Establishment of Hybridoma Colonies Secreting Monoclonal Antibodies Specific for Tanapox and Swinepox Early Secretory Proteins
Utarnachitt, Richard B.
Poxviruses employ proteins called "virokines" to help overcome the host's immune system. Virokines share significant homology with certain cytokine receptors. This allows virokines to bind to cytokines and prevent their interaction with the native receptors. Neutralizing this interaction between cytokines and their native receptors helps disable the host's immune system. These virokines are usually classified as early proteins because they appear before the replication of the viral genome (Fields and Knipe, 1991). Studying the biological mechanism of these early proteins would serve to expand our understanding of how viruses evade the immune system. Tanapox virus (TPV) and swinepox virus (SPV) are two poxviruses used to study the activity of these proteins. TPV produces an early secretory protein with a mass of approximately 38 kDa which binds to IL-2, IL-5 and IFN-y (Essani et al., 1994) Neutralization of these cytokines would allow TPV to incapacitate the TH I cell mediated and TH2 associated immune response, crippling specific anti-viral immunity. Spy produces an uncharacterized early secretory protein with a mass of approximately 34 kDa which may be involved in the mimicry of IFN-y receptors and G-protein coupled receptors. Interfering with the function of these proteins would inhibit normal immune responses including antibody production, T cell function, expression of cell surface antigens, regulation of natural killer cells, macrophage function and G protein function (Mas sung et al., 1993). The first step in elucidating the function of these viral secretory proteins is to isolate pure protein from the media or supernatant surrounding virus infected cells. The monospecific binding capability of monoclonal antibodies have proven to be useful in isolation of a variety of antigens. In this experiment, monoclonal antibodies were generated against the 38 kDa protein produced by SPY and the 34 kDa protein produced by TPV. Hybridoma were produced by fusion of spleen cells from immunized Balb/c mice with myeloma cells (p63Ag8.653). The clones were screened for reactivity to viral antigen using indirect ELISA. The results of these tests were scored visually. Due to time constraints, only preliminary results were obtained from this study: Three TPV hybridoma colonies and four SPY hybridoma colonies reacted to viral antigen. Although it could not be determined whether the reactive colonies were producing antibody which recognized the two proteins of interest, the hybridoma developed are some of the first monoclonal antibodies to recognize antigenic determinants for TPV and SPV.
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