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 a s low as ca 10"laque forming units (PFUs) ml-' of cell culture fluid, which corresponds to about 102 PFUs spotted onto the immunoblot assay matrix. Time course studies showed that the detection of IHNV antigen in cell culture by inlmunoblot assay was directly related to the concentration of virus in the inoculum and that positive in~munoblot 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 0. mykiss were assayed by the immunoblot method, false positive reactions results from the binding of monoclonal antibody to some of the ovarian fluid proteins. The advantages and limitations of the assay are discussed.

Monoclonal-Antibody-Based Immunodot Assay Distinguishes between Viral Hemorrhagic Septicemia Virus (VHSV) and Infectious Hematopoietic Necrosis Virus (IHNV)

“…We have described a sensitive immunoblot method, based on two monoclonal antibodies, for differentiation between IHNV and VHSV. Several other investigators (Hsu and Leong 1985;McAllister and Schill 1986;Schultz et al 1989) have investigated the immunoblot assay for detection of rhabdoviruses and have found that it is a rapid and sensitive method for identification of salmonid viruses. McAllister and Schill (1986), using polyclonal antisera raised against IHNV on a dot 1 2 3 4 5 6 7 8 9 10 11 12 H FIGURE 1.-Nitrocellulose spotted with 81 isolates of IHNV, 8 isolates of VHSV, and 4 nonsalmonid fish rhabdoviruses stained with anti-IHNV nucleoprotein antibody 1NDW14D.…”

“…c ND -not determined; the electropherotype of this isolate has not been classified by the method described by Hsu et al (1986). blot, showed that their assay was sensitive to about 10 5 pfu/mL of virus. In a further modification of this assay, Schultz et al (1989) produced a dot blot system for the detection of IHNV with a biotinylated anti-IHNV monoclonal antibody. Biotin conjugated to the antibody reacted with avidinlabeled peroxidase and detected the virus in the range of 10 4 pfu/mL, a value comparable to that displayed in our system.…”

Techniques for diagnosing viral diseases of salmonid fish

“…The minimum titre of IPNV necessary to show a positive reaction was 106 T C I D~~ ml-' in cell culture (Kimura et al 1984) and lo3 TCID,o g-' in trout undergoing the disease (Table 1). The IPNV antigen could be detected after an 80-fold Schultz et al (1989) Enzyme immunoassay (ELISA)…”

“…Other techniques either require instruments or cornplicated manipulations, or have low sensivitity, or are time-consuming , Schultz et al 1989). Those reasons have been invoked in favour of the more simple, speedy and sensitive immunodot technique for detection of 20 ng ml-' or 105 TCID,o ml-' of IPNV (Table 1).…”

“…MAbs have been developed and used in several investigations , Lilipun et al 1989 for antigenic characterization and improved diagnosis of IPNV with the immunodot test. Schultz et al (1989) developed an immunodot assay for detection of IHNV by using MAbs that detected ~O ' T C I D~~. Because the immunodot detects binding Abs rather than neutralizing Abs, identification of the 3 serotypes of VHSV was also made possible by using a unique antiserum to one of them (McAllister & Owens 1987).…”