ELISA-based Antibody Quantification

Immunoglobulin G reactivity against recombinant proteins was measured by enzyme-linked immunosorbent assay as described elsewhere [31 (link)]. In brief, 96-well, flat-bottom, microtiter plates (Nunc MaxiSorp; Thermo Fisher Scientific) were coated with 100ng/well recombinant protein in Dulbecco’s phosphate-buffered saline ([PBS] Sigma-Aldrich) and incubated overnight at 4°C. After blocking (washing buffer with 1% Ig-free bovine serum albumin [BSA]), plasma samples (1:400) were added in duplicate, followed by horseradish peroxidase-conjugated rabbit antihuman IgG (1:3000; Dako). Bound antibodies were detected by adding TMB PLUS2 (Eco-Tek), and the reaction was stopped by adding 0.2 M H₂SO₄. The optical density (OD) was read at 450nm (VERSAmax microplate reader; Molecular Devices), and the specific antibody levels were calculated in arbitrary units (AUs) using the equation 100 × [(OD_SAMPLE-OD_BLANK)/(OD_POS.CTRL-OD_BLANK)], essentially as described elsewhere [31 (link)]. Negative cutoff values were calculated as the mean AU values plus 2 standard deviations (SD) obtained with the negative control samples described above. Individuals were considered responders if their specific antibody level was higher than the cutoff. The breadth of antibody response was defined as the number of antigens recognized by an individual [36 (link)].