Measurement of total and free prostate specific antigen (PSA) in human serum samples using an ultra-microanalytical system

•PSA detection and quantification is used as a tumor marker.•UMELISA PSA is an assay for measurement of total and free PSA in human serum samples.•Two-in-one test kit capable of quantifying and differentiating total and free PSA.•Fluorescent assay that uses only 10 µL of sample and reagents.•Cost-effective diagnostic kit implemented in the Cuban Public Health System. [Display omitted] Prostate specific antigen (PSA) is a serine protease used for the screening of prostate cancer. The total portion of PSA (tPSA) can be found in its free form (fPSA), or bound to other proteins forming a stable complex. A heterogeneous sandwich-type UltraMicro Enzyme-Linked ImmunoSorbent Assay (UMELISA) has been developed for the measurement of tPSA and fPSA in human serum samples. Strips coated with a high affinity monoclonal antibody (MAb) directed against PSA are used as solid phase, to ensure the specificity of the assay. Biotinylated MAbs specific for tPSA and fPSA ensured sensitivity, given the high affinity binding to streptavidin. The assay was completed in 1.5 h, with a measuring range 0.019–20 µg/L (tPSA), and 0.009–20 µg/L (fPSA). The intra- and inter-assay CV were lower than 9%. Recovery percentages were 96–105%. High correlations were found between the values of the UMELISA PSA standards and the International Reference Standards 96/670 (R2 = 0.9996) and 96/688 (R2 = 0.9989). The assay did not recognize any of the interfering molecules tested. Regression analysis of serum samples showed a good correlation with Roche Elecsys total PSA (n = 631, R2 = 0.986, ρc = 0.992), BioMérieux VIDAS TPSA (n = 631, R2 = 0.989, ρc = 0.993) and Roche Elecsys free PSA (n = 164, R2 = 0.973, ρc = 0.979), all with a relative difference below 15%, and a p