A miniaturized assay for influenza neuraminidase-inhibiting antibodies utilizing reverse genetics-derived antigens

Abstract

Background: Antibodies to neuraminidase (NA) contribute to protection during influenza virus infection, but NA inhibition (NI) titers are not routinely analyzed in vaccine trials. One reason is the cumbersome nature of the conventional thiobarbituric acid (TBA) NI assay, which uses chemical methods to quantify free sialic acid following incubation of NA with substrate in the presence of serum. In addition, the assay is complicated by the need to use virus of a hemagglutinin (HA) subtype novel to the host to detect NA-specific antibodies only.

Objectives: Our primary objectives were to miniaturize the colorimetric NI assay to a format suitable for quantitative analysis of large numbers of samples, and validate the specificity and sensitivity of the miniaturized format with ferret and human sera. An additional aim was to use reverse genetics to construct HA-mismatched viral reagents bearing NA of recent influenza A vaccine strains and H6 HA.

Results: Analysis of ferret antisera by the miniaturized assay demonstrated sensitivity and specificity comparable with the conventional assay. Similar increases in the NI titers in sera from vaccinated human volunteers were measured in miniaturized and conventional assays. Inactivated and live-attenuated vaccines increased NI titers against a given subtype at approximately the same rate.

Conclusions: The reagents and miniaturized format of the TBA method described here provide a platform for practical serological monitoring of functional antibodies against NA.

Figure 1

Colorimetric detection of sialic acid in a miniaturized assay format. (A) Serial dilutions of purified N‐acetyl neuraminic acid were added to duplicate wells of a 96‐well PCR microplate, converted to chromophore by the miniaturized periodate‐thiobarbituric acid reactions, extracted, and measured in terms of absorbance at 550 nm. (B) Allantoic fluid stocks of reverse genetics‐derived viruses H6N1_NewCal/99 and H6N2_Wis/05 were serially diluted and incubated overnight with fetuin substrate in duplicate microplate wells. Liberated sialic acid was assayed in the same manner as N‐acetyl neuraminic acid was assayed in (A). For subsequent NI antibody analysis, each virus was diluted to the concentration which yielded a reading of 1·0. Error bars represent the standard deviation (SD) of replicate wells. At some data points, the SD is not large enough for bars to be visible.

Figure 2

Analysis of ferret antiserum NI activity against homologous and heterosubtypic NA antigens in miniaturized colorimetric assay. Serial dilutions of normal ferret serum (NFS), ferret anti‐A/New Caledonia/20/1999, and ferret anti‐A/Wisconsin/67/2005 were incubated for 30 minutes with (A) H6N1_NewCal/99 or (B) H6N2_Wis/05 antigens in duplicate wells of a 96‐well microplate. Fetuin substrate was added, and samples were incubated 15 hours at 37°C. Sialic acid cleavage from the substrate was quantified using the miniaturized colorimetric assay.