Innoscan 1100al microarray scanner

Macaque serum samples from a subset of pregnant dams (n=11) were screened for both IgM and IgG antibody binding to the peptide array at specific post-infection timepoints: 13-17 and 21-24 DPI for IgM and 27-31 and 108-135 DPI for IgG (Supplementary Table 1). This subset was selected based on the dams that had completed pregnancy at the time the peptide array was performed. Samples were diluted 1:50 in binding buffer (0.01M Tris-Cl, pH 7.4, 1% alkali-soluble casein, 0.05% Tween-20). Diluted serum aliquots and negative controls (binding buffer only) were bound to arrays overnight for 16–20 hours at 4 °C. After binding, the arrays were washed 3x in wash buffer (1x TBS, 0.05% Tween-20), 10 minutes per wash. Primary sample binding was detected via a fluorescently labeled anti-primate IgM or IgG antibody (Supplementary Table 2). The secondary antibody was diluted in secondary binding buffer (1x TBS, 1% alkali-soluble casein, 0.05% Tween20) and incubated with arrays for 3 hours at room temperature, then washed 3x in wash buffer (10 minutes per wash) and 30 seconds in reagent-grade water. The IgM antibody was diluted 1:20,000 and the IgG antibody was diluted 1:40,000. Fluorescent signal of the secondary antibody was detected by scanning at 635 nm at 2 µm resolution and 4% gain, using an InnoScan 1100 AL microarray scanner (Innopsys).

Recombinant HA trimers (50 μg ml⁻¹ final) were pre-complexed with the anti-His mouse antibody (Thermo Fisher Scientific) and the Alexa488-linked anti-mouse IgG (Thermo Fisher Scientific) at 4:2:1 molar ratio for 15 min on ice in 50 μl PBS-T. This complex was incubated on the array surface in a humidified chamber for 60 min before washing and analysis. For GNL detection of whole viruses, virus stocks diluted to 256 HAU (final) in PBS, 3% BSA were incubated on the array surface in a humidified chamber for 60 min, followed by washing and incubation with either GNL:Streptavidin-Alexa488 for a final 60 min. Following final washing, all arrays were scanned using an Innoscan 1100AL microarray scanner (Innopsys). A complete list of the glycans on the array is presented in Supplementary Data S1, glycan schematics/cartoons shown are according to the Symbol Nomenclature for Glycans recommended by the NLM.^{67 ,68 (link)} Fully processed glycan array plots are presented in Supplementary Data S2, while full descriptions of the microarray experiment and datasets are presented in Supplementary Data S5 and S6 according to the MIRAGE consortium format.^{78 (link),79 (link)}

Recombinant trimeric HA0 was expressed in HEK293S GnTI^-/- (American Type Culture Collection), purified and analyzed on glycan array as previously described [35 (link)]. Briefly, soluble trimeric HA (50 μg mL^-1) was pre-complexed with the anti-HIS mouse antibody (MA1-21315, Thermo Fisher Scientific, Waltham, MA) and the Alexa647-linked anti-mouse IgG (A-21235, Thermo Fisher Scientific) at 4:2:1 molar ratio for 15 min on ice in 50 μL PBST. This complex was incubated on the array surface in a humidified chamber for 60 min before washing and analysis using an Innoscan 1100AL microarray scanner (Innopsys, Chicago, IL). Fluorescent signal intensity was measured using Mapix (Innopsys) and mean intensity minus mean background of 4 replicate spots was calculated. A complete list of the glycans on the array is presented in S11 Fig.