Label‐Free HPLC Method to Evaluate Neuraminidase Activity in Biological Fluids Containing the Newcastle Disease Virus

ABSTRACT Newcastle disease virus utilizes its multifunction hemagglutinin neuraminidase (HN) protein for sialic acid recognition and its cleavage from the adjacent lactose unit. Detection of neuraminidase activity of HN is crucial for studying NDV infection biology. Traditional neuraminidase assays predominantly employ synthetic fluorogenic substrates such as 2′‐(4‐methylumbelliferyl)‐α‐D‐N‐acetylneuraminic acid. However, existing methods are limited by their inability to accurately mimic the natural environment of glycan substrates. Although the majority of neuraminidase assays in current use have been optimized for influenza neuraminidases, methods specifically developed and validated for HN of NDV are limited. Here, we present an optimized high‐performance liquid chromatography‐based protocol tailored for NDV‐HN protein that utilizes an Aminex HPX‐87H carbohydrate column for analyzing enzyme activity and sialollactose as a physiologically relevant substrate. Upon enzymatic cleavage by hemagglutinin‐neuraminidase protein, free sialic acid, cleaved from the sialollactose moiety, is directly detected at 210 nm, bypassing the need for derivatization with chromogenic or fluorogenic agents. With the help of standard curves, the quantity of sialic acid released can be efficiently measured under different conditions such as at different pH or in the presence of an inhibitor. It could be crucial for studies evaluating viral infectivity, drug efficacy, and mutant phenotypes of the NDV‐HN protein.