DBC Free Prostate Specific Antigen (fPSA) 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 specific monoclonal antibodies: A monoclonal antibody specific for fPSA is immobilized onto the microplate and another monoclonal antibody specific for a different region of fPSA is conjugated to horse radish peroxidase (HRP). fPSA 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 fPSA in the sample. A set of standards is used to plot a standard curve from which the amount of fPSA in patient samples and controls can be directly read.

CLINICAL APPLICATIONS 
Prostate specific antigen (PSA) is a 33-kDa glycoprotein secreted by epithelial cells of the prostate gland. In human serum, PSA is primarily complexed with α1-antichymotrypsin, and to a lesser extent with other serum proteins. Only a small fraction of PSA is present as the free form (free PSA). The expected normal level of PSA in male serum is lower than 4 ng/mL. A rise in the concentration of PSA indicates prostate pathology, including benign prostatic hyperplasia (BPH) and prostate cancer. Free PSA (fPSA) has been studied in attempts to help distinguish BPH from untreated prostate cancer. These studies have shown that the ratio of free PSA / total PSA is lower in untreated prostate cancer than in patients with BPH.

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 15 ng/mL, then dilute it with assay buffer at a dilution of no more than 1:8. The result obtained should be multiplied by the dilution factor.