Journal of Virological Methods 191 (2013) 88–94 Contents lists available at SciVerse ScienceDirect Journal of Virological Methods jou rn al hom epage: www.elsevier.com/locate/jviromet Comparative assay of fluorescent antibody test results among twelve European National Reference Laboratories using various anti-rabies conjugates E. Robardet a,∗ , S. Andrieu a , T. Bruun Rasmussen b , M. Dobrostana c , D.L. Horton d , P. Hostnik e , I. Jaceviciene f , T. Juhasz g , T. Müller h , F. Mutinelli i , A. Servat a , M. Smreczak j , E. Vanek k , S. Vázquez-Morón l , F. Cliquet a a French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Nancy, Laboratory for Rabies and Wildlife, WHO Collaborating Centre for Research and Manage- ment in Zoonoses Control, OIE Reference Laboratory for Rabies, European Union Reference Laboratory for Rabies, European Union Reference Institute for Rabies Serology, Technopôle Agricole et Vétérinaire, BP 40009, 54220 Malzéville cedex, France b National Veterinary Institute, Section of Virology, Technical University of Denmark, Denmark c Institute of Food Safety, Animal Health and Environment “BIOR”, Lejupes Str.3, 1076 Riga, Latvia d Animal Health and Veterinary Laboratories Agency, Virology Department, Woodham Lane, New Haw, Addlestone, Surrey, United Kingdom e National Veterinary Institute, Gerbiˇ ceva 60, 1000 Ljubljana, Slovenia f National Food and Veterinary Risk Assessment Institute, Laboratory Department of Republic of Lithuania, J. Kairiukscio str.10, 08409 Vilnius, Lithuania g Central Veterinary Institute, 1149 Budapest, Virological Department, Tabornok utca 2, Hungary h Federal Research Institute for Animal Health, Institute of Epidemiology, Friedrich-Loeffer Institut, Seestr.55, D-16868 Wusterhausen, Dosse, Germany i Istituto Zooprofilattico Sperimentale delle Venezie, NRL for Rabies, Viale dell’Universita, 10, 35020 Legnaro, PD, Italy j National Veterinary Research Institute, Ul. Partyzantow 57, 24-100 Pulawy, Poland k AGES, Institute for Veterinary Disease Control, Robert Koch-Gasse 17, 2340 Mödling, Austria l Public Health Institute Carlos III, Service of Diagnostic Microbiology, Ctra. Majadahonda-Pozuelo s/n, Majadahonda, Madrid 28220, Spain Article history: Received 23 November 2012 Received in revised form 19 March 2013 Accepted 25 March 2013 Available online 8 April 2013 Keywords: Rabies Diagnosis Fluorescent antibody test Fluorescein isothiocyanate Conjugate a b s t r a c t Twelve National Reference Laboratories (NRLs) for rabies have undertaken a comparative assay to assess the comparison of fluorescent antibody test (FAT) results using five coded commercial anti-rabies con- jugates (Biorad, Bioveta, Fujirebio, Millipore, and SIFIN conjugates). Homogenized positive brain tissues infected with various lyssavirus species as well as negative samples were analyzed blindly using a stan- dardized FAT procedure. Conjugates B, C, D, and E were found to be significantly more effective than conjugate A for GS7 (French RABV) diluted samples (1/8 and 1/100) while the frequency of concordant results of conjugates C and D differ significantly from conjugates A, B and E for CVS 27. For detection of EBLV-1 strains, conjugates C and D also presented a significantly lower frequency of discordant results compared to conjugates A, B and E. Conjugates B, C and D were found to be significantly more effective than conjugates E and A for EBLV-2 and ABLV samples. In view of these results, conjugates C and D set themselves apart from the others and appeared as the most effective of this 5-panel conjugates. This study clearly demonstrates that the variability of conjugates used by National Reference Laboratories can potentially lead to discordant results and influence assay sensitivity. In case of false negative results this could have a dramatic impact if the animal under investigation is responsible for human exposure. To avoid such situations, confirmatory tests should be implemented. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. 1. Introduction Considering the efforts that are being undertaken to eliminate canine and wildlife rabies in the world, rabies surveillance is of the utmost importance. A condition precedent to the systematic ongo- ing collection, analysis and interpretation of rabies data and the dissemination of information is a reliable rabies diagnosis (Cliquet et al., 2010). The latter is also an indispensable condition in human ∗ Corresponding author. Tel.: +33 3 83 298 95; fax: +33 3 83 298 958. E-mail address: emmanuelle.robardet@anses.fr (E. Robardet). medicine as regards the administration of adequate and timely post-exposure prophylaxis (WHO, 2005). Various methods are used for the detection of lyssavirus of the family rhabdoviridae. The fluorescent antibody test (FAT) remains the “gold standard” and consequently the most commonly used rabies diagnostic technique (Meslin et al., 1996; OIE, 2012). Rabies virus antigens are detected in the brain tissue of infected animals using Fluorescein isothio- cyanate (FITC) labelled anti-rabies antibodies (Dean et al., 1996). Because of its high sensitivity and specificity this simple technique provides reliable results on fresh specimens within a few hours in 98–100% of cases (OIE, 2012). However, small variations in the procedure, including the area of the brain examined (Bingham and 0166-0934/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.03.027