Minimum Distance Between Two Epitopes in Sandwich Immunoassays for Small Molecules

The pursuit of the limit between dimensionalities is a scientific goal with high 16 applicability. Sandwich immunoassay, usually based on two antibodies binding two 17 epitopes, is one of the most popular mainstay tools in both academic and industrial 18 fields. Herein, we determined and evaluated the minimum distance of two epitopes in 19 sandwich immunoassays for small molecules. Briefly, nine model analytes comprising 20 two hapten epitopes, i.e., melamine (MEL) and p -nitroaniline (NIA), were designed by 21 increasing the linear chain linkers brick by brick. Two groups of monoclonal antibodies 22 (mAbs) were produced with different recognition properties toward MEL and NIA 23 using 12 new haptens with different spacer arms. The results indicated that two epitopes 24 of the analyte with a distance of only 2.4 Å could be simultaneously bound by two 25 mAbs, which is the known limit of epitope distance in sandwich immunoassays thus 26 far. We further found that an epitope distance of below 8.8 Å for the analyte generally 27 induces noticeable steric hindrance of antibodies, preventing a sandwich immunoassay 28 with high probability. These observations were investigated and evaluated by molecular 29 docking, molecular dynamics, and surface plasmon resonance and using model and real 30 analytes. Altogether, we determined the minimum distance of two epitopes and 31 explored the molecular mechanism of the antibody–analyte–antibody ternary complex 32 in sandwich immunoassays, providing a theoretical basis for hapten design, antibody 33 discovery and development, and sandwich immunoassay establishment for small 34 molecules. The 0.6 Å 7A3–MN9–17B3 0.4 Å 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.