Rapid and sensitive chemiluminescent enzyme immunoassay for measuring tumor markers

Abstract

Abstract We developed a chemiluminescent enzyme immunoassay (CLEIA) to quantify such tumor markers as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CA19-9, and CA125. We used a novel chemiluminescent substrate, a derivative of 1,2-dioxetane phosphate (AMPPD), to measure alkaline phosphatase as a labeling enzyme to Fab' fragments of antibody. Regardless of the solid phase, i.e., polystyrene beads (6 mm diameter) or ferrite-coated particles (0.3 microns diameter), the standard curves within the dynamic ranges of the conventional RIA or enzyme immunoassay (EIA) were linear in all cases except for those with AFP. Use of the ferrite particles further shortens the immunoreaction, so the assay can be performed in 30 min. In addition, the relationships between concentrations of the marker and chemiluminescent signals for CA19-9, CA125, and CEA were linear up to concentrations about 10-fold greater than the ordinary dynamic ranges. Intra- and interassay CVs (averages for individual analyte) were 2.2%-4.9% and 2.0%-5.8%, respectively. In an analysis of serum samples, results of the CLEIA correlated reasonably well with those of RIA or EIA. The lower limit of detection by CLEIA with ferrite particles was 390 arb. units/L for CA19-9, 990 arb. units/L for CA125, 0.06 micrograms/L for CEA, and 0.03 micrograms/L for AFP. Thus, the sensitivity increased to between two- and 10-fold that of RIA or colorimetric EIA, depending on the analytes.