Detection of infectious hematopoietic necrosis virus in cell culture fluid using immunoblot assay and biotinylated monoclonal antibody

Abstract

Affinity purified monoclonal antibody was biotinylated and used with avidin-labeled peroxidase in an immunoblot assay to detect infectious hematopoietic necrosis virus (IHNV). The procedure proved capable of detecting the virus a titers as low as ca 10 super(4) plaque forming units (PFUs)/ml of cell culture fluid, which corresponds to about 10 super(2) PFUs spotted onto the immunoblot assay matrix. Time course studies showed that the detection of IHNV antigen in cell culture by immunoblot assay was directly related to the concentration of virus in the inoculum and that positive immunoblot reactions coincided with the appearance of cytopathic changes in inoculated cell cultures. We used the immunoblot assay to detect and identify IHNV antigen in medium from cells inoculated with ovarian fluids. Immunoblot assay was not suitable for detecting IHNV in raw ovarian fluid. When ovarian fluids from IHNV-negative chinook salmon Oncorhynchus tshawytscha) and steelhead trout O. mykiss were assayed by the immunoblot method, false positive reactions results from the binding of monoclonal antibody to some of the ovarian fluid proteins.