Iron level in blood is a reliable diagnostic indicator of various disease states. Increased levels of iron concentration in blood are associated with blood loss, increased destruction of red blood cells (e.g. hemorrhage) or decreased blood cell survival, acute hepatitis, certain sideroachrestic anemias, ingestion of iron-rich diets, defects in iron storage (e.g. pernicious anemia). Decreased levels of blood iron may result from insufficient iron ingestion from diets, chronic blood loss pathologies, or increased demand on iron storage as during normal pregnancy. Simple, direct and automation-ready procedures for measuring iron concentrations find wide applications in research, drug discovery and environmental monitoring. BioAssay Systems' iron assay kit is designed to measure total iron directly in serum without any pretreatment. The improved method utilizes a chromogen that forms a blue colored complex specifically with Fe2+. Fe3+ in the sample is reduced to Fe2+, thus allowing the assay for total iron concentration. The intensity of the color, measured at 590nm, is directly proportional to the iron concentration in the sample. The optimized formulation substantially reduces interference by substances in the raw samples.

Simple and high-throughput. The procedure involves addition of a single working reagent and incubation for 40 min. Can be readily automated as a high-throughput assay for thousands of samples per day.

Improved reagent stability and versatility. The optimized formulation has greatly enhanced reagent and signal stability. Cuvet or 96-well plate assay.

Low interference in biological samples. No pretreatments are needed. Assays can be directly performed on serum samples.

Drug Discovery/Pharmacology: effects of drugs on iron metabolism.

Environmental Monitoring: iron in soil extracts, mineralized samples.

Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

Reagent: Prepare enough Working Reagent by mixing 20 volumes of Reagent A, 1 volume Reagent B and 1 volume Reagent C. Fresh reconstitution is recommended. Equilibrate to room temperature before assay.

1. Standards. Prepare 400 μL 1000 μg/dL Premix by mixing 40 μL 10 mg/dL standard and 360 μL distilled water. Dilute standards as follows.

2. Set up standards and samples. Transfer 50 μL diluted Standards and 50 μL samples into wells of a clear bottom 96-well plate. As a sample blank control, add 200 μL Reagent A to sample "blank" wells. Add 200 μL Working Reagent to all other wells. Tap plate to mix. Store diluted standards at 4 °C for future use.

3. Incubate 40 min at room temperature and read optical density at 510- 630nm (peak absorbance at 590nm).

1. Prepare standards as in 96-well assay. Set up centrifuge tubes labeled Standards and Samples ("Sample" and sample "Blank"). Transfer 250 μL Standards and Samples to tubes.

3. Transfer to cuvets and read OD at 590nm (510nm-630nm).

Subtract blank OD (water, #8) from the standard OD values and plot the OD against standard iron concentrations. Determine the slope using linear regression fitting. Iron concentration of the sample is calculated as

Conversions: 1 mg/dL Fe equals 179 μM, 0.001% or 10 ppm.

Note: (1). Iron chelators (e.g. EDTA) interfere wit this assay and should be avoided in sample preparation. (2).This kit can be applied to measure Fe2+ (vs. total iron) content. Prepare Working Reagent by mixing 20 vol of Reagent A, 1 vol of water and 1 vol Reagent C (no reductant in the Working Reagent). The procedure is the same as described for the total iron assay.

Mice serum, fetal bovine serum (Invitrogen), and goat serum (Invitrogen) were assayed using the 96-well plate assay protocol. The iron concentrations were 173 ± 2 (n = 4), 149 ± 1 (n = 4), 88 ± 2 μg/dL (n = 4), respectively. Coefficient of variance < 2%.

1. Habel, M-E. and Jung, D. (2006). c-Myc over-expression in Ramos Burkitt’s lymphoma cell line predisposes to iron homeostasis disruption in vitro. Biochem. Biophys Res Comm 341(4):1309-1316.

2. Wu, Y. J. et al (2007). In vivo leukocyte labeling with intravenous ferumoxides/protamine sulfate complex and in vitro characterization for cellular magnetic resonance imaging. Am J Physiol Cell Physiol 293: C1698-C1708.