AbstractAn ELISA on microplate was established for the total serum PSA. We selected the monoclonal antibody for the assay from commercial sources making certain that it reacted with both free PSA and PSA–α1‐antichymotrypsin (PSA–ACT) complex from human serum with similar affinity (so‐called “equimolar”). We also chose a test format with polyclonal anti‐PSA antibodies coated on the well and monoclonal anti‐PSA antibodies for quantification to gain higher test sensitivity. Two different sample volumes from each specimen, 5 and 50 m̈l, were used for the assay in order not only to further increase test sensitivity and improve precision at both low and highly elevated PSA concentrations, but also to widen the assay concentration range (0–500 ng total PSA per ml). Using two sample volumes also reduces any hook effect and shortens the turn‐around time because repeated determinations are usually required when specimens contain highly elevated PSA concentrations. The use of pooled sera containing approximately 95% PSA–ACT complex and 5% free PSA as a calibrator allows for a close matching of the calibrator with serum specimens in immunoreactivity and PSA composition. Moreover, our assay shows no hook effect up to 15,000 ng/ml. The within‐day precision (% CV) in the critical concentration range of 4–12 ng/mL is approximately 5%. The PSA values obtained from this assay correlate well with that of the Hybritech kit (γ = 0.998, slope equals to 1.033), indicating that this kit can replace the Hybritech Tandem E PSA kit for serum PSA determination in clinical laboratories.