Application of Enzyme-Linked Lectin Assay for Evaluation of Neuraminidase and Neuraminidase Inhibition Activity of Avian Orthoavulavirus 1 (AOAV-1) and AOAV-1 Antibody.

Detection of antibody response to Newcastle disease virus (NDV) or other avian orthoavulaviruses 1 (AOAVs-1) can serve as a useful tool for monitoring infection and response to vaccination. Two commonly used serologic tests for AOAV-1 are the hemagglutinin inhibition (HI) test and ELISA. Enzyme-linked lectin assay (ELLA) is based on the ability of the neuraminidase (NA), which is also part of the hemagglutinin-neuraminidase (HN) protein of AOAV-1, to cleave sialic acid residues from a substrate coated on the solid surface, and the assay is performed similarly to ELISA. The same ELLA can be applied to detect and quantify the neuraminidase inhibition (NI) antibody present in the sample. In this study, we first evaluated ELLA for measuring NA activity of different AOAV-1 strains on fetuin substrate. In contrast to influenza A virus, which shows optimal NA activity in neutral pH, all AOAV-1 strains tested showed higher NA activity under lower pH and the preference of specific pH varied by the strain. We established assay parameters, including the pH and temperature conditions, for optimal NA activity of two AOAV-1 strains and conducted ELLA to evaluate NI antibody (NI-ELLA). NI-ELLA was highly specific with antigen dose of 95% effective concentrations (EC₉₅), and showed less than 10% background nonspecific NI reactivity with all AOAV-1-negative sera collected from different poultry species. NI-ELLA was conducted to determine the endpoint titer of sera collected from birds vaccinated with Newcastle disease vaccine. The NI antibody titer determined by NI-ELLA correlated well with the HI antibody titer against the same antigen used for both tests, while showing a detection limit more than 20 times higher. Our study shows great potential of NI-ELLA as a functional antibody assay like the HI test to evaluate vaccine-induced antibody response against challenge virus, with the added advantages of being more sensitive and having a higher-throughput screening capability than the HI test.