Recently, we have developed synthetic polymer-based antibody mimetics called iBodies that can successfully replace antibodies in many biochemical applications including ELISA (enzyme-linked immunosorbent assay), flow cytometry, immunocytochemistry, affinity purification, and confocal microscopy. In this study, we focused primarily on the effect of a linker between the polymer backbone and biotin, and then on the influence of the number of biotins per polymer chain on the efficacy of the ELISA or pull-down assays. In addition, the biotin position on the polymer chain was investigated. Moreover, we developed a novel chain transfer agent suitable for RAFT polymerization, which enables the functionalization of specifically the polymer precursors and simplifies the synthesis of semitelechelic antibody mimetic materials. By employing optimized iBodies the sensitivity of the ELISA with a lengthened linker between the polymer backbone and biotin was increased up to 5 times. Importantly, we found that one biotin at the end of the polymer chain can replace up to 12 biotins located along the polymer chain and maintain the signal level in the ELISA, as well as in the pull-down assay.