In both cases, antigens were arrayed in square patterns as indicated by the yellow boxes in (a); a mouse IgG Fc domain was included as negative control (red boxes). of an expanded label-free hemagglutinin microarray to the analysis of avian serum samples. Samples from influenza virus challenge experiments in mallards yielded strong, selective detection of antibodies to the challenge antigen in most cases. Samples acquired in the field from mallards were also analyzed, and compared with viral hemagglutinin inhibition and microneutralization assays. We find that the AIR hemagglutinin microarray can provide a simple and robust alternative to standard methods, offering substantially greater information density from a simple workflow. == Introduction == Current methods of influenza serology including hemagglutination inhibition (HI), microneutralization (MN), and enzyme-linked immunosorbant assays (ELISA) have proven to be broadly useful in the clinical laboratory [1]. In the context of surveillance and evaluation of vaccine efficacy applications, however, the limitations Betamethasone acibutate of these assays including their complexity and ability to only provide information about a single antibodyantigen response at a time have proven problematic. Because of this, it is widely recognized that there is a need for new methods for detecting influenza antibodies [2]. Ideally, such technologies should be able to provide quantitative information about several antibody responses to different antigens simultaneously (i.e. a multiplex test) while doing so in a fast, reagentless, sample-conserving way (as, particularly for avian surveillance, limited volumes of sample are available), and independently of the host species tested. As alternatives to the traditional serologic assays, these Betamethasone acibutate new methods could dramatically simplify the process of analyzing samples acquired in the field. We have recently developed a technology that should prove useful in addressing this goal. Arrayed Imaging Reflectometry, or AIR, is a label-free biosensor technique able to provide quantitative information on 10s to 100s of analytes simultaneously, while requiring low sample volumes (< 25 microliters) and simple instrumentation. In brief, AIR relies on the creation of a near-perfect antireflective condition on the surface of a silicon chip [3]. When target molecules bind to immobilized probes (antibodies or antigens) on the surface of the chip, this causes a disturbance in the antireflective condition, producing a change in the reflected light that quantitatively and sensitively reports the amount of the target analyte present in a sample. As a label-free technique, AIR utilizes a simple work flow involving only application of the diluted sample to the chip, incubation, and a final rinse and dry step prior to imaging. This system can be implemented using an imaging system that has no moving parts, no need for temperature control, and an estimated component cost of under $5000. Further details of the Rabbit Polyclonal to OR5W2 method, and its application to a broad range of targets, have been reported elsewhere [4]. In preliminary studies focused on influenza antibody detection, we examined the performance of an AIR array consisting of 5 hemagglutinins, with human samples derived from a trial of a candidate Betamethasone acibutate H5N1 flu vaccine [5]. We found that this array readily enabled us to profile relative antibody responses (many of which were cross-reactive) in human serum, and differentiate subjects receiving placebo from those to whom the candidate vaccine had been administered. Other groups, using labeled approaches (in which a fluorophore-tagged secondary antibody is incubated with the array post sample incubation, allowing readout via a fluorescence microarray scanner), have similarly examined the utility of influenza antigen microarrays for assessing responses to infection or vaccination [610]. A random peptide library has also been employed in this context [11]. Following these initial Betamethasone acibutate validation experiments, we sought to determine if the AIR Betamethasone acibutate hemagglutinin microarray could be used in the context of influenza surveillance in avian species. In particular, the label-free aspect of the AIR hemagglutinin array is attractive with this software, since the simplified workflow of such a device potentially allows for the production of field-deployable, self-contained devices. Such devices would allow for the immediate analysis of samples in the field, rather than requiring their transport back to a centralized facility. To that end, we statement here the extension of the Air flow HA array to a larger quantity of HA isoforms, and its use in the context of analyzing avian serum samples. == Materials and Methods == == Ethics Statement == All animal work was examined and authorized by the University or college of Georgia Institution Animal Care and Use Committee (AUP#: A2013 05021) == Label-free substrates == Amine-reactive Air flow chips were purchased from Adarza BioSystems, Inc. == Recombinant hemagglutinins == The following recombinant hemagglutinins (Table 1) were purchased from Sino Biological, Inc., and used as supplied. == Table 1. Recombinant hemagglutinins.