A kind of theophylline homogeneous enzyme immunoassay test kit and preparation method thereof
A technology for theophylline and reagents, applied in the field of theophylline homogeneous enzyme immunoassay kit and its preparation, can solve the problem of poor sensitivity and specificity of the kit, affect the sensitivity and specificity of the kit, and fail to reflect the overall theophylline Immunity and other issues, to achieve good stability and repeatability, easy operation, high sensitivity
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Embodiment 1
[0053] Example 1 Synthesis of Theophylline Derivatives
[0054] Step 1: first dissolve 100mmol of compound 1 and 120mmol of compound 2 in 150mL of absolute ethanol, then add 150mmol of triethylaluminum to obtain the first reaction solution, heat the first reaction solution under reflux for 16 hours, and concentrate the product in a vacuum after cooling. Recrystallization gave compound 3 in a yield of 33%.
[0055] Compound 3:
[0056] ESI-MS: 344.08[M-H] -
[0057] Elemental analysis: theoretical value / measured value, C (48.85 / 48.97), H (7.41 / 7.34), N (20.29 / 20.40), O (23.45 / 23.30).
[0058]
[0059] Step 2: Add 15ml of 2N NaOH solution to the product of the previous step, and reflux in an oil bath at 75°C for 1.5 hours. After the reaction, cool down and adjust the pH to 3 with 6N hydrochloric acid. A large amount of precipitation occurs, and the solid is recovered by filtration. Then recrystallization by hot water gave 17 mg of compound 4 (theophylline derivative).
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Embodiment 2
[0065] 1. Synthesis of theophylline artificial antigen
[0066] a. Dissolve 250 mg of bovine serum albumin (BSA) in 100 mL of 0.1 mol / L, pH 8.5 phosphate buffer;
[0067] b. Add the following components into the beaker and stir to dissolve: 100mg theophylline derivative prepared in Example 1, 2.5mL dimethylamide (DMF), 4.5mL ethanol, 7.0mL (10mmol / L, pH5.0) phosphoric acid Potassium buffer, 200mg 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide, 50mg N-hydroxysulfosuccinimide (Sulfo-NHS), stirred and dissolved for 30min; pH5.5;
[0068] Add the dissolved solution in b above dropwise to the BSA solution in a above, and stir overnight at 2-8°C to obtain the antigen;
[0069] The synthesized antigen was purified by dialysis to obtain theophylline artificial antigen.
[0070] 2. Preparation of anti-theophylline-specific antibody
[0071] Dilute the synthetic theophylline artificial antigen to 1.0 mg / mL with PBS phosphate buffer solution, then mix 1.0 mL of the antigen solution w...
Embodiment 3
[0084] Embodiment 3 utilizes automatic biochemical analyzer to carry out sample test
[0085] (1). Collection of serum samples: collect serum samples according to conventional methods;
[0086] (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;
[0087] (3). After the instrument is running normally, put the reagents R1 and R2 into the reagent chambers of R1 and R2 in turn, and put the serum sample into the sample plate 1 (S1), 0μg / mL, 2.5μg / mL, 5.0μg / mL, 10.0 Put the theophylline calibration solution of μg / mL, 20.0μg / mL, and 40.0μg / mL into the designated position of the sample tray 2 (S2);
[0088] (4). When the instrument is in the Standby state, set the operation procedure and inspection parameters of theophylline. The specific inspection parameters are shown in Table 1: ...
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