Microplate Chemiluminescent Enzyme Immunoassay for the Quantitative Analysis of Free Prostate-Specific Antigen in Human Serum

A microplate chemiluminescent enzyme immunoassay method was established with high sensitivity for the quantitative analysis of free prostate specific antigen (f-PSA) in human serum. The immunoassay used alkaline phosphatase (ALP) as the labeled enzyme and highly sensitive ALP-(3-(2′-spiroadamantane)-4-methoxy-4-(3”-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD) chemilumincence reaction as detection system. Several physicochemical parameters such as incubation time, immunoreaction sequence, and detection time were optimized. The linear range of proposed method for f-PSA was 0.15–20 ng ml–1 with a correlation coefficient of 0.998. The detection limit was 0.01 ng ml–1. Inter-assay and intra-assay C.V. was below 7%. The recovery was 88%–108%. Cross-reactivity of normal tumor markers in human serum, such as CA50, CA125, CA15-3, CA242, CEA and AFP, were also studied. It showed that the specificity was good. The stability of these markers was examined for 3 days/5 days/7 days under 4 °C and 37 °C to ensure the feasibility of commercial use: C.V. was below 6%, liner correlation coefficient was above 0.998. Compared with CLIA kit, which purchased from Monobind Inc., USA, and these markers showed good correlation. Analysis results showed that this method was stable and reliable. It can be used for the development of commercial kit and also in the clinical analysis of f-PSA for the auxiliary diagnosis of prostate cancer.