Preparing hemagglutinating antigen from isolates of infectious bronchitis virus

The hemagglutinating (HA) activity of 14 strains of infectious bronchitis virus (IBV) was investigated. The optimal conditions for IBV antigen preparation include inoculation of 10- or 11-day-old specific pathogen-free embryonated eggs and incubation for 30 hours at 37 C. Embryos were inoculated via the allantoic cavity with 0.1 ml of a low embryonic passage of the virus (10(7) to 10(8) EID50/ml). Allantoic fluid was harvested and pooled, and a 100-fold concentration of virus particles was achieved by centrifugation for 3 hours at 30,000 x g. Virus pellets were resuspended in Tris-hydrochloride buffer containing 3 units of phospholipase-C (type-1) enzyme/ml and incubated for 2 hours at 37 C. All IBV strains tested demonstrated positive HA activity with chicken red blood cells. The antigen was stored in liquid state or lyophilized at 4 C.