IM-CCA
CCA020-140718
718E
FOR RESEARCH USE ONLY
Catalog #CCA-0#020

MC Reagent Biochemical Assay Series

Calcium Assay Kit
Chlorophosponaz
Chlorophosponazo-III Chromogenic method
Biochemical
Bioc mical Significance and Test Summary
Calcium is the most abundant and one of the most important minerals in the human body. Approximately 99% ofbody calcium is found in bones. The calcium level in the extracellular space is in dynamic equilibrium with bone
calcium. Calcium ions affect the contractility of heart and skeletal muscles and are essential for the function of thenervous system. Calcium ion
ions play an important role in blood clotting and bone minimization.
miization. In plasma, calcium
is bound to a considerable extent to proteins, 10 % is in the form of inorganic complexes and 50 % is present as
free ion species
species.. The calcium homeostasis regulated by the parathyroid hormone (PTH), calcitriol (CT), andcalcitonin. A decrease in albumin level causes a decrease in serum calcium. Low levels of calcium are found in
hypoparathyroidism, pseudohypoparathyroidism, vitamin D deficiency, malnutrition and intestinal malabsortion.
Among causes of hypercalc
hypercalcemia
emia are cancers, large intake of vitamin D, enhanced renal retention, osteoporosis,sarcosidosis, thyrotoxicosis,hyrotoxicosis, hyperparathyroidism.
hyperparathyroidism.
This product is a direct colorimetric assay kit without deproteinization
eproteinization of the sample. Calcium withChlorophosphonazo
Chlorophosphonazo-III (as chelator) at neutral pH, yields a blue colored complex. The intensity of the color
formed is proportional to the calcium concentration in the samplesample.

1. Kit contents (20000 tests)
R-1RSTD

Chelate color
10 mg/dL
g/dL Ca Standard

1 x 50 mL
1 x 1.0 mL

Ready to use
Ready to use*Storage conditions: Store at 22-8°C. Don’t freeze
freeze.
*Expiration: 1 year. After the vials are opened, the kit should be used in one month.
month*Measuring range: 0.05-20
20 mg/dL

2. Materials required but not provided(1) Distilled water(2) Micropipettors and pipette tips
(3) Clear flatflat-bottom 96-well
well plate(4) Microplate reader with 690 nm capability

3. Assay preparation
Bring all reagents to room temperature before use.

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) Dilute the sample 1/2 in distilled water. Add 6M HCl to the diluted sample andadjust pH 2.0
2.0-3.0 (e.g. 5--10 µL of 6M HCl/1mL of lysatelysate).
). Centrifuge at 6,000 rpm for 15 min
minutes.. Collect thesupernatant and use it for assay.
Biological fluid: Add 6M HCl to the sample and adjust pH 2.0
2.0-3.0 (e.g. 5--10 µL of 6M HCl/HCl/1mL
1mL of lysate).
Centrifuge
Centrifuge at 6,000 rpm ffor 15 minutes
nutes.. Collect the supernatant and use it for assay.
Tissue: Add 3%% TCA solution, vortex
vo tex for 1 minuteute and incubate for 30 minutes
minat 4-8°C.
8°C. Centrifuge at 6,000 rpm
for 15 minutes
minutes.. Collect the supernatant and use it for assay.
Note: Sample pH should be between pH 2 and pH 8.

5. Assay protocol(1) Add 5 µL of Distilled water(Blank)
water(Blank)//STD(Standard)/(Standard)/Sample
ample into each well.
(2) Add 250 µµL of R-1
1 to each well and incubate for 10 min
minutesutes at room temperature.

(3) Read the absorbance at 690 nm (680680-700 nm) and 750 nm (740-800800 nm,nm reference wavelengthwavelength)) . ----- OD

6.. Calculations
ΔODStandard = ODStandard - ODBlank, ∆ODSample = OD Sample - ODBlank
Calcium ((mg/dL) =ΔOD
ODSample/ΔODStandard x 10
Calcium ((mM) =ΔODSample/ΔODStandard x 2.495(Assay example)
DW (Blank)
Standard
SampleOD (690(69 nm)
0.505
0.991
0.837

OD (750 nm)
0.066
0.078
0.073

OD
0.439
0.913
0.764

ΔOD
0.474
0.325

Calcium (mg/dL)(m6.86(a) Measurement at 690 nm and 750 nm (reference wavelength):
OD = OD (690 nm) - OD (750 nm)
ΔODStandard = (0.991 - 0.078) - (0.505 - 0.066) = 0.913
ΔODSample = (0.837 - 0.073)
0.0- (0.5050.505 - 0.066)) = 0.764
0.325/ 0.474
0.474) x 10 = 6.86 (mg/dL)(mg/dL)
CalciumSample = ∆ODSample/∆ODStandard x 10 = (0.325/
CalciumSample (mM)
M) = ∆OD∆ Sample/∆OD∆ODStandard x 2.495.495 = (0.325/0.325/ 0.474)
0.474 x 2.495 = 1.71 (mM)(b) Measurement at 690 nm:
ΔODStandard = 0.991 - 0.505 = 0.486
0.
ΔODSample = 0.837 - 0.505 = 0.332
0.332/ 0.4860.486) x 10 = 6.83 (mg/dL)
CalciumSample = ∆ODSample/∆ODStandard x 10 = (0.332/
CalciumSample (µM)M) = ∆ODSample/∆OD∆ODStandard x 2.495.495 = (0.332/0.332/ 0.486)
0.486 x 2.495 = 1.70 (mM)

7.. Interferences
EDTA inhibits calcium to chromogenic system. The test is not affected by presence of bilirubin-F and bilirubin-C
C up to 40 mg/dL,
hemoglobin up to 1 g/dL and chyle up to 1,000 FTU.

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

9.. Reference.
(1)1) J. W. Ferguson, J. J. Richard, J. W. O'laughlin and C. V. Banks: Simultaneous Spectrophotometric Determination ofCalcium and Magnesium with Chlorophosphonazo-III,
Chlorophosphonazo III, Anal. Chem,, 36, 796.2 (1962).
(2)) D. S. Howell, J. C. Pita, J. F. Marquez, "Ultramicro Spectrophotometric Determination of Calcium in Biologic Fluids", Anal.
Chem,, 38, 434 (1966).
(3)) Fujita. T, Noguchi. K, Terashima. I : Apoplastic mesophyll signals induce rapid stomatal responses to CO 2 in Commelina
communis, New Phytol
Phytol,, 199(2), p395-406
p395 406 (2012).

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 water.
(3) Accuracy to the microliter is important to obtain good results. Ensure maximum precision when pipetting.
(4) Temperature for the chromogenic
chromoge
reaction may affect the optical density.
densi It may be necessary to adjust the reaction timedepending on the room temperature.
(5) HighHigh concentration of proteins or lipid inn cell lysate or in tissue extract may affect the observed value. Please remove themby ultrafiltration or centrifugation.
(6)6) 24 hour pooled urine
urine:: Dilute the sample 1/2
1/ in distilled water. Mix. Multiply results by 2 (dilution factor) for assay sample.

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)