It was shown that it distinguishes between brain tissue samples from CJD – affected and nonCJD-affected patients reacting only with the native PrPSc deposits in immunohistochemical assays. Because of these properties, it has a great potential to be used in immunodiagnostic procedures or prion research. It was already used to induce anti-idiotypic response in mice and chicken in order to produce anti-idiotypic antibodies, which represented a new experimental approach in prion research. To better understand the narrow selectivity of V5B2, interaction between the antibody and P1 peptide was investigated, but only the most recent studies revealed that V5B2 selectively recognizes the newly discovered C-terminally truncated PrP, named PrP226*. To prepare a more suitable form of V5B2 for further immunotherapeutic development, recombinant single-chain antibody fragments have been derived from the mAb. However, murine antibody fragments are immunogenic, which is the major obstacle to their clinical application. For that reason, we decided to reduce its immunogenicity by humanization. The present data describe the humanization of the antibody single-chain fragment V5B2 and its characterization. To our knowledge, this is the first report of an anti-PrP mAb being humanized. We rationally designed four variants of humanized scFvs V5B2 by resurfacing of variable regions guided by computer modelling. By site-directed mutagenesis, human amino acid residues were stepwise introduced into murine variable regions. After being produced in E. coli using pMD204 expression vector, humanized antibody fragments were purified from the periplasm and their antigen-binding LEE011 molecular weight properties were analysed. The optimized construct was a scFv with 13 mutations introduced at key positions in the structure, which retained stability, binding specificity and affinity of the parent antibody. We believe that the recombinant humanized scFv with preserved functional properties of V5B2 could be used for designing new compounds with potentially diagnostic and therapeutic anti-prion properties. Single-chain Fvs are much smaller than whole antibodies, besides they do not require glycosylation and can be produced in a bacterial expression system. Such production is simpler, faster and significantly reduces production costs. Therefore, instead of full-length V5B2 antibody the scFv form was chosen to be humanized and produced. We have successfully humanized a single-chain antibody fragment of the murine monoclonal antibody V5B2, specific for PrPScfragment associated with prion-infected samples. We constructed over 30 scFvs on the DNA level and expressed, purified and examined a total of 14 humanized constructs: 4 for basic stages and 10 intermediates between different stages in search of destabilizing mutations. Construct with optimal binding properties and maximal possible amino acid replacements was denoted huScFv V5B2.