ABSTRACTwere mixed with 1 X 106 trypsinized MCB2 cells, together with ultraviolet-inactivated Sendai virus (400-1000 hemagglutinating units per ml). These mixtures were kept at 4°C for 15 min and then incubated at 37°C for 20 min with frequent shaking. After washing, they were grown in minimum essential medium containing 10% fetal calf serum and 10% tryptose phosphate broth for 24 hr and further incubated in growth medium supplemented with 100 ,uM hypoxanthine, 0.4 ,uM aminopterin, and 16 ,tM thymidine (HAT selective medium).Hybrid cell colonies were obtained in 2-3 weeks and then seeded into semisolid agar medium (13). A number of hybrid clones and subclones were isolated from several different colonies.Detection of EBNA. The anticomplement immunofluorescence test was carried out for the detection of EBNA (2). Cells grown on coverslips were dried at room temperature and fixed in acetone/methanol (1:1, vol/vol) at -20°C. The coverslips were treated with heat-inactivated standard EBV-seropositive serum from a normal person (anti-EBNA of 1:640), in parallel with inactivated seronegative serum (anti-EBNA of <1:5), containing complement (seronegative serum with anti-EBNA of <1:5) at 37°C for 50 min. They were washed four times with balanced salt solution, stained with fluorescein Abbreviations: EBV, Epstein-Barr virus; EBNA, EBV-determined nuclear antigen; BrdUrd, 5-bromodeoxyuridine.