Microplate Chemiluminescent Enzyme Immunoassay for the Quantitative Analysis of Free Prostate-Specific Antigen in Human Serum
A microplate chemiluminescence enzyme immonoassay with high sensitivity for the quantitative analysis of free prostate specific antigen (f-PSA) in human serum was established. The immunoassay utilized alkaline phosphatase (ALP) as the labeled enzyme, and the highly sensitive ALP-AMPPD (4-methoxy-4-(3"-phosphate-phenyl)-spiro-(1, 2-dioxetane-3, 2'-adamantane)) chemiluminescence reaction as the detection system. Several physicochemical parameters such as incubation time, immunoreaction sequence, and detection time were studied and optimized. A one-step sandwiched reaction mode was applied with incubation at 37 degrees C for 2 h and chemiluminescent detection with 50 mu L AMPPD for 30-80 min. The linear range of the proposed method for f-PSA was 0.15-20 mu g/L with r = 0.998. The detection limit was 0. 0 1 mu g/L. Inter-assay and intra-assay RSD were both below 7%. The recovery was 88%-108%. The cross-reactivity of six normal tumor markers in human serum, including CA50, CA125, CA15-3, CA242, CEA and AFP, was studied. It was shown that this method exhibited high specificity to f-PSA. The stability of the method was evaluated by identifying its analytical performance after stored at 4 degrees C and 37 degrees C for 3, 5, 7 days with RSD lower than 6% and correlation coefficient higher than 0. 998, which showed satisfied reliability and stability. Compared with CLIA kit purchased from Monobind. Inc. USA, there was a good correlation between the two methods. The proposed method has shown great potential in the clinical analysis of f-PSA for prostate cancer diagnosis, as well as in the commercial kits development.