Figure 4.

Detection of glycan-specific antibodies by indirect ELISA (see details in the “Materials and Methods” section). (a) Optimization of protein concentration (in pg/ml) and serum dilutions for ELISA using serum sample 19–5577_37 and purified CtxB-BCAL2737a produced in B. cenocepacia K56-2 (glycosylated protein) and ΔpglL (unglycosylated negative control) as a protein antigen. The results indicate the mean ± range of duplicate assays (b) ELISA results using a panel of glanders positive and negative horse serum samples (see Table 1 for additional details). Dotted line indicates the negative cutoff OD₄₅₀ value for background color in the ELISA determined using 15 CFT negative serum samples. PNA (peanut agglutinin lectin) and PBS were also used as additional positive and negative controls, respectively. ELISA were done with unadsorbed and adsorbed sera (using unglycosylated CtxB-BCAL2737a, as described in Results. Arrows indicate serum samples that showed OD values higher than the cutoff value with unabsorbed sera and lower than the cutoff value with absorbed sera. Three technical replicates were performed for each sample