Autor(es):
Rollett, Alexandra 
; Thallinger, Barbara ; Ohradanova-Repic, Anna ; Machacek, Christian ; Walenta, Evelyn ; Paulo, Artur Cavaco ; Birner-Gruenberger, Ruth ; Bogner-Strauss, Juliane G. ; Stockinger, Hannes ; Guebitz, G. M.
Data: 2013
Identificador Persistente: http://hdl.handle.net/1822/27462
Origem: RepositóriUM - Universidade do Minho
Descrição
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.
