Advent of innovative chemiluminescent enzyme immunoassay

Using 1,1'-oxalyldiimidazole (ODI) chemiluminescence detection, a new chemiluminescent enzyme immunoassay (CLEIA) was developed to quantify prostate specific antigen (PSA) in human serum. The results observed in ODI CLEIA were compared with those obtained in commercially available enzyme linked immunosorbent assay (ELISA), fluorescence enzyme immunoassay (FEIA), and luminol CLEIA. PSA complex-conjugated horseradish peroxidase (HRP) was formed from one-step sandwich immunoreaction of PSA, PSA primary antibody and PSA secondary antibody-conjugated HRP for 15 min in a strip-well at 36.5°C. CL substrate solution (Amplex Red and H2O2 in PBS buffer, pH 7.4) was added in the washed strip-well and incubated for 10 min at room temperature. When resorufin formed in this process was mixed with 1,1'-oxalyldi-4-methylimidazole and H2O2 in a testing tube, rapid and bright CL was observed. Detection limit (0.035 ng/ml) of PSA in ODI CLEIA was much lower than those (0.50 and 0.25 ng/ml) in commercially available ELISA and luminol CLEIA even though total incubation time of the former (25 min) was shorter than those of the latter (45 and 35 min). Also, the dynamic range (0-100 ng/ml, R2=0.9996) of ODI CLEIA was wider than those of other EIAs. In conclusion, the excellent correlation (r=0.9767) between ODI CLEIA and Advia Centaur XP Immunoassay System indicates that the accurate, precise, and rapid ODI CLEIA can be applied as a novel CLEIA capable of diagnosing and monitoring various diseases.