Immunoassay method for nano-colloidal gold marker of tadalafil and analogs thereof

A technology of tadalafil and nanocolloids, which is applied in the field of nanocolloidal gold-labeled immunoassays of tadalafil and its analogues, achieving the effects of strong reliability, stable signals and stable methods

Active Publication Date: 2015-04-08
SHAOGUAN COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0015] The purpose of the present invention is to overcome the deficiency that the existing immunoassay technology can only detect one target substance, and through targeted antigen design, immunized animals can induce the production of not only tadalafil, but also all potential tadalafil The general antibody of non-structural analogues provides a nano-colloidal gold-labeled immunoassay method capable of tadalafil and its analogues, which is applied in the field of food and drug safety testing, changing the traditional illegal addition of multiple steps Lock detection problem, to achieve one-step rapid detection of illegal addition of tadalafil and its analogues

Method used

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  • Immunoassay method for nano-colloidal gold marker of tadalafil and analogs thereof
  • Immunoassay method for nano-colloidal gold marker of tadalafil and analogs thereof
  • Immunoassay method for nano-colloidal gold marker of tadalafil and analogs thereof

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Experimental program
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Effect test

Embodiment 1

[0046] Highlight the common group of tadalafil and its analogs artificial immune antigen synthesis method: (1) Weigh 2mg of tadalafil and dissolve it in 2mL of anhydrous pyridine solution, add 5mg of carboxymethyl hydroxylamine, shake at room temperature overnight . The next day, evaporate pyridine to dryness with a rotary evaporator, dissolve the reactant with 5 mL of sodium bicarbonate solution, and discard the precipitate. Then adjust the pH value to 3.0 with hydrochloric acid, and extract with ethyl acetate. The aqueous phase was discarded. The collected ethyl acetate was evaporated to dryness with a rotary evaporator to obtain tadalafil oximide. (2) Using the modified active ester method to synthesize the immune antigen, dissolve 0.5 mg of hapten in 0.5 mL of anhydrous methanol, then add 0.05 g of carbodiimide (EDC) and 0.01 g of N-hydroxysuccinimide (NHS), at room temperature After overnight reaction, the precipitate was separated by centrifugation, and the supernatan...

Embodiment 2

[0048] Then add 400 μg of the isolated and purified monoclonal antibody to the pre-prepared 40nm colloidal gold solution (pH 8.2), react at room temperature for 15 minutes, add polyethylene glycol (PEG) to a final concentration of 1%, and continue stirring for 15 minutes . Centrifuge at 8000r / min for 30 minutes, suck off the supernatant, and the resulting precipitate is the purified gold-labeled antibody. Spray the gold-labeled antibody conjugate on the glass cellulose membrane, spray the tadalafil and its analogue-OVA conjugate on the NC membrane as a test line, and spray goat anti-rabbit IgG on the NC membrane as a test line. Quality control line, and then paste the sample absorbent pad, glass fiber sprayed with colloidal gold antibody, NC film sprayed with test line and quality control line, and water-absorbing glass fiber on a plastic bottom plate (5mm×6mm) in sequence.

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Abstract

The invention discloses an immunoassay method for a nano-colloidal gold marker of tadalafil and analogs thereof. The determination method comprises the following steps: coupling oximate tadalafil with bovine serum albumin by adopting an activated ester method, synthesizing artificial immunity antigen which is used for immunizing animals, and preparing specific tadalafil and analogs thereof; in addition, coupling the oximate tadalafil with egg albumin to be used for construct an immunity detection method; separating, purifying and labeling the tadalafil and analogs thereof to the nano-colloidal gold, and then respectively curing the tadalafil and analogs detection antibodies thereof and goat-anti-rabbit IgG on a carrier, and adding a detection antigen into a detection solution. According to the method, the lowest detection limit of the tadalafil and analogs thereof legally added into health products and Chinese patent medicines is 10mu g / kg, and the fast detection can be completed within 5 minutes. The method is stable, fast and accurate, and suitable for one-step fast detection on legally added medicines.

Description

technical field [0001] The invention relates to the technical field of food and drug safety detection, in particular to a nano colloidal gold-labeled immunoassay method for tadalafil and its analogues. Background technique [0002] Functional foods and Chinese patent medicines produced from plant materials are considered safe and have no side effects, and have been widely favored by consumers all over the world in recent years. Among them, natural aphrodisiacs is the most active product type. However, the illegal addition of phosphodiesterase-5 inhibitors (PDE-5 inhibitors) in natural aphrodisiac products to increase the aphrodisiac effect to promote sales and seek illegal benefits has become a global problem. [0003] In the early 1990s, a PDE-5 inhibitor drug used by Pfizer for the treatment of coronary heart disease was found to be effective in the treatment of impotence in phase II and III clinical trials. No". Afterwards, the other two PDE-5 inhibitor drugs, vardenaf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/02G01N33/15G01N33/68
Inventor 郭杰标
Owner SHAOGUAN COLLEGE
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