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

Abstract

A technique was developed with flow cytometry to quantify the two immediate-early proteins ZEBRA and Rta, which are involved in the activation of Epstein-Barr virus replication. We evaluated four monoclonal antibodies on four cell lines (B95-8, RAJI, Namalwa, and P3HR1) with varying levels of expression of these replication-phase antigens. The Namalwa lymphoma cell line was used as a negative control. Four fixation-permeabilization procedures were 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 procedure allowed ZEBRA antigen to be detected in 4.85% of peripheral blood mononuclear cells from a transplant recipient with a lymphoproliferative disease.

FIG. 1

Flow cytometry analysis of ZEBRA (MAb AZ125, 1 μg/assay) and Rta (MAb AR5A9, 0.5 μg/assay) antigen expression on three cell lines stimulated by TPA plus n-butyrate and prepared by three different fixation-permeabilization procedures: (left to right) isotype control, PF/M, Intraprep, and Fix&Perm. Percentages are the portions of cells expressing the related antigen. Mean fluorescence intensity is in parentheses.

FIG. 2

RT-PCR amplification of ZEBRA mRNA (182 bp) in PBMCs obtained from a bone marrow transplant recipient with EBV-associated B-cell lymphoproliferative disease (lane 3). Lanes 1 and 2, negative and positive controls, respectively; lane 4, size marker V (Boehringer).

FIG. 3

Biparametric analysis by forward angle light scatter (FSC-H) versus FITC fluorescence intensity (FL1-H) of MAb AZ125 and related isotype staining on PBMCs from a bone marrow transplant recipient with EBV-associated B-cell lymphoproliferative disease. The windows identify EBV-positive PBMCs.