PRODUCTION OF MONOCLONAL ANTIBODIES AGAINST INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS (HYBRIDOMA, IMMUNOBLOT, FISH, ELISA)

Infectious hematopoietic necrosis (IHN) virus, a member of the Rhabdovirus group, is a major pathogen of salmonid fish. Because of its poor immunogenicity, hybridoma technology was used in an attempt to produce a standardized immunologic reagent. Fusion of a mouse myeloma line, P3-X63-Ag8.653 cells, with splenic lymphocytes from BALB/c mice immunized with purified IHN virus resulted in the production of a stable hybridoma cell line that continually secreted monoclonal antibodies against the virus. The cell line can be routinely subcultivated at 3 day intervals and has undergone more than 130 passages. The cells can be stored in liquid nitrogen and readily recovered with no reduction in growth rate or level of antibody production.; The immunoglobulin produced by the hybridoma cell line was isotyped as IgG 2b, with a kappa light chain. Antibodies in cell culture fluids were concentrated by Protein A Sepharose CL-4B affinity chromotography with average yields of 16 mg of IgG from 350 mL of culture fluids. Attempts to obtain higher levels of antibody by inducing the production of tumors and ascitic fluids in mice were unsuccessful. Athough the monoclonal antibody produced by the hybridoma had strong binding activity, it had poor neutralizing activity giving a titer of only 1:50 against 50 tissue culture infectious doses of virus. No cross reaction was noted by enzyme-linked immunosorbent assay (ELISA) when the monoclonal antibody was tested against viral hemorrhagic septicemia virus, infectious pancreatic necrosis virus, eel virus European, vesicular stomatitis virus, and Herpes simplex virus type 1.; Monoclonal antibody purified by affinity chromatography was biotinylated for 45 minutes. The biotinylated monoclonal antibody was incorporated into a solid phase support assay (immunoblot). This assay system could detect virus less than 10('5) plaque forming units per mL. Time course studies done to determine the optimal point to assay for IHN virus in cell culture, showed that virus could be detected within 72 hours.; The immunoblot assay requires a solid phase support of Whatman 541 ashless filter paper, the biotinylated monoclonal antibody preparation, an avidinated horseradish peroxidase and a substrate of o-dianisidine dihydrochloride. Nonspecific reactions of virus-free ovarian fluids were found by western blot analysis to be due to a binding reaction between the monoclonal antibody and some of the proteins in the ovarian fluid. Inoculation of ovarian fluids onto cell culture, followed by immunoblot assay of culture fluids within 72 hours was determined to be the best method to detect virus in the ovarian fluid samples.; This detection system was used to determine whether virus was present in two different groups of ovarian fluid samples. This detection system requires about three hours to perform and does not utilize any special or expensive equipment.