Application

This kit can be used to measure the concentration of iron in serum samples

Detection significance

Serum iron is an essential element in human body. Iron with physiological activity mainly exists in the form of ferroheme and transferrin in plasma. 65% of the iron in the body is bound up in hemoglobin molecules in red blood cells. About 4% is bound up in myoglobin molecules. Around 30% of the iron in the body is stored as ferritin or hemosiderin in the spleen, the bone marrow and the liver. Small amounts of iron can be found in other molecules in cells throughout the body. None of this iron is directly accessible by testing the serum. However, some iron is circulating in the serum. Transferrin is a molecule produced by the liver that binds one or two iron (III) ions, i.e. ferric iron, Fe3+. Transferrin is essential if stored iron is to be moved and used.

Detection principle

Under the action of acidic solution and reductant, ferric ions can be separated from transferrin in serum, and reduced into ferrous ions (Fe2+). The latter then bind to bipyridine and form pink complexes. The concentration of iron can be calculated by measuring the OD value at 520 nm indirectly.

Experimental instruments

Test tubes, Vortex Mixer, Centrifuge, Water bath, Spectrophotometer (520 nm）

Preparation of sample

It is recommended to take 2~3 samples which expected large difference to do pre-experiment before formal experiment:

Serum: Detect the sample directly. (The serum must be centrifuged before test if it is turbid.)

Operation steps

(1)  Blank tube: Add 0.5 mL of Double-distilled water into a 5 mL centrifuge tube.

Standard tube: Add 0.5 mL of 2 mg/mL Iron Standard working solution into a 5 mL centrifuge tube.

Sample tube: Add 0.5 mL of Sample into a 5 mL centrifuge tube.

(2)  Add 1.5 mL of Iron Chromogenic agent, mix fully with vortex mixer, then incubate in 100℃ water bath for 5 min. (Blank tube and standard tube can be treated without water bath.)

(3)  Cool the tubes with running water, centrifuge the tubes at 3500 rpm for 10 min. (If the supernatant is still turbid, take the turbid supernatant into another centrifuge tube and centrifuge again.)

(4)  Take 1.0 mL supernatant. Set to zero with double-distilled water, and measure the OD value of each tube with spectrophotometer at 520 nm with 0.5 cm optical path quartz cuvette.

      Note: It can be refer to the following operating table

Mix fully with vortex mixer, then incubate in 100℃ water bath for 5 min. Cool the tubes with running water, centrifuge the tubes at 3500 rpm for 10 min. Take 1.0 mL supernatant. Set to zero with double-distilled water, and measure the OD value of each tube with spectrophotometer at 520 nm with 0.5 cm optical path quartz cuvette.

[Note]: When taking the supernatant for colorimetry measurement, it is suggested to take the supernatant carefully with the pipette to avoid adding sediment to optical path quartz cuvette and affect the OD value.

Technical parameters

1.  The sensitivity of the kit is 0.08 mg/L.

2.  The intra-assay CV is 2.98% and the inter-assay CV is 3.19%.

3.  The recovery of the kit is 98.1 %.

4.  The linear range of the kit is 0.08-50 mg/L.

Notes

1. The kit is for scientific research only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The validity of kit is 6 months.

4. Do not use components from different batches of kit.