Phenobarbital homogeneous-phase enzyme immunoassay reagent kit and preparation method thereof
A detection kit and a homogeneous enzyme immunoassay technology, applied in the field of medical testing, can solve the problems of semi-quantitative, complex operation, long cycle, etc., and achieve high accuracy, high sensitivity, and strong specificity
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Embodiment 1
[0037] a. Synthesis of bovine serum albumin-phenobarbital derivative immune antigen:
[0038] 1. Preparation of BSA solution, weigh 20 mg of bovine serum albumin (BSA) and dissolve it in 5 mL of 0.2 mol / L, pH 8.5 phosphate buffer at room temperature;
[0039] 2. Activation of phenobarbital derivatives, weigh 10 mg of specific phenobarbital derivatives in a small beaker, and add 350 μL dimethylamide (DMF), 350 μL ethanol, 700 μL 10 mmol / L, pH5.0 Potassium phosphate buffer, 40 mg 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, and 5 mg N-hydroxysulfosuccinimide (NHS), these chemicals were stirred and dissolved at room temperature React for 30 minutes;
[0040] 3. Coupling and purification of the antigen. The activated phenobarbital derivative is added dropwise to the BSA solution and stirred overnight at 2-8° C. to obtain the antigen; and the coupled antigen is dialyzed and purified.
[0041] B, the preparation of anti-phenobarbital derivative antibody, the steps are as follo...
Embodiment 2
[0052] The detection experiment of embodiment diphenobarbital detection kit product
[0053] 1. Calibration experiment
[0054] see figure 2 , the phenobarbital calibrator was dissolved in blank serum and prepared into 6 kinds of calibrating solutions with a series concentration of 0, 5, 10, 20, 40, and 80 μg / mL. The working volume of the calibrating solution was 10-35 μL, and then Add 100-200 μL of reagent R1 and 100-200 μL of reagent R2, and use the two-point rate method to detect the absorbance change rate at the main wavelength of 340 nm and the secondary wavelength of 405 nm, and establish and optimize the phenobarbital homogeneous enzyme immunoassay calibration curve ( figure 2 ), the establishment and optimization of the calibration curve was completed on a Hitachi 7180 automatic biochemical analyzer.
[0055] 2. Recovery experiment
[0056] Using the established phenobarbital calibration curve, determine low (20 μg / mL), medium (40 μg / mL)) and high (80 μg / mL) serum...
Embodiment 3
[0077] Embodiment 3 Utilize automatic biochemical analyzer to carry out sample test
[0078] (1). Collection of serum samples: collect serum samples according to conventional methods;
[0079] (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;
[0080] (3). After the detection of the instrument is running normally, put the reagents R1 and R2 into the reagent chambers of R1 and R2 in turn, put the serum sample into the sample tray 1 (S1), put 0μg / mL, 5μg / mL, 10μg / mL, 20μg / mL Put the phenobarbital calibration solution of mL, 40 μg / mL, and 80 μg / mL into the designated position of the sample plate 2 (S2);
[0081] (4). When the instrument is in the Stand by state, set the operating procedures and detection parameters of phenobarbital, and the specific detection parameters are show...
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