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Chapter 6 : Immunofluorescence

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Immunofluorescence, Page 1 of 2

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Abstract:

The early immunofluorescence assays (FA) used noncommercial preparations of polyclonal antisera directed against the target virus and a secondary reagent coupled with either rhodamine or fluorescein. Now, more than 60 years after the first report, immunofluorescence remains one of the primary technologies used by diagnostic virology laboratories. A wide variety of fluorochromes have been chemically modified so that they can be directly coupled to proteins. The excitation-emission spectra of one of the most commonly used fluorochromes, fluorescein isothiocyanate (FITC), are illustrated. Direct detection of viruses in clinical specimens has an advantage over culture isolation due to more timely reporting of results. Immunofluorescence has an advantage over enzyme immunoassays, as specimen adequacy can be determined and more than one virus can be detected. Specimens that are considered most appropriate for analysis by immunofluorescence include nasopharyngeal swabs, aspirates, or washes, bronchoalveolar lavage samples, swabs (including the recently introduced flocked swab specimen processing technology) or scrapings from vesicular lesions, tissue biopsy specimens (e.g., lung, liver, and brain), blood leukocytes, conjunctival cells, corneal scrapings, and urine sediment. Quality assurance establishes standard operating procedures for all aspects of testing, including specimen collection and processing, assay protocols, validation requirements, and quality control (QC) for all tests performed in a laboratory. Critical components, such as swabs, buffers, and transport media, etc., should also be included, along with package inserts from the commercial kits used in the laboratory.

Citation: Schutzbank T, McGuire R, Scholl D. 2009. Immunofluorescence, p 77-88. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch6
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Figures

Image of FIGURE 1
FIGURE 1

Excitation and emission spectra of FITC.

Citation: Schutzbank T, McGuire R, Scholl D. 2009. Immunofluorescence, p 77-88. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch6
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Image of FIGURE 2
FIGURE 2

Optical system of an epifluorescence upright microscope. See text for details. Courtesy of Douglas Kline, Department of Biological Sciences, Kent State University, Kent, OH.

Citation: Schutzbank T, McGuire R, Scholl D. 2009. Immunofluorescence, p 77-88. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch6
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Image of FIGURE 3
FIGURE 3

Schematic representation of IFA (A) and DFA (B).

Citation: Schutzbank T, McGuire R, Scholl D. 2009. Immunofluorescence, p 77-88. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch6
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Image of FIGURE 4
FIGURE 4

Analysis of respiratory specimens known to be negative (A) and positive (B) for influenza A virus using FITC-labeled influenza-specific MAb reagents. Images 1 and 3: primary image capture using Evans blue and FITC filters, respectively; images 2 and 4: software algorithm analysis of images 1 and 3. The boxed cells in images 2 and 4 were analyzed for colocalized fluorescence signal. Seven influenza-positive cells were identified (panel B, image 4), thus indicating a “positive” result.

Citation: Schutzbank T, McGuire R, Scholl D. 2009. Immunofluorescence, p 77-88. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch6
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Tables

Generic image for table
TABLE 1

Performance characteristics of DFAs versus viral culture

Citation: Schutzbank T, McGuire R, Scholl D. 2009. Immunofluorescence, p 77-88. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch6
Generic image for table
TABLE 2

Problems most frequently encountered with FA a

Citation: Schutzbank T, McGuire R, Scholl D. 2009. Immunofluorescence, p 77-88. In Specter S, Hodinka R, Young S, Wiedbrauk D (ed), Clinical Virology Manual, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815974.ch6

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