Journal of Clinical Virology 47 (2010) 280–281 Contents lists available at ScienceDirect Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv My Favourite Assay A staining control for the HCMV pp65 antigen test Andreas Osterman 1 , Rudolf Haase 1 , Nasim Motamedi, Hans Nitschko, Gundula Jaeger, Armin Baiker ∗ Max von Pettenkofer-Institute, Department of Virology, Ludwig-Maximilians-University, Pettenkoferstrasse 9a, D-80336 Munich, Germany article info Article history: Received 10 November 2009 Received in revised form 18 December 2009 Accepted 22 December 2009 Keywords: Staining control pp65 antigenemia Cell line Immunofluorescence test 1. Reasons for developing the assay The HCMV phosphoprotein 65 (pp65) antigen test represents a fast, cheap and sensitive method for the routine diagnosis of an active HCMV infection. 1 However, the lack of economic and easy accessible (immuno-)staining control slides for the validation of this test has been considered as one problem for its standardization and quality assurance. Approaches to manufacture suitable stain- ing control slides were manifold, including their preparation out of pp65 positive patient peripheral blood leukocyte (PBL) samples or their in vitro-generation by co-cultivation of donor PBLs with HCMV infected endothelial cells. 2 Commercially available HCMV pp65 antigen test providers supply staining control slides that are based on HCMV infected and uninfected fibroblasts (ARGENE, France), or based on donor PBLs preparations mixed with defined ratios of recombinant, pp65 expressing SF9 insect cells (CMV Brite TM Kit, IQ Products, The Netherlands). The disadvantages of these existing rather sophisticated, labor intensive and thus expensive methods are the lack of unlimited amounts of biological material, the depen- dency on infectious HCMV and differences in staining intensity of recombinant pp65, respectively. 3 2. Methods used in designing the assay We describe our in house method to produce HCMV pp65 stain- ing control slides, based on a genetically modified HEK293 cell line expressing recombinant pp65. ∗ Corresponding author. Tel.: +49 89 5160 5231; fax: +49 89 5160 5279. E-mail address: baiker@mvp.uni-muenchen.de (A. Baiker). 1 Equal contribution. 3. Protocol For the generation of a recombinant pp65 expressing cell line, the cDNA encoding HCMV pp65 was amplified by PCR from the laboratory adapted HCMV strain AD169 and inserted into a mod- ified, blasticidin selectable pEPI-1 plasmid vector. 4 The resulting vector, pEPI-BSD-pp65, was transfected into HEK293 cells (ATCC CRL-1573) using standard protocols. Transfected HEK293 cells were selected in selection media (growth media supplemented with 5 g/ml blasticidin) in order to obtain the stable cell line: HEK293/pp65. Aliquots of the resulting cell line were prepared and cryopreserved until further usage. For the generation of in house HCMV pp65 staining control slides, frozen HEK293/pp65 cells were reconstituted and expanded in selection media. A total of 100,000 cells were attached to glass slides by cytocentrifugation. After cytospin, glass slides were air-dried for 10 min, fixed, per- meabilized and washed using CINAkit (ARGENE, France) reagents according to the manufacturer’s instructions. After washing, glass slides could be stored at -80 ◦ C up to several months. For HCMV pp65 detection by indirect immunofluorescence microscopy, glass slides were equilibrated for 5 min in washing buffer and further processed using CINAkit reagents and protocol. Red counterstain- ing was achieved by incubation of the secondary (FITC conjugated) antibody in the presence of 0.01% Evans blue. 4. Validation data Within our routine HCMV pp65 antigen test read out, patient samples and control slides are examined at a 20× magnification under a Leica DML microscope after excitation with blue light. HCMV pp65 positive cells can be identified by the green FITC signal. Fig. 1A depicts the typical picture of a pp65 antigenemia nega- 1386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2009.12.017