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Methods for detecting phenytoin

A detection method and phenytoin technology, applied in the biological field, can solve the problems of not reaching the effective blood drug concentration, narrow therapeutic window, large individual differences, etc., and achieve the effects of convenient operation, strong specificity and high sensitivity

Inactive Publication Date: 2012-08-15
JIAXING JIUQIJIU BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional antiepileptic drug phenytoin has a narrow therapeutic window, large individual differences, and the efficacy and toxicity are closely related to the blood concentration. Clinicians often fail to achieve the effective blood concentration when administering drugs only based on experience.

Method used

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  • Methods for detecting phenytoin
  • Methods for detecting phenytoin
  • Methods for detecting phenytoin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1 Synthesis of phenytoin immunogen

[0078] The chemical structure of the phenytoin derivative used in the following examples is shown in formula (III).

[0079]

[0080] Formula (III)

[0081] 1. the synthesis of phenytoin derivatives, its chemical structural formula is as shown in formula (III)

[0082] (1) Synthetic Chemicals 3

[0083]

[0084] 1) Accurately weigh 5.3g, 26.6mmol of compound 1 (hydroxybenzophenone) and 1.9g, 14mmol of anhydrous potassium carbonate (K 2 CO 3 ), these two compounds were added to the flask.

[0085] 2) Add 150 mL of DMF and sonicate for 15 min to dissolve the compound.

[0086] 3) Add 6.5g, 31.2mmol of chemical compound 2 (ethyl 5-bromopentanoate), and stir at 55°C for 72h.

[0087] 4) Filter the solution after cooling down to room temperature, dissolve the reactants with 5mL DMF, and dry in vacuo.

[0088] 5) Use 150mL concentrated sodium bicarbonate (NaHCO 3 ) solution to dissolve the residue, extract with 150mL ...

Embodiment 2

[0116] Example 2 Preparation of Anti-Phenytoin Specific Antibody

[0117] (1) Dilute the synthetic phenytoin immunogen to 1.5 mg / ml with PBS to obtain an antigen solution, then mix the antigen solution with Freund's complete adjuvant, and inject it into rabbits;

[0118] (2) After 2 to 3 weeks, inject the rabbit once with 1.0ml of the same antigen solution mixed with Freund's incomplete adjuvant, and then once every four weeks, twice in total, and extract the antiserum of the rabbit to obtain effective antibodies.

Embodiment 3

[0119] Example 3 A homogeneous enzyme immunoassay for phenytoin

[0120] 1. Preparation of homogeneous enzyme immunoassay reagents

[0121] (1) Preparation of R1 reagent

[0122] Dilute the prepared antibody into R1 buffer, the homogeneous R1 buffer contains 50mM Tris, 0.25% BSA, 50mM G-6-P and 50mM NAD. The volume ratio of antibody to R1 buffer was 1:2000.

[0123] (2) Preparation of R2 reagent

[0124] 1) Preparation of G6PDH-phenytoin

[0125] a) Weigh 15mg G6PDH, dissolve it in 12ml, 0.05M Tris buffer, add 100mg NADH, 0.5ml carbitol and 1ml DMF in sequence and mix well;

[0126] b) Dissolve 10 mg of phenytoin derivative in 420 μl DMSO and 180 μl DMF, add 6 μl tributylamine and 3 μl isochloroformate, and stir for 30 minutes at 2-8°C;

[0127] c) Stir overnight at 2-8° C., and purify the obtained G6PDH-phenytoin.

[0128] 2) Dilute the prepared G6PDH-phenytoin into R2 buffer. R2 buffer was 100 mM Tris, 0.25% BSA. The volume ratio of antibody to R2 buffer was 1:3000. ...

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Abstract

The invention discloses two methods for detecting phenytoin. The methods comprise the following steps of: making a sample to be detected in contact with an anti-phenytoin specific antibody; and judging the phenytoin content of the sample according to the combination condition of the substance in the sample to be detected and the antibody, wherein the anti-phenytoin specific antibody is obtained from a phenytoin immunogen immune animal. The two detecting methods are high in specificity, can accurately measure the content of the phenytoin, and provide a proof for real-time monitoring of the clinical medicament concentration of the phenytoin.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to two detection methods for phenytoin, including a homogeneous enzyme immunoassay for phenytoin and an enzyme-linked immunosorbent assay for phenytoin. Background technique [0002] Phenytoin (5,5-Diphenyl-2,4-imidazolidinedione), the structural formula of which is shown in formula (II). [0003] [0004] Formula (II) [0005] Phenytoin is an antiepileptic drug. Epilepsy is a common neurological disease, and it is also the second most persistent disease among neurological diseases in my country after cerebrovascular diseases. At present, antiepileptic drugs are still the main means of controlling epileptic seizures. The traditional anti-epileptic drug phenytoin has a narrow therapeutic window, large individual differences, and the efficacy and toxicity are closely related to the blood concentration. Clinicians often fail to reach the effective blood concentration when administering d...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/543G01N33/577G01N33/535G01N33/531
Inventor 虞留明田军袁红霞孟雷蔡江丽
Owner JIAXING JIUQIJIU BIOLOGICAL TECH
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