Anti human iga

Fc array analysis was performed blinded to group as described (47 (link), 48 (link)) to evaluate polyclonal Ab responses. Briefly, plasma was centrifuged for two min at 14.8 x g, then aliquoted and analyzed for binding to a panel of antigens listed in Supplementary Table 1. Plasma was diluted 1:1,000 for detection reagents (Supplementary Table 2) of tetramerized Fcγ receptors (Source: Duke Protein Production Facility) and anti-rhesus IgG (Source: Southern Biotech). For increased sensitivity, detection reagents C1q (Source: Sigma) and anti-human IgA (Source: Southern Biotech) were collected with a plasma dilution of 1:250, along with a replicate of the anti-rhesus IgG. Data collection was performed using Luminex Exponent version 4.2 software.

For immunofluorescence, tissues were immediately isolated, fixed for 2 h with 4% paraformaldehyde in PBS at 4 °C and soaked in 30% sucrose in PBS overnight at 4 °C. Tissues were then embedded in Tissue-Tek OCT blocks (Sakura) and frozen in liquid nitrogen. The frozen samples were sectioned at a thickness of 10 μm by cryostat (Leica, CM3050S). The following antibodies were used: anti-mouse CD3ε (BioLegend, clone 145-2C11), anti-mouse B220 (eBioscience, clone RA3-6B2) and anti-mouse CD11c (SouthernBiotech, polyclonal). Tissue sections were counterstained with DAPI (Sigma-Aldrich) and mounted with Fluoromount-G antifade reagent (SouthernBiotech).
For human tissue staining, tissues were freshly isolated, embedded in Tissue-Tek OCT blocks (Sakura) or SCEM (SECTION-LAB) and frozen in liquid nitrogen. The frozen samples were sectioned at a thickness of 10 μm by cryostat (Leica, CM3050S). The following antibodies were used for immunohistochemistry staining: anti-human CD68 (eBioscience, clone 815CU17), anti-human CD19 (Abcam, clone EPR5906) and anti-human IgA (SouthernBiotech, polyclonal). Fluorescence images were obtained using a BZ-X700 fluorescence microscope (Keyence).

EIA was performed according to a comprehensive published method (Protocol 3.3.1. in CPM [15 (link)]) with the following modifications (a step-by-step protocol is provided in S1 Text). Briefly, purified recombinant proteins quantified by the Lowry protein assay kit (Thermo Fisher Scientific, Waltham, MA) or whole-cell extract of Leptospira diluted in 1X sodium carbonate coating buffer was used to coat Nunc MaxiSorp flat-bottom EIA plates (eBioscience, San Diego, CA) at 0.5–1 μg/ml (protein) or 10⁵-10⁸cells/well (Leptospira extract) overnight at 4°C. The following day the plates were washed with 1XPBS, blocked with 1% BSA for 2h, washed again, and incubated with human or murine serum diluted at 1:50 or 1:100. Goat anti-mouse or goat anti-human IgG conjugated to HRP diluted at 1:10000 (Jackson ImmunoResearch, West Grove, PA) was used as the secondary antibody. The OD₄₅₀ was read on a SpectraMax Plus EIA reader (Molecular Devices). For determination of Ig class and IgG isotypes the secondary antibody conjugated to HRP was diluted as follows: anti-human -IgG1, -IgG2, -IgG3, -IgG4 (1:1000, Invitrogen), anti-human IgA (1:8000, Southern Biotech), anti-human IgD (1:1200, Southern Biotech) and IgM (1:10,000, Jackson ImmunoResearch Laboratories, Inc.). The EIA cutoff was set at three standard deviations above the average OD₄₅₀ of all healthy control samples.