Application of enzyme-linked lectin assay for evaluation of neuraminidase and neuraminidase inhibition activity of Avian Orthoavulavirus 1 (AOAV-1) and AOAV-1 antibody

Authors: Lee, Chang; Gladney, Jessica

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/24/2025
Publication Date: 8/29/2025
Citation: Lee, C.W., Gladney, J.D. 2025. Application of enzyme-linked lectin assay for evaluation of neuraminidase and neuraminidase inhibition activity of Avian Orthoavulavirus 1 (AOAV-1) and AOAV-1 antibody. Journal of Virological Methods. Avian Diseases, 69(3) : 326-333. https://doi.org/10.1637/aviandiseases-D-25-00049 .
DOI: https://doi.org/10.1637/aviandiseases-D-25-00049

Interpretive Summary: Detection of antibody response to Newcastle disease virus (NDV) or other avian orthoavulaviruses 1 (AOAVs-1) can serve as a useful tool for monitoring the infection and response to vaccination. Two commonly used serologic tests for AOAV-1 are hemagglutinin inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA). Enzyme linked lectin assay (ELLA) is based on the ability of the neuraminidase (NA), which is part of the hemagglutinin-neuraminidase (HN) protein of AOAV-1, to cleave sialic acid residues from a substrate coated on the surface of 96-well plates. The same assay can be applied to measure 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 virus which shows optimal NA activity in neutral pH, all AOAV-1 strains tested showed higher NA activity in lower pH condition and the preference of specific pH varied by the strain. We established assay parameters including the pH and temperature condition for optimal NA activity of AOAV-1 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 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 more than 20 times higher detection limit. Our study shows great potential of NI-ELLA as a functional antibody assay like HI test to evaluate vaccine induced antibody response against challenge virus with an added advantage of higher sensitivity and high-throughput screening capability of ELISA.

Technical Abstract: Newcastle disease (ND) is caused by infections with virulent strains of avian orthoavulavirus 1 (AOAV-1) and is one of reportable diseases to the World Organization for Animal Health due to its devastating impact on poultry industry. The ND virus cause a systemic infection, spread throughout the body, and produce up to 100% mortality in non-vaccinated chickens. ARS researchers established enzyme-linked lectin assay (ELLA) as a functional analysis tool to evaluate and characterize the hemagglutinin-neuraminidase (HN) protein activity of different AOAV-1 strains. ARS scientists further demonstrated that the new assay can evaluate neuraminidase inhibition antibody titers in sera collected from birds vaccinated with ND vaccine which correlated well with hemagglutination inhibition (HI) antibody titer (the most common assay for evaluation of immune response) with higher sensitivity. This study shows that the ELLA carries the advantage of two commonly used serologic tests, HI test and ELISA, and the newly established assay can serve as useful tool for monitoring the AOAV-1 infection and antibody response to vaccination.