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
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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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