QuantiChrom Calcium Assay Kit

We have whole blood samples. Does your assay work?

Yes, our QuantiChrom^TM Calcium Assay Kit (DICA-500) can be used on whole blood samples. To correct for interference in the sample matrix, two internal standard methods have been validated. Protocol A is quicker whereas Protocol B is slightly more involved, but requires less sample and is, thus, recommended for customer's that have a limited quantity of sample. Additionally, protocol B requires less Reagent because each sample requires one well rather than three separate wells per sample. Please note that 20 mM EDTA is needed for this experiment and is not provided. The customer should prepare this solution, or is available for purchase upon request.

PROTOCOL A: 3 Separate wells needed for each sample

A1. Whole Blood samples require an internal standard and need three separate reactions: 1) Sample plus Standard 2) Sample alone and 3) Sample Blank. For the internal standard prepare 250 uL 10 mg/dL Ca²⁺ Standard by mixing 125 uL 20 mg/dL Standard and 125 uL dH₂O.

Transfer 5 uL whole blood sample to three separate wells. Add 5 uL of 10 mg/dL Ca²⁺ to the 1) Sample plus Standard well, 5 uL dH₂O to 2) Sample alone well and 5 uL 20 mM EDTA to 3) sample Blank well.

A2. Add 200 uL Working Reagent and tap lightly to mix. Note: If any particulates or turbidity are seen pipette up and down to dissolve.

A3. Incubate 3 min at room temperature and read optical density at 570-650 nm (peak absorbance at 612 nm).

PROTOCOL B: 1 Well needed for each sample

B1. Dilute standard to 10 mg/dL Ca²⁺ by mixing 125 uL 20 mg/dL Standard and 125 uL dH₂O.

B2. Transfer 5 uL whole blood sample to a well.

B3. Add 200 uL Working Reagent and tap lightly to mix. Note: If any particulates are seen pipette up and down to dissolve.

B4. Incubate 3 min at room temperature and read optical density at 570-650 nm (peak absorbance at 612 nm). OD_SAMPLE

B5. Carefully transfer 5 uL of 10 mg/dL standard to the sample well from step 2. Tap plate to mix. Repeat Step 4. OD_STANDARD

B6. Add 5 uL of 20 mM EDTA to the same well from step 2. Tap plate to mix. Repeat step 4. OD_BLANK

CALCULATION
The whole blood sample concentration is computed as follows:

[Ca²⁺] = (OD_SAMPLE - OD_BLANK)/(OD_STANDARD - OD_SAMPLE) x 10 x n (mg/dL)

where OD_SAMPLE, OD_BLANK, and OD_STANDARD are the OD readings of the Sample, Sample Blank, and the Sample plus Standard respectively, 10 is the concentration of the standard in mg/dL, and n is the sample dilution factor. If the calculated calcium concentration is greater than 10 mg/dL, dilute sample in dH₂O and repeat assay. Multiply result by the dilution factor n.

EXAMPLE
One human blood sample was assayed using the two methods. The Ca²⁺ concentration was 8.48 mg/dL using Protocol A and 8.38 mg/dL using Protocol B.

Is the calcium assay compatible with acids?

Yes, our QuantiChrom^TM Calcium Assay is compatible with acids, such as 0.5 M HCl.

Do you know if phosphate in the sample will interfere with the calcium assay or if calcium in the sample will interfere with the phosphate assay?

Phosphate (at least up to 30 mM) in the sample does not interfere with the calcium assay.

Does freezing of serum have any impact on serum Ca compared to using fresh serum?

Our assay kit measures the total calcium content of samples. In serum about half of the calcium is free ("ionized") and the other half is bound to proteins, especially albumin (ca.40%) or anions (ca.10%). Repeated freeze-thaw cycles may cause precipitation of proteins which would alter the outcome of the assay. We recommend avoiding repeated freeze thaw cycles for serum samples.


For more detailed product information and questions, please feel free to Contact Us. Or for more general information regarding our assays, please refer to our General Questions.

RT