DBC Total Prostate Specific Antigen (PSA Total) Elisa

PRINCIPLE OF THE TEST 
The principle of the following enzyme immunoassay test follows a typical two-step capture or ‘sandwich’ type assay. The assay makes use of two highly specific monoclonal antibodies: A monoclonal antibody specific for total PSA is immobilized onto the microplate and another monoclonal antibody specific for a different region of PSA is conjugated to horse radish peroxidase (HRP). Total PSA from the sample and standards are allowed to bind to the plate, washed, and subsequently incubated with the HRP conjugate. After a second 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 total PSA in the sample. 
A set of standards is used to plot a standard curve from which the amount of total PSA 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 portion of PSA is present as the free form (free PSA). The expected normal level of total PSA in male serum is lower than 4 ng/mL. A rise in the concentration of PSA indicates prostate pathology, including benign prostatic hyperplasia and prostate cancer.

SPECIMEN COLLECTION AND STORAGE 
Approximately 0.1 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.