The Epstein-Barr virus (EBV) is the etiological agent underlying infectious mononucleosis (Rezk and Weiss, 2007) and is associated with several forms of cancer (Kutok and Wang, 2006) as well as onset of chronic fatigue syndrome (Klimas and Koneru, 2007) and multiple sclerosis (Holmey and Hestvik, 2008). The ability to detect EBV viral particles in patient tissues is critical to the study and diagnosis of such diseases. Here I outline the development of an immunohistochemical method identifying EBV in extracted tonsil tissue. Steps include the identification of a candidate viral protein antigen, generation of an antibody and use of the antibody for immunohistochemisry.
The first step in screening histochemically for the presence of EBV in tissue is to develop an antibody against one or more EBV protein antigens. An ideal target for any immunohistochemical assay is a protein readily accessible by an antibody. For EBV a prime candidate protein is latent membrane protein 1 (LMP1), which is situated on the surface of plasma membranes in host cells (Hennessy et al., 1984). LMP1 is also of great pathological interest as it is an oncoprotein capable of transforming healthy cells in which it is expressed to a cancerous state (Wang et al., 1985).
The next step in identifying EBV histochemically is the development of an antibody specific for LMP1. As the gene encoding LMP1 has been identified (Wang et al., 1985), its coding sequence can be cloned into an expression vector to allow production of recombinant protein in bacteria (Sambrook and Russell, 2001). The protein can then be extracted, purified and injected into a rabbit to generate LMP1-specific antibodies. Rabbits are then bled and serum collected by removing blood cells through centrifugation. The resulting serum should contain antibodies specific for the EBV LMP1 protein.
Next the rabbit serum collected following injection with LMP1 must be characterized to ensure the presence and specificity of antibodies targeting the viral protein. This can be accomplished by using the antibody to probe immunoblots containing protein from cells infected with EBV (Hennessy et al., 1984). By incubating the immunoblot with the antibody and then performing an immunodetection procedure involving secondary antibodies conjugated to fluorophores one should be able to visualize LMP1 protein as a fluorescent band (Sambrook and Russell, 2001). In the case of multiple bands due to cross-reactivity of the antibody with non-viral proteins, comparison of banding patterns between infected and uninfected cells on the same immunoblot will allow identification of EBV-derived protein.
The final step in developing an immunohistochemical assay to detect EBV in human tonsils is to use the antibody for immunocytochemistry on biological tissue. Because antibodies can bind non-specifically to molecules in situ (Harlow and Lane, 1999) it is important to ensure that the LMP1 antibody will generate accurate, unambiguous signals during histochemistry. Due to the ubiquity of EBV and its tendency to remain latent in many tissues (Robertson, 2005) it is not possible to acscertain whether a given tonsil sample lacks EBV to serve as a negative control for the tests. Thus using tissue dissected from mouse models is desirable to ensure antibody specificity. A useful mouse strain for these purposes is the LMP1 transgenic line described by Kulwichit et al. (1998). These mice have been engineered to produce LMP1 in B-lymphocytes and thus immunohistochemistry on tissues rich in lymphocytes such as the spleen should give signal when probed with the LMP1 antibody. In contrast, control spleens from mice that have not been genetically modified should not produce signal when probed with the antibody.
Conducting the histochemistry assay involves a number of steps (Harlow and Lane, 1999). First, spleens should be dissected from experimental and control mice, fixed with paraformaldehyde and then embedded in paraffin wax. Wax blocks are then trimmed and sectioned using a microtome. Alternatively, fixed tissue may be frozen and sectioned with a cryostat. Once sections are on slides, wax must be dissolved by passing slides through an organic substance such as xylene followed by rehydration through an ethanol:water series. Next the tissue is blocked, typically using bovine serum albumin or goat serum, which aids in reducing non-specific interactions between the antibody and tissue/proteins. Following blocking, sections are incubated with the LMP1 primary antibody and then washed with Tris-buffered saline. The tissue is then blocked again and treated with a secondary antibody conjugated to a fluorescent molecule. Since the LMP1 antibody was made in mice the secondary antibody must target mouse antibodies. Such secondary antibodies are readily available commercially. Following secondary antibody incubation tissue is washed again and then dehydrated, dried and mounted in an organic medium. Slides are then ready for viewing using a fluorescent or confocal microscope. These microscopes excite the fluorophore conjugated to the secondary antibody and then detect the photons of light released. The spatial pattern of fluorescence observed should directly reflect the position of the LMP1 protein since the primary antibody is bound to the protein and the fluorescing secondary antibody is bound to the primary.
Once immunohistochemistry using the LMP1 antibody on transgenic LMP1 mouse tissue has shown authentic signal relative to the negative control the antibody is ready for use on human tonsil tissue as an indicator of the presence of EBV. Treatment of tonsil tissue, probing with the antibody and detection and visualization of signal would use identical procedures to those described above for mouse tissue.
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