Dual‐labeled chemiluminescence enzyme immunoassay for simultaneous measurement of total prostate specific antigen (TPSA) and free prostate specific antigen (FPSA)

The specificity for early diagnostic of prostate‐specific antigen (PSA) is low because the current technology mostly allows the detection of only one biomarker at one time. In this work, a dual‐labeled chemiluminescence enzyme immunoassay (CLEIA) for simultaneous measurement of total PSA (TPSA) and free PSA (FPSA) was proposed. Anti‐PSA McAb (Mab1) was immobilized on a microplate as the solid phase, horseradish peroxidase (HRP)‐labeled anti‐TPSA monoclonal antibody (McAb2) and alkaline phosphatase (ALP)‐labeled anti‐FPSA McAb3 were used as detection antibodies. Two chemiluminescence reactions of HRP with luminol and ALP with 4‐methoxy‐4‐(3‐phosphate‐phenyl)‐spiro‐(1,2‐dioxetane‐3,2′‐adamantane) (AMPPD) were used as the signal detecting system. Based on a sandwich model, the amount of FPSA and TPSA could be determined simultaneously. The effects of several physico‐chemical parameters were studied and optimized. Cross‐reactivities of six common tumor markers in serum were studied. The proposed method presented the sensitivity of 0.03 ng ml^?1 and 0.05 ng ml^?1 for FPSA and TPSA respectively, with low cross‐reactivities. Compared with the results from commercial chemiluminescent kits there was good correlation, indicating that this established method could be used to simultaneously to measure the concentrations of FPSA and TPSA in one serum sample and also could greatly facilitate the early diagnosis for PCa in clinical practice.