Artificial antigen of tadalafil and analogues thereof, antibody and ELISA kit thereof

A technology of tadalafil, artificial antigen, applied in immunoglobulin, measuring device, animal/human protein, etc.

Active Publication Date: 2016-04-13
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to overcome the above-mentioned defects in the detection method of tadalafil in the prior art, and provide a kind of artificial hapten of tadalafil

Method used

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  • Artificial antigen of tadalafil and analogues thereof, antibody and ELISA kit thereof
  • Artificial antigen of tadalafil and analogues thereof, antibody and ELISA kit thereof
  • Artificial antigen of tadalafil and analogues thereof, antibody and ELISA kit thereof

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preparation example Construction

[0067] The preparation method of reagent used in the embodiment is as follows:

[0068] Coating solution: obtained by dissolving 1.69g of sodium carbonate and 2.95g of sodium bicarbonate in 1L of double distilled water.

[0069] Blocking solution: Dissolve 0.1g BSA (bovine serum albumin), 5g glycine and 5g sucrose in 100mL PBS solution (0.01mol / LpH7.4).

Embodiment 1

[0070] The synthesis of embodiment 1 hapten

[0071] Weigh 78mg (0.2mM) of aminotadalafil and 18mg (0.2mM) of maleic anhydride into a round-bottomed flask, and dissolve the mixture with a small amount of anhydrous DMF, reflux at 60°C for 2h, developer methanol: chloroform 1 : 10 for reaction monitoring. After the reaction, DMF was removed by vacuum rotary evaporation at 75°C. The residue was dissolved with a small amount of ethyl acetate, recrystallized by adding n-hexane dropwise, filtered and dried, and the obtained light yellow powder was tadalafil- Maleic anhydride hapten.

Embodiment 2

[0072] Synthesis of embodiment 2 artificial antigen

[0073] Weigh the tadalafil-maleic anhydride hapten 0.02mM, NHS3.22mg (0.028mM) and DCC5.78mM (0.028mM) obtained in Example 1, dissolve them in 200μl DMF, stir overnight at 4°C, and dissolve the reaction solution Centrifuge at 8000r / min for 10min, take the supernatant to get solution A; weigh 13.6mg BSA (or 4.5mgKLH, 9mgOVA) and dissolve it in 4mL PBS buffer, stir at 4°C, add solution A dropwise to the BSA solution (or KLH solution or OVA solution), and react overnight in the dark. The reaction solution was put into a dialysis bag and dialyzed against PBS for three days. Ultraviolet scanning confirmed that the coupling was successful, and the tadalafil-maleic anhydride antigen was obtained.

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Abstract

The invention discloses an artificial antigen of tadalafil and analogues thereof, an antibody and an ELISA kit thereof. The kit comprises an enzyme label plate coated with a tadalafil antigen, a tadalafil antibody operating fluid, a horseradish peroxidase labelled secondary antibody, a tadalafil standard-substance operating fluid, a substrate liquid, a substrate buffer, a reaction stopping liquid and a condensation washing liquid. The invention also discloses a method for detecting tadalafil and analogues thereof in health-care food by utilizing the kit. The provided kit for detecting tadalafil and analogues thereof employs an indirect competitive enzyme-linked immunosorbent analysis technology, possesses the maximum detection scope of 0.12-7.45 ng / mL, the half-inhibitory concentration of 0.933 ng / ml, the detection limit of 0.095 ng / mL and the recovery rate of 83.0-119.2%, is low in detection limit, high in sensitivity, good in stability and low in cost, is extremely suitable for primary screening on a large amount of samples, and possesses practical significance on rapid detection on illegally-added tadalafil in health-care food.

Description

technical field [0001] The invention relates to the technical field of food safety immunoassay, and more specifically relates to an artificial antigen of tadalafil and structural analogues thereof, an antibody and an enzyme-linked immunoassay kit thereof. Background technique [0002] Tadalafil (tadalafil) is the second-generation FDA-approved prescription drug for the treatment of male functional erectile dysfunction following sildenafil. Like sildenafil, it is a PDE5 inhibitor. Compared with sildenafil No, tadalafil has the advantage of long duration of drug effect. [0003] Tadalafil, like sildenafil, is a non-selective PDE-5, which inhibits PDE6 while inhibiting PDE5, thereby causing visual and hearing damage. And if it is taken in excess, it will cause headache, indigestion, color resolution decline and other adverse reactions. When taken together with nitrate drugs, it will lead to severe hypotension and even sudden death. Therefore, PDE-5 drugs must be prescribed by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D471/14G01N33/543C07K14/765C07K14/77C07K14/795C07K16/44
CPCC07D471/14C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/543
Inventor 沈玉栋邓丽华杨金易徐振林华彦涛王弘肖治理雷红涛孙远明
Owner SOUTH CHINA AGRI UNIV
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