Sequential use of paraformaldehyde and methanol as optimal conditions for the direct quantification of ZEBRA and Rta antigens by flow cytometry

A technique was developed with flow cytometry to quantify the two immediate -early proteins ZEBRA and Rta, which are involved in the activation of Epst ein-Barr virus replication. We evaluated four monoclonal antibodies on four cell lines (B95-8, RAJI, Namalwa, and P3HR1) with varying levels of expres sion of these replication-phase antigens. The Namalwa lymphoma cell line wa s used as a negative control, Four fixation-permeabilization procedures wer e compared, The preparation of cells with paraformaldehyde and methanol in sequence, and antigen detection with AZ125 and AR 5A9 monoclonal antibodies , were found to be the optimal conditions in these cell lines. Our procedur e allowed ZEBRA antigen to be detected in 4.85% of peripheral blood mononuc lear cells from a transplant recipient with a lymphoproliferative disease.