Development of Highly Sensitive Ru-Chelate Based ECL Immunoassay 2: Electrochemical and Immunochemical Studies on Homogeneous and Heterogeneous ECL Excitation

Studies on the electrochemical and immunochemical reaction kinetics of the heterogeneous type of ECL excitations were made comparatively with homogeneous types of ECL excitations by measuring human IgG (hIgG) using an antibody labeled with a ruthenium(II) tris(bipyridyl) (Ru-chelate) as the luminophore (Ru-Ab). Solid-phase sandwich-type immunoassays were carried out on the surface of magnetic micro-beads (MB) with a diameter of 4.5 µm. In the ECL measurement, two types of ECL excitation methods were compared. One was a homogeneous ECL excitation, where the reacted MB together with non-reacted Ru-Ab were excited in a suspending phase without any bind/free (B/F) separation procedure. The other was a heterogeneous one where the reacted MB were excited over the electrode after being collected by a magnet following the B/F separation to remove the non-reacted Ru-Ab. In electrochemical studies, it was revealed that the Ru-Ab reacted with hIgG decreased the ECL emission efficiency. The decreasing ratio was inversely correlated with the cubic root of the luminophore molecular weight. In homogenous ECL excitation for the reacted matrix containing both the reacted MB and the non-reacted Ab, however, a reverse correlating dose response curve appeared only in the area beyond the antigen-antibody equivalent point, the so-called antigen excess zone; as a result the S/N ratio of the ECL signal was as small as only 1.3. In contrast, the heterogeneous ECL excitation for the reacted MB, with the non-reacted Ru-Ab removed by B/F separation, demonstrated 1000 or more times the S/N ratio in the area before the antigen-antibody equivalent point. Thus, this heterogeneous ECL excitation with B/F separation improved the detection sensitivity dramatically up to 1000 or more times higher than that of the homogeneous ECL excitation. Consequently, the sensitivity of heterogeneous ECLIA could be competitive with that of the conventional chemiluminescence immunoassay.