Microarray Analysis of Antibody Responses

The subset of sera from the original study was analysed with a microarray as described earlier [5] (link)–[8] (link). Briefly, recombinant proteins were produced in human embryonic kidney cells (HEK293) and purified by HIS-tag purification (purity more than 95%), as specified by the manufacturer (Immune Technologies, New York, USA). Oncyte avid nitrocellulose film-slides containing 64 pads per slide were used (Grace bio-labs, Bend, USA), and spot signals were quantified by the use of a Scanarray scanner (Perkin Elmer, Waltham, USA) using an adaptive circle quantification method. Finally, conjugates consisted of goat anti-human IgG (Fc-fragment specific) conjugated with Dylight649-fluorescent dye (Jackson Immuno Research, West Grove, PA, USA).
Table 1 shows the antigens included in the study. Notice that next to the antibody response against the A/2009 (H1N1) pandemic virus, we tested the samples against a range of other antigens, among which A/1918 (H1N1). The hemagglutinin of H1N1 virus of 1918 is genetically and antigenically related to the 2009 virus [11] (link)–[14] (link). Readers of each test (HI and microarray) were blind to results of the other tests, and had no access to ancillary information (age, sex).