earticle

영어

Microsphere-based flow cytometric assay is commonly used immune assay for the analysis of mixed analytes in solution. It is based on antibodies conjugated fluorescence micro beads and uses the flow cytometer for detection. In this study, we developed a new flow cytometric assay by using fluorescence and autodisplaying proteins co-expressing E.coli. The autodisplay technology is one of the surface display methods for proteins or peptides at the E.coli outer membrane. In the autodisplay technology, proteins are expressed as recombinant proteins in a poly protein precursor for the autotransporter secretion pathway and automatically aligned on the outer membrane surface[1]. These aligned proteins can be used a affinity layer for capturing the analyte[2]. The autotranspoter vector and fluorescence protein expressing vector were transfected into the UT5600(DE3)　E.coli. For the fluorescence proteins, eGFP and tdTomato were used and Z-domains were autodisplayed on the surface of E.coli[3]. APC conjugated antibodies were treated to measure the activity of autodisplayed Z-domains and the activity Z-domain and expressed fluorescence proteins was measured by using a fluorescnece photometer and a flow cytometer. the result of fluorescence photometer shows that intracellular expressed eGFP and tdTomato have a excitation/emission wavelength of 488/507 and 554/581 nm. The flow cytometer result shows a clear increasement of fluorescence intensity after APC conjugated antibodies treatment.