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Study of epstein‐barr virus‐determined nuclear antigen (EBNA) by chromatography on fixed cell nuclei

Low ionic strength (50 to 100 mM NaCl) and pH 6.0 were found to be optimal conditions for in vitro conversion of Epstein‐Barr virus (EBV)‐determined nuclear antigen (EBNA)‐negative nuclei to EBNA‐positive nuclei by addition of the complement‐fixing (CF) antigen extracted from Raji cells. In vitro conversion of nuclei to EBNA‐positivity was sensitive to DNase but not to RNase treatment. This suggests that nuclear DNA is a specific target substance to which EBV‐CF antigen binds. If nuclei were fixed with methanol/acetic acid and subsequently treated with 0.6 M NaCl, EBNA could be eluted from in vitro‐converted Ramos nuclei with 0.3 and 0.4 M NaCl. The same conditions were also found to be optimal for the adsorption and elution of EBV‐CF antigen in DNA‐cellulose chromato‐graphy. This indicates that the DNA‐binding properties of EBNA antigen can be studied by “chro‐matography” on fixed nuclei followed by the ACIF test. The obvious advantages of this method over chromatography on DNA‐cellulose are its simplicity, the possibility of testing many samples in one experiment and, especially, the use of minimal amounts of material. Significant differences in elution patterns for EBNA were found when nuclei derived from different cell lines (Ramos, Raji, and P3HR‐1) were converted in vitro to EBNA‐positivity. EBNA is eluted from in vitro‐converted nuclei of EBV genome‐positive P3HR‐1 cells at an almost 0.1 M higher concentration of NaCl than is necessary for a similar degree of elution from nuclei of EBV genome‐negative Ramos cells.