Development of a rapid immunoassay system: Luminescent detection of antigen-associated antibody-luciferase in the presence of a dye that absorbs light from free antibody-luciferase

Abstract

In this report, we developed a rapid immunoassay system, designated the bioluminescent interference gathering optical (BINGO) assay, which required no time-consuming washing steps for removal of unbound antibodies. This system employed a luciferase (Luc)-conjugated antibody (LucAb) and a dye that absorbed light from the LucAb. The antigen-associated LucAb was localized by transfer of an antigen to the detector-side of a chamber where a detector photomultiplier tube (PMT) was installed. In contrast, the free LucAb was distributed throughout the solution, and the light emitted by the free LucAb was absorbed by the dye. Therefore, only light from LucAb associated with antigen could be detected by the PMT. The new system could be used to rapidly detect the amount of antigen-antibody-Luc complex by collecting steps, such as centrifugation or magnetic collection of antibody-coated magnetic beads. Proof-of-principle experiments were performed using a model system with streptavidin beads and biotinylated Luc. The feasibility of the system was demonstrated using magnetic beads coated with anti-Escherichia coli O157 antibody, enabling detection of 4 × 10³ cells in only 15 min. Thus, this system may have applications in a variety of biomedical fields.

Keywords: Antibody localization; Dye; Luciferase; Luminescence; Rapid immunoassay.