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Clinical and Diagnostic Laboratory Immunology, March 2000, p. 206-211, Vol. 7, No. 2
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Sequential Use of Paraformaldehyde and Methanol as Optimal Conditions for the Direct Quantification of ZEBRA and Rta Antigens by Flow Cytometry

Berthe-Marie Imbert-Marcille,1,* Marianne Coste-Burel,1 Nelly Robillard,2 Jacqueline Foucaud-Gamen,3 Sylviane Billaudel,1 and Emmanuel Drouet3

Virology Laboratory, EA-1156,1 and Flow Cytometry Department,2 Biology Institute of University Hospital, Nantes, and Viral Immunology Laboratory, CNRS ESA-5082, Grenoble,3 France

Received 17 September 1999/Returned for modification 10 November 1999/Accepted 6 January 2000

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.

* Corresponding author. Mailing address: Laboratoire de Virologie EA-1156, Institut de Biologie CHU Nantes, 9 quai Moncousu, 44093 Nantes Cedex, France. Phone: 33/2/40-08-41-23. Fax: 33/2/40-08-41-39. E-mail: bmimbert{at}sante.univ-nantes.fr.

Clinical and Diagnostic Laboratory Immunology, March 2000, p. 206-211, Vol. 7, No. 2
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.




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Copyright © 2000 by the American Society for Microbiology. All rights reserved.