The BI-EIA-PSA free is atwo-step immunoassay, based on principle of sandwich method. The assay system utilizes a high affinity and specificity monoclonal antibodies (enzymeconjugated and immobilized) directed against a distinct antigenic determinants of the intact f PSA molecule. The BI-EIA-PSA free plates are coated with anti-PSA monoclonal antibody. Serum to be tested is diluted and incubated with the precoated plate. In this step PSA binds to immobilized monoclonal antibody. Non specific antibodies are removed bywashing. Anti-free PSA antibody conjugated to horseradish peroxidase (HRP) is added and incubated. In this step the HRP-conjugate is bound to the prebound antigen - anti body complex. Unbound conjugate is removed by washing. A solution of TMB-Substrate is added and incubated, resulting in the development of a bluecolor. The color development is stopped with the addition of Stopping Reagent,changing the color to yellow. The color intensity of the test sample is directly proportional to the concentration of f PSA.