Enzymatic synthesis of antibody-human serum albumin conjugate for targeted drug delivery using tyrosinase from Agaricus bisporus

• Autor(a) principal: Rollett, Alexandra
• Data de Publicação: 2013
• Outros Autores: Thallinger, Barbara, Ohradanova-Repic, Anna, Machacek, Christian, Walenta, Evelyn, Paulo, Artur Cavaco, Birner-Gruenberger, Ruth, Bogner-Strauss, Juliane G., Stockinger, Hannes, Guebitz, G. M.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/1822/27462
• topic: Science & Technology

Highly specific targeted drug delivery devices can be obtained with antibody-human serum albumin (mAb-HSA) conjugates. However, their conventional production involves several reaction steps including chemical modification and activation of both proteins followed by cross-linking often involving toxic chemicals. Here, we describe the enzymatic synthesis of mAb-HSA conjugates for targeted drug delivery devices using tyrosinase from Agaricus bisporus under mild reaction conditions (pH 6.8, 25 [degree]C). Reaction conditions were optimized by using fluorescence labeled HSA to facilitate SDS-PAGE analysis with fluorescence scanning. Enzymatic cross-linking in the presence of natural low molecular weight phenolic compounds (e.g. caffeic acid) resulted in reaction products in the molecular weight range of [similar]216 kDa, corresponding to mAb-HSA conjugates. The composition of the conjugates was confirmed with tryptic digestion followed by LC-MS/MS analysis of the resulting peptide fragments. Successful binding of mAb-HSA conjugates (in contrast to free HSA) to MHC II molecules, located on antigen-presenting cells, was demonstrated by both ELISA and flow cytometry analysis.