A reagent, a kit and a method for detecting the concentration of aripiprazole and dehydro-aripiprazole based on a homogeneous enzyme method

Abstract

The present application relates to the technical field of immunological detection, and specifically discloses a reagent, a kit and a method for detecting the concentration of aripiprazole and dehydrogenated aripiprazole based on a homogeneous enzyme method. The reagent comprises: a reagent R1 comprising an antibody and a homogeneous enzyme substrate and a reagent R2 comprising an enzyme-labeled conjugate, wherein the enzyme-labeled conjugate is formed by coupling a hapten as shown in 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; and the antibody is generated in response to the antigen. The kit comprises the reagent. The method comprises the following steps: first adding the reagent R1 to the sample to be tested and reacting, then adding the reagent R2 and reacting to obtain a detection solution; and determining the absorbance change rate of the detection solution at a working wavelength of 340 nm. The present application improves the specificity and sensitivity of simultaneous detection of aripiprazole and dehydrogenated aripiprazole.

Description

Technical Field

[0001] The present application relates to the technical field of immunological detection, and in particular to a reagent, a kit and a method for detecting the concentration of aripiprazole and dehydro-aripiprazole based on a homogeneous enzymatic method. Background Art

[0002] Schizophrenia is a group of chronic diseases with unknown etiology. It usually develops slowly or subacutely in young and middle-aged people. Clinically, it often manifests as a syndrome with different symptoms, involving multiple disorders of perception, thinking, emotion and behavior, as well as incoordination of mental activities. Patients are generally conscious and have basically normal intelligence, but some patients will experience cognitive impairment during the course of the disease. The course of the disease is generally prolonged, with repeated attacks, aggravation or deterioration. Some patients eventually decline and become mentally disabled, but some patients can remain cured or basically cured after drug treatment and psychotherapy.

[0003] Aripiprazole is a new type of atypical antipsychotic drug. It has a bidirectional regulatory effect on the dopamine (DA) nervous system. It is a stabilizer of DA transmitters and has a high affinity for D2, D3, 5-HT1A and 5-HT2A receptors. Aripiprazole produces anti-schizophrenia effects through partial agonism of D2 and 5-HT1A receptors and antagonism of 5-HT2A receptors. The peak time of aripiprazole blood concentration after oral administration is 3 to 5 hours, and the half-life is 48 to 68 hours.

[0004] The structural formulas of aripiprazole and its metabolites are shown below:

[0005]

[0006] Among them, dehydroaripiprazole is the main active metabolite of aripiprazole. Therefore, when detecting the concentration of aripiprazole, it is necessary to measure aripiprazole and dehydroaripiprazole at the same time. However, there are large inter-individual and intra-individual differences in the concentrations of aripiprazole and dehydroaripiprazole, and patients with mental illness have poor compliance and do not take drugs on time and in the right amount, delaying treatment; therefore, monitoring the concentrations of aripiprazole and dehydroaripiprazole plays an important role in judging the efficacy of aripiprazole, evaluating the therapeutic effect, avoiding side effects, and adjusting individualized medication regimens.

[0007] At present, the concentration of aripiprazole and dehydro-aripiprazole is mostly determined by liquid phase secondary mass spectrometry (LC-MS-MS), but the LC-MS-MS method requires repeated extractions, and has the disadvantages of cumbersome operation, complexity, time-consuming and labor-intensive, and low throughput, which cannot meet the clinical high-throughput, rapid and accurate detection needs. For example, the patent document with application number CN201910630169.8 discloses a method for detecting aripiprazole and dehydro-aripiprazole in blood, which is based on the determination of aripiprazole and dehydro-aripiprazole in blood by liquid chromatography-mass spectrometry, and requires pretreatment of blood samples. For example, the patent document with application number CN201910671626.8 discloses a blood aripiprazole drug concentration monitoring kit and its detection method, which is developed based on multi-dimensional online solid phase extraction liquid chromatography analysis technology.

[0008] For example, the patent document with application number CN201380054988.3 discloses an antibody to aripiprazole hapten and its use. The antibody prepared can simultaneously recognize aripiprazole and dehydrogenated aripiprazole; however, the patent document does not disclose the specificity of the antibody in testing clinical samples, and the cross-reactivity to aripiprazole inactive metabolites (hydroxylation and N-dealkylation aripiprazole). At the same time, the patent document with application number CN201911043578.4 discloses an artificial aripiprazole antigen and its preparation method. The antibody prepared using the artificial antigen can be used to detect the content of aripiprazole, but the patent document does not disclose the specificity of the antibody to aripiprazole metabolites.

[0009] Therefore, obtaining high-quality antibodies against aripiprazole and dehydroaripiprazole in order to establish immunological detection methods for aripiprazole and dehydroaripiprazole with strong specificity and sensitivity is of great significance for formulating individualized dosage regimens for aripiprazole and evaluating its clinical efficacy and safety. Summary of the invention

[0010] In order to improve the specificity and sensitivity of simultaneous detection of aripiprazole and dehydro-aripiprazole, the present application provides a reagent, a kit and a method for detecting the concentrations of aripiprazole and dehydro-aripiprazole based on a homogeneous enzymatic method.

[0011] In the first aspect, the present application provides a reagent for detecting the concentration of aripiprazole and dehydro-aripiprazole based on a homogeneous enzymatic method, which adopts the following technical scheme:

[0012] A reagent for detecting the concentration of aripiprazole and dehydro-aripiprazole based on a homogeneous enzymatic method, the reagent comprising

[0013] Reagent R1: contains antibodies and homogeneous enzyme substrate;

[0014] Reagent R2: comprising an enzyme-labeled conjugate formed by coupling a hapten and an enzyme marker through a coupling method;

[0015] The structural formula of the hapten is shown in formula (I):

[0016]

[0017] The hapten and the carrier protein are coupled by a coupling method to form an antigen;

[0018] The antibodies are produced in response to the antigen.

[0019] In some embodiments, in the antigen, the molar ratio of the hapten to the carrier protein is 1:(0.010-0.020), for example 1:0.014.

[0020] In some embodiments, in the antigen, the hapten and the carrier protein are coupled by a coupling agent to obtain the antigen. The molar ratio of the hapten to the coupling agent is 1:(2-3), for example, 1:2.43. Wherein, the coupling agent is a carbodiimide coupling agent. The carbodiimide coupling agent includes but is not limited to N,N'-dicyclohexylcarbodiimide (abbreviated as DCC, CAS No. 538-75-0), N,N'-diisopropylcarbodiimide (abbreviated as DIC, CAS No. 693-13-0), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (abbreviated as EDC, CAS No. 25952-53-8) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (abbreviated as EDCI, CAS No. 7084-11-9).

[0021] In some embodiments, the method for preparing the antigen comprises the following steps:

[0022] S2-1, dissolving the hapten in dimethyl sulfoxide to obtain a dimethyl sulfoxide solution of the hapten;

[0023] S2-2, dissolving the coupling agent in water to obtain an aqueous solution of the coupling agent;

[0024] S2-3, mixing the dimethyl sulfoxide solution of the hapten obtained in step S2-1 and the aqueous solution of the coupling agent obtained in step S2-2, and reacting them at room temperature for 0.5 to 2 hours to obtain a semi-coupling reaction solution;

[0025] S2-4, dissolving the carrier protein in a buffer to obtain a carrier protein solution;

[0026] S2-5, mixing the carrier protein solution obtained in step S2-4 and the semi-coupling reaction solution obtained in step S2-3, and stirring at room temperature for 1 to 3 hours to obtain a coupling reaction solution;

[0027] S2-6. Dialyze the coupling reaction solution obtained in step S2-5 into a buffer solution to obtain the antigen.

[0028] In some embodiments, in step S2-1, the concentration of the hapten in the dimethyl sulfoxide solution of the hapten can be (15-25) mmol / L, for example, 21.441 mmol / L.

[0029] In some embodiments, in step S2-2, the concentration of the coupling agent in the aqueous solution of the coupling agent may be (500-600) mmol / L, for example, 521.644 mmol / L.

[0030] In some embodiments, in step S2-4, the concentration of the carrier protein in the carrier protein solution may be (0.050-0.100) mmol / L, for example 0.060 mmol / L. The carrier protein may be selected from bovine serum albumin, bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, human serum albumin, and rabbit serum albumin. The buffer may be PBS buffer.

[0031] In some embodiments, in step S2-6, the buffer may be PBS buffer. The dialysis is performed multiple times, such as 2 times, 3 times, 4 times, 5 times, 6 times, etc. The volume of the buffer used in each dialysis is (400-600) times, such as 500 times, of the coupling reaction solution.

[0032] In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody, wherein the preparation of the monoclonal antibody and the polyclonal antibody can adopt the technology well known in the art, that is, the classical hybridoma cell fusion technology.

[0033] In some embodiments, the mass percentage concentration of the antibody in the reagent R1 is (0.01-0.1)%, for example, 0.05%.

[0034] In some embodiments, the method for preparing the monoclonal antibody specifically comprises the following steps:

[0035] S3-1. Inoculation of antigens into hosts (e.g., mice, rabbits, goats, sheep, etc.): dilute the antigens of aripiprazole and dehydro-aripiprazole to 1 mg / mL using PBS buffer, add an equal volume of Freund's complete adjuvant, emulsify completely, and perform the first immunization of the host at a dose of (0.01-0.2) mg / host; after an interval of four weeks, weigh 1 mg of the antigens of aripiprazole and dehydro-aripiprazole and 1 mg of Freund's incomplete adjuvant, mix them, and stir at a stirring speed of 2000 rpm / min for 2 hours to complete the emulsification, and perform booster immunization of the host after the first immunization at a dose of (0.01-0.2) mg / host;

[0036] S3-2. The spleen cell line from the inoculated host was fused with Sp2 / 0 cells, and the antigens of aripiprazole and dehydro-aripiprazole were used to coat the ELISA 96-well plate. The fused cells were subjected to titer and competition assays using indirect ELISA and indirect competitive ELISA, respectively. Antibodies that could specifically recognize aripiprazole and dehydro-aripiprazole and had no cross-reaction with hydroxylation-aripiprazole and N-dealkylation-aripiprazole were produced.

[0037] In some embodiments, the method for preparing the polyclonal antibody specifically comprises the following steps:

[0038] First immunization: antigen inoculation into the host (such as mice, rabbits, goats, sheep, etc.): dilute the antigen of aripiprazole and dehydro-aripiprazole to 1 mg / mL using PBS buffer, add an equal volume of Freund's complete adjuvant, emulsify completely, and perform the first immunization of the host at a dose of (0.01-0.2) mg / head;

[0039] Enhanced immunization: After an interval of (2 to 6) weeks, weigh 1 mg of aripiprazole and dehydro-aripiprazole antigens and mix with 1 mg of Freund's incomplete adjuvant and stir at a stirring speed of 2000 rpm / min for 2 hours to complete emulsification. The hosts that have undergone the initial immunization are given a booster immunization at a dose of (0.01 to 0.2) mg / host, and blood is collected to determine the serum titer and specificity.

[0040] The booster immunization is repeated multiple times, for example, 4 times or 5 times. The time interval between two adjacent booster immunizations is (2 to 6) weeks, for example, 4 weeks.

[0041] In some embodiments, the homogeneous enzyme substrate is prepared by dissolving β-nicotinamide adenine dinucleotide and glucose-6-phosphate in a buffer; wherein the concentration of β-nicotinamide adenine dinucleotide in the homogeneous enzyme substrate is (10-30) mmol / L, and the concentration of glucose-6-phosphate in the homogeneous enzyme substrate is (20-40) mmol / L.

[0042] In some embodiments, the concentration of the enzyme-labeled conjugate in the reagent R2 is (4-6) U / mL, for example, 5 U / mL.

[0043] In some embodiments, the enzyme marker is glucose-6-phosphate dehydrogenase.

[0044] In some embodiments, in the enzyme-labeled conjugate, the amount of the enzyme marker is (500-700) U / 0.5 mg hapten, such as 600 U / 0.5 mg hapten.

[0045] In some embodiments, in the enzyme-labeled conjugate, the hapten and the enzyme marker are coupled by a coupling agent to obtain the enzyme-labeled conjugate. In the enzyme-labeled conjugate, the weight ratio of the hapten and the coupling agent is 1: (1.5-2.5), for example, 1: 2. Wherein, the coupling agent is a carbodiimide coupling agent. The carbodiimide coupling agent includes but is not limited to N, N'-dicyclohexylcarbodiimide (abbreviated as DCC, CAS No. 538-75-0), N, N'-diisopropylcarbodiimide (abbreviated as DIC, CAS No. 693-13-0), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (abbreviated as EDC, CAS No. 25952-53-8) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (abbreviated as EDCI, CAS No. 7084-11-9).

[0046] In some embodiments, the method for preparing the enzyme-labeled conjugate comprises the following steps:

[0047] S4-1, dissolving glucose-6-phosphate dehydrogenase (abbreviated as G6PDH, CAS No. 56-73-5, 600 U) in PBS buffer (1 mL) to obtain a glucose-6-phosphate dehydrogenase solution;

[0048] S4-2, dissolving the antigen (0.5 mg) in dimethyl sulfoxide (DMSO, 100 uL) to obtain an antigen solution;

[0049] S4-3, after the glucose-6-phosphate dehydrogenase solution and the antigen solution are evenly mixed, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (abbreviated as EDCI, 1 mg) is added and mixed at room temperature for 2 h;

[0050] S4-4. Dialyze into PBS buffer to obtain enzyme-labeled conjugate.

[0051] In some embodiments, in step S4-1, the concentration of the glucose-6-phosphate dehydrogenase in the glucose-6-phosphate dehydrogenase solution is (500-700) U / mL.

[0052] In some embodiments, in step S4-2, the concentration of the antigen in the antigen solution is 3-7 mg / mL, for example, 5 mg / mL.

[0053] In some embodiments, the homogeneous enzyme substrate is prepared by dissolving β-nicotinamide adenine dinucleotide and glucose-6-phosphate in a buffer. The concentration of β-nicotinamide adenine dinucleotide in the homogeneous enzyme substrate is (10-30) mmol / L, for example, 20 mmol / L. The concentration of glucose-6-phosphate in the homogeneous enzyme substrate is (20-40) mmol / L, for example, 30 mmol / L. The buffer may be a Tris buffer, for example, a 55 mmol / L, pH=8.0 Tris buffer.

[0054] In a second aspect, the present application provides a kit for detecting the concentration of aripiprazole and dehydro-aripiprazole based on a homogeneous enzymatic method, which adopts the following technical scheme:

[0055] A kit for detecting the concentration of aripiprazole and dehydro-aripiprazole based on a homogeneous enzyme method, the kit comprising the reagent.

[0056] In some embodiments, the kit can be used for the detection of urine samples or blood samples.

[0057] In a third aspect, the present application provides a method for detecting the concentration of aripiprazole and dehydro-aripiprazole based on a homogeneous enzymatic method, which adopts the following technical scheme:

[0058] A method for detecting the concentration of aripiprazole and dehydro-aripiprazole based on a homogeneous enzymatic method, the method comprising the following steps:

[0059] First, the reagent R1 is added to the sample to be tested and reacted for (3-8) min (e.g., 5 min), and then the reagent R2 is added and reacted for (3-8) min (e.g., 5 min) to obtain a detection solution;

[0060] The absorbance change rate of the detection solution was measured at a working wavelength of 340 nm.

[0061] In some embodiments, the volume ratio of the sample to be tested, the reagent R1 and the reagent R2 is 1:(2-3):(2-3), for example, 1:2.5:2.5.

[0062] In some embodiments, the device used in the method is a biochemical analyzer.

[0063] In some embodiments, the sample to be tested may be urine or serum.

[0064] In summary, this application has the following beneficial effects:

[0065] Since the antigens and antibodies used in the present application have the ability to simultaneously identify aripiprazole and its active metabolite dehydro-aripiprazole, and have antibody specificity superior to that of related technologies, i.e., they have no cross-reaction with the inactive metabolites of aripiprazole, Hydroxylation-aripiprazole and N-Dealkylation-aripiprazole. Therefore, the reagents, kits and methods for the homogeneous enzymatic detection of aripiprazole and dehydro-aripiprazole concentrations of the present application improve the specificity and sensitivity of the simultaneous detection of aripiprazole and dehydro-aripiprazole. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 is the H NMR spectrum of the hapten in the present application;

[0067] Figure 2 It is a Native-PAGE electrophoresis identification diagram of bovine serum albumin and antigen in this application;

[0068] Figure 3 It is a comparison of the detection results of the method constructed based on the homogeneous enzyme method of the present application with the HPLC-MS method. DETAILED DESCRIPTION

[0069] The present application is further described in detail below with reference to the accompanying drawings and embodiments.

[0070] Preparation of haptens for detecting aripiprazole and dehydroaripiprazole concentrations

[0071] The structural formula of the hapten is:

[0072] The synthetic route of hapten is:

[0073]

[0074] The preparation method of the hapten is as follows: the aripiprazole represented by formula (i) and the alkyl alcohol represented by formula (ii) undergo amide alcoholysis reaction to generate the compound represented by formula (iii); the compound represented by formula (iii) is then subjected to hydrolysis reaction and neutralization reaction in sequence to obtain the hapten represented by formula (I).

[0075] When the alkyl alcohol represented by formula (ii) is methanol, the method for preparing the hapten specifically comprises the following steps:

[0076] S1-1. Synthesis of the compound represented by formula (iii):

[0077]

[0078] Aripiprazole (4.5 g, 10 mmol) and methanol (20 mL, 15.836 g, 494 mmol) were weighed and stirred to dissolve, and then added to a 50 mL three-necked flask. Concentrated sulfuric acid (1.5 g) was slowly added dropwise at a temperature of 5 to 15 ° C., and then the temperature was raised to 40 to 45 ° C. and reacted at 40 to 45 ° C. for 2 hours. After the reaction was completed, 60 mL of water was added, the temperature was lowered to 5 to 10 ° C., and the pH was adjusted to 8.5 with liquid alkali, and 40 mL of ethyl acetate was added, stirred for 20 minutes, and the mixture was allowed to stand for stratification to obtain an organic layer (i.e., an ethyl acetate solution of the compound represented by formula (iii)).

[0079] S1-2. Synthesis of hapten represented by formula (I):

[0080]

[0081] The organic layer obtained in step S1-1 was concentrated under reduced pressure to remove ethyl acetate, and 50 mL of water was added after evaporating the ethyl acetate; then, the pH was adjusted to 12 with liquid alkali, and the hydrolysis reaction was carried out at room temperature for 30 minutes; 30 mL of ethyl acetate was added, and the pH was adjusted to 4.5 with dilute sulfuric acid, and the mixture was stirred for 20 minutes and allowed to stand for stratification to obtain an organic layer. 100 mL of dichloromethane was added to the organic layer, and the temperature was lowered to 0-5°C, stirred for crystallization for 2 hours, filtered, and the filter cake was dried to obtain the hapten represented by formula (I) (3.5 g, yield 75.2%).

[0082] Characterization of haptens: The H NMR spectrum of haptens is as follows Figure 1 As shown. Figure 1 It can be seen that the hapten of aripiprazole represented by formula (I) was successfully prepared.

[0083] Preparation of antigens for detecting aripiprazole and dehydroaripiprazole concentrations

[0084] The method for preparing the antigen specifically comprises the following steps:

[0085] S2-1. Weigh the above hapten (molecular weight 466.4007, 10 mg) and dissolve it in dimethyl sulfoxide (1 mL) to obtain a dimethyl sulfoxide solution of the hapten (concentration 21.441 mmol / L);

[0086] S2-2, weigh 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (abbreviated as EDCI, CAS No. 7084-11-9, molecular weight 191.7016, 10 mg) and dissolve it in water (100 uL) to obtain an aqueous solution of the coupling agent (concentration 521.644 mmol / L);

[0087] S2-3, mixing the dimethyl sulfoxide solution of the hapten obtained in step S2-1 and the aqueous solution of the coupling agent obtained in step S2-2, and reacting them at room temperature for 1 hour to obtain a semi-coupling reaction solution;

[0088] S2-4, dissolving bovine serum albumin (BSA, CAS No. 9048-46-8, molecular weight 66.446 KDa, 20 mg) in 5 mL of PBS buffer to obtain a carrier protein solution (concentration 0.060 mmol / L);

[0089] S2-5, mixing the carrier protein solution obtained in step S2-4 and the reaction solution obtained in step S2-3, and stirring at room temperature for 2 hours to obtain a coupling reaction solution;

[0090] S2-6. The coupling reaction solution obtained in step S2-5 was dialyzed four times with PBS buffer, and the volume ratio of the coupling reaction solution to PBS buffer during each dialysis was 1:500, to obtain the antigen.

[0091] Characterization of antigen: Bovine serum albumin (BSA) and the antigen (BSA-aripiprazole derivative) coupled with hapten and bovine serum albumin were identified by Native-PAGE electrophoresis. Figure 2 As shown. Figure 2 It can be seen that the coupled antigen exhibits a faster electrophoretic speed than the bovine serum albumin alone, indicating that the antigen preparation is successful.

[0092] Preparation of antibodies for detecting aripiprazole and dehydroaripiprazole concentrations

[0093] The antibody may be a monoclonal antibody or a polyclonal antibody, wherein the preparation of the monoclonal antibody or the polyclonal antibody may adopt the technology known in the art, i.e., the classical hybridoma cell fusion technology.

[0094] The method for preparing a monoclonal antibody specifically comprises the following steps:

[0095] S3-1. Inoculation of antigens into the host: dilute the antigens of aripiprazole and dehydro-aripiprazole to 1 mg / mL using PBS buffer, add an equal volume of Freund's complete adjuvant, emulsify completely, and perform the first immunization of mice at a dose of 0.1 mg / mouse; after an interval of four weeks, weigh 1 mg of the antigens of aripiprazole and dehydro-aripiprazole and 1 mg of Freund's incomplete adjuvant, mix them, and stir at a stirring speed of 2000 rpm / min for 2 hours to complete emulsification, and perform booster immunization of mice that have undergone the first immunization at a dose of 0.1 mg / mouse;

[0096] S3-2. The spleen cell line from the inoculated host was fused with Sp2 / 0 cells, and the antigens of aripiprazole and dehydro-aripiprazole were used to coat the ELISA 96-well plate. The fused cells were subjected to titer and competition assays using indirect ELISA and indirect competitive ELISA, respectively. The three cell lines with the best competition for aripiprazole were screened and named 45F2, 1B2, and 21G7, respectively.

[0097] Among them, the 45F2 cell line was deposited in the General Microbiology Center of China Microorganism Culture Collection (abbreviated as: CGMCC; address: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Postal Code 100101) on May 19, 2022, and the deposit number is CGMCC NO.45162.

[0098] Table 1: Indirect competitive ELISA results of cell supernatants of 45F2 cell line

[0099]

[0100] Table 2: Indirect competitive ELISA results of cell supernatants of 1B2 cell line

[0101]

[0102] Table 3: Indirect competitive ELISA results of cell supernatants of 21G7 cell line

[0103]

[0104] The calculation formula for the cross-reaction rate is:

[0105] From Tables 1 to 3, it can be seen that the antibodies produced by the 45F2 cell line showed almost the same recognition efficiency for aripiprazole and its active metabolite dehydro-aripiprazole, and had very low cross-reactivity to the inactive metabolites of aripiprazole, Hydroxylation-aripiprazole and N-Dealkylation-aripiprazole. Therefore, the antibodies produced by the 45F2 cell line are suitable for simultaneously detecting the concentrations of aripiprazole and dehydro-aripiprazole in the sample to be tested.

[0106] This may be because the derivatization site of the hapten of the present application is far away from the common characteristic structure of aripiprazole and dehydro-aripiprazole, so the antibody of the present application can equivalently recognize aripiprazole and its active metabolite dehydro-aripiprazole. In addition, the antibody of the present application has no cross-reaction to the inactive metabolites Hydroxylation-aripiprazole and N-Dealkylation-aripiprazole. Moreover, the characteristic structure of dehydro-aripiprazole is innovatively removed from the hapten of the present application, and an extremely short linker is used to couple with the carrier protein, so aripiprazole and dehydro-aripiprazole can be recognized at the same time, which is very beneficial for the determination of aripiprazole and its active metabolite dehydro-aripiprazole.

[0107] In summary, the antigen and antibody of aripiprazole provided in the present application have the ability to simultaneously recognize aripiprazole and dehydro-aripiprazole, and have antibody specificity superior to that of related technologies, i.e., no cross-reaction with hydroxylation-aripiprazole and N-dealkylation-aripiprazole. Therefore, the antigen and antibody provided in the present application can construct a method for accurately monitoring the concentration of aripiprazole and dehydro-aripiprazole in blood and urine.

[0108] A method for detecting aripiprazole and dehydroaripiprazole based on homogeneous enzyme immunoassay

[0109] Reagents for the determination of aripiprazole and dehydroaripiprazole concentrations based on homogeneous enzymatic assay

[0110] The reagents for detecting the concentration of aripiprazole and dehydro-aripiprazole based on the homogeneous enzyme method include: 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.

[0111] Wherein, the preparation of reagent R1:

[0112] The preparation method of reagent R1 comprises the following steps:

[0113] β-Nicotinamide adenine dinucleotide (NAD, CAS No. 53-84-9, molecular weight 663.425, 13.2685 g, 20 mmol) and glucose-6-phosphate (G6P, CAS No. 112898-35-8, molecular weight 260.136, 7.8041 g, 30 mmol) were dissolved in Tris buffer (55 mmol / L, pH=8.0, 1 L) to prepare homogeneous enzyme substrates;

[0114] Add antibody 45F2 to the homogeneous enzyme substrate to obtain reagent R1. The mass percentage concentration of antibody 45F2 in reagent R1 can be (0.01-0.1)%; in this specific embodiment, the mass percentage concentration of antibody 45F2 in reagent R1 is 0.05%.

[0115] Preparation of reagent R1:

[0116] The preparation method of the enzyme-labeled conjugate comprises the following steps:

[0117] S4-1, dissolving glucose-6-phosphate dehydrogenase (abbreviated as G6PDH, CAS No. 56-73-5, 600 U) in PBS buffer (1 mL) to obtain a glucose-6-phosphate dehydrogenase solution (concentration 600 U / mL);

[0118] S4-2, dissolving the antigen (0.5 mg) in dimethyl sulfoxide (DMSO, 100 uL) to obtain an antigen solution;

[0119] S4-3, after the glucose-6-phosphate dehydrogenase solution and the antigen solution are evenly mixed, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (abbreviated as EDCI, CAS No. 7084-11-9, molecular weight 191.7016, 1 mg) is added and mixed at room temperature for 2 h;

[0120] S4-4. Dialyze into PBS buffer to obtain enzyme-labeled conjugate.

[0121] The preparation method of reagent R1 comprises the following steps:

[0122] The enzyme-labeled conjugate is added to a Tris buffer (0.1 mol / L, pH=8.5) to obtain reagent R2. The concentration of the enzyme-labeled conjugate in reagent R2 may be (4-6) U / mL; in this specific embodiment, the concentration of the enzyme-labeled conjugate in reagent R2 is 5 U / mL.

[0123] Method for detecting the concentration of aripiprazole and dehydroaripiprazole based on homogeneous enzymatic method

[0124] The method for detecting the concentration of aripiprazole and dehydro-aripiprazole based on homogeneous enzymatic method comprises the following steps:

[0125] First, add reagent R1 (50uL) to the sample to be tested (20uL) and react for 5min, then add reagent R2 (50uL) and react for 5min to obtain a detection solution;

[0126] The absorbance change rate of the detection solution was measured at a working wavelength of 340 nm.

[0127] In this specific embodiment, the method for detecting the concentration of aripiprazole and dehydro-aripiprazole based on the homogeneous enzymatic method uses a Hitachi 7170 biochemical analyzer.

[0128] 40 clinical urine samples were selected for testing, and the concentrations of aripiprazole and dehydroaripiprazole in the 40 urine samples were determined by the method constructed in the present application and the HPLC-MS method, respectively.

[0129] Comparing the detection results of the method constructed based on the homogeneous enzyme method of the present application with those of the HPLC-MS method, the results are as follows Figure 3 As shown. Figure 3 It can be seen that the method constructed based on the homogeneous enzymatic method in the present application for detecting the concentrations of aripiprazole and dehydro-aripiprazole has a good correlation with the HPLC-MS method and meets clinical needs.

[0130] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A reagent for detecting the concentration of aripiprazole and dehydro-aripiprazole based on a homogeneous enzymatic method, characterized in that: The reagents include Reagent R1: contains antibodies and homogeneous enzyme substrate; Reagent R2: comprising an enzyme-labeled conjugate formed by coupling a hapten and an enzyme marker through a coupling method; The structural formula of the hapten is shown in formula (I): The hapten and the carrier protein are coupled by a coupling method to form an antigen; The antibodies are produced in response to the antigen.

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

3. The reagent according to claim 1, characterized in that The antibody is a monoclonal antibody or a polyclonal antibody.

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

5. The reagent according to claim 1, characterized in that The homogeneous enzyme substrate is prepared by dissolving β-nicotinamide adenine dinucleotide and glucose-6-phosphate in a buffer solution; wherein the concentration of the β-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 according to claim 1, characterized in that In the enzyme-labeled conjugate, the dosage of the enzyme marker is (500-700) U / 0.5 mg hapten.

7. The reagent according to claim 1, characterized in that The enzyme marker is glucose-6-phosphate dehydrogenase.

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

9. A kit for detecting the concentration of aripiprazole and dehydro-aripiprazole based on a homogeneous enzymatic method, characterized in that: The kit comprises the reagent according to any one of claims 1 to 8.

10. A method for detecting the concentration of aripiprazole and dehydro-aripiprazole based on a homogeneous enzymatic method, characterized in that: The method is based on the reagent according to any one of claims 1 to 8; the method comprises the following steps: First, add the reagent R1 to the sample to be tested and react for (3-8) min, then add the reagent R2 and react for (3-8) min, obtaining a detection solution; The absorbance change rate of the detection solution is measured at a working wavelength of 340 nm; The volume ratio of the sample to be tested, the reagent R1 and the reagent R2 is 1:(2-3):(2-3); The sample to be tested is derived from urine or serum.

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