Figure 1.

Saturation binding curve at 4°C for Antarctic fur seal (data from Delehanty et al., 2019). Specific binding (by CBG) is the difference between total binding and nonspecific binding. Visual inspection suggests that the asymptote is almost reached at ~5 nM free cortisol, and by 15 nM free cortisol the nonspecific binding exceeds specific binding. Using this data, we chose to add 10 nM cortisol to plasma samples in the MCBC assay for Antarctic fur seals. Note that in practice, because total and nonspecific binding counts increase steadily as cortisol concentrations increase, small pipetting errors will result in increasing errors in calculating the specific binding. Therefore, we use the point where the nonspecific binding crosses the specific binding line as a rough maximum GC concentration for the MCBC assay. This is largely an issue for the DCC (used for this curve) and dialysis methods, as the harvester method has much lower nonspecific binding counts