Numerous techniques are available to detect viruses, such as cell culture-based diagnostics and polymerase chain reaction-based techniques, most of which are quite time consuming. Immunoassay using monoclonal antibodies is also a standard tool for detection of disease-causing organisms. We propose here a paramagnetic bead-based imaging immunoassay for the rapid and sensitive detection of viruses. We show proof-of-concept experiments detecting MS2 phage viruses using a novel M13 bacteriophage label as the analytical reporter with low-nonspecific binding. The M13 labels consist of biotinylated M13 phage attached to biotinylated anti-MS2 antibodies through avidin. These M13 phage reporters are highly detectable using fluorescence microscopy, at the single phage level when labeled with fluorescent dyes such as Alexa-555. In this assay, MS2 phage are enriched on the magnetic beads and sandwiched by the M13 phage reporter. Then, every bead is magnetophoresed to the coverslip surface using a permanent magnet. After microscopic imaging, the beads bearing M13 phage reporters are selectively identified using automated high-throughput image analysis. By counting individual magnetic beads, the sensitivity and speed of the proposed assay can be improved, compared to ordinary colorimetric immunoassays.

Sensitive, rapid, amplification-free immunoassay employing fluorescently labeled bacteriophage, magnetic capture beads, and image processing technology

247th National Meeting (2014)

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