Hirotaka Miyachi

High-sensitive flow-based kinetic exclusion assay for okadaic acid assessment in shellfish samples.

Episodes of shellfish contamination with okadaic acid (OA) are a human health threat that is causing increasing concern. As a way to overcome the shortcomings involved in the reference methods of analysis set by legislations, alternative procedures are envisaged. This paper describes the development of different immunosensors for the analysis of OA, focusing on the comparison of their sensitivity, precision, ease of use and sample matrix effects. Initially, a surface plasmon resonance (SPR)-based immunosensor was developed, which enabled the quantification of the toxin in mussel samples at concentrations in the range of the 160 microg kg(-1) European regulatory limit with good percentages of recovery. Nevertheless, calibration curves with spiked mussel samples showed that matrix effects could not be neglected. Alternatively, a flow-immunosensing system based on kinetic exclusion measurements was developed achieving the theoretical lowest limit of detection enabled by the affinity of the anti-OA antibody (IC(70)=0.03 microg L(-1) in the assay solution). This highly sensitive automated system allows rapid and reliable OA quantification, with no significant matrix effects for the analysis of spiked mussel and scallop samples. Performance features such as high sensitivity and precision, low limits of detection and simplicity of the analysis protocol, shows the biosensing-systems based on kinetic exclusion measurements for toxin detection in shellfish samples as highly performing tools for rapid and continuous screening.

Simultaneous detection of DNA-binding proteins using exo-Taq-based reaction

The DNA-binding protein (DBP) has a wide range of roles such as those in DNA repair, recombination, and gene expression. Recently, a microarray-based method has been developed for the high-throughput analysis of DNA-protein interactions. However, to maximize the advantages of this method, the detection process should be improved so that the method can be applied to many proteins without the use of antibody or sample labeling. Previously, we presented a primary report on the detection of DBP, which is applicable to the microarray format. The system consists of three steps: first, the target DBP in the sample solution is incubated with a probe DNA; second, the probe is digested with Exo (Exonuclease) III; finally, the probe is extended withTaq DNA polymerase using fluorescent dye-labeled dUTP as a substrate. The binding DBP protects the probe from digestion by Exo III. Therefore, only the DBP-bound probe allows the following extension. In this study, the simultaneous detection of multiple DBPs was examined, and then the DBPs were analyzed using a crude extract of the cultured cells to demonstrate the general applicability of the method. Our method can be applied to many DBPs using the same procedure and components, whereas in the antibody-based method, the same number of antibodies as DBPs is needed to detect target DBPs in ELISA (enzyme-linked immunosorbent assay). These results suggest that our method is useful for the high-throughput detection of DBPs in the microarray format.