A combined pseudoreplica-immunochemical technique for research and diagnostic virology

Pseudoreplica and immunochemical techniques were combined in a single protocol for identification of virus in research and clinical specimens. Stock preparations of vesicular stomatitis virus (VSV), cytomegalovirus (CMV), and herpes simplex virus (HSV) were used to develop the technique. Traditional pseudoreplicas of viral stock solutions were prepared but not negatively stained. The virus was then immunolabeled in two stages. Virus-specific polyclonal antisera were used in the first stage; colloidal gold conjugated antibodies were used as indicator antibody in the second stage. After immunolabeling, the grids were negatively stained. As a demonstration of the clinical usefulness of this approach, it was employed to antenatally identify human parvovirus B19 particles in ascites from a 22 week gestational fetus with nonimmune hydrops fetalis. The combined pseudoreplica-immunochemical approach offers several advantages over both the pseudoreplica and immunochemical methods when used in isolation. Advantages include relative purification of samples, concentration of virus, morphological preservation, and enhanced diagnostic specificity.