Oncyte avid

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).

Antigens were diluted in protein array buffer (Maine Manufacturing, USA) and protease inhibitor (1:500, BioVision, USA). Antigens were diluted to a final concentration of 0.5 µg/µl and 75 ng/µl for the P particles and the non-structural proteins, respectively. Antigens were spotted in duplicates into 24-pad nitrocellulose coated slides (Oncyte avid, Grace bio-labs, USA) using a sciFLEXARRAYER SX and a Pierce Dispense capillary, PDC coating type2 (SCIENION, Germany). Slides were blocked with 125 µl blocking buffer (ThermoFisher) at 37 °C for 1 h. Sera and antibodies were diluted in blocking buffer substituted with 0.1% Tween 20 Surfactant (ThermoFisher). For all washing steps, PBS was used. After blocking, slides were incubated for 1 h at 37 °C with 125 µl of sera in fourfold dilution starting at 1:20 or 1:40, as stated in the text. Slides were washed six times and incubated with goat anti-human IgG, conjugated with Alexa Fluor 647 (1:1300, Jackson Immuno Research) and Cy3-AffiniPure Goat Anti-Human IgA (1:200, Jackson Immuno Research). Slides were washed with PBS, then with water, and dried. Bound dye was quantified using the PowerScanner (TECAN, Switzerland). As positive control, human immunoglobulin G ((100 mg/ml), Privigen, Germany) was used per slide in the same dilutions as the children’s sera.

RPPA were constructed with microdissected breast tissue lysates diluted to 0.25 µg/mL, commercial cell line lysates, and bovine serum albumin (BSA, Pierce #23209) [36 –38 ]. Lysates were printed in technical replicates onto nitrocellulose coated glass slides (Oncyte Avid, Grace Bio-Labs), using an Aushon 2470 arrayer (Quanterix) equipped with 350 μm solid pins. SK-BR-3 nuclear extract (Santa Cruz Biotechnology sc-2134, breast adenocarcinoma), MCF7 + EGF + β-estradiol (Santa Cruz Biotechnology sc-24730, breast adenocarinoma), and BT-474 (ATCC HTB-20) cell lysates were printed on each array as quality control samples. BSA was printed in a calibration curve to quantify total protein per spot [37 , 39 ] using Sypro Ruby protein blot stain (Invitrogen/Molecular Probes) per manufacturer’s directions and scanned using a Cy3 laser (Tecan Power Scanner).

RPPA experiments were performed as previously described [33 (link),34 (link),35 (link)]. Briefly, cell lysates from three biological replicates for each condition were spotted in nitrocellulose-coated glass slides (Oncyte Avid, Grace-Biolabs, Bend, OR, USA) in technical triplicates using the Aushon 2470 contact printer equipped with 185 µm solid pins. All primary antibodies used were previously validated using Western bloting to prove specificity. Signal intensities of spots were quantified using GenePixPro 7.0 (Molecular Devices) and RPPA raw data preprocessing and quality control were performed using the RPPanalyzer R-package [36 (link)]. Intensity values were log2 transformed and plotted using Morpheus software (https://software.broadinstitute.org/morpheus). Log2 transformed values are listed in Table S3 and antibodies used in the study are listed in Table S4.