A Novel Synthetic Receptor-Based Immunoassay for Influenza Vaccine Quantification

Abstract

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.

A monoclonal antibody‐based immunoassay for measuring the potency of 2009 pandemic influenza H1N1 vaccines

“…Other studies have also evaluated the feasibility of an ELISA approach to determining influenza vaccine potency . Indeed, the idea of using a mAb‐based ELISA as a potency assay for inactivated influenza vaccines was proposed nearly 30 years ago .…”

Collaborative studies on the development of national reference standards for potency determination of H7N9 influenza vaccine

“…While several clinical trials have shown that SRID data correlate well with sero-conversion and vaccine efficacy. [25][26][27] Our future plan is to collect sera of patients enrolled in the clinical trials on these H7N9 vaccines and investigate the correlation of sero-conversion to potency of the vaccines determined by SRID using our reagents. Interested investigators may contact the authors for the antigen and antibody reagents in their exploratory research.…”

Standardisation of inactivated influenza vaccines—Learning from history

“…To take advantage of this property, receptor‐binding alternative potency assays have been developed in ELISA45 and SPR46 formats. The advantages of a receptor‐binding approach are that it is selective for conformationally correct antigen and does not require strain‐specific antibody reagents for the assay set‐up.…”