Optimization and diagnostic evaluation of monoclonal antibody-based blocking ELISA formats for detection of neutralizing antibodies to Hendra virus in mammalian sera

•Two Hendra virus monoclonal antibody based ELISAs for multispecies serology diagnosis have been developed.•The two monoclonal antibodies exhibited differing functional characteristics, which enabled identification of false positive reactions and achievement of higher diagnostic specificity.•The two assays exhibited analytical sensitivity characteristics comparable to virus neutralisation test.•Analytical specificity testing revealed strong cross-reactivity with the related Nipah virus, but cross-reactions with other paramyxoviruses were not detected.•The assays were validated for detection of antibody in equine, canine and to a lesser extent to feline sera. However, partial evaluation of megachiropteran sera, suggests a potential application of the assays also for bats. Maintenance of Hendra virus (HeV) in pteropid bat populations has been associated with spillover events in horses, humans and dogs. Experimental studies have demonstrated infections for several other species including guinea pigs, cats and ferrets. The criteria of a sensitive and specific serological test that is effective for a range of species, but which does not require use of live virus, has not been satisfactorily addressed by currently available tests. We have evaluated the use of two HeV neutralizing monoclonal antibodies (mAbs) in a blocking format enzyme-linked immunosorbent assay (bELISA) to detect serum antibody against a recombinant expressed HeV G protein (sol G) in several animal species. The human mAb m102.4 neutralises both HeV and the closely related Nipah virus (NiV); the mouse mAb 1.2 neutralises only HeV. Given these functional differences, we have investigated both antibodies using a bELISA format. Diagnostic sensitivity (DSe) and diagnostic specificity (DSp) were optimized using individual thresholds for mAb 1.2 and m102.4. For mAb 1.2 the positive threshold of >33% inhibition yielded DSe and DSp values of 100% (95% CI 95.3–100.0) and 99.5 (95% CI 98.8–99.8) respectively; for mAb m102.4 a positive threshold of >49% inhibition gave DSe and DSp values of 100 (95% CI 95.3–100.0) and 99.8 (95% CI 99.2–100.0) respectively. At these thresholds the DSe was 100% for both tests relative to the virus neutralization test. Importantly, the occurrence of false positive reactions did not overlap across the assays. Therefore, by sequential and selective application of these assays, it is possible to identify false positive reactions and achieve a DSp that approximates 100% in the te