CN115181076B - Hapten, antigen, cell strain, antibody, reagent and kit for detecting concentration of aripiprazole and dehydroaripiprazole - Google Patents
Hapten, antigen, cell strain, antibody, reagent and kit for detecting concentration of aripiprazole and dehydroaripiprazole Download PDFInfo
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- CN115181076B CN115181076B CN202210727200.1A CN202210727200A CN115181076B CN 115181076 B CN115181076 B CN 115181076B CN 202210727200 A CN202210727200 A CN 202210727200A CN 115181076 B CN115181076 B CN 115181076B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
- C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
- C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
- C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
- C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
Abstract
The application relates to the technical field of immunological detection, and particularly discloses a hapten, an antigen, a cell strain, an antibody, a reagent and a kit for detecting the concentration of aripiprazole and dehydroaripiprazole. The structural formula of the hapten is shown as a formula (I). The antigen is a conjugate formed by coupling the hapten and a carrier protein through a coupling method. The cell line is produced in response to the antigen. The antibody is generated in response to the antigen. The reagent comprises the antibody. The kit comprises the antibody. The application improves the specificity and sensitivity of simultaneously detecting the aripiprazole and the dehydroaripiprazole.
Description
Technical Field
The application relates to the technical field of immunological detection, in particular to a hapten, an antigen, a cell strain, an antibody, a reagent and a kit for detecting the concentration of aripiprazole and dehydroaripiprazole.
Background
Schizophrenia is a group of chronic diseases with unknown etiology, which mostly develops slowly or subacute in young and strong years, and is clinically manifested as syndromes with different symptoms, involving various disorders such as sensory perception, thinking, emotion and behavior, and uncoordinated mental activities. The patient is generally aware of the clear and normal intelligence, but some patients suffer from impairment of cognitive function during the course of the disease. The disease course is usually prolonged and repeated attacks, aggravations or deteriorations appear, and some patients finally suffer from decline and mental disabilities, but some patients can keep a recovery state or a basic recovery state after drug treatment and psychological treatment.
Aripiprazole is a novel atypical anti-schizophrenia drug which has a bidirectional regulating effect on Dopamine (DA) nerval system, is a stabilizer of DA transmitter, and has high affinity with D2, D3, 5-HT1A and 5-HT2A receptors. Aripiprazole exerts an anti-schizophrenia effect by partial agonism at D2 and 5-HT1A receptors and antagonism at 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.
The structural formula of the aripiprazole and its metabolites is shown below:
among them, dehydroaripiprazole is a major active metabolite of aripiprazole. Therefore, in detecting the concentration of aripiprazole, it is necessary to simultaneously measure aripiprazole and dehydroaripiprazole. However, the concentration of aripiprazole and dehydroaripiprazole varies greatly between individuals and within individuals, and the compliance of patients with mental diseases is poor, and the drugs are not taken in time in a dose, which delays the treatment; therefore, monitoring the concentration of aripiprazole and dehydroaripiprazole plays an important role in judging the efficacy of aripiprazole, evaluating the therapeutic effect, evading side effects, and adjusting the personalized medication regimen.
At present, a liquid phase secondary mass spectrometry (LC-MS-MS) method is mostly adopted for determining the concentration of aripiprazole and dehydroaripiprazole, but the LC-MS-MS method needs repeated extraction for many times, has the defects of complex operation, time and labor waste, low flux and the like, and cannot meet the clinical high-flux, rapid and accurate detection requirements. For example, patent document CN201910630169.8 discloses a method for detecting aripiprazole and dehydroaripiprazole in blood, which is based on the combined liquid chromatography and mass spectrometry method for measuring aripiprazole and dehydroaripiprazole in blood and requires pretreatment of blood samples. For example, patent document CN201910671626.8 discloses a kit for monitoring the concentration of aripiprazole drug in blood and a detection method thereof, wherein the detection method is developed based on multidimensional online solid phase extraction liquid chromatography analysis technology.
For example, patent document CN201380054988.3 discloses an antibody of aripiprazole hapten and application thereof, and the prepared antibody can simultaneously recognize aripiprazole and dehydroaripiprazole; however, the patent document does not disclose the specificity of the antibody test clinical specimen, and the cross-reactivity to the inactive metabolites of aripiprazole (Hydroxylation and N-Dealkylation aripiprazole). Meanwhile, patent document CN201911043578.4 discloses an aripiprazole artificial antigen and a preparation method thereof, and an antibody prepared by using the artificial antigen can be used for detecting aripiprazole content, but the patent document does not disclose the specificity of the antibody to aripiprazole metabolites.
Therefore, the high-quality aripiprazole and dehydroaripiprazole antibodies are obtained, the immunological detection method of the aripiprazole and the dehydroaripiprazole with strong specificity and high sensitivity is established, and the method has very important significance for formulating an individual dosage scheme of the aripiprazole and evaluating the clinical curative effect and safety of the aripiprazole.
Disclosure of Invention
In order to improve the specificity and sensitivity of the simultaneous detection of aripiprazole and dehydroaripiprazole, the present application provides a hapten, an antigen, a cell line, an antibody, a reagent and a kit for detecting the concentration of aripiprazole and dehydroaripiprazole.
In a first aspect, the present application provides a hapten for detecting aripiprazole and dehydroaripiprazole concentrations, which adopts the following technical scheme:
a hapten for use in detecting the concentration of aripiprazole and dehydroaripiprazole, said hapten having the formula (I):
in some embodiments, the method of preparing the hapten comprises the following steps:
s1-1, aripiprazole shown in formula (i) is dissolved in alkyl alcohol shown in formula (ii) and amide alcoholysis reaction is carried out to obtain compound shown in formula (iii);
s1-2 and the compound shown in the formula (iii) are subjected to hydrolysis reaction and neutralization reaction sequentially to obtain the hapten shown in the formula (I).
In some embodiments, in step S1-1, R in the alkyl alcohol ROH represents a C1-C8 alkyl group. Wherein the alkyl alcohol may be selected from methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, and octanol.
In some embodiments, in step S1-1, the reaction temperature of the amidoalcoholysis reaction is from 40 to 45 ℃.
In some embodiments, in step S1-1, the molar ratio of the aripiprazole to the alkyl alcohol is 1 (40-60).
In some embodiments, in step S1-2, the pH of the hydrolysis reaction is between 11 and 12.
In some embodiments, in step S1-2, the reaction temperature of the hydrolysis reaction is 20 to 30 ℃.
In some embodiments, in step S1-2, the pH of the neutralization reaction is 4 to 5.
In a second aspect, the present application provides an antigen for detecting aripiprazole and dehydroaripiprazole concentrations, which adopts the following technical scheme:
an antigen for detecting the concentration of aripiprazole and dehydroaripiprazole, said antigen being a conjugate formed by coupling said hapten and a carrier protein by a coupling method.
In some embodiments, the molar ratio of hapten to carrier protein is 1 (0.010 to 0.020), e.g., 1.
In some embodiments, in the antigen, the hapten and the carrier protein are conjugated via a coupling agent to provide the antigen. The molar ratio of the hapten to the coupling agent is 1 (2-3), for example 1. Wherein the coupling agent is a carbodiimide coupling agent. The carbodiimide coupling agents include, but are not limited to, N '-dicyclohexylcarbodiimide (DCC for short, CAS number 538-75-0), N' -diisopropylcarbodiimide (DIC for short, CAS number 693-13-0), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC for short, CAS number 25952-53-8), and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI for short, CAS number 7084-11-9).
In some embodiments, the carrier protein is selected from the group consisting of bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, human serum albumin, and rabbit serum albumin.
In some embodiments, the method of preparing the antigen comprises the steps of:
s2-1, dissolving the hapten in dimethyl sulfoxide to obtain a dimethyl sulfoxide solution of the hapten;
s2-2, dissolving a coupling agent in water to obtain an aqueous solution of the coupling agent;
s2-3, mixing the dimethyl sulfoxide solution of the hapten obtained in the step S2-1 with the coupling agent aqueous solution obtained in the step S2-2, and reacting at room temperature for 0.5-2 hours to obtain a semi-coupling reaction solution;
s2-4, dissolving carrier protein in a buffer solution to obtain a carrier protein solution;
s2-5, mixing the carrier protein solution obtained in the step S2-4 with the semi-coupling reaction solution obtained in the step S2-3, and stirring at room temperature for 1-3 hours to obtain a coupling reaction solution;
s2-6, dialyzing the coupling reaction solution obtained in the step S2-5 into a buffer solution to obtain the antigen.
In some embodiments, in step S2-1, the concentration of the hapten in the dimethylsulfoxide solution of the hapten can be (5-20) mg/mL, such as 10mg/mL.
In some embodiments, in step S2-2, the concentration of the coupling agent in the aqueous solution of the coupling agent may be (50-200) mg/mL, such as 100mg/mL.
In some embodiments, in step S2-4, the concentration of the carrier protein in the carrier protein solution may be (2-10) mg/mL, such as 4mg/mL.
In some embodiments, in step S2-4, the buffer may be a PBS buffer. The carrier protein can be selected from bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, human serum albumin, rabbit serum albumin and the like.
In some embodiments, in step S2-6, the buffer may be a PBS buffer. The number of times of dialysis is multiple, e.g., 2, 3, 4, 5, 6, etc. The volume of the buffer solution used in each dialysis is 400 to 600 times, for example 500 times, the volume of the coupling reaction solution.
In a third aspect, the present application provides a cell line for detecting aripiprazole and dehydroaripiprazole concentrations, which adopts the following technical scheme:
a cell line for detecting aripiprazole and dehydroaripiprazole concentrations, said cell line produced in response to said antigen.
In some embodiments, the cell line is named 45F2 cell line, and is preserved in CGMCC (CGMCC for short; address: siro 1 of Beijing university, suzhou province, north Chen, xilu No. 3, the institute of microbiology, china academy of sciences; postal code 100101) with the preservation number of CGMCC NO.45162 at 19.05 and 19.2022.
In a fourth aspect, the present application provides an antibody for detecting aripiprazole and dehydroaripiprazole concentrations, which adopts the following technical scheme:
an antibody for detecting the concentration of aripiprazole and dehydroaripiprazole, said antibody being produced in response to said antigen.
In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody. Both monoclonal and polyclonal antibodies can be prepared by techniques known in the art, i.e., classical hybridoma cell fusion techniques.
In some embodiments, the method for preparing the monoclonal antibody specifically comprises the following steps:
s3-1, vaccination of antigen to host (e.g. mouse, rabbit, goat, sheep, etc.): diluting the antigen of the aripiprazole and the dehydrogenated aripiprazole to 1mg/mL by adopting a PBS buffer solution, adding equivalent volume of Freund's complete adjuvant, completely emulsifying, and carrying out primary immunization on a host according to the dose of (0.01-0.2) mg/unit; after four weeks, weighing 1mg of aripiprazole and dehydroaripiprazole antigens and 1mg of Freund incomplete adjuvant, mixing, stirring at 2000rpm/min for 2 hours to complete emulsification, and performing boosting immunization on the host immunized for the first time according to the dose of (0.01-0.2) mg/host;
s3-2, fusing a splenocyte line from an inoculated host with Sp2/0 cells, coating an ELISA 96 pore plate with antigens of aripiprazole and dehydroaripiprazole, and performing titer and competition measurement on the fused cells respectively by an indirect ELISA method and an indirect competition ELISA method, wherein the antibody can specifically recognize the aripiprazole and the dehydroaripiprazole and simultaneously has no cross with the Hydroxylation-aripiprazole and the N-Dealkylation-aripiprazole.
In some embodiments, the method for preparing the polyclonal antibody specifically comprises the following steps:
the first immunization: vaccination of the host (e.g. mouse, rabbit, goat, sheep, etc.): diluting the antigen of the aripiprazole and the dehydrogenated aripiprazole to 1mg/mL by adopting a PBS buffer solution, adding equivalent volume of Freund's complete adjuvant, completely emulsifying, and carrying out primary immunization on a host according to the dose of (0.01-0.2) mg/unit;
and (3) enhancing immunity: after the interval of 2 to 6 weeks, 1mg of aripiprazole and dehydroaripiprazole antigen and 1mg of Freund incomplete adjuvant are weighed and mixed, the mixture is stirred for 2 hours under the condition that the stirring speed is 2000rpm/min to complete emulsification, the host after the first immunization is subjected to enhanced immunization according to the dose of (0.01 to 0.2) mg/host, and blood is collected to determine the serum titer and specificity.
Wherein the enhancing immunity is repeated for a plurality of times, e.g. 4 times, 5 times. The time interval between two adjacent booster immunizations is (2 to 6) weeks, for example: 4 weeks.
In a fifth aspect, the present application provides a reagent for detecting aripiprazole and dehydroaripiprazole concentrations, which adopts the following technical scheme:
a reagent for detecting aripiprazole and dehydroaripiprazole concentrations, said reagent comprising said antibody.
In a sixth aspect, the present application provides a kit for detecting aripiprazole and dehydroaripiprazole concentrations, which adopts the following technical scheme:
a kit for detecting aripiprazole and dehydroaripiprazole concentrations comprising the antibody.
In some embodiments, the kit can be used for the detection of urine samples or blood samples.
In summary, the present application has the following beneficial effects:
the antigen and the antibody provided by the application have the capability of simultaneously recognizing aripiprazole and active metabolite dehydroaripiprazole, and have antibody specificity superior to the related technology, namely, the antigen and the antibody have no cross reaction with inactive metabolites of aripiprazole, namely, hydroxylation-aripiprazole and N-Dealkylation-aripiprazole. Therefore, the antigen and the antibody provided by the application can be used for constructing an immunological detection method for accurately monitoring the concentration of the aripiprazole and the dehydroaripiprazole in blood and urine.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a hapten in the present application;
FIG. 2 is a Native-PAGE electrophoresis of bovine serum albumin and antigen in the present application.
Detailed Description
The present application will be described in further detail with reference to the following drawings and examples.
Preparation of haptens for detecting aripiprazole and dehydroaripiprazole concentrations
The structural formula of the hapten is:
the synthetic route of the hapten is as follows:
the preparation method of the hapten comprises the following steps: carrying out amide alcoholysis reaction on aripiprazole shown in a formula (i) and alkyl alcohol shown in a formula (ii) to generate a compound shown in a formula (iii); and (5) sequentially carrying out hydrolysis reaction and neutralization reaction on the compound shown in the formula (iii) to obtain the hapten shown in the formula (I).
When the alkyl alcohol shown in the formula (ii) is methanol, the preparation method of the hapten comprises the following steps:
(iv) synthesis of S1-1, a compound represented by formula (iii):
aripiprazole (4.5g, 10mmol) and methanol (20mL, 15.836g, 494mmol) are weighed, stirred and dissolved, added into a 50mL three-necked bottle, concentrated sulfuric acid (1.5 g) is slowly dropped at the temperature of 5-15 ℃, and then heated to 40-45 ℃ and reacted for 2 hours at the temperature of 40-45 ℃. After the reaction, 60mL of water was added, the temperature was reduced to 5 to 10 ℃, then pH =8.5 was adjusted with liquid alkali, and 40mL of ethyl acetate was added, stirred for 20 minutes, and left to stand for separation to obtain an organic layer (i.e., an ethyl acetate solution of the compound represented by formula (iii)).
S1-2, synthesis of hapten shown in formula (I):
evaporating ethyl acetate from the organic layer obtained in the step S1-1 by vacuum concentration, evaporating the ethyl acetate to dryness, and adding 50mL of water; then, firstly, adjusting the pH to be =12 by using liquid alkali, and carrying out hydrolysis reaction for 30 minutes at room temperature; further, 30mL of ethyl acetate was added, and pH =4.5 was adjusted with dilute sulfuric acid, and the mixture was stirred for 20 minutes and allowed to stand for separation to obtain an organic layer. 100mL of methylene chloride was added to the organic layer, the temperature was reduced to 0 to 5 ℃ and the mixture was stirred for crystallization for 2 hours, followed by filtration and drying of the cake to obtain a hapten for aripiprazole of the formula (I) (3.5 g, yield 75.2%).
Characterization of the hapten: the nuclear magnetic resonance hydrogen spectrum of the hapten is shown in figure 1. As can be seen from FIG. 1, the hapten preparation of aripiprazole represented by formula (I) was successful.
Preparation of antigens for detecting aripiprazole and dehydroaripiprazole concentrations
The preparation method of the antigen specifically comprises the following steps:
s2-1, weighing 10mg of hapten of the aripiprazole, and dissolving the hapten in 1mL of dimethyl sulfoxide to obtain a dimethyl sulfoxide solution of the hapten;
s2-2, weighing 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and dissolving the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in 100ul of water to obtain a water solution of the coupling agent;
s2-3, mixing the dimethyl sulfoxide solution of the hapten obtained in the step S2-1 with the aqueous solution of the coupling agent obtained in the step S2-2, and reacting at room temperature for 1 hour to obtain a semi-coupling reaction solution;
s2-4, weighing 20mg of carrier protein, and dissolving the carrier protein in 5mL of PBS buffer solution to obtain a carrier protein solution; in this embodiment the carrier protein is bovine serum albumin;
s2-5, mixing the carrier protein solution obtained in the step S2-4 with the reaction solution obtained in the step S2-3, and stirring at room temperature for 2 hours to obtain a coupling reaction solution;
and S2-6, dialyzing the coupling reaction solution obtained in the step S2-5 by using a PBS buffer solution for four times, wherein the volume ratio of the coupling reaction solution to the PBS buffer solution is 1.
Characterization of the antigen: native-PAGE electrophoresis of Bovine Serum Albumin (BSA) and an antigen (BSA-aripiprazole derivative) coupled with a hapten and BSA was performed, and the results are shown in FIG. 2. As can be seen from FIG. 2, the conjugated antigen showed a faster electrophoretic speed than bovine serum albumin alone on electrophoresis, indicating that the antigen was successfully prepared.
Preparation for detecting aripiprazole and dehydroaripiprazole concentrations
The antibody may be a monoclonal antibody or a polyclonal antibody. Both monoclonal and polyclonal antibodies can be prepared by techniques known in the art, i.e., classical hybridoma cell fusion techniques.
The preparation method of the monoclonal antibody specifically comprises the following steps:
s3-1, inoculation of antigen to host: diluting the antigen of the aripiprazole and the dehydrogenated aripiprazole to 1mg/mL by adopting a PBS buffer solution, adding equivalent volume of Freund's complete adjuvant, completely emulsifying, and immunizing a mouse for the first time according to the dose of 0.1 mg/mouse; after four weeks, weighing 1mg of aripiprazole and dehydroaripiprazole antigens and 1mg of Freund incomplete adjuvant, mixing, stirring at 2000rpm/min for 2 hours to complete emulsification, and performing boosting immunization on the mice immunized for the first time according to the dose of 0.1 mg/mouse;
s3-2, fusing a spleen cell line from an inoculated host with Sp2/0 cells, coating an ELISA 96 pore plate with antigens of aripiprazole and dehydroaripiprazole, performing titer and competition measurement on the fused cells respectively by an indirect ELISA method and an indirect competition ELISA method, and screening to obtain 3 cell strains which compete best for the aripiprazole, wherein the cell strains are respectively named as 45F2, 1B2 and 21G7.
Wherein the 45F2 cell strain is preserved in China general microbiological culture Collection center (CGMCC for short; address: no. 3 of Xilu No. 1 of Beijing, chaoyang, institute of microbiology, china academy of sciences; zip code 100101) at 19.05.19.2022 years, and the preservation number is CGMCC NO.45162.
Table 1: indirect competitive ELISA results for cell supernatants of 45F2 cell lines
Table 2: indirect competitive ELISA results for cell supernatants of 1B2 cell lines
Table 3: indirect competitive ELISA results for cell supernatants of 21G7 cell lines
The cross-reactivity is calculated by the formula:
as can be seen from tables 1 to 3, the antibodies produced by the 45F2 cell line exhibited almost uniform recognition efficiency of dehydroaripiprazole, an active metabolite thereof, and very low cross-reaction between Hydroxylation-aripiprazole and N-dealkylation-aripiprazole on inactive metabolites of aripiprazole. Therefore, the antibody produced by the 45F2 cell line is suitable for simultaneously detecting the concentration of aripiprazole and dehydroaripiprazole in a sample.
This is probably due to the fact that the hapten of the present application is derived at a site remote from the common characteristic structures of aripiprazole and dehydroaripiprazole, and therefore the antibodies of the present application are capable of recognizing aripiprazole and its active metabolite dehydroaripiprazole equally. Furthermore, the antibodies to aripiprazole of the present application do not cross-react with the inactive metabolites, hydroxylation-aripiprazole and N-dealkylation-aripiprazole. Moreover, the characteristic structure of the dehydroaripiprazole is creatively removed from the hapten of the application, and an extremely short connecting arm is adopted to be coupled with a carrier protein, so that the aripiprazole and the dehydroaripiprazole can be simultaneously identified, and the method is very favorable for the determination of the aripiprazole and the active metabolite dehydroaripiprazole.
In summary, the antigen and antibody provided by the present application have the ability to simultaneously recognize aripiprazole and its active metabolite dehydroaripiprazole, and simultaneously have antibody specificity superior to that of the related art, i.e., no cross reaction with the inactive metabolites of aripiprazole, namely, hydroxylation-aripiprazole and N-Dealkylation-aripiprazole. Therefore, the antigens and antibodies provided herein enable the construction of immunological detection methods for accurately monitoring aripiprazole and dehydroaripiprazole concentrations in blood and urine.
It will be understood that the above embodiments are merely exemplary embodiments taken to illustrate the principles of the present invention, which is not limited thereto. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit and substance of the invention, and these modifications and improvements are also considered to be within the scope of the invention.
Claims (9)
1. A hapten for use in detecting the concentration of aripiprazole and dehydroaripiprazole, wherein the hapten has the structural formula shown in formula (I):
2. the method of producing the hapten according to claim 1, wherein the method of producing the hapten comprises the steps of:
s1-1, aripiprazole shown in formula (i) is dissolved in alkyl alcohol shown in formula (ii) and amide alcoholysis reaction is carried out to obtain compound shown in formula (iii); r in the alkyl alcohol ROH represents C1-C8 alkyl;
and (5) sequentially carrying out hydrolysis reaction and neutralization reaction on the compounds shown in the S1-2 and the formula (iii) to obtain the hapten shown in the formula (I).
3. The method according to claim 2, wherein the reaction temperature of the amide alcoholysis reaction in step S1-1 is 40 to 45 ℃.
4. The method according to claim 2, wherein the hydrolysis reaction has a pH of 11 to 12 in step S1-2.
5. The method according to claim 2, wherein the neutralization reaction is carried out at a pH of 4 to 5 in step S1-2.
6. An antigen for use in detecting the concentration of aripiprazole and dehydroaripiprazole, wherein said antigen is a conjugate formed by coupling the hapten and the carrier protein according to claim 1 by a coupling method.
7. The antigen according to claim 6, wherein the molar ratio of the hapten to the carrier protein is 1 (0.010-0.020).
8. The antigen of claim 7, wherein the hapten and the carrier protein are conjugated via a coupling agent to provide the antigen.
9. A cell line for detecting aripiprazole and dehydroaripiprazole concentrations, wherein said cell line is produced in response to the antigen of any one of claims 6 to 8;
the cell strain is named as 45F2 cell strain and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO.45162.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104822660A (en) * | 2012-08-21 | 2015-08-05 | 詹森药业有限公司 | Haptens of aripiprazole and their use in immunoassays |
| CN110054694A (en) * | 2012-08-21 | 2019-07-26 | 詹森药业有限公司 | The antibody and application thereof of Aripiprazole haptens |
| CN110922357A (en) * | 2019-10-30 | 2020-03-27 | 杭州博拓生物科技股份有限公司 | Aripiprazole artificial antigen and preparation method thereof |
| WO2022078524A2 (en) * | 2021-11-03 | 2022-04-21 | Hangzhou Dac Biotech Co., Ltd. | Specific conjugation of an antibody |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104822660A (en) * | 2012-08-21 | 2015-08-05 | 詹森药业有限公司 | Haptens of aripiprazole and their use in immunoassays |
| CN110054694A (en) * | 2012-08-21 | 2019-07-26 | 詹森药业有限公司 | The antibody and application thereof of Aripiprazole haptens |
| CN110922357A (en) * | 2019-10-30 | 2020-03-27 | 杭州博拓生物科技股份有限公司 | Aripiprazole artificial antigen and preparation method thereof |
| WO2022078524A2 (en) * | 2021-11-03 | 2022-04-21 | Hangzhou Dac Biotech Co., Ltd. | Specific conjugation of an antibody |
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