Principles of Virology, Volume 1. Jane Flint

Principles of Virology, Volume 1 - Jane Flint


Скачать книгу
such as a fluorescent dye or an enzyme (Fig. 2.12). A more sensitive approach is indirect immunostaining, in which a second antibody is coupled to the indicator. The second antibody recognizes a common region on the virus-specific antibody.

      Multiple second-antibody molecules bind to the first antibody, resulting in an increased signal from the indicator compared with that obtained with direct immunostaining. Furthermore, a single indicator-coupled second antibody can be used in many assays, avoiding the need to purify and couple an indicator to multiple first antibodies.

      In practice, virus-infected cells (unfixed or fixed with acetone, methanol, or paraformaldehyde) are incubated with polyclonal or monoclonal antibodies (Box 2.7) directed against viral antigen. Excess antibody is washed away, and in direct immunostaining, cells are examined by microscopy. For indirect immunostaining, the second antibody is added before examination of the cells by microscopy. Commonly used indicators fluoresce on exposure to UV light. Filters are placed between the specimen and the eyepiece to remove blue and UV light so that the field is dark, except for cells to which the antibody has bound, which emit light of distinct colors (Fig. 2.12). Today’s optics are much better at keeping the wavelengths separated, permitting the use of different colors to detect various components in the same specimen. Antibodies can also be coupled to molecules other than fluorescent indicators, including enzymes such as alkaline phosphatase, horseradish peroxidase, and β-galactosidase, a bacterial enzyme that in a test system converts the chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) to a blue product. In these instances, excess antibody is washed away, a suitable chromogenic substrate is added, and the presence of the indicator antibody is revealed by the development of a color that can be visualized.

      Immunostaining has been applied widely in the research laboratory for determining the sub-cellular localization of cellular and viral proteins (Fig. 2.12), monitoring the synthesis of viral proteins, determining the effects of mutation on protein production, localizing the sites of viral genome replication in animal hosts, and determining the effect of infection on structure of the tissue. It is the basis of the fluorescent-focus assay.

      Immunostaining of viral antigens in smears of clinical specimens may be used to diagnose viral infections. For example, direct and indirect immunofluorescence assays with nasal swabs or washes can detect a variety of viruses, including influenza virus and measles virus. Viral proteins or nucleic acids may also be detected in infected animals by immunohistochemistry. In this procedure, tissues are embedded in a solid medium such as paraffin, and thin slices are produced using a microtome. Viral antigens can be detected within the cells in the sections by direct and indirect immunofluorescence assays.

       Fluorescent Proteins

       Fluorescence Microscopy

      Fluorescence microscopy allows virologists to study all steps of virus reproduction, including cell surface attachment, cell entry, trafficking, replication, assembly, and egress. Single virus particle tracking can be achieved by inserting the coding sequence for a fluorescent protein into the viral genome, often


Скачать книгу