earticle

영어

Recently, Z-domain was expressed as a fusion protein at the outer membrane of E.coli by using autodisplay technology. Because of the binding activity towards the Fc region of IgG, theZ-domain has been used for the orientation control of the antibodies in order to improve the sensitivity of immunoassays. By immobilizing detection antibodies, the E.coli cell with autodisplayed Z-domains could be applied for “direct immunoassay” and this type of immunoassays could achieve hyper sensitive results. For the direct immunoassay, however, the E.coli should be centrifuged repeatedly for washing steps and reagent chaging steps. In this work, magnetic beads were used as a solid support of the E.coli cells with autodisplayed Z-domains in order to perform the direct immunoassay without centrifugation steps. By using super-paramagnetic beads, the centrifugation step was simply replaced to the separation step by using an external magnet. For the immobilization of E.coli cells to the magnetic beads, the surface of magnetic beads was modified with poly-L-lysine to bind the negatively charged E.coli cells. In this work, the changes in surface charge and diameter of the magnetic beads were analyzed during the modification steps to confirm the construction of E.coli -magnetic bead complex. The formation of E.coli-magnetic bead complex was also analyzed by using SEM images. For the feasibility test of magnetic bead based immunoassay, horseradish peroxidase (HRP) was used as a model analyte and a biomarker for inflammatory diseases called C-reactive protein (CRP) was used for demonstration of an application to medical diagnosis.