Total Iron Binding Capacity (TIBC) Assay Kit (Ferrozine, Colorimetric Method)

Cat. No.: T1509330

GRADE & PURITY BioReagent

Overview

Iron is an essential trace element in the human body, with a total content of approximately 3270 mg. Iron is widely distributed; 67.6% of it exists as a cofactor in hemoglobin molecules, participating in oxygen transport. About 2.59% and 4.15% are found in bones and myoglobin, respectively, while stored iron accounts for approximately 25.37%. In serum, iron exists exclusively in the ferric (Fe³⁺) ion form bound to transferrin. Therefore, when measuring serum iron, it is first necessary to separate Fe³⁺ from transferrin. Total Iron-Binding Capacity (TIBC) refers to the total amount of iron that transferrin in the serum can bind.

Detection Principle

TIBC is detected using ferrozine as the substrate. An excess of iron is added to the test serum or plasma, allowing iron to bind to the available transferrin. Free iron is then removed by adsorption using an iron adsorbent. In an acidic medium, the serum iron bound to transferrin is released and reduced to Fe²⁺ by a reducing agent. The Fe²⁺ then reacts with ferrozine to form a purplish-red complex. The absorbance at 562 nm is measured using a spectrophotometer. This method is suitable for detecting the total iron content in serum and plasma samples, which represents the Total Iron-Binding Capacity (TIBC). This detection method is a direct assay and requires a serum blank to correct for the sample's inherent color. Iron content is calculated according to the formula. This detection kit shows a good linear relationship up to 140 μmol/L. Triglycerides ≤ 3.39 mmol/L and bilirubin ≤ 171 μmol/L cause minimal interference with this method. This kit is for research use only and is not suitable for clinical diagnosis or other purposes.

Applicable Samples: Serum, Plasma

Reagents, consumables and Equipments not provided

• Spectrophotometer (capable of measuring absorbance at 562 nm)
• Centrifuge tubes or test tubes, cuvettes

Operating Steps

1. Preparation of Sample Treatment Iron Solution and Iron Standard Working Solution

Take an appropriate amount of Iron Standard (100 μg/mL). Prepare the Iron Standard (10 μg/mL) by mixing Iron Standard (100 μg/mL) with TIBC Iron Standard Diluent at a ratio of 1:9. This serves as the Sample Treatment Iron Solution.

Simultaneously, prepare the Iron Standard (2 μg/mL) by mixing Iron Standard (100 μg/mL) with TIBC Iron Standard Diluent at a ratio of 1:49. This serves as the Iron Standard Working Solution.

Store at 4°C protected from light. Stable for 3 months.

2. Sample Preparation

Plasma and serum, prepared by conventional methods, can be directly used for detection with this kit. Store at -20°C for TIBC detection.

Use dry, stoppered test tubes or disposable sterile polyethylene centrifuge tubes with lids that have been treated with dilute hydrochloric acid and cleaned with deionized water.

Add 0.45 mL of serum or plasma, 0.25 mL of Iron Standard (10 μg/mL), and 0.2 mL of ddH₂O. Mix thoroughly and incubate at room temperature for 10 minutes.

Add 50 mg of Iron Adsorbent, mix, and incubate at room temperature for 10 minutes, vortexing 4 times during this period.

Centrifuge at 3000 g for 10 minutes. Collect the supernatant for use.

3. TIBC Sample Addition

Use dry test tubes or disposable sterile polyethylene centrifuge tubes treated with dilute hydrochloric acid and cleaned with deionized water.

Set up blank, standard, and test tubes according to the table below. Add solutions in the specified order, taking care to avoid bubbles. If the iron ion concentration in the sample is too high, reduce the sample volume or dilute appropriately before measurement. It is recommended to set up duplicate tubes for sample detection.

Substance Added (mL)	Blank Tube	Standard Tube	Test Tube
ddH₂O	0.45	—	—
Iron Standard (2 μg/mL)	—	0.45	—
Supernatant	—	—	0.45
TIBC Assay Buffer	1.2	1.2	1.2

Mix well. Read the absorbance of the test tube at 562 nm using the blank tube for zeroing (this is the serum blank, A_{Serum Blank}).

Ferrozine Chromogenic Solution

0.05

0.05

0.05

4. TIBC Measurement

Mix well and let stand at room temperature for 15 minutes or incubate at 37°C for 10 minutes. Using the blank tube for zeroing, measure the absorbance of the standard and test tubes at 562 nm using a spectrophotometer (recorded as A_Standard and A_Test). Colorimetric measurement should be completed within 1 hour.

5. Result Calculation

Plasma/Serum Total Iron-Binding Capacity (TIBC) (μmol/L) = { [A_Test − (A_{Serum Blank} × 0.97)] / A_Standard } × 71.6

Parameter Explanation:

A_Test: Absorbance of the test tube measured after adding Ferrozine Chromogenic Solution.

A_{Serum Blank}: Absorbance of the test tube measured before adding Ferrozine Chromogenic Solution.

A_Standard: Absorbance of the standard tube.

Unit Conversion

Iron Standard (2 μg/mL) = Iron Standard (35.8 μmol/L)

μg/dL = μmol/L / 0.179

Plasma/Serum Unsaturated Iron-Binding Capacity (UIBC) (μmol/L) = TIBC − Serum Iron Content

Precautions

1. Hemolyzed samples interfere with detection; avoid using hemolyzed samples whenever possible.

2. If the sample concentration is too high, dilute with distilled water and repeat the measurement. Multiply the result by the dilution factor.

3. The water used in the experiment should not be ordinary distilled water; use high-purity deionized water whenever possible.

4. Glassware should be soaked in 10% hydrochloric acid for 24 hours, then rinsed with deionized water before use.

5. Avoid contact with ironware to prevent iron contamination.

6. The color of the standard is stable for 24 hours. The color of the serum sample is stable within 30 minutes. The color will gradually deepen over time, so measurement should be completed within 1 hour.

7. 0.97 is the volume correction factor.

8. The intra-assay coefficient of variation (CV) for this method is ≤ 3.1%; the inter-assay CV is ≤ 2.6%.

9. Please use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.

Appendix

Standard Curve Preparation:

Following the manual instructions at room temperature, measure the absorbance of a series of standards (0, 0.5, 1, 2, 4, 6, 8, 10 μg/mL) at 562 nm using a spectrophotometer. The standard curve is as follows (for reference only):

Note: Due to differences in detection instruments, operational techniques, and other conditions, the standard curve may vary. These values are for reference only. Based on testing experience, the standard curve may deviate for iron standards below 0.1 μg/mL and above 40 μg/mL.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Room temperature

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions. T1509330A, T1509330D: Store at 2-8℃ long term (12 months). Store in the dark. T1509330B, T1509330E, T1509330F: Store at room temperature long term (12 months).   T1509330C: 

Contents & Storage

T1509330	Component	50T	Storage
T1509330A	Iron Standard (100 μg/mL)	3 mL	2-8℃. Store in the dark.
T1509330B	TIBC Iron Standard Diluent	30 mL	RT.
T1509330C	TIBC Assay Buffer	60 mL	2-8℃.
T1509330D	Ferrozine Chromogenic Solution	3 mL	2-8℃. Store in the dark.
T1509330E	ddH₂O	10 mL	RT.
T1509330F	Iron Adsorbent	3 g	RT.