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Preparation method of immunodetection reagent of carbamazepine homogeneous enzyme

The technology of immunodetection and carbamazepine is applied in the field of carbamazepine average phase enzyme immunoassay reagent and its detection, which can solve the problem that high-performance liquid chromatography takes a long time, requires high technical level of testing personnel, and requires pretreatment and operation. The process is complex and other problems, to achieve high-throughput and rapid determination, easy implementation and popularization, and low requirements.

Active Publication Date: 2015-05-06
苏州博源医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

High-performance liquid chromatography takes a long time, and the sample pretreatment and operation process is extremely complicated, which requires high technical level of the testing personnel; the radioactive rays of the radioimmunoassay method have caused great harm to the health of the operators. Rarely used; the reagents required by the fluorescence polarization method are mainly imported and extremely expensive, and expensive analytical instruments need to be purchased at the same time

Method used

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  • Preparation method of immunodetection reagent of carbamazepine homogeneous enzyme
  • Preparation method of immunodetection reagent of carbamazepine homogeneous enzyme
  • Preparation method of immunodetection reagent of carbamazepine homogeneous enzyme

Examples

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Comparison scheme
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Embodiment 1

[0044] Example 1: Synthesis and structure confirmation of carbamazepine derivatives

[0045] The chemical structure of the carbamazepine derivative used in the following examples is shown in formula (IV):

[0046]

[0047] Formula (IV)

[0048] The synthetic route of this carbamazepine derivative is as follows:

[0049]

[0050] Concrete synthetic steps are as follows:

[0051] Synthesis of Compound 2

[0052]

[0053] 1) Accurately weigh 10.0 g (51.8 mmol) of compound 1 5H-dibenzo[b,f]azepine, which was purchased from Sigma;

[0054] 2) Dissolve compound 1 in 1500mL of dichloromethane, add 5.4g (18.1mmol) of triphosgene for treatment, then add 100ml (126.6mmol) of pyrimidine, and stir overnight at room temperature to obtain a mixture;

[0055] 3) Rinse the above mixture with water and brine, add Na 2 SO 4 , concentrated by vacuum drying and purified by a chromatographic column filled with silica gel, the mobile phase is a mixed mobile phase of petroleum ether / e...

Embodiment 2

[0070] Embodiment two: the synthesis of BSA-carbamazepine immunogen

[0071] The BSA-carbamazepine immunogen consists of bovine serum albumin BSA and the carbamazepine derivative-(CH 2 ) n The -COO- group is connected. In this example, the synthesis method of the immunogen is described in detail by taking n=4 as an example. The specific steps are as follows:

[0072] 1) Dissolve 20mg of BSA in 5ml of 0.2M, pH8.5 phosphate buffer solution (Phosphate buffer solution, PBS), and place the above solution in beaker A;

[0073] 2) Add the following chemicals into beaker B and stir to dissolve: 20mg carbamazepine derivative, 0.35ml dimethylformamide (DMF), 0.35ml ethanol, 0.7ml 10mM potassium phosphate buffer solution at pH 5.0. 40mg 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 5mg N-hydroxysuccinimide (N-hydroxysuccinimide, Sulfo-NHS), stirred and dissolved at room temperature, reacted for 30 minute;

[0074] 3) Add the solution in beaker B to beaker A dropwise to obtain a mi...

Embodiment 3

[0076] Embodiment three: the preparation of anti-carbamazepine specific antibody

[0077] The BSA-carbamazepine immunogen prepared above was inoculated into experimental animal rabbits by conventional methods, and the antiserum was taken after booster immunization, and the specific steps were as follows:

[0078] Dilute the above-mentioned synthesized BSA-carbamazepine immunogen to 1.0 mg / ml with PBS to obtain an antigen solution, then mix 1.0 ml of the antigen solution with Freund's complete adjuvant, and inject it into rabbits;

[0079] After 2 to 3 weeks, the rabbits were injected with 1.0ml of the same antigen solution and Freund's incomplete adjuvant, and then once every four weeks, for a total of 4 injections.

[0080] Blood was collected from the above-mentioned experimental animal rabbit, and the specific antibody against carbamazepine was obtained by separation and purification. The titer of the antibody was determined to be 1:30000.

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Abstract

The invention discloses a carbamazepine immunogen, a carbamazepine specific antibody directly obtained through the immunogen, a detection reagent containing the specific antibody and a method for detecting the content of the carbamazepine in samples to be tested. The immunodetection reagent and the detection method have the advantages that the carbamazepine immunogen is strong in specificity and high in immunogenicity; the prepared carbamazepine immunogen is strong in specificity and high in titer and has no cross reaction with 45 common drugs; the homogeneous enzyme immunodetection reagent containing the carbamazepine specific antibody can conveniently, fast and accurately determine the content of the carbamazepine in the samples and can measure a plurality of samples simultaneously on a full-automatic biochemical analyzer, so that high-throughput and fast measuring of the carbamazepine are achieved, the accuracy is high, the specificity is strong, the accuracy and the detection efficiency are greatly improved compared with the prior art, full automation in the detection process is achieved simultaneously, the requirements on detection staff is not high, and the immunodetection reagent and the detection method are easy to achieve, popularize and use.

Description

technical field [0001] The invention relates to a detection reagent and a detection method, in particular to a kammazime mean-phase enzyme immunoassay reagent and a detection method thereof. Background technique [0002] Carbamazepine (Carbamazepine) structural formula is shown in (III): [0003] [0004] Formula (III) [0005] Carbamazepine is a common psychiatric drug with antiepileptic, antitrigeminal neuralgia, antiglossopharyngeal neuralgia, prevention and treatment of manic depression and antiarrhythmia. However, due to the narrow range of its effective therapeutic concentration, the therapeutic effect cannot be achieved when the concentration is low, and side effects such as dizziness, lethargy, fatigue, vomiting, aplastic anemia, toxic hepatitis and shock can be caused after overdose. Therefore, it is very necessary to monitor drug concentration during treatment. [0006] At present, the methods for monitoring drug concentration of carbamazepine mainly include ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/96G01N33/531
Inventor 虞留明田军蔡江丽
Owner 苏州博源医疗科技有限公司
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