The nucleocapsid protein of the Gray strain of infectious bronchitis virus (IBV) is highly immunogenic and cross-reactive among various distinct serotypes. Recombinant nucleocapsid polypeptide expressed in bacteria with a histidine tag at the amino terminus has been used as antigen for developing an assay to detect IBV-specific antibody. This fusion protein was produced readily in bacteria and easily purified with a nickel column which bound to the histidine tag. Conditions were optimized for using these preparations for an IBV-specific ELISA. Although differences in optical densities could be detected between pre-immune and positive sera for the Ark, Mass, and Gray strains with antigen concentrations between 50 and 0.1 microg per well, the greatest differences could be detected with 3 and 1.5 microg of protein per well. Maximum differences in optical densities between pre-immune and positive sera were obtained using 2.4 microg per well of protein and sera diluted between 1:80 and 1:160. In addition, as little as 30 ng/dot of recombinant nucleocapsid consistently detected IBV-specific sera in immunoblot assays which have convenient field applications.