Highly sensitive and rapid tandem bioluminescent immunoassay using aequorin labeled Fab fragment and biotinylated firefly luciferase

Abstract

We established a simultaneous bioluminescent assay utilizing aequorin (Aq) and biotinylated firefly luciferase (b-Luc); furthermore, we developed a highly sensitive and rapid tandem bioluminescent immunoassay (BLIA) involving the Aq-labeled Fab fragment and b-Luc–streptavidin complex. Minimum detection limits of Aq and b-Luc were 9.4 × 10 −21 mol assay −1 (blank + 3S.D.) and 3.6 × 10 −19 mol assay −1 (blank + 3S.D.), respectively. Measurements of two luminescent proteins were completed in 4 s with a single assay medium. In this study, prostatic acid phosphatase (PAP) and prostate specific antigen (PSA), which served as analytes, were measured in the tandem BLIA. PAP and PSA were detected by the Aq-labeled anti-Dig Fab fragment and b-Luc–streptavidin complex, respectively. The measurable ranges of PAP and PSA were 0.04–100 and 0.2–200 ng mL −1, respectively. This technique was also applied to the simultaneous measurement of PSA and α-fetoprotein (AFP). Measurable ranges of PSA and AFP were 0.2–200 and 1.95–1000 ng mL −1, respectively. Levels of PAP and PSA or PSA and AFP in human serum could be accurately determined with the proposed BLIA. Satisfactory correlations were observed between results obtained from the proposed BLIA and those derived from commercial kits.