Immunoprecipitation of Epstein-Barr virus EBNA1 protein using human polyclonal serum.

ABSTRACT

We have developed a method that permits the use of human polyclonal serum to immunoprecipitate BamH1-K EBNA(EBNA1) from EBV transformed cell lines and from cells transfected with an expression vector containing the Bam K region of EBV. Serum from healthy seropositive donors is preabsorbed once with lysate of EBV-negative Burkitt lymphoma cells, then fractionated by gel filtration. The main IgG fraction is then used for the immunoprecipitations. Immunoprecipitated material is visualized by immunoblotting using the same serum. Two proteins with apparent molecular weights of 74 and 62 kD are specifically precipitated from extracts of B95-8 cells. Several proteins are immunoprecipitated from cells transfected with the Bam K containing vector, the apparent molecular weights of the 4 major bands are 74, 68, 62 and 57 kD. Labelling of transfected cells with [3H]glycine and [32P]orthophosphate shows that the 74 and 62 kD proteins can be labelled with both isotopes.