In the above sandwich immunoassay experiment (Figs. and), the sensitivity of the sandwich immunoassay based on 7A3-17B3 for MN5 was significantly better than that for MN1-MN4 but far worse than that for MN6-MN9, indicating that the epitope distance of the analyte is critical for the sensitivity of the sandwich immunoassay. We then studied the correlation between the analyte epitope distance and the sensitivity of the sandwich immunoassay using MN5, MN7, and MN9 with the same 7A3-17B3 mAb pair. Steric hindrance was observed when MN5 was clamped by 7A3 and 17B3 simultaneously in the real binding process, as indicated by the relatively lower sensitivity in the sandwich immunoassay with a LOD of 265.4 nM (Fig. ). Thus, we analyzed the recognition mechanism of the 7A3-MN5-17B3 ternary complex to gain insight into the interactions. The binding cavity of the complex was mainly maintained by hydrogen bonds provided by G307 of 7A3 as well as R55 and N40 of 17B3. For MN7, the epitope distance of 13.9 Å prevents damage to the sensitivity of the sandwich immunoassay from steric hindrance to the greatest extent (Fig. ). The 7A3-MN7-17B3 ternary complex affording the most sensitivity sandwich immunoassay with a LOD of 0.00035 nM, was maintained by conventional hydrogen bonds and hydrophobic forces from Y379, D327, E319, L439, and S370 of 7A3 as well as Y210, Y212, and Y152 of 17B3 (Fig. and Supplementary Fig. ). Surprisingly, a hydrogen bond formed between the NIA epitope and S370 of 7A3 from hapten M4, indicating that the 7A3-MN7 binding cavity was tighter than that of 7A3-MN5. Thus, increased interaction forces made the 7A3-MN7-17B3 complex more stable compared to the 7A3-MN5-17B3 complex, improving its sensitivity in sandwich immunoassays. MN9, with the longest epitope distance of 19.0 Å, provided a 2,500-fold worse sensitivity than that of MN7 in the sandwich immunoassay. Following molecular dynamics study, the expected stable ternary complex 7A3-MN9-17B3 was obtained at 20 ns. The lower sensitivity of the sandwich immunoassay for MN9, with a LOD of 0.9 nM, can be attributed to the higher flexibility of MN9 due to its longer epitope distance, increasing the stretch degree between the ends of the two mAbs (Supplementary Fig. , Fig. , and video). The RMSD of 0.6 Å for the 7A3-MN9-17B3 complex was higher than the 0.4 Å for the 7A3-MN7-17B3 complex, further showing that long epitope distances of an analyte may not always be conducive to sandwich immunoassays with high sensitivity. The epitope folding of the analyte caused by long flexible chains should be considered to develop a robust sandwich immunoassay for small molecules. These results show that an analyte epitope distance of at least 8.8 Å can bind two mAbs with acceptable steric hindrance, and an epitope distance of ~13.9 Å was suggested to develop sandwich immunoassays with high sensitivity.