Each hapten-BSA was used to immunize a set of 10 BALB/c mice three times at 21-day intervals. The mice antisera of 12 groups respectively immunized with haptens M1-M6 and N1-N6 were monitored from the second immunization. MEL or NIA was used to determine the antibody titer and affinity by ncELISA and ciELISA. In the study, the antibody titer is defined as the antisera dilution that furnished an ODmax between 1.5 and 2.0, while antibody affinity is expressed as the inhibition ratio calculated according to Eq. S2 at a MEL or NIA concentration of 10 μg/mL as described in the Supplementary Information. In short, a higher antibody titer or antibody affinity indicates a stronger antibody response. All haptens with various spacer arm lengths generated specific antibody responses to MEL or NIA in all mice. In addition, the levels of antibody response varied significantly depending on the degree of exposure of the epitope to the immune system via the spacer arm (Supplementary Table ). The study aimed to produce mAbs that maximally recognize the two epitopes (MEL and NIA) with as much diversity as possible to facilitate the development of sandwich immunoassays for the model analytes. Thus, mice with the highest affinity to MEL or NIA from all groups were chosen to produce mAbs (Supplementary Table ). Notably, the mAbs prepared from haptens M1, M5, and M6 failed due to their low titer and poor affinity. In total, 17 mAbs were obtained, including 6 anti-MEL mAbs and 11 anti-NIA mAbs (Supplementary Table ). Specifically, mAbs induced by haptens M3 and M4, possessing a moderate spacer arm length, showed comparable affinities to MEL with IC50 values ranging from 16 to 79 ng/mL. mAbs from haptens N3 and N4 showed high affinities toward NIA, with IC50 values ranging from 81 to 601 ng/mL, which were nearly 2-67 times better than those of mAbs from haptens N1, N2, N5, and N6 with short or long spacer arm lengths.