An immunoassay for multiple pde-5 inhibitor drugs

A technology of PDE-5 and inhibitors, applied in the field of immunoassay, to achieve broad application prospects, rapid detection, and better sensitivity

Active Publication Date: 2020-08-11
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is to overcome the defects and deficiencies of the multiple PDE-5 inhibitor drug detection techniques in the above-mentioned prior art, and provide a method for targeting multiple PDE-5 drugs (including sildenafil, vardenafil, etc.) , reddenafil, mironafil and their structural analogues) haptens, artificial antigens, antibodies, thereby constructing an immunoassay method for a variety of PDE-5 inhibitor drugs

Method used

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  • An immunoassay for multiple pde-5 inhibitor drugs
  • An immunoassay for multiple pde-5 inhibitor drugs
  • An immunoassay for multiple pde-5 inhibitor drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The preparation of embodiment 1 immunogen / coating original

[0049] 1. The present invention has synthesized a novel small molecule compound by analyzing the spatial structure of several PDE-5 inhibitor drugs, as shown in formula (I), it can be used as a hapten for preparing artificial antigens and antibodies for detecting multiple A PDE-5 inhibitor drug.

[0050]

[0051] 2. In the preparation method of the immunogen used in the present invention and the coating original, the difference lies in the use type of the carrier protein, the immunogen carrier protein mainly adopts bovine serum albumin (BSA), and the described coating former carrier protein mainly adopts egg white Protein (OVA), the coupling method used is active ester method. Take the preparation method of the following immunogen as an example.

[0052] (1) Active ester method: Dissolve 5 mg (0.015 mol) of the hapten Norneovardenafil in 0.3 mL of DMF, add 4.78 mg of DCC (0.023 mol), 2.65 mg of NHS (0.023...

Embodiment 2

[0058] Example 2 Antibody Preparation and Identification

[0059] The prepared immunogen was evenly emulsified with an equal amount of immune adjuvant (incomplete Freund's adjuvant was used for the first immunization, and incomplete Freund's adjuvant was used for subsequent booster immunizations), and animals were immunized. New Zealand white rabbits weighing 2.5 to 3 kg were immunized by subcutaneous injections on the back, subcutaneous injections in various parts, leg muscles, and ear veins. The second immunization was performed 4 weeks later, and the immunization was added every 3 weeks thereafter. One week after the fourth booster immunization, blood was collected from the ear vein, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises, the ear vein is used to boost the immunization. One week later, blood was collected from the heart, bathed in water for 0.5-1 hour, centrifuged at 10,000°C for 15 minutes at 4°C, and the supernata...

Embodiment 3

[0061] The specificity and sensitivity of embodiment 3 antibody

[0062] 1. According to the above effect, use antiserum to draw enzyme-linked immunoassay (ELISA) standard curve; use phosphate Tween buffer (PBST, 0.1mol / L, pH=7.4) as the diluent of all samples: 50 μL of serial concentrations Drug standards and 50 μL of multispecific antibodies with appropriate dilution times were added to a 96-well microplate plate, and the absorbance (OD) was measured by a microplate analyzer after the competition reaction. Take the OD value as the vertical axis, and the corresponding logarithm value of the standard substance concentration as the horizontal axis, and use the four parameters of origin9.1 software to perform curve fitting on the function: y=(A-D) / [1+(X / C)B]+D

[0063] Among them, A and D represent the minimum and maximum absorbance values ​​(OD) of the drug concentration, respectively, and C is the midpoint concentration. When the standard concentration is equal to C, the OD va...

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Abstract

The invention discloses an immunodetection method for various PDE-5 inhibitor drugs. A novel micromolecule structure is synthesized at first, and is used as hapten to prepare immunogen and coating antigen, therefore, an antibody is prepared, and the immunodetection method for various PDE-5 inhibitor drugs is constructed. The antibody and the immunodetection method can be used for detecting various PDE-5 inhibitor drugs, various PDE-5 inhibitor drug residues in samples such as health food can be detected simultaneously for the first time, the valence of the antibody can reach 1: 500,000, and the immunodetection method for various PDE-5 inhibitor drugs has high detection specificity on PDE-5 inhibitor drugs such as sildenafil, vardenafil, hongdenafil and mirodenafil and analogues thereof, and is good in sensitivity and high in accuracy; and moreover, the detection speed is high, and therefore, the immunodetection method for various PDE-5 inhibitor drugs is used for rapidly detecting residual PDE-5 inhibitors in food, and has wide application prospects.

Description

technical field [0001] The invention belongs to the technical field of immunoassay. More specifically, it relates to an immunoassay method for various PDE-5 inhibitor drugs (including sildenafil, vardenafil, reddenafil, mironafil and structural analogues thereof). Background technique [0002] Drugs such as sildenafil, vardenafil, reddenafil, and mironafil are PDE-5 inhibitors, which are used to treat male erectile dysfunction and are prescription drugs. 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, waldenafil, red field to anti-fatigue products Nafei, etc. pose a serious threat to the health of consumers. At present, there are many health foods in the market that claim to have functions such as invigorating the kidney and strengthening yang, anti-fatigue, and enhancing immunity. The illegal addition of chemicals is shocking. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D487/04C07K14/765C07K14/77C07K14/795C07K16/44G01N33/535
CPCC07D487/04C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/535
Inventor 沈玉栋华彦涛徐振林杨金易王弘孙远明雷红涛
Owner SOUTH CHINA AGRI UNIV
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