Ceruloplasmin Colorimetric Activity Kit

Catalog No. ABIN2815079

Product Details

Target: Ceruloplasmin (CP) (Ceruloplasmin (Ferroxidase) (CP))

Detection Method: Colorimetric

Host: Panda

Application: Activity Assay (AcA)

Purpose: The DetectX® Ceruloplasmin Activity Kit is designed to quantitatively measure ceruloplasmin ac- tivity in diluted serum and urine samples.

Brand: DetectX®

Sample Type: Serum, Urine

Components: Clear 96 well Half Area Plates 2 Plates
Ceruloplasmin Standard* 20 μL 200 Units/mL of human ceruloplasmin in a special stabilizing solution. *New Standard Range
Assay Buffer Concentrate 28 mL A 5X concentrate containing detergents and stabilizers.
Ceruloplasmin Colorimetric Substrate 2 Vials Ceruloplasmin substrate lyophilized from a special stabilizing solution.
Plate Sealers 2 Each
* Ceruloplasmin activity is based upon the published determination by G. Curzon and L.Vallet, Biochem. J., 1960, 74:279-287.
The definition of a unit of activity was arbitrarily defined as the ac- tivity of an amount of ceruloplasmin giving an OD of 0.10 at 550nm under their defined conditions.
The activity of our standard is determined using the Curzon and Vallet method.

Material not included: Repeater pipet with disposable tips capable of dispensing 25 μL.
An incubator capable of maintaining 30 °C. 96 well plate reader capable of reading optical density at 560 nm.
Software for converting optical density readings from the plate reader and carrying out four pa- rameter logistic curve (4PLC) fitting.

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Protocol: A human ceruloplasmin standard is provided to generate a standard curve for the as- say and all samples should be read off of the standard curve.
Samples are diluted in the provided Assay Buffer and added to the wells of a half area clear plate.
The reconstituted Ceruloplasmin Substrate is added and the plate is incubated at 30 °C for 60 minutes.
The ceruloplasmin in the standards and samples reacts with the substrate to produce a colored product.
The optical den- sity is read at 560 nm.
Increasing levels of ceruloplasmin in the samples causes an increase in the fuchsia (pink-purple) product.
The activity of the ceruloplasmin in the sample is calculated after making a suitable correction for any dilution, using software available with most plate readers.
The results are expressed in terms of milliunits (mU) of ceruloplasmin activity per mL.
Ceruloplasmin activity is based upon the published determination by G.
Curzon and L.
Vallet, Biochem.
J., 1960, 74:279-287.
Due to our optimized reagents and incubation times the optical density generated by 1 U/mL of ceruloplasmin is substantially higher than 0.1 OD.

Reagent Preparation:

Allow the kit reagents to come to room temperature for 30-60 minutes.
We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine Cerulo- plasmin activities.
Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit.
Assay Buffer Dilute Assay Buffer Concentrate 1:5 by adding one part of the concentrate to four parts of deion- ized water.
Once diluted this is stable at 4 °C for 3 months.
Standard Preparation New Standard Range Standards are prepared by labeling seven tubes as #1 through #7.
Add 995 μL of Assay Buffer to tube #1.
Pipet 300 μL of Assay Buffer into tubes #2 to #6.
Carefully add 5 μL of the Ceruloplasmin Stock from the vial to tube #1 and vortex completely.
Take 300 μL of the Cp solution in tube #1 and add it to tube #2 and vortex completely.
Repeat the serial dilutions for tubes #3 through #6.
The ceruloplas- min activity in tubes 1 through 8 will be 1,000, 500, 250, 125, 62.5, and 31.25 mU/mL.
Use all Standards within 2 hours of preparation.

Sample Preparation:

Serum and Urine Samples Serum and urine samples should be diluted at least 1:20 fold in the Assay Buffer supplied. 7 Allow the kit reagents to come to room temperature for 30-60 minutes. We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine Cerulo- plasmin activities. Ensure that all samples have reached room temperature and have been diluted as appropriate prior to running them in the kit. Assay Buffer Dilute Assay Buffer Concentrate 1:5 by adding one part of the concentrate to four parts of deion- ized water. Once diluted this is stable at 4 °C for 3 months. Standard Preparation Standards are prepared by labeling seven tubes as #1 through #7. Add 995 µL of Assay Buffer to tube #1. Pipet 300 µL of Assay Buffer into tubes #2 to #7. Carefully add 5 µL of the Ceruloplasmin Stock from the vial to tube #1 and vortex completely. Take 600 µL of the Cp solution in tube #1 and add it to tube #2 and vortex completely. Re- peat the serial dilutions for tubes #3 through #7. The ceruloplasmin activity in tubes 1 through 8 will be 5, 3.33, 2.22, 1.48, 0.988, 0.658, and 0.439 U/mL. Use all Standards within 2 hours of preparation. Preparation Add 3 mL of water to the vial and mix thoroughly. This solution can be stored at 4 °C for up to 2 weeks. The solution can also be stored at -20 °C for up to the expiration date on the kit label.
Samples that need to be stored after collection should be stored at -70 °C or lower,preferably after being frozen in liquid nitrogen. This kit should be stored at -20 °C until the expiration date of the kit. Once opened the kit can be stored at 4 °C up to the expiration date on the kit label, except for the Cerulo- plasmin Standard and reconstituted Ceruloplasmin Substrate, which must be stored at -20 °C.

Assay Procedure:

Use the plate layout sheet on the back page to aid in proper sample and standard identification. Set plate parameters for a 96-well Corning Costar 3695 plate. See: http://www.ArborAssays.com/resources/lit.asp for plate dimension data.
1. Pre-warm incubator to 30 °C.
2. Pipet 100 μL of diluted samples or appropriate standards into duplicate wells in the plate.
3. Pipet 100 μL of Assay Buffer into duplicate wells as the Zero standard.
4. Add 25 μL of the reconstituted Cp Substrate solution to each well using a repeater pipet.
5. Incubate at 30 °C for 60 minutes.
6. Read the optical density generated from each well in a plate reader capable of reading at 560 nm. NOTES 37 °C Incubation Incubation at 37 °C will increase the optical density of the reaction by about 44 % . Room Temperature Incubation Incubation at room temperature (~21-23 °C) will decrease the optical density of the reaction by about 50 %. This measurement was made with room temperature at 22.3 °C.

Calculation of Results:

Average the duplicate OD readings for each standard and sample.
Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean OD for the zero standard.
The sample activity obtained should be multiplied by the dilution factor to obtain neat sample values.
Or use the online tool from www.myassays.com/arbor-assays-ceruloplasmin-colorimetric-activity- kit.assay to calculate the data. *The MyAssays logo is a registered trademark of MyAssays Ltd. typical data - new Standard range Sample Mean OD Mean Net OD Ceruloplasmin Activity (mU/mL) Standard 1 1.471 1.36 1,000 Standard 2 0.826 0.715 500 Standard 3 0.445 0.335 250 Standard 4 0.26 0.149 125 Standard 5 0.176 0.065 62.5 Standard 6 0.14 0.029 31.25 Zero 0.111 0 0 Sample 1 0.767 0.657 460.6 Sample 2 0.488 0.378 277.6 Always run your own standard curves for calculation of results.
Do not use these data.
Ceruloplasmin Unit Definition Ceruloplasmin activity is based upon the published determination by G.
Curzon and L.
Vallet, Biochem.
J., 1960, 74:279-287.
The definition of a unit of activity is arbitrarily de- fined as the activity of an amount of ceruloplasmin giving an OD of 0.10 at 550nm under the published defined conditions.

Assay Precision: Two panda urine samples diluted in Assay Buffer were run in replicates of 20 in an assay.
Inter Assay Precision:
Two panda urine samples diluted in Assay Buffer were run in duplicates in sixteen assays run over multiple days by three operators.

Restrictions: For Research Use only

Handling

Precaution of Use: As with all such products, this kit should only be used by qualified personnel who have had labo- ratory safety instruction.
The complete insert should be read and understood before attempting to use the product.
The Ceruloplasmin Standard supplied in this kit is purified from human blood.
The source of the protein was tested and found to be negative for hepatitis and HIV.
However please treat the standard as a potentially infectious sample.

Storage: -20 °C,4 °C,RT

Storage Comment: This kit must be stored at -20°C until the expiration date of the kit. Once opened the kit can be stored at 4°C up to the expiration date on the kit label, except for the Cerulo- plasmin Standard and reconstituted Ceruloplasmin Substrate, which must be stored at -20°C.

References

Costantini, Casasole, Eens: "Does reproduction protect against oxidative stress?" in: The Journal of experimental biology, Vol. 217, Issue Pt 23, pp. 4237-43, (2014) (PubMed).

Mistry, Gill, Kurlak, Seed, Hesketh, Méplan, Schomburg, Chappell, Morgan, Poston et al.: "Association between maternal micronutrient status, oxidative stress, and common genetic variants in antioxidant enzymes at 15 weeks? gestation in nulliparous women who subsequently develop ..." in: Free radical biology & medicine, Vol. 78, pp. 147-55, (2014) (PubMed).

Target Details

Target: Ceruloplasmin (CP) (Ceruloplasmin (Ferroxidase) (CP))

Alternative Name: Ceruloplasmin (CP Products)

Synonyms: CP-2 Kit, fi23f10 Kit, wu:fi23f10 Kit, D3Ertd555e Kit, CERP Kit, CP Kit, ceruloplasmin Kit, CP Kit, cp Kit, Cp Kit, LOC100533122 Kit

Background: Ceruloplasmin (Cp) is a multicopper oxidase enzyme involved in the safe handling of oxygen in some metabolic pathways of vertebrates. Discovered in 1948, a blue protein from the a2-globulin fraction of human serum possessing oxidase activity towards aromatic diamines and catechol was purified by Holmberg and Laurell1. It was denoted ceruloplasmin, literally meaning 'a blue substance from plasma'. Specialized copper sites have been recruited during evolution to provide long-range electron transfer reactivity and oxygen binding and activation in proteins destined to cope with oxygen reactivity in different organisms. Ceruloplasmin belongs to the family of multi- copper oxidases which are among the few enzymes able to bind molecular oxygen to perform its complete reduction to water2,3. Ceruloplasmin contains 95 % of the copper in serum4. Cp found in serum is expressed in the liver, but it is also expressed in the brain, lung, spleen and testis. Aceruloplasminaemia is an autosomal recessive disorder of iron metabolism characterized by the complete absence of ceruloplasmin5,6. The role of Cp in tissue iron overload and the subsequent clinical findings of diabetes, retinal degeneration and neurodegeneration has been associated with iron overload in aceruloplasminaemic patients7. Thus it is clearly indicated that ceruloplasmin plays an essential role in iron metabolism. Ceruloplasmin is also associated with reproduction. Copper-deficient female rats seem to be protected against mortality. This protection has been suggested to be provided by estrogens, since estrogens alter the subcellular distribution of cop- per in the liver, an increase in plasma copper levels and subsequent ceruloplasmin synthesis8