Clone ga 1

Total S-IgA, IgA1, IgA2 and SC determined by sandwich ELISA (Bosch et al., 2001 (link)). All assays used the same capture antibody that was directed against the Secretory Component (Clone GA-1, Sigma-Aldrich, Saint Louis, MO, USA). The high specificity and quality of this monoclonal antibody has been established in a multicenter WHO/NIUIS study (Mestecky et al., 1996 (link)) and corroborated in our laboratory by Western blot and ELISA. For detection, we used HRP-conjugated mouse anti-human monoclonal antibodies directed against S-IgA, IgA1, IgA2, or SC (all obtained from Nordic Immunology, Tilburg, Netherlands). These monoclonal antibodies have been determined to be of high specificity in several comparative studies (de Fijter et al., 1995 (link); Mestecky et al., 1996 (link)). All standards (for S-IgA, IgA1, IgA2, and SC) were also obtained from Nordic. The intra-assay variability of each assay was <4% (inter-assay CV% < 6%). The ELISAs were all found to be of very high sensitivity, with upper detection limits (>200pg/ml) that were several thousand-fold above the lowest observed concentrations.

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).