Fig. 1. Illustration of the enzyme-linked lectin assay (ELLA) for comparing NA antigenicity.

Step A1, viruses are titrated using fetuin-coated plates. Step A2, NA exposed galactose residues on the fetuin glycans are bound by peanut agglutinin linked to horseradish peroxidase (PNA-HRP). Step A3, PNA-HRP binding is determined using the substrate OPD and measuring the absorbance (Abs) at 490 nm. Step B1, Ferret antisera are titrated with a fixed virus dilution from the linear range and added to fetuin-coated plates and processed similarly for PNA-HRP binding (Step B2) and development (Step B3). NA inhibition (NAI) titres are determined by inversely plotting the NA activity data and calculating the reciprocal sera dilution that corresponds to the 50% inhibitory concentration (IC₅₀).