CN115267171A - Reagent, kit and method for detecting aripiprazole and dehydroaripiprazole concentrations based on homogeneous enzymatic method - Google Patents

Abstract

The application relates to the technical field of immunological detection, and particularly discloses a reagent, a kit and a method for detecting aripiprazole and dehydroaripiprazole concentrations based on a homogeneous enzyme method. The reagent comprises: the kit comprises a reagent R1 containing an antibody and a homogeneous enzyme substrate and a reagent R2 containing an enzyme-labeled conjugate, wherein the enzyme-labeled conjugate is formed by coupling a hapten shown in a formula (I) and glucose-6-phosphate dehydrogenase through a coupling agent; the hapten and the carrier protein are coupled through a coupling agent to form an antigen; the antibody is generated in response to the antigen. The kit comprises the reagent. The method comprises the following steps: adding the reagent R1 into a sample to be detected, reacting, adding the reagent R2, and reacting to obtain a detection solution; the rate of change of absorbance of the test solution was measured at an operating wavelength of 340 nm. The application improves the specificity and sensitivity of simultaneously detecting the aripiprazole and the dehydroaripiprazole.

Description

Reagent, kit and method for detecting aripiprazole and dehydroaripiprazole concentrations based on homogeneous enzyme method

Technical Field

The application relates to the technical field of immunological detection, in particular to a reagent, a kit and a method for detecting aripiprazole and dehydroaripiprazole concentrations based on a homogeneous enzyme method.

Background

Schizophrenia is a group of chronic diseases with unknown etiology, which usually starts slowly or subacute in young and old years, and is clinically manifested as syndromes with different symptoms, involving various disorders such as sensory perception, thinking, emotion and behavior, and incoordination of 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 bilateral regulating effect on the Dopamine (DA) ergic nervous system, is a stabilizer of DA transmitters, 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 formulas of aripiprazole and its metabolites are shown below:

among them, dehydroaripiprazole is the main active metabolite of aripiprazole. Therefore, in detecting the concentration of aripiprazole, it is necessary to simultaneously measure aripiprazole and dehydroaripiprazole. However, the aripiprazole and dehydroaripiprazole concentrations vary 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 certain amount, which delays treatment; therefore, monitoring aripiprazole and dehydroaripiprazole concentrations plays an important role in judging aripiprazole therapeutic effects, evaluating therapeutic effects, avoiding side effects, and adjusting personalized medication regimens.

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 requirements of high-flux, rapid and accurate detection. For example, patent document CN201910630169.8 discloses a method for detecting aripiprazole and dehydroaripiprazole in blood, which is based on the liquid chromatography-mass spectrometry method for measuring aripiprazole and dehydroaripiprazole in blood and requires pretreatment of blood sample. For example, patent document No. CN201910671626.8 discloses a kit for monitoring concentration of aripiprazole drug in blood and a detection method thereof, wherein the detection method is developed based on a multi-dimensional online solid-phase extraction liquid chromatography analysis technology.

For example, patent document CN201380054988.3 discloses an antibody of aripiprazole hapten and its use, 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, 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 simultaneously detecting the aripiprazole and the dehydroaripiprazole, the application provides a reagent, a kit and a method for detecting the concentration of the aripiprazole and the dehydroaripiprazole based on a homogeneous enzyme method.

In a first aspect, the reagent for detecting the concentrations of aripiprazole and dehydroaripiprazole based on a homogeneous enzymatic method provided by the application adopts the following technical scheme:

a reagent for detecting the concentration of aripiprazole and dehydroaripiprazole based on a homogeneous enzymatic method, said reagent comprising

Reagent R1: comprising an antibody and a homogeneous enzyme substrate;

and (3) reagent R2: comprises forming enzyme-labeled conjugate by coupling hapten and enzyme label through coupling method;

the structural formula of the hapten is shown as the formula (I):

coupling the hapten and the carrier protein by a coupling method to form an antigen;

the antibody is generated in response to the antigen.

In some embodiments, in the antigen, the molar ratio of the hapten to the carrier protein in the antigen is 1 (0.010-0.020), for example 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 method of preparing the antigen comprises the steps of:

s2-1, dissolving the hapten in dimethyl sulfoxide to obtain 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 half-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 (15 to 25) mmol/L, e.g., 21.441mmol/L.

In some embodiments, in step S2-2, the concentration of the coupling agent in the aqueous solution of the coupling agent may be (500 to 600) mmol/L, such as 521.644mmol/L.

In some embodiments, in step S2-4, the concentration of the carrier protein in the carrier protein solution may be (0.050 to 0.100) mmol/L, such as 0.060mmol/L. Wherein the carrier protein can be selected from bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, human serum albumin, rabbit serum albumin, and the like. The buffer may be PBS buffer.

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 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 mass percentage concentration of the antibody in the reagent R1 is (0.01 to 0.1)%, for example 0.05%.

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 dehydroaripiprazole to 1mg/mL by adopting a PBS buffer solution, adding an isovolumetric Freund's complete adjuvant, completely emulsifying, and carrying out primary immunization on a host according to the dosage of (0.01-0.2) mg/body; 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 spleen cell line from an inoculated host with Sp2/0 cells, coating an ELISA 96-well plate with the antigen of aripiprazole and dehydroaripiprazole, and performing titer and competition determination on the fused cells by adopting an indirect ELISA method and an indirect competition ELISA method respectively, wherein the fused cells can specifically recognize the aripiprazole and the dehydroaripiprazole and simultaneously have no cross antibody 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: inoculation of antigen to a 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: for 4 weeks.

In some embodiments, the homogeneous enzyme substrate is made by dissolving beta-nicotinamide adenine dinucleotide and glucose-6-phosphate in a buffer; wherein the concentration of the beta-nicotinamide adenine dinucleotide in the homogeneous enzyme substrate is (10-30) mmol/L, and the concentration of the glucose-6-phosphate in the homogeneous enzyme substrate is (20-40) mmol/L.

In some embodiments, the concentration of the enzyme-labeled conjugate in the reagent R2 is (4-6) U/mL, such as 5U/mL.

In some embodiments, the enzyme label is glucose-6-phosphate dehydrogenase.

In some embodiments, the amount of the enzyme label in the enzyme-labeled conjugate is (500 to 700) U/0.5mg hapten, such as 600U/0.5mg hapten.

In some embodiments, in the enzyme-labeled conjugate, the hapten and the enzyme-labeled conjugate are conjugated by a coupling agent to obtain the enzyme-labeled conjugate. In the enzyme-labeled conjugate, the weight ratio of the hapten to the coupling agent is 1 (1.5-2.5), such as 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 method for preparing the enzyme-labeled conjugate comprises the following steps:

s4-1, dissolving glucose-6-phosphate dehydrogenase (G6 PDH for short, CAS No. 56-73-5, 600U) in PBS buffer solution (1 mL) to obtain glucose-6-phosphate dehydrogenase solution;

s4-2, dissolving the antigen (0.5 mg) in dimethyl sulfoxide (DMSO for short, 100 uL) to obtain an antigen solution;

s4-3, uniformly mixing the glucose-6-phosphate dehydrogenase solution and the antigen solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI for short, 1 mg), and uniformly mixing at room temperature for 2 hours;

s4-4, dialyzing the mixture into PBS buffer solution to obtain the enzyme-labeled conjugate.

In some embodiments, in step S4-1, the concentration of the glucose-6-phosphate dehydrogenase in the glucose-6-phosphate dehydrogenase solution is (500 to 700) U/mL.

In some embodiments, in step S4-2, the concentration of the antigen in the antigen solution is 3 to 7mg/mL, such as 5mg/mL.

In some embodiments, the homogeneous enzyme substrate is prepared by dissolving beta-nicotinamide adenine dinucleotide and glucose-6-phosphate in a buffer. The concentration of said beta-nicotinamide adenine dinucleotide in said homogeneous enzyme substrate is (10-30) mmol/L, e.g., 20mmol/L. The concentration of glucose-6-phosphate in the homogeneous enzyme substrate is (20-40) mmol/L, e.g., 30mmol/L. Wherein the buffer may be a Tris buffer, for example 55mmol/L Tris buffer at pH = 8.0.

In a second aspect, the present application provides a kit for detecting aripiprazole and dehydroaripiprazole concentrations based on a homogeneous enzymatic method, which adopts the following technical scheme:

a kit for detecting the concentration of aripiprazole and dehydroaripiprazole based on a homogeneous enzymatic method, said kit comprising said reagents.

In some embodiments, the kit can be used for the detection of urine samples or blood samples.

In a third aspect, the present application provides a method for detecting aripiprazole and dehydroaripiprazole concentrations based on a homogeneous enzymatic method, which adopts the following technical scheme:

a method for detecting aripiprazole and dehydroaripiprazole concentrations based on a homogeneous enzymatic method, comprising the steps of:

adding the reagent R1 into a sample to be detected, and reacting for 3-8 min (for example, 5 min), and then adding the reagent R2, and reacting for 3-8 min (for example, 5 min), so as to obtain a detection solution;

the rate of change of absorbance of the test solution was measured at an operating wavelength of 340 nm.

In some embodiments, the volume ratio of the sample to be tested, the reagent R1 and the reagent R2 is 1 (2-3) to (2-3), for example 1.

In some embodiments, the method uses a device that is a biochemical analyzer.

In some embodiments, the test sample may be urine or serum.

In summary, the present application has the following beneficial effects:

the antigen and the antibody adopted 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 reagent, the kit and the method for detecting the concentration of the aripiprazole and the dehydroaripiprazole by the homogeneous enzyme method improve the specificity and the sensitivity of simultaneously detecting the aripiprazole and the dehydroaripiprazole.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a hapten in the present application;

FIG. 2 is Native-PAGE electrophoresis of bovine serum albumin, antigen in this application;

FIG. 3 is a graph showing the results of HPLC-MS method compared with the method based on homogeneous enzyme method.

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 the aripiprazole shown in the formula (i) and the alkyl alcohol shown in the formula (ii) to generate a compound shown in the formula (iii); and (iv) 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 as 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 the pH =4.5 was adjusted with dilute sulfuric acid, and the mixture was stirred for 20 minutes, left standing, and layered to obtain an organic layer. To the organic layer was added 100mL of dichloromethane, and the mixture was cooled to 0 to 5 ℃ and crystallized with stirring for 2 hours, followed by filtration and drying of the cake to obtain the hapten 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 the hapten (with the molecular weight of 466.4007, 10 mg) and dissolving the hapten in dimethyl sulfoxide (1 mL) to obtain a dimethyl sulfoxide solution (with the concentration of 21.441 mmol/L) of the hapten;

s2-2, weighing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI for short, CAS number 7084-11-9, molecular weight 191.7016, 10 mg) and dissolving in water (100 uL) to obtain an aqueous solution (concentration 521.644 mmol/L) 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, dissolving bovine serum albumin (BSA for short, CAS No. 9048-46-8, molecular weight 66.446KDa, 20mg) in 5mL of PBS buffer solution to obtain a carrier protein solution (the concentration is 0.060 mmol/L);

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 of antibody for detecting aripiprazole and dehydroaripiprazole concentrations

The antibodies may be monoclonal antibodies and polyclonal antibodies. 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 dehydroaripiprazole to 1mg/mL by adopting a PBS buffer solution, adding an isovolumetric Freund 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 for aripiprazole and its active metabolite dehydroaripiprazole, and very low cross-reactivity for aripiprazole inactive metabolites, namely, hydroxylation-aripiprazole and N-Dealkylation-aripiprazole. Therefore, the antibody produced by the 45F2 cell line is suitable for simultaneously detecting the aripiprazole and dehydroaripiprazole concentrations in a sample to be tested.

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 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 in 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 aripiprazole antigen and antibody provided by the present application have the ability to recognize aripiprazole and dehydroaripiprazole simultaneously, and have antibody specificity superior to that of the related art, i.e., no cross reaction with Hydroxylation-aripiprazole and N-Dealkylation-aripiprazole. Accordingly, the antigens and antibodies provided herein enable the construction of methods for accurately monitoring aripiprazole and dehydroaripiprazole concentrations in blood and urine.

Method for detecting aripiprazole and dehydroaripiprazole based on homogeneous enzyme method immune construction

Reagent for detecting aripiprazole and dehydroaripiprazole concentration based on homogeneous enzyme method

The reagent for detecting the concentration of aripiprazole and dehydroaripiprazole based on the homogeneous enzyme method comprises the following components: a reagent R1 comprising an antibody and a homogeneous enzyme substrate and a reagent R2 comprising an enzyme-labeled conjugate formed by coupling a hapten and glucose-6-phosphate dehydrogenase via a coupling agent.

Wherein, the preparation of the reagent R1:

the preparation method of the reagent R1 comprises the following steps:

dissolving beta-nicotinamide adenine dinucleotide (NAD for short, CAS No. 53-84-9, molecular weight 663.425, 13.2685g, 20mmol) and glucose-6-phosphate (G6P for short, CAS No. 112898-35-8, molecular weight 260.136,7.8041g, 30mmol) in Tris buffer (55 mmol/L, pH =8.0, 1L) to prepare homogeneous enzyme substrate;

antibody 45F2 was added to the homogeneous enzyme substrate to give reagent R1. Wherein, the mass percentage concentration of the antibody 45F2 in the reagent R1 can be (0.01-0.1)%; in this embodiment, the concentration of antibody 45F2 in reagent R1 is 0.05% by mass.

Preparation of reagent R1:

the preparation method of the enzyme-labeled conjugate comprises the following steps:

s4-1, dissolving glucose-6-phosphate dehydrogenase (G6 PDH for short, CAS number 56-73-5, 600U) in PBS buffer solution (1 mL) to obtain glucose-6-phosphate dehydrogenase solution (concentration 600U/mL);

s4-2, dissolving the antigen (0.5 mg) in dimethyl sulfoxide (DMSO for short, 100 uL) to obtain an antigen solution;

s4-3, uniformly mixing the glucose-6-phosphate dehydrogenase solution and the antigen solution, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI for short, CAS number 7084-11-9, molecular weight 191.7016, 1mg), and uniformly mixing for 2h at room temperature;

s4-4, dialyzing the mixture into PBS buffer solution to obtain the enzyme-labeled conjugate.

The preparation method of the reagent R1 comprises the following steps: the method comprises the following steps:

the enzyme-labeled conjugate was added to Tris buffer (0.1 mol/L, pH = 8.5) to obtain reagent R2. Wherein, the concentration of the enzyme-labeled conjugate in the reagent R2 can be (4-6) U/mL; in this embodiment, the concentration of the enzyme-labeled conjugate in the reagent R2 is 5U/mL.

Method for detecting aripiprazole and dehydroaripiprazole concentration based on homogeneous enzyme method

The method for detecting the concentration of aripiprazole and dehydroaripiprazole based on the homogeneous enzyme method comprises the following steps:

adding a reagent R1 (50 uL) into a sample to be detected (20 uL) and reacting for 5min, and adding a reagent R2 (50 uL) and reacting for 5min to obtain a detection solution;

the rate of change of absorbance of the test solution was measured at an operating wavelength of 340 nm.

In the present embodiment, the method for detecting the concentration of aripiprazole and dehydroaripiprazole based on the homogeneous enzymatic method employs a Hitachi 7170 biochemical analyzer.

Clinical 40 samples of urine to be detected are selected, and the aripiprazole and dehydroaripiprazole concentrations in the 40 samples of urine to be detected are determined by the construction method and the HPLC-MS method of the application respectively.

Comparing the detection results of the method based on the homogeneous enzyme method and the HPLC-MS method, the results are shown in FIG. 3. As can be seen from FIG. 3, the correlation between the HPLC-MS method and the method for detecting the aripiprazole and dehydroaripiprazole concentrations based on the homogeneous enzyme method is good, and the clinical requirements are met.

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 scope of the invention, and such modifications and improvements are also considered to be within the scope of the invention.

Claims (10)

1. A reagent for detecting the concentration of aripiprazole and dehydroaripiprazole based on a homogeneous enzymatic method, comprising a reagent R1: comprising an antibody and a homogeneous enzyme substrate;

and (3) reagent R2: comprises forming enzyme-labeled conjugate by coupling hapten and enzyme label through coupling method;

the structural formula of the hapten is shown as the formula (I):

coupling the hapten and the carrier protein by a coupling method to form an antigen;

the antibody is generated in response to the antigen.

2. The reagent according to claim 1, wherein the molar ratio of the hapten to the carrier protein in the antigen is 1 (0.010-0.020).

3. The reagent according to claim 1, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

4. The reagent according to claim 1, wherein the mass percentage concentration of the antibody in the reagent R1 is (0.01-0.1)%.

5. The reagent of claim 1, wherein the homogeneous enzyme substrate is prepared by dissolving β -nicotinamide adenine dinucleotide and glucose-6-phosphate in a buffer; wherein the concentration of the beta-nicotinamide adenine dinucleotide in the homogeneous enzyme substrate is (10-30) mmol/L, and the concentration of the glucose-6-phosphate in the homogeneous enzyme substrate is (20-40) mmol/L.

6. The reagent as claimed in claim 1, wherein the amount of the enzyme label in the enzyme-labeled conjugate is (500 to 700) U/0.5mg of hapten.

7. The reagent according to claim 1, wherein the enzyme label is glucose-6-phosphate dehydrogenase.

8. The reagent of claim 1, wherein the concentration of the enzyme-labeled conjugate in the reagent R2 is (4-6) U/mL.

9. A kit for detecting aripiprazole and dehydroaripiprazole concentrations based on a homogeneous enzymatic method, comprising the reagent according to any one of claims 1 to 8.

10. A method for detecting aripiprazole and dehydroaripiprazole concentrations based on a homogeneous enzymatic method, comprising the steps of:

adding the reagent R1 into a sample to be detected, reacting for 3-8 min, adding the reagent R2, and reacting for 3-8 min to obtain a detection solution;

measuring the absorbance change rate of the detection solution under the working wavelength of 340 nm;

preferably, the volume ratio of the sample to be detected, the reagent R1 and the reagent R2 is 1 (2-3) to (2-3);

preferably, the test sample is derived from urine or serum.

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