Theophylline homogeneous enzyme immunoassay kit and preparation method thereof

A detection kit and homogeneous enzyme immunoassay technology, applied in the field of medical testing, can solve the problems of high price, short validity period, cumbersome operation, etc., and achieve the effects of good stability and repeatability, rapid detection, and high sensitivity

Inactive Publication Date: 2011-11-23
济南金域医学检验中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The highly automated FPIA method has become the most commonly used method for clinical monitoring of theophylline blood concentration due to its simplicity and rapidity, but its kits rely on imports, expensive and short validity period are its unavoidable shortcomings
However, the HPLC method often has the disadvantages of cumbersome operation, low efficiency, long measurement cycle and high analysis cost, which limit its wide application in clinical drug concentration monitoring.

Method used

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  • Theophylline homogeneous enzyme immunoassay kit and preparation method thereof
  • Theophylline homogeneous enzyme immunoassay kit and preparation method thereof
  • Theophylline homogeneous enzyme immunoassay kit and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0036] 1. Synthesis of Theophylline Immunogen

[0037] a. Dissolve 200 mg of bovine serum albumin (BSA) in 50 mL of 0.2 mol / L, pH 8.5 phosphate buffer;

[0038] b. Add the following components into a beaker and stir to dissolve: 100mg theophylline derivatives, 3.5mL dimethylamide (DMF), 3.5mL ethanol, 7.0mL (10mmol / L, pH5.0) potassium phosphate buffer, 400mg1 -Ethyl-3-(-3-dimethylaminopropyl)carbodiimide, 50mg of N-hydroxysulfosuccinimide, stirred and dissolved for 30min;

[0039] Add the solution dissolved in b above dropwise to the BSA solution in a above, and stir overnight at 2-8°C to obtain the antigen;

[0040] The synthesized antigen is purified by dialysis to obtain theophylline immunogen.

[0041] 2. Preparation of anti-theophylline-specific antibody

[0042] Dilute the synthetic theophylline immunogen to 1.0 mg / mL with PBS phosphate buffer solution, then mix 1.0 mL of antigen solution with Freund's complete adjuvant, and inject it into rabbits; 14-21 days later, u...

Embodiment 2

[0054] Embodiment 2 utilizes automatic biochemical analyzer to carry out sample test

[0055] (1) Collection of serum samples, collect serum samples according to conventional methods;

[0056] (2) According to the operating instructions of the Hitachi 7180 automatic biochemical analyzer, turn on the instrument, perform the optical density detection of the instrument and clean the probe, and check whether the instrument is operating normally;

[0057] (3) After the instrument is running normally, put the reagents R1 and R2 into the reagent compartments of R1 and R2 in turn, and put the serum sample into the sample tray 1 (S1), 0.0μg / mL, 2.5μg / mL, 5.0μg / mL, 10.0μg / mL Put the theophylline calibration solution of μg / mL, 20.0μg / mL, and 40.0μg / mL into the designated position of the sample plate 2 (S2);

[0058] (4) When the instrument is in the Stand by state, set the operation procedure and inspection parameters of theophylline, and the specific inspection parameters are shown in ...

Embodiment 3

[0064] 1. Preparation of theophylline homogeneous enzyme immunoassay calibration curve

[0065] see figure 1 , the homogeneous enzyme immunoassay of theophylline was carried out with an automatic biochemical analyzer, and the reaction system could be further optimized by adjusting the ratio of reagent R1 and reagent R2. mL, 5.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 40.0 μg / mL. The working volume of the calibration solution is 10-35 μL, then add 100-200 μL reagent R1 and 100-200 μL reagent R2, use the two-point rate method to detect the change rate of absorbance at the main wavelength of 340 nm and the secondary wavelength of 405 nm, establish and optimize the tea Alkaline homogeneous enzyme immunoassay calibration curve, the establishment and optimization of the calibration curve was completed on Hitachi 7180 automatic biochemical analyzer. Get a working standard curve as figure 1 shown.

[0066] 2. Sensitivity test

[0067] Add unequal amount of theophylline standard substance ...

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Abstract

The invention aims to provide simple, rapid, high-sensitivity and full-automation theophylline drug concentration detection equipment, and a preparation method thereof. The invention provides a theophylline homogeneous enzyme immunoassay kit and a preparation method thereof. The preparation method comprises the following steps: synthesizing a theophylline immunogen; preparing a theophylline-resisting specific antibody; preparing an enzyme labeling conjugate; and determining the sample. The equipment provided by the invention has high sensitivity, and good stability and repetitiveness; the sensitivity can reach 0.1 mu g / mL which is far lower than theophylline clinical drug-used range of 10-20 mu g / mL; the average intraassay and interassay precision is less than 2.0%; and the sample recovery rate is 103.4+ / -1.55%. The equipment provided by the invention can be applied on an automatic biochemical analyzer, is simple in operation, can be used for carrying out high-flux rapid sample detection, and is suitable for clinical detection of conventional treatment drug concentration. The theophylline antibody provided by the invention has strong specificity, has no cross reaction on 31 kinds of tested conventional drugs and compounds in the drug cross reaction test, and is suitable for clinically inspecting the blood concentration of the theophylline.

Description

technical field [0001] The invention belongs to the field of medical testing, in particular to a theophylline homogeneous enzyme immunoassay kit and a preparation method thereof. Background technique [0002] Theophylline is a derivative of methylxanthines, which is commonly used clinically to treat bronchial asthma, asthmatic chronic bronchitis and cardiogenic asthma. But theophylline also has many toxic side effects, including: nausea, headache, diarrhea, vomiting, gastrointestinal bleeding, epilepsy and cardiac arrhythmia. Since the effective concentration of theophylline has a narrow therapeutic window (the reference range of therapeutic serum concentration is 10-20 μg / mL), and the incidence of toxic and side effects is closely related to its blood concentration, the serum concentration of theophylline in patients is detected during treatment. Very important. [0003] Currently, there are mainly high-performance liquid chromatography (HPLC) and fluorescence polarizatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58G01N33/531G01N33/535
Inventor 虞留明梁晓翠蔡江丽陈瑞东梁耀铭
Owner 济南金域医学检验中心有限公司
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