An antigen-capiure enzyme-linked immunosorbent assay (ELISA) has been developed lo delect antigens of Australian ... brain preparations at titres as low as 1000 TCIDSQ/KX) H]. ... These included iwo flavivirus group- reactive .... family (Burke, personal communication), .... diagnosis of Ross River virus in human blood.
(Submitted June 18, 1986. Accepted for publication September 25, 1986.) Summary. An antigen-capiure enzyme-linked immunosorbent assay (ELISA) has been developed lo delect antigens of Australian llaviviruses in mosquito pooh, suckling mouse brain and infected celt culture supernatant fluid. A monoclonal antibody reaciivc lo an epiiope on the envelope glycoprotein common to all llaviviruses was used as the capture aniibody. Purilied rabbit IgG, produced againsi Murray Valley encephalitis (MVE) virus, which reacted with eight Australian llaviviruses in haemagglutination inhibiiion (H!) and in an indirect fluorescent antibody test, was used as the indicator antibody in direci and indirect antigen-capture ELISA. A monoclonal aniibody specific for a subgroup of encephalitic flaviviruses was conjugated to horseradish peroxidase and used as the indicator aniibody to distinguish MVE, Kunjin and Alfuy viruses from the remainder tested. This ELISA could detect viral aniigen in mosquito cell culture fluids and suckling mouse brain preparations at titres as low as 1000 TCIDSQ/KX) H]. Viral aniigen in a single mosquilo infected with MVE could be detected in a pool of 5(X).
INTRODUCTION Techniques currently used to detect arbovirus antigens in arthropods and clinical specimens include inoculation of mammahan and arthropod cell culture or the inoculation of suckling mice (1; 2; 3). Identification of viral isolates usually involves immunofiuorescent staining with specific monoclonal antibodies (4; 5; 3) or neutralisation of the virus in cell culture or suckling mice by hyperimmune mouse serum (6; 7). These techniques require extensive cell culture facilities and animal accommodation and are therefore impractical for routine clinical assays or for use in the field. An antigen-capture ELISA has been described which uses monoclonal antibodies to detect yellow fever virus in samples of human serum (8). An analogous test has been applied to the detection of alphaviruses in pools of mosquitoes (9). Abbreviations used in this paper ELISA. Enzymelinked immunosorbent assay; MVE, Murray Valley encephalitis: HI, haemagglutination inhibition; IFAT, indirect fluorescent antibody test; TCID. tissue culture infective dose; SMIC, suckling mouse inlracerebral.
Here we describe an antigen-capture sandwich-ELISA which will detect antigens common to all the Australian flaviviruses and specifically identify a subgroup of encephalitic flaviviruses using monoclonal antibodies. MATERIALS AND METHODS Viruses The following virus strains were obtained from the Queensland Institute of Medical Research Arbovirus reference centre and prepared as 20% suckling mouse brain suspensions in borate-buffered saline pH 9-0 + 7-5'('o bovine serum albumin; MVE strain F3/51; Kunjin (KUN) strain MRM16; Alfuy (ALF) strain MRM3929; Siratford (STR) strain C338; Edge Hilt
