A reagent, a kit and a method for detecting the concentration of aripiprazole and dehydro-aripiprazole based on magnetic particle chemiluminescence

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 dehydro-aripiprazole based on a magnetic particle chemiluminescence method. The reagent includes: an enzyme label working solution: comprising a hapten-enzyme marker conjugate obtained by coupling a hapten and an enzyme marker through a coupling method; a magnetic label working solution: comprising an antibody-magnetic marker conjugate obtained by coupling an antibody and a magnetic marker through a coupling method; an indicator solution: comprising an indicator for quantitatively detecting the concentration of an enzyme marker; wherein the structural formula of the hapten is as shown in formula (I), the hapten and the carrier protein are coupled to obtain an antigen through a coupling method; and the antibody is generated in response to the antigen. The kit includes the reagent. The method includes: S10, establishment of a standard curve; S20, detection of the concentration of aripiprazole and dehydro-aripiprazole. The present application improves the specificity and sensitivity of simultaneous detection of aripiprazole and dehydro-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 magnetic particle chemiluminescence 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 magnetic particle chemiluminescence method.

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

[0012] A reagent for detecting the concentration of aripiprazole and dehydro-aripiprazole based on magnetic particle chemiluminescence method, the reagent comprising:

[0013] Enzyme label working solution: comprising a hapten-enzyme marker conjugate obtained by coupling a hapten and an enzyme marker through a coupling method;

[0014] Magnetic label working solution: comprising an antibody-magnetic marker conjugate obtained by coupling an antibody and a magnetic marker through a coupling method;

[0015] Indicator solution: containing an indicator for quantitatively detecting the concentration of the enzyme marker;

[0016] Wherein, the structural formula of the hapten is shown in formula (I):

[0017]

[0018] The hapten and carrier protein are coupled to obtain an antigen;

[0019] The antibodies are produced in response to the antigen.

[0020] In some embodiments, in the hapten-enzyme marker conjugate, the enzyme marker is alkaline phosphatase, and the indicator is a luminescent substrate for quantitatively detecting the content of alkaline phosphatase.

[0021] In some embodiments, in the hapten-enzyme marker conjugate, the weight ratio of the hapten to the enzyme marker is 1:(1.5-2.5), for example, 1:2.

[0022] In some embodiments, in the hapten-enzyme marker conjugate, the hapten and the enzyme marker are coupled by a coupling agent to obtain the hapten-enzyme marker conjugate. Wherein, the weight ratio of the hapten to the coupling agent is 1: (1.5 to 2.5), for example, 1: 2. 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).

[0023] In some embodiments, the concentration of the hapten-enzyme marker conjugate in the enzyme label working solution is (0.5-5) ug / mL, such as 1 ug / mL, 2 ug / mL, 3 ug / mL, 4 ug / mL.

[0024] In some embodiments, the method for preparing the hapten-enzyme marker comprises the following steps:

[0025] S4-1, dissolving the enzyme marker in PBS buffer to obtain an enzyme marker solution;

[0026] S4-2, dissolving the above hapten in dimethyl sulfoxide to obtain a hapten solution;

[0027] S4-3, the alkaline phosphatase solution obtained in step S1-1 and the hapten solution obtained in step S1-2 are mixed evenly, a coupling agent is added, and mixed evenly at room temperature for (1 to 3) hours; thereafter, dialyzed into PBS buffer to obtain the hapten-enzyme marker conjugate.

[0028] In some embodiments, the method for preparing the enzyme labeling working solution comprises the following steps:

[0029] The hapten-enzyme marker conjugate was diluted with 50mmol / L MES 0.9% Nacl 5mg / mL BSA 1mM Mgcl2 pH=6.7 to obtain an enzyme label working solution.

[0030] In some embodiments, in step S4-1, the concentration of the enzyme marker in the enzyme marker solution is (0.1-2) mg / mL, for example, 1 mg / mL. The buffer may be a PBS buffer.

[0031] In some embodiments, in step S4-2, the concentration of the hapten in the hapten solution is (4-6) mg / mL.

[0032] 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.

[0033] 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).

[0034] In some embodiments, the method for preparing the antigen comprises the following steps:

[0035] S2-1, dissolving the hapten in dimethyl sulfoxide to obtain a dimethyl sulfoxide solution of the hapten;

[0036] S2-2, dissolving the coupling agent in water to obtain an aqueous solution of the coupling agent;

[0037] 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;

[0038] S2-4, dissolving the carrier protein in a buffer to obtain a carrier protein solution;

[0039] 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;

[0040] S2-6. Dialyze the coupling reaction solution obtained in step S2-5 into a buffer solution to obtain the antigen.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] In some embodiments, the mass percentage concentration of the antibody in the reagent R1 is (0.01-0.1)%, for example, 0.05%.

[0047] In some embodiments, the method for preparing the monoclonal antibody specifically comprises the following steps:

[0048] 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 emulsification, and perform booster immunization of the host after the first immunization at a dose of (0.01-0.2) mg / host;

[0049] 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.

[0050] In some embodiments, the method for preparing the polyclonal antibody specifically comprises the following steps:

[0051] 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;

[0052] 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.

[0053] 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.

[0054] In some embodiments, the magnetic marker is magnetic microparticles, such as Dynal beads M280 Tosyl magnetic beads.

[0055] In some embodiments, in the magnetic label working solution, the weight ratio of the antibody to the magnetic particles is 1:(40-60), for example, 1:50.

[0056] In some embodiments, the concentration of the antibody-magnetic marker conjugate in the magnetic label working solution is 0.1 mg / mL to 1 mg / mL, for example, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL.

[0057] In some embodiments, the method for preparing the magnetic scale working solution comprises the following steps:

[0058] S5-1, dilute 50 mg of Dynal beads M280 Tosyl magnetic particles as a magnetic marker in 2 mL of BB buffer (50 mmol / L, pH=8.0) to obtain a magnetic marker solution;

[0059] S5-2, adding the antibody to the magnetic marker solution and mixing, and then shaking at (30-40)° C. (e.g., 37° C.) for (5-10) h (e.g., 8 h) to obtain an antibody-magnetic marker conjugate solution;

[0060] S5-3, remove the supernatant by magnetic suction, add TBST buffer (50mmol / L Tris 0.9%Nacl 0.1%TW20, pH=7.4) and adjust the concentration of magnetic particles to 0.5mg / mL, and react at 37°C for 2h;

[0061] S5-4. Remove the supernatant by magnetic suction, add TBST buffer (50 mM Tris 0.9% Nacl 0.1% TW20, pH=7.4) and adjust the concentration of magnetic particles to 0.4 mg / mL to obtain a magnetic label working solution.

[0062] In some embodiments, in step S5-1, the concentration of the magnetic marker in the magnetic marker solution is (20-30) mg / mL.

[0063] In a second aspect, the present application provides a kit for detecting the concentration of aripiprazole and dehydro-aripiprazole based on a magnetic particle chemiluminescence method, which adopts the following technical scheme:

[0064] A kit for detecting aripiprazole and dehydro-aripiprazole based on magnetic particle chemiluminescence method, the kit comprising the reagent.

[0065] In some embodiments, the kit can be used for the detection of urine samples or blood samples.

[0066] In a third aspect, the present application provides a method for detecting aripiprazole and dehydro-aripiprazole based on magnetic particle chemiluminescence, which adopts the following technical solution:

[0067] A method for detecting the concentration of aripiprazole and dehydro-aripiprazole based on magnetic particle chemiluminescence, the method is based on the agent; the method comprises the following steps:

[0068] S10. Establishment of standard curve

[0069] Using aripiprazole and / or dehydroaripiprazole as a standard, a standard solution is prepared;

[0070] The standard solution, magnetic microparticle liquid working solution and enzyme label working solution are mixed evenly, and after incubation reaction and washing, the indicator solution is added to develop color and collect the relative light unit of the 400-550nm light signal;

[0071] The concentration of the standard solution is used as the abscissa, and the relative light unit of the standard solution is used as the ordinate to draw a standard curve, and the linear regression equation is calculated;

[0072] S20, detection of aripiprazole and dehydroaripiprazole concentrations

[0073] The sample to be tested, magnetic microparticle liquid working solution and enzyme label working solution are mixed evenly, and after incubation reaction and washing, the indicator solution is added for color development and the relative light unit of the 400nm-550nm light signal is collected;

[0074] According to the linear regression equation, the concentration of the relative light unit of the sample to be tested corresponding to the horizontal coordinate on the standard curve is calculated.

[0075] In some embodiments, the concentration range of the standard solution is 0 ng / mL to 1200 ng / mL.

[0076] In some embodiments, the test sample is derived from blood or urine.

[0077] In summary, this application has the following beneficial effects:

[0078] Since the antigen and antibody 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., there is no cross-reaction with the inactive metabolites of aripiprazole, Hydroxylation-aripiprazole and N-Dealkylation-aripiprazole. Therefore, the reagent, kit and method for detecting the concentration of aripiprazole and dehydro-aripiprazole based on the magnetic particle chemiluminescence method of the present application improve the specificity of simultaneous detection of aripiprazole and dehydro-aripiprazole, and have the advantages of high sensitivity and good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 is the H NMR spectrum of the hapten in the present application;

[0080] Figure 2 It is the result of Native-PAGE electrophoresis identification of hapten, bovine serum albumin and antigen in this application;

[0081] Figure 3 is the standard curve of aripiprazole concentration in this application;

[0082] Figure 4 It is the standard curve of the standard solution of dehydro-Aripiprazole in this application. DETAILED DESCRIPTION

[0083] The present application is further described in detail below with reference to the accompanying drawings and embodiments.

[0084] Preparation of haptens for detecting aripiprazole and dehydroaripiprazole concentrations

[0085]

[0086] The synthetic route of hapten is:

[0087]

[0088] 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).

[0089] When the alkyl alcohol represented by formula (ii) is methanol, the method for preparing the hapten specifically comprises the following steps:

[0090] The method for preparing the hapten of aripiprazole specifically comprises the following steps:

[0091] S1-1. Synthesis of the compound represented by formula (iii):

[0092]

[0093] 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 the mixture was 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 allowed to stand for stratification to obtain an organic layer (i.e., an ethyl acetate solution of the compound represented by formula (iii)).

[0094] S1-2. Synthesis of hapten represented by formula (I):

[0095]

[0096] The organic layer was concentrated under reduced pressure to remove ethyl acetate, and 50 mL of water was added after evaporating the ethyl acetate to dryness; 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 mixture was cooled to 0-5°C and stirred for crystallization for 2 hours, and then filtered. After the filter cake was dried, the hapten represented by formula (I) (3.5 g, yield 75.2%) was obtained.

[0097] 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.

[0098] Preparation of antigens for detecting aripiprazole and dehydroaripiprazole concentrations

[0099] The method for preparing the antigen specifically comprises the following steps:

[0100] S2-1, weigh 10 mg of aripiprazole hapten and dissolve it in 1 mL of dimethyl sulfoxide to obtain a dimethyl sulfoxide solution of the hapten;

[0101] S2-2, weigh 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and dissolve it in 100 ul of water to obtain an aqueous solution of the coupling agent;

[0102] S2-3, mixing the dimethyl sulfoxide solution of the hapten in step (1) and the aqueous solution of the coupling agent in step (2), and reacting them at room temperature for 1 hour to obtain a semi-coupling reaction solution;

[0103] S2-4, weigh 20 mg of carrier protein and dissolve it in 5 mL of PBS buffer to obtain a carrier protein solution; in this specific embodiment, the carrier protein is bovine serum albumin;

[0104] S2-5, mixing the carrier protein solution of step (4) and the reaction solution of step (3), and stirring at room temperature for 2 hours to obtain a coupling reaction solution;

[0105] S2-6. The coupling reaction solution of step (5) was dialyzed four times with PBS buffer, and the volume ratio of the coupling reaction solution to PBS buffer was 1:500 during each dialysis, to obtain the antigen.

[0106] Characterization of antigen: Bovine serum albumin (BSA) and 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 was successfully prepared.

[0107] Preparation of antibodies for detecting aripiprazole and dehydroaripiprazole concentrations

[0108] 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.

[0109] The method for preparing a monoclonal antibody specifically comprises the following steps:

[0110] 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;

[0111] 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.

[0112] Among them, the 45F12 hybridoma 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.

[0113] Table 1: Indirect competitive ELISA results of cell supernatants of 45F2 cell line

[0114]

[0115] Table 2: Indirect competitive ELISA results of cell supernatants of 1B2 cell line

[0116]

[0117]

[0118] Table 3: Indirect competitive ELISA results of cell supernatants of 21G7 cell line

[0119]

[0120] The calculation formula for the cross-reaction rate is:

[0121] 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 the simultaneous detection of aripiprazole and dehydro-aripiprazole concentrations.

[0122] 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 equally recognize aripiprazole and the 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.

[0123] In summary, the antigens and antibodies provided in the present application have the ability to simultaneously recognize 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 antigens and antibodies of aripiprazole provided in the present application can establish an immunological detection method for accurately monitoring the concentrations of aripiprazole and dehydro-aripiprazole in blood and urine.

[0124] Construction of a method for detecting the concentration of aripiprazole and dehydroaripiprazole based on magnetic microparticle chemiluminescence

[0125] Reagent for detecting the concentration of aripiprazole and dehydroaripiprazole based on magnetic microparticle chemiluminescence

[0126] The reagents for detecting the concentration of aripiprazole and dehydroaripiprazole based on magnetic particle chemiluminescence assay include:

[0127] Enzyme label working solution: comprising a hapten-enzyme marker conjugate obtained by coupling a hapten and an enzyme marker through a coupling method;

[0128] Magnetic label working solution: comprising an antibody-magnetic marker conjugate obtained by coupling an antibody and a magnetic marker through a coupling method;

[0129] Indicator solution: Contains the indicator used to quantitatively detect the concentration of the enzyme marker.

[0130] The method for preparing the hapten-enzyme marker conjugate comprises the following steps:

[0131] S4-1, dissolving alkaline phosphatase (ALP, 1 mg) in PBS buffer (1 mL) to obtain an enzyme marker solution;

[0132] S4-2, dissolving the above hapten (0.5 mg) in dimethyl sulfoxide (DMSO, 100 uL) to obtain a hapten solution;

[0133] S4-3. Evenly mix the alkaline phosphatase solution obtained in step S1-1 and the hapten solution obtained in step S1-2, add 1 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, CAS No. 7084-11-9), and mix at room temperature for 2 h; then, dialyze into PBS buffer to obtain a hapten-enzyme marker conjugate.

[0134] The preparation method of the enzyme labeling working solution comprises the following steps:

[0135] Then, the hapten-enzyme marker conjugate was diluted to 1 ug / mL with 50 mmol / L MES 0.9% Nacl 5 mg / mL BSA 1 mM Mgcl2 pH=6.7 to obtain the enzyme label working solution.

[0136] The preparation method of the magnetic scale working solution comprises the following steps:

[0137] S5-1, dilute 50 mg of Dynal beads M280 Tosyl magnetic particles in 2 mL of BB buffer (50 mM, pH = 8.0) to obtain a magnetic marker solution;

[0138] S5-2, adding 1 mg of aripiprazole antibody 45F2 to the magnetic marker solution and mixing, and then shaking and reacting at 37° C. for 8 h to obtain a magnetic particle-antibody mixture;

[0139] S5-3, remove the supernatant by magnetic suction, add TBST buffer (50mmol / L Tris 0.9%Nacl 0.1%TW20, pH=7.4) and adjust the concentration of magnetic particles to 0.5mg / mL, and react at 37°C for 2h;

[0140] S5-4. Remove the supernatant by magnetic suction, add TBST buffer (50 mM Tris 0.9% Nacl 0.1% TW20, pH=7.4) and adjust the concentration of magnetic particles to 0.4 mg / mL to obtain a magnetic label working solution.

[0141] The indicator solution is: commercial AMPPD luminescent solution.

[0142] Method for detecting the concentration of aripiprazole and dehydroaripiprazole based on magnetic particle chemiluminescence The method for detecting aripiprazole and dehydro-aripiprazole based on magnetic particle chemiluminescence method comprises the following steps: S10, establishing a standard curve; S20, detecting the concentrations of aripiprazole and dehydro-aripiprazole.

[0143] S10. Establishment of standard curve

[0144] Aripiprazole and dehydroaripiprazole standards were used as standards, and calf serum matrix was used to prepare standard solutions. The concentrations of the standard solutions were 0 ng / mL, 50 ng / mL, 150 ng / mL, 300 ng / mL, 600 ng / mL, and 1200 ng / mL, respectively.

[0145] 20uL of standard solution, 40uL of magnetic microparticle liquid working solution and 50uL of enzyme label working solution were mixed evenly, and the reaction was incubated at 37°C for 5min, washed (specific steps included magnetically removing the supernatant, adding TBST buffer to resuspend, and magnetically removing the supernatant again), and the indicator solution was added for color development and the relative light unit RLU of the light signal of 400-550nm was collected by a photomultiplier tube. The collection results are shown in Table 4.

[0146] Table 4 Collection results of concentrated standard solution of aripiprazole and standard solution of dehydrogenated aripiprazole

[0147]

[0148] Draw a standard curve of the standard solution with the concentration of the standard solution as the horizontal axis and the relative light unit of the standard solution as the vertical axis.

[0149] The standard curve of aripiprazole concentration is as follows: Figure 3 As shown, the linear regression equation is calculated as follows: Y = (A - D) / [1 + (X / C) B ]+D, A =103108568.924163, B =1.07257075717918, C =13.1743174321519, D =128379.691512192, correlation coefficient R2 =0.999999906310124.

[0150] The standard curve of dehydroaripiprazole is as follows Figure 4 As shown, the linear regression equation is calculated as: Y = (A - D) / [1 + (X / C) B ]+D, A=102948270.120879, B=1.07080983164846, C=13.1814135327384, D=116511.776675287, correlation coefficient R2=0.9999.

[0151] The repeatability of low-concentration aripiprazole and high-concentration aripiprazole was tested respectively, and the results are shown in Table 5.

[0152] Table 5 Repeatability of low-concentration aripiprazole and high-concentration aripiprazole

[0153]

[0154] The detection limit LOB of aripiprazole was tested. The results are shown in Table 6.

[0155] Table 6 Detection limit LOB of aripiprazole

[0156]

[0157] As can be seen from Tables 5 and 6, the detection of aripiprazole and dehydro-aripiprazole concentrations using antibodies produced by the 45F2 cell line based on the magnetic particle luminescence method has the advantages of high sensitivity and good repeatability.

[0158] S20, detection of aripiprazole and dehydroaripiprazole concentrations

[0159] 20uL of the sample to be tested, 40uL of magnetic microparticle liquid working solution and 50uL of enzyme label working solution were mixed evenly, and after incubation and washing, the indicator solution was added for color development and the relative light units of the 400nm-550nm light signal were collected;

[0160] According to the linear regression equation, the concentration of the relative light unit of the sample to be tested corresponding to the horizontal coordinate on the standard curve is calculated.

[0161] 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 skilled 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 magnetic particle chemiluminescence method, characterized in that: The reagents include: Enzyme label working solution: comprising a hapten-enzyme marker conjugate obtained by coupling a hapten and an enzyme marker through a coupling method; Magnetic label working solution: comprising an antibody-magnetic marker conjugate obtained by coupling an antibody and a magnetic marker through a coupling method; Indicator solution: containing an indicator for quantitatively detecting the concentration of the enzyme marker; Wherein, the structural formula of the hapten is shown in formula (I): The hapten and carrier protein are coupled to obtain an antigen; The antibodies are produced in response to the antigen.

2. The reagent according to claim 1, characterized in that In the hapten-enzyme marker conjugate, the enzyme marker is alkaline phosphatase, and the indicator is a luminescent substrate for quantitatively detecting the content of alkaline phosphatase.

3. The reagent according to claim 1, characterized in that In the hapten-enzyme marker conjugate, the weight ratio of the hapten to the enzyme marker is 1:(1.5-2.5).

4. The reagent according to claim 1, characterized in that The concentration of the hapten-enzyme marker conjugate in the enzyme label working solution is (0.5-5) ug / mL.

5. The reagent according to claim 1, characterized in that In the antigen, the weight ratio of the hapten to the carrier protein is 1:(1-3).

6. The reagent according to claim 1, characterized in that The magnetic marker is magnetic particles.

7. The reagent according to claim 1, characterized in that In the magnetic label working solution, the weight ratio of the antibody to the magnetic particles is 1:(40-60).

8. The reagent according to claim 1, characterized in that The concentration of the antibody-magnetic marker conjugate in the magnetic marker working solution is (0.1-1) mg / mL.

9. A kit for detecting the concentration of aripiprazole and dehydro-aripiprazole based on magnetic particle chemiluminescence 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 magnetic particle chemiluminescence, 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: S10. Establishment of standard curve Using aripiprazole and / or dehydroaripiprazole as a standard, a standard solution is prepared; The standard solution, magnetic microparticle liquid working solution and enzyme label working solution are mixed evenly, and after incubation reaction and washing, the indicator solution is added to develop color and collect the relative light unit of the 400-550nm light signal; The concentration of the standard solution is used as the abscissa, and the relative light unit of the standard solution is used as the ordinate to draw a standard curve, and the linear regression equation is calculated; S20, detection of aripiprazole and dehydroaripiprazole concentrations The sample to be tested, magnetic microparticle liquid working solution and enzyme label working solution are mixed evenly, and after incubation reaction and washing, the indicator solution is added for color development and the relative light unit of the 400nm-550nm light signal is collected; According to the linear regression equation, the concentration of the relative light unit of the sample to be tested corresponding to the horizontal coordinate on the standard curve is calculated; The concentration range of the standard solution is (0-1200) ng / mL; The sample to be tested is derived from blood or urine.

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