact with the beginning, a material that includes the regions with their ends in contact with the beginning but is not in fluid communication, or a material that includes the regions overlapping with each other (e.g., without limitation, from top to bottom). The general structure of a lateral flow test strip is shown in FIG. 4.
In addition to this, the antibodies prepared from the aripiprazole artificial antigen of the present invention can also be used for immunotherapy for various diseases caused by aripiprazole, for example, an immunological agent for the treatment of addiction to aripiprazole. The antibody is thus used as a pharmaceutical agent.
The aripiprazole artificial antigen and the antibody prepared in the embodiment 1 are used as main raw materials to prepare a colloidal gold lateral chromatography detection test strip, and the preparation method of the colloidal gold lateral chromatography detection test strip is the existing known preparation method in the field; the test strip is used for detecting the aripiprazole standard substance, and the using amount of a detection sample is 50 mul; 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 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, when the concentration of aripiprazole is 0ng/mL, the result is negative, and all the results of 30 detections are negative, that is, the number of negative detections is 30, the accuracy is 100%.
TABLE 1 test results
The detection results in table 1 show that the aripiprazole artificial antigen and antibody prepared by the invention have higher specificity, and the test strip has very high sensitivity in the detection of aripiprazole, in the embodiment, the detected sensitivity is 100ng/ml, and completely meets the market detection requirement.
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 aripiprazole artificial antigen and antibody prepared in example 1 were used as main raw materials to prepare a colloidal gold lateral chromatography test strip, and the standard substance of the substance in table 2 was tested, with a sample usage of 50 μ l.
TABLE 2 Cross-reaction results
Note: "+" indicates cross-reaction and "-" indicates no reaction.
The cross-reaction results in Table 2 show that the aripiprazole artificial antigen prepared by the invention has high specificity and 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. An aripiprazole hapten, which has the structure shown in formula (I):
2. an aripiprazole artificial antigen, which is characterized by having a structure shown in formula (II): wherein, p is carrier protein, n represents the number of hapten coupled on one carrier protein;
3. an aripiprazole artificial antigen according to 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 an aripiprazole hapten, for preparing the hapten according to claim 1, comprising the steps of:
weighing aripiprazole, adding the aripiprazole into a reaction container, then adding pyridine and carboxymethyl hydroxylamine hemihydrochloride, and stirring and reacting for 2-20 hours at room temperature; after the reaction is finished, evaporating the solvent to dryness, filtering, extracting the filtrate with ethanol, filtering again, collecting the filtrate, spin-drying to obtain a product, and purifying by thin-layer chromatography to obtain the hapten; wherein, the aripiprazole: the ratio of the amount of the substance to the amount of the carboxymethylhydroxylamine hemihydrochloride is 1:1 to 1: 4.
5. A method for preparing an aripiprazole artificial antigen, for preparing the artificial antigen of any one of claims 2-3, comprising the steps of:
(1) preparation of aripiprazole hapten:
weighing aripiprazole, adding the aripiprazole into a reaction container, then adding pyridine and carboxymethyl hydroxylamine hemihydrochloride, and stirring and reacting for 2-20 hours at room temperature; after the reaction is finished, evaporating the solvent to dryness, filtering, extracting the filtrate by using ethanol, filtering again, collecting the filtrate, spin-drying to obtain a product, and purifying by using a thin-layer chromatography to obtain the aripiprazole hapten;
aripiprazole: carboxymethyl hydroxylamine hemihydrochloride is 1:1 to 1:4, and the ratio is the ratio of the amount of the substance;
(2) preparation of aripiprazole artificial antigen:
a. weighing the prepared aripiprazole hapten into a 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 the supernatant as solution A; aripiprazole hapten: n-hydroxysuccinimide: n, N-dicyclohexylcarbodiimide in a ratio of 1:1 to 3, the ratio being a ratio of the amounts of the substances;
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, wherein the protein concentration is 2-20mg/ml, and recording the obtained solution as solution B;
d. dripping the A liquid into the B liquid under stirring, and standing the obtained mixed liquid at 4 ℃ overnight to obtain artificial antigen mixed liquid;
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 aripiprazole artificial antigen.
6. An antibody obtained by immunizing an animal with the aripiprazole 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.
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| CN115181076A (en) * | 2022-06-24 | 2022-10-14 | 北京丹大生物技术有限公司 | Hapten, antigen, cell strain, antibody, reagent and kit for detecting concentration of aripiprazole and dehydroaripiprazole |
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