CHARACTERIZATION OF THE INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS GLYCOPROTEIN USING NEUTRALIZING MONOCLONAL-ANTIBODIES

To study the antigenic nature of the glycoprotein (G protein) of infec tious hematopoietic necrosis virus (IHNV), 31 neutralizing monoclonal antibodies (MAbs) were produced against a reference isolate of the vir us. The MAbs were compared using a neutralization assay, an enzyme-lin ked immunosorbent assay (ELISA), and by immunoblotting of the G protei n in the native, reduced, and deglycosylated forms. Hybridoma culture fluids of the various MAbs could be diluted from 1:2 to 1:512 and stil l completely neutralize 1 X 10(4) plaque-forming units of IHNV. Simila rly, the end point dilutions that produced optical density readings of 0.1 or greater in the ELISA were 1:40 to 1:10240. Western blotting sh owed that all of the MAbs reacted with the G protein in the unreduced (i.e. native) conformation; however, only 9 nine of the MAbs were able to react with the G protein following reduction by 2-mercaptoethanol. Deglycosylation of the protein did not influence the binding ability of any of the MAbs. These data indicate that all the MAbs recognized a mino acid sequences on the protein itself and that the IHNV glycoprote in contains linear as well as conformation-dependent neutralizing epit opes. When rainbow trout Oncorhynchus mykiss fingerlings were passivel y immunized with MAbs against either a linear or a conformation-depend ent epitope, the fish were protected against challenge with wild-type IHNV.