Carbamazepine chemiluminescence immunoassay reagent and preparation and detection method thereof

Abstract

This invention discloses a carbamazepine chemiluminescent immunoassay reagent and its preparation and detection method. The advantages of this invention are: the chemiluminescent immunoassay reagent containing anti-carbamazepine specific antibodies can conveniently, rapidly, and accurately determine the carbamazepine content in a sample, and can simultaneously measure multiple samples on a fully automated chemiluminescent immunoassay analyzer, achieving high-throughput and rapid determination of carbamazepine. It offers high accuracy, strong specificity, and significantly improved precision and detection efficiency compared to previous methods. Furthermore, it achieves full automation of the detection process, requiring less skill from testing personnel, and is easy to implement and widely adopt.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a carbamazepine chemiluminescent immunoassay reagent and its preparation and detection method. Background Technology

[0002] Therapeutic Drug Monitoring (TDM) is a process guided by clinical pharmacology, pharmacokinetics, and clinical chemistry principles. It utilizes advanced drug analysis techniques to measure drug concentrations in blood or other body fluids, obtaining relevant pharmacokinetic parameters to guide safe and rational drug use in clinical practice. This helps clinicians develop individualized dosing regimens to avoid or reduce side effects, improve treatment efficacy, and increase cure rates. Carbamazepine (CBZ) is a common antiepileptic drug, primarily used clinically to treat glossopharyngeal neuralgia, epileptic seizures, and trigeminal neuralgia. While effective for epilepsy, carbamazepine has a narrow effective concentration range. The minimum effective concentration is close to the toxic concentration; below this concentration, it has no therapeutic effect, while exceeding it can cause varying degrees of adverse reactions, such as dizziness, drowsiness, vomiting, and skin allergies, as well as serious adverse reactions like heart failure, liver damage, leukopenia, ataxia, and increased seizure frequency. Furthermore, the bioavailability and pharmacokinetic properties of carbamazepine at therapeutic doses vary greatly among individuals, necessitating the integration of blood drug concentration monitoring results for rational drug use in clinical practice. Therefore, monitoring the blood concentration of carbamazepine in patients receiving the drug can ensure safe and rational clinical drug use and reduce the occurrence of side effects and adverse reactions.

[0003] Currently, the main methods for monitoring carbamazepine concentration include high-performance liquid chromatography (HPLC), radioimmunoassay (RIA), and fluorescence polarization assay. HPLC is time-consuming, and the sample pretreatment and operation processes are extremely complex, requiring a high level of technical expertise from the testing personnel. RIA poses a significant health hazard to operators due to its radioactive radiation, and is therefore rarely used internationally. Fluorescence polarization assay requires imported reagents that are extremely expensive, and also necessitates the purchase of costly analytical instruments.

[0004] This invention employs a competitive immunoassay principle, using magnetic microparticles as the solid phase of the immunoassay reaction. It utilizes chemiluminescence immunoassay in conjunction with a fully automated chemiluminescence immunoassay analyzer to determine the carbamazepine content in human samples. The sample, carbamazepine-alkaline phosphatase conjugate, and paramagnetic particles coated with anti-carbamazepine specific antibodies are added to the reaction vessel. Carbamazepine in the sample competes with the carbamazepine-alkaline phosphatase conjugate for a limited number of binding sites on the anti-carbamazepine specific antibody. After incubation at 37°C in the reaction vessel, carbamazepine and the carbamazepine-alkaline phosphatase conjugate bound to the specific antibody on the solid-phase magnetic beads are held in the magnetic field, while unbound carbamazepine and the carbamazepine-alkaline phosphatase conjugate are washed away. Then, substrate solution for the fully automated immunoassay system is injected, and the intensity of the chemiluminescent photons is detected. The intensity of the light is inversely proportional to the concentration of carbamazepine in the sample. The amount of analyte in the sample is determined by a stored multi-point calibration curve. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a detection reagent that is safe, rapid, efficient, sensitive, and accurate in detecting the carbamazepine content in a sample, as well as a detection method that can be used in conjunction with various types of automated chemiluminescence immunoassay analyzers and does not require highly skilled personnel.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A chemiluminescent immunoassay reagent for carbamazepine is provided, comprising: an anti-carbamazepine specific antibody, and an indicator for detecting the anti-carbamazepine specific antibody-carbamazepine complex; the anti-carbamazepine specific antibody is obtained by immunizing animals with a carbamazepine immunogen, the structural formula of which is shown in formula (I):

[0008]

[0009] In the formula, R is a linking group, and the carrier is immunogenic; the above indicator reagents are selected from chemiluminescent reagents, enzyme reagents, radioisotope reagents, and fluorescent reagents.

[0010] In the aforementioned carbamazepine chemiluminescent immunoassay reagent, R stands for -(CH2). n -CO-, where n is an integer between 1 and 20; preferably, the above R is -(CH2)4-CO-.

[0011] In the aforementioned carbamazepine chemiluminescent immunoassay reagent, the carrier is an immunogenic protein, preferably bovine serum albumin.

[0012] The aforementioned carbamazepine chemiluminescent immunoassay reagent includes an indicator selected from chemiluminescent reagents, comprising: a carbamazepine-alkaline phosphatase conjugate and a luminescent substrate; the luminescent substrate is 2-chloro-5-(4-methoxyspiro-1,2-dioxane-3,2′-(5-chlorotricyclo3.3.1.13.7decane)-4-yl-1-phenylphosphate disodium.

[0013] The aforementioned carbamazepine chemiluminescent immunoassay reagent, wherein the carbamazepine-alkaline phosphatase conjugate is formed by coupling alkaline phosphatase with a carbamazepine derivative, the structural formula of which is shown in formula (II):

[0014]

[0015] The R mentioned above is -(CH2). n -CO-, where n is an integer between 1 and 20.

[0016] In the aforementioned carbamazepine chemiluminescent immunoassay reagent, R is -(CH2)4-CO-.

[0017] The method for detecting carbamazepine using the aforementioned test reagents includes the following steps:

[0018] 1) Contact the sample to be tested with the above-mentioned anti-carbamazepine specific antibody;

[0019] 2) Based on the binding of carbamazepine to the above-mentioned anti-carbamazepine specific antibody in the sample to be tested, the content of carbamazepine in the sample is determined by indicator reagent.

[0020] The aforementioned method for detecting carbamazepine uses physiological samples as the test samples.

[0021] In the aforementioned method for detecting carbamazepine, the physiological sample is serum or plasma.

[0022] The advantages of this invention are: the chemiluminescent immunoassay reagent containing the above-mentioned anti-carbamazepine specific antibody can conveniently, quickly, and accurately determine the carbamazepine content in a sample, and can simultaneously measure multiple samples on a fully automated chemiluminescent immunoassay analyzer, realizing high-throughput and rapid determination of carbamazepine. It has high accuracy, strong specificity, and significantly improved precision and detection efficiency compared to previous methods. At the same time, it realizes the full automation of the detection process, has low requirements for testing personnel, and is easy to implement and promote. Attached Figure Description

[0023] Figure 1 This is the standard curve of the chemiluminescent immunoassay of carbamazepine;

[0024] Figure 2 This is a linear analysis graph of the chemiluminescent immunoassay of carbamazepine. Detailed Implementation

[0025] The technical solution adopted in this invention is:

[0026] Carbamazepine immunogen, the structural formula of which is shown in formula (I):

[0027]

[0028] In the formula, R is a linking group, which can be -(CH2). n -CO-, where n is an integer between 1 and 20, and specifically, R is -(CH2)4-CO-; the carrier is immunogenic, preferably an immunogenic protein. Although other sufficiently large immunogenic substances can also be used as carriers, proteins are generally chosen. The most commonly used immunogenic carriers include serum proteins, hemocyanin (KLH), and thyroglobulin. The choice of carrier is basic common sense for those skilled in the art.

[0029] An anti-carbamazepine specific antibody was obtained by immunizing animals with the carbamazepine immunogen shown in formula (I).

[0030] The term "antibody" as used in this invention refers not only to complete antibody molecules but also to antibody fragments or derivatives that retain the specific binding ability of a complete antibody. The antibodies of this invention can be polyclonal or monoclonal antibodies, but are preferably polyclonal antibodies.

[0031] The method for obtaining polyclonal antibodies involves immunizing an animal at one or more sites using the carbamazepine immunogen shown in formula (I), with or without adjuvant. Host animals include rabbits, goats, mice, sheep, guinea pigs, or horses. Continuous immunization continues until the antibody titer reaches its peak. Blood is collected from the animal periodically to obtain appropriate amounts of specific antiserum, which can be purified.

[0032] Monoclonal antibodies can be produced using somatic cell hybridization techniques.

[0033] A carbamazepine chemiluminescent immunoassay reagent comprises: the aforementioned anti-carbamazepine specific antibody and an indicator reagent for detecting the anti-carbamazepine specific antibody-carbamazepine complex. The indicator reagent is selected from chemiluminescent reagents, enzyme reagents, radioisotope reagents, and fluorescent reagents. Preferably, the indicator reagent is a chemiluminescent reagent comprising: a carbamazepine-alkaline phosphatase conjugate and a luminescent substrate, which can be obtained by chemical synthesis.

[0034] The method for detecting carbamazepine includes the following steps:

[0035] 1) Contact the sample to be tested with the above-mentioned anti-carbamazepine specific antibody;

[0036] 2) Based on the binding of carbamazepine to the above-mentioned anti-carbamazepine specific antibody in the sample to be tested, the content of carbamazepine in the sample is determined by indicator reagent.

[0037] The test sample can be any physiological sample, such as serum, plasma, urine, saliva, etc. Preferably, the test sample is serum or plasma.

[0038] The present invention will be further described below with reference to specific embodiments.

[0039] Example 1: Synthesis and structural confirmation of carbamazepine derivatives

[0040] The chemical structures of the carbamazepine derivatives used in the following examples are shown in formula (IV):

[0041]

[0042] The synthetic route for this carbamazepine derivative is as follows:

[0043]

[0044] The specific synthesis steps are as follows:

[0045] (1) Synthesis of compound 2

[0046]

[0047] 1) Accurately weigh 10.0 g (51.8 mmol) of compound 1 5H-dibenzo[b,f]azapyrrolidone, which was purchased from Sigma-Aldrich.

[0048] 2) Compound 1 was dissolved in 1500 mL of dichloromethane, treated with 5.4 g (18.1 mmol) of triphosgene, and then 100 mL (126.6 mmol) of pyrimidine was added. The mixture was stirred overnight at room temperature to obtain a mixture.

[0049] 3) The mixture was rinsed with water and brine, Na2SO4 was added, the mixture was dried under vacuum and concentrated, and purified by a silica gel-packed column. The mobile phase was a mixture of petroleum ether and ethyl acetate in a volume ratio of 10:1. Finally, the mixture was dried to obtain 12.9 g of white solid compound 2, namely 5H-dibenzo[b,f]azapyro-5-carbonyl chloride, with a yield of 95%.

[0050] (2) Synthesis of carbamazepine derivatives

[0051]

[0052] 1) Weigh 4.65 g (39.6 mmol) of ε-aminovaleric acid and dissolve it in a mixed solution of 2800 mL toluene and 120 mL triethylamine;

[0053] 2) Add 9.22 g (36 mmol) of compound 2 at room temperature and stir overnight at 90 °C;

[0054] 3) Cool to room temperature, concentrate under vacuum, add 500 mL of dichloromethane, wash with water and brine, add Na2SO4 to dry, concentrate, and obtain a yellow compound;

[0055] 4) The above yellow compound was crystallized with ethyl acetate / petroleum ether at a volume ratio of 1:10, and dried to obtain 1.636 g of white solid purified product, namely the carbamazepine derivative shown in formula (IV), with a yield of 12%.

[0056] (3) Structural identification of the above-mentioned white solid purified product.

[0057] 1) The above-mentioned white solid compound was subjected to nuclear magnetic resonance spectroscopy using a Bruker Avance III plus 400MHz spectrometer, with TMS as an internal standard. The results are as follows: 1 ¹H NMR (400MHz, DMSO-d6): δ 1.39–1.30 (m, 4H), 2.14 (2H, t, J = 7.4Hz), 2.93 (2H, q, J = 6.0Hz), 5.53 (2H, t, J = 5.6Hz), 7.00 (s, 1H), 7.36–7.48 (m, 8H). Characterized as a carbamazepine derivative as shown in formula (IV).

[0058] 2) The obtained derivatives were analyzed and identified using chromatography / mass spectrometry (LCMS) with an Agilent Technologies LC / MSD1200 series tandem quadrupole mass spectrometer in either positive or negative ionization mode. The column specifications were: Welchrom XB-C18 (50×4.6mm, 5μm), column temperature 30℃, flow rate 1.5mL / min, mobile phase 95% water and 5% acetonitrile, 6 min, followed by a final 0.5 min under these conditions.

[0059] LCMS results showed: purity 98.62%; retention time 2.682 min; molecular weight 336.4; molecular weight 337.2 (M+1). + ).

[0060] Based on the above results, it can be determined that the white solid compound is a carbamazepine derivative represented by formula (IV).

[0061] Similarly, when ε-aminovaleric acid analogs are used in the reaction, analogs of carbamazepine derivatives as shown in formula (IV) can be obtained, differing only in the number of -CH2- groups, n. Here, ε-aminovaleric acid and its analogs are both denoted as compound A. The relationship between the amount of compound A added and the amount of carbamazepine derivative obtained when n takes different values ​​is shown in Table 1.

[0062] Table 1. Required compound mass and yield for different n values.

[0063]

[0064]

[0065] Example 2: Synthesis of Carbamazepine Immunogen

[0066] Carbamazepine immunogen is derived from bovine serum albumin (BSA) and the carbamazepine derivative represented by formula (II) with -(CH2). n The immunogen is formed by linking -CO- groups. In this embodiment, taking n=4 as an example, the synthesis method of the immunogen is described in detail. The specific steps are as follows:

[0067] Dissolve 20 mg BSA in 5 ml of 0.2 M, pH 8.5 phosphate buffer solution (PBS), and place the solution in beaker A.

[0068] Add the following chemicals to beaker B and stir to dissolve: 20 mg carbamazepine derivative, 0.35 ml dimethylformamide (DMF), 0.35 ml ethanol, 0.7 ml 10 mM potassium phosphate buffer (pH 5.0), 40 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and 5 mg N-hydroxysuccinimide (Sulfo-NHS). Dissolve by stirring at room temperature and react for 30 minutes.

[0069] The solution in beaker B was added dropwise to beaker A to obtain a mixed solution, which was stirred overnight at 2–8 °C. The mixed solution was purified by dialysis with neutral phosphate buffer (4 × 4 L) to obtain BSA-carbamazepine immunogen, which was stored at -20 °C.

[0070] Similarly, when n takes other integers in the range of 1 to 20, the carbamazepine immunogen shown in formula (I) can be prepared using the same method. Of course, the carrier is still an immunogenic protein, which can be serum protein, hemocyanin (KLH), or thyroglobulin. Preferably, the carrier is bovine serum albumin.

[0071] Example 3: Preparation of anti-carbamazepine specific antibody

[0072] The BSA-carbamazepine immunogen prepared above was inoculated into experimental rabbits using standard methods. After booster immunization, antiserum was collected. The specific steps are as follows:

[0073] The synthesized BSA-carbamazepine immunogen was diluted to 1.0 mg / ml with PBS to obtain an antigen solution. Then, 1.0 ml of the antigen solution was mixed with Freund's complete adjuvant and injected into rabbits.

[0074] Two to three weeks later, the rabbits were injected again with 1.0 ml of the same antigen solution and Freund's incomplete adjuvant. This was repeated every four weeks for a total of four injections.

[0075] Blood was collected from the above-mentioned experimental rabbits, and anti-carbamazepine specific antibodies were isolated and purified. The titer of the antibody was determined to be 1:30000.

[0076] Example 4: Preparation of Carbamazepine Chemiluminescent Immunoassay Reagent

[0077] Preparation of chemiluminescent reagent 1: Magnetic microparticles coated with anti-carbamazepine specific antibody were added to the magnetic bead base solution until the final concentration of the magnetic microparticles coated with anti-carbamazepine specific antibody was 0.1 mg / mL. The mixture was vortexed for 1 minute. The pH of the solution was adjusted to 7.10-7.30 with 6N HCl or 3M NaOH. The volume was brought up to 1L with the magnetic bead base solution. After the volume was brought up, the mixture was stirred for another 10 minutes.

[0078] Preparation of chemiluminescent reagent 2: Add carbamazepine-alkaline phosphatase conjugate to alkaline phosphatase base solution until the final concentration of carbamazepine-alkaline phosphatase conjugate is 1 KU / L. Stir with a magnetic stirrer for 25-35 minutes. Adjust the pH of the solution to 7.50-7.70 with 6N HCl or 3M NaOH. Make up the volume to 1L with alkaline phosphatase base solution. After making up the volume, continue stirring for ten minutes.

[0079] Preparation of chemiluminescent reagent 3: Add 90% purified water (v / v), 8.47g of 145mmol / L sodium chloride, and 0.5mL of 0.05% preservative Prucinine 300 (v / v) and stir for 25-35 minutes. Adjust the pH of the solution to 7.10-7.30 with 6N HCl or 3M NaOH and bring the volume to 1L. After bringing the volume to 1L, continue stirring for 10 minutes.

[0080] The preparation method of the magnetic microparticles coated with anti-carbamazepine specific antibody is as follows: 10 mL of 5 mg / mL magnetic beads, 90 mL of activation solution, 20 μg / mg of anti-carbamazepine specific antibody, 500 mg / mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 500 mg / mL of N-hydroxythiosuccinimide sodium salt are placed in a rotary incubator and mixed by rotation. The magnetic beads are coated for 120 minutes. The coated magnetic beads are washed three times with washing solution. 100 mL of magnetic bead blocking solution is added and mixed by rotation. The magnetic beads are blocked for 60 minutes. The blocked magnetic beads are washed three times with washing solution. 100 mL of magnetic bead base solution is added to the washed magnetic beads to preserve them.

[0081] The preparation method of the activation solution is as follows: 90% purified water (volume fraction), 19.52g of 100mmol / L morpholine ethanesulfonic acid, 0.5mL of 0.05% Tween-20, and 0.5mL of 0.05% preservative Precambriconazole 300 are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 4.90-5.10 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes.

[0082] The magnetic bead blocking solution is prepared as follows: 90% purified water (volume fraction), 20.0g of 2% bovine serum albumin, 0.5mL of 0.05% Tween-20, 0.5mL of 0.05% preservative Precambriconazole 300, and 6.057g of 50mmol / L tris(hydroxymethyl)aminomethane are stirred and mixed for 25–35 minutes. The pH of the solution is adjusted to 7.10–7.30 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes.

[0083] The cleaning solution is prepared as follows: 90% purified water (volume fraction), 8.5g of 145mmol / L sodium chloride, 0.95g of 6.66mmol / L disodium hydrogen phosphate, 0.45g of 3.33mmol / L potassium dihydrogen phosphate, 0.5mL of 0.05% preservative Precinctine 300, and 0.5mL of 0.05% Tween-20 are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 7.10-7.30 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After bringing the volume to 1L, stirring is continued for ten minutes.

[0084] The preparation method of the magnetic bead base solution is as follows: 90% purified water (volume fraction), 6.057g of 50mmol / L tris(hydroxymethyl)aminomethane, 2.5g of 0.25% bovine serum albumin, 0.5mL of 0.05% Tween-20, and 0.5mL of 0.05% preservative Precambriconazole 300 are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 6.90-7.10 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes.

[0085] The preparation method of the carbamazepine-alkaline phosphatase conjugate is as follows: 5.6 μL of 1 KU / mL alkaline phosphatase is added to 0.5 mL of alkaline phosphatase basal solution to prepare an alkaline phosphatase solution. 2 mg of 2 mg / mL carbamazepine derivative, 0.5 mL of activation solution, 2 mg / mL of 2 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 2 mg / mL of 2 mg / mL N-hydroxythiosuccinimide sodium salt are placed in a magnetic stirrer and stirred for 5 minutes to activate. The activated carbamazepine derivative is added to the above alkaline phosphatase solution and coupled at 2-8℃ for 120 minutes. The coupled carbamazepine-alkaline phosphatase conjugate is dialyzed 4 times with dialysis buffer, each dialyzing for 8 hours.

[0086] The alkaline phosphatase base solution is prepared as follows: 90% purified water (v / v), 1.25g of 0.125% bovine serum albumin, 0.20g of 1mmol / L magnesium chloride, 13.63mg of 0.1mmol / L zinc chloride, 8.5g of 145.5mmol / L sodium chloride, 6.057g of 50mmol / L tris(hydroxymethyl)aminomethane, and 0.5mL of 0.05% preservative Precambriconazole 300 are stirred and mixed for 25–35 minutes. The pH of the solution is adjusted to 7.50–7.70 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After bringing the volume to 1L, stirring is continued for ten minutes.

[0087] The preparation method of the activation solution is as follows: 90% purified water (volume fraction), 19.52g of 100mmol / L morpholine ethanesulfonic acid, 0.5mL of 0.05% Tween-20, and 0.5mL of 0.05% preservative Precambriconazole 300 are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 4.90-5.10 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes.

[0088] The preparation method of the dialysate is as follows: 90% purified water, 8.47g of 145mmol / L sodium chloride, 1.15g of 8.1mmol / L disodium hydrogen phosphate, 0.2g of 1.47mmol / L potassium dihydrogen phosphate, and 0.5mL of 0.05% preservative are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 7.30-7.50 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes.

[0089] Example 5: Carbamazepine Chemiluminescent Immunoassay

[0090] (1) Set the reaction parameters of the fully automated chemiluminescence immunoassay analyzer (Table 1).

[0091] Table 1. Reaction parameters for carbamazepine detection using a fully automated chemiluminescence immunoassay analyzer.

[0092]

[0093] (2) Reagent performance evaluation experiment

[0094] The performance of the carbamazepine chemiluminescent immunoassay reagent prepared in Example 4 was validated. The main performance indicators tested included appearance, fill volume, limit of detection, linearity, repeatability, batch-to-batch difference, accuracy, calibrator accuracy and homogeneity, and quality control accuracy and homogeneity. Experimental parameters were set according to the methods described above. The reagent performance validation results are as follows:

[0095] Appearance

[0096]

[0097]

[0098]

[0099] Filling volume

[0100]

[0101] Filling volume

[0102]

[0103] Filling volume

[0104]

[0105]

[0106] Linearity: 1.00–20.00 μg / mL

[0107]

[0108] Repeatability unit: μg / mL

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] Calibrator

[0116] Accuracy unit: μg / mL

[0117]

[0118] Uniformity unit: μg / mL calibrator 1

[0119]

[0120]

[0121] Uniformity

[0122] Calibrator 2 units: μg / mL

[0123]

[0124] Uniformity

[0125] Calibrator 3 units: μg / mL

[0126]

[0127]

[0128]

[0129] Uniformity

[0130] Quality control sample 1 unit: μg / mL

[0131]

[0132]

[0133] Quality control sample, 2 units: μg / mL

[0134]

[0135] Quality control product 3

[0136]

[0137]

[0138] It should be noted that the above description is only an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A carbamazepine chemiluminescent immunoassay reagent, characterized in that, include: Anti-carbamazepine specific antibody, used as an indicator for detecting the anti-carbamazepine specific antibody-carbamazepine complex; the above-mentioned anti-carbamazepine specific antibody was obtained by immunizing animals with a carbamazepine immunogen, the structural formula of which is shown in formula (I): ； In the formula, R is a linking group, and the carrier is immunogenic; the above indicator reagents are selected from chemiluminescent reagents, enzyme reagents, radioisotope reagents, and fluorescent reagents.

2. The carbamazepine chemiluminescent immunoassay reagent according to claim 1, characterized in that, The R mentioned above is -(CH2). n -CO-, where n is an integer between 1 and 20.

3. The carbamazepine chemiluminescent immunoassay reagent according to claim 2, characterized in that, The R mentioned above is -(CH2)4-CO-.

4. The carbamazepine chemiluminescent immunoassay reagent according to claim 1, characterized in that, The aforementioned carrier is an immunogenic protein, preferably bovine serum albumin.

5. The carbamazepine chemiluminescent immunoassay reagent according to claim 1, characterized in that, The above indicator reagent is selected from chemiluminescent reagents, including: carbamazepine-alkaline phosphatase conjugate and luminescent substrate; the above luminescent substrate is 2-chloro-5-(4-methoxyspiro-1,2-dioxane-3,2′-(5-chlorotricyclo3.3.1.13.7decane)-4-yl-1-phenylphosphate disodium.

6. The carbamazepine chemiluminescent immunoassay reagent according to claim 5, characterized in that, The above carbamazepine-alkaline phosphatase conjugate is formed by coupling alkaline phosphatase with a carbamazepine derivative, the structural formula of which is shown in formula (II): ； The R mentioned above is -(CH2). n -CO-, where n is an integer between 1 and 20.

7. The carbamazepine chemiluminescent immunoassay reagent according to claim 6, characterized in that, The R mentioned above is -(CH2)4-CO-.

8. A method for detecting carbamazepine using the detection reagent according to any one of claims 1 to 7, characterized in that, Includes the following steps: 1) Contact the sample to be tested with the above-mentioned anti-carbamazepine specific antibody; 2) Based on the binding of carbamazepine to the above-mentioned anti-carbamazepine specific antibody in the sample to be tested, the content of carbamazepine in the sample is determined by indicator reagent.

9. The method for detecting carbamazepine according to claim 8, characterized in that, The samples to be tested are physiological samples.

10. The method for detecting carbamazepine according to claim 9, characterized in that, The physiological samples mentioned above are serum or plasma.

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