[Overview] A representative immunoassay method, ELISA, requires laborious and time-consuming washing steps. Immunochromatography strip-test, which is rapid and easy handled, is used in various fields such as food safety and diagnostics. However, it is not suitable for quantitative detection and its sensitivity is relatively low. To overcome such drawbacks of these assays, we developed a novel homogeneous assay using luciferase-antibody complex (LucAb) and dye that absorbs the light from LucAb. In this system, when antigen is absent, luminescence of free LucAb is absorbed by dye, which uniformly dispersed in the solution (Figure). On the other hand, when antigen is localized on the neighboring area of the PMT, a portion of LucAb rapidly moves to the antigen around the PMT, therefore the light of the LucAb gathered on the antigen reach to the PMT without attenuation. The new system can rapidly detect the amount of the antigen-antibody-luc complex. [Results] We selected blue dextran as a suitable dye for the system, because it absorbs most of the luminescence wavelength of luc, but not inhibits luc activity enzymatically. Then, feasibility of this system was investigated by a model experiment, using biotinylated Luc (b-Luc) and streptavidin beads (SA beads). After mixing reagents and placing the tube on a magnet to localize “b-Luc bound-SA beads” for 10 s, the signal intensity was 7.8 times higher than that of mock using non-binding carboxylic acid beads. Furthermore, the detection of E. coli O157 was also examined. After incubation of 4 X 105 cells of E. coli O157 and LucAb for 30 min followed by centrifugation, the PMT signal was 4.7 times higher than that without E. coli O157 cells, indicating the applicability of the rapid immune assay system to food poisoning bacteria. [Acknowledgement] Aichi Science and Technology Foundation and Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) financially supported this work.

Development of a novel immunosorbent homogeneous assay system using luciferase-antibody complex and dye for luminescence absorption

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.