To validate the epitope distance required by establishing a successful sandwich immunoassay for small molecules, we designed and synthesized two new model analytes using typical antibacterial agents, SBA and NOR, that are widely used in human and veterinary clinics. The synthesized analytes of SBA-C2-NOR and SBA-C10-NOR are composed of SBA and NOR, separated by epitope distances of 3.8 and 13.9 Å, respectively (Fig. ). The mAbs against SBA and NOR, i.e., 4D11 and N2H3A8, were previously prepared by our group and used to establish sandwich immunoassays. Both model analytes worked successfully in the established sandwich immunoassays, with LODs of 0.30 and 0.02 nM for SBA-C2-NOR and SBA-C10-NOR, respectively (Fig. ). The results showed that successful sandwich immunoassays could be achieved for small molecules even with short epitope distances above 2.4 Å. The effect of epitope distance of analyte on the sensitivity of the sandwich immunoassay was also further observed. The LOD of the sandwich immunoassay for SBA-C10-NOR with the suggested epitope distance of 13.9 Å was more than 10-fold that of SBA-C2-NOR with a short epitope distance of 3.8 Å (Fig. and Supplementary Table ). Moreover, the sandwich immunoassays provide clear advantages in sensitivity, detection range, and specificity compared to competitive immunoassays for new model analytes, especially for SBA-C10-NOR, where the LOD improved nearly 30-fold (Supplementary Table ). For assay specificity, the sandwich immunoassays showed extremely high specificities toward the target analyte due to the simultaneous use of two mAbs (Supplementary Table ), while competitive immunoassays provided structural analogs with high cross-reactivities, such as sulfaguanidine and ciprofloxacin, with the potential risk of false positives.