AN IMMUNOPEROXIDASE PLAQUE-ASSAY FOR RETICULOENDOTHELIOSIS VIRUS AND ITS APPLICATION TO A SENSITIVE SERUM NEUTRALIZATION ASSAY

A rapid assay for the enumeration of reticuloendotheliosis virus (REV) is described. Chicken embryo fibroblast monolayer cultures were infec ted with REV and incubated 6 days under an agar overlay. After removal of the overlay, cells were fixed with acetone/ethanol. Foci of infect ion (hereafter referred to as plaques) were detected using either an a nti-REV envelope monoclonal antibody or convalescent chicken serum as the primary antibody; the secondary antibody was either horseradish pe roxidase-conjugated goat anti-mouse IgG (for use with monoclonals) or goat anti-chicken IgG (for use with chicken serum). Staining with a su bstrate solution containing diaminobenzidine, CoCl2, and H2O2 revealed individual dark plaques on a light gray background. This method worke d equally well for the SNV, CSV, and REV-T strains of REV; further, it detected all six field isolates tested. Results indicate that this im munoperoxidase technique is a rapid and reliable method for detection and titration of REV as well as for the assay of neutralizing antibody in chicken serum.