Abstract
BackgroundAlternative methods are being sought to measure the potency of influenza vaccines. Label-free technologies that do not require the use of hemagglutinin (HA)-specific antisera are particularly attractive as the preparation of antiserum delays availability of potency reagents. The objective of these experiments was to evaluate the use of a Corning Epic® label-free method to quantify functional influenza hemagglutinin in rHA preparations. The method was optimized to quantify recombinant HA (rHA) of B/Brisbane/60/2008 (B/BR/08). Fetuin was immobilized onto plates and the change in wavelength of refracted light measured using an Enspire (Perkin Elmer) instrument.ResultsThe change in wavelength measured in response to addition of rHA of B/BR/08 was proportional to its concentration and was optimal in the presence of native rHA conformations. However, the assay was strain-dependent and did not correlate with HAU measured using turkey red blood cells.ConclusionsThe Corning Epic® label-free method is suitable for quantifying the native forms of rHA for B/BR/08 and A/Brisbane/59/2007 (H1N1) and A/Hangxhou/3/2013 (H7N9). This method is a useful tool for research purposes but further investigation is needed to identify suitable glycoproteins to use as ligands that allow quantification of HAs from a broader range of virus strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-015-0019-5) contains supplementary material, which is available to authorized users.
Highlights
Alternative methods are being sought to measure the potency of influenza vaccines
single radial immunodiffusion (SRID) is suitable as a potency assay because it clearly distinguishes between antigen that induces hemagglutination inhibiting (HAI) antibodies and denatured antigen that induces poor HAI titers, the antiserum needed for this assay can take many weeks to produce, when there are difficulties in purifying the HA immunogen
Optimization of conditions for quantitation of HA-receptor binding by label-free Epic® The steps conducted to quantify ligand with capacity to bind receptor are shown in Figure 1: (i) fetuin immobilization step: this is the chemical conjugation of the receptor-containing glycoprotein to pre-activated plates; (ii) wash step: following wash and equilibration in the ligand buffer, the plate is read to establish a baseline reading; (iii) HA binding step: the ligand is added and the wavelength of refracted light is measured by a sensor in the portion of the well containing immobilized fetuin and an internal control sensor located in a portion of the well that has no capacity to immobilize fetuin
Summary
Label-free technologies that do not require the use of hemagglutinin (HA)-specific antisera are attractive as the preparation of antiserum delays availability of potency reagents The objective of these experiments was to evaluate the use of a Corning Epic® label-free method to quantify functional influenza hemagglutinin in rHA preparations. SRID is suitable as a potency assay because it clearly distinguishes between antigen that induces hemagglutination inhibiting (HAI) antibodies and denatured antigen that induces poor HAI titers, the antiserum needed for this assay can take many weeks to produce, when there are difficulties in purifying the HA immunogen Since this bottleneck could impact timely availability of seasonal and pandemic influenza vaccines, alternative potency assays that do not require the use of antisera are being sought [2]. The ease of measuring interactions of the native molecules without the use of labeled antigen-specific antibodies or other staining techniques, make label-free systems ideal for investigating functional interactions between large multimeric glycoproteins and cellular receptors
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