Abstract The tumor-associated structure N-acetyl-galactosamine-O-Ser/Thr (Tn antigen), which is overexpressed in various tumor cell types, notably of the breast, ovary and colon, is an interesting determinant that is useful for cancer diagnosis and follow-up. The aim of this research was to study different assay strategies in order to determine the most sensitive system for further application in epitope characterization and binding assessment. The tetrameric isolectin obtained from Vicia villosa seeds (VVLB 4) shows high affinity for the tumor-associated structure. A monoclonal antibody against VVLB 4, MabVV 34, was generated, and the interaction between MabVV 34 and VVLB 4 was studied by means of binding and inhibition assays. Several synthetic peptides (10 amino acid sequences) designed from the amino acid sequence of VVLB 4 and obtained from trypsin digestion were tested to determine which amino acids were involved in the interaction between MabVV 34 and VVLB 4. The further unraveling of this epitope was investigated by inhibition using designed synthetic peptides as well as mixtures mimicking variable density effect. Under the experimental circumstances, MabVV 34 was able to inhibit the binding of VVLB 4 to Tn. Two of the four peptide sequences assayed showed better inhibition properties. Finally, mixtures containing these selected sequences allowed the evaluation of binding and inhibition as a function of Tn density. We conclude that the present study facilitates the further development of a specific Tn marker and may contribute to the development of Tn-like radiolabelled peptides or Tn-specific radiolabelled fragments providing a highly selective tool for cancer diagnosis and treatment. This strategy may contribute to characterize the new generation of radiopharmaceuticals for diagnosis and therapy based on biomolecules like antibodies, fragments or peptides, whose application is directly guided by their specific molecular recognition.