Monoclonal antibody-based ELISA for detection of antibodies against H5 avian influenza viruses

Diagnostic and containment measures are essential for the management of avian influenza. In this study, a monoclonal antibody (MAb)-based competitive ELISA for detecting antibodies against H5 avian influenza viruses was developed and validated. Twenty-five anti-H5 MAbs were characterised using competitive, indirect and sandwich ELISAs, immunofluorescence, Western blotting and virus neutralisation and haemagglutination inhibition assays. One MAb (5D8) with wide intra-subtype cross-reactivity was selected and characterised using escape mutant selection. Epitope analysis showed that this MAb recognises a conformational epitope comprising amino acid residues in positions 170, 235 and 240 located in the receptor binding domain. The diagnostic performance of the test was evaluated by ROC analysis using a panel of 950 known sera collected from different avian species, including chickens, turkeys, ducks, pheasants, wild Anseriformes and ostriches. The competitive ELISA had excellent diagnostic performance and discriminatory power with high Se and Sp values (Se: 99.6-95% CI 98.0-100; Sp: 99.4-95% CI 98.5-99.8). In addition to its excellent diagnostic performance, properties of the competitive ELISA, such as high feasibility of testing sera without pre-treatment and potential for automation and instrument-mediated detection, make it ideal for screening samples, confirming positive HI assay results or analysing samples that are difficult to test using the HI assay.