Vaccination is the most effective prophylactic method for preventing
influenza. Quantification of
influenza vaccine antigens is critically important before the
vaccine is used for human immunization. Currently the
vaccine antigen quantification relies on
hemagglutinin content quantification, the key antigenic component, by single radial immunodiffusion (SRID) assay. Due to the inherent disadvantages associated with the traditional SRID; i.e. low sensitivity, low throughput and need for annual
reagents, several approaches have been proposed and investigated as alternatives. Yet, most alternative methods cannot distinguish native
hemagglutinin from denatured form, making them less relevant to antigenic analyses. Here, we developed a quantitative immunoassay based on the
sialic acid binding property of
influenza vaccine antigens. Specifically, we chemically synthesized human and
avian influenza virus receptors analogues, N-acetylneuraminic acid-2,6-lactose and N-acetylneuraminic acid-2,3-lactose derivatives with an azidopropyl aglycon, using α-2,6- and α-2,3-sialyltransferases, respectively. The azido group of the two
sialyllactose-derivatives was reduced and conjugated to mouse
serum albumin through a squarate linkage. We showed that the synthetic α-2,6- and α-2,3-receptors selectively bound to human and avian-derived
hemagglutinins, respectively, forming the basis of a new, and robust assay for
hemagglutinin quantification.
Hemagglutinin treated at high temperature or low pH was measured differentially to untreated samples suggesting native conformation is dependent for optimal binding. Importantly, this receptor-based immunoassay showed excellent specificity and reproducibility, high precision, less turnaround time and significantly higher sensitivity and throughput compared with SRID in analyzing multiple
influenza vaccines.