Optimization of Two Immunofluorescent Antibodies for the Detection of <Emphasis Type="Italic">Escherichia coli</Emphasis> Using Immunofluorescent Microscopy and Flow Cytometry |
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Authors: | Moira McCarthy Sarah C Culloty |
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Institution: | (1) Department of Zoology, Ecology and Plant Science, University College Cork, The Cooperage, Distillery Fields, North Mall, Cork, Ireland;(2) Department of Zoology, Ecology and Plant Science, University College Cork, Cork Enterprise Centre, Distillery Fields, North Mall, Cork, Ireland |
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Abstract: | Two commercially available fluorescein isothiocyanate (FITC) -conjugated anti-Escherichia coli antibodies, tested for immunofluorescence were assessed for their suitability in screening E. coli using flow cytometry. Staining efficacy was initially tested using immunofluorescent microscopy; and further optimization
was carried out using flow cytometry. Initially, an acetone fixation step was utilized; however, it was determined statistically
that the step could be omitted without impacting the assay and thus reduce the time involved. There was no statistical difference
between the staining proficiency of the two antibodies employed. The percentage staining was quite low, approximately 10%
for the two antibodies, which indicated that both were equally sensitive but ultimately, more specific antibodies are required
for the detection of E. coli. Known proportions of target-E. coli (105, 106, and 107 cells/ml) were mixed with large quantities of non-target bacteria; there was a significant correlation among all
the antibodies at the different bacterial cell concentrations. Therefore, despite the low staining percentage achieved on
the bacterial cultures, there is a representative and comparative level of staining occurring, between samples and between
bacterial strains. |
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Keywords: | |
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