Microbiota-reactive IgA antibodies are abundant in the adult human small intestine. (A) IgA-free microbes were isolated from feces of SPF Rag2-deficient mice. Gates were set according to in vitro–cultured GFP-expressing E. coli and fecal material isolated from germ-free mice (top row). GFP signal or Syto9 nucleic acid dye were used to identify bacteria (bottom row). Anti-human IgG1 antibody was used to detect mAb-positive fecal bacteria. Representative staining is depicted for previously described low (mGO53) and high (ED38) polyreactive control antibodies (Meffre et al., 2004; Wardemann et al., 2003) and staining with supernatant from nontransfected HEK-cells (Med CTRL) on feces from SPF Rag2-deficient mice. (B and C) Two collections of antibodies derived from IgA- or IgG-expressing plasmablasts/plasma cells from HDs (Benckert et al., 2011) or CD patients were screened for reactivity to microbes isolated from SPF Rag2-deficient mice. (B) Symbols represent individual antibodies (162 HD mAbs and 118 CD mAbs) assayed in one representative experiment. Binding to <1% of bacteria was considered background (dashed line). Groups were compared by Kruskal–Wallis and post hoc Mann–Whitney U test (**, P < 0.001; ***, P < 0.0001). (C) Antibodies were categorized according to the following binding capacities: no/low (0–1% of bacteria), intermediate (1–5%), and high (≥5%). FSC-A, forward scatter area; SSC-A, side scatter area; SSC-H, side scatter height.