Over 90% of mAb pairs allowed for the efficient sandwich immunoassays of analytes MN7-MN9 with an epitope distance of at least 13.9 Å, indicating successful simultaneous binding for most mAb pairs. Notably, unsuccessful mAb pairs were observed in all model analytes, showing that the production of mAbs with diverse recognition properties is important and necessary to establish sandwich immunoassays for small molecules. Despite this, the overall sandwich formation rates of mAbs from haptens N1 and N2 are much lower than those of mAbs from haptens N3 and N4 with moderate spacer arm lengths. The results further implied that haptens with extra-long or short spacer arms should be avoided to produce a suitable antibody and establish efficient sandwich immunoassays for small molecules.