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ABSTRACT
Two novel and high-throughput enzyme-linked immunosorbent assays (ELISA) were developed for determining tonalide in human blood samples. For establishing the proposed methods, the tonalide hapten and immunogen primarily were prepared. After the immunization, the polyclonal antibody and biotinylated polyclonal antibody were obtained. The biotin-streptavidin system and polyamidoamine-gold nanoparticle conjugation were applied respectively to establish thebiotin-streptavidin-ELISA and Au-polyamidoamine-ELISA. For reducing the background interference, some factors and procedures were also discussed and optimized. Under the optimal conditions, the IC10 of biotin-streptavidin-ELISA was 0.046 µg/L and the IC10 of Au-polyamidoamine-ELISA was 0.016 µg/L. The Au-polyamidoamine-ELISA was used to determine tonalide in human blood and the recovery values and coefficients of variation were acceptable. In this study, tonalide was detected in 92.2% of the samples; the median and the maximum values were 0.62 and 1.76 µg/L, respectively. In general, due to the specificity of the antibody and novel nanoprobe design, this Au-polyamidoamine-ELISA simplified the sample preparation and provided a useful and potential way for trace determination of tonalide. The results also suggested that tonalide concentrations were not significantly relative to gender, but the high concentrations of tonalide in human blood should encourage further toxicological studies.