Calibration of iron concentration versus fluorescence.

A series of concentrations of iron, ranging from 0 to 400 μmol/L (described in “Materials and methods”), was prepared in HBS buffer containing either BSA (circles) or apo-Tf (triangles). A 20-μL sample from each solution (“input sample”) was mixed with either HBS containing 2 mmol/L NaHCO₃ (empty symbols) or oxalate-Mn reagent (filled symbols) (final volume, 250 μL). Aliquots of 100 μL were transferred to DFO-coated plates and processed in the NTBI assay as described in “Materials and methods.” The fluorescence was read after 2 hours of incubation with CA-Fe and plotted semilogarithmically against the concentration of iron in the original 20-μL input sample (A). The most sensitive region of the calibration curve (0-12.5 μmol/L Fe) was plotted separately as a linear graph (B). Bars indicate standard deviation of the mean of 4 individual samples.