Serum samples from all subjects were screened for the presence of the IgM, IgG, IgA, and secretory IgA (sIgA) isotypes of anti-MAA antibodies as previously described (Anderson et al., 2014 (link)). Briefly, ELISA plates were coated with 2 μg/well of either MAA-Albumin or unmodified Albumin. Additional wells were coated with known concentrations of human IgM, IgG, IgA, or sIgA isotype standards (Sigma) from which the relative antibody concentrations were extrapolated. Plates were incubated overnight at 4°C and then washed, blocked with 2% bovine serum albumin, and incubated with 200 μL subject serum (diluted 1:1000). Following incubation at 37°C for 1 hr, a secondary horseradish peroxidase-conjugated goat anti-human antibody specific for IgM (5μ Fc fragment–specific), IgG (Fcγ-specific), IgA (α-chain–specific; Jackson ImmunoResearch), or sIgA (mouse anti-human secretory component, Clone GA-1; Sigma) was added. Plates were developed using TMB substrate, and the absorbance at 450 nm was determined using an MRX II microplate reader (Dynatech).