Title:
Capture antibody targeted fluorescence in situ hybridization (CAT-FISH): dual labeling allows for increased specificity in complex samples.

Abstract:
Pathogen detection using biosensors is commonly limited due to the need for sensitivity and specificity in detecting targets within mixed populations. These issues were addressed through development of a dual labeling method that allows for both liquid-phase fluorescence in situ hybridization (FISH) and capture antibody targeted detection (CAT-FISH). CAT-FISH was developed using Escherichia coli O157:H7 and Staphylococcus aureus as representative bacteria, and processing techniques were evaluated with regard to FISH intensities and antibody recognition. The alternative fixative solution, methacarn, proved to be superior to standard solid-phase paraformaldehyde fixation procedures, allowing both FISH labeling and antibody recognition. CAT-FISH treated cells were successfully labeled with FISH probes, captured by immunomagnetic separation using fluorescent cytometric array beads, and detected using a cytometric array biosensor. CAT-FISH treated cells were detectable with LODs comparable to the standard antibody-based technique, (~10(3)cells/ml in PBS), and the technique was also successfully applied to two complex matrices. Although immunomagnetic capture and detection using cytometric arrays were demonstrated, CAT-FISH is readily applicable to any antibody-based fluorescence detection platform, and further optimization for sensitivity is possible via inclusion of fluorescently tagged antibodies. Since the confidence level needed for positive identification of a detected target is often paramount, CAT-FISH was developed to allow two separate levels of specificity, namely nucleic acid and protein signatures. With proper selection of FISH probes and capture antibodies, CAT-FISH may be used to provide rapid detection of target pathogens from within complex matrices with high levels of confidence.