Rapid and simple quantitation of methamphetamine by using a homogeneous time-resolved fluoroimmunoassay based on fluorescence resonance energy transfer from europium to Cy5

A rapid and simple homogeneous time-resolved fluoroimmunoassay based on fluorescence resonance energy transfer from europium (Eu) to cyanine dye (Cy5) has been developed for the quantitation of methamphetamine. In this assay, Eu chelate was labeled to a conjugate of methamphetamine and bovine serum albumin (MA-BSA), as an energy donor, and Cy5 was labeled to anti-MA as an energy acceptor. The close proximity between the two labels in the immunocomplex permits energy transfer from the excited Eu(3+) donor. Therefore, by measuring the sensitized emission of Cy5 with the time-resolved assay, immunocomplex of MA-BSA and anti-MA can be measured in the homogeneous solution without separation steps within 30 min. By a competitive immunoassay, MA could be assayed in the range 0.1-1,000 ng/mL. The intra-assay variations were 5.4-14.8% at 5 different concentrations. When urine or serum samples were examined, the quenching of Eu fluorescence was observed, but the acceptor-to-donor ratio constantly depended upon the dilution of samples. Twenty urine samples were assayed, and the data showed a good correlation to those obtained by gas chromatography (r = 0.94). The homogeneous assay using Eu-Cy5 energy transfer is time-saving without any washing procedures and is suitable for screening drugs that are commonly abused.