IM-CFE
CFE010-140718
718E
FOR RESEARCH USE ONLY
Catalog #CFE-010#010

MC Reagent Biochemical Assay Series

Iron Assay Kit
Nitroso-PSAP
PSAP Chromogenic method
Biochemical
Bioc mical Significance and Test Summary
Iron is one of the most important elements,
element , which function as enzyme cofactor. Iron in the blood is bound withtransferrin and transported throughout the body to synthesize globin proteinsprotein suchh as myoglobin and hemoglobin.
Iron is crucial for synthesis of oxygen-transporttransport protein. Its deficiency causes iron deficiency anemia, chronichemorrhagic anemia and infectious anemia.
This product is a direct colorimetric assay kit without deproteinization
proteinization of the sample. Dissociated iron from thetransferrin-irontransferrin iron complex by weakly acid buffer and reduced by means of reductant (:Ferric(:Ferric→Ferrous).
Ferrous). Ferrous
ions give a blue colored complex with Nitroso-PSAP
Nitroso PSAP (as chromogen). The intensity of the colo
coloredred complex is
proportional to the iron concentration in the sample. The color intensity is proportional to the amount of ironpresent in the sample.

1. Kit contents (100 tests)
R-1
R-2
R-3
STD

Bufferuffer
Bufferuffer
Chelate color
200 µg/dLµg/dL Fe Standard

1 x 16 mL
1 x 7 mL
1 x 0.7mL
1 x 1.5 mL

Ready to use
Ready to use
Ready to use
Ready to use*Storage conditions: Store at 2
2-8°C. Don’t freeze
freeze.
*Expiration : 1 year. After the vials are opened, the kit should be used in one month.
month*Measuring range
range: 10-1000
000 µg/dL

2. Materials required but not provided(1)(1) Distilled water(2)( Micropipettor
icropipettors and pipette tips(3)(3 Clear flat--bottom 96-wellwell plate(4)(4 Micro plate
te reader with 750 nm capability

3. Assay preparation(1) Bring all reagents to room temperature before use.
(2) Prepare working reagent: Mix 70 µLµ of R-2 and 7 µL of R-3
3 for 1 test.
(e.g.) Preparation for 50 testssR
R-2: 70 µL x 50 tests
test = 3.5.5 mL
R
R-3: 7 µL x 50 tests
test = 350
0 µL
Mix 3.5 mL of R-2 and 350
50 µL of R-3 in a vessel.

4. Sample preparation
Serum/ Plasma: Insoluble substances in serum and plasma samples should be removed by filtration orcentrifugation. EDTA
EDTA-plasma
plasma cannot be used.
Urine (24 hour pooled urine)/ Biological fluid: Add 6M HCl to the sample and adjust pH 2.0-3.0 (e.g. 5-10µL
L
6M HCl/1mL
HCl/1mL of lysatelysate).
). Centrifuge at 6,000 rpm for 15 min
minutesutes.. Collect the supernatant and use it for assay.
Tissue: Add
d 5% TCA solution, vovortex
tex for 1 minuteute and incubate for 30 minutes
minat 4-8°C.
8°C. Centrifuge at 6,000 rpmfor 15 minutes
minutes.. Collect the supernatant and use it for assay.
Note: Sample
Sample pH should be between pH 2 and pH 8.

5. Assay protocol(1) Add 15 µL of Distilled
istilled water (Blank)/STD(Standard(Blank) TD(Standard)/Sample
ample into each well.
well(2) Add 160 µL
L of R-1 to each well and incubate
ifor 10 minutes
utes at room temperature.
temperature(3) Add 75 µL
L of Working
orking reagent to each well and incubate for 5 minutes at room temperaturetemperature.
(4) Read the absorbance at 750 nm (740-760 nm).. ----- OD

6.. Calculations
Calculations
ΔODStandard = ODStandard - ODBlank, ∆ODSample = OD Sample - ODBlank
Iron (µg/dL)
g/dL) = ΔODSample/ΔODStandard x 200
Iron (µM)M) ==ΔODSample/ΔODStandard x 35.8
(Assay example)
DW (Blank)
StandardSample

OD (750 nm)
0.048
0.107
0.081

ΔOD
0.059
0.033

Iron (µg/dL)112

ΔODStandard = ODStandard (750 nm) - ODBlank (750 nm) = 0.107 - 0.048
0.0 = 0.059
ΔODSample = ODSample (750 nm) - ODBlank (750nm) ==0.081 - 0.048 = 0.033
IronSample (µg/dL) = ∆ODSample/∆ODStandard x 200 = ((0.033/ 0.059)) x 200 = 112 (µg/dL)
IronSample (µM) = ∆ODSample
0.033/ 0.0590.059) x 35.8 = 20.0 (µM)(ample/∆ODStandard x 35.8 = (0.033/7.. Interferences
EDTA inhibits iron to chromogenic system. The test is not affected by presence of bilirubin-F
bilirubin and bilirubin-C
C up to 40 mg/dL,
hemoglobin up to 0.
0.1 g/dL and chyle up to 500 FTU.

8.. Quality Control
Use of control sera is recommended to monitor the quality of assay results.

9.. Reference(1)1) Yamada M, Tanioka K, Kishi K, Hatanaka U, Ohnishi M. An automated method for measurement of serum iron and unsaturunsatur
ated iron binding capacity using nitroso--PSAP. Jpn J Lab Auto,Auto,13,13, p659-63
p659 63 (1988).
(2)2) Nakaya. M, Tajima. M, Kosako. H, Nakaya. T, Hashimoto. A, Watari. K, Nishihara. H, Ohba. M, Komiya. S, Tani. N, Nishida.
M, Taniguchi. H, Sato. Y, Matsumoto. M, Tsuda. M, Kuroda. M, Inoue. K, Kurose. H:
H GRK6 deficiency in mice causes
autoimmune disease due to impaired apoptotic cell clearance, Nature Commun,Commun 4, p1532 (2013).
(3)3) Ikeda. Y, Tajima. S, Izawa
Izawa--Ishizawa.
Ishizawa. Y, Kihira. Y, Ishizawa. K, Tomita. S, Tsuchiya. K, Tamaki. T: Estrogen regulates
hepcidin expression via GPR30
GPR30-BMP6-dependent
dependent signaling in hepatocytes, PLoS One,One, 7(7) (2012).
(4)4) Tsugawa. H, Suzuki. H, Matsuzaki. J, Hirata. K, Hibi. T: FecA1, a bacterial iron transporter, determines the survival ofHelicobacter pylori in the stomach, Free Radic Biol Med
Med.. 15, 52(6), p1003-10
p1003 10 (2012).
(5)5) Hayashi. K, Nakamura. M, Sakamoto. W, Yogo. T, Miki. H, Ozaki. S, Abe. M, Matsumoto.
Matsumoto . T, Ishimura. K:
Superparamagnetic nanoparticle clusters for cancer theranostics combining magnetic resonance imaging andhyperthermia treatment, Theranostics.
Theranostics. 23, 3(6), p366p366-76 (2013).

10.
10. Technical support & troubleshooting(1) Unstableness of incubation temperature may result in unstable results.
(2) Use disposable test tube and glassware washed with 1M HNO 3 or 1M HCl, and rinse with distilled waterwater.
(3) Accuracy to the microliter is important to obtain good results. Ensure maximum precision when pipetting.
(4) Temperature for the chromogenic
chromogen reaction may affect the optical density.
density It may be necessary to adjust the reaction timedepending on the room temperature.
(5) Highigh concentration of proteins or lipid inn cell lysate or in tissue extract may affect the observed value. Please remove themby ultrafiltration or centrifugation.
(6) Species of heme
heme-iron cannot be analyzed using this assay kit.

Japan Institute for the Control of Aging (JaICA),
Nikken SEIL Co., Ltd.
Haruoka 710-1,710 1, Fukuroi, Shizuoka 437
437-0122,0122, Japan
TEL: +81-538-49+81-0125 FAX:
FAX +81-538-49
49-1267
www.jaica.com/e/ E-mail: biotech@jaica.com(Supported languages: Japanese and English)