Figure 4.

Detection of the target proteins using aptamer variants as the primary detector agents in ELONA. The target was directly immobilized on the plate surface by an incubation with the target solution (100 μl per well) at each indicated concentration, in the presence of 1 μg/ml of BSA in 0.1 M carbonate buffer (pH 9.6), for 2 h. After the incubation, the plate surfaces were further coated with blocking solution (1% BSA in 1× d-PBS(–), 300 μl per well) for 2 h. After the blocking reaction, 100 μl of the detector solution (10 nM each aptamer derivative in 1× binding buffer) was added to each well and then the binding reaction was performed for 30 min. After the incubation, 100 μl of the secondary detector solution (50 ng/ml HRP-conjugated streptavidin in 1× binding buffer) was added to each well, and then incubated for 30 min. After washing the wells, the TMB reaction (100 μl per well) was performed for 5 min (left, VEGF₁₆₅) or 15 min (right, IFNγ). The sample size is two per each combination set, and the error bars represent one standard deviation. The bars with wavy lines indicate that at least one of the two sample wells showed overflow (OD₄₅₀ > 4.000).