(582cs) Sensitive, Rapid, Ampli?cation-Free Immunoassay Using Labeled Bacteriophage, Magnetic Capture Beads and Image Processing Techniques

An amplification-free, imaging immunoassay is designed using labeled bacteriophage M13 as highly detectable “phage tags” and 1 μm magnetic beads as the immunosensing surface. The immunoassay sandwich is formed by a capture antibody immobilized on the surface of magnetic beads, the analyte and then a second anti-analyte antibody coupled to the phage tag. The phage tags are stained specifically with FITC-labeled anti-M13 antibodies for fluorescence detection at the level of a single label. Magnetophoresis is used to transport magnetic beads toward the coverslip surface in two minutes, which reduces the duration of the assay and improves its sensitivity. The magnetic beads and the fluorescently-labeled complexes are imaged at the same focus area using brightfield and fluorescence microscopy, respectively. By comparing the locations of beads and fluorescent complexes, the beads with attached phage tags are selectively identified using automated high-throughput image analysis. The target concentration is directly estimated from the fraction of beads bearing phage tags without the need for experimental standard curves.  This assay can be easily modified to detect a wide range of proteins and pathogens.