Copper Content Assay Kit (Cuprizone, Micro Method)

Cat. No.: C1507278

GRADE & PURITY BioReagent

Descripción general

Copper is an essential trace element in the human body and a key component of enzymes such as ceruloplasmin (ferroxidase), superoxide dismutase, and cytochrome oxidase. The total body copper content is approximately 90 mg, with 90–95% bound to globulin as ceruloplasmin and 5–10% bound to albumin, which serves as a plasma copper carrier. Detection methods for copper include spectrophotometry, atomic absorption spectroscopy, and neutron activation analysis.

Detection Principle

In an acidic medium, copper bound to albumin dissociates. After protein precipitation, copper ions chelate with Cuprizone to form a stable blue complex. The absorbance is measured at 620 nm using a microplate reader. The intensity of the blue color is linear with the copper concentration in serum or plasma. The copper content is calculated by comparing the sample absorbance to a standard curve. This kit demonstrates good linearity up to 62.8 μmol/L, high specificity, and considerable sensitivity. It is intended for research use only and not suitable for clinical diagnosis or other purposes.

C1507278	Component	100T	Storage
C1507278A	Copper Standard (100 μg/mL)	1 mL	2-8℃
C1507278B	Copper Standard Diluent	5 mL	RT
C1507278C	Cu Acidification Solution	15 mL	RT
C1507278D	Lea Protein Precipitation Solution	25 mL	RT. Store in the dark.
C1507278E	Cu Assay Buffer	15 mL	2-8℃
C1507278F	Cuprizone Chromogenic Solution	1.2 mL	2-8℃. Store in the dark.
C1507278G	ddH₂O	10 mL	RT

Materials Required but Not Provided

1.5 mL centrifuge tubes or test tubes, Centrifuge, Microplate reader, 96-well plate

Experimental Procedure

1. Sample Preparation

Plasma or serum samples prepared by standard methods can be used directly. Store at -20°C for Cu detection.

2. Preparation of Copper Standard Working Solution

Dilute the Copper Standard (100 μg/mL) with Copper Standard Diluent at a ratio of 1:24 (Copper Standard : Diluent) to obtain a Copper Standard Working Solution (4 μg/mL). Store at 4°C protected from light; stable for 3 months.

3. Cu Assay Setup

It is best to use dry test tubes cleaned with dilute hydrochloric acid and deionized water, or disposable sterile 1.5 mL polyethylene centrifuge tubes, along with a clean 96-well plate. If these conditions cannot be met, avoid using copper-contaminated utensils. Proceed according to the table below.

Reagent (μL)	Blank Tube/Well	Standard Tube/Well	Test Tube/Well
ddH₂O	200	—	—
Copper Standard (4 μg/mL)	—	200	—
Test Sample	—	—	200
Cu Acidification Solution	140	140	140

Mix thoroughly and incubate at room temperature for 10 min.

Lea Protein Precipitation Solution

200

200

200

Mix thoroughly, incubate at room temperature for 10 min, then centrifuge at 3000 × g for 10 min. Transfer the supernatant from each tube to a 96-well plate as follows:

Resulting Supernatant	100	100	100
Cu Assay Buffer	140	140	140
Cuprizone Chromogenic Solution	10	10	10

4. Cu Measurement

Mix well and incubate at room temperature for 20 minutes. Using the Blank to zero the instrument, measure the absorbance of the Standard and Test wells at 620 nm (recorded as A_standard and A_test ).

5. Calculation of Results

Plasma/Serum Copper (μmol/L) = (A_test  / A_standard ) × 62.8

Parameter Description:

• A_test : Absorbance of the test well

• A_standard : Absorbance of the standard well

• Copper Standard (4 μg/mL) is equivalent to 62.8 μmol/L

• Unit Conversion: μg/dL = μmol/L / 0.157

Reference Interval:

Sample Type	Serum Copper Concentration
Healthy Adult Males	10.99 ~ 21.98 μmol/L (70 ~ 140 μg/dL)
Healthy Adult Females	12.56 ~ 23.55 μmol/L (80 ~ 150 μg/dL)

Precautions

1. If the sample concentration is too high, dilute with deionized water and re-assay, multiplying the result by the dilution factor.

2. Use high-purity deionized water throughout the procedure, not ordinary distilled water.

3. Glassware should ideally be soaked in 10% hydrochloric acid for 24 hours and rinsed thoroughly with deionized water before use.

4. Avoid copper contamination from instruments, test tubes, syringes, etc., during the experiment.

5. The measurement wavelength can be selected between 600–640 nm with minimal impact on results.

6. The color developed by this kit is stable and can be maintained for 1 hour at 4–20°C.

7. Color may fade or disappear over time; complete measurements within 1 hour.

8. Store Cu Assay Buffer sealed tightly immediately after use to prevent evaporation of active components.

9. The classical standard uses a Copper Standard (2 μg/mL). If instrument precision is high and interference is low, this standard can be used, and the calculation formula becomes: Plasma/Serum Copper (μmol/L) = (A_test  / A_standard ) × 31.4.

10. Use reagents promptly after opening to prevent potential effects on subsequent experiments.

Appendix

Reference Standard Curve Range: (Zeroed with Blank) 

• Absorbance (OD) for the 2 μg/mL Copper Standard typically ranges from 0.05 to 0.07 when measured by spectrophotometer.

• Absorbance (OD) for the 4 μg/mL Copper Standard typically ranges from 0.10 to 0.13.

  The standard curve obtained by measuring the absorbance of copper standards at 0.1, 0.5, 1, 2, 4, 8, and 16 μg/mL is shown below:

Note: Reference ranges may vary due to differences in detection instruments and operational techniques. These values are for reference only. For precise copper quantification, multi-point calibration using a standard curve is recommended. Empirical data suggests deviations in the standard curve may occur for standard concentrations below 0.5 μg/mL or above 50 μg/mL.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Store at 2-8°C,Protected from light,Room temperature

Enviado en

Wet ice

Contents & Storage

C1507278	Component	100T	Storage
C1507278A	Copper Standard (100 μg/mL)	1 mL	2-8℃
C1507278B	Copper Standard Diluent	5 mL	RT
C1507278C	Cu Acidification Solution	15 mL	RT
C1507278D	Lea Protein Precipitation Solution	25 mL	RT. Store in the dark.
C1507278E	Cu Assay Buffer	15 mL	2-8℃
C1507278F	Cuprizone Chromogenic Solution	1.2 mL	2-8℃. Store in the dark.
C1507278G	ddH₂O	10 mL	RT