In humans, both humoral and cellular immune effector mechanisms play important roles in host protection against influenza. However, the humoral immunity response is the most reliable correlate of protection against influenza in humans and includes mucosal and serum antibody responses. Response to the HA protein is most important since HA-specific antibodies can neutralize the virus by preventing it from initiating an infection. Neutralization involves blocking binding of the virus to host cells but may also work at other steps involved in the entry and uncoating of the virus (21
These assays in which the biosensor probe is loaded with HA analyze the polyclonal antibody response to HA but are different from the HI and MN assays in that they measure the total antibody response and cannot distinguish between antibodies binding near the RBS (as in the case of the HI test) and other nonneutralizing antigenic sites. Although nonspecific effects such as binding serum glycoproteins and glycolipids containing sialic acid may affect this assay, we minimize these effects by both RDE treatment and dilution (at 1:80) of the sera prior to analysis. In addition the assay could be focused further to highlight only bound antibodies by utilizing an additional step using an anti-human Ig polyclonal reagent, similar to the isotyping experiment shown in Fig. . However, comparison of the reactivity of ferret sera in this assay to that in a corresponding HI assay revealed a good correlation, although there was, in general, increased detection in the biosensor assay (from 2- to 8-fold depending on the HA being analyzed). Comparing results from H1, H3, and H5 revealed the best correlation to be that with the H3 HAs analyzed (Table ).
The increased disparity between the detection level observed and HI results for the H1 and H5 HAs may be due to the presence of non-HI antibodies. Results with the H5-VN04 HA suggest this to be the case, as although binding of the sera was detected down to dilutions of 1:640 to 1,280, fetuin binding to the receptor-binding site could be inhibited effectively only at dilutions of 1:160 (Fig. ). Thus, our H5 (and H1) antisera appear to contain a higher proportion of non-HI antibodies than do antisera to H3 HA. The ability of an antibody to block access to the receptor-binding site, as described for Fig. , is the basis of an HI assay. However, this assay requires an available HA, and currently, it takes 4 to 5 weeks to produce recombinant HA in quantities sufficient for analysis. Thus, such a biosensor assay cannot replace the HI in its current format, particularly in a pandemic situation. However, its future development to define reagents that can bind to human-adapted seasonal and pandemic virus HAs could be useful in situations where it can complement current assays such as analyzing a patient's immune response to infection and/or vaccination.
The assay platform was further characterized by a limited analysis of human sera from vaccine efficacy trials with both TIV and LAIV, as well as anonymous human serum samples from the Red Cross (collected during November and December 2008). Although the two vaccines stimulate different compartments of the immune system (3
), both prevent laboratory-confirmed influenza (3
) and reduce viral shedding and the severity of respiratory symptoms (5
). TIV vaccines produce a significantly higher level of neutralizing serum IgG (particularly IgG1) and IgA antibody responses than do LAIV vaccines (6
). Our results are consistent with all these observations in that a greater difference between pre- and postvaccination sera was seen with the sera collected from people receiving the TIV vaccine (Fig. ). Interestingly, a number of the sera also from the general population had appreciable binding to H3-BR07 and H1-BR07 (e.g., GP4 and GP6), suggesting that these people may have been vaccinated, while one sample (GP5) bound to H3-BR07 only, suggesting possible exposure to a circulating H3 virus.
The serum antibody response to influenza virus infection or vaccination is commonly measured using the HI test and can be detected in approximately 80% of subjects after natural influenza virus infection with an HI titer of ≥40 (14
). Indeed, an HI titer of 40 is correlated with protective immunity to antigenically homologous viruses (14
). Based on this pilot study comprising only a limited number of positive samples, it is not possible to establish a reliable correlation between biosensor binding data and HI titers. However, as more reagents and human sera are screened, assay performance can be subjected to a more rigorous examination and accuracy/significance will be determined.
With respect to influenza, the next generation of assays should be able to detect antibodies in serum and plasma, with high sensitivity and minimal sample processing, while minimizing cross-reactivity and nonspecific binding. Biolayer interferometry technology was selected for study/development because this platform satisfies a number of these criteria. In addition, there are no long incubation steps in the analysis, which improves sample throughput. Indeed, assuming a 5-min analysis time to assess antibody binding to the recombinant HA, up to 96 samples (including controls) can be analyzed in approximately 1 h using the current system. By using such a short analysis time, multiple HAs on separate probes can be used to analyze a single sample in the same assay to look at cross-reactivity to other HAs from similar and different subtypes. Finally, biosensors are disposable, thus reducing the logistics for biosensor regeneration, which might lead to cross-contamination and/or loss of performance.
In order to compare results with different sera and HAs, we thus chose to present binding during each run calculated as a percentage of the maximum binding observed with homologous ferret sera at a 1:80 dilution. In this way, variation between experiments can be normalized using sera as a positive control. Interestingly, not all of the homologous ferret sera resulted in a maximum binding response for all HAs tested (for example, in Fig. , anti-H3-WI05-L binds consistently better to the H3-BR07 HA than does the homologous anti-H3-BR07 serum). Similarly, human sera from vaccinees (TIV4 and TIV6) and the general population (GP6) all bound to H1-SI06 at a level higher than did the ferret sera normalized at the 100% level (Fig. ). Thus, other antibody sources such as those in hyperimmunized sheep sera with maximal-avidity binding could be considered possible alternatives to ferret sera.
Ultimately, the usefulness of any assay for medium-/high-throughput analysis in the public health arena requires reagents to be in a ready-to-use format and preferably have a long shelf life. Preliminary tests on H5 HA stability proved that recombinant HA can be prepared on probes and stored at room temperature for more than 2 months without loss of performance, as measured by neutralizing monoclonal antibody binding as well as binding of fetuin, a sialic acid-containing receptor surrogate. Further development of this methodology for other HAs is required; this technology could help to standardize methodology by minimizing inter- and intralaboratory variability, improving reagents, and introducing assay automation. Future development of such reagents and alternative technologies to include multiplexing might also offer a robust platform for future influenza surveillance activities.