Highly sensitive immunoassay for direct diagnosis of viral hemorrhagic septicemia which uses antinucleocapsid monoclonal antibodies

Abstract

An antigen capture enzyme-linked immunosorbent assay (ELISA) based on the detection of the viral nucleocapsid (anti-N system) was developed for the diagnosis of viral hemorrhagic septicemia. Four monoclonal antibodies directed against the viral nucleocapsid were produced; they all recognized the four viral hemorrhagic septicemia virus (VHSV) serotypes. Three of these monoclonal antibodies were used in a new antigen capture ELISA. The efficiency of the anti-N system in detecting purified and crude viruses as well as the virus in infected-organ extracts and infected blood was compared with that of a recently described antigen capture ELISA based on the detection of viral envelope glycoprotein Gp (anti-G system). For the detection of purified virus, the anti-N system was found to be as sensitive as the anti-G system (detection limit, 1 ng of total viral protein per ml), but the anti-N system was much more sensitive than the anti-G system for the detection of crude VHSV I (detection limits, 1 x 10(4) PFU/ml versus 5 x 10(5) PFU/ml). In organ extracts, VHSV I could be detected by both systems 3 days postinfection. The signal for the assay of VHSV I in blood 24 h postinfection was higher with the anti-N system than the anti-G system. Furthermore, VHSV I could be detected in 80% of the brain samples of surviving trout by the anti-N system and also by the anti-G system, but with a lower signal. In conclusion, we have developed a highly sensitive immunoassay for VHSV I that is more rapid and easier to perform than the currently used plaque assay.