In the present study, we address these questions using two typical hapten epitopes, melamine (MEL) and p-nitroaniline (NIA), that were connected by linkers with an increasing linear alkane length of 1-14 methylenes to create nine model analytes. Two groups of monoclonal antibodies (mAbs) against MEL and NIA were separately produced based on 12 newly designed haptens, including six MEL haptens and six NIA haptens. Numerous mAb pairings were studied to investigate combinatorial associations and determine the minimum two-epitope distance of the analyte that could still be simultaneously bound by two distinct mAbs. The molecular mechanism of antibody-analyte-antibody recognition in sandwich immunoassay was further investigated by molecular docking, molecular dynamics, and surface plasmon resonance (SPR). Finally, model analytes, i.e., sulfabenzamide-norfloxacin (SBA-NOR) conjugates, and a real analyte, namely abamectin (ABM), were used to evaluate the conclusions of this study. Microtiter plates and cell culture plates were purchased from Corning Inc. (New York, NY, USA). The mAbs were conjugated with HRP by using an HRP conjugation kit from Abcam (Cambridge, MA, USA). The buffer and solution used in this work can be found in the Supplementary Information.