An Enzyme Immunoassay to Detect Australian Flaviviruses and Identify the Encephalitic Subgroup using Monoclonal Antibodies

Abstract

Summary An antigen‐capture enzyme‐linked immunosorbent assay (ELISA) has been developed to detect antigens of Australian flaviviruses in mosquito pools, suckling mouse brain and infected cell culture supernatant fluid. A monoclonal antibody reactive to an epitope on the envelope glycoprotein common to all flaviviruses was used as the capture antibody. Purified rabbit IgG, produced against Murray Valley encephalitis (MVE) virus, which reacted with eight Australian flaviviruses in haemagglutination inhibition (HI) and in an indirect fluorescent antibody test, was used as the indicator antibody in direct and indirect antigen‐capture ELISA. A monoclonal antibody specific for a subgroup of encephalitic flaviviruses was conjugated to horseradish peroxidase and used as the indicator antibody to distinguish MVE, Kunjin and Alfuy viruses from the remainder tested. This ELISA could detect viral antigen in mosquito cell culture fluids and suckling mouse brain preparations at titres as low as 1000 TCID50/100μ1. Viral antigen in a single mosquito infected with MVE could be detected in a pool of 500.

Epitope Analysis of the Envelope and Non-structural Glycoproteins of Murray Valley Encephalitis Virus

“…This cell line produces an IgG2a MAb (4G2) that reacts with a flavivirus group-specific epitope and has neutralizing and HI activity (Henchal et al, 1985). Production and purification of rabbit antibodies to MVE have been previously described (Hall et al, 1987).…”

“…Fourteen hybridomas were selected for further study on the basis of their specificity and were cloned at least twice by limiting dilution before antibody was produced as ascitic fluid. Each MAb was retested for viral specificity in EIA and examined for haemagglutination inhibition (HI) and neutralizing activity to MVE/3/51 by standard methods (see Hall et al, 1987). The isotype of each antibody was determined using the Mouse Typer kit (Bio-Rad).…”

Type‐specific monoclonal antibodies produced to proteins of Murray Valley encephalitis virus

“…Mou.se hyperimmune antiserurn was prepared as described previously (9). A monoclonal antibody reacting to the E protein of dengue 2 virus, D1-4G2-415 (4G2).…”

“…(18). Conjugation of purified antibodies to horseradish peroxidase (Sigma type VI) by a modification of the method of Wilson and Nakane (19) has been described previously (9).…”

“…ELISA and neutralisation tests. These assays were performed as described above and previously (9). A description of the MVE isolates used is given in Table 1.…”