Copper is essential to all living organisms as a trace dietary mineral because it is a key constituent of the respiratory enzyme complex cytochrome c oxidase. In molluscs and crustaceans, copper is a constituent of the blood pigment hemocyanin, replaced by the iron-complexed hemoglobin in fish and other vertebrates. In humans, copper is found mainly in the liver, muscle, and bone. The adult body contains between 1.4 and 2.1 mg of copper per kilogram of body weight.

1.   Serum and plasma samples should be clarified and avoid hemolysis. Heparin is recommended as anticoagulant.

2.   Carry the assay in a ventilated place.

                                                                           [Note]: A-H, standard wells; S1-S40, sample wells.

1)   Standard tube: Take 20 μL of Standard solution with different concentrations into the wells.

Sample tube: Take 20 μL of tested Sample into the wells.

2)   Add 300 μL of Chromogenic agent into each tube of Step 1.

3)   Cover the plate with sealer and incubate at 37℃ for 5 min.

4)   Measure the OD value at 580 nm with Microplate Reader. 

Dilute 100 μmol/L Copper ion standard with double distilled water to a serial concentration. The recommended dilution gradient is as follows: 60, 50, 40, 30, 20, 10, 5, 0 μmol/L. Then carry the assay according to the operation table.

Plot the standard curve by using OD value of standard and correspondent concentration as y-axis and x-axis respectively. Create the standard curve with graph software (or EXCEL). The concentration of the sample can be calculated according to the formula based on the OD value of sample. The standard curve is: y= ax + b.