Preparation method of phenytoin homogeneous enzyme immunoassay kit and phenytoin polyclonal antibodies

A polyclonal antibody and detection kit technology, applied in the field of medical testing, can solve the problems of complex operation, high reagent cost, high detection cost, etc., and achieve the effects of strong specificity, high accuracy and high sensitivity

Inactive Publication Date: 2012-12-05
重庆金域医学检验所有限公司
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  • Abstract
  • Description
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Problems solved by technology

The HPLC method requires sample pretreatment, the operation is relatively complicated, and the detection cost is expensive; the ELISA detection method has high sensitivity, but cannot be accurately quantified; fluorescence polarization immunoassay (FPIA) has the advantages of simple operation and high sensitivity, but the cost of reagents is high. It is not suitable for the detection of conventional therapeutic drugs, hindering the promotion of personalized treatment

Method used

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  • Preparation method of phenytoin homogeneous enzyme immunoassay kit and phenytoin polyclonal antibodies
  • Preparation method of phenytoin homogeneous enzyme immunoassay kit and phenytoin polyclonal antibodies
  • Preparation method of phenytoin homogeneous enzyme immunoassay kit and phenytoin polyclonal antibodies

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Preparation of phenytoin-specific polyclonal antibody:

[0036] a. Synthesis of phenytoin immune antigen

[0037] 1) Activation of phenytoin derivatives. Weigh 20 mg of specific phenytoin derivatives into a small beaker, and add 700 μL of pure dimethylformamide (DMF), 700 μL of absolute ethanol, and 1.4 mL of 10 mM pH Potassium phosphate buffer solution of 5.0, 80mg1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide and 10mgN-hydroxyl sulfosuccinimide (N-hydroxysuccinimide, Sulfo-NHS), will These chemicals were stirred and dissolved for 30 minutes at room temperature;

[0038] 2) Preparation of BSA solution, weighing 40mg of bovine serum albumin (Bovine Serum Albumin, BSA) and dissolving it in 10mL of 0.2M pH 8.5 phosphate buffer at room temperature;

[0039] 3) Coupling and purification of the antigen, the activated phenytoin derivative is added dropwise to the BSA solution, stirred overnight at 2-8°C to obtain the antigen; and the coupled antigen is dialyzed and purifie...

Embodiment 2

[0044] The preparation of phenytoin homogeneous enzyme immunoassay kit includes the following steps: preparation of phenytoin derivative-specific polyclonal antibody, preparation of glucose hexaphosphate dehydrogenase-labeled phenytoin conjugate, preparation of calibrator Preparation, preparation of homogeneous enzyme immunoassay reagents and sample testing using an automatic biochemical analyzer

[0045] a. Prepare phenytoin-specific polyclonal antibody according to the method of Example 1.

[0046] b. Preparation of phenytoin enzyme-labeled antigen

[0047] 1) Weigh 30mg of glucose hexaphosphate dehydrogenase, dissolve it in 24mL, 0.05M Tris buffer, then add 200-500mg of NADH, 1.0-2.0mL of carbitol and 2-6.0mL of dimethyl methylene Sulfone mix;

[0048] 2) Dissolve 20 mg of phenytoin derivative in 840 μL dimethyl sulfoxide and 360 μL DMF, add 12 μL tributylamine and 6 μL isobutylchloroformate, and stir at 2-8 °C 30min;

[0049] 3) The above two solutions were mixed, stir...

Embodiment 3

[0069] Inspection experiment of phenytoin test kit products

[0070] a. Recovery experiment

[0071] Using the established phenytoin calibration curve, determine three concentrations of phenytoin serum samples: low (2.5 μg / mL), medium (10 μg / mL) and high (40 μg / mL) concentrations, and repeat the measurement 5 times for each sample .

[0072] Table 2: Recovery experiment of phenytoin kit

[0073]

[0074] The specific results are shown in Table 2. The average recovery rate (Recovery) of the sample test is greater than 95%, the standard deviation (SD) is less than 1.44, and the correlation coefficient deviation (CV) is less than 8%.

[0075] b. Drug interference experiment

[0076] Select 32 commonly used compounds and drugs, adjust their working concentration to 10.0 μg / mL, and conduct interference test on Hitachi 7180 automatic biochemical analyzer. The test results are shown in Table 3:

[0077] Table 3: Drug Interference Experiment of Phenytoin Kit

[0078]

[007...

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Abstract

The invention aims at providing a preparation method of high-specificity phenytoin polyclonal antibodies and a phenytoin homogeneous enzyme immunoassay kit. The preparation method comprises the steps of preparing phenytoin specific polyclonal antibodies, preparing phenytoin conjugates labeled by glucose-6-phosphate dehydrogenase, and preparing calibration solution. The specificity of the phenytoin polyclonal antibodies is high, the valence is high, the phenytoin polyclonal antibodies almost do not crossly react with tested thirty-two kinds of common drugs and compounds during drug cross reaction testing, and the phenytoin polyclonal antibodies are suitable for the clinical detection of blood concentration of phenytoin. The sensitivity of the phenytoin drug concentration homogeneous enzyme immunoassay kit is high, samples with concentration being 10ng / ml can be accurately detected and the effective detection range of phenytoin drug concentration is 20ng / ml to 40mug / ml. The kit provided by the invention has the advantages of high accuracy, high recovery rate above 90 percent, convenience, rapidness, low cost, high sensitivity and full automation.

Description

technical field [0001] The invention belongs to the field of medical examination, and in particular relates to a phenytoin homogeneous enzyme immunoassay kit and a preparation method of a polyclonal antibody thereof. Background technique [0002] 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 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. Therefore, the monitoring of phenytoin plasma concentration has important clinical guiding significance for the diagnosis and treatment of epilepsy. [0003] At present, the met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44G01N33/536G01N33/532G01N33/535
Inventor 虞留明陈瑞东李冬王金文燕启江
Owner 重庆金域医学检验所有限公司
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