Enzyme-Linked Immunosorbent Assay for Antibody Detection

Enzyme-linked immunosorbent assay (ELISA) was performed as previously described [32 (link)]. Briefly, the peptide (20μg/mL in 100 μl of Phosphate Buffer Saline, i;e. PBS) was coated for 150 minutes at 37°C into Maxisorp plates (Nunc, Roskilde, Denmark). Plates were blocked by Protein-Free Blocking-Buffer (Pierce, Thermo Scientific, France). Each eluate was incubated in triplicate at 4°C overnight at 1/20 dilution in PBS-Tween 1%. Mouse biotinylated Ab to human IgG (BD Biosciences, San Diego, CA) was incubated at a 1/1000 dilution in PBS-Tween 1% and peroxidase-conjugated streptavidin (GE Healthcare, Orsay, France) was added (1/1000 dilution in PBS-Tween 1%). Colorimetric development was carried out using 2, 2’-azino-bis (3-ethylbenzthiazoline 6-sulfonic acid) diammonium (ABTS; Thermo Scientific, France) and absorbance (OD) was measured at 405 nm. Individual results were expressed as the ΔOD value calculated according to the formula ΔOD = ODx − ODn, where ODx represented the mean of individual OD values in the two wells containing antigen and ODn the OD value in well without antigen. A subject was considered as an “immune responder” if ΔOD was higher than the cut-off (Cut-off = mean (ΔOD_unexposed) + 3SD = 0.181) calculated from specific IgG level in negative “not exposed” controls (n = 10).