Thereafter, MN1-MN9 with various epitope distances were superimposed onto the shared binding cavity of 7A3-17B3 with extended conformations. The results showed that MN1-MN4 with epitope distances of 2.4-6.3 Å were too short to be superimposed onto the shared binding cavity, meaning that stable antibody-analyte-antibody ternary complexes could not be formed using these model analytes (Fig. ). Steric hindrance occurs when MN1-MN4 bind to two mAbs at the same time, and only weak sandwich immunoassays could be achieved as a result (Fig. ). MN5 with an epitope distance of 8.8 Å, comparable to the distance of the shared binding cavity of 7A3-17B3, was then superimposed onto the shared binding cavity, and the initial ternary complex was successfully generated as expected. Molecular dynamics study provided the stable conformation of the 7A3-MN5-17B3 ternary complex, with a root mean square deviation (RMSD) of 0.6 Å in 60-100 ns (Fig. and Supplementary Fig. ). Thus, the epitope distance of ~8.8 Å for the analyte was considered the shortest for establishing sandwich immunoassays with high success rates for small molecules, as demonstrated both experimentally and through molecular dynamics study.