Compared to 7A3-17B3, with the best performing sandwich immunoassay, 7A3-10A9 and 7A3-7F2, which are representative of medium-and low-performing mAb pairs, exhibited lower success rates in the sandwich immunoassays for model analytes with varied epitope distances (Fig. ). Besides the epitope distance, the success of sandwich immunoassay was found to heavily depend on the recognition property of the two mAbs employed. Thus, we chose 7A3-10A9 and 7A3-7F2 to address the importance of the selected mAbs for the performance of sandwich immunoassay and carried out docking and molecular dynamics of the ternary complexes with MN7. The mAbs 10A9 and 7F2 with different recognition properties were produced from MEL haptens N1 and N5, respectively. Similar to the 7A3-MN7-17B3 ternary construction, NIA was first docked to 10A9 and 7F2. The epitope distances of the 7A3-10A9 and 7A3-7F2 shared cavities were measured as 8.3 and 11.9 Å, respectively, indicating that the 10A9 and NIA binding cavities were shallower than those of 7F2 and NIA (Supplementary Fig. ). It can be assumed that 10A9 from hapten N1, possessing the shortest spacer arm, may only recognize the fractional epitope of NIA due to the shielding effect of the carrier and then forms a shallow binding cavity (Fig. ). In contrast, 7F2 from hapten N5, with a longer spacer arm, recognizes not only NIA epitopes but also part of the spacer arms to form a deep binding cavity. Subsequently, molecular dynamics analysis of the two mAb pairs, 7A3-10A9 and 7A3-7F2, was conducted, and stable ternary complexes were obtained (Supplementary Fig. , Fig. , and video). Interaction between 10A9 and NIA occurs only at the nitro group end in the 7A3-MN7-10A9 complex, as the shallow binding cavity of 10A9 causes the flexible linker of MN7 to become more exposed. This causes the analyte to fold and accounts for the medium performance of the sandwich immunoassay (Fig. ). For 7A3-MN7-7F2, the NIA epitope of MN7 is deeply inserted into the binding cavity of 7F2 and forms hydrogen bonds with D338, Q443, and Y445 in 7F2 (Fig. ). This is unfavorable to the simultaneous binding of the other mAbs to the analytes due to steric hindrance and leads to the poor performance of the sandwich immunoassay. The results showed that the recognition properties of mAbs significantly affect the performances of sandwich immunoassays for analytes and are closely related to the designed hapten structure. Compared to 17B3 from hapten N4, which performed the best in sandwich immunoassay, 10A9 and 7F2 from haptens with too short or long spacer arms usually formed shallow or deep binding cavities and induced steric hindrance, which is not suitable for the sandwich immunoassay of small molecules. The study provides significant reference value for the selection of paired mAbs that may be useful to create sandwich immunoassays with less steric hindrance.