The chromatic interference suppression of the Synermed® UIBC method is based upon the higher sensitivity of the chromogen used and the detection at a red wavelength, thus optimizing the signal to noise ratio.
Albumin binds with bromcresol green to form a colored complex. The absorbance of the complex is quantitated at 660 nm and is directly proportional to the albumin concentration in the sample.
In the Synermed UIBC reagent system, a known amount of ferrous iron is added, in excess, to the serum at an alkaline pH to enable saturation of the unoccupied iron binding sites on the transferrin molecule.
transferrin + Fe+2 -> transferrin-Fe complex + excess Fe +2
The ferrous ion that remains unbound to transferrin is then quantitated by complexing with the chromogen 3-(2-Pyridyl)-5,6-bis(2-[5-furylsulfonic Acid]-1,2,4-triazine to form a blue chromophore which absorbs in the near-infrared region of the spectrum. The absorbance of the blue iron-dye complex is proportional to UIBC concentration and is read spectrophotometrically at 600 nm.
excess Fe+2 + chromogen -> Fe+2-chromogen complex
Then by subtracting the amount of unbound iron thus measured from the total amount of iron originally added to the sample, the unsaturated iron-binding capacity may be determined.
To calculate the TIBC, perform a serum iron assay on the sample. Take the result obtained for the iron assay and add the UIBC result to obtain a value for TIBC.