Detecting viral proteins in infected cells or tissues by immunostaining

Many virological techniques are based on the specificity of the antibody-antigen reaction. Examples in our virology toolbox include western blot analysis and ELISA. While very useful, these methods cannot be used to visualize viral proteins in infected cells or tissues. To do that we must turn to immunostaining.

In direct immunostaining (illustrated), an antibody that recognizes a viral antigen is coupled directly to an indicator (a fluorescent dye or an enzyme). Indirect immunostaining is a more sensitive method because a second antibody is coupled to the indicator. The second antibody recognizes a common epitope on the virus-specific antibody. Multiple second antibodies can bind to the first antibody, leading to an increased signal from the indicator compared to direct immunostaining.

To carry out immunostaining, virus-infected cells are fixed to preserve cell morphology or tissue architecture. This step is usually accomplished with acetone, methanol, or paraformadehyde. After incubation of fixed cells with the appropriate antibody, excess antibody is removed by washing, followed by microscopy. Common indicators that are coupled to antibody molecules include fluorescein and rhodamine, which fluoresce on exposure of the cells to ultraviolet light. Filters are placed between the specimen and the eyepiece to remove blue and ultraviolet light; this ensures that the field is dark, except for cells that have bound antibody. These emit green (fluorescein) or red (rhodamine) light.

Antibodies can be coupled to indicators other than fluorescent molecules. Examples are enzymes such as alkaline phosphatase, horseradish peroxidase, and β-galactosidase. These enzymes can convert an added substrate to a colored dye. For example, the bacterial enzyme β-galactosidase converts the chromogenic substrate X-gal to a blue product, which can be visualized by microscopy.

Immunostaining is widely used in the research laboratory to determine subcellular location of proteins in cells. An example is the location of the herpes simplex viral protein VP22 in the nucleus of infected cells. To produce this image, virus-infected cells were stained with an antibody against VP22 and a mouse monoclonal antibody against α-tubulin, a cellular protein. Second antibodies bound to indicator molecules were then added: fluorsecein-conjugated anti-rabbit antibody, and Texas red-conjugated anti-mouse antibody (Texas red is another red fluorescent dye). The stained cells were then photographed with a microscope using ultraviolet light. The results show that VP22 (green) is located in the cell nucleus. Cellular α-tubulin is stained red. Photo courtesy of John Blaho.

Other uses of immunostaining include monitoring the synthesis of viral proteins, determining the effects of mutation on protein production, and investigating the sites of virus replication in animal hosts. Immunostaining of viral antigens in clinical specimens is also used to diagnose viral infections. Direct and indirect immunofluorescence assays with nasal swabs or washes are routine for diagnosis of infections with respiratory syncytial virus, influenza virus, parainfluenza virus, measles virus, and adenovirus.

When cultured cells are examined by this technique it is called immunocytochemistry; when tissues are studied, the procedure is immunohistochemistry. Flow cytometry is yet another way to use immunostaining to study the synthesis of one or more proteins in cells.

Detection of antigens or antibodies by ELISA

A more rapid method than Western blot analysis to detect a specific protein in a cell, tissue, organ, or body fluid is enzyme-linked immunosorbent assay, or ELISA. This method, which does not require fractionation of the sample by gel electrophoresisis, is based on the property of proteins to readily bind to a plastic surface.

To detect viral proteins in serum or clinical samples, a capture antibody, directed against the protein, is linked to a solid support such as a plastic 96 well microtiter plate, or a bead. The clinical specimen is added, and if viral antigens are present, they will be captured by the bound antibody. The bound viral antigen is then detected by using a second antibody linked to an enzyme. A chromogenic molecule – one that is converted by the enzyme to an easily detectible product – is then added. The enzyme amplifies the signal because a single catalytic enzyme molecule can generate many product molecules.

To detect antibodies to viruses, viral protein is linked to the plastic support, and then the clinical specimen is added. If antibodies against the virus are present in the specimen, they will bind to the immobilized antigen. The bound antibodies are then detected by using a second antibody that binds to the first antibody.

ELISA is used in both experimental and diagnostic virology. It is a highly sensitive assay that can detect proteins at the picomolar to nanomolar range (10-12 to 10-9 moles per liter). It is the mainstay for the diagnosis of infections by many different viruses, including HIV-1, HTLV-1, adenovirus, and cytomegalovirus.