SPR analysis was further used to validate the specific binding of antibodyanalyte-antibody ternary structures formed in the sandwich immunoassays based on the 7A3-17B3 pair. First, mAb 7A3 was immobilized on a chip, and then, each model analyte was added at 10 µM, except for MN1 that was added at 100 µM. It should be stated that MN3 and MN8 could not be analyzed by SPR due to their poor solubility in PBS. All tested model analytes demonstrated increased signals at step I, indicating successful binding between 7A3 and the model analytes (Fig. ). Different degrees of dissociation were then observed over time in step II as the 7A3-analyte complex gradually became stable. Afterward, mAb 17B3 was added to bind the 7A3-analyte complex, and the signal was expected to increase further. The signal still increased significantly during step III, including for the smallest analyte MN1. To confirm whether the increased signal was specific, we set up a control to verify the binding between the two mAbs without adding any model analyte but PBS. The PBS signal was maintained at the baseline through the analytical procedure, indicating that no specific binding had occurred between the two mAbs and proving that the increased signal observed in step III was caused by the binding of 17B3 to the 7A3-analyte complex (Fig. ). For MN1, MN2, and MN4, the signals increased slightly by 10-20 units after the addition of 17B3, while those for MN6, MN7, and MN9 increased more noticeably by 20-40 units, which is consistent with the performance of the model analytes in the established sandwich immunoassay. Furthermore, MN7, achieving the highest sensitivity in the established sandwich immunoassay, was analyzed by SPR at concentrations of 1-10 μM. The 7A3-MN7 signal gradually increased in step I, followed by varying degrees of dissociation as the MN7 concentration increased during step II (Fig. ). After the addition of 17B3 in step III, the elevated signal of the 7A3analyte-17B3 ternary complex was observed, again indicating the formation of a stable antibody-analyte-antibody ternary complex.