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Sildenafil and its analogue immunodetection method

An immunological detection method, the technology of sildenafil, which is applied in the field of immunological detection of sildenafil and its structural analogues, can solve the problems of hindering popularization and application, long detection time, and high degree of instrumentation, and achieve shortened detection time, Detect fast, sensitive effects

Active Publication Date: 2016-02-24
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Sildenafil and its analogue immunodetection method
  • Sildenafil and its analogue immunodetection method
  • Sildenafil and its analogue immunodetection method

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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 times concentrated washing solution: pH7.40.4mol / L phosphate buffer solution containing 0.5% Tween20 by volume fraction, diluted to 1 times 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 / LpH7.4).

[0052] Sildenafil standard solution: use N,N-dimethylformamide to dilute the sildenafil standard to 1mg / mL for later use; then dilute the sildenafil standard to a concentration of 0.01mol / LPBS buffer Sildenafil standard solution o...

Embodiment 1

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

[0055] 1. Synthesis of hapten

[0056] Weigh 0.49g of mercaptosildenafil, add 0.5g of anhydrous potassium carbonate, add 3-5ml of DMF after dehydration, add a small amount of potassium iodide as catalyst, then add 0.18g of methyl bromoacetate and dissolve in 5-10mL 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 dichloromethane The...

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 41.67ng / 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 0.01mol / LPBS buffer solution to obtain sildenafil standard solutions with concentrations of 0, 0.0002, 0.002, 0.02, 0.2, 2, 20, and 200ng / mL.

[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 the sample to...

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Abstract

The invention belongs to the technical field of immunodetection and discloses a sildenafil and its analogue immunodetection method. An artificial antigen is prepared from a mercapto-sildenafil hapten, and an antibody is prepared by the artificial antigen and is used for detecting sildenafil and its analogue. The method solves the problems of the existing sildenafil detection technology, has the largest sildenafil detection range of 0.024-1.21ng / mL, sensitivity of 0.17ng / mL, a detection limit of 0.008ng / mL and a recovery rate of 86.0-90.8%, has a fast detection rate, greatly shortens detection time, is not influenced by an operator proficiency degree, can be finished in about 80min, and has a low detection limit and high 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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IPC IPC(8): G01N33/02G01N33/53
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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