DBC Ferritin Elisa

PRINCIPLE OF THE TEST 
he principle of the following enzyme immunoassay test follows a typical one-step capture or ‘sandwich’ type assay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specific for ferritin is immobilized onto the microplate and another monoclonal antibody specific for a different region of ferritin is conjugated to horse radish peroxidase (HRP). Ferritin 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 ferritin in the sample. A set of standards is used to plot a standard curve from which the amount of ferritin in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
Ferritin is a protein which serves as a storage center for iron. It is found in many tissues but the highest concentrations are in the liver, spleen and bone marrow. The total body iron stores in normal people correlate well with the concentration of ferritin in serum. 
If there is a deficiency in iron, that is the concentration of iron is low in the blood, the ferritin result will be decreased. Likewise, an overload of iron indicates an increase in the level of ferritin. However, in some conditions like liver disease ferritin will be elevated.

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 800 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.