DBC C-Peptide Elisa

PRINCIPLE OF THE TEST 
The principle of the following enzyme immunoassay test follows a typical one-step capture or ‘sandwich’ type assay. The assay makes use of two highly specifi c monoclonal antibodies: A monoclonal antibody specifi c for C-peptide is immobilized onto the microplate and another monoclonal antibody specifi c for a different region of C-peptide is conjugated to horse radish peroxidase (HRP). C-peptide from the sample and standards are allowed to bind simultaneously to the plate and to the HRP conjugate. The washing and decanting steps remove any unbound HRP conjugate. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. The absorbance is measured on a microtiter plate reader. The intensity of the colour formed by the enzymatic reaction is directly proportional to the concentration of C-peptide in the sample. 
A set of standards is used to plot a standard curve from which the amount of C-peptide in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
Human C-peptide of insulin is an amino acid chain of 3000 molecular weight, joining the A and B chains of insulin. It is secreted from the granules in the islet beta cells at equimolar concentrations with insulin. It is however, not as rapidly degraded by the liver as in the case of insulin, and therefore is more stable in the blood. These characteristics make the determination of C-peptide an advantageous test as an indicator to quantify insulin. It is therefore used in evaluating hypoglycemia and insulnoma.

SPECIMEN COLLECTION AND STORAGE 
Approximately 0.2 mL of serum is required per duplicate determination. Collect 4–5 mL of blood into an appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4°C for up to 24 hours or at -10°C or lower if the analyses are to be done at a later date. Consider all human specimens as possible biohazardous materials and take appropriate precautions when handling.

CALCULATIONS 
1. Calculate the mean optical density of each calibrator duplicate. 
2. Calculate the mean optical density of each unknown duplicate. 
3. Subtract the mean absorbance value of the “0” calibrator from the mean absorbance values of the calibrators, controls and serum samples. 
4. Draw a calibrator curve on log-log paper with the mean optical densities on the Y-axis and the calibrator concentrations on the X-axis. If immunoassay software is being used, a 4-parameter or 5-parameter curve is recommended. 
5. Read the values of the unknowns directly off the calibrator curve. 
6. If a sample reads more than 16 ng/mL then dilute it with calibrator A at a dilution of no more than 1:8. The result obtained should be multiplied by the dilution factor.