A kind of immunoassay method for sildenafil and structural analog thereof

An immunodetection method and technology for sildenafil, which are applied in the field of immunodetection of sildenafil and its structural analogs, can solve problems such as hindering popularization and application, long detection time, high detection cost, etc. The effect of fast detection and low detection limit

Active Publication Date: 2017-10-20
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
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Problems solved by technology

Although the detection accuracy of the above-mentioned several instrumental analysis methods is high, their popularization and application are hindered by the high degree of instrumentation, long detection time, cumbersome process, and high detection cost. Therefore, they are usually used as laboratory confirmation methods, but cannot Satisfy on-site or large-scale rapid screening and testing
However, immunoassay is worthy of popularization as a commonly used screening method because of its low cost, simple operation, fast speed, large sample size and low degree of instrumentation. The prior art lacks an antibody for simultaneous detection of sildenafil and its Immunization approach for structured drugs

Method used

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  • A kind of immunoassay method for sildenafil and structural analog thereof
  • A kind of immunoassay method for sildenafil and structural analog thereof
  • A kind of immunoassay method for sildenafil and structural analog thereof

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preparation example Construction

[0047] The preparation method of reagent used in the specific embodiment is as follows:

[0048] Coating solution: obtained by dissolving 1.69g of sodium carbonate and 2.95g of sodium bicarbonate in 1L of double distilled water.

[0049] 20-fold concentrated washing solution: pH7.4 0.4mol / L phosphate buffer containing 0.5% Tween20 by volume, diluted to 1 time with deionized water before use.

[0050] PBS: Dissolve 5.8 g of sodium chloride, 2.9 g of disodium hydrogen phosphate dodecahydrate, 0.2 g of potassium dihydrogen phosphate and 0.2 g of potassium chloride in 1 L of double distilled water.

[0051] Blocking solution: Dissolve 0.1g BSA (bovine serum albumin), 5g glycine, and 5g sucrose in 100mL PBS solution (0.01mol / L pH7.4).

[0052] Sildenafil standard solution: use N,N-dimethylformamide to dilute the sildenafil standard to 1mg / mL for later use; then use 0.01 mol / L PBS buffer to dilute the sildenafil standard to Sildenafil standard solutions with concentrations of 0, 0...

Embodiment 1

[0054] The preparation of embodiment 1 hapten, artificial antigen and antibody

[0055] 1. Synthesis of hapten

[0056] Weigh 0.49 g of mercaptosildenafil, add 0.5 g of anhydrous potassium carbonate, add 3 to 5 ml of DMF after dehydration, add a small amount of potassium iodide as a catalyst, then add 0.18 g of methyl bromoacetate and dissolve in 5 to 10 mL of DMF , after reacting for half an hour, adjust the pH to 1 with concentrated hydrochloric acid, terminate the reaction, and evaporate DMF to dryness, a yellow thick oily substance appears, dissolve the yellow oily substance with 10 mL each of diethyl ether and distilled water, extract at least 3 times with diethyl ether, take the water layer, and Shake the water layer while dripping enhanced sodium hydroxide solution, add dropwise with concentrated NaOH until the white precipitate no longer increases, the pH is about 11, dissolve and extract the precipitate with dichloromethane, extract at least 3 times and take dichlorom...

Embodiment 2

[0074] Embodiment 2 direct competition chemiluminescent ELISA method

[0075] 1. The detection method includes the following steps:

[0076] (1) Coat the mercaptosildenafil antigen with the molecular structure shown in formula III on a microwell plate, dilute the coated antigen to 41.67 ng / mL with coating solution, add 100 μL of coating solution to each well, and keep at 37°C Incubate overnight, pour off the liquid in the well, wash twice with washing solution, and pat dry. Then add 120 μL of blocking solution to each well, incubate at 37°C for 3 hours, pour off the liquid in the well, and dry in an oven at 37°C.

[0077] (2) Dilute the sildenafil standard with 0.01 mol / L PBS buffer solution into sildenafil standard solutions with concentrations of 0, 0.0002, 0.002, 0.02, 0.2, 2, 20, and 200 ng / mL, respectively.

[0078] (3) Take the microwell plate coated in step (1), add 50 μL of sildenafil standard solution of different concentrations in the standard well, add 50 μL of th...

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Abstract

The invention belongs to the technical field of immune detection, and discloses an immunological detection method for sildenafil and its structural analogues, that is, an artificial antigen is prepared with a thiol sildenafil hapten, and then an antibody is prepared, and used for detection of sildenafil Non and its structural analogues, this method overcomes the defects and steps of the existing technology for detecting sildenafil, the maximum detection range of sildenafil is 0.024~1.21 ng / mL, the sensitivity is 0.17 ng / mL, and the detection The detection limit is 0.008 ng / mL, and the recovery rate is 86.0-90.8%. This method is fast and greatly shortens the detection time. Regardless of the influence of the detection personnel's operating proficiency, the entire detection process only takes about 80 minutes to complete, and the detected Lower limit and higher sensitivity.

Description

technical field [0001] The invention relates to the technical field of immunoassay, in particular to an immunoassay method for sildenafil and its structural analogues. Background technique [0002] Sildenafil is a PDE-5 inhibitor used to treat erectile dysfunction in men and is a prescription drug. However, some health food manufacturers illegally add chemical drugs to health food in order to highlight the efficacy of their products, such as illegally adding sildenafil citrate, tadalafil, vadenafil, etc. to anti-fatigue products. The health of consumers poses a serious threat. [0003] The phenomenon that chemical drugs are illegally added to health food for nourishing kidney and strengthening yang and anti-fatigue in the market is shocking. The main ingredients added are: sildenafil, tadalafil, vardenafil, among which illegal addition of sildenafil is more common. In late March 2006, 2 of the 7 types of health food investigated and dealt with added sildenafil, and 5 type...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D487/04G01N33/53
Inventor 沈玉栋邓丽华朱彬杨金易李瑞婷徐振林王弘雷红涛孙远明
Owner SOUTH CHINA AGRI UNIV
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