Strip-based immunochromatographic assay using specific egg yolk antibodies for rapid detection of morphine in urine samples

Using specific egg yolk antibodies (IgY), a strip-based immunochromatographic assay was developed for rapid detection of morphine in urine samples. IgY type antibody against morphine was generated by immunizing chickens with well-characterized monoacetyl morphine–protein conjugate. The antibody was labeled with gold nanoparticles and used as an immunoprobe in the dipstick format for the visual detection of morphine in urine samples. The dipstick was developed using three membranes: an application pad made of glass fiber membrane to hold the tracer, a signal generation test line on nitrocellulose membrane (detection zone) and a cellulose membrane used as an absorption pad. Analytes of interest (morphine and its analogues) added to the sample well, dissolved the labeled antibody (tracer), and the antigen–antibody complex formed was transported by the flow caused by capillary action to the test line. The color signal of test line in proportion to the morphine concentration in urine samples was measured using a detector. The developed dipstick assay format was optimized, showing the average IC50 values of morphine as low as 9.45 ng/mL, the detection range of 1–1000 ng/mL and the lowest detection limit 2.5 ng/mL under optimal conditions of analysis. The correlation between the developed dipstick and ELISA was 0.948 in the analysis of urine samples spiked with morphine. The developed dipstick could be a highly sensitive and convenient tool for rapid detection of opiate drugs in samples with high degree of stability.