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Chapter 8 : Viral Antigen Detection

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Viral Antigen Detection, Page 1 of 2

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Abstract:

Laboratorians continually seek methodologies that yield accurate results in a timely fashion, are cost effective, and require less technical expertise. Diagnosis of viral infections via viral antigen detection methods such as immunofluorescence (FA), immunochromatography (lateral flow) (IC), and enzyme immunoassays (EIA) offer many of these attractive features and are useful for direct detection of viral antigens in an array of clinical specimens and for identification of cultivated viruses. Whether the detection method is FA, rapid IC, or EIA, detection of antigens of the common respiratory viruses (i.e., adenovirus; influenza virus [Flu] A and B; parainfluenza virus [PIV] −1, −2, and −3 and respiratory syncytial virus [RSV]), has been shown to be more useful in patient management than either traditional virus isolation (1, 2, 3) or viral detection in rapid culture using centrifugation-enhanced inoculation (4). There is considerable variability in the sensitivity, specificity, technical considerations, and turnaround time among the various methods, and each method may perform differently depending on the viral target. This chapter deals with principles of FA, IC, and EIA and their contemporary applications in viral antigen detection.

Citation: Leland D, Relich R. 2016. Viral Antigen Detection, p 95-104. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch8
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Figures

Image of FIGURE 1
FIGURE 1

DFA staining with FITC-labeled monoclonal antibodies of virus-infected cells, 400x. (A) influenza B–infected cells, (B) herpes simplex–infected cells, and (C) respiratory syncytial virus–infected cells. Green fluorescence indicates antigen detected. Red fluorescence is background material stained with Evans Blue counterstain included in the stain preparation. Photos courtesy of Indiana Pathology Images.

Citation: Leland D, Relich R. 2016. Viral Antigen Detection, p 95-104. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch8
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Image of FIGURE 2
FIGURE 2

Diagnostic Hybrids (DHI) proficiency panel samples stained with the DHI D FastPoint L-DFA method, 200x. (A) parainfluenza (yellow) and adenovirus (green), (B) human metapneumovirus (green), and (C) influenza A (yellow) and B (green). Red fluorescence is uninfected cells and debris stained with propidium iodide and Evans Blue counterstain. Photos courtesy of Indiana Pathology Images.

Citation: Leland D, Relich R. 2016. Viral Antigen Detection, p 95-104. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch8
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Image of FIGURE 3
FIGURE 3

IC (lateral flow) testing mechanism. (A) Viral antigen in the specimen is added to the sample pad of a nitrocellulose strip; labeled antiviral antibodies are bound nonspecifically on the sample pad. The nitrocellulose strip also includes a test area of unlabeled antiviral antibodies and a control area of unlabeled animal antihuman IgG; (B) Labeled antiviral antibodies on the sample pad bind to viral antigen in the sample, and the complexes migrate along the strip; (C) Migration continues; (D) Viral antigens, in complex with labeled antiviral antibodies, are recognized and captured by the unlabeled antiviral antibodies in the test area of the strip, forming a visible line; excess labeled antiviral antibodies continue to migrate and are captured at the control line by anti-IgG. This control ensures that the specimen migrated the entire length of the strip and that the strip is functioning properly.

Citation: Leland D, Relich R. 2016. Viral Antigen Detection, p 95-104. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch8
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Image of FIGURE 4
FIGURE 4

Membrane EIA testing mechanism and gross appearance. Testing mechanism: (A) viral antigen in specimen is nonspecifically adhered to the membrane by filtration through a focusing device; (B) enzyme-labeled antiviral antibody is added and binds to viral antigen present on the membrane; (C) a substrate solution is added and changes color when acted upon by the enzyme. Gross appearance: (D) a test area is defined within the cassette—this is colorless at the beginning of the assay; (E) the test area changes color due to the action of the enzyme on the substrate solution. The colored dot in the center of the test area is a built-in control to ensure proper function of the device.

Citation: Leland D, Relich R. 2016. Viral Antigen Detection, p 95-104. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch8
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Image of FIGURE 5
FIGURE 5

Immunohistochemical staining reveals the presence of viral antigens (dark brown areas) in tissue-thin sections. (A) Adenovirus-infected hepatocyte (center of image) demonstrating both nuclear and cytoplasmic staining. Scattered throughout the tissue are coarse, refractile, and brown-staining bile pigment granules; (B) BK virus–infected renal tubular epithelial cells demonstrating nuclear staining; (C) Cytomegalovirus-infected lung tissue reveals viral antigens in both nuclear and cytoplasmic compartments. Original magnification 400x. Photos courtesy of Indiana Pathology Images.

Citation: Leland D, Relich R. 2016. Viral Antigen Detection, p 95-104. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch8
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Tables

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TABLE 1

Characteristics of Viral Antigen Detection Methods

Citation: Leland D, Relich R. 2016. Viral Antigen Detection, p 95-104. In Loeffelholz M, Hodinka R, Young S, Pinsky B (ed), Clinical Virology Manual, Fifth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555819156.ch8

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