Detection of Antibody-Forming Cells Directed Against Newcastle Disease Virus and Their Immunoglobulin Class by Double Immunoenzyme Histochemistry

Abstract Antibody-forming cells (AFCs) against Newcastle disease virus (NDV) and their immunoglobulin (Ig) class were demonstrated by a double immuno-enzyme histochemical technique. The AFCs were stained and quantified in spleen sections of chickens euthanatized at day 7 postexposure to the Roakin strain of NDV. The sections were incubated with NDV to determine the specificity of the AFCs. Bound virus was subsequently visualized with a primary monoclonal antibody (MAb), a secondary horseradish peroxidase–conjugated MAb, and 3-amino-9-ethylcarbazole as substrate. IgM and IgA were stained with MAbs and an alkaline phosphatase (AP)-conjugated secondary antibody. IgG class antibodies were demonstrated with an AP-conjugated rabbit serum. The final substrate for the three Igs was naphthol AS-MX-phosphate and fast blue BB. About 64–159/mm2 AFCs against NDV were detected. Of these virus-binding cells, about 55% produced IgM, 37% produced IgG, and the remainding 8% produced IgA.