Optimization of two immunofluorescent antibodies for the detection of Escherichia coli using immunofluorescent microscopy and flow cytometry
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Published on: March 5, 2019
Moira McCarthy1, Sarah C Culloty
1Department of Zoology, Ecology and Plant Science, University College Cork, The Cooperage, Distillery Fields, North Mall, Cork, Ireland. moira.mccarthy@ucc.ie
Insights
This study evaluated fluorescein isothiocyanate (FITC)-conjugated antibodies for detecting Escherichia coli (E. coli) via flow cytometry. While antibodies showed comparable sensitivity, higher specificity is needed for reliable E. coli screening.
Area of Science:
- Microbiology
- Immunology
- Biotechnology
Background:
- Flow cytometry is a powerful tool for bacterial analysis.
- Accurate detection of Escherichia coli (E. coli) is crucial in various fields.
- Fluorescein isothiocyanate (FITC)-conjugated antibodies are commonly used for bacterial identification.
Purpose of the Study:
- To assess the suitability of two commercial FITC-conjugated anti-E. coli antibodies for E. coli screening using flow cytometry.
- To optimize the staining protocol for improved efficiency.
- To evaluate the specificity and sensitivity of the selected antibodies.
Main Methods:
- Immunofluorescent microscopy was used for initial staining efficacy testing.
- Flow cytometry was employed for further optimization and analysis.
- Acetone fixation step was evaluated for its necessity, with statistical analysis performed.
- Known concentrations of E. coli were mixed with non-target bacteria for specificity testing.
Main Results:
- No statistically significant difference was observed between the staining proficiency of the two tested antibodies.
- The percentage of E. coli staining was low (approximately 10%), indicating good sensitivity but a need for greater specificity.
- The omission of the acetone fixation step did not statistically impact the assay results, reducing processing time.
- A significant correlation was found between antibody staining and bacterial cell concentrations (10⁵, 10⁶, 10⁷ cells/ml) when E. coli was mixed with non-target bacteria.
Conclusions:
- The evaluated FITC-conjugated anti-E. coli antibodies demonstrate comparable sensitivity but limited specificity for E. coli detection via flow cytometry.
- Optimization by omitting the acetone fixation step enhances assay efficiency without compromising results.
- Further development of more specific antibodies is recommended for improved E. coli screening.
- Despite low staining percentages, the assay provides representative and comparative staining, suitable for relative quantification and sample comparison.
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.
