All of the iron in serum is bound to transferrin, an iron transport protein. About one-third of transferrin is bound to iron, and the remainder exists as free transferrin that is not bound to iron. The total iron-binding capacity (TIBC) is the iron-binding capacity of all transferrin in serum, and the unsaturated iron-binding capacity (UIBC) is the transferrin that is not bound to iron. The relationship between TIBC and UIBC is "TIBC = UIBC + serum iron." TIBC fluctuates with iron metabolism disorders, blood diseases, liver diseases, tumors, inflammation, etc.

UIBC is known to be elevated in iron deficiency and low in infection, inflammation, malignancy, nephrotic syndrome, and low protein symptoms.

This method determines the Unsaturated Iron Binding Capacity (UIBC) by observing the color development in the visible region due to the formation of a chelate complex between bathophenanthroline and iron. When a buffer solution containing a known excess of iron is added to the sample, the unsaturated transferrin in the sample binds to the iron in the buffer solution to form saturated transferrin.

When a chromogenic reagent (bathophenanthroline/reducing agent) is then added, the residual iron that is not bound to transferrin turns red.

The UIBC can be obtained by observing the absorbance at 546 nm as the main wavelength and 600 nm as the secondary wavelength, and subtracting the amount of residual iron from the known excess iron.

◯ Samples that can be directly quantified with this kit

Examples: serum, plasma

The denaturing agent in the kit dissociates iron from protein and causes coloration. No special pretreatment is required.

※ Serum or plasma can be directly used as samples for this assay.

※ Do not use blood collection tubes containing EDTA as it may affect the assay value.

(1) Add 20 µl of DIW, standard solution, and assay sample to wells of 96-well plate.

(2) Apply 200 µl of R-A solution to each well.

(3) Incubate the plate at room temperature for 5 minutes.

(4) Measure absorbance OD1 at the wavelength of 546 nm(main) and 600 nm (secondary).

(5) Add 30 µl of R-R solution to each well.

(6) Incubate the plate at room temperature for 5 minutes.

(7) Measure absorbance OD2 at the wavelength of 546 nm(main) and 600 nm (secondary).

(8) Calculate the iron concentration using the following formula:

(1) Measurement of control serum with this kit

Figure 1: Results of UIBC measurement of control serum with known iron concentration.

• Yoshinaga M et al. Regnase-1 Maintains Iron Homeostasis via the Degradation of Transferrin Receptor 1 and Prolyl-Hydroxylase-Domain-Containing Protein 3 mRNAs. Cell Rep. 2017 May. PubMed PMID: 28538180.

• Oshima K et al. Iron suppresses erythropoietin expression via oxidative stress-dependent hypoxia-inducible factor-2 alpha inactivation. Lab Invest. 2017 Mar. PubMed PMID:28263291.

• Ramsay,W.N.M. The determination of total iron-binding capacity of serum :Clin.Chim.Acta,2.221-226(1957)

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