Scheme of the NTBI assay.
The scheme depicts the 4 basic steps of the assay for both normal (left) and NTBI-containing (right) sera. The iron is depicted as a full circle and the transferrin molecules denoted by T. Step 1: A serum sample, which has been premixed with 100 mmol/L oxalate, 2.5 mmol/L MnCl2, and 20 mmol/L HEPES pH 7.4, is added to DFO-coated wells. The mobilized NTBI binds to the DFO on the plastic during a 2-hour incubation. Step 2: Only the DFO-bound iron remains after the wells are washed. Step 3: Calcein-Fe complex (CA-Fe) is added to the wells. CA-Fe is nonfluorescent because of quenching of calcein by the bound iron. Step 4: The fluorescence is measured after a 2-hour incubation, during which the remaining available DFO molecules withdraw iron from CA-Fe, causing the released calcein to become fluorescent. For normal serum, a maximum level of fluorescence is attained, whereas for NTBI-containing serum, fluorescence is relatively lower. The fluorescence generated is inversely proportional to the concentration of NTBI in the original sample.
