Piezorray spotter

IgG levels against different human and avian influenza HA types were measured using a protein microarray platform as described previously30 (link)31 (link)32 (link)33 (link)34 (link)35 (link). Briefly, recombinant proteins of the HA1 part of HA of different influenza virus subtypes (see supplementary material S2) were printed onto nitrocellulose-coated glass slides (64pad, Oncyte Avid, Grace Biolabs, Bend, USA) using a non-contact Piezorray spotter (Perkin Elmer, Waltham, USA). Subsequently, dried blood spots were eluted as described previously and samples were tested at a 1:80 dilution30 (link). A Dylight649-labelled goat-anti-human IgG (Fc-fragment specific, Jackson ImmunoResearch) was used to bind to serum antibodies and fluorescence was quantified by means of a microarray scanner (ScanArray, Perkin Elmer). The protein microarray technique allows simultaneous and standardized detection of antibodies against different influenza subtypes in a minute serum quantity. It has also been used to measure influenza IgG titers in humans35 (link).

Purified P particles and VLPs were diluted in protein array buffer (Maine Manufacturing) and protease inhibitor (BioVision), with final concentration of 1 mg ml⁻¹ (determined by checkerboard titration, data not shown). Proteins were spotted in triplicate with two 333 pL spots onto 64-pad nitrocellulose-coated slides (Oncyte avid, Grace bio-labs) using a non-contact Piezorray spotter (PerkinElmer) as described previously (Koopmans et al., 2012 (link)). Slides were incubated with Blotto blocking buffer (Thermo Fisher Scientific) to avoid non-specific nitrocellulose binding, and subsequently with serial fourfold diluted human sera starting at a 1 : 40 dilution. Rabbit sera and B-cell supernatants were tested at a single dilution (1 : 20 for rabbit sera, 1 : 8 for B-cell supernatant pools and 1 : 4 for individual cultures). After washing, slides were incubated with goat anti-human IgG or anti-rabbit IgG (Fc-fragment specific), conjugated with Alexa Fluor 647 fluorescent dye (Jackson Immuno Research). Bound dye was quantified using a ScanArray Gx Plus microarray scanner (PerkinElmer).