Olivier Cabre* , Marc Grandadam†, Jean-Lou Marié‡, Patrick Gravier†, Aurélie Prangé†, Yan Santinelli§, Vincent Rous¶, Olivier Bourry#, Jean-Paul Durand†, Hugues J. Tolou†, and Bernard Davoust**
Author affiliations: *École du Val-de-Grâce, Paris, France; †Institut de Médecine Tropicale du Service de Santé des Armées, Marseille, France; ‡Secteur Vétérinaire de Marseille, Marseille, France; §Service Vétérinaire du Régiment de Cavalerie de la Garde Républicaine, Paris, France; ¶Secteur Vétérinaire de Lyon, Lyon, France; #Centre International de Recherches Médicales, Franceville, Gabon; **Direction Régionale du Service de Santé des Armées de Toulon, Toulon, France
Figure 2. West Nile virus (WNV) Western blot as a validation of ELISA-positive results. Proteins from WNV-infected cells lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 13% acrylamide gels and transferred on polyvinylidene difluoride membranes. Strips were cut and blocked with nonfat milk (5%). Serum samples diluted (1:200) in phosphate-buffered saline (PBS), 5% nonfat milk, and 0.1% Tween 20 were loaded on strips and incubated for 1 h with slow shaking. Strips were washed 4 times in PBS, 0.5% Tween, and incubated for 1 h in anti-horse immunoglobulin G (IgG) peroxidase (1:8,000). After 4 washes, Western blots were incubated for 5 min with trimethylbenzidine substrate (with specific enhancer). Strips were washed once with water to stop the staining. Results were obtained within 24 h. MW, molecular weight; T+, horse from Chad with positive Western blot results; serum samples 1–11, horse from southeast France, IgG positive by ELISA but negative by neutralization; NS5, nonstructural protein 5; E, envelope; PreM, premembrane; C, capsid.
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